Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level

APPLICABILITY OF LIGHT EMITTING DIODE

IRRADIATION IN PHYSIOTHERAPY

Elke Vinck

Thesis submitted in fulfilment of the requirements for the degree of Doctor in Motor Rehabilitation and Physiotherapy

Promotor prof dr D Cambier

Department of Rehabilitation Sciences and Physiotherapy Faculty of Medicine and Health Sciences

Ghent University Academic Year 2005-2006

Promotor

Prof dr D Cambier Ghent University Belgium

Examination Board

Prof dr P Calders Artevelde University College Belgium


Prof dr M Cornelissen Ghent University Belgium

Prof dr M De Muynck Ghent University Belgium

Prof dr M Dyson University of London UK

Prof dr P Lievens Free University Brussels Belgium

Prof dr K Peers Catholic University Leuven Belgium

Prof dr G Vanderstraeten Ghent University Belgium

Process Supervisory Board





VII

TABLE OF CONTENTS

GENERAL INTRODUCTION 1

Background 3

Physical characteristics 6

Mechanisms of action 12

Aims and outline 15

PART I WOUND HEALING 25

Chapter 1 Do infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment

27

Chapter 2 Increased fibroblast proliferation induced by light emitting diode and low level laser irradiation

47

Chapter 3 Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level

61

PART II ANALGESIA 73

Chapter 4 Evidence of changes in sural nerve conduction mediated by light emitting diode irradiation

75

Chapter 5 Pain reduction by infrared light emitting diode irradiation a pilot study on experimentally induced delayed-onset muscle soreness in humans

91

GENERAL DISCUSSION 111

Summary 113

Clinical implications and future research directions 118

Final conclusion 123

NEDERLANDSTALIGE SAMENVATTING 129

Do not attempt to do a thing unless you are sure of yourself

but do not relinquish it simply because someone else is not sure of you

(Stewart E White)

IX

ACKNOWLEDGEMENTS

I wish to thank those people who supported me over the years and who helped me to

shape my life and work

First of all I would like to express my gratitude towards my promotor prof dr D

Cambier as without his encouraging words criticism inspiration and unremitting

support I would still be floundering about the contents of chapter 1 Thank you for

your good advice when I needed it the most

The members of the supervisory committee prof dr M Cornelissen prof dr M De

Muynck and prof dr G Vanderstraeten thank you for your assistance and helpful

feedback during the process of formation of this thesis

I also gratefully acknowledge the external members of the examination board prof dr

P Calders prof dr M Dyson prof dr P Lievens and prof dr K Peers for their

constructive reflections which contributed to the improvement of this thesis

I am greatly indebted to my special mentor prof dr J Anders of the Uniformed

Services University of Bethesda Maryland for the research suggestions she made as

well as for her unlimited belief in the value of my work

I wish to thank prof dr L Deridder for providing access to the laboratory of

Histology the Centre of Sports Medicine of the Ghent University Hospital for

allowing me to use their equipment as well as MDB-Laser Belgium for generously

providing the light emitting diode equipment

Sincere appreciation is extended to the volunteers that participated in this study and to

Tom and Roel for their valuable technical assistance in the collection of the data as

well as for their useful input into the research design of the investigation described in

chapter 5

X

Warm thanks go to the colleagues of the department of Human Anatomy

Embryology Histology and Medical Physics for providing the culture medium for the

technical support for the helpful discussions and principally for the amusing pastime

aseptic chats

In addition I also want to thank my colleagues of the associated institute Kinesitherapie

Gent and above all the colleagues of the department of Rehabilitation Sciences and

Physiotherapy Ann Axel Barbara Bart Bert Bihiyga Bruno Carine Damien Els2

Erik Frike Helga Inge James Karlien Kathy Kat(h)leen Koen Marc Martine

Mieke Paris (Nele) Patrick Roland Sophie Stefan Stijn (Veerle) Wiene Wim and

Youri thank you for the organisation and your attendance at many memorable

sidelines such as the survival-weekend the first department-day Fata Revaki our

legendary Thursday-sport activities and Friday-afternoon coffee breaks St Nicholas

visit skiing holiday New Yearrsquos lunch with quiz bowling spinning the introduction of

ldquosubetelyrdquo during our conspicuous presence at WCPT 2003 et cetera We shared many

treasured moments thanks to you a common working day often turned out to be very

pleasant I know that it will be impossible to find a comparable team of colleagues to

work with in the future

I especially want to thank Barbara to remind me on a regularly basis of my deadlines

to listen to my grieves and joy and to be willing to offer me a window-seat in our

office Kurt (although you abandoned at a certain moment) for solving my computer

problems Pascal for assistance with the statistical analyses Lieven for your motivating

interest and finally Fabienne Tine and Kim as loyal and appreciated friends who

worn-out several sports shoesbathing suits to supply in the weekly portion of sports I

needed to remain physically and mentally fit

I also extend my appreciation to my family and friends for their interest in my research

activities permanent mental support for the adoption of the surviving chickens but in

particular for looking after Louka and for the numerous relaxing moments Special

thanks are going to Katelijne and Frank for the delicious hot meals on lonely evenings

XI

Thomas Stijn and Johan thank you for the repeated (mostly futile) attempts to

convince me to do something together Sebastiaan each time during the past few years

when I doubted about the sense of my work it was your ridiculous story about a man

who wanted to invent superglue but instead invented the well-known yellow post-it

which stimulated me to continue my scientific quest

Of course I owe most gratitude to Luc my most devoted supporter Dearest I know

that since august 2004 you lived a solitary life in Dubai Although I think it was

possibly easier not to live under the same roof with me these last stressful months I

am aware that it was very difficult for you not to be able to play with Louka and to

miss some precious months of her life

Louka thank you for your radiant smile and daily baby speeches I am sorry that you

had to miss your daddy I promise that we will be reunited very soon

Elke Vinck

Ghent March 2006

GENERAL INTRODUCTION

General introduction

3

BACKGROUND

The use of light for therapeutic purposes reaches far back in time Current interest for

photomedicine with his its biological and medical effects relies fundamentally on two

major evolutions in the given field (1) the research results regarding the use of

ultraviolet (UV) radiation by the end of the 19th century and (2) the developments in

the light amplification by stimulated emission of radiation (laser)-technology The production

of the first laser the ruby pulsed laser was rapidly succeeded by the development of

the helium-neon laser and other lasers like the argon the neodymium-glass and the

neodymium-yttrium-aluminium-garnet lasers1

As in the mid-1990s semiconductor and diode-based lasers gained popularity the

principally massive gas and dye lasers were rendered obsolete Therapeutic light

technology further continued to evolve and todayrsquos therapeutic light source is as likely

to be a light emitting diode (LED) or polarized light as a semiconductor or diode

laser1

Technological advancement and variation of the light sources necessitate a

concomitant update and revision of research in the respective domains of application

Unfortunately this logical and rational necessity has rarely been fulfilled From a

historical perspective this lack of appropriate research has led to disenchanting

evolutions in the use of light especially in physiotherapy The experience exists in this

medical field that light sources were promoted and commercialised for a vast regimen

of indications without foregoing scientific backup Consequently research developed

often after the commercial introduction in physiotherapy As these investigations

frequently gave rise to conflicting results for certain indications scepticism arose and

the use of the given modality knew a waning popularity for all its indications The final

result of such an inappropriate frame of promotion commercialisation and research is

a growing clinical disuse of a given modality even for motivated indications In view of

the actual increasing interest in LED treatment and based on former ascertainment

one has to state that a literature review for the given source reveals that research

mostly covers only low level laser (LLL) studies23 Although recently a number of

papers can be noted that report on the effects of LEDs and polarized light still

4

numerous source-specific-questions need to be answered as research concerning

mechanisms of action and efficacy of the current light sources remains limited in view

of a substantiated clinical application4-17

The reason for the contemporary light-oriented interest in physiotherapeutic practice

for LED devices is in essence based on several advantages of LED in comparison with

LLL For example the use of LEDs is esteemed to be safer as the delivered power

does not damage tissue LEDs can be made to produce multiple wavelengths thereby

stimulating outright a broader range of tissue types and probes that cover a large

treatment area are available18 In addition from a commercial point of view LEDs are

far more interesting as they are a good deal cheaper than laser diodes and they have a

long life span as these solid devices stand robust handling

As a result of the above-mentioned lack of literature on LED some providers of these

devices have taken for granted that the biological response of tissue to light irradiation

cannot be equated merely to a light source They declare that a given response solely

depends on the extent of absorption of radiated light by the tissue19 Consequently

these providers state that it is acceptable to extrapolate scientific findings of LLL

studies for explaining the mechanisms of action and detailing the efficacy of LED and

other alternative light sources Thus actually without appropriate scientific support

equal biological effects are attributed to LED as to LLL Nevertheless prudence is

called for such an extrapolation firstly because it is irrespective of the mentioned

dissimilarities and by simple projection one ignores a number of physical differences

between LLL and LED (eg coherence and degree of collimation or divergence)

Secondly LLL therapy is still not yet an established and evidence-based clinical tool20

Notwithstanding the historical efforts there still remains a considerable amount of

ignorance scepticism and controversy concerning the use and clinical efficacy of

LLL321-26 This ascertainment can be attributed to the broad spectrum of proposed

parameters for irradiation as well as to the difficult objective measurement of possible

irradiation effects and even to the exceptional range of unsubstantiated indications for


5

which light therapy was promoted27-29 A lack of theoretical understanding can also be

responsible for the existing controversies as the evaluation and interpretation of

research results would be simplified largely when the appropriate knowledge about the

mechanisms of light action would be available

LLL literature can undoubtedly be used as basis for research on LED and as a

comparative reference for these given investigations However to guarantee evidence-

based use of LED within physiotherapy the need for specific research in view of an

accurate consumption of LED is definite especially for potential promising clinical

applications in physiotherapy according to LLL literature mainly wound healing and

analgesia3031

Hitherto the most substantial research concerning the use of LED for improvement

of wound healing is provided by Whelan and colleagues1832-34 As healing is retarded

under the influence of prolonged exposure to microgravity (eg during long-term space

flights) and in case of absence of exposure to sunlight such as in submarine

atmospheres they performed wound healing experiments for military application in the

given circumstances3233 In vitro experiments revealed that LED treatment increased

proliferation of 3T3 fibroblasts and L6 rat skeletal muscle cell lines doubled DNA

synthesis in LED treated fibroblasts and accelerated growth rate of fibroblasts and

osteoblasts during the initial growing phase183233 Animal in vivo wound healing studies

demonstrated therapeutic benefits of LED in speeding the early phase of wound

closure and in changing gene expression in a type 2 diabetic mouse model183234

Human studies noted 50 faster healing of lacerations a return of sensation and

increased tissue granulation as a result of LED irradiation1833

Associates of the Rehabilitation Sciences Research Group of the Ulster University in

Northern Ireland extensively investigated the effectiveness of light in the treatment of

pain The emphasis was laid primarily on the analysis of the effects of various low level

laser light sources35-44 However in the year 2001 two studies gave an account on the

efficacy of non-laser sources the so-called multisource diode arrays4546 Noble et al46

6

noticed relatively long-lasting neurophysiological effects a significant change of the

nerve conduction characteristics (decrease of the negative peak latency) was mediated

by a monochromatic multisource infrared diode device Glasgow et al45 suggested that a

comparable multisource diode device was ineffective in the management of delayed-

onset of muscle soreness (DOMS)

Despite the major value of these described trials a definitive answer regarding the

ability of LED in influencing wound healing or pain is not forthcoming cardinally

because a number of aspects are not yet investigated Consequently more research is

required in order to avoid inaccurate use of LED As inaccuracy leads inevitable to the

formerly mentioned scepticism regarding the effectiveness of a medium and possibly

to the undeserved fall into disuse of the treatment modality which happened in a way

with LLL therapy

PHYSICAL CHARACTERISTICS

This chapter supplies a short but comprehensive review of opto-physics A brief

description of the physical characteristics of the LED source used is essential as the

physical properties of light play an important part in the ultimate efficacy of treatment

According to the International Electrotechnical Commission (IEC 60825-1) an LED

can be defined as

Any semiconductor p-n junction device which can be made to produce electromagnetic radiation by

radiative recombination in the wavelength range of 180 nm to 1 mm produced primarily by the process

of spontaneous emission1947

The LED device used for this doctoral thesis is depicted in figure 1 (BIO-DIO

preprototype MDB-Laser Belgium) This illustration shows that a probe consists of

32 single LEDs disseminated over a surface of 18 cm2


7

Figure 1 LED device and three available probes (infrared red and green)

Three highly monochromatic probes were available each emitting light of a different

wavelength within the above-defined range (table 1)2748 The wavelength of the light

emitted and thus its colour depends on the band gap energy of the materials forming

the p-n junctiona This light property is a key determinant to obtain maximum

photochemical or biological responses as light absorption by tissue molecules is

wavelength specific27 Only by absorbing radiation of the appropriate wavelength

(namely the wavelengths equal to the energy states of the valence electrons)

photoacceptor molecules will be stimulated resulting in a direct photochemical

reaction284849 The wavelengths of radiation that a molecule can absorb the so-called a A p-n junction is the interface at which a p-type semiconductor (dominated by positive electric charges) and n-type semiconductor (dominated by negative electric charges) make contact with each other The application of sufficient voltage to the semiconductor results in a flow of current allowing electrons to cross the junction into the p region When an electron moves sufficiently close to a positive charge in the p region the two charges re-combine For each recombination of a negative and a positive charge a quantum of electromagnetic energy is emitted in the form of a photon of light44750

8

absorption spectrum of a particular molecule is limited absorption often only occurs

over a waveband range of about 40-60 nm274851 Nevertheless the absorption

spectrum at cell or tissue level is broad because cells are composed of many different

molecules

Besides its influence on the absorption by means of tissue molecules there is a crucial

link between wavelength and penetration depth of the irradiated light Penetration into

tissue decreases as the wavelength shortens hence green light penetrates less than red

light which at his turn penetrates less into tissue than infrared light2748 Detailed

principles of light penetration will be discussed below

The LED device used emits non-coherent light In the 1980s the observed biological

responses after laser irradiation were generally thought to be attributable to the

coherenceb of the light485253 Though currently the clinical and biological significance

of coherence is seriously questioned54 According to several authors coherence does

not play an essential role in laser-tissue interactions firstly as it was proven that both

coherent and non-coherent light clinically show equal efficacy75556 Secondly as

according to some authors almost immediately after transmission of light through the

skin coherence is lost due to refraction and scattering282948 Nevertheless Tuner et

al1957 state that both findings are incorrect coherence is not lost in tissue due to the

phenomenon of scattering and non-coherent light is not as efficient as coherent light

This lack of consensus makes it necessary to mention whether or not light is

coherent2758

Further decisive characteristics to accomplish phototherapeutic efficacy are the power

exposure time output mode and beam area Based on these parameters both

irradiancec and radiant exposured can be calculated According to numerous authors

some of these parameters are more crucial than others to determine whether

b Coherence is a term describing light as waves which are in phase in both time and space Monochromaticity and low divergence are two properties of coherent light48

c Irradiance can be defined as the intensity of light illuminating a given surface The radiometric unit of measure is Wm2 or factors there of (mWcm2)48

d The radiant exposure is a function of irradiance and exposure time thus it is a measure of the total radiant energy applied to an area the unit of measure is Jcm248


9

absorption of light will lead to a photobiological event192728485455 However the

literature yields several controversial findings as not all authors attribute an equal

importance to a given parameter For example according to Nussbaum et al59

irradiance was the determinant characteristic in the biomodulation of Pseudomonas

aeruginosa rather than the expected radiant exposure Moreover van Breugel et al49

found that in order to stimulate tissue cell proliferation a specific combination of

irradiance and exposure time are more important than the actual radiant exposure Low

et al3940 on the contrary highlighted the critical importance of the radiant exposure in

observing neurophysiological effects Whereas Mendez et al60 reported that both

parameters influence the final results of light therapy

Koutna et al61 even suggested that the output mode of light applications plays a more

prominent role in the treatment outcome than the wavelength of the used light source

Nevertheless this finding could not be confirmed by other research results Besides

more controversial findings have been published regarding the output mode although

the repetition rate in a pulsed mode was considered as an important treatment

parameter several investigations failed to prove its value19272840414461-64

Based on these findings it was opted within the investigations of this doctoral thesis to

irradiate in a continuous mode The remaining dosimetric parameters (wavelength

exposure time and power) depended on the purpose of each investigation they are

described in the respective chapters The data necessary for the calculation of the

radiant exposure for the equipment used in the respective trials are summarized in

table 1

Table 1 Wavelength and power-range properties of the three available LED probes Wavelength (nm) Power (mW) Low Medium High

Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10

The radiant exposure of the used LED can be calculated as follows65

RE =

Radiant Exposure [Jcm2]

T = Treatment time [min] P = Power [mW] (see table 1) S = Square irradiated zone[18 cm2]

PRE = α S T

α = 006 (continuous mode) or

003 (pulsed mode)

The parameters commented on so far can be considered as the external dosimetry

involving all parameters directly controlled by the operator limited by the apparatus

used Furthermore there is the so-called internal dosimetry referring to (1) several

physical phenomena (reflection transmission scattering and absorption) influencing

the light distribution within the tissue during energy transfer (2) the optical

characteristics of the irradiated tissue as well as (3) the relation between the external

dosimetry and these respective elements5466

This internal dosimetry determines to a considerable extend the penetration of light

into tissue Penetration can be defined as the tissue depth at which the radiant

exposure is reduced to 37 of its original value1948 However this definition only

accounts for the absolute penetration depth resulting in direct effects of light at that

depth In addition there is also a relative penetration depth leading up to effects

deeper in the irradiated tissue and even in certain degree throughout the entire

body1967 These so-called systemic effects can be caused by chemical processes initiated

at superficial levels at their turn mediating effects at a deeper tissue level57

Involvement of several forms of communication in the tissue such as blood circulation

and transport of transmitters or signal substances is possible1967 This means that light

sources with poor absolute penetration do not necessarily give inferior results than

those with a good absolute penetration19

In the same context it should be noted that calculation and even measurement of the

exact light distribution during irradiation is highly complicated principally as tissues

have complex structures and also because the optical properties of tissues vary largely

inter-individual2768


11

Studies regarding actual penetration depth of LED light are scarce consequently the

knowledge on the topic of penetration depth of LED light is based on literature

originating from LLL research19 These findings established with various LLL sources

revealed that there is an obvious relation between penetration depth and

wavelength27486769-71

Three final remarks can be made on the dosimetry First of all it should be noted that

partly as a result of the above-mentioned contrasting findings on dosimetry ideal light

source characteristics for effective treatment of various medical applications are not yet

established and probably never really will be28 Therefore in the attempt to offer

sufficient guidelines for correct use of treatment parameters one should always try to

provide detailed description of light source properties used in any trial so the

practitioner can interpret the scientific results adequately and accordingly draw the

correct conclusions for his clinical practice

A second comment is based on the mentioned possible influence of the external and

internal dosimetric parameters on the photobiological effectiveness of light the

intrinsic target tissue sensitivity the wavelength specificity of tissue and the relation

between radiated wavelength and penetration depth19546572 So it should be

emphasized that caution is recommended when comparing research results of light

sources with different wavelengths or other dissimilar dosimetric parameters

A third and final remark considers the extrapolation issue Comparison of the

therapeutic usefulness of the same light source used on different species should occur

cautiously So simply extrapolating the dosage used for one species to another is

inadvisable in addition direct conversion of dosimetry from in vitro research to in vivo

clinical practice is inappropriate So purposive and specific research is the prerequisite

to produce safe and correct use of light as a therapeutic modality27

12

MECHANISMS OF ACTION

In the past decennia several mechanisms of action for biostimulation and pain

inhibition have been proposed and investigated73 Research was primarily based on

studies at the molecular and cellular levels and as a second resort investigations

occurred at the organism level resulting in numerous possible explanatory

mechanisms272858

It is the common view that light triggers a cascade of cellular and molecular reactions

resulting in various biological responses Thus different mechanisms of whom the

causal relationships are very difficult to establish- underlie the effects of light3448557475

To illustrate this complex matter the various mechanisms of action will be summarised

by means of a comprehensive model (fig 2) Detailed discussion about the different

individual components of the proposed model and other effects than those regarding

wound healing or analgesia were not provided as this was beyond the scope of this

general introduction

As depicted in figure 2 exposure to light leads to photon absorption by a

photoacceptor molecule causing excitation of the electronic state or increased

vibrational state of the given molecule275173 This process is followed by primary

photochemical reactions7475 Several key mechanisms have been discussed in the

literature Respiratory chain activation is the central point and can occur by an

alteration in redox properties acceleration of electron transfer generation of reactive

oxygen species (namely singlet oxygen formation and superoxide generation) as well as

by induction of local transient heating of absorbing chromophores192848515576-83 It is

supposed that each of these respective mechanisms plays a part in obtaining a

measurable biological effect It is yet not clear if one mechanism is more prominent

and decisive than another nevertheless recent experimental evidence has revealed that

mechanisms based on changes in redox properties of terminal enzymes of respiratory

chains might be of crucial importance2848517679

The primary mechanisms occurring during light exposure are followed by the dark

reactions (secondary mechanisms) occurring when the effective radiation is switched


13

off2851 Activation of respiratory chain components is followed by the initiation of a

complicated cellular signalling cascade or a photosignal transduction and amplification

chain associated with eg changes in the cellular homeostasis alterations in ATP or

cAMP levels modulation of DNA and RNA synthesis membrane permeability

alterations alkalisation of cytoplasm and cell membrane depolarisation283251557184-87

The sequence of events finally results in a range of physiological effects essential for

the promotion of the wound healing process for supplying analgesia or other

advantageous responses (acceleration of inflammatory processes oedema re-

absorption increased lymph vessel regeneration or increased nerve

regeneration)12181927486188-93

Photostimulation of the wound healing process can be mediated by increased

fibroblast proliferation enhanced cell metabolism increased (pro)collagen synthesis

and transformation of fibroblasts into myofibroblasts19276173798594-98 Investigations

have been especially focussed on fibroblasts but other possible physiological effects

attributing to an accelerated wound healing were also observed suppression and

alteration of undesirable immune processes increased leukocyte activity new

formation of capillaries increased production of growth factors and enzymes while

monocytes and macrophages can provide an enlarged release of a variety of substances

related to immunity and wound healing1619277376

As pain and nociception are even less understood than wound healing the possible

mechanisms in obtaining pain relief by the use of light are less underpinned However

it is established that light therapy influences the synthesis release and metabolism of

numerous transmitter signal substances involved in analgesia such as endorphin nitric

oxide prostaglandin bradykinin acetylcholine and serotonin In addition to these

neuropharmacological effects there is experimental evidence for diminished

inflammation decreased C-fibre activity increased blood circulation and reduced

excitability of the nervous system1927848899

One should be aware that a large amount of research regarding the possible

mechanisms of light action was conducted at the cellular level The described cascade

of reactions at the organism level is possibly even more complex as in contradiction to

14

the in vitro situation in vivo a range of supplementary interactions can influence the

sequence of effects and accordingly the final responses Besides it needs to be

mentioned that this summary did not take into account the origin of the light or the

external dosimetry thus the description is based on investigations performed with

various light sources and different dosages

Figure 2 Model summarizing the identified mechanisms of light action

Secondarymechanisms

Primary mechanisms

Final effects

Trigger

Stimulated wound healing Analgesia

Exposure to light

Photon absorption by photoacceptors

Respiratory chain activation

Accelerated electrontransfer

Reactive oxygen generation

Heating of absorbing chromophores

Altered redox properties

darr inflammation uarr oedema resorption

uarr lymph vessel regenerationuarr blood circulation

Photosignal transduction and amplification chain

uarr fibroblast proliferation uarr cell metabolism uarr collagen synthesis uarr myofibroblast transformation uarr release of growth factors uarr release of enzymes uarr capillary formation

darr C-fibre activity darr nervous excitability neuropharmacological effects


15

Regardless of the large number of previous investigations identification of underlying

mechanisms of light action remains an important issue as these are not yet fully

understood and because probably not all mechanisms of action are currently

identified Convincing explanation of the mechanisms in normal as well as in

pathological tissue could banish the existing suspicion concerning the use of light as a

treatment modality2732547678

AIMS AND OUTLINE

The introduction of LED in medicine and in physiotherapy more specifically requires

particular scientific research especially within the fields of its clinical potential

application wound healing and analgesia The above described gaps in literature

regarding the use of LED laid the foundation of this doctoral thesis

Consequently the general purpose of this thesis is to explore a scientific approach for

the supposed biostimulatory and analgesic effect of LED and to formulate an answer

in view of an evidence-based clinical use of this treatment modality

The detailed objectives can be phrased as follows

Aim 1 To assess the biostimulatory effectiveness of LED

irradiation under normal in vitro conditions

Aim 2 To investigate the value of LED treatment to ameliorate

in vitro cell proliferation under conditions of impaired healing

Aim 3 To examine the effectiveness of LED in changing the

nerve conduction characteristics in view of analgesia

Aim 4 To determine the efficacy of LED irradiation as an

analgesic treatment modality in a laboratory setting

Part I investigates the influence of LED on wound healing In pursuit of the first aim

chapter 1 reports the results of a twofold study The first part consisted of an in vitro trial

16

measuring fibroblast proliferation with a Buumlrker hemocytometer Proliferation of these

cells needs to be considered as an exponent of the wound healing process as

fibroblasts fulfil a crucial role in the late inflammatory phase the granulation phase

and early remodelling100 Secondly an in vivo case study exploring the postulation that

LED irradiation could accelerate and ameliorate the healing of a surgical incision was

described

The results contrasted sharply with the findings of the in vitro part Two fundamental

causes were proposed in order to explain the different biological effect of LED

irradiation observed in vitro and in vivo the used irradiation parameters and evaluation

method

The experiment described in chapter 2 endeavoured to explore these considerations A

similar study was therefore performed but as distinctive characteristics different light

source properties an adapted irradiation procedure and the use of a colorimetric assay

based on 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT) for the

counting of the cells were used

As stimulation of the wound healing process is virtually mainly indicated under

conditions of impaired healing (resulting in a situation which threatens to become

chronic and debilitating) proper attention for this matter is warranted192855 Besides

the medical consequences the costs involved with impaired healing yield also a socially

relevant problem to tackle Impaired healing will become even more common as the

world population continues to age After all senescence of systems and age-committed

comorbid conditions are commonly the culprits responsible for poor wound healing101

Thus finding cost-effective time-sparing non-invasive and practical treatment

modalities to cure wounds is a necessity

Aiming to assess the biostimulative effects by means of LED in these circumstances a

third study was conducted with respect of the previous results regarding irradiation

parameters and cell proliferation analysis The irradiation experiment described in

chapter 3 analysed the fibroblast cultivation in medium supplemented with glucose

This medium modification serves as a pattern for cell proliferation in diabetic patients


17

a population for whom stimulation of the wound healing process is a clinical relevant

feature

In part II the potential analgesic effects of LED treatment (aim 3 and 4) were explored

by means of two studies A first investigation (chapter 4) evaluated the influence of LED

on the sensory nerve conduction characteristics of a human superficial peripheral

nerve as a potential explanatory mechanism of pain inhibition by LED which is based

on the putative neurophysiological effects of this treatment modality The experimental

hypothesis postulated that LED generates an immediate decrease in conduction

velocity and increase in negative peak latency In addition it was postulated that this

effect is most prominent immediately after the irradiation and will weaken as time

progresses

The values of nerve conduction velocity and negative peak latency of a baseline

antidromic nerve conduction measurement were compared with the results of five

identical recordings performed at several points of time after LED irradiation

Chapter 5 illustrates a study assessing the analgesic efficiency of LED in a laboratory

setting To guarantee an adequate standardized and controlled pain reduction study

there was opted to observe a healthy population with experimentally induced DOMS

Induction of DOMS has been described in a number of studies as a representative

model of musculoskeletal pain and stiffness because it can be induced in a relatively

easy and standardised manner the time course is quite predictable and the symptoms

have the same aetiology and are of transitory nature4445102-105

The treatment as well as the assessment procedure was performed during 4

consecutive days The first day isokinetic exercise was performed to induce pain

related to DOMS Subsequently the volunteers of the experimental group received an

infrared LED treatment and those of the placebo group received sham-irradiation

Evaluation of the effect of the treatment on perceived pain was registered by a visual

analog scale and by a mechanical pain threshold these observations occurred every day

18

prior to and following LED irradiation Eccentricconcentric isokinetic peak torque

assessment took place daily before each treatment

For the analysis of the results three different factors were taken into consideration

time (day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group

(placebo or experimental)

In completion of this thesis the most prominent findings are summarized and the

clinical implications are discussed The general discussion also includes some future

research directions and a final conclusion


19

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radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-92 3 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy

in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austriaca 33(3)132-137

4 Stahl F Ashworth S Jandt K and Mills R (2000) Light-emitting diode (LED) polymerisation of dental composites flexural properties and polymerisation potential Biomaterials 21(13)1379-1385

5 Mills R Jandt K and Ashworth S (1999) Dental composite depth of cure with halogen and blue light emitting diode technology Br Dent J 186(8)388-391

6 Vreman H Wong R Stevenson D Route R Reader S Fejer M Gale R and Seidman D (1998) Light-emitting diodes a novel light source for phototherapy Pediatr Res 44(5)804-809

7 Pontinen P Aaltokallio T and Kolari P (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118

8 Schmidt M Reichert K Ozker K Meyer G Donohoe D Bajic D Whelan N and Whelan H (1999) Preclinical evaluation of benzoporphyrin derivative combined with a light-emitting diode array for photodynamic therapy of brain tumors Pediatr Neurosurg 30(5)225-231

9 Schmidt M Bajic D Reichert K Martin T Meyer G and Whelan H (1996) Light-emitting diodes as a light source for intraoperative photodynamic therapy Neurosurgery 38(3)552-557

10 Sommer A Pinheiro A Mester A Franke RP and Whelan H (2001) Biostimulatory windows in low-intensity laser activation lasers scanners and NASAs light-emitting diode array systems J Clin Laser Med Sur 19(1)29-33

11 Vinck E Vinck H Cagnie B and Cambier D (2004) Photodynamic therapy of feline superficial squamous cell carcinoma of the nasal planum Vlaams Diergeneeskund Tijds 73424-428

12 Whelan H Smits R Buchman E Whelan N Turner S Margolis D Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Philippi A Graf W Hodgson B Gould L Kane M Chen G and Caviness J (2001) Effect of NASA light-emitting diode irradiation on wound healing J Clin Laser Med Sur 19(6)305-314

13 Ojeda A Redondo E Gonzalez Diaz G and Martil I (1997) Analysis of light-emission processes in light-emitting diodes and semiconductor lasers Eur J Phys 18(2)63-67

14 Monstrey S Hoeksema H Saelens H Depuydt K Hamdi M Van Landuyt K and Blondeel P (2002) Conservative approach for deep dermal burn wounds using polarised-light therapy Br J Plast Surg 55(5)420-426

15 Monstrey S Hoeksema H Depuydt K Van Maele G Van Landuyt K and Blondeel P (2002) The effect of polarized light on wound healing Eur J Plast Surg 24377-382

16 Bolton P Dyson M and Young S (1992) The effect of polarized light on the release of growth factors from the U-937 macrophage-like cell line Laser Ther 233-42

17 Stasinopoulos D (2005) The use of polarized polychromatic non-coherent light as therapy for acute tennis elbowlateral epicondylalgia A pilot study Photomed Laser Surg 23(1)66-69

18 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43

19 Tuner J Hode L (2004) The laser therapy handbook Tallinn Prima Books AB 20 Allendorf J Bessler M Huang J Kayton M Laird D Nowygrod R and Treat M (1997) Helium-

neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3)340-345

21 Basford J (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8)671-675

20

22 Schindl A Heinze G Schindl M Pernerstorfer Schon H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2)240-246

23 Lagan K Clements B McDonough S and Baxter G (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1)27-32

24 Schlager A Kronberger P Petschke F and Ulmer H (2000) Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1)39-42

25 Nemeth A J (1993) Lasers and wound healing Dermatol Clin 11(4)783-789 26 Lowe A Walker M OByrne M Baxter G and Hirst D (1998) Effect of low intensity

monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5)291-298

27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science

Publishers 29 Basford J (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg

Med 16(4)331-342 30 Baxter G Bell A Allen J and Ravey J (1991) Low level laser therapy Current clinical practice in

Northern Ireland Physiotherapy 77(3)171-178 31 Cambier D and Vanderstraeten G (1997) Low-level laser therapy The experience in flanders

Eur J Phys Med Rehabil 7(4)102-105 32 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D

Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15

33 Whelan H Buchmann E Whelan N Turner S Cevenini V Stinson H Ignatius R Martin T Cwiklinski J Meyer G Hodgson B Gould L Kane M Chen G and Caviness J (2001) NASA Light Emitting Diode medical applications from deep space to deep sea Space Technology and Applications International Forum

34 Whelan H Buchmann E Dhokalia A Kane M Whelan N Wong-Riley M Eells J Gould L Hammamieh R Das R and Jett M (2003) Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice J Clin Laser Med Sur 21(2)67-74

35 Basford J Hallman H Matsumoto J Moyer S Buss J and Baxter G (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6)597-604

36 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol (Lond) 43563

37 Baxter G Allen J Walsh D Bell A and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol (Lond) 446445

38 Baxter G Walsh D Allen J Lowe A and Bell A (1994) Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79227-234

39 Lowe A Baxter G Walsh D and Allen J (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46

40 Lowe A Baxter G Walsh D and Allen J M (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Lasers Med Sci 10(4)253-259

41 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Lasers Surg Med 26(5)485-490


21

42 Baxter G Effect of combined phototherapylow intensity laser therapy upon experimental ischaemic pain Potential relevance of experimental design 14th World Congress Physical Therapy Barcelona Spain 2004 Proceedings CD

43 Craig J Barron J Walsh D and Baxter G (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230

44 Craig J Barlas P Baxter G Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380

45 Glasgow P Hill I McKevitt A Lowe A and Baxter G (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1)33-39

46 Noble J Lowe A Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295

47 IEC 60825-1 (1993) International Electrotechnical Commission Safety of laser products Part 1 Equipment classification requirements and users guide

48 Nussbaum E Baxter G and Lilge L (2003) A review of laser technology and light-tissue interactions as a background to therapeutic applications of low intensity lasers and other light sources Phys Ther Rev 831-44

49 van Breugel H and Bar P (1992) Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5)528-537

50 Nakamura S (1998) The roles of structural imperfections in InGaN-based blue light-emitting diodes and laser diodes Science 281956-961

51 Karu Tiina (consulted on 2005 august 22) Cellular mechanism of low-power laser therapy httpwwwtinnitusustiinakarupresentationhtml

52 Mester E Mester A and Mester A (1985) The biomedical effects of laser application Lasers Surg Med 5(1)31-39

53 Moore K Calderhead G (1991) The clinical application of low incident power density 830 nm GaAlAs diode laser radiation in the therapy of chronic intractable pain A historical and optoelectronic rationale and clinical review Int J Optoelectron 6503-520

54 King P (1989) Low level laser therapy A review Lasers Med Sci 4141-148 55 Karu T (1989) Photobiology of low-power laser effects Health Phys 56(5)691-704 56 Simunovic Z (2000) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical

Application of Low Energy-Laser Laser Therapy LLLT Rijeka Vitgraf 57 Hode L and Tuner J (2000) Low level laser therapy (LLLT) contra light emitting diode therapy

(LEDT) - What is the difference Proceedings of SPIE 416690-97 58 Kitchen S and Partridge C (1991) A review of low level laser therapy Part I Background

physiological effects and hazards Physiotherapy 77(3)161-163 59 Nussbaum E Lilge L and Mazzulli T (2003) Effects of low-level laser therapy (LLLT) of 810 nm

upon in vitro growth of bacteria Relevance of irradiance and radiant exposure J Clin Laser Med Sur 21(5)283-290

60 Mendez T Pinheiro A Pacheco M Nascimento P and Ramalho L (2004) Dose and wavelength of laser light have influence on the repair of cutaneous wounds J Clin Laser Med Sur 22(1)19-25

61 Koutna M Janisch R and Veselska R (2003) Effects of low-power laser irradiation on cell proliferation Scripta Medica 76(3)163-172

62 Al-Watban F and Zhang X (2004) The comparison of effects between pulsed and CW lasers on wound healing J Clin Laser Med Sur 22(1)15-18

63 Panjehpour M Overholt B DeNovo R Petersen M and Sneed R (1993) Comparative study between pulsed and continuous wave lasers for photofrin photodynamic therapy Lasers Surg Med 13(3)296-304

64 Dyson M (2005) The significance of pulsing in the stimulation of tissue repair by light A review Lasers Med Sci 20S21

22

65 MDB Laser (2000) Bio Dio (Preprotoype) User guide Belgium Ekeren 66 Fisher J (1992) Photons physiatrics and physicians A practical guide to understanding laser light

interaction with living tissue Part I J Clin Laser Med Sur 10(6)419-426 67 Tuner J and Hode L (2000) Depth of penetration of laser light in tissue Laser Partner

Clinixperience 151-3 68 Anderson R and Parrish J (1981) The optics of human skin J Invest Dermatol 77(1)13-19 69 De Cuyper H and Lambert H (1991) Penetration depth of infrared- and helium-neon-laserlight

An assessment in vivo Eur J Phys Med Rehabil 1(5)133-137 70 Oshiro T Ogata H Yoshida M Tanaka Y Sasaki K and Yoshimi K (1996) Penetration depths

of 830nm diode laser irradiation in the head and neck assessed using a radiographic phanom model and wavelength-specific imaging film Laser Ther 8197-204

71 Kolarova H Ditrichova D and Wagner J (1999) Penetration of the laser light into the skin in vitro Lasers Surg Med 24(3)231-235

72 Chen Q Huang Z Chen H Shapiro H Beckers J and Hetzel F (2002) Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy Photochem Photobiol 76(2)197ndash203

73 Conlan M Rapley J and Cobb C (1996) Biostimulation of wound healing by low-energy laser irradiation A review J Clin Periodontol 23(5)492-496

74 Parrish J (1981) New concepts in therapeutic photomedicine photochemistry optical targeting and the therapeutic window J Invest Dermatol 7745-50

75 Smith K (1981) Photobiology and photomedicine the future is bright J Invest Dermatol 772-7 76 Rochkind S Rousso M Nissan M Villarreal M Barr Nea L and Rees D (1989) Systemic effects

of low-power laser irradiation on the peripheral and central nervous system cutaneous wounds and burns Lasers Surg Med 9(2)174-182

77 Polo L Presti F Schindl A Schindl L Jori G and Bertoloni G (1999) Role of ground and excited singlet state oxygen in the red light-induced stimulation of Escherichia coli cell growth Biochem Biophys Res Commun 257(3)753-758

78 Bertoloni G Sacchetto R Baro E Ceccherelli F and Jori G (1993) Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 6328 nm light J Photochem Photobiol B-Biol 18191-196

79 Lubart R Friedmann H Sinykov M and Grossman N (1995) Biostimulation of photosensitized fibroblasts by low incident levels of visible light energy Laser Ther 7101-106

80 Harris D (1991) Biomolecular mechanisms of laser biostimulation J Clin Laser Med Sur 9277-280

81 Grossman N Schneid N Reuveni H Halevy S and Lubart R (1998) 780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures involvement of reactive oxygen species Lasers Surg Med 22212-218

82 Oren D Charney D Lavi R Sinyakov M and Lubart R (2001) Stimulation of reactive oxygen species production by an antidepressant visible light source Biol Psychiatry 49464-467

83 Lavi R Shainberg A Friedmann H Shneyvays V Rickover O Eichler M Kaplan D and Lubart R (2003) Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells 278(42)40917-40922

84 Reddy K (2004) Photobiological basis and clinical role of low-intensity lasers in biology and medicine J Clin Laser Med Sur 22(2)141-150

85 Greco M Guida G Perlino E Marra E and Quagliariello E (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser Biochem Biophys Res Commun 163(3)1428-1434

86 Vacca R Marra E Quagliariello E and Greco M (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation Biochem Biophys Res Commun 195(2)704-709

87 Vacca R Marra E Quagliariello E and Greco M (1994) Increase of both transcription and translation activities following separate irradiation of the in vitro system components with He-Ne laser Biochem Biophys Res Commun 203(2)991-997

88 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral


23

nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200

89 Anders J Borke R Woolery S and Van de Merwe W (1993) Low power laser irradiation alters the rate of regeneration of the rat facial nerve Lasers Surg Med 13(1)72-82

90 Rochkind S Nissan M Barr-Nea L Razon N Schwartz M and Bartal A (1987) Response of peripheral nerve to He-Ne laser Experimental studies Lasers Surg Med 7441-443

91 Lievens P (1986) Lasertherapie (deel II) De invloed van laser-bestralingen op het lymfesysteem en de wondheling Ned T Fysiotherapie 96140-142

92 Lievens P (1991) The effect of a combined HeNe and IR laser treatment on the regeneration of the lymphatic sysytem during the process of wound healing Lasers Med Sci 6193-199

93 Lievens P (1991) The effect of IR laser irradiation on the vasomotricity of the lymphatic system Lasers Med Sci 6189-191

94 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin J and Martin P (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1)171-178

95 Bosatra M Jucci A Olliaro P Quacci D and Sacchi S (1984) In vitro fibroblast and dermis fibroblast activation by laser irradiation at low energy An electron microscopic study Dermatologica 168(4)157-162

96 Enwemeka C Rodriquez O Gall N and Walsh N (1990) Morphometrics of collagen fibril populations in HeNe laser photostimulated tendons J Clin Laser Med Sur 847-52

97 Abergel P Lam T Meeker C Castel C Dwyer R and Uitto J (1984) Biostimulation of procollagen production by low energy lasers in human skin fibroblast culturesJ Invest Dermatol 82(4)395

98 Abergel P Lam T Meeker C Dwyer R and Uitto J (1984) Low energy lasers stimulate collagen production in human skin fibroblast cultures Clin Res 32(1)16A

99 Shimoyama N Lijima K Shimoyama M and Mizuguchi T (1992) The effects of helium-neon laser on formalin-induced activity of dorsal horn neurons in the rat J Clin Laser Med Sur 1091-94

100 Kloth L McCulloch J and Feedar J (1990) Wound healing Alternatives in management USA FA Davis Company

101 Lanzafame R (2004) Revolutions and revelations J Clin Laser Med Surg 22(1)1-2 102 Ciccone C Leggin B and Callamaro J (1991) Effects of ultrasound and trolamine salicylate

phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-678 103 Craig J Cunningham M Walsh D Baxter G and Allen J (1996) Lack of effect of transcutaneous

electrical nerve stimulation upon experimentally induced delayed onset muscle soreness in humans Pain 67(2-3)285-289

104 Minder P Noble J Alves-Guerreiro J Hill I Lowe A Walsh D and Baxter G (2002) Interferential therapy Lack of effect upon experimentally induced delayed onset muscle soreness Clin Physiol Funct Imaging 22(5)339-347

105 Ernst E (1998) Does post-exercise massage treatment reduce delayed onset muscle soreness A systematic review Br J Sports Med 32(3)212-214

PART I WOUND HEALING

CHAPTER 1

DO INFRARED LIGHT EMITTING DIODES HAVE A

STIMULATORY EFFECT ON WOUND HEALING FROM AN IN

VITRO TRIAL TO A PATIENT TREATMENT

Elke Vincka Barbara Cagniea Dirk Cambiera and Maria Cornelissenb

a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium

b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium

Proceedings of SPIE 2002 4903 156-165

Chapter 1

28

ABSTRACT

Variable effects of different forms of light therapy on wound healing have been

reported This preliminary study covers the efficacy of infrared light emitting diodes

(LED) in this domain

Cultured embryonic chicken fibroblasts were treated in a controlled randomised

manner LED irradiation was performed three consecutive days with a wavelength of

950 nm and a power output of 160 mW at 06 cm distance from the fibroblasts Each

treatment lasted 6 minutes resulting in a surface energy density of 32 Jcm2

The results indicated that LED treatment does not influence fibroblast proliferation at

the applied energy density and irradiation frequency (p=0474)

Meanwhile the effects of LED on wound healing in vivo were studied by treating a

surgical incision (6 cm) on the lateral side of the right foot of a male patient The

treatment started after 13 days when initial stitches were removed The same

parameters as the in vitro study were used but the treatment was performed five times

The healing could only be evaluated clinically the irradiated area (26 cm) showed a

more appropriate contraction less discoloration and a less hypertrophic scar than the

control area (34 cm)

The used parameters failed to demonstrate any biological effect of LED irradiation in

vitro although the case study on the other hand illustrated a beneficial effect

Keywords Light Emitting Diodes Fibroblasts Wound healing

From an in vitro trial to a patient treatment

29

INTRODUCTION

Various beneficial effects of lasers and photodiodes at relatively low intensities have

been reported involving treatment of neurological impairments12 treatment of pain3-5

treatment of soft tissue injuries67 wound healing8-10 et cetera In particular the

enhancement of wound healing has been a focus of contemporary research11-16 It

seems a logic tendency while according to Baxter17 Photobiostimulation of wound healing

remains the cardinal indication for therapeutic laser in physiotherapy he concluded this on the

basis of a questionnaire about low power laser (LPL) in the current clinical practice in

Northern Ireland18 Cambier et al19 confirmed these findings in a comparable survey

into clinical LPL experience in Flanders

Nevertheless there remains a considerable amount of ignorance scepticism and

controversial issues concerning the use and clinical efficacy of LPL even in the domain

of wound healing12152021 This is at least in part a consequence of the inability to

measure and control operating variables related to connective tissue repair and of the

wide range of suitable parameters for irradiation

Thus LPL therapy is not yet an established clinical tool11 as LPLs have some inherent

characteristics which make their use in a clinical setting problematic including

limitations in wavelength capabilities and beam width The combined wavelength of

light optimal for wound healing cannot be efficiently produced and the size of

wounds which may be treated by LPLs is limited Some companies offer an

alternative to LPLs by introducing light emitting diodes (LEDrsquos) These diodes can be

made to produce multiple wavelengths and can have probes with large surface area

allowing treatment of large wounds Still one can not accept this light source as an

alternative for LPL therapy based on the cited advantages without proper investigation

regarding its biostimulatory effects

The effectiveness of this possible alternative for LPLs must be studied in vitro and in

addition in animal models or in humans because the effects of LED at the cellular level

do not necessarily translate to a noticeable effect in vivo The small amount of previous

investigations demonstrate that LED effects are as difficult to isolate162223 as LPL

Chapter 1

30

effects and the results are conflicting just like the results in literature specific on the

use of LPL121520

The purpose of the first part of this study is to examine the hypothesis stating that

LED irradiation can influence fibroblast proliferation Therefore a comparison of the

proliferation from fibroblasts in irradiated and control wells was performed The in vitro

investigation was linked with an in vivo case study This part enquired the assumption if

LED irradiation could accelerate and ameliorate the healing of a surgical incision

IN VITRO INVESTIGATION

MATERIALS AND METHODS

The complete procedure from isolation to proliferation analysis was executed twice

(trial A and B) Trial A involved 30 irradiated petri dishes and the same number of

control dishes The second trial consisted of 27 irradiated and 27 control dishes

Cell isolation and culture procedures

Primary fibroblast cultures were initiated from 8-days old chicken embryos Isolation

and disaggregating of the cells occurred with warm trypsin (NV Life Technologies

Belgium) according the protocol described by Ian Freshney (1994)24 The primary

explants were cultivated at 37degC in Hanksrsquo culture Medium (NV Life Technologies

Belgium) supplemented with 10 Fetal Calf Serum (Invitrogen Corporation UK)1

Fungizone (NV Life Technologies Belgium) 1 L-Glutamine (NV Life

Technologies Belgium) and 05 Penicillin-Streptomycin (NV Life Technologies

Belgium) When cell growth from the explants reached confluence cells were detached

with trypsine and subcultured during 2 days in 80-cm2 culture flasks (Nunc NV

Life Technologies Belgium) with 12 ml of primary culture medium After 24 hours the

cells were removed from the culture flasks by trypsinization and counted by

hemocytometry Secondary subcultures were initiated in 215 cm2 petriplates (Nunc


31

NV Life Technologies Belgium) The fibroblasts were seeded at a density of

70000cm2 resulting in 1505000 cells per well After adding 5 ml primary culture

medium the cells were allowed to attach for 24 hours in a humidified incubator at

37degC

Properties of the Light Emitting Diode

Prior to LED treatment all dishes were microscopically checked to guarantee that the

cells are adherent and to assure that there is no confluence nor contamination The

dishes were divided randomly into the treated or the control group Medium was then

removed by tipping the dishes and aspirating with a sterile pipette Following the

aspiration 2 ml fresh medium was added and treatment started

A Light Emitting Diode (LED) device (BIO-DIO preprototype MDB-Laser

Belgium) with a wavelength of 950 nm (power-range 80-160 mW frequency-range 0-

1500 Hz) was used The surface of the LED probe was 18 cm2 and it consisted of 32

single LEDrsquos For the treatments in this study an average power of 160 mW at

continuous mode was applied The irradiation lasted 6 minutes resulting in an energy

density of 32 Jcm2 The distance to the fibroblasts numbered 06 cm and as a result

of the divergence in function of this distance the surface of the LED (18 cm2) covered

the complete surface of the used petriplates (215 cm2)

After these manipulations 3 ml medium was added to each dish followed by 24 hours

incubation

One LED irradiation was performed daily during three consecutive days according

this procedure Control cultures underwent the same handling during these three days

but were sham-irradiated

Proliferation analysis

After the last treatment a trypsination was performed to detach the cells from the

culture dishes followed by centrifugation Once the cells were isolated from the used

trypsine they were resuspended in 4 ml fresh medium The number of fibroblasts

Chapter 1

32

within this suspension as reflection for the proliferation was quantified by means of a

Buumlrker Chamber or hemocytometry

The counting of the cells involved amalgamating 200 microl Trypan blue (01 Sigma-

Aldrich Corporation UK) and 100 microl cell suspension in an Eppendorf (Elscolab

Belgium) Approximately 40 microl of this suspension (cellsTrypan blue) was pipetted on

the edge of the coverslip from the Buumlrker Chamber Finally a blinded investigator

using an inverted light microscope counted the number of cells in 25 small squares

In order to calculate the number of cells one should multiply the amount of cells

counted in the Buumlrker Chamber with the volume of 25 small squares (10000 mm3) and

the dilution factor (the amount of Trypan blue suspended with the cells 21=3)

Statistical methods

The data were analysed statistical in order to examine the hypothesis that LED

irradiation enhances fibroblast proliferation They were processed as absolute figures

for both trials separately In a second phase the counted cell numbers were converted

in relative figures so the data of both trials could be analysed as the data of one test

These relative figures were obtained by expressing each figure as a percentage from the

highest figure (=100) of that trial and this for each assay separately

A Kolmogorov-Smirnoff test of normality was performed on the data followed by a

Mann-Whitney-U test when the test of normality was significant and otherwise a T-

test Differences were accepted as significant when plt005 For this analysis SPSSreg

100 was used

RESULTS

The descriptive data for both trials are depicted in figure I The mean number of cells

in trial A is higher than in trial B for the controls as for the treated wells There is a

mean difference of 1252500 fibroblasts between the controls and 1223000 between

the irradiated wells of trial A and B The averages of both trials show that in control

cultures there are slightly more fibroblasts than in the treated cultures Nevertheless no


33

statistically significant difference could be found between the two groups in either trial

nor in the combined data The test of normality (Kolmogorov-Smirnoff) was not

significant for trial A (p=020) nor trial B (p=020) Only the combined data from both

trials were significant (plt001) for normality Further analysis respectively T-test for

the single trials (trial A p=0412 trial B p=0274) or Mann-Whitney-U test for the

combined data (p=0474) revealed no statistical significant differences

DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06

Trial A Trial A Trial B Trial B

Mea

n n

um

ber

of

cells

Control

Irradiated

Figure I Mean number of fibroblasts within control and irradiated wells for trail A and B

DISCUSSION

Biostimulatory effectiveness of lasers and photodiodes at relatively low intensities

(lt500 mW) in vitro have been analysed by evaluating various factors involving

(pro)collagen production25-27 cell viability2829 growth factor production28 and

myofibroblast formation30 Fibroblast proliferation also is an important factor to

consider In accordance with wound healing fibroblasts fulfil an essential role especially

in the late inflammatory phase and the early granulation phase31 Despite the failure of

some studies to demonstrate beneficial effects of LPL irradiation on fibroblast

proliferation (determined by cell counting)3233 Webb et al34 provided evidence of very

Chapter 1

34

significantly higher cell numbers in irradiated wells (with an increase ranging from 89 -

208 ) Atabey et al35 also revealed a significant increase in cell number two or more

irradiations resulted in an increased fibroblast proliferation Several other studies

confirmed these positive findings25263637

The results of this present in vitro study indicate that LED treatment does not

influence fibroblast proliferation Although the dosimetric parameters (in particular the

arbitrary energy density of 32 Jcm2) used in this study are well within the

recommended limits (energy density 0077 Jcm2 ndash 73 Jcm2) described in previous

studies about LPL therapy raising enhanced fibroblast proliferation252634-37

Van Breugel et al36 gave a possible explanation for these controversial results

According to them the fibroblast proliferation is not inherent at the energy density

They provide evidence that independent of the energy density the power density and

the exposure time determine the biostimulative effects of LPL irradiation LPL with a

power below 291 mW could enhance cell proliferation while a higher power had no

effect

Some authors also argued that the absorption spectrum of human fibroblasts show

several absorption peaks and pointed out that a wavelength of 950 nm is far above the

highest peak of about 730 nm3638 At longer wavelengths they determined a general

decrease in absorption Despite these results several investigators pose biostimulative

effects on fibroblast cell processes by using LPLs with a wavelength of 830 nm2739 or

even 904 nm2526 Loevschall et al29 note the absorption spectrum from fibroblasts is

ranged from 800 nm to 830 nm principally because of the presence of cytochrome

oxidase in the cells Furthermore the supposed superior absorption of the fibroblasts

at lower wavelengths is restricted by an inferior skin transmission than at higher

wavelengths38

Beside the used probe the Bio-Dio has a 570 nm and a 660 nm probe emitting

respective green and red light The 950 nm beam of light was used for its high power

density but according to a range of remarks mentioned above the effects of the two

other probes must be as well evaluated


35

Another factor one can not ignore is that besides fibroblast proliferation other

processes or morphologic changes were not analysed although several authors have

posed that those changes and processes could be responsible for the biostimulative

effect of low power light resulting in enhanced wound healing17 Pourreau-Schneider et

al30 for example described a massive transformation of fibroblasts into myofibroblasts

after LPL treatment These modified fibroblasts play an important role in contraction

of granulation tissue30 A second example is an increased (pro)collagen production

after low power light therapy25-27 which is also considered as a responsible factor for

accelerated wound healing25-2736 and is often unrelated to enhanced fibroblast

proliferation3640

It may be wondered if the light sources mostly LPL in the consulted literature are

representative for the LED used in this study although this LPL literature is often

used for that purpose As in the early days of LPL the stimulative effects upon

biological objects were explained by its coherence the beam emitted by the Bio-Dio on

the contrary produces incoherent light Nowadays contradictory research results are

responsible for a new discussion the clinical and biological significance of coherence

The findings of some authors172341-43 pose that the coherence of light is of no

importance of LPL and its effects although the opposite has also been stated4445

Therefore it is unclear whether the property lsquoincoherencersquo of the LED can be

accounted for the non-enhanced fibroblast proliferation in this trial

Another possible explanation for the absence of biostimulative effect is related to the

moment of analysis of the proliferation The evaluation one day after the last

irradiation did not allow a delayed enhancement of proliferation while it is determined

in numerous investigations that the effects occur more than 24 hours after the last

treatment273746 and that they weaken after a further undefined period of time34

The fluctuation in cell numbers between both trials despite the use of an identical

protocol was remarkable Hallman et al33 noticed comparable fluctuations due to poor

reproducibility of their technique In this study the fluctuations are attributable to the

counting of the cells by Buumlrker hemocytometer before seeding According to some

authors Buumlrker hemocytometer is not sufficiently sensitive47 it suffers from large

Chapter 1

36

variability48 and it is often difficult to standardize48 Overestimation of the cell

concentration also occurs with Buumlrker hemocytometer49 The insufficient sensitivity

was contradicted by Lin et al50 moreover satisfactory correlations with flow-

cytometer48 and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide assay

for cell counting (MTT)51 were determined

An automated counter can be a reliable alternative to the Buumlrker hemocytometer as it

provides accurate cell counts in a short period of time with less intervention from the

investigator52

These remarks and controversies point out the possible deficiencies from the used

proliferation analyses and the relativity from the obtained results Other analyse

methods and analyses from different cell processes and morphologic changes could be

considered for further investigation

IN VIVO INVESTIGATION


The effects of LED on wound healing in vivo were studied by treating a postsurgical

incision A male patient received chirurgical treatment for the removal of a cyst

situated approximately 15 cm posterior from the lateral malleolus of his right foot For

removal of the cyst an incision of 6 cm was made The incision was sutured and 12

days after the surgery the stitches were removed Visual inspection demonstrated that

the healing process of the wound proceeded well but not equally over the whole 6

centimetres (figure II) Epithelialization and wound contraction appeared to have

progressed better in the upper part (approximately 3 cm) of the cicatrice than at the

lower part (covered with eschar) No evidence of infection was noted in either part

LED treatment started the 13th day The incision was treated partially the lowest part

(26 cm) with the inferior epithelialization and wound contraction was irradiated the

remaining 34 cm served as control area This control area was screened from radiation

with cardboard and opaque black cling film


37

The light source destinated for the treatment was the same device used for the in vitro

irradiation namely the BIO-DIO a Light Emitting Diode from MDB-Laser LED

output parameters were identical with those applicated in the preceding in vitro

investigation In particular a continuous wave at an average power of 160 mW and 6

minutes of treatment duration corresponding to an energy density of 32 Jcm2 An

equal distance from the probe to the target tissue as from the probe to the culture

medium was respected A plastic applicant of according height guaranteed constant

distance of 06 cm from the surface of the skin

Figure II Surgical incision before the first treatment 13 days after initial stitching

Therapy was performed once a day during five consecutive days repeatedly at the same

time resulting in an extension of the duration of the in vitro therapy with two days

Visual macroscopic observations were accomplished 6 52 and 175 days after the first

treatment

Comparison of the cutaneous sensitivity at the irradiated area and the control area was

accomplished with a Semmes-Weinstein aesthesiometer (Smith amp Nephew Rolyan) 175

days after the first treatment A control measurement also occurred at the same region

Chapter 1

38

on the left foot The aesthesiometer used in this study consisted of five hand-held

nylon monofilaments with a length of 38 mm and varying diameter

Sensitivity threshold is traced by presenting a monofilament of a certain diameter

vertically to the skin The monofilament bends when a specific pressure has been

reached with a velocity proportional to its diameter Measurements allow mapping

areas of sensitivity loss and assessing the degree of loss Sensitivity levels are classified

from lsquonormal sensitivityrsquo attributed when the patient is able to discriminate the smallest

filament (00677 grams) over lsquodiminished light touchrsquo (04082 grams) lsquodiminished

protective sensationrsquo (20520 grams) and lsquoloss of protective sensationrsquo (3632 grams) to

finally lsquountestablersquo (4470 grams) when the patient did not respond to any of the

filaments

RESULTS

Visual estimation at any point of time after irradiation divulged no occurrence of

problems with dehiscence or infection in either part of the wound During the five

days of therapy the irradiated area looked dryer than the control area After the last

irradiation this was no longer recorded

Figure III Surgical incision 6 days after initiating LED treatment The lower 26 cm was irradiated the upper 34 cm served as control area


39

Figure III representing the first evaluation six days after the initial treatment

illustrates that the wound healing has evolved slightly in both parts Though the lower

irradiated part remains of inferior quality as regards to epithelialization and wound

contraction In the course of the reparative process the influence of light exposures

were registered At 52 days after the first irradiation beneficial effects of LED

treatment are clearly present (Figure IV)

Figure IV Surgical incision 52 days after initiating LED treatment

The irradiated area (26 cm) showed a more appropriate contracture than the control

area (34 cm) characterized by less discoloration at scar level and a less hypertrophic

scar A similar trend was noticed at a third visual observation 175 days after the initial

treatment At that moment no impairments at cutaneous sensitivity level were stated

and the sensitivity showed no differences between left or right foot nor between the

two areas of the cicatrice

Chapter 1

40

DISCUSSION

The results of this case study indicate that LED had a positive influence on wound

healing in humans as determined by visual observations Many investigators

examining the effects of LPL on wound healing by means of a range of observation

and treatment methods reported accelerated and enhanced wound healing8-10 others

described comparable outcomes using LEDrsquos16222353 However several LPL12-15 and

LED21 studies were unable to repeat these results

The late but beneficial findings in this study seem to be to the credit of LED-therapy

Though several authors establish positive results in an earlier stage of the wound

healing process8-1020 one should question why the differences did not occur at the first

evaluation on day 6 An explanation can be found in the start of the treatment Most

investigators start LPL irradiation within 24 hours after traumatizing the skin8-1014 so

they influence a first cellular and vascular reaction with the production of chemical

mediators of inflammation resulting in an enhanced collagen production9 tremendous

proliferation of capillaries and fibroblasts8 and stimulation of growth factors9 By the

time the first treatment in this study took place the traumatized tissue was in an

overlapping stage between an almost finished inflammatory phase and a scarcely

initiated re-epithelialization and wound contraction phase At that moment an infiltrate

of fibroblasts is present So fibroblast proliferation a possible mechanism of the

biostimulative effect had already occurred and could no longer be influenced Growth

factor production and collagen deposition have also decreased at that stage

Granulation tissue formation and fibroplasia in the contrary are initiating by that time

Those prolonged and slow processes with belated results are of significant importance

for the course of the final stage of wound healing and for the outlook of the future

scar31

The experimental findings revealed that the sensitivity of the skin according to the

threshold detection method of Semmes and Weinstein was normal at all the

investigated areas (treated and not treated) Bell et al54 claimed the 283 filament is a

good and objective predictor of normal skin sensitivity No other LPL nor LED

studies investigating this quality of the skin were found


41

CONCLUSION

This study demonstrates that although LED application at the applied energy density

and irradiation frequency failed to stimulate the fibroblast proliferation it does seem to

have beneficial biostimulative effects on wound healing in human skin confirmed by

the favourable re-epithelialization and contracture

These results are discussed in the context of other experimental findings but no

reasonable explanation for this discrepancy could be found The literature on wound

healing after LED treatment in animal models or in humans is presently very limited

and contradictory The diversity in used radiation parameters and the absence of

references on how the wounds were measured or evaluated or what the end point was

for lsquocompletersquo healing cause difficulties in interpreting laboratory results The in vitro

investigations are better standardised nevertheless these results show a number of

conflicts One can conclude that until today the controversial findings are characteristic

for many results obtained with light photobiomodulation

However the postponed favourable results in the case study confirm some facts of the

discussion Namely the short period of incubation 24 hours in the in vitro part of the

study can be responsible for the lack of enhanced fibroblast proliferation It also

confirms that other cell processes and morphologic changes possibly are responsible

for biostimulative effects in vivo other observation methods should be considered for

future in vivo experiments

Despite these remarks we believe that LED application on cutaneous wounds of

human skin is useful with a single flash daily at the dose applied in this study for at

least three days

Furthermore future investigation is necessary to explain the mechanisms of LED

biomodulation and to provide sufficient guidelines in the use of the most effective

parameters for LED treatment Subsequently resolving the lack of scientific evidence

and nullifying the controversial acknowledgements of the effect of LED can bring

about a widespread acceptance for the use of LED in clinical settings

Chapter 1

42

ACKNOWLEDGMENTS

The authors wish to acknowledge Prof De Ridder for supplying the laboratory and the

material necessary for this study as well as Ms Franccedilois laboratory worker for

providing the culture medium and for the technical support


43

REFERENCES

1 G Baxter D Walsh J Allen A Lowe and A Bell Effects of low intensity infrared laser irradiation upon conduction in the human median nerve in vivo Exp Physiol 79(2) 227-34 (1994)

2 J Basford H Hallman J Matsumoto S Moyer J Buss and G Baxter Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Lasers Surg Med 13(6) 597-604 (1993)

3 J Stelian I Gil B Habot et al Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy J Am Geriatr Soc 40(1) 23-6 (1992)

4 A Honmura A Ishii M Yanase J Obata and E Haruki Analgesic effect of Ga-Al-As diode laser irradiation on hyperalgesia in carrageenin-induced inflammation Lasers Surg Med 13(4) 463-9 (1993)

5 D Cambier K Blom E Witvrouw G Ollevier M De Muynck and G Vanderstraeten The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15 195-200 (2000)

6 G Reddy L Stehno Bittel and C Enwemeka Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-7 (1998)

7 K Moore N Hira I Broome and J Cruikshank The effect of infrared diode laser irradiation on the duration and severity of postoperative pain a double blind trial Laser Therapy 4 145-148 (1992)

8 A Amir A Solomon S Giler M Cordoba and D Hauben The influence of helium-neon laser irradiation on the viability of skin flaps in the rat Br J Plast Surg 53(1) 58-62 (2000)

9 W Yu J Naim and R Lanzafame Effects of photostimulation on wound healing in diabetic mice Lasers Surg Med 20(1) 56-63 (1997)

10 L Longo S Evangelista G Tinacci and A Sesti Effect of diodes-laser silver arsenide-aluminium (Ga-Al-As) 904 nm on healing of experimental wounds Lasers Surg Med 7(5) 444-7 (1987)

11 J Allendorf M Bessler J Huang et al Helium-neon laser irradiation at fluences of 1 2 and 4 Jcm2 failed to accelerate wound healing as assessed by both wound contracture rate and tensile strength Lasers Surg Med 20(3) 340-5 (1997)

12 K Lagan B Clements S McDonough and G Baxter Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 28(1) 27-32 (2001)

13 A Schlager K Oehler K Huebner M Schmuth and L Spoetl Healing of burns after treatment with 670-nanometer low-power laser light Plast Reconstr Surg 105(5) 1635-9 (2000)

14 D Cambier G Vanderstraeten M Mussen and J van der Spank Low-power laser and healing of burns a preliminary assay Plast Reconstr Surg 97(3) 555-8 discussion 559 (1996)

15 A Schlager P Kronberger F Petschke and H Ulmer Low-power laser light in the healing of burns a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group Lasers Surg Med 27(1) 39-42 (2000)

16 A Gupta J Telfer N Filonenko N Salansky and D Sauder The use of low-energy photon therapy in the treatment of leg ulcers - a preliminary study J Dermat Treatment 8 103-108 (1997)

17 GD Baxter and J Allen Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone (1994)

18 G Baxter A Bell J Allen and J Ravey Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77(3) 171-178 (1991)

19 DC Cambier and G Vanderstraeten Low-level laser therapy The experience in flanders Eur J Phys Med Rehab 7(4) 102-105 (1997)

20 A Nemeth J Lasers and wound healing Dermatol Clin 11(4) 783-9 (1993)

Chapter 1

44

21 A Lowe M Walker M OByrne G Baxter and D Hirst Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23(5) 291-8 (1998)

22 H Whelan J Houle N Whelan et al The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum 37-43 (2000)

23 P Pontinen T Aaltokallio and P Kolari Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18 (1996)

24 Freshney R Ian Culture of animal cells a manual of basic technique New york Wiley-Liss (1994)

25 R Abergel R Lyons J Castel R Dwyer and J Uitto Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2) 127-33 (1987)

26 S Skinner J Gage P Wilce and R Shaw A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-92 (1996)

27 E Ohbayashi K Matsushima S Hosoya Y Abiko and M Yamazaki Stimulatory effect of laser irradiation on calcified nodule formation in human dental pulp fibroblasts J Endod 25(1) 30-3 (1999)

28 K Nowak M McCormack and R Koch The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors a serum-free study Plast Reconstr Surg 105(6) 2039-48 (2000)

29 H Loevschall and D Arenholt Bindslev Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro Lasers Surg Med 14(4) 347-54 (1994)

30 N Pourreau Schneider A Ahmed M Soudry et al Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137(1) 171-8 (1990)

31 L Kloth J McCulloch and J Feedar Wound healing Alternatives in management USA FA Davis Company (1990)

32 MA Pogrel C Ji Wel and K Zhang Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20(4) 426-432 (1997)

33 H Hallman J Basford J OBrien and L Cummins Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8(2) 125-9 (1988)

34 C Webb M Dyson and WHP Lewis Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301 (1998)

35 A Atabey S Karademir N Atabey and A Barutcu The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 18 99-102 (1995)

36 H van Breugel and P Bar Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12(5) 528-37 (1992)

37 N Ben-Dov G Shefer A Irinitchev A Wernig U Oron and O Halevy Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3) 372-380 (1999)

38 F Al-Watban and XY Zhang Comparison of the effects of laser therapy on wound healing using different laser wavelengths Laser therapy 8 127-135 (1996)

39 Y Sakurai M Yamaguchi and Y Abiko Inhibitory effect of low-level laser irradiation on LPS-stimulated prostaglandin E2 production and cyclooxygenase-2 in human gingival fibroblasts Eur J Oral Sci 108(1) 29-34 (2000)

40 P Abergel T Lam C Meeker R Dwyer and J Uitto Low energy lasers stimulate collagen production in human skin fibroblast cultures Clinical Research 32(1) 16A (1984)

41 Karu Tiina The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers (1998)

42 J Kana G Hutschenreiter D Haina and W Waidelich Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116(3) 293-6 (1981)


45

43 J Basford Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16(4) 331-42 (1995)

44 M Boulton and J Marshall He-Ne laser stimulation of human fibroblast profileration and attachment in vitro Lasers Life Sci 1(2) 125-134 (1986)

45 E Mester A Mester F and A Mester The biomedical effects of laser application Lasers Surg Med 5(1) 31-9 (1985)

46 N Pourreau Schneider M Soudry M Remusat J Franquin and P Martin Modifications of growth dynamics and ultrastructure after helium-neon laser treatment of human gingival fibroblasts Quintessence Int 20(12) 887-93 (1989)

47 P Rebulla L Porretti F Bertolini et al White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2) 128-33 (1993)

48 V Deneys A Mazzon A Robert H Duvillier and M De Bruyere Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2) 172-7 (1994)

49 A Mahmoud B Depoorter N Piens and F Comhaire The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2) 340-5 (1997)

50 D Lin F Huang N Chiu et al Comparison of hemocytometer leukocyte counts and standard urinalyses for predicting urinary tract infections in febrile infants Pediatr Infect Dis J 19(3) 223-7 (2000)

51 Z Zhang and G Cox MTT assay overestimates human airway smooth muscle cell number in culture Biochem Mol Biol Int 38(3) 431-6 (1996)

52 D Haskard and P Revell Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3) 319-22 (1984)

53 H Whelan J Houle D Donohoe et al Medical applications of space light-emitting diode technology - Space station and beyond Space technology and applications international forum 3-15 (1999)

54 J Bell Krotoski E Fess J Figarola and D Hiltz Threshold detection and Semmes-Weinstein monofilaments J Hand Ther 8(2) 155-62 (1995)

CHAPTER 2

INCREASED FIBROBLAST PROLIFERATION INDUCED BY

LIGHT EMITTING DIODE AND LOW LEVEL LASER

IRRADIATION

Elke Vinck a Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk

Cambiera

a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Human Anatomy Embryology Histology and Medical Physics Ghent University Belgium

Lasers in Medical Science 2003 18(2) 95-99

Chapter 2

48

ABSTRACT

Background and Objective As Light Emitting Diode (LED) devices are

commercially introduced as an alternative for Low Level Laser (LLL) Therapy the

ability of LED in influencing wound healing processes at cellular level was examined

Study DesignMaterials and Methods Cultured fibroblasts were treated in a

controlled randomized manner during three consecutive days either with a infrared

LLL or with an LED light source emitting several wavelengths (950 nm 660 nm and

570 nm) and respective power outputs Treatment duration varied in relation to

varying surface energy densities (radiant exposures)

Results Statistical analysis revealed a higher rate of proliferation (plt0001) in all

irradiated cultures in comparison with the controls Green light yielded a significantly

higher number of cells than red (plt0001) and infrared LED light (plt0001) and than

the cultures irradiated with the LLL (plt0001) the red probe provided a higher

increase (plt0001) than the infrared LED probe and than the LLL source

Conclusion LED and LLL irradiation resulted in an increased fibroblast proliferation

in vitro This study therefore postulates possible stimulatory effects on wound healing

in vivo at the applied dosimetric parameters

Keywords Biostimulation Fibroblast proliferation Light Emitting Diodes Low

Level Laser Tetrazolium salt

LED induced increase of fibroblast proliferation

49

INTRODUCTION

Since the introduction of photobiostimulation into medicine the effectiveness and

applicability of a variety of light sources in the treatment of a wide range of medical

conditions [1-5] has thoroughly been investigated in vitro as well as in vivo The results

of several investigations are remarkably contradictory This is at least in part a

consequence of the wide range of indications as well as the wide range of suitable

parameters for irradiation and even the inability to measure the possible effects after

irradiation with the necessary objectivity [457] A lack of theoretical understanding

can also be responsible for the existing controversies In fact theoretical understanding

of the mechanisms is not necessary to establish effects though it is necessary to

simplify the evaluation and interpretation of the obtained results As a consequence

the widespread acceptance of especially Low Level Laser (LLL) therapy in the early

seventies is faded nowadays and biostimulation by light is often viewed with scepticism

[8] According to Baxter [49] contemporary research and consumption in

physiotherapy is in particular focused on the stimulation of wound healing Tissue

repair and healing of injured skin are complex processes that involve a dynamic series

of events including coagulation inflammation granulation tissue formation wound

contraction and tissue remodelling [10] This complexity aggravates research within this

cardinal indication

Research in this domain mostly covers LLL studies but the current commercial

availability of other light sources appeals research to investigate as well the effects of

those alternative light sources eg Light Emitting Diode (LED) apparatus

The scarcity of literature on LED is responsible for consultation of literature

originating from LLL studies [11] but it may be wondered if this literature is

representative for that purpose As in the early days of LLL therapy the stimulating

effects upon biological objects were explained by its coherence [1213] while the beam

emitted by LEDrsquos on the contrary produces incoherent light Though the findings of

some scientists [914151617] pose nowadays that the coherence of the light beam is

not responsible for the effects of LLL therapy Given that the cardinal difference

between LED and LLL therapy coherence is not of remarkable importance in

Chapter 2

50

providing biological response in cellular monolayers [5] one may consult literature

from LLL studies to refer to in this LED studies

The purpose of this preliminary study is to examine the hypothesis that LED

irradiation at specific output parameters can influence fibroblast proliferation

Therefore irradiated fibroblasts cultures were compared with controls The article

reports the findings of this study in an attempt to promote further discussion and

establish the use of LED



Fibroblasts were obtained from 8-days old chicken embryos Isolation and

disaggregation of the cells was performed with warm trypsin according the protocol

described by Ian Freshney (1994) [18] The primary explants were cultivated at 37degC in

Hanksrsquo culture Medium supplemented with 10 Fetal Calf Serum 1 Fungizone 1

L-Glutamine and 05 Penicillin-Streptomycin When cell growth from the explants

reached confluence cells were detached with trypsine and subcultured during 24 hours

in 80-cm2 culture flasks (Nunc) in 12 ml of primary culture medium After 72 hours

the cells were removed from the culture flasks by trypsinization and counted by Buumlrker

hemocytometry For the experiment cells from the third passage were plated in 96-well

plates (Nunc) with a corresponding area of 033 cm2 they were subcultured at a

density of 70000 cellcm2 Cultures were maintained in a humid atmosphere at 37deg C

during 24 hours

All supplies for cell culture were delivered by NV Life Technologies Belgium except

for Fetal Calf Serum (Invitrogen Corporation UK)

Irradiation sources

In this study two light sources a Light Emitting Diode (LED) device and a Low Level

Laser (LLL) device were used in comparison to control cultures

The used LLL was an infrared GaAlAs Laser (Unilaser 301P MDB-Laser Belgium)


51

with an area of 0196 cm2 a wavelength of 830 nm a power output ranging from 1-400

mW and a frequency range from 0-1500 Hz

The Light Emitting Diode device (BIO-DIO preprototype MDB-Laser Belgium)

consisted of three wavelengths emitted by separate probes A first probe emitting

green light had a wavelength of 570 nm (power-range 10-02 mW) the probe in the

red spectrum had a wavelength of 660 nm (power-range 80-15 mW) and the third

probe had a wavelength of 950 nm (power-range 160-80 mW) and emitted infrared

light The area of all three probes was 18 cm2 and their frequency was variable within

the range of 0-1500 Hz

Exposure regime

Prior to irradiation the 96-well plates were microscopically verified to guarantee that

the cells were adherent and to assure that there was no confluence nor contamination

Following aspiration of 75 Hanksrsquo culture Medium irradiation started The remaining

25 (50 microl) medium avoided dehydration of the fibroblasts throughout irradiation

The 96-well plates were randomly assigned in the treated (LLL or green red or infrared

LEDrsquos) or the control group

For the treatments in this study the continuous mode was applied as well for the LLL

as for the three LED-probes The distance from light source to fibroblasts was 06 cm

LLL therapy consisted of 5 seconds irradiation at a power output of 40 mW resulting

in a radiant exposure of 1 Jcm2 The infrared and the red beam delivered a radiant

exposure of 053 Jcm2 and the green beam emitted 01 Jcm2 corresponding to

exposure-times of respectively 1 minute 2 minutes or 3 minutes and a respective

power output of 160 mW 80 mW or 10 mW

After these handlings the remaining medium was removed and new Hanksrsquoculture

medium was added followed by 24 hours of incubation

One irradiation (LLL or LED) was performed daily during three consecutive days

according to the aforementioned procedure Control cultures underwent the same

handling but were sham-irradiated

Chapter 2

52

Determination of cell proliferation

The number of cells within the 96-well plates as a measure for repair [19] was

quantified by a sensitive and reproducible colorimetric proliferation assay [2021] The

colorimetric assay was performed at two different points of time to determine the

duration of the effect of the used light sources

This assay exists of a replacement of Hanksrsquoculture medium by fresh medium

containing tetrazolium salt 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium

bromide (MTT) 24 or 72 hours after the third irradiation for MTT analysis as

described by Mosmann (1983) [22] Following a 4 hour incubation at 37degC the MTT

solution was substituted by lysing buffer isopropyl alcohol The plates were

temporarily shaken to allow dissolution of the produced formazan crystals After 30

minutes of exposure to the lysing buffer absorbance was measured The absorbance at

400 to 750 nm which was proportional to fibroblast proliferation was determined

using an ELtimes800 counter (Universal Microplate Reader Bio-Tek Instruments INC)

The complete procedure from isolation to MTT assay was executed six times (Trial A

B C D E and F) while it was impossible to irradiate all the investigated number of

wells with the same LED apparatus on one day All the trials included as much control

as irradiated wells but the number of control and irradiated wells in each trial varied

depending on the number of available cells after the second subculturing A further

consequence of the available number of cells is the number of probes examined per

trial Varying from 4 probes in trial A and F to 1 probe in trial B C D and E

Incubation period before proliferation analyses numbered 24 hours To investigate if

the stimulatory effect tends to occur immediately after irradiation or after a longer

period of time incubation in trial F lasted 72 hours

An overview of the followed procedures regarding incubation time before proliferation

analysis number of analysed wells for each trial and the number of probes examined

per trial is given in table 1 As a consequence of the differences in procedures followed

and because each trial started from a new cell line the results of the five trials must be

discussed separately


53

Statistical analysis

Depending on the amount of groups to be compared within each trial and depending

on the p-value of the Kolmogorov-Smirnov test of normality a T-test or one-way

ANOVA was used for parametrical analyses and a Kruskal-Wallis or Mann-Whitney-U

test was used for nonparametrical comparisons Statistical significance for all tests was

accepted at the 005 level For this analysis Statistical Package for Social Sciences 100

(SPSS 100) was used

RESULTS

The results presented in table 1 show that cell counts by means of MTT assay

revealed a significant (plt0001) increase in the number of cells in comparison to their

respective sham-irradiated controls for all the irradiated cultures of trial A B C D

and E except the irradiated groups in trial F

Moreover the results of trial A showed that the effect of the green and red LED probe

was significantly (plt0001) higher than the effect of the LLL probe With regard to the

amount of proliferation the green probe yielded a significantly higher number of cells

than the red (plt0001) and the infrared probe (plt0001) Furthermore the red probe

provided a higher increase in cells (plt0001) than the infrared probe

The infrared LED source and the LLL provided a significant (plt0001) higher number

of cells than the control cultures but no statistical significant difference was recorded

between both light sources

The trials A B C D and E regardless of the number of probes used in each trial

were analysed after 24 hours of incubation after the last irradiation The incubation

period of trial F lasted 72 hours

The means of trial F illustrated that the effect was opposite after such a long

incubation The control cultures had significantly (plt0001) more fibroblasts than the

irradiated cultures with the exception of the LED-infrared group that showed a not

significant increase of cells Further analysis revealed that the green probe yielded a

significantly lower number of cells than the red (plt0001) and the infrared probe

(plt0001) and that the red probe provided a higher decrease (plt0001) than the

Chapter 2

54

infrared probe Laser irradiation induced a significant decrease of fibroblasts in

comparison to the infrared irradiated cultures (plt0001) and the control cultures

(p=0001) LED irradiation with the green and the red probe revealed no statistical

significant differences

Table 1 Fibroblast proliferation after LED and LLL irradiation

Groups

Absorbency (proportional to the number of fibroblasts)a

Trial A n=64 Control 595 plusmn 056 TP=24h Irradiated (LLL) 675 plusmn 050

Irradiated (LED-infrared) 676 plusmn 049 Irradiated (LED-red) 741 plusmn 059 Irradiated (LED-green) 775 plusmn 043 Trial B n=368 Control 810 plusmn 173 TP=24h Irradiated (LLL) 881 plusmn 176 Trial C n=368 Control 810 plusmn 173 TP=24h Irradiated (LED-infrared) 870 plusmn 178 Trial D n=192 Control 886 plusmn 084 TP=24h Irradiated (LED-red) 917 plusmn 066 Trial E n=192 Control 818 plusmn 075 TP=24h Irradiated (LED-green) 891 plusmn 068 Trial F n=64 Control 482 plusmn 049 TP=72h Irradiated (LLL) 454 plusmn 065 Irradiated (LED-infrared) 487 plusmn 044 Irradiated (LED-red) 446 plusmn 044 Irradiated (LED-green) 442 plusmn 035 a Absorbency proportional to the number of fibroblasts as determined by MTT analysis plusmn SD and significances ( plt0001) in comparison to the control group n = number of analysed wells for each group within a trial TP = Time Pre-analysis incubation time before proliferation analysis

DISCUSSION

Despite the failure of some studies [223] to demonstrate beneficial effects of laser and

photodiode irradiation at relatively low intensities (lt500mW) on fibroblast


55

proliferation this study provides experimental support for a significant increased cell

proliferation Therefore these results confirm previous studies that yielded beneficial

stimulating effect [1152425] Remarkably though is the higher increase noted after

irradiation at lower wavelengths (570 nm) Van Breughel et al [26] observed a general

decrease in absorption at longer wavelengths and concluded that several molecules in

fibroblasts serve as photoacceptors resulting in a range of absorption peaks (420 445

470 560 630 690 and 730 nm) The wavelength of the used lsquogreenrsquo LED probe is the

closest to one of these peaks

Karu [5] also emphasises that the use of the appropriate wavelength namely within the

bandwidth of the absorption spectra of photoacceptor molecules is an important

factor to consider

In this particular context penetration depth can almost be ignored as virtually all

wavelengths in the visible and infrared spectrum will pass through a monolayer cell

culture [12] The irradiance (Wcm2) on the contrary could have had an important

influence on the outcome of this study The higher increased proliferation by the lower

wavelengths is possibly a result of the lower irradiance of these wavelengths Lower

irradiances are confirmed by other experiments to be more effective than higher

irradiances [111626]

The used radiant exposures reached the tissue interaction threshold of 001 Jcm2 as

described by Poumlntinen [17] but in the scope of these results it also needs to be noticed

that there is a substantial difference in radiant exposure between the LLL (1 Jcm2)

the green LED probe (01 Jcm2) and the remaining LED probes (053 Jcm2)

Consequently the results of especially trial A and F must be interpreted with the

necessary caution It is possible that the determined distinction between the used light

sources and the used probes is a result from the various radiant exposures applied

during the treatments of the cultures

Notwithstanding the increased proliferation revealed with MTT analysis 24 hours after

the last irradiation this study was unable to demonstrate a stimulating effect when

analysis was performed 72 hours after the last irradiation Moreover this longer

incubation period even yielded an adverse effect Although a weakening of the

Chapter 2

56

photostimulating influence over time is acceptable it can not explain a complete

inversion Especially in the knowledge that a considerable amount of authors still

ascertain an effect after a longer incubation period [2427] In an attempt to illuminate

this finding one can suppose that the circadian response of the cells triggered by the

LED and the LLL [1228] forfeited after a prolonged period (72 hours) in the dark

The most obvious explanation is even though a decreased vitality and untimely cell

death in the irradiated cell cultures as a result of reaching confluence at an earlier point

of time than the control cultures The cells of a confluent monolayer have the tendency

to inhibit growth and finally die when they are not subcultured in time No other

reasonable explanations could be found for this discrepancy

Photo-modulated stimulation of wound healing is often viewed with scepticism The

real benefits of Light Emitting Diodes if any can only be established by histological

and clinical investigations performed under well controlled protocols Despite these

remarks this study suggests beneficial effects of LED and LLL irradiation at the

cellular level assuming potential beneficial clinical results LED application on

cutaneous wounds of human skin may be assumed useful at the applied dosimetric

parameters but future investigation is necessary to explain the mechanisms of LED





Persons in good health rarely require treatment for wound healing as posed by Reddy

et al [13] light has a possible optimal effect under conditions of impaired healing

Postponed wound healing is a time-consuming and often expensive complication

Thus future prospects must remind to examine the therapeutic efficacy of LED on

healing-resistant wounds


57

ACKNOWLEDGMENTS

The authors are grateful to Prof Deridder for supplying the laboratory as well as the

material necessary for this investigation and to Ms Franccedilois laboratory worker for


Chapter 2

58

REFERENCES

1 Reddy GK Stehno Bittel L Enwemeka CS (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-55

2 Pogrel MA Ji Wel C Zhang K (1997) Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes Lasers Surg Med 20426-32

3 Reddy GK Stehno Bittel L Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-7

4 Baxter GD Allen J Therapeutic lasers theory and practice 1st ed Edinburgh Churchill Livingstone 1994

5 Karu T The science of low-power laser therapy 1st ed New Delhi Gordon and Breach Science Publishers 1998

6 Lagan KM Clements BA McDonough S Baxter GD (2001) Low intensity laser therapy (830nm) in the management of minor postsurgical wounds a controlled clinical study Lasers Surg Med 2827-32

7 Basford JR (1995) Low intensity laser therapy still not an established clinical tool Lasers Surg Med 16331-42

8 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61671-5

9 Baxter G Bell A Allen J Ravey J (1991) Low level laser therapy Current clinical practice in Northern Ireland Physiotherapy 77171-8

10 Karukonda S Corcoran Flynn T Boh E McBurney E Russo G Millikan L (2000) The effects of drugs on wound healing part 1 Int J Dermatol 39250-7

11 Lowe AS Walker MD OByrne M Baxter GD Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Lasers Surg Med 23291-8

12 Boulton M Marshall J (1986) He-Ne laser stimulation of human fibroblast proliferation and attachment in vitro Lasers in the Life Science 1125-34

13 Mester E Mester AF Mester A (1985) The biomedical effects of laser application Lasers Surg Med 531-9

14 Pontinen PJ Aaltokallio T Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21105-18

15 Whelan HT Houle JM Whelan NT Donohoe DL Cwiklinski J Schmidt MH Gould L Larson DL Meyer GA Cevenini V Stinson H (2000) The NASA Light-Emitting Diode medical program - Progress in space flight terrestrial applications Space Technology and Applications International Forum pp 37-43

16 Kana JS Hutschenreiter G Haina D Waidelich W (1981) Effect of low-power density laser radiation on healing of open skin wounds in rats Arch Surg 116293-6

17 Poumlntinen P (2000) Laseracupunture In Simunovic Z (ed) Lasers in Medicine and Dentistry Part One Basic Science and Up-to-date Clinical Application of Low Energy-Laser Laser Therapy LLLT 1st ed Rijeka Vitgraf 2000pp 455-475

18 Freshney I Culture of animal cells A manual of basic technique New York Wiley-Liss 1994 19 Savunen TJ Viljanto JA (1992) Prediction of wound tensile strength an experimental study Br J

Surg 79401-3 20 Freimoser F Jakob C Aebi M Tuor U (1999) The MTT [3-(45-Dimethylthiazol-2yl)-

25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 653727-9

21 Alley MC Scudiero DA Monks A Hursey ML Czerwinski MJ Fine DL et al (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay Cancer Res 48589-601

22 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Methods 6555-63


59

23 Hallman HO Basford JR OBrien JF Cummins (1988) LA Does low-energy helium-neon laser irradiation alter in vitro replication of human fibroblasts Lasers Surg Med 8125-9

24 Webb C Dyson M Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22294-301

25 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11783-9 26 van Breugel HH Bar PR (1992) Power density and exposure time of He-Ne laser irradiation are

more important than total energy dose in photo-biomodulation of human fibroblasts in vitro Lasers Surg Med 12528-37

27 Pourreau Schneider N Ahmed A Soudry M Jacquemier J Kopp F Franquin JC et al (1990) Helium-neon laser treatment transforms fibroblasts into myofibroblasts Am J Pathol 137171-8

28 Pritchard DJ (1983) The effects of light on transdifferentiation and survival of chicken neural retina cells Exp Eye Res 37315-26

CHAPTER 3

GREEN LIGHT EMITTING DIODE IRRADIATION ENHANCES

FIBROBLAST GROWTH IMPAIRED BY HIGH GLUCOSE LEVEL

Elke Vincka Barbara Cagniea Maria Cornelissenb Heidi Declercqb and Dirk

Cambiera


Photomedicine and Laser Surgery 2005 23(2) 167-171

Chapter 3

62

ABSTRACT

Background and Objective The chronic metabolic disorder diabetes mellitus is an

important cause of morbidity and mortality due to a series of common secondary

metabolic complications such as the development of severe often slow healing skin

lesions

In view of promoting the wound-healing process in diabetic patients this preliminary

in vitro study investigated the efficacy of green light emitting diode (LED) irradiation on

fibroblast proliferation and viability under hyperglycemic circumstances

Materials and Methods To achieve hyperglycemic circumstances embryonic chicken

fibroblasts were cultured in Hanksrsquo culture medium supplemented with 30 gL

glucose LED irradiation was performed on 3 consecutive days with a probe emitting

green light (570 nm) and a power output of 10 mW Each treatment lasted 3 min

resulting in a radiation exposure of 01 Jcm2

Results A Mann-Whitney test revealed a higher proliferation rate (p=0001) in all

irradiated cultures in comparison with the controls

Conclusion According to these results the effectiveness of green LED irradiation on

fibroblasts in hyperglycemic circumstances is established Future in vivo investigation

would be worthwhile to investigate whether there are equivalent positive results in

diabetic patients

Keywords Light Emitting Diodes middot Fibroblast proliferation middot Diabetes

Fibroblast proliferation under hyperglycemic circumstances

63

INTRODUCTION

The chronic metabolic disorder diabetes mellitus is found worldwide It exhibits wide

geographic variation in incidence and prevalence generally 11 of the world

population is affected and worldwide it is the twelfth leading cause of death1 Those

figures may be higher for urban regions as well as for industrialized countries Due to

multiple factors involving the aging process of the population and lifestyle changes

(such as reduced physical activity hypercaloric eating habits and concomitant obesity)

these figures may increase in the future2-6 Therefore diabetes mellitus could become

the most common chronic disease in certain regions as stated by Gale it ldquotargets the

rich in poor countries and the poor in rich countriesrdquo6

The harmful disruption of the metabolic equilibrium in diabetes mellitus results in

characteristic end-organ damage that occurs in various combinations and that follows

an unpredictable clinical pathway

Accordingly the major consequence of diabetes mellitus in terms of morbidity

mortality and economic burden principally concerns macroangiopathies or

arteriosclerosis and microangiopathies including nephropathy neuropathy and

retinopathy7-10

One of these devastating consequences which often appears in time is the

development of various skin defects that are frequently resistant to healing and that

tend to be more severe than similar lesions in nondiabetic individuals Diabetes

mellitus even increases the risk of infection by an increased susceptibility to bacteria

and an impaired ability of the body to eliminate bacteria1112

Skin problems are a severe complication in diabetic individuals and require a

comprehensive and appropriate multidisciplinary approach to prevention and

treatment12

Hyperglycemia is the key metabolic abnormality in diabetes mellitus that is believed to

play the most prominent role in the development of diabetic complications With the

development of insulin treatment for type I diabetes and various oral hypoglycemic

agents for type 2 diabetes a reduction in the development of skin defects due to

hyperglycemia should be noted913-15 Nevertheless once a lesion appears simply

Chapter 3

64

waiting for that lesion to heal spontaneously is often unsatisfactory Wounds in

diabetic patients often need special care in comparison to those persons in good

health who rarely require treatment for wound healing1617 Special care is directed

besides of course toward optimal diabetes regulation toward patient education

maximum pressure relief controlling infection recovery of circulation in case of

ischemia and different modalities of intensive wound treatment18

In the last few years various therapies have been introduced with varying success An

example of such a therapy is the photo-modulated stimulation of diabetic lesions In

vitro as well as in vivo the effectiveness of especially low level lasers (LLL) has been

subject of extensive investigation1920 Due to contradictory research results LLL-

photobiostimulation of injured skin is often viewed with scepticism20-22 As the use of

light in the domain of wound healing is less time-consuming less expensive less

invasive than many of the other introduced treatment modalities and practical to use

however it seems worthwhile to investigate the value and benefits of a newly

introduced and alternative light source the light emitting diodes (LEDrsquos)

Preliminary research has proved that green LED with particular properties (an

exposure time of 3 minutes a power output of 10 mW and a radiant exposure of 01

Jcm2) revealed positive stimulatory effects on fibroblast proliferation in vitro23 These

results may be of great importance to the diabetic patient because as posed by Reddy et

al light has a possible beneficial effect in the case of impaired healing1617

To obtain insight into the ability of LED to stimulate fibroblast proliferation under

diabetic-specific conditions of impaired healing the proliferation was assessed in

irradiated and control cultures cultivated in medium with a high quantity of glucose

MATERIAL amp METHODS

Cell cultivation

Primary fibroblast cultures were established by outgrowth from 8-day-old chicken

embryos After isolation and disaggregating as described by Freshney (1994)24 the cells

were grown to confluence at 37degC in Hanksrsquo culture medium supplemented with 10


65

fetal calf serum 1 fungizone 1 L-glutamine and 05 penicillin-streptomycin

Secondary cultures were initiated by trypsinization followed by plating of the cells in

80-cm2 culture flasks (Nunc) and cultivation during 72 h The fibroblasts were

disaggregated by trypsinization and counted by Buumlrker hemocytometry Subsequently

231 x 104 fibroblasts (corresponding to a density of 70 x 104 cellscm2) from the third

passage were plated in the wells of 96-well tissue culture plates (Nunc) For 24 h the

cells were maintained in 200 microl supplemented Hanksrsquo culture medium and a humidified

atmosphere at 37deg C to allow them to attach to the bottom of the wells

Light source specifications and illumination procedure

To control adherence of the cells and to assure that there was no confluence or

contamination the 96-well plates were microscopically examined before irradiation

Subsequently the tissue culture plates were randomly assigned for use in the treated

and control groups Immediately before irradiation 75 of the Hanksrsquo culture medium

was aspirated The remaining 25 (50 microl) medium avoided dehydration of the

fibroblasts throughout irradiation

Irradiation was performed with a light emitting diode (LED) device The LED device

(BIO-DIO preprototype) emitted green light with a wavelength of 570 nm (power

range 02-10 mW) The area of the probe was 18 cm2 and its frequency was variable

within the range of 0-1500 Hz

The investigation used the following illumination properties the continuous mode a

distance of 06 cm from light source to fibroblasts and a beam emission of 01 Jcm2

radiant exposure This procedure resulted in an exposure time of 3 min and a power

output of 10 mW Immediately after irradiation the remaining medium was aspirated

and the cells were restored in 200 microl Hanksrsquo culture medium containing 1667 mM

glucose (30 gL) and incubated at 37deg C

Irradiation and medium changes occurred at 1-day intervals so one irradiation was

implemented each 24 h for 3 days in a row and from the first irradiation onwards all

medium renewals occurred with glucose-supplemented Hanksrsquo culture medium

Control cultures were handled in the same manner but were sham-irradiated

Chapter 3

66

Proliferation assay

Fibroblast survival and proliferation were determined by a sensitive and reproducible

colorimetric assay the assay which detects merely living cells and the signal generated

bears a constant ratio to the degree of activation of the fibroblasts and the number of

fibroblasts25-27 It is a reliable method as it considers all cells in a sample rather than

only a small subsample26

Subsequent to an incubation period of 24 h the 200 microl glucose-supplemented

Hanksrsquoculture medium was substituted by the same amount of Hanksrsquoculture medium


bromide (MTT)2627 After 4 h of incubation at 37deg C the pale yellow MTT solution

was replaced by the lysing buffer isopropyl alcohol and the plates were shaken during

30 min to dissolve the dark-blue formazan crystals and to produce a homogeneous

solution The optical density of the final solution was measured on an ELtimes800 counter

(Universal Microplate Reader Bio-Tek Instruments Prongenbos Belgium) using a test

wavelength varying from 400 to 750 nm

The used light source was provided by MDB-Laser (Ekeren Belgium) and all supplies

for cell culture were delivered by NV Life Technologies (Merelbeke Belgium) except

for fetal calf serum supplied by Invitrogen Corporation (Paisley UK)

Data analysis

On account of the p-value of the Kolmogorov-Smirnov test of normality (p=034) a

Mann-Whitney U test was performed for nonparametrical comparison of the results

Statistical significance for all tests was accepted at the 005 level For this analysis the

Statistical Package for the Social Sciences (SPSS 100 SPSS Inc Chicago IL) was used

RESULTS

The MTT measurements from each of the 256 control wells and 256 irradiated wells

and the subsequent nonparametrical analysis from the optical densities obtained

disclosed a significantly (p=0001) higher rate of proliferation in hyperglycemic


67

circumstances after irradiation than in the same circumstances without irradiation (Fig

1)

Fig 1 Significantly (p=0001) higher fibroblast proliferation in the irradiated group after green LED irradiation as determined by MTT analysis (plusmn SD)

DISCUSSION

The outcome of these in vitro experiments based on the above-described light source

properties and the illumination procedure described clearly demonstrated the

stimulatory potential of LED on fibroblast proliferation and the cell viability of

fibroblasts cultured in hyperglycemic medium Preliminary research has already

demonstrated that under these conditions (an exposure time of 3 min a wavelength of

570 nm a power output of 10 mW and a radiant exposure of 01 Jcm2) this

procedure allowed the highest number of living cells The nature of the light and the

usual questions concerning coherence wavelength power output and radiant

exposures have been discussed previously23

Although these findings confirm the results previously found one cannot ignore the

important methodological difference between previous investigations and the current

study as the cells in this experiment were cultured in hyperglycemic medium2328-30

Absorbency - Proportional to the number of fibroblasts

621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68

After a growth period with normal Hanksrsquo culture medium a necessary step to ensure

normal growth of these secondary subcultures and normal attachment to the bottom

of the wells the Hanksrsquo culture medium was supplemented with glucose

Several earlier studies have established that exposure to glucose concentrations (20-40

mM) mimicing hyperglycemia of uncontrolled diabetes results in a restraint of human

vascular endothelial cell proliferation1531-34 This restraint is more pronounced for

higher glucose15 concentrations and is expressed especially after protracted exposure to

high glucose levels31 A similar restraint was found for cultured fibroblasts by

Hehenberger et al3536 According to some authors however cultured fibroblasts

conversely have been shown to maintain responsiveness to ambient high glucose323738

As there are some ambiguities in literature regarding normal or inhibited growth of

fibroblasts in medium supplemented with glucose39 a pilot study was performed to

determine the amount of glucose necessary to inhibit normal growth after 72 h of

culturing in 96-well plates at a density of 70times104 cellscm2 This pilot study

demonstrated that an amount of 1667 mM glucose was necessary to cause a decrease

of cell viability and to bring about a decline in fibroblast proliferation

This concentration resulted in a remarkable reduction of cell viability and a noteworthy

decrease in the proliferation rate in comparison to control cultures grown in 55 mM

glucose although this concentration is too high to mimic severe diabetic

hyperglycaemia (20-40 mM31-33) This high antiproliferative effect was necessary to

investigate the effect of LED in distinct destructive conditions in order to obtain an

incontrovertible result

In addition it is possible that the present investigation needed a higher amount of

glucose to result in a remarkable reduction of proliferation as exposure to glucose was

limited to 72 h and as previous studies revealed that the antiproliferative effect of high

glucose was more pronounced with prolonged exposure with a maximal inhibition

attained by 7-14 days1531

Moreover with regard to the in vivo situation it must be stated that in vitro and in vivo

cell growth are too complex to compare A key question is whether fibroblast

senescence in tissue culture and in the intact organism are similar Cristofalo et al40


69

reported that this is not the case as fibroblasts have a finite ability to divide and

replicate but apparently the pathway or the morphologic characteristics leading to the

replicative senescence is not identical in vivo compared to in vitro

Furthermore extrinsic aging related to environmental damage which in diabetic

patients is mainly due to a chronic exposure to high levels of glucose during life is

unachievable in vitro

Unless a number of questions regarding the mechanism according to which LED

stimulates fibroblast proliferation in this particular condition remain unanswered the

results ascertain the potential effects of LED on fibroblast proliferation and viability

CONCLUSION

The current results should be interpreted with caution However these results

demonstrate the effectiveness of green LED irradiation at the above-described light

source properties and the illumination procedure described on cells in hyperglycemic

circumstances

The findings of the present study using an experimental in vitro model indicate that the

use of LED irradiation to promote wound healing in diabetic patients may have

promising future results As the present study establishes the possibility of using LED

irradiation in experimental in vitro situations it would be a worthwhile extension to

perform in vivo investigations to determine whether these in vitro observations were

relevant to the physiological situation and to determine the effect of these LED

properties on human tissue response

ACKNOWLEDGMENTS

The authors are greatly indebted to P Coorevits for assistance with the statistical

analysis and to Professor L Deridder and Ms N Franccedilois of the department of

Human Anatomy Embryology Histology and Medical Physics for providing access to

the laboratory and for helpful discussions

Chapter 3

70

REFERENCES

1 World Health Organisation The world health report of 2002 Statistical Annex 2002 pp 179-201

2 van Ballegooie E and van Everdingen J (2000) CBO-richtlijnen over diagnostiek behandeling en preventie van complicaties bij diabetes mellitus retinopathie voetulcera nefropathie en hart- en vaatziekten Ned Tijdschr Geneeskd 144(9) 413-418

3 Weiss R Dufour S Taksali SE et al (2003) Prediabetes in obese youth a syndrome of impaired glucose tolerance severe insulin resistance and altered myocellular and abdominal fat partitioning Lancet 362(9388) 951-957

4 Stovring H Andersen M Beck Nielsen H Green A and Vach W (2003) Rising prevalence of diabetes evidence from a Danish pharmaco-epidemiological database Lancet 362(9383) 537-538

5 Barcelo A and Rajpathak S (2001) Incidence and prevalence of diabetes mellitus in the Americas Pan Am J Public Health 10(5) 300-308

6 Gale E (2003) Is there really an epidemic of type 2 diabetes Lancet 362(9383) 503-504 7 Reiber GE Lipsky BA and Gibbons GW (1998) The burden of diabetic foot ulcers Am J

Surg 176(2A Suppl) 5S-10S 8 American Diabetes Association (1999) Consensus development conference on diabetic foot

wound care Diabetes Care 22(8)1354-1360 9 Wang PH Lau J and Chalmers TC (1993) Meta-analysis of effects of intensive blood-

glucose control on late complications of type I diabetes Lancet 341(8856) 1306-1309 10 Vermeulen A(1982) Endocriene ziekten en stofwisselingsziekten Gent Story Scientia 11 Laing P (1998) The development and complications of diabetic foot ulcers Am J Surg 176(2A

Suppl) 11S-19S 12 Kozak G (1982) Clinical Diabetes Mellitus Philadelphia Saunders Company p 541 13 Groeneveld Y Petri H Hermans J and Springer M (1999) Relationship between blood

glucose level and mortality in type 2 diabetes mellitus a systematic review Diabet Med 16(1) 2-13

14 Reichard P Nilsson BY and Rosenqvist U (1993) The effect of long-term intensified insulin treatment on the development of microvascular complications of diabetes mellitus N Engl J Med 329(5) 304-309

15 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4) 491-501

16 Reddy GK Stehno Bittel L and Enwemeka CS (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22(5) 281-287

17 Reddy K Stehno-Bittel L and Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9(3) 248-255

18 Bakker K and Schaper NC(2000) Het diabetisch voetulcus nieuwe ontwikkelingen in de behandeling Ned Tijdschr Geneeskd 144(9) 409-412

19 Skinner SM Gage JP Wilce PA and Shaw RM (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3) 188-192

20 Schindl A Schindl M Pernerstorfer-Schoumln H and Schindl L (2001) Low intensity laser therapy in wound healing - a review with special respect to diabetic angiopathies Acta Chir Austr 33(3) 132-137

21 Basford JR (1986) Low-energy laser treatment of pain and wounds hype hope or hokum Mayo Clin Proc 61(8) 671-675

22 Schindl A Heinze G Schindl M Pernerstorfer-Schoumln H and Schindl L (2002) Systemic effects of low-intensity laser irradiation on skin microcirculation in patients with diabetic microangiopathy Microvasc Res 64(2) 240-246

23 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D(2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18 95-99


71

24 Freshney I (1994) Culture of animal cells A manual of basic technique New York Wiley-Liss 25 Alley MC Scudiero DA Monks A et al (1988) Feasibility of drug screening with panels of

human tumor cell lines using a microculture tetrazolium assay Cancer Res 48(3) 589-601 26 Freimoser F Jakob C Aebi M and Tuor U(1999) The MTT [3-(45-Dimethylthiazol-2yl)-

25Diphenyltetrazolium Bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities Appl Environ Microbiol 65(8) 3727-3729

27 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays J Immunol Meth 65 55-63

28 Webb C Dyson M and Lewis WHP (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5) 294-301

29 Nemeth AJ (1993) Lasers and wound healing Dermatol Clin 11(4) 783-789 30 Whelan H Houle J Whelan N et al (2000) The NASA light-emitting diode medical program -

progress in space flight terrestrial applications Space Technol Applic Intern Forum 504 37-43 31 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of

cultured human endothelial cells Diabetes 36(11) 1261-1267 32 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA

damage in cultured human endothelial cells J Clin Invest 77(1) 322-325 33 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in

culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7) 621-627 34 Stout RW (1982) Glucose inhibits replication of cultured human endothelial cells Diabetologia

23(5) 436-439 35 Hehenberger K Hansson A Heilborn JD Abdel Halim SM Ostensson CG and Brismar

K (1999) Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat a spontaneous model of non-insulin dependent diabetes mellitus Wound Repair Regen 7(1) 65-71

36 Hehenberger K Heilborn JD Brismar K and Hansson A (1998) Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate Wound Repair Regen 6(2) 135-141

37 Turner JL and Bierman EL (1978) Effects of glucose and sorbitol on proliferation of cultured human skin fibroblasts and arterial smooth-muscle cells Diabetes 27(5) 583-588

38 Hayashi JN Ito H Kanayasu T Asuwa N Morita I Ishii T and Murota S (1991)Effects of glucose on migration proliferation and tube formation by vascular endothelial cells Virchows Arch B Cell Pathol Incl Mol Pathol 60(4) 245-252

39 Maquart FX Szymanowicz AG Cam Y Randoux A and Borel JP (1980) Rates of DNA and protein syntheses by fibroblast cultures in the presence of various glucose concentrations Biochimie 62(1) 93-97

40 Cristofalo VJ Allen RG Pignolo RJ Martin BG and Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture a reevaluation Proc Natl Acad Sci USA 95(18) 10614-10619

PART II ANALGESIA

CHAPTER 4

EVIDENCE OF CHANGES IN SURAL NERVE CONDUCTION

MEDIATED BY LIGHT EMITTING DIODE IRRADIATION

Elke Vincka Pascal Coorevitsa Barbara Cagniea Martine De Muynckb Guy

Vanderstraetenab and Dirk Cambiera

a Department of Rehabilitation Sciences and Physiotherapy Ghent University Belgium b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium


Chapter 4

76

ABSTRACT

The introduction of light emitting diode (LED) devices as a novel treatment for pain

relief in place of low-level laser warrants fundamental research on the effect of LED

devices on one of the potential explanatory mechanisms peripheral neurophysiology in

vivo

A randomised controlled study was conducted by measuring antidromic nerve

conduction on the peripheral sural nerve of healthy subjects (n=64) One baseline

measurement and five post-irradiation recordings (2 min interval each) were performed

of the nerve conduction velocity (NCV) and negative peak latency (NPL)

Interventional set-up was identical for all subjects but the experimental group (=32)

received an irradiation (2 min at a continuous power output of 160 mW resulting in a

radiant exposure of 107 Jcm2) with an infrared LED device (BIO-DIO preprototype

MDB-Laser Belgium) while the placebo group was treated by sham irradiation

Statistical analysis (general regression model for repeated measures) of NCV and NPL

difference scores revealed a significant interactive effect for both NCV (p=0003) and

NPL (p=0006) Further post hoc LSD analysis showed a time-related statistical

significant decreased NCV and an increased NPL in the experimental group and a

statistical significant difference between placebo and experimental group at various

points of time

Based on these results it can be concluded that LED irradiation applied to intact skin

at the described irradiation parameters produces an immediate and localized effect

upon conduction characteristics in underlying nerves Therefore the outcome of this in

vivo experiment yields a potential explanation for pain relief induced by LED

Keywords Light Emitting Diodes middot Sural nerve middot Conduction velocity middot Negative

peak latency middot Analgesic effect

Nerve conduction characteristics

77

INTRODUCTION

Since the introduction of photobiostimulation into medicine the light sources used

have advanced technologically and varied in characteristics over the years

Advancement and variation of the sources implicate a concomitant necessity to revise

research results in the respective domains of application Research and clinical

applications in the past particularly focused on the effectiveness of low-level lasers

have shifted now to novel treatment units such as light emitting diode (LED) devices

The efficacy and applicability of LED irradiation within the field of wound healing has

already partially been substantiated in vitro [12] as well as in vivo [3-6] However LED

is not only promoted for its beneficial effects on the wound-healing process it is also

suggested to be potentially effective in the treatment of pain of various aetiology

although this claim has not yet been investigated thoroughly either experimentally or

clinically The putative analgesic effects of LED remain to be further explored

As the basic vehicle of pain is the neuronal system [7] measuring the

neurophysiological effect of LED treatment would be an appropriate experimental

approach to investigate the efficacy of LED on pain inhibition Nerve conduction

studies have become a technique for investigating the neurophysiologic effects of light

therapy [8-9]

Review of literature regarding standard nerve conduction studies revealed that previous

human studies on the influence of various light sources on peripheral nerves have

utilized different methods which hampers a comprehensive comparison In general

this research was performed on the superficial radial nerve [10-13] described by Shin J

Oh [14] as an uncommon nerve conduction technique or on the mixed median nerve

[891315-17] Following the method of Cambier et al [18] the authors of this study

decided to investigate the effect of the light source used on the conduction

characteristics of the sural nerve By investigating this solely sensory nerve interaction

of motor nerve fibres (motor response can easily be provoked by antidromic nerve

stimulation [19]) can be avoided and given the superficial nature of the nerve it should

be sufficiently amenable to the effects of percutaneous LED irradiation

Chapter 4

78

A second major difference between the trials and therefore also hindering an

appropriate comparison between the results is the wide range of used light sources

HeNe lasers [121316] and GaAlAs lasers [8-101718] or a monochromatic infrared

multisource treatment unit [15]

With respect to the potential importance of LED irradiation for the treatment of pain

the current investigation was designed to assess the putative neurophysiological effects

of LED on the sensory nerve conduction of the human superficial peripheral sural

nerve and to establish a time course of the supposed phenomenon

The experimental hypothesis postulates that LED generates an immediate decrease in

conduction velocity and increase in negative peak latency In addition it can be

postulated that this effect is most prominent immediately after the irradiation and will

weaken as time progresses

STUDY DESIGN

The study was approved by the Ethical Committee of the Ghent University Hospital

After explanation of the experimental procedure a written informed consent was

obtained from each subject

Subjects

After screening based on a brief medical history excluding subjects with

contraindications to LED irradiation (such as light hypersensitivity fluctuating blood

pressure insufficient blood circulation fever inflammation of the skin) or conditions

that might affect sensory nerve conduction (such as diabetes peripheral neuropathy

radicular syndrome peripheral nerve damage neuromuscular disorders or peripheral

edema) eligible subjects were enrolled Sixty-four healthy volunteers 24 males and 40

females (mean age 26plusmn6 years range 18-42 years) participated in this study The body

mass index (BMI) of each subject varied within the normal range (=185-249) [20]

(mean BMI 216plusmn17 range 186-249) Subjects were randomly allocated to a placebo


79

or an experimental group Each group of 32 subjects was composed of 12 males and

20 females

Experimental Procedure and Data Acquisition

In order to be able to quantify the negative peak latency (NPL) (measured from the

start of the stimulus artefact to the peak of the negative portion of the nerve action

potential) and nerve conduction velocity (NCV) of the sural nerve a rigid protocol was

followed

With respect to the known relationship between nerve conduction characteristics and

temperature the ambient temperature was kept constant (23ordmC-26ordmC room

temperature) during the investigation In view of this temperature issue the

standardized protocol started with 10 min of accommodation during which the

subjects rested in prone position on a treatment table

Immediately before this adjustment period the skin over the dorsolateral aspect of the

left calf and foot was cleaned with alcohol to remove surface lipids This preparation of

the treatment area was followed by the placement of the electrodes (TECA

Accessories Oxford Instruments Medical Systems Division Old Woking UK) as

described by Delisa et al [21]

The two-posted (2 cm separation anode distal) surface caption electrode was placed

distal and posterior of the lateral malleolus on the skin covering the sural nerve The

fixation of the earth electrode (Medelec Oxford Instruments Medical Systems

Division Old Woking UK) occurred 12 cm above the caption electrode according to

the description of Delisa et al [21] A standard bipolar stimulator was used at 14 cm

above the caption electrode to map the ideal stimulation point To level off

intraindividual variations in the amount of sensory response attributable to the

successive placement of the bipolar stimulator in course of the investigation a two-

posted (2 cm separation cathode distal) bar stimulating electrode was attached at the

point where the maximal response was obtained

This placement of the electrodes allows antidromic stimulation of the sural nerve

Electrophysiological stimulation and recordings were obtained with a Medelec

Chapter 4

80

Sapphire Premiere (Vickers Medical Old Woking UK) providing a monophasic pulse

of 01 ms A supramaximal stimulus intensity with a nominal voltage of 72-295 was

used to produce each evoked sensory response

Baseline measurements of NPL and NCV were immediately followed by treatment of

the subjects according the protocol detailed below Recordings were subsequently

repeated at 2-min intervals over an 8-min period resulting in five recordings (one

immediately after the completion of the treatment and one at 2 4 6 and 8 min after

irradiation) Skin temperature was recorded concomitantly throughout the procedure

at the time of baseline measurement immediately after LED irradiation at the time of

the first recording and consequently at 2-min intervals together with the four final

electrophysiological recordings For this a surface digital C9001 thermometer

(Comark UK) sensitive to temperature changes of 01degC was used at the same point

of LED administration namely at 7 cm above the caption electrode The procedure

was identical for both conditions but subjects in the placebo group received a sham

LED irradiation

Light Characteristics and Irradiation Procedure

Irradiation was administrated with a light emitting diode device (BIO-DIO

preprototype MDB-Laser Belgium) The probe used emitted infrared light with a

wavelength of 950 nm (power range 80-160 mW) The area of the probe was 18 cm2

and the frequency was variable within the range of 0-1500 Hz

Preceding baseline measurement the treatment point was marked on the skin overlying

the course of the sural nerve at 7 cm above the capture electrode ie the exact mid-

point between the stimulation and capture electrode The LED probe was held in

contact with the skin perpendicular to the skin surface during the complete irradiation

procedure LED treatment consisted for all subjects of the experimental group out of 2

minutes lasting irradiation The LED was set to deliver a continuous energy density of

107 Jcm2 at a power output of 160 mW These parameters were selected as they are

appropriate for the treatment of pain in a clinical setting First of all because the


81

duration of the treatment is clinically feasible and secondly because the parameters are

within the scope of previously described light source characteristics [1-36915]

Statistics

Although superficial skin temperature did not change significantly in course of the

investigation the influence of the measured skin temperature on NPL and NCV was

taken into account by using a correction factor of respectively 02 msdegC and 147

ms degC All corrections were calculated towards a reference skin temperature of 32degC

Difference scores ie the variation between baseline measurements and each post-

irradiation recording were used as the basis for statistical analysis A General

Regression Model for repeated measures with one within-subjects factor (time 0

min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) and

one between-subjects factor (group placebo or LED irradiated) was performed

followed by appropriate pairwise comparisons (post hoc LSD or post hoc Least

Significant Difference) to determine whether any differences between baseline

measurements and post-irradiation recordings were statistically significant

The Statistical package for social sciences (SPSS 110) was used for analysis and

statistical significance for all tests was accepted at the 005 level

RESULTS

Figure 1 shows NCV mean difference scores of the placebo and the LED irradiated

group plotted against time in minutes The values of the irradiated subjects decrease

directly after the irradiation and reach a first low point 2 min after finishing LED

treatment This decrease is followed by a marginal increase at 4 and 6 min and again an

important decrease at 8 min Statistical analysis (general regression model for repeated

measures) of these data indicated a significant interactive effect (P=0003)

Chapter 4

82

Fig 1 Mean difference scores (ms variation between baseline measurements and post-treatment recordings) of the nerve conduction velocity plotted against time (minutes) (meansplusmnSD n=32)

Post hoc LSD further showed significant differences between baseline measurements

and all post-treatment recordings (Table 1) Mutual comparison of the values from the

post-treatment recordings did not reveal any significant difference In addition there

was no significant difference determined in the placebo group in course of time

Table 1 Summary of the influence of LED irradiation on nerve conduction velocity

Minutes Placeboa Time-Related Significance b

LEDa Time-Related Significance b

Group Significance c

0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001

8 0021plusmn0386 0932 -1146plusmn1881 0003 0001 a Mean difference scores and standard deviations of the recorded nerve conduction velocity of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)

Nerve Conduction Velocity

-14

-12

-1

-08

-06

-04

-02

0

02

04

Baseline 0 min 2 min 4 min 6 min 8 min

Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

A similar representation was used for the results of the NPL Figure 2 reproduces NPL

plotted against time in minutes revealing for the irradiated group an increased latency

with two important peaks one at 4 min and one at 8 min

Fig 2 Mean difference scores (variation between baseline measurements and post-treatment recordings) of the negative peak latency plotted against time (minutes) (meansplusmnSD n=32)

Statistical analysis of the mean difference scores again indicated a significant interactive

effect (P=0006) Further post hoc LSD analysis of the data presented in Table 2

showed significant differences between baseline measurements and all post-treatment

recordings of the experimental group The mean difference score of the first post-

treatment recording of this same group (LED irradiated) differed significantly with the

recording 4 min (P=0003) 6 min (P=0018) and 8 min (Plt0001) after LED

irradiation As well as the recording 2 min after irradiation which differed significantly

(P=0013) with the 8 min post-treatment recording As observed for the NCV the

NPL of the placebo group did not reveal any significant difference in time course

At the time of the final recording the NCV and NPL mean difference scores of the

irradiated group did not return to their respective baseline values

Negative Peak Latency

-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84

Furthermore post hoc LSD analysis also presented in Tables 1 and 2 (group

significance) revealed statistical differences between the experimental and the placebo

group for NCV as well as for NPL NCV and NPL were statistical significant between

both groups at all points of time except from the NPL recording immediately after

finishing irradiation

DISCUSSION

Notwithstanding the above-mentioned difficulties in comparing results between

different trials on nerve conduction we attempt to discuss the current findings in view

of the results of the previous studies

This investigation revealed that percutaneous LED irradiation at feasible and current

clinical parameters generates measurable and significant changes in human sural nerve

antidromic conduction latency and velocity These results thus support previous

findings of light-mediated nerve conduction latency shifts in vivo [8101218]

although there are several important issues to be discussed

Table 2 Summary of the influence of LED irradiation on negative peak latency




0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034

8 0014 plusmn0052 0264 0064plusmn0088 lt0001 0007 a Mean difference scores and standard deviations of the recorded negative peak latency of the placebo and the LED irradiated group b P-values of the pairwise comparison (post hoc LSD) between baseline measurements and all post-treatment recordings (baseline=preceding irradiation 0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) of the placebo and the LED irradiated group c P-values of the pairwise comparison (post hoc LSD) between placebo and irradiated subjects for all post-treatment recordings (0 min=immediately after irradiation 2 4 6 and 8 min following LED irradiation) Significant P-values (Plt005)


85

A first comment deals with the progress of the NCV and NPL in function of time As

postulated the NCV decreases significantly immediately after irradiation

corresponding with a significant increased NPL However this effect does not weaken

as time progresses both variables remain significant throughout the 8 min during

observation period

Cambier et al [18] noted a similar significant effect of GaAlAs laser irradiation on the

conduction characteristics until 15 minutes post-treatment as did Walsh et al [10]

although this slight increase in NPL was not significant at any moment Two other

studies [822] with a GaAlAs laser even registered comparable effects over a period of

55 [8] and 60 min [22] post-irradiation respectively Given the results of these previous

studies post-treatment conduction measurements should be extended in time At

present for all studies it remains unclear at what point of time the effect extinguishes

although the interval of time during which LED treatment remains effective is

clinically important when treating pain

Noble et al [15] also noticed relatively long-lasting neurophysiological effects (at least

45 min) mediated by a monochromatic multisource infrared diode device although it

needs to be mentioned that this study performed with a comparable light source as the

current investigation revealed a significant decrease of the NPL These inverse results

between the study of Noble et al [15] and the current investigation could be attributed

to the concomitant increase of the skin temperature [15] As it has been well

recognised that a variation in tissue temperature causes a corresponding alteration in

nerve conduction velocities and peak latencies [91523-27] the temperature changes

may indeed provide an explanation for the observed findings In an attempt to analyse

the influence of a direct photobiological effect on sural nerve conduction

characteristics rather than working out the effects based upon thermal mechanisms

the present study corrected the skin temperature towards a reference temperature of

32degC This correction was performed notwithstanding the fact that the superficial skin

temperature did not change significantly before and after LED irradiation as well as

despite the fact that influencing nerve temperature takes place long after affecting skin

temperature [23] and thus being (almost) impossible after 2 min of irradiation

Chapter 4

86

followed by 8 min of registration Introduction of the correction factor implies likewise

that eventual influence on nerve conduction by cooling of the limb due to inactivity as

described by Greathouse et al [11] can be excluded

These facts suggest that temperature changes did not contribute to the demonstrated

effects of LED on nerve conduction Nevertheless the underlying mechanism of the

observed effects remains indistinct

A following remark regarding the fluctuation of NCV and NPL in function of time

considers the fact that both the NCV and the NPL do not change in a constant way up

to eight minutes after LED irradiation (Figs 1 2) The decrease in NCV and the

increase in NPL display a small though not significant inversion of the effect at 4 and

(NCV) or 6 (NPL) min This is probably attributable to the fact that some degree of

fluctuation is to be expected when measuring NCV and NPL besides there is a similar

variation in the placebo groups

Although investigating dose dependency was not intended an additional remark

considers the fact that the use of optimal irradiation parameters is essential to obtain

the observed neurophysiological effect Nevertheless it is impossible to determine

ideal light source characteristics for effective treatment as the range of used

wavelengths (632-950 nm) radiant exposures (107-96 Jcm2) and even frequency

(pulsed or continuous) are not sufficiently similar between the different studies It can

only be concluded that a pulsing light source [91028] does not provide the postulated

results Radiant exposure exposure time power range and wavelength are not yet

established but based on this study and previously described assays it can be

speculated that the ranges of these parameters are quite large

In comparison with other studies where the number of subjects is 10 or less [8-

1115162229] (with the exception of the studies from Cambier et al [18] and Snyder-

Mackler et al [12] who respectively tested 15 and 24 subjects) a relatively large number

of subjects (n=32) was investigated in each group In spite of the large investigated

population it should be noted that the magnitude of the described changes in NCV

and NPL can simply be replicated by lowering the temperature of the extremity as the

observed changes are within the expected physiological ranges making the clinical


87

significance of the change questionable (This fact does not implement that the

decrease and the significant changes were temperature mediated)

A key question and meanwhile the initial impetus for future investigation is whether

the measured effects can be extrapolated to the actual nociceptive afferents namely the

myelinated Aδ-fibres(12-30 ms [14]) and unmyelinated C-fibres (05-2 ms [14])

respectively conducting acute and chronic pain The functional testing of these

nociceptive pathways has recently been extensively evaluated The currently accepted

neurophysiological method of assessing nociceptive pathways relies on laser-evoked

potentials (LEPs) [30] as they selectively activate Aδ-fibres and C-fibres [31]

As up till now LEP is not available in this or any surrounding research centre the

investigators of this study had to perform a standard nerve conduction study (assessing

the large myelinated Aβ afferents) Therefore the current and previous beneficial

results of low level light therapy on conduction characteristics of nerves in vivo should

initiate measurements of clinical effectiveness first of all in laboratory settings and

afterward at a clinical level

CONCLUSION

Despite these remarks and the limited knowledge regarding the underlying mechanism

the present findings enable the following conclusions to be drawn LED irradiation at

clinical applied energy densities produces an immediate and localized effect upon

conduction characteristics in underlying nerves More specifically it is proven that

LED treatment lowers the NCV and augments the NPL resulting in a reduced

number of impulses per unit of time Therefore the outcome of this in vivo experiment

assumes that LED possibly induces pain relief

In order to encourage a widespread acceptance for the use of this non-invasive pain-

reducing modality in clinical settings prospective research should establish the precise

relationship between LED and pain relief as well as determine the ideal irradiation

parameters and verify which painful conditions can be treated with this treatment unit

Chapter 4

88

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr S Rimbaut for explaining the handling of the

equipment and MDB-Laser Belgium for generously providing the Light Emitting

Diode equipment


89

REFERENCES

1 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Laser Med Sci 18(2)95-9

2 Vinck E Cagnie B Cornelissen M Declercq H and Cambier D (2005) Green light emitting diode irradiation enhances fibroblast growth impaired by high glucose level Journal of Photomedicine and Laser Surgery (In Press)

3 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electrother Res 21(2) 105-18

4 Lowe AS Walker MD OByrne M Baxter GD and Hirst DG (1998) Effect of low intensity monochromatic light therapy (890 nm) on a radiation-impaired wound-healing model in murine skin Laser Surg Med 23(5) 291-8

5 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M et al (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space technology and applications international forum pp 37-43

6 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in Biomedical Optics and Imaging 3(28 Proceedings of SPIE 4903) 156-65

7 Bromm B and Lorenz J (1998) Neurophysiological Evaluation of Pain Electroencephalography and Clinical Neurophysiology 107(4)227-53


9 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Laser Surg Med 14(1)40-6

10 Walsh D Baxter G and Allen J (2000) Lack of effect of pulsed low-intensity infrared (820 nm) laser irradiation on nerve conduction in the human superficial radial nerve Laser Surg Med 26(5)485-90

11 Greathouse DG Currier DP and Gilmore RL (1985) Effects of clinical infrared laser on superficial radial nerve conduction Phys Ther 65(8)1184-7

12 Snyder-Mackler L and Bork CE (1988) Effect of helium-neon laser irradiation on peripheral sensory nerve latency Phys Ther 68(2)223-5

13 Basford JR Daube JR Hallman HO Millard TL and Moyer SK (1990) Does low-intensity helium-neon laser irradiation alter sensory nerve active potentials or distal latencies Laser Surg Med 10(1)35-9

14 Oh SJ (1993) Clinical electromyography Nerve conduction studies Williams and Wilkins Baltimore

15 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Surg 19(6)291-5

16 Walker JB and Akhanjee LK (1985) Laser-induced somatosensory evoked potentials evidence of photosensitivity in peripheral nerves Brain Res 344(2)281-5

17 Basford JR Hallman HO Matsumoto JY Moyer SK Buss JM and Baxter GD (1993) Effects of 830 nm continuous wave laser diode irradiation on median nerve function in normal subjects Laser Surg Med 13(6)597-604

18 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Laser Med Sci 15195-200

19 Aydin G Keles I Demir SO Baysal AI (2004) Sensitivity of Median Sensory Nerve Conduction Tests in Digital Branches for the Diagnosis of Carpal Tunnel Syndrome Am J Phys Med Rehab 83(1)17-21

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20 National Institutes of Health National Heart Lung and Blood Institute (1998) Clinical guidelines on the identification evaluation and treatment of overweight and obesity in adults the evidence report NIH publication no 98-4083

21 DeLisa J MacKenzie K and Baran E (1987) Manual of nerve conduction velocity and somatosensory evoked potentials New York Raven Press

22 Baxter GD Allen JM and Bell AJ (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63

23 Geerlings A and Mechelse K (1985) Temperature and nerve conduction velocity some practical problems Electromyogr Clin Neurophysiol 25(4)253-9

24 DHaese M and Blonde W (1985) The effect of skin temperature on the conductivity of the sural nerve Acta Belg Med Phys 8(1)47-9

25 Halar E DeLisa J and Brozovich F (1980) Nerve conduction velocity relationship of skin subcutaneous and intramuscular temperatures Arch Phys Med Rehabil 61(5)199-203

26 Bolton CF Sawa GM and Carter K (1981) The effects of temperature on human compound action-potentials J Neurol Neurosur and Psychiatry 44(5)407-13

27 Hlavova A Abramson D Rickert B and Talso J (1970) Temperature effects on duration and amplitude of distal median nerve action potential J Appl Physiol 28(6)808-12

28 Lowe AS Baxter GD Walsh DM and Allen JM (1995) The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nmpulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve Laser Med Sci 10(4)253-9

29 Baxter GD Allen JM Walsh DM Bell AJ and Ravey J (1992) Localization of the effect of low-energy laser irradiation upon conduction latencies in the human median nerve in vivo J Physiol-London 446P445

30 Truini A Romaniello A Galeotti F Iannetti GD and Cruccu G (2004) Laser Evoked Potentials for Assessing Sensory Neuropathy in Human Patients Neurosci Lett 361(1-3)25-8

31 Bentley DE Watson A Treede RD Barrett G Youell PD Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-56

CHAPTER 5

PAIN REDUCTION BY INFRARED LIGHT EMITTING DIODE

IRRADIATION A PILOT STUDY ON EXPERIMENTALLY

INDUCED DELAYED-ONSET MUSCLE SORENESS IN HUMANS

Elke Vincka Barbara Cagniea Pascal Coorevitsa Guy Vanderstraetenab and Dirk

Cambiera


b Department of Physical Medicine and Orthopaedic Surgery Ghent University Belgium

Accepted for publication in Lasers in Medical Science December 2005

Chapter 5

92

ABSTRACT

Objective The present pilot study investigated the analgesic efficacy of light emitting

diode (LED) In view of a standardised and controlled pain reduction study design this

in vivo trial was conducted on experimentally induced delayed-onset muscle soreness

(DOMS)

Design Thirty-two eligible human volunteers were randomly assigned to either an

experimental (n=16) or placebo group (n=16) Immediately following the induction of

muscle soreness perceived pain was measured by means of a visual analog scale (VAS)

followed by a more objective mechanical pain threshold (MPT) measurement and

finally an eccentricconcentric isokinetic peak torque (IPT) assessment The

experimental group was treated with infrared LED at one of both arms the other arm

served as control Irradiation lasted 6 min at a continuous power output of 160 mW

resulting in an energy density of 32 Jcm2 The subjects of the placebo group received

sham irradiation at both sides In post-treatment a second daily assessment of MPT

and VAS took place The treatment and assessment procedure (MPT VAS and IPT)

was performed during 4 consecutive days

Results Statistical analysis (a general linear model followed by post hoc least

significant difference) revealed no apparent significant analgesic effects of LED at the

above-described light parameters and treatment procedure for none of the three

outcome measures However as the means of all VAS and MPT variables disclose a

general analgesic effect of LED irradiation in favour of the experimental group

precaution should be taken in view of any clinical decision on LED

Conclusion Future research should therefore focus on the investigation of the

mechanisms of LED action and on the exploration of the analgesic effects of LED in a

larger randomised clinical trial and eventually in more clinical settings

Keywords Light Emitting Diode middot Infrared middot Analgesic effect middot Delayed-onset

muscle soreness middot Musculus biceps brachii

Delayed-onset muscle soreness

93

INTRODUCTION

The analgesic efficacy of light emitting diode (LED) irradiation is recently being

investigated by means of a nerve conduction study on the superficial peripheral sural

nerve [1] It was demonstrated that LED irradiation at clinical applied densities

produces an immediate and localized effect upon conduction characteristics in

underlying nerves More specific LED induces a decreased number of sensory

impulses per unit of time thus possibly inducing pain relief [1]

Given the established influence of this treatment modality on the nerve conduction

velocity and thereby its potential analgesic ability the current investigation was

designed

Studies investigating the efficacy of a therapeutic modality on pain often experience

difficulties regarding standardisation of the population as analysis or comparison of

pain with different aetiologies is almost impossible Therefore we opted to measure the

analgesic effects of LED in a laboratory setting on a sample with experimentally

induced delayed-onset of muscle soreness (DOMS)

Muscle soreness usually occurs at the musculotendinous junction 8-24 h after the

induction exercise and then spreads throughout the muscle [2-4] The correlates of

DOMS reach peak intensity at 24-48 h with symptoms disappearing around days 5-10

[2 3 5-10] The cardinal signs characterising DOMS are reduced muscle force

decreased range of motion and in particular muscle pain which is more pronounced

during movement and palpation [8 11] Despite the large volume of research that has

been undertaken to identify the underlying pathophysiology of DOMS the precise

mechanism is not yet universally accepted Several theories such as the torn-tissue

theory the connective tissue damage theory the muscle spasm theory and the

inflammation theory still remain viable though the current opinion states that DOMS

arises from a sequence of events in which several theories occupy an important place

[2 6 12 13]

DOMS has been used as a representative model of musculoskeletal pain and stiffness

in a number of studies [4 7 11 14 15] as it has a number of advantages it can be

induced in a relatively easy and standardised manner in a group of healthy subjects the

Chapter 5

94

time-course is relatively predictable and the symptoms have the same aetiology and are

of transitory nature [14 16] Nevertheless it should be emphasised that the use of this

particular experimental model to test the effectiveness of LED does not mean that this

treatment modality is necessarily advocated for the treatment of DOMS but merely

that it may be helpful in documenting the efficacy of LED in a clinical model of

musculoskeletal pain and stiffness In addition studies based on the induction of

DOMS under carefully controlled laboratory conditions can not replace research

involving actual patients but offer the opportunity to assess the effectiveness of

particular therapeutic interventions and might help to define additional clinical research

[14]

The experimental hypothesis of the current study postulates that infrared LED reduces

pain and muscle sensitivity associated with DOMS


The study was approved by the ethical committee of the Ghent University Hospital

After providing information regarding the study design and possible consequences

related to participation at the study written informed consent was obtained from each

subject

Subjects

Healthy human volunteers were recruited from the university population Individuals

with any upper limb pathology neurological deficit and recent injury to either upper

extremity or undiagnosed pain were excluded Other exclusion criteria were


pressure insufficient blood circulation fever and inflammation of the skin) or

conditions in which physical exertion is contraindicated (such as cardiovascular deficits

hypertension and respiratory problems)

Thirty-two eligible subjects (16 males and 16 females) aged 19-35 years (mean age

23plusmn4 years) were enrolled All subjects were randomly assigned using a random table


95

of numbers to the experimental or placebo group Each group of 16 subjects

consisted by stratification of equal numbers of men and women Age height and

weight did not differ significantly between the three groups

All subjects were physically active however none performed on a regular basis any type

of upper body weight-training Subjects were requested to refrain from any form of

strenuous physical activity and they were asked to avoid any form of medication

including anti-inflammatory agents as well as alcohol for 2 days before testing and for

the duration of the study

Overview of experimental design

The study lasted 4 consecutive days On day 1 isokinetic exercise was performed to

induce pain related to DOMS Immediately following induction exercise an initial

assessment of the outcome measures (visual analog scale or VAS mechanical pain

threshold or MPT and isokinetic peak torque or IPT) occurred Subsequently the

subjects were treated under blinded conditions according to the randomised group

allocation In post-treatment the MPT was re-recorded and perceived pain was

reassessed with a VAS Contrary to these outcome measures the muscle strength was

only measured in pre-treatment at the one hand because short-term effects of LED

on muscle strength were not postulated and on the other hand because post-

treatment muscle strength can be influenced by too many different physiological

factors related to the pre-treatment measurement On the succeeding days (day 2 3

and 4) the treatment and assessment procedure was similar with approximately 24 h

separating each treatment

In both of the groups the two arms of the participants were included in the study In

the experimental group an equal number of dominant and non-dominant arms were

treated The non-treated arm served as control arm In the placebo group also an equal

number of dominant and non-dominant arms were considered as treated arm and the

other arm was classified in the non-treated group The procedure was identical for

both conditions but the subjects in the placebo group received sham LED irradiation

on both arms

Chapter 5

96

Specific aspects of the experimental design and procedures are detailed below

Pain induction

Muscle soreness was induced in a standardised fashion via a daily calibrated computer-

operated Biodex isokinetic dynamometer (Biodex Medical Systems Inc Shirley NY

USA) Induction occurred separately and in random order in the elbow flexors of both

arms Therefore the subjects were seated as described by the usersrsquo manual of Biodex

Prior to induction of DOMS the subjects were allowed an initial familiarization session

to become comfortable performing maximum voluntary contractions at the required

angular velocities This was immediately followed by determination of the maximum

eccentric and concentric peak torque at an angular velocity of 60degs and 120degs

Subsequently four sessions of eccentricconcentric work were performed with each

arm The first two sessions consisted of elbow flexion at an angular velocity of 60degs

first of all along an arch of 120deg from full extension (designated as 0deg) through 120deg

and second of all elbow flexion over a range of 60deg from 30deg to 90deg of flexion (mid-

range) followed by two sessions at an angular velocity of 120degs again the first time

along an arch of 120deg and followed by the mid-range performance The subjects were

asked to accomplish maximum voluntary contractions during all the sessions Each

session was performed until exhaustion which was defined as the point when the

subject lost 70 of the initial eccentric and concentric peak torque There was a 1-

minute rest between each session This procedure was based on a pilot study and

previously described induction protocols [17-21]

Outcome measures

Outcome measures of subjective pain measurements MPT and muscle strength were

measured in this order on days 1-4 Subjective pain measurements and MPT occurred

immediately prior to and following irradiation whereas muscle strength measurements

only took place before LED treatment

Measurement of subjective pain Perceived muscle soreness was measured

subjectively by means of a 100-mm VAS A series of scales were completed separately


97

for each arm pain at rest followed by pain perception associated with full extension of

the arms and finally with maximal flexion of the arms The subjects were not allowed

to compare one VAS result with another

This assessment tool commonly used in measuring experimentally induced pain [22

23] has been found to be a reliable and valid method [24-26]

MPT Tenderness MPT used as a more objective correlate of muscle tenderness

has been demonstrated to be a reliable method to measure experimental induced

muscle soreness [27] This outcome measure was assessed by using a handheld

pressure algometer with a 12-cm diameter head (Microfet 2 Hoggan Health Industries

South Jordan USA) On day 1 three points were marked at 4-cm intervals [8] along a

line from the radial insertion of the musculus biceps brachii at the elbow to the

intertubercular groove of the humerus thus resulting in three measure points one at

the musculotendinous junction (4 cm) and two at the muscle belly (8 cm and 12 cm) A

pressure of 4Ns was delivered The subjects were instructed to say yes at the exact

moment the pressure perceived became painful Each point was recorded three times

in pre-treatment as well as in post-treatment The average MPT score for each point in

pre- and post-treatment was used for statistical analyses

Muscle strength assessment Eccentric and concentric IPT were measured on the

same computerised dynamometer as was used for the induction of pain and an

identical standardisation procedure regarding positioning was followed

A warm-up session of two maximum voluntary contractions at the required angular

velocities was followed by determination of the eccentric and concentric peak torque

The first session at 60degs consisted of three repetitions followed by a 1-min during

rest and for the second session at 120degs five repetitions were performed The

subjects were instructed to flex and extend the elbow through the entire range of

motion as forcefully and rapidly as possible for each repetition The maximum

eccentric and concentric torque produced during the respective repetitions was used

for statistical analysis

Chapter 5

98

Light source specifications and treatment procedure

Light treatment was applied daily according to group allocation Irradiation occurred

with an LED device (BIO-DIO preprototype MDB-Laser Ekeren Belgium) The

probe used emitted infrared light with a wavelength of 950 nm (power range 80ndash160

mW) The area of the probe was 18 cm2 and consisted of 32 single LEDs The

frequency was variable within the range of 0ndash1500 Hz

During the complete irradiation procedure the LED probe was held in contact with

the skin perpendicular to the skin surface and at the exact mid-point between the MPT

mark at 4 cm and the one at 8 cm Light source properties were identical for all

subjects of the experimental group and consisted out of irradiation of 6-min lasting

duration at a continuous power output of 160 mW resulting in an energy density of

32 Jcm2 To conceal the treated side and condition the subjects were blinded to the

treatment status For the experimental condition a probe was held in contact with each

arm but only one of the two probes was attached to the LED device The subjects of

the placebo group received sham irradiation at both sides

The selected parameters are within the scope of previously described light source

characteristics for pain reduction [1 28-30] and they are appropriate for the treatment

of pain in a clinical setting because the duration of the treatment is clinically feasible


The three outcome measures were analysed separately For the VAS and MPT

measurements the same procedure was followed a general linear model (GLM) for

repeated measures with two within-subjects factors (time days 1 2 3 and 4 and pre-

post preceding and following LED irradiation) and one between-subject factor (group

placebo or infrared LED irradiated) was performed If necessary the GLM was

followed by appropriate pairwise comparisons (post hoc least significant difference or

LSD) to determine whether any differences between measurements were statistically

significant A similar model was carried out separately for both the treated and the

control arm


99

In contrast to MPT and VAS the muscle strength was analysed differently The peak

torque values recomputed towards body weight of the subjects were statistically

analysed using a GLM for repeated measures This model consisted of one within-

subject factor (time day 1 2 3 and 4) and one between-subject factor (group placebo

or infrared LED irradiated) The model was completed twice first for the treated arm

and consequently for the control arm

The statistical package for social sciences (SPSS 110 SPSS Inc Chicago IL USA)

was used for analysis and statistical significance for all tests was accepted at the 005

level

RESULTS

Statistical analysis of all variables of the three outcome measures revealed no significant

interactive effects of the main interaction (time times group times pre-post) The means and

standard deviations of the variables for both the treated and the control arm are

outlined in Table 1 for the VAS Table 2 for the MPT and Table 3 for the IPT The

means of all VAS and MPT variables disclose a non-statistical significant general

analgesic effect of LED irradiation conveyed by lower subjective pain rates and higher

MPT values in the irradiated group than in the placebo group The lower VAS rates are

present from day 1 until the last day of the study but they are more clearly present

from day 3 pre-treatment The higher MPT values are present from day 1 post-

irradiation until the last day and they are more visible at 4 cm followed by 12 cm and

finally at 8 cm In addition to the analgesic influence of LED an increased

convalescence of muscle strength was noted It should be remarked that this outcome

is similar for the treated as well as for the control arm of the irradiated group The

findings are also illustrated by Fig 1 2 and 3 depicting the time-course for both arms

of VAS at rest MPT at 4 cm and IPT for eccentric contraction at 60degs respectively

Graphical presentation of the other variables shows a similar course

Chapter 5

100

Table 1 Mean scores and standard errors for the visual analog scale of the treated and the control arm of the placebo and the LED irradiated group

Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post

Treated arm Placebo Rest 55plusmn28 61plusmn27 68plusmn26 52plusmn21 125plusmn42 114plusmn35 99plusmn37 84plusmn30 Eccentric 79plusmn42 108plusmn39 267plusmn38 216plusmn40 396plusmn57 384plusmn51 325plusmn529 296plusmn47 Concentric 92plusmn35 104plusmn35 176plusmn34 180plusmn33 318plusmn46 313plusmn40 251plusmn47 241plusmn42 Irradiated Rest 51plusmn28 51plusmn27 66plusmn26 44plusmn21 86plusmn42 56plusmn35 49plusmn37 31plusmn30 Eccentric 91plusmn42 78plusmn39 233plusmn38 191plusmn40 300plusmn57 230plusmn51 222plusmn529 168plusmn47 Concentric 82plusmn35 74plusmn35 132plusmn34 122plusmn33 208plusmn46 166plusmn40 142plusmn47 103plusmn42

Control arm Placebo Rest 32plusmn25 44plusmn24 74plusmn23 46plusmn16 132plusmn42 105plusmn34 88plusmn37 68plusmn24 Eccentric 64plusmn42 64plusmn32 234plusmn42 176plusmn34 355plusmn48 355plusmn49 301plusmn48 28plusmn43 Concentric 81plusmn36 94plusmn34 184plusmn33 164plusmn30 302plusmn44 272plusmn42 215plusmn42 208plusmn36 Irradiated Rest 51plusmn25 48plusmn24 56plusmn23 36plusmn16 69plusmn42 49plusmn34 37plusmn37 26plusmn24 Eccentric 98plusmn42 79plusmn32 218plusmn42 195plusmn34 284plusmn48 246plusmn49 172plusmn48 161plusmn43 Concentric 89plusmn36 70plusmn34 122plusmn33 10630 192plusmn44 161plusmn42 111plusmn42 94plusmn36

Figure 1 Mean visual analog scale (VAS) score (at rest) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)

Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)

Placebo - treated armIrradiated - treated armPlacebo - control armIrradiated - control arm


101

Table 2 Mean scores and standard errors for the mechanical pain threshold of the treated and the control arm of the placebo and the LED irradiated group

Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post

Treated arm Placebo 4 cm 1577plusmn147 1284plusmn159 1045plusmn252 1194plusmn237 1098plusmn194 1201plusmn274 1436plusmn217 1515plusmn2128 cm 1761plusmn147 1659plusmn156 1289plusmn246 1390plusmn245 1351plusmn185 1421plusmn227 1711plusmn205 1780plusmn21412 cm 1704plusmn184 1674plusmn210 1119plusmn286 1212plusmn280 1202plusmn239 1309plusmn282 1587plusmn245 1538plusmn257Irradiated 4 cm 1515plusmn147 1851plusmn159 1738plusmn252 1852plusmn237 1719plusmn194 1925plusmn274 2004plusmn217 2005plusmn2128 cm 1708plusmn147 1675plusmn156 1640plusmn246 1682plusmn245 1478plusmn185 1620plusmn227 1824plusmn205 1967plusmn21412 cm 1697plusmn184 1775plusmn210 1637plusmn286 1642plusmn280 1540plusmn239 1663plusmn282 1864plusmn245 2021plusmn257Control arm Placebo 4 cm 1556plusmn160 1294plusmn177 1112plusmn202 1246plusmn239 1091plusmn229 1184plusmn224 1434plusmn217 1404plusmn2088 cm 1768plusmn152 1660plusmn149 1369plusmn205 1416plusmn221 1366plusmn206 1442plusmn248 1783plusmn254 1798plusmn20912 cm 1732plusmn190 1566plusmn190 1177plusmn239 1292plusmn310 1255plusmn248 1283plusmn298 1671plusmn285 1550plusmn249Irradiated 4 cm 1520plusmn160 1850plusmn177 1587plusmn202 1725plusmn239 1718plusmn229 1778plusmn224 2068plusmn217 2026plusmn2088 cm 1697plusmn152 1752plusmn149 1506plusmn205 1586plusmn221 1563plusmn206 1646plusmn248 2158plusmn254 1958plusmn20912 cm 1712plusmn190 1835plusmn190 1432plusmn239 1670plusmn310 1555plusmn248 1707plusmn298 1981plusmn285 2056plusmn249

Figure 2 Mean mechanical pain threshold (MPT) score (at 4 cm) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)

Mechanical Pain Threshold

0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102

Table 3 Mean scores and standard errors for the isokinetic peak torque of the treated and the control arm of the placebo and the LED irradiated group

Day 1 Day 2 Day 3 Day 4

Treated arm Placebo Eccentric 60degsec 50plusmn05 54plusmn05 47plusmn04 49plusmn05 Concentric 60degsec 43plusmn04 39plusmn04 40plusmn04 40plusmn04 Eccentric 120degsec 45plusmn04 48plusmn04 48plusmn05 46plusmn05 Concentric 120degsec 37plusmn04 37plusmn03 34plusmn04 34plusmn04 Irradiated Eccentric 60degsec 50plusmn05 57plusmn05 54plusmn04 58plusmn05 Concentric 60degsec 42plusmn04 43plusmn04 41plusmn04 45plusmn04 Eccentric 120degsec 47plusmn04 51plusmn04 52plusmn05 57plusmn05 Concentric 120degsec 41plusmn04 38plusmn03 38plusmn04 41plusmn04

Control arm Placebo Eccentric 60degsec 54plusmn05 54plusmn05 48plusmn05 53plusmn06 Concentric 60degsec 44plusmn04 45plusmn04 40plusmn04 43plusmn05 Eccentric 120degsec 49plusmn04 54plusmn05 48plusmn05 51plusmn04 Concentric 120degsec 40plusmn04 35plusmn03 35plusmn04 40plusmn04 Irradiated Eccentric 60degsec 55plusmn05 54plusmn05 57plusmn05 59plusmn56 Concentric 60degsec 44plusmn04 43plusmn04 38plusmn04 46plusmn05 Eccentric 120degsec 48plusmn04 54plusmn05 55plusmn05 58plusmn04 Concentric 120degsec 38plusmn04 36plusmn03 42plusmn04 43plusmn04

Figure 3 Mean isokinetic peak torque (IPT) score (eccentric at 60degs) of the treated and the control arm of the placebo and the LED irradiated group plotted against time (day and pre-post)

Isokinetic Peak Torque

04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

Despite the absence of significant main interaction effects the remaining interactions

as well as the main effects were statistically significant for some variables Only the

significant interactions including the between-subject factor group as well as the main-

effect group will be discussed The other interactions and effects establish the successful

induction of DOMS but are not relevant in view of the postulated hypothesis

The interaction between group and time is significant (p=014) for the VAS in

association with full extension for the control arm Post hoc LSD reveals no difference

between both groups a significant effect over time for both groups is found

Consequently this will not be further evaluated

A second significant interaction (p=0002) is the one among the within-subject factor

pre-post and the between-subject factor group for the MPT at 12 cm for the control arm

Post hoc LSD (p=0001) reveals that the LED-irradiated subjects tolerate more

pressure after than before the treatment whereas in the placebo group a not

significant decrease of supported pressure is noted

Finally GLM analysis revealed that at the treated arm the irradiated group tolerates

significantly (p=0047) more pressure than the placebo group (MPT at 4 cm)

DISCUSSION

It has previously been demonstrated that the LED source used might assist in

accelerating wound healing [31] that it has a direct cellular effect [3233] and that it

changes nerve conduction characteristics [1] Nevertheless LED-treated experimental

induced DOMS failed to prove the analgesic efficacy of LED at the above-described

light parameters and treatment procedure The current outcome concurs with other

research that demonstrated a lack of effect of various forms of light therapy on DOMS

[8 11 15] However despite the absence of an apparent and overall definitive finding

the present results cannot exclude favourable effects of LED treatment on pain Since

first of all an isolated statistical significant pre-post difference between groups (control

arm MPT at 12 cm) and a significant group difference (treated arm MPT at 4 cm)

revealed that subjects of the irradiated group tolerate more pressure than the subjects

of the placebo group Second of all the overall means identified generally lower VAS

Chapter 5

104

scores higher MPT values and higher peak torques in the irradiated group This

implied that the treated subjects experienced noticeable less pain supported more

pressure on the painful muscle and generated more force than the non-treated

participants However these results are not statistically significant consequently it is

possible that these differences were found by coincidence and that there is no

relationship between the treatment and the described results of the three outcome

measures though it should be mentioned that the absence of significant findings is

more probably attributable to the small sample size involved in this study This

assumption is based on a post hoc power analysis It was calculated that for the small

effect size measured after treatment and for the measured control group event rate a

sample size of 80 subjects in each group was required at α=005 and power=080

(two-sided) to reveal significant results

Another factor conceivably responsible for the lack of solid evidence of the beneficial

effects of LED treatment upon DOMS-associated pain is related to the size of the

treatment effect in relation to the severity of the induced DOMS It is possible that by

using multiple exhaustive sets of exercise severe DOMS were induced which masked

relatively small but apparent treatment effects [4 11] In this same context it is

possible that the results only become significantly different after a prolonged treatment

and follow-up period as previous research noticed that recuperation subsequent to

DOMS induction can last up to 10 days [8]

Although it needs to be stressed that these results are not statistically significant critical

analysis of the overall means leads up to three additional remarks A primary comment

relates to the pre- and post-treatment courses of the results Starting at day 2 a clear

reduction of pain and muscle sensitivity was observed immediately post-treatment

Still one cannot conclude that this is indicative for the analgesic effect of LED

irradiation as a similar decrease in VAS and increase in MPT values was noted in the

treated and the control arm of the placebo group Perhaps this was caused by placebo

effect as reported by Pollo et al [34] the expectation of the participant can easily result

in pain relief but it can only be elucidated by implementation of a control group


105

Nevertheless in the current study this particular finding can be most probably

attributed to the physiological effects of the peak torque measurement performed

between the pre- and post-treatment recordings of VAS and MPT on the painful

flexor muscle of the upper arm For the assessment of muscle strength two short

series of alternative concentric and eccentric efforts were performed in succession

involving elevation of muscle blood flow [20] Increase of muscle blood flow can assist

in the removal of inflammatory markers and exudate consequently reducing local

tenderness [4] In addition the force assessment can be considered as a form of active

warming-up resulting in an increased muscle temperature which can reduce muscle

viscosity [35] bring about smoother muscle contractions and diminish muscle stiffness

[3536] thus decreasing the sensitivity of the muscle and moderating pain during

movement In any case the beneficial influence of LED immediately after irradiation

can not be securely interpreted due to the sequential assessment of the outcome

measures

A second additional remark considers the fact that both arms of the irradiated subjects

demonstrated evidence of the beneficial effects of LED as a similar reduction of pain

and muscle sensitivity and higher peak torques were found in course of time at the

treated arm as well as at the control arm of the irradiated subjects This ascertainment

points to the possibility that infrared LED induces systemic effects [3738] Ernst [16]

stated that in case LED works via systemic effects the use of the contralateral side as a

control arm might be ill-advised Thus reinforcing that future research should include a

control group to bring clarification [4 7 16]

Finally it needs to be mentioned that although the extent of DOMS was probably

relatively high for investigating the postulated hypothesis the time-course of the

present study corresponds to that reported by other investigators [2 3 5-10]

Significant time effects in many of the variables revealed that muscle damage was

evident diffuse muscle soreness became progressively worse 24-48 h after DOMS

induction followed by a small amelioration after 72 h [35910] After 72 h the follow-

Chapter 5

106

up was ceased consequently further regain of force and attenuation of pain and

muscle sensitivity could not be evaluated Extending the duration of the assessment

period could be useful in assessing any longer-term effects of LED treatment

particularly because as mentioned above differences between both groups are more

clearly present from day 3 pre-treatment and also because DOMS may last for up to 10

days when induced with the described protocol [715]

Lack of knowledge regarding both the precise mechanism of action of LED and the

specific pathophysiology of DOMS hampers the way to offer a definitive explanation

for the absence of more obvious statistically significant differences Still the small

number of significant findings and the mean values suggest that possible analgesic

effects of infrared LED may not be excluded yet but to be able to estimate the real

value of LED further research is necessary A large-scaled randomised clinical trial

which takes the above-mentioned remarks into consideration should be performed

CONCLUSION

Regardless of the reasons for the absence of statistical significant effects reported here

and although LED may have some potential in the management of pain and functional

impairment associated with DOMS its effectiveness at the applied densities has not

been established

Future research should focus on evaluation of the appropriateness of DOMS as an

experimental model of pain and muscle damage Validation of this model would

enhance the ability to study various modalities for their potential effects on pain and

muscle injuries Besides the mechanisms of LED action are not known thus further

fundamental investigations need to address the underlying mechanism and

physiological basis of pain modulation utilizing LED treatment

Once LED irradiation has finally proven its treatment value in an experimental model

the most important prospect considers establishing the effectiveness of LED to reduce

pain in clinical settings


107

ACKNOWLEDGMENTS

The authors would like to thank Mr T Barbe and Mr R Deridder for their technical

assistance in the collection of the data as well as for their valuable input into the

research design Sincere appreciation is extended to the volunteers that participated in

this study and to MDB-Laser (Belgium) for generously providing the light emitting

diode equipment The authors also gratefully recognize Prof Dr G Van Maele for

assistance with the statistical analysis and for helpful discussion

Chapter 5

108

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2 Cheung K Hume PA and Maxwell L (2003) Delayed Onset Muscle Soreness - Treatment Strategies and Performance Factors Sports Med 33(2)145-164

3 MacIntyre DL Reid WD and McKenzie DC (1995) Delayed muscle soreness The inflammatory response to muscle injury and its clinical implications Sports Med 20(1)24-40

4 Minder PM Noble JG Alves-Guerreiro J Hill ID Lowe AS Walsh DM et al (2002) Interferential Therapy Lack of Effect Upon Experimentally Induced Delayed Onset Muscle Soreness Clin Physiol Funct Imaging 22(5)339-347

5 Clarkson PM and Tremblay I (1988) Exercise-induced muscle damage repair and adaptation in humans J Appl Physiol 65(1)1-6

6 Cleak MJ and Eston RG (1992) Delayed onset muscle soreness mechanisms and management J Sports Sci 10(4)325-341

7 Craig JA Cunningham MB Walsh DM Baxter GD and Allen JM (1996) Lack of Effect of Transcutaneous Electrical Nerve Stimulation Upon Experimentally Induced Delayed Onset Muscle Soreness in Humans Pain 67(2-3)285-289

8 Craig JA Barron J Walsh DM and Baxter GD (1999) Lack of effect of combined low intensity laser therapyphototherapy (CLILT) on delayed onset muscle soreness in humans Lasers Surg Med 24(3)223-230

9 Ebbeling CB and Clarkson PM (1989) Exercise-Induced Muscle Damage and Adaptation Sports Med 7(4)207-234

10 Tiidus PM and Ianuzzo CD (1983) Effects of Intensity and Duration of Muscular Exercise on Delayed Soreness and Serum Enzyme-Activities Med Sci Sports Exerc 15(6)461-465

11 Glasgow PD Hill ID McKevitt AM Lowe AS and Baxter D (2001) Low intensity monochromatic infrared therapy a preliminary study of the effects of a novel treatment unit upon experimental muscle soreness Lasers Surg Med 28(1) 33-39

12 Armstrong RB (1984) Mechanisms of exercise-induced delayed onset muscular soreness a brief review Med Sci Sports Exerc 16(6)529-538

13 Rodenburg JB Steenbeek D Schiereck P and Bar PR (1994) Warm-up stretching and massage diminish harmful effects of eccentric exercise Int J Sports Med 15(7)414-419

14 Ciccone CD Leggin BG and Callamaro JJ (1991) Effects of ultrasound and trolamine salicylate phonophoresis on delayed-onset muscle soreness Phys Ther 71(9)666-675 discussion 675-678

15 Craig J Barlas P Baxter D Walsh D and Allen J (1996) Delayed-onset muscle soreness Lack of effect of combined phototherapylow-intensity laser therapy at low pulse repetition rates J Clin Laser Med Sur 14(6)375-380


17 Dvir Z (2003) Isokinetics muscle testing interpretation and clinical applications Churchill Livingstone Edinburgh

18 Lambert MI Marcus P Burgess T and Noakes TD (2002) Electro-Membrane Microcurrent Therapy Reduces Signs and Symptoms of Muscle Damage Med Sci Sports Exerc 34(4)602-607

19 Sumida K Greenberg M and Hill J (2003) Hot gel packs and reduction of delayed-onset muscle soreness 30 minutes after treatment J Sport Rehabil 12221-228

20 Tiidus PM and Shoemaker JK (1995) Effleurage massage muscle blood flow and long-term post-exercise strength recovery Int J Sports Med 16(7)478-483

21 Zhang J Clement D and Taunton J (2000) The efficacy of Farabloc an electromagnetic shield in attenuating delayed-onset muscle soreness Clin J Sport Med 10(1)15-21

22 Fitzgerald GK Rothstein JM Mayhew TP and Lamb RL (1991) Exercise-Induced Muscle Soreness After Concentric and Eccentric Isokinetic Contractions Phys Ther 71(7)505-513


109

23 Nosaka K and Clarkson PM (1997) Influence of Previous Concentric Exercise on Eccentric Exercise-Induced Muscle Damage J Sports Sci 15(5)477-483

24 Jensen MP Karoly P and Braver S (1986) The Measurement of Clinical Pain Intensity - a Comparison of 6 Methods Pain 27(1)117-126

25 Price DD Mcgrath PA Rafii A and Buckingham B (1983) The Validation of Visual Analog Scales as Ratio Scale Measures for Chronic and Experimental Pain Pain 17(1)45-56

26 Revill SI Robinson JO Rosen M and Hogg MIJ (1976) Reliability of a Linear Analog for Evaluating Pain Anaesthesia 31(9)1191-1198

27 Nussbaum EL and Downes L (1998) Reliability of Clinical Pressure-Pain Algometric Measurements Obtained on Consecutive Days Phys Ther 78(2)160-169

28 Lowe AS Baxter GD Walsh DM and Allen JM (1994) Effect of low intensity laser (830 nm) irradiation on skin temperature and antidromic conduction latencies in the human median nerve relevance of radiant exposure Lasers Surg Med 14(1)40-46

29 Noble J Lowe A and Baxter G (2001) Monochromatic infrared irradiation (890 nm) effect of a multisource array upon conduction in the human median nerve J Clin Laser Med Sur 19(6)291-295

30 Pontinen PJ Aaltokallio T and Kolari PJ (1996) Comparative effects of exposure to different light sources (He-Ne laser InGaAl diode laser a specific type of noncoherent LED) on skin blood flow for the head Acupunct Electro-Ther Res 21(2)105-118

31 Vinck E Cagnie B Cambier D and Cornelissen M (2001) Does infrared light emitting diodes have a stimulatory effect on wound healing From an in vitro trial to a patient treatment Progress in biomedical optics and imaging 3(28 Proceedings of SPIE 4903)156-165

32 Vinck EM Cagnie BJ Cornelissen MJ Declercq HA and Cambier DC (2003) Increased fibroblast proliferation induced by light emitting diode and low power laser irradiation Lasers Med Sci 18(2)95-99

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GENERAL DISCUSSION

General discussion

113

SUMMARY

As outlined in the general introduction the overall objective of this doctoral thesis is to

develop the current knowledge about the mechanisms of LED action in view of the

eventual provision of evidence-based support for the clinical use of LED as a

biostimulatory and analgesic treatment modality especially in the field of

physiotherapy

Part I Wound healing

The investigations described in chapter 1 and 2 were conducted to gain insight into the

potential biostimulation of LED irradiation under normal in vitro conditions (aim 1) As

fibroblasts are principal cells for biostimulation (in view of growing and dividing in

healing wounds) the influence of LED irradiation on fibroblast proliferation was

assessed1

The first investigation consisted of a pilot study performed in order to evaluate the

appropriateness of the cell isolation technique cell culture protocol and proliferation

analysis as well as to appraise the feasibility of the light source properties and

illumination procedure

Data analysis revealed no statistically significant differences between the infrared LED

irradiated and control petri dishes for the used parameters (table 1) Considering this

outcome other experimental findings disclose that the absence of stimulatory effects of

LED irradiation on fibroblast proliferation can partly be attributed to the use of

inappropriate light source properties However the applied external dosimetric

parameters are well within the recommended spectrum described by previous studies

investigating fibroblast proliferation influenced by light2-7 Nevertheless it cannot be

excluded that changes in the illumination procedure (such as the use of lower power

shorter exposure times wavelengths with finer coverage of the absorption spectrum of

the irradiated cells and a longer incubation period between the last irradiation and cell

counting) could still result in an increased fibroblast proliferation467 Of equal

importance in interpreting the lack of distinctive results are the imperfections of the

applied proliferation analysis (Buumlrker hemocytometer) This analysis device entails

114

considerable intervention from the investigator compromising the reliability of the

method It is also a time-consuming technique with an insufficient sensitivity for some

purposes and it shows a considerable variability in intra- and inter-tester cell counts8-11

To avoid contamination of the results by these modifiable remarks a similar

experiment (chapter 2) was performed in which wavelength power and output mode of

the infrared LED source were not modified (table 1) only the exposure time was

reduced resulting in a lower radiant exposure In addition the effect of two other

emission spectra was evaluated These probes emitting red and green light had a

shorter wavelength than the infrared LED source and the power was half or a

sixteenth of the power from the infrared probe Consequently the red LED irradiation

occurred with a different exposure time than the infrared one in order to attain the

same radiant exposure (053 Jcm2) With respect to the green LED it was not

endeavoured to achieve the same radiant exposure as 16 min of irradiation is not

feasible for in vitro or clinical application

Finally also an LLL light source was integrated Although it was not attempted to

analyse the effectiveness of LED in comparison to LLL enclosure of this modality was

interesting in order to join in with the available literature covering mostly LLL studies

To bypass the described problems regarding analysis of fibroblast proliferation

counting of the cells was carried out this time by means of a colorimetric MTT assay

This method provides more accurate cell counts in a short period of time and therefore

can be considered as a more reliable alternative to Buumlrker hemocytometer11

MTT assay 24 h after the last irradiation revealed a significantly increased number of

cells in the irradiated wells in comparison to their (respective sham-irradiated) controls

Although the study supplied experimental support for a significantly increased cell

proliferation by all external dosimetric properties based on the results of the

comparative trial with an incubation period of 24 hours irradiation with the green

LED source yielded the highest number of fibroblasts Thus it can be concluded that

the wavelength of the green LED is probably within the bandwidth of the absorption

spectrum of some photoacceptor molecules in embryonic chicken fibroblasts and that

General discussion

115

the chosen dosimetric parameters are largely apposite to irradiate monolayer fibroblast

cultures in vitro612

Table 1 External dosimetric properties summarized for each chapter

Wavelength Power Exposure

time Output mode

Radiant exposure

PART I Chapter 1

In vitro part

LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2

In vivo part LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2 Chapter 2


LED-red 660 nm 80 mW 2 min continuous 053 Jcm2

LED-green 570 nm 10 mW 3 min continuous 01 Jcm2 LLL 830 nm 40 mW 5 sec continuous 1 Jcm2

Chapter 3 LED-green 570 nm 10 mW 3 min continuous 01 Jcm2

PART II Chapter 4


Chapter 5 LED-infrared 950 nm 160 mW 6 min continuous 32 Jcm2

The next aim of the first part of this doctoral thesis was to explore whether LED

treatment could ameliorate in vitro cell proliferation under conditions of impaired

healing In the pursuit of this aim fibroblasts were cultured in medium supplemented

with glucose -mimicking cell proliferation in diabetic patients (chapter 3) Based on a

pilot study the amount of glucose necessary to inhibit normal growth was determined

In order to attain an important reduction of cell viability and decreased proliferation

rate a relatively high concentration of glucose (1667 mM) was necessary in

comparison to the in vivo concentration in conditions of severe diabetic hyperglycaemia

(20-40 mM)13-16 This is in essence a consequence of the glucose exposure dissimilarity

between both circumstances in vitro limited to 72 h whereas the human tissue of a

diabetic patient in vivo is chronically exposed to glucose

Treatment of the fibroblasts occurred in respect of the previously described results

with the same irradiation parameters and illumination procedure (chapter 2)

Accordingly green LED irradiation labelled as the most appropriate treatment for

116

irradiation of a monolayer fibroblast culture in vitro was used at an equal dosage as in

the previous study (table 1)

Analysis of the cell proliferation by means of MTT measurements yielded a

significantly higher rate of proliferation in hyperglycaemic circumstances after

irradiation than in the control conditions (ie hyperglycaemic circumstances without

irradiation) Thus this outcome supported the stimulatory potential of green LED

irradiation on fibroblast proliferation and cell viability of fibroblasts cultured in a

considerable destructive hyperglycaemic medium

Finally although the results of the in vivo part of chapter 1 were persuasive and

encouraging they will not be further discussed in this summary of part I as it was not

aimed in this doctoral thesis to investigate the wound healing process in vivo However

the results of this case study can be a valuable hold for future in vivo research

The possible clinical implications of these results and future research directions in the

scope of wound healing will be discussed below

Part II Analgesia

In the second part two studies investigated the effects of LED irradiation as a

potential intervention mode in one of the most important fields in physiotherapy

practice analgesia Chapter 4 describes the influence of LED treatment on changing

sensory nerve conduction characteristics of a human superficial peripheral nerve

Altering nerve conduction characteristics may not be the sole beneficial purpose to

attain with LED irradiation in view of analgesia but the advantage of nerve conduction

characteristics is that they are objective measurable physical variables and changes in

these characteristics provide a potential explanatory mechanism of pain inhibition by

LED treatment17

The results showed that percutaneous LED irradiation at feasible clinical parameters

can generate a significant decrease in NCV and increase in NPL for all recordings post-

treatment in comparison to the baseline measurement The data in the placebo group

did not reveal any significant difference in the same course of time Statistical analysis

General discussion

117

revealed significant differences between the experimental and the placebo group for

NCV as well as for NPL at all time-points of observation with exception of the NPL

recording immediately after finishing irradiation

It was also observed that the noted effects did not weaken as time progressed It can

be concluded that post-treatment conduction measurements should be extended in

time which is in accordance with the findings of some previous studies18-21 Clarifying

the point of time at which the effect extinguishes is necessary and clinically relevant

when treating pain by means of LED irradiation Besides obtaining the desired

neurophysiological effects ideally the optimal irradiation parameters should be

applied The most favourable dosimetric properties are not yet determined but based

on this study and previously described assays it can be speculated that the dosimetric

window is quite large

Regardless of these clinically important remarks the present findings allow to draw the

following conclusion LED irradiation at clinically applied densities can generate an

immediate and localized effect upon conduction characteristics in underlying nerves as

LED treatment results in lowering the NCV and augmenting the NPL Therefore the

outcome of this in vivo experiment assumes a potential pain relief by means of LED

treatment and justifies further research regarding its clinical effectiveness in laboratory

settings and at a clinical level

The fourth and final aim was to determine the efficacy of LED irradiation as an

analgesic treatment modality in a laboratory setting In pursuit of this aim chapter 5

illustrates a clinical study observing the effect of LED treatment on a model

comprising experimentally induced DOMS in a healthy population The progress of

pain perception and peak torque was evaluated during 4 consecutive days commencing

on the day of DOMS induction The effect of infrared LED treatment at the light

parameters described (table 1) was assessed with regard to three different factors time

(day 1 2 3 and 4) pre-post (preceding or following LED irradiation) and group

(placebo or experimental) Statistical analysis of all variables of the 3 outcome measures

(VAS MPT and IPT) revealed no significant interactive effects of the main interaction

118

(timegrouppre-post) For the remaining interactions and for the main effects only a

few significant findings were relevant in view of the postulated hypothesis

Notwithstanding the absence of an apparent and overall statistically significant finding

the present results indicate favourable trends of LED treatment on pain as the means

of all VAS and MPT variables show a statistically nonsignificant general analgesic

effect of infrared LED irradiation expressed by lower subjective pain rates and higher

MPT values in the irradiated group In addition to the analgesic influence of LED an

augmented restoration of muscle strength was noted The lack of solid statistically

significant evidence for these beneficial effects of LED treatment upon DOMS-

associated pain can possibly be attributed to the small sample size in this study or even

to the size of the treatment effect in relation to the severity of the induced DOMS as

induction of severe DOMS can mask relatively small but apparent treatment

effects2223 A final possibility is that the results only become significantly different after

a prolonged treatment and follow up period as previous research demonstrated that

recuperation subsequent to DOMS induction can last up to 10 days24

It should also be noted that the described general analgesic effect of LED irradiation

was identical for the treated as well as for the control arm in the irradiated group

proposing that infrared LED might induce systemic effects 2526 However it needs to

be stressed that these results were not statistically significant

Regardless of the absence of statistically significant findings the mean values suggest a

potential role for infrared LED irradiation in the management of pain and functional

impairment associated with DOMS Notwithstanding this postulation future research

is absolutely required to establish the effectiveness of LED treatment to reduce pain as

well at the applied densities as for other dosimetric parameters

CLINICAL IMPLICATIONS AND FUTURE RESEARCH DIRECTIONS

In the course of the past years during the process of the genesis of this thesis

therapeutic physical agents in general and phototherapeutic modalities in particular

became less important as physiotherapeutic modes of treatment than during the

preceding two decades The diminished use of these treatment modalities in the

General discussion

119

physiotherapy practice is to a certain degree a consequence of the controversial

research findings regarding the use of these physical agents This issue of controversy

led to less support for the use of these treatment modalities and a growing scepticism

regarding the effectiveness of these physical agents within the scope of the growing

climate of evidence-based practice A second responsible protagonist for the loss of

popularity of physical agents is linked with the current tendency within physiotherapy

emphasising active remedial therapy The establishment of this development was based

on various experiments mainly performed during the last decade demonstrating that

active treatment modalities are for numerous impairments and disabilities preferable to

more passive forms of therapy In Belgium the prevailing nomenclature which came

into use on 1 May 2002 went along with this tendency In the appendix to the Royal

decree of 14 September 1984 towards settlement of the nomenclature of medicinal

treatments concerning compulsory insurance for medical care and allowances the

personal involvement of the physical therapist during the physiotherapeutic session

was emphasized and it was even defined that massage physical techniques within the

framework of electrotherapy ultrasonic therapy laser therapy and several other techniques for thermal

application can only be remunerated when they are applied supplementarily and not as a sole therapy

This implies that passive treatment modalities should not be used as sole method of

treatment and should always be considered as an adjunct to an active treatment

program This development needs to be applauded in many cases such as various

painful musculoskeletal problems functional instability rehabilitation of neurological

patients re-activation of the elderly population psychomotor rehabilitation

cardiovascular and respiratory convalescence Nevertheless it would be erroneous to

entirely reject physical agents including LED treatment Based on the findings of the

above described experiments it needs to be stressed that for some purposes especially

within the scope of impaired wound healing LED irradiation could be a suitable

therapeutic measure This statement is founded on the results of part I of the present

thesis they provided satisfactory fundamental evidence for the advantageous effects of

LED treatment on a crucial exponent of the wound healing process namely fibroblast

proliferation The beneficial findings are the result of basic in vitro research As it is

120

inaccurate to simply extrapolate these results to the clinical practice the clinical use of

LED irradiation for wound healing needs to be preceded by purposive and specific in

vivo investigations to substantiate these basic research findings27

The case study described in chapter 1 indicates a foundation for further in vivo research

Visual appraisal of the surgical incision revealed (from the 65th day in the course of the

reparative process onwards) that the irradiated area -which initially showed inferior

epithelialization and wound contraction- showed a more appropriate contracture than

the control area characterized by less discoloration at scar level and a less hypertrophic

scar These clear beneficial effects of LED treatment on a human cutaneous wound

can serve as preliminary impetus for further research into the clinical applicability of

LED therapy although this case study is insufficient in order to guarantee a safe

correct and effective use of LED as a therapeutic modality

Despite these remarks it tentatively can be concluded that based on a detailed analysis

of the available data of the present in vitro studies and the given case report in

combination with the small number of previously published human studies the

beneficial effects of LED irradiation at the cellular level are obvious and therefore a

potentially favourable outcome can be assumed in clinical practice28-30 LED-

modulated stimulation of wound healing can be gradually and vigilantly implemented

clinically Nevertheless the real benefits of LED irradiation within the scope of wound

healing can only be established by additional clinical trials as thus far clinical

application and stipulation of dosimetry still occurs on a trial-and-error basis which is

not conducive to a generally accepted clinical use of LED To lend more credibility to

the treatment of wounds by means of LED irradiation and to expel the existing

controversy and scepticism surrounding this topic in vivo investigations on wound

healing using a number of different animal models and adequately controlled human

studies are necessary In addition these studies should be performed preferably on a

population suffering from impaired healing as a consequence of diabetes mellitus or as

a result of any other debilitating reason because as posed by Reddy et al3132 and as

mentioned above light has possible optimal clinical effects in the treatment of healing-

resistant wounds

General discussion

121

Drawing general conclusions and formulating clinical implications for analgesia is

obviously less manifest first of all because only a limited number of possible

mechanisms of action in order to obtain analgesia were highlighted and secondly

because both studies did not come to a joint or complementary conclusion The

outcome of the first study revealed that LED treatment lowers the NCV and augments

the NPL resulting in a slower stimulus conduction and consequently a reduced number

of sensory pulses per unit of time Thus it could be assumed that LED induces pain

relief but the results of the study describing the effect of LED treatment on

experimentally induced DOMS failed to prove the analgesic efficacy of LED therapy

In addition it needs to be emphasised that the first study (chapter 4) measured the effect

of LED irradiation on the large myelinated Aβ afferents A noteworthy question and

meanwhile a stimulus for future investigation is whether the measured effects can be

extrapolated from these sensory nerve fibres to the actual nociceptive afferents

notably the myelinated Aδ-fibres and unmyelinated C-fibres The functional testing of

these nociceptive pathways relies on laser-evoked potentials which selectively activate

Aδ-fibres and C-fibres3334 This technique was presently not available therefore a

standard sensory nerve conduction study was performed

Whereas stimulation of wound healing by means of LED irradiation can be cautiously

implemented in the clinical practice at this stage it is too early to promote LED

irradiation as a treatment modality for pain To make this possible it is essential to

conduct numerous studies with regards to the use of LED in the field of analgesia

Future research should focus on fundamental investigations in order to discover the

underlying mechanisms and physiological basis of pain modulation utilizing LED

treatment Furthermore the evaluation of the appropriateness of DOMS as an

experimental model of pain is an important prospect to consider as validation of this

model would enhance the ability to study various modalities for their potential effects

on pain Irrespective of the difficulties regarding standardisation of the research

population and evaluation of soreness inextricably linked with clinical pain studies the

122

ultimate objective of future research should be the establishment of the effectiveness

of LED irradiation to reduce pain of miscellaneous origin in a clinical setting

Regardless of the encouraging results of the described studies and besides the earlier

proposed specific directions for future research (directed towards wound healing or

pain relief) it is necessary in the interest of the patientrsquos well being and to the

advantage of the prospective clinical use of LED to highlight a few more issues for

future research Therefore one has to deal with some limitations of the performed

investigations A first limitation concerns the fact that only two mechanisms of LED

action were investigated (notably changed fibroblast proliferation and alteration of the

nerve conduction characteristics) So one can conclude that for further and better

understanding of the mechanisms of action it is necessary to perform more basic

research Answering the questions regarding the functioning of LED irradiation will

simplify the evaluation and reinforce the interpretation of the obtained results and

ultimately contribute to a more widespread and well definded acceptance of the use of

LED in clinical settings

A second general limitation of this doctoral thesis is the substantial difference in the

used external dosimetric parameters between the different chapters and even within

one and the same study (illustrated in table 1) this complicates the comparison

between the different trials In each trial the dosimetry was individually ascertained

based on previous studies within the given field As not for every application the same

dosimetry is suggested in literature a range of dosages were used Another important

factor in deciding on the dosimetry was the clinical applicability of the dosage as it is

useless to investigate the appropriateness of a treatment modality at a clinically

unrealistic dose As a result of this limitation the current findings do not fully

contribute to the explanation regarding the ideal parameters one should use although

this was not set as a principal purpose Based on this thesis and previously described

assays it can be speculated that the possible window for these parameters is quite large

the ideal irradiation parameters and proper timing or sequencing of LED irradiation

General discussion

123

for example to the various phases of wound healing and to different painful conditions

are therefore possibly unattainable

The establishment of an appropriate dosimetry should also consist of investigating the

absolute and relative penetration depth of LED irradiation into human tissue This is

less crucial within the scope of wound healing but it is of key importance while

treating deep-seated tissue (eg nerve fibres muscles circulatory components et

cetera)

Finally this thesis only investigated the efficiency of LED in a very limited number of

conditions notebly wound healing and pain Within the scope of physiotherapy and

medicine in general there are numerous other purposes for which LED irradiation is

promoted such as oedema arthritis miscellaneous orthodontic applications seasonal

affective disorder neonatal jaundice photodynamical therapy et cetera2835-41

In summary additional work on establishing proper dosimetry and identifying the

biochemical or photobiologic phenomena that are responsible for improving wound

healing and reducing pain or even other effects within a broader spectrum of

conditions remains to be done in order to answer unreciprocated questions Until that

time the potential clinical usefulness and actual value of LED irradiation for wound

healing and even to a larger extent for analgesia should always be approached with

appropriate professionalism and even caution

FINAL CONCLUSION

LED devices are promoted for clinical use but the currently available scientific

documentation regarding effectiveness of this physical agent is rather scarce Through

providing scientific support for the biostimulatory and analgesic effectiveness of LED

irradiation this doctoral thesis attempted to bridge in some degree this gap

The conducted studies revealed that LED irradiation undeniably has potential

beneficial effects on wound healing and to a lesser degree within the scope of

analgesia However based on the present results it can be corroborated that light

124

therapy in the guise of LED irradiation is not magic but these results can raise some

corrective doubts in fundamental disbelievers and antagonists

Nevertheless we have to join the queue of scientists who have found beneficial results

but cannot elucidate with certainty how this outcome was established Thus although

the present results are encouraging a continuing development and integration of new

knowledge based on further research is necessary in various domains of intervention

Therefore several directions for future investigations were proposed in order to cover

as many existing gaps and to answer the utmost number of remaining questions as

possible Still one ought to be aware not to carry future fundamental research at a

disproportional level and the inevitable quest for mechanisms of LED action should

not hypothecate the potential clinical value implying that at a certain point it should be

appropriate to make the transfer from science to the application of the available

knowledge in clinical practice

The described findings regarding LED irradiation are comparable to the results of

previously published studies performed with other light sources Consequently as

postulated by some LED providers it can be speculated that the biological response of

tissue to light irradiation can probably not be equated merely to a light source but

rather to a broad photo-energy window

General discussion

125

REFERENCES

1 Whelan H Houle J Donohoe D Bajic D Schmidt M Reichert K Weyenberg G Larson D Meyer G and Caviness J (1999) Medical applications of space light-emitting diode technology - Space station and beyond Space Technology and Applications International Forum 3-15

2 Abergel R Lyons R Castel J Dwyer R and Uitto J (1987) Biostimulation of wound healing by lasers experimental approaches in animal models and in fibroblast cultures J Dermatol Surg Oncol 13(2)127-133

3 Skinner S Gage J Wilce P and Shaw R (1996) A preliminary study of the effects of laser radiation on collagen metabolism in cell culture Aust Dent J 41(3)188-192

4 Webb C Dyson M and Lewis W (1998) Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts Possible mechanisms for increase in cell counts Lasers Surg Med 22(5)294-301

5 Atabey A Karademir S Atabey N and Barutcu A (1995) The effects of the helium neon laser on wound healing in rabbits and on human skin fibroblasts in vitro Eur J Plast Surg 1899-102


7 Ben-Dov N Shefer G Irinitchev A Wernig A Oron U and Halevy O (1999) Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro Biochim Biophys Acta - Mol Cell Res 1448(3)372-380

8 Rebulla P Porretti L Bertolini F et al (1993) White cell-reduced red cells prepared by filtration a critical evaluation of current filters and methods for counting residual white cells Transfusion 33(2)128-133

9 Deneys V Mazzon A Robert A Duvillier H and De Bruyere M (1994) Reliable and very sensitive flow-cytometric method for counting low leucocyte numbers in platelet concentrates Vox Sang 67(2)172-177

10 Mahmoud A Depoorter B Piens N and Comhaire F (1997) The performance of 10 different methods for the estimation of sperm concentration Fertil Steril 68(2)340-345

11 Haskard D and Revell P (1984) Methods of assessing the synovial fluid cell count Clin Rheumatol 3(3)319-322

12 Karu T (1998) The science of low-power laser therapy New Delhi Gordon and Breach Science Publishers

13 Lorenzi M Nordberg JA and Toledo S (1987)High glucose prolongs cell-cycle traversal of cultured human endothelial cells Diabetes 36(11)1261-1267

14 Lorenzi M Montisano DF Toledo S and Barrieux A (1986) High glucose induces DNA damage in cultured human endothelial cells J Clin Invest 77(1)322-325

15 Lorenzi M Cagliero E and Toledo S(1985) Glucose toxicity for human endothelial cells in culture Delayed replication disturbed cell cycle and accelerated death Diabetes 34(7)621-627

16 Kamal K Du W Mills I and Sumpio BE (1998) Antiproliferative effect of elevated glucose in human microvascular endothelial cells J Cell Biochem 71(4)491-501

17 Kramer J and Sandrin M (1993) Effect of low-power laser and white light on sensory conduction rate of the superficial radial nerve Physiotherapy Canada 45(3)165-170

18 Baxter G Allen J and Bell A (1991) The effect of low-energy-density laser irradiation upon human median nerve-conduction latencies J Physiol-London 435P63


20 Cambier D Blom K Witvrouw E Ollevier G De Muynck M and Vanderstraeten G (2000) The influence of low intensity infrared laser irradiation on conduction characteristics of peripheral nerve A randomised controlled double blind study on the sural nerve Lasers Med Sci 15195-200

126







27 Baxter G and Allen J (1994) Therapeutic lasers Theory and practice Edinburgh Churchill Livingstone 28 Whelan H Houle J Whelan N Donohoe D Cwiklinski J Schmidt M Gould L Larson D Meyer

G Cevenini V and Stinson H (2000) The NASA light-emitting diode medical program - progress in space flight terrestrial applications Space Technology and Applications International Forum 37-43



31 Reddy G Stehno Bittel L Enwemeka C (2001) Laser photostimulation accelerates wound healing in diabetic rats Wound Repair Regen 9248-255

32 Reddy G Stehno Bittel L Enwemeka C (1998) Laser photostimulation of collagen production in healing rabbit Achilles tendons Lasers Surg Med 22281-287


34 Bentley D Watson A Treede R Barrett G Youell P Kulkarni B et al (2004) Differential Effects on the Laser Evoked Potential of Selectively Attending to Pain Localisation Versus Pain Unpleasantness Clin Neurophysiol 115(8)1846-1856




38 Uumlşuumlmez S Buumlyuumlkyilmaz T Karaman A-I (2004) Effect of light-emitting diode on bond strength of orthodontic brackets Angle Orthod 74(2)259-263

39 Yun Sil C Jong Hee H Hyuk Nam K Chang Won C Sun Young K Won Soon P Son Moon S Munhyang L (2005) In vitro and in vivo efficacy of new blue light emitting diode phototherapy compared to conventional halogen quartz phototherapy for neonatal jaundice J Korean Med Sci 2061-64

40 Lam R (1998) Seasonal affective disorder diagnosis and management Prim Care Psychiatry 463-74

General discussion

127

41 Glickman G Byrne B Pineda C Hauck WW Brainard GC (2005)Light Therapy for Seasonal Affective Disorder with Blue Narrow-Band Light-Emitting Diodes (LEDs) Biol Psychiatry DOI 101016Article in Press

NEDERLANDSTALIGE SAMENVATTING

Nederlandstalige samenvatting

131


Het medicinale gebruik van licht met therapeutische doeleinden gaat ver terug in de

tijd Ook in het domein van de kinesitherapie zijn hiervan duidelijke sporen terug te

vinden met vooreerst de toepassing van ultraviolette (UV) stralen en later de applicatie

van laagvermogen laserlicht (LVL) ter behandeling van een gamma aan aandoeningen

Vandaag is het gebruik van UV-licht in de kinesitherapie nagenoeg verdwenen en rest

enkel nog de sporadische klinische toepassing van LVL Deze tanende populariteit is

ongetwijfeld terug te koppelen aan een mentaliteitsverandering op klinisch zowel als

op wetenschappelijk vlak Klinisch is er duidelijk sprake van het toenemende belang

van de actieve betrokkenheid van de patieumlnt in zijn herstelproces waardoor passieve

interventies zoals lichttherapie elektrotherapie en massage geleidelijk in onbruik raken

Deze klinische tendens is onlosmakelijk verbonden met het hedendaagse klimaat van

ldquoop evidentie gestoelderdquo strategieeumln en daarin blijken deze passieve therapievormen

moeilijk te verantwoorden

Deze passieve interventies werden ontwikkeld en geiumlntroduceerd in een periode waarin

de vooropgestelde wetenschappelijke eisen duidelijk secundair waren aan andere

overtuigingen en belangen Het ongeloof en scepticisme (gevoed vanuit

methodologische tekorten nauwelijks reproduceerbare onderzoeksdesigns gebrek aan

consistente resultaten en ongebreidelde indicatie-extrapolatie naar klinische settings) in

de academische wereld aangaande bijvoorbeeld het klinisch gebruik van laagvermogen

laser vormden aldus geen rationele hinderpaal voor een veralgemeende aanvaarding in

de kinesitherapie Recente rigoureuze (meta)analyses stellen deze mentaliteit vandaag

aan de kaak en leiden onherroepelijk tot het verlaten van heel wat passieve interventies

inclusief het gebruik van licht

Deze mentaliteitswijziging is op zich toe te juichen maar impliceert ook het risico dat

de potentieumlle klinische waarde van bijvoorbeeld laser voor selectieve en onderbouwde

doelstellingen of indicaties waarvoor wel evidentie kan worden aangeleverd in eacuteeacuten en

dezelfde pennentrek worden opgeofferd Een typisch gevolg wanneer bewijskracht

komt na de commercialisatie en zo het spreekwoordelijke kind met het badwater wordt

geloosd

132

De technologie stond intussen niet stil en de ontwikkeling van nieuwe lsquotherapeutischersquo

lichtbronnen bleef evolueren zodat vandaag ook light emitting diodes (LEDs) en

gepolariseerd licht als fysische bronnen kunnen worden aangewend Teneinde te

anticiperen op het gekenschetste karakteristieke verloop en jammerlijke herhalingen te

voorkomen lijkt een gerichte en rationele a priori aanpak conform de

wetenschappelijke eisen van het huidig medisch klimaat absoluut aangewezen

Te meer daar grondige literatuurstudie leert dat men ten behoeve van de

werkingsmechanismen en effectiviteit van deze recente toepassingen van lichttherapie

zich beroept op dezelfde (soms gecontesteerde) onderzoeken van laagvermogen laser

De fysische verschillen tussen LED en LVL laten bovendien vermoeden dat de

extrapolatie vanuit deze literatuur ten onrechte gebeurt en dat voorzichtigheid hierbij is

geboden De introductie van alternatieve lichtbronnen in de huidige

kinesitherapeutische praktijk met voornamelijk LEDs blijkt dus wetenschappelijk

weerom nagenoeg niet onderbouwd en de nakende commercialisatie dreigt aldus

eenzelfde bedenkelijke levenscyclus van deze lichtbronnen te gaan induceren Nood

naar specifiek wetenschappelijk onderzoek met betrekking tot het evidence-based

gebruik van LEDs in de kinesitherapie is dan ook bijzonder pertinent in het bijzonder

binnen de domeinen van haar potentieel beloftevolle klinische toepassingen

wondheling en analgesie

Een eerste deel van dit doctoraal proefschrift handelt over de invloed van LED op de

wondheling Aan de hand van drie in vitro onderzoeken die werden uitgevoerd op

prominente protagonisten van de wondheling de fibroblasten werd getracht het

fundamenteel biostimulatoir effect van LED bestralingen te beoordelen Fibroblasten

zorgen voor de vorming van een essentieumlle bindweefselmatrix die onderzoek naar de

proliferatie van deze cellen cruciaal maakt in het kader van wondheling Dit opzet werd

respectief geconcretiseerd onder normale in vitro omstandigheden en in een toestand

waarbij de normale celgroei werd verstoord

In een eerste onderzoek (hoofdstuk 1) werd aan de hand van Buumlrker hemocytometrie het

effect van LED op de proliferatie en viabiliteit van fibroblasten nagegaan Statistische


133

data-analyse leverde geen significante resultaten op Dit kan mogelijk enerzijds worden

verklaard door het gebruik van een inadequate LED dosering en anderzijds een

methodologisch cruciaal storende factor de evaluatiemethode Buumlrker hemocytometrie

vergt namelijk een aanzienlijke en tijdrovende interventie van de onderzoeker wat de

precisie en betrouwbaarheid van de techniek hypothekeert met een grote intra- en

inter-tester variabiliteit tot gevolg

In een poging aan deze kritische bemerkingen tegemoet te komen werd hetzelfde

onderzoek gereproduceerd (hoofdstuk 2) met weliswaar modificatie van de

bestralingsparameters (dosering) De effecten van de drie verschillende LED

golflengtes en eacuteeacuten LVL lichtbron op de proliferatie en viabiliteit van de fibroblasten

werden hierbij geanalyseerd door middel van een meer betrouwbare en minder

subjectieve analyse de colorimetrische meting op basis van 3-(45-dimethylthiazol-2-

yl)-25-diphenyl tetrazolium bromide (MTT)

De studieresultaten uit dit gemodificeerd design toonden het significant biostimulatoir

effect van alle LED doseringen aan evenals van de LVL bron Deze data vormden

tevens een basis voor meer coherente en relevante inzichten aangaande de globale

bestralingsparameters (golflengte stralingsintensiteit en stralingsduur)

Gezien de stimulatie van de wondheling in se pas geiumlndiceerd is in condities waarbij het

wondhelingsproces verstoord verloopt en bijgevolg een chronisch en invaliderend

karakter dreigt te kennen en pas in dergelijke omstandigheden een biostimulatoire hulp

rechtvaardigt werd in een derde fase het in vitro onderzoek onder gemanipuleerde

vorm uitgevoerd ter enscenering van een verstoord helingsproces (hoofdstuk 3) De

fibroblasten werden hierbij gecultiveerd in een gemodificeerd cultuurmedium met

extreem hoge concentraties glucose Deze modificatie van het medium staat model

voor de celproliferatie bij de diabetespatieumlnt een populatie waarbij in de klinische

praktijk de stimulatie van het wondhelingsproces een klinisch relevante interventie kan

vormen Ook onder deze hyperglycemische omstandigheden vertoonde LED met de

gehanteerde parameters gunstige cellulaire effecten op het vlak van viabiliteit en

proliferatie

134

Het tweede deel van dit proefschrift exploreert het domein van het potentieel

analgetisch effect van LED binnen de kinesitherapie aan de hand van twee

fundamentele onderzoeken

In het eerste onderzoek (hoofdstuk 4) werd het effect nagegaan van LED op de perifere

sensorische zenuwgeleidingskarakteristieken van de nervus suralis Als experimentele

hypothese werd vooropgesteld dat LED een daling van de zenuwgeleidingssnelheid en

een stijging van de negatieve pieklatentie veroorzaakt wat een mogelijke neurale

verklaring van een analgetisch effect van het medium zou kunnen belichamen

Hiervoor werden de resultaten van een eerste antidrome elektroneurografische (ENG)

meting vergeleken met de resultaten van een identieke ENG meting uitgevoerd op vijf

verschillende tijdstippen (0 2 4 6 en 8 min) na LED bestraling De resultaten tonen

aan dat percutane LED bestraling onder de gehanteerde parameters een onmiddellijke

significante daling van de zenuwgeleidingssnelheid en gelijktijdig een stijging van de

negatieve pieklatentie genereert overeenkomstig met de geponeerde experimentele

hypothese

Een tweede studie (hoofdstuk 5) evalueerde de effectiviteit van LED als pijnstillend

fysisch agens bij een experimenteel geiumlnduceerd pijnmodel waarbij musculoskeletale

pijn en stijfheid centraal staan delayed-onset muscle soreness (DOMS) Met behulp

van een gestandaardiseerd protocol voor een isokinetisch concentrischexcentrische

krachtsinspanning werden DOMS uitgelokt Na inductie van DOMS werd een LED

behandeling (met een klinisch haalbare dosering) uitgevoerd De behandeling werd vier

keer herhaald met een interval van 24 h tussen elke behandeling Het effect van LED

op het verloop van DOMS werd in de loop van deze periode (4 dagen) geeumlvalueerd

(zowel voor als na de LED behandeling) aan de hand van een gestandaardiseerde

isokinetische krachtmeting en een registratie van de waargenomen spierpijn De

spierpijn en -gevoeligheid werd beoordeeld via een visueel analoge schaal en met

behulp van een kwantitatieve hand-hold algometer

Analyse van de bekomen data bracht geen significante verschillen tussen de

controlegroep en de experimentele groep aan het licht Op basis van de gemiddelden


135

kon descriptief en zonder statistische pretenties voorzichtig worden afgeleid dat LED

behandeling gunstige effecten bleek te genereren ten overstaan van de recuperatie van

de spierkracht de mate van spiergevoeligheid en de hoeveelheid pijn die door de

proefpersonen werd waargenomen gedurende de evaluatieperiode De algemene

afwezigheid van significanties kan mogelijk worden verklaard door de relatief kleine

proefgroep die werd onderzocht enof door de grootte van het behandeleffect in

verhouding tot de ernst van de geiumlnduceerde DOMS Ernstige DOMS kunnen immers

een aanwezig behandeleffect gemakkelijk maskeren Een uitbreiding van de follow-up

kan hierbij eventueel een oplossing bieden Gezien de resultaten is hier evenwel

absolute omzichtigheid geboden en moet deze visie louter als speculatief worden

beschouwd

Als gevolg van de beschreven klinische en wetenschappelijke trends binnen de

kinesitherapie is het gebruik van fysische middelen en lichttherapie in het bijzonder de

laatste jaren aanzienlijk afgenomen

De positieve resultaten van de verschillende in vitro studies in het kader van wondheling

vormen een cruciale en belovende voedingsbodem opdat ook de klinische toepassing

vooral bij (diabetes)patieumlnten met chronische -slecht helende- wonden potentieel

gunstige resultaten kan opleveren Bijgevolg vormt het eerste deel van dit doctoraat een

belangrijke basis tot design voor verder in vitro maar vooral ook klinisch onderzoek

Gelijkaardige besluitvorming met betrekking tot analgesie is een meer delicate kwestie

Er werden immers slechts een beperkt aantal aspecten van het pijnmechanisme

onderzocht en bovendien kwamen beide studies niet tot een eenduidig noch

complementair resultaat Verder onderzoek ter exploratie van de mogelijke

onderliggende mechanismen en fysiologische basis voor pijnmodulatie is daarom

onontbeerlijk om de klinische toepassing van LED in het kader van pijnstilling op

termijn wetenschappelijk te rechtvaardigen

136

LED tovenarij trend of therapie

LED mag geen magische krachten worden toegemeten maar verdient het lot van een

kort leven en een vroegtijdige dood niet De onderzoeksresultaten vormen een

wetenschappelijk platform opdat LED binnen de kinesitherapie de kans zou krijgen

zich te ontwikkelen tot een therapie maar weliswaar voor relevante en heel specifieke

indicaties

Light thinks it travels faster than anything but it is wrong No matter how fast light travels it finds

the darkness has always got there first and is waiting for it

(Terry Pratchett Reaper Man 1991)

Promotor


Examination Board

Prof dr P Calders Artevelde University College Belgium




Prof dr M Dyson University of London UK

Prof dr P Lievens Free University Brussels Belgium

Prof dr K Peers Catholic University Leuven Belgium


Process Supervisory Board





VII

TABLE OF CONTENTS


Background 3



Aims and outline 15



27


47


61



75


91


Summary 113






(Stewart E White)

IX

ACKNOWLEDGEMENTS























chapter 5

X




aseptic chats

























XI













Elke Vinck

Ghent March 2006



3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties




VII

TABLE OF CONTENTS


Background 3



Aims and outline 15



27


47


61



75


91


Summary 113






(Stewart E White)

IX

ACKNOWLEDGEMENTS























chapter 5

X




aseptic chats

























XI













Elke Vinck

Ghent March 2006



3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties






(Stewart E White)

IX

ACKNOWLEDGEMENTS























chapter 5

X




aseptic chats

























XI













Elke Vinck

Ghent March 2006



3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties




IX

ACKNOWLEDGEMENTS























chapter 5

X




aseptic chats

























XI













Elke Vinck

Ghent March 2006



3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties




X




aseptic chats

























XI













Elke Vinck

Ghent March 2006



3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties




XI













Elke Vinck

Ghent March 2006



3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties






3

BACKGROUND













laser1

















4






























5










analgesia3031



















6












with LLL therapy






can be defined as








7











8




molecules

















coherent2758









9






















table 1


Infrared 950 80 120 160 Red 660 15 46 80

Green 570 02 42 10

10


RE =



PRE = α S T


003 (pulsed mode)

























11


























12






mechanisms272858

























13































14







Secondarymechanisms

Primary mechanisms

Final effects

Trigger


Exposure to light













15







AIMS AND OUTLINE



















16






described




method





















17


feature









progresses

















18










19

REFERENCES






















20






















21























22

























23




















CHAPTER 1








Chapter 1

28

ABSTRACT





















29

INTRODUCTION





























Chapter 1

30


use of LPL121520

























31




37degC
















incubation








Chapter 1

32











Statistical methods










100 was used

RESULTS







33







DESCRIPTIVE DATA

1730000181750029530003070000

00E+00

50E+05

10E+06

15E+06

20E+06

25E+06

30E+06

35E+06

40E+06


Mea

n n

um

ber

of

cells

Control

Irradiated


DISCUSSION









Chapter 1

34















effect










wavelengths38






35










proliferation3640





















Chapter 1

36








investigator52





















37













treatment




Chapter 1

38











filaments

RESULTS







39














Chapter 1

40

DISCUSSION
























scar31







41

CONCLUSION






















least three days






Chapter 1

42

ACKNOWLEDGMENTS





43

REFERENCES





















Chapter 1

44
























45













CHAPTER 2



IRRADIATION


Cambiera



Chapter 2

48

ABSTRACT




















49

INTRODUCTION


















cardinal indication












Chapter 2

50





















during 24 hours



Irradiation sources





51










Exposure regime



















Chapter 2

52































53







(SPSS 100) was used

RESULTS






















Chapter 2

54






Groups




DISCUSSION




55











factor to consider




















Chapter 2

56




























57

ACKNOWLEDGMENTS




Chapter 2

58

REFERENCES
























59







CHAPTER 3




Cambiera



Chapter 3

62

ABSTRACT




lesions














diabetic patients



63

INTRODUCTION
















retinopathy7-10








treatment12






Chapter 3

64

























Cell cultivation





65






























Chapter 3

66

Proliferation assay


















Data analysis





RESULTS





67


1)


DISCUSSION














621 x 10-1 682 x 10-1

0010203040506070809

1

Control Irradiated

Groups

Ab

sorb

ency

Chapter 3

68
































69










CONCLUSION




circumstances








ACKNOWLEDGMENTS





Chapter 3

70

REFERENCES























71


















PART II ANALGESIA

CHAPTER 4







Chapter 4

76

ABSTRACT




vivo














points of time








77

INTRODUCTION

















therapy [8-9]












Chapter 4

78













STUDY DESIGN




Subjects











79


20 females





followed























Chapter 4

80















LED irradiation















81



Statistics















RESULTS







Chapter 4

82










0 0171plusmn0353 0329 -0752plusmn1348 0002 lt0001

2 -0008plusmn0357 0969 -0915plusmn1520 0004 0002

4 0111plusmn0377 0647 -0908plusmn1898 0021 0004

6 0055plusmn0397 0770 -0809plusmn1301 0002 lt0001



-14

-12

-1

-08

-06

-04

-02

0

02

04


Time Course

Dif

fere

nce

Sco

re (

m

s)

PlaceboLED


83

















-001

0

001

002

003

004

005

006

007


Time course

Dif

fere

nce

Sco

re (

ms)

PlaceboLED

Chapter 4

84






DISCUSSION













0 0004 plusmn0053 0755 0029plusmn0080 0019 0145

2 -0002 plusmn0046 0856 0044plusmn0100 0002 0021

4 0001 plusmn0056 0925 0058plusmn0090 lt0001 0004

6 0015 plusmn0054 0216 0052plusmn0079 lt0001 0034



85





observation period


























Chapter 4

86
































87
















CONCLUSION












Chapter 4

88

ACKNOWLEDGMENTS



Diode equipment


89

REFERENCES




















Chapter 4

90













CHAPTER 5





Cambiera




Chapter 5

92

ABSTRACT




(DOMS)
























93

INTRODUCTION









designed

















[2 6 12 13]




Chapter 5

94










[14]







subject

Subjects











95





























on both arms

Chapter 5

96


Pain induction




















Outcome measures








97





























Chapter 5

98




























control arm


99









level

RESULTS

















Chapter 5

100


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




Visual Analog Scale

0

02

04

06

08

1

12

14

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n V

AS

scor

e (a

t re

st)



101


Day 1 pre

Day 1 post

Day 2 pre

Day 2 post

Day 3 pre

Day 3 post

Day 4 pre

Day 4 post




0

5

10

15

20

25

Day

1 -

Pre

Day

1 -

Post

Day

2 -

Pre

Day

2 -

Post

Day

3 -

Pre

Day

3 -

Post

Day

4 -

Pre

Day

4 -

Post

Time course

Mea

n M

PT

sco

re (

at 4

cm)


Chapter 5

102







04

045

05

055

06

065


Time course

Mea

n I

PT

sco

re (

ecce

ntr

ic a

t 60

degse

c)



103

















DISCUSSION













Chapter 5

104































105














measures















Chapter 5

106














CONCLUSION




been established











107

ACKNOWLEDGMENTS







Chapter 5

108

REFERENCES
























109

















GENERAL DISCUSSION

General discussion

113

SUMMARY





physiotherapy






assessed1


















114






























General discussion

115





time Output mode

Radiant exposure

PART I Chapter 1

In vitro part







PART II Chapter 4

















116















Part II Analgesia









LED treatment17





General discussion

117






























118






























General discussion

119































120






























resistant wounds

General discussion

121





























122





























General discussion

123







cetera)













FINAL CONCLUSION








124



















General discussion

125

REFERENCES





















126





















General discussion

127




131






























geloosd

132































133





























proliferatie

134















hypothese
















135











beschouwd
















136






indicaties




Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level

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Transcript of Green Light Emitting Diode Irradiation Enhances Fibroblast Growth Impaired by High Glucose Level