©2011 MARIA NURIA ROYO GASCON ALL RIGHTS RESERVED

©2011

MARIA NURIA ROYO GASCON

ALL RIGHTS RESERVED

NEUROPLASTICITY OF SPINAL CORD NEURONS BASED ON PIEZOELECTRIC

STIMULATION AND ELECTROPHYSIOLOGICAL ANALYSIS AFTER STEM CELL-

DERIVED PROGENITOR TRANSPLANT

By

MARIA NURIA ROYO GASCON

A Dissertation submitted to the

Graduate School − New Brunswick

Rutgers, The State University of New Jersey

and

The Graduate School of Biomedical Sciences

University of Medicine and Dentistry of New Jersey

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Graduate Program in Biomedical Engineering

written under the direction of

Dr. William Craelius

and approved by

________________________________

________________________________

________________________________

________________________________

New Brunswick, New Jersey

October, 2011

ii

ABSTRACT OF THE DISSERTATION

Neuroplasticity of spinal cord neurons based on piezoelectric stimulation and electrophysiological

analysis after stem cell-derived progenitor transplant

By MARIA NURIA ROYO GASCON

Dissertation Director:

William Craelius

Repair strategies in the context of spinal cord injury cover a broad amount of fields. Different

approaches have been considered ranging from the chemical, mechanical, pharmacological,

material sciences, electrical and chemical engineering sphere. There is much interest in

combinational therapies since many approaches yield promising results yet none is beneficial

enough in functional terms. There is also a necessity of properly evaluate the improvement

these therapies pose from a functional point of view and do it with sufficient resolution to target

and invest in strategies with higher potential.

This thesis is born with the interest of contributing to the spinal cord injury field at those two

levels. On one side, I have developed an injury model and techniques to test the efficacy of a

therapy for spinal cord repair as compared to controls. On the other, I have proposed a

combinational therapy in the form of a scaffold that combines biomaterials and electrical fields to

stimulate regeneration

In the first part of this thesis, I have developed an animal model to test effectiveness of treatment

by analyzing the electromyography signal of the intercostal muscles. The respiratory system is a

good test bench, usually neglected in regeneration studies. I have used a mix of engineering

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approaches from signal processing to animal physiology analysis to provide the test with enough

resolution to identify improvement. Then, I have proved the efficacy of the model by using a stem

cell therapy.

In the second part of the thesis, I have tested piezoelectric polymers as a useful platform to

deliver electrical fields to neurons. I have shown the resulting increase in neuronal growth upon

exposure to alternating electrical fields, in concrete, in neuronal branching. These results

encourage the use of biocompatible piezoelectric polymers which are very versatile in nature, as a

source for combinational therapies. Future studies will translate this in vitro model into an in vivo

treatment which will be assessed with the strategy explained in the first part.

iv

Dedication

Lluís Montal, beyond time, distance and across borders of life, I hope you are proud.

This goes to you

Lluís Montal, més enllà del temps, la distància i les fronteres d’aquest món, espero que

n’estiguis orgullós. Aquesta va per tu.

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ACKNOWLEDGMENTS

This PhD would have not been possible without the help of many people. I especially thank my

advisor, Dr. William Craelius. He got into this academic adventure with an electrical engineer

with a passion to cure Spinal Cord Injury. Bill, you are an impressive scientist but more

impressive person. Thank you for believing in me.

I also thank Dr. Jerry Scheinbeim, who opened the world of polymers to me with patience and

understanding and who always cared about my well-being beyond anything else.

I thank Dr. Bonnie Firestein, for offering her laboratory, ideas and friendship. Her passion for

science is contaminating. I am one of the lucky ones to be infected. I also thank all the people

from her lab, for the knowledge and discussion on tissue culture. I never annoyed them enough!!

You would not have this thesis in your hands if not for Dr. Troy Shinbrot. He stirred the fun for

discovery and gave me good advice: do what you enjoy in life because time will pass no matter

what.

I thank Dr. Dave Shreiber who always challenged me to improve and excel.

I thank Dr. Hans Keirstead, a driven scientist and a person of freedom, who inspired me in so

many ways. I also thank everybody in his lab for teaching all there is to know about animal work,

with amusement and style.

I thank the people at California Stem Cell, Inc, especially Dr. Monica Siegenthaler, for the cells,

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the expertise and the discussion on the stem cells studies. It was challenging and exciting!

I thank all my friends who have worked in the Rurehablab through the years, for being all such a

supportive team. Especially, and no words will be enough, for my dear friend Dr. Mike Wininger.

He is a brilliant scientist, a real mentor, and a true friend from whom I’ve learnt in so many

aspects of my career. He has been a guiding light. This message he will read next to an open

Matlab window. Mike, I owe you big!

I thank all my friends in Barcelona, New Jersey and California or anywhere life has tricked them

to go now. If I come out of this stronger and amazingly crazy is because of you. Especially have

suffered this thesis and need special mention the following:

Alberto and Rocío, my adoptive parents in the US. Mercedes, my home away from home. Nachi,

my Argentinian twin sister. Carles, who taught me that if I don’t believe in me, the battle is

already lost. Lavanya Peddada who has walked this long path with me. This PhD would make

sense if just for getting her in my life.

I thank all the people with Spinal Cord Injury and Spinal Muscular Atrophy, because they are the

example and inspiration for us working in the benches. We will make it happen!

My warmest gratitude goes to my family, in concrete my dear grandma. Last but not least, I thank

my parents and siblings. If I could make it so far and away, through all the happy and the rough

times, is because they are always with me. If I can live the life I’ve chosen, is because they have

my back. Mom, dad, Cèlia, Jaume and Roger, this and anything I will ever accomplish belongs to

you. You make me strong.

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Finalment, agraeixo als meus pares i germans el seu recolzament durant tots aquests anys. Si me

n’he sortit, tan lluny de casa, en els moments feliços i en els difícils. és perquè sempre sou amb

mi.. Gràcies a vosatres, tinc la vida que he escollit. Mama, papa, Cèlia, Jaume i Roger, aquest

doctorat i qualsevol cosa que aconsegueixi en aquest món, us pertany. Sou vosaltres que em feu

forta.

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TABLE OF CONTENTS

ABSTRACT....………………………………………………………………………………...…………….ii

DEDICATION…………………………..………………………………………………………………….iv

ACKNOWLEDGMENTS………………...………………………………………………………………...v

TABLE OF CONTENTS…………………………………………………………………………………viii

LIST OF TABLES…………………………………………………………………………………………xii

LIST OF ILLUSTRATIONS…………………………………………………………………………….xiii

CHAPTER 1 INTRODUCTION .................................................................................................................. 1

1.1. HYPOTHESIS ......................................................................................................................................... 3

1.2. CONTRIBUTIONS TO THE FIELD ............................................................................................................. 3

CHAPTER 2 ASSESSMENT OF TREATMENTS IN SPINAL CORD INJURY .................................. 5

2.1. GOAL OF THIS CHAPTER ........................................................................................................................ 5

2.2. ETIOLOGY OF SPINAL CORD INJURY ..................................................................................................... 5

2.3. CONSIDERATIONS ON PRIORITIES IN THE CONTEXT OF SPINAL CORD INJURY ......................................... 6

2.4. HISTOLOGY, MOLECULAR BIOLOGY AND GENETIC TESTS ...................................................................... 7

2.5. BEHAVIORAL TESTS .............................................................................................................................. 8

2.6. FUNCTIONAL TESTS ............................................................................................................................ 10

2.7. SIGNIFICANCE OF ELECTROPHYSIOLOGICAL ANALYSIS OF TREATMENT .............................................. 12

CHAPTER 3 ELECTROMYOGRAPHY ................................................................................................. 13

3.1. GOAL OF THIS CHAPTER ...................................................................................................................... 13

3.2. ELECTROPHYSIOLOGY OF THE SKELETAL MUSCLE .............................................................................. 13

3.3. AUTOMATIC MUP ANALYSIS ............................................................................................................. 18

3.4. IP ANALYSIS (IPA) ............................................................................................................................. 19

3.5. ELECTROPHYSIOLOGICAL PARAMETERS AFTER NERVE INJURY ........................................................... 24

3.6. ELECTROPHYSIOLOGICAL DIFFERENCES BETWEEN RATS AND HUMANS .............................................. 26

ix

3.7. SUMMARY .......................................................................................................................................... 27

CHAPTER 4 EMG SOFTWARE DEVELOPMENT .............................................................................. 28


4.2. NEED FOR SOFTWARE MODIFICATIONS ............................................................................................... 28

4.3. ELECTROCARDIOGRAM REMOVAL ...................................................................................................... 30

4.4. AUTOMATIC ZERO-CROSSINGS TRACKING .......................................................................................... 32

4.5. TURNS/AMPLITUDE ANALYSIS (TAA) ................................................................................................. 32

4.6. ANALYSIS OF THE ELECTROMYOGRAPHIC BURST ................................................................................ 36

4.7. SUMMARY .......................................................................................................................................... 37

CHAPTER 5 RAT MODEL OF INJURY ................................................................................................ 39


5.2. PHYSIOLOGY OF THE RESPIRATORY SYSTEM ....................................................................................... 39

5.3. ANATOMY OF INTERCOSTAL MUSCLES ................................................................................................ 41

5.4. RESPIRATORY FUNCTION OF INTERCOSTAL MUSCLES ......................................................................... 42

5.5. INNERVATION OF INTERCOSTAL MUSCLES .......................................................................................... 44

5.6. INJURY LOCATION AND TYPE .............................................................................................................. 45

5.7. EXPERIMENTAL PROTOCOL ................................................................................................................ 46

5.8. SUMMARY .......................................................................................................................................... 48

CHAPTER 6 RESULTS ON A STEM CELL REPAIR THERAPY ...................................................... 50


6.2. BACKGROUND .................................................................................................................................... 51

6.3. METHODS ........................................................................................................................................... 52

6.4. ELECTROPHYSIOLOGICAL RESULTS .................................................................................................... 56

6.5. BEHAVIORAL RESULTS ....................................................................................................................... 58

6.6. HISTOLOGICAL RESULTS ..................................................................................................................... 58

6.7. DISCUSSION ........................................................................................................................................ 64

6.8. SUMMARY .......................................................................................................................................... 65

x

CHAPTER 7 EFFECTS OF ELECTRICAL AND MECHANICAL STIMULI IN NEURONS ......... 66


7.2. STRATEGIES FOR SPINAL CORD INJURY .............................................................................................. 67

7.3. CHEMICAL GROWTH FACTORS ............................................................................................................ 68

7.4. STRUCTURAL SUPPORT ....................................................................................................................... 68

7.5. STIFFNESS ........................................................................................................................................... 70

7.6. MECHANICAL STIMULI ........................................................................................................................ 70

7.7. ELECTRICAL STIMULI ......................................................................................................................... 75

7.8. ELECTRICALLY ACTIVE POLYMERS ..................................................................................................... 80

7.9. SIGNIFICANCE ..................................................................................................................................... 82

CHAPTER 8 PIEZOELECTRICITY ....................................................................................................... 83


8.2. HISTORY ............................................................................................................................................. 83

8.3. PIEZOELECTRIC CLASSES .................................................................................................................... 84

8.4. CONSTITUTIVE EQUATIONS FOR A PIEZOELECTRIC MATERIAL ............................................................. 85

8.5. PIEZOELECTRIC POLYMERS ................................................................................................................. 88

8.6. SUMMARY .......................................................................................................................................... 92

CHAPTER 9 EXPERIMENTAL DESIGN AND CHARACTERIZATION OF PIEZOELECTRIC

MATERIALS ............................................................................................................................................... 93


9.2. FABRICATION OF PIEZOELECTRIC FILMS ............................................................................................. 94

9.3. CHARACTERIZATION OF MATERIALS ................................................................................................... 94

9.4. CELL CULTURE ................................................................................................................................... 97

9.5. SETUP AND STIMULATION PROTOCOL ................................................................................................ 97

9.6. IMMUNOCYTOCHEMISTRY ................................................................................................................ 101

9.7. CELL ANALYSIS AND IMAGING .......................................................................................................... 101

9.8. STATISTICAL ANALYSIS .................................................................................................................... 103

xi

9.9. SUMMARY ........................................................................................................................................ 103

CHAPTER 10 GROWTH OF CELLS ON PIEZOELECTRIC POLYMER FILMS ........................ 104

10.1. GOAL OF THIS CHAPTER .................................................................................................................. 104

10.2. EFFECTS OF PIEZOELECTRIC POLYVINYLIDENE FLUORIDE IN SPINAL CORD NEURONS ..................... 104

10.3. DISCUSSION .................................................................................................................................... 109

10.4. SUMMARY ...................................................................................................................................... 112

CHAPTER 11 CONCLUSION ................................................................................................................ 113

CHAPTER 12 BIBLIOGRAPHY ............................................................................................................ 115

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LIST OF TABLES

Table 1- MUP features ............................................................................................................. 16

Table 2- MUP parameters obtained by a Multi-MUP Analysis and criteria used by it. .......... 19

Table 3- Parameters obtained from analysis of the IP signal. ................................................. 24

Table 4-IPA parameters provided by EMGvet software. ......................................................... 30

Table 5- Comparison of criteria for IPA parameters between Keypoint and EMGvet, where d

is duration of segments and amp is amplitude of segments. ................................................. 38

Table 6- Animal groups, condition,outcome measuremes, sacrifice points and number of

subjects (n) .............................................................................................................................. 52

Table 7-Rheolograph measurements for PZ films fabricated as described in methods ......... 95

xiii

LIST OF ILLUSTRATIONS

Figure 1.Schematic shows 2 motor unit pools and the patterns of innervation of each

neuron in different colors. ...................................................................................................... 14

Figure 2. Schematic of SFAP potentials recorded at short distance (A) and far distance (B). 15

Figure 3. Schematic representation of motor unit potential (MUP) generation and recording

by needle electrode. ................................................................................................................ 16

Figure 4. Main features of a MUP. .......................................................................................... 17

Figure 5- EMG signal of the IIIrd intercostal muscle before (A) and after (B) being processed

by the cleansing algorythm. In (A) it was also included the digitalized signal, which is green

for only EKG, magenta for respiratory burst, and red for smoothed signal. .......................... 32

Figure 6-Turns are depicted as black asterisk. Maximum are depicted in red dots for

maximum and green for minimum.in a time series (vector) .................................................. 33

Figure 7- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter

plots of the values of the duration versus the amplitude of the individual segments (red),

and threshold values for activity (black) for two different animals (A and B) 1 week after

injury. ....................................................................................................................................... 34



and threshold values for activity (black) for two different animals (A and B) 4 weeks after

injury. ....................................................................................................................................... 34


xiv


and threshold values for activity (black) for two different animals (A and B) 5 weeks after

injury ........................................................................................................................................ 35

Figure 10- Inspiratory burst with activity regions based on definition of EMGvet. Activity is

presented in red while the general inspiratory burst in blue. ................................................ 35


presented in red while the general inspiratory burst in blue ................................................. 36

Figure 12- (A) raw and digitized EMG signals of the IIIrd intercostal muscle for several

inspirations before processing. Digitalized signal was also included, green for only EKG,

magenta for respiratory burst, and red for smoothed signal. (B) EMG during a single

inspiration after processing by the cleansing algorithm. ........................................................ 37

Figure 13- IP analysis of EMG signal from intercostal muscles 2 (a) and 3 (b) presented as

turns per second. Data expressed as mean and standard error. Student t-test with *p<0.05,

**p<0.005. ............................................................................................................................... 57

Figure 14-BBB locomotor scores for MotorGraft treated and vehicle control animals with a

T3 SCI. Testing was performed at 5 days post injury prior to MotorGraft transplantation for

pretreatment comparison (5D Post-injury/Pre-MG). Testing was preformed again at 1 week,

2 weeks, 20 days, and 27 days after MotorGraft transplantation (1 Wk Post-MG, 2 Wks Post-

MG, 20 D Post-MG, and 27 D Post-MG, respectively). The locomotor capability of

MotorGraft and vehicle control treated animals did not differ. Data are expressed as mean ±

standard error. ........................................................................................................................ 58

Figure 15-MotorGraft cells survive and begin to express ChAT. a, d) Human nuclei (green)

xv

were detected in the spinal cords of transplanted animals. b, c, e, f) Hoechst nuclear counter

stain (blue) (b and e) colocalizes with human nuclear staining (c and f). g, h, i) ChAT positive

staining (red) is localized with MotorGraft (human nuclei in green) and surrounding cells

(Hoechst in blue). Images taken at 200X magnification. ........................................................ 59

Figure 16-a) The average number of neurons in a quadrant of spinal cord cross sections from

vehicle control (ave= 29) and MotorGraft transplanted animals(ave=38). There were

significantly greater number of neurons in transplanted animals (ave=38). b) The average

number of motor neurons in a ventral horn of vehicle control (ave= 7) and MotorGraft

transplanted animals.(ave=12) rostral to the injury epicenter. c, d) Representative NeuN

staining (red) in transplanted (c) and vehicle control (d) spinal cord sections. e, f)

Representative ChAT staining (red) in transplanted (e) and vehicle control (f) spinal cord

sections. Images taken at 200X magnification ....................................................................... 61

Figure 17- Comparison of serotonergic fiber sprouting (Stained by 5-HT) in vehicle control

and MotorGraft transplanted groups. MotorGraft transplantation resulted in increased

integrated density of 5-HT fibers (red) 2mm rostral (+2) to the injury epicenter. Analysis is at

2mm and 1mm rostral (+2 and +1, respectively) and 2mm and 1mm caudal (-2 and -1,

respectively) to the injury epicenter. Images taken at 200X magnification. Data is expressed

as mean ± standard error. *p<0.05. ........................................................................................ 63

Figure 18- Nomenclature of axis ............................................................................................. 87

Figure 19-PVDF Molecule ........................................................................................................ 89

Figure 20- Axis nomenclature for the piezoelectric matrix ..................................................... 90

Figure 21- PLLA molecule. The asterisk indicates the chiral carbon atom. ............................. 91

xvi

Figure 22- DSC graphic showing the melting temperature of PVDF. ...................................... 95

Figure 23- FTIR spectra of non-polarized PVDF (PV, top) and piezoelectric PVDF (PZ, bottom).

The characteristic peaks of the α-phase and β-phase have been marked for convenience. . 96

Figure 24- Set-up of the well plate for seeding. ...................................................................... 97

Figure 25- VIBES: Polycarbonate base for stimulation of well plate cell cultures. ................. 98

Figure 26- Mechanical characterization of well-plate stimulation system: contour plot of

frequency (left) and amplitude (right) across the well plate. ................................................. 99

Figure 27: Mechanical characterization of well plate stimulation at 80Hz, 50Hz and 20Hz. 100

Figure 28- Centrifugal labeling scheme. Processes attached to the soma (green circle) have

an order of 1. At each branch point the order is increased by one. ..................................... 102

Figure 29- Spinal cord cultures immunostained with a MAP2 antibody after 5 DIV in the four

conditions: (A) US-PV, (B) S-PV, (C) US-PZ and (D) S-PZ. Scale bar=30 µm. .......................... 105

Figure 30- Comparison of branching features between (A) US-PV and S-PV and (B) US-PZ and

S-PZ. *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-Whitney test). Standard error

depicted. ................................................................................................................................ 106

Figure 31- Comparison of average number of processes per cell between (A) US-PV and S-PV

and (B) US-PZ and S-PZ. * p<0.05, *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-

Whitney test). Standard error depicted. ............................................................................... 107


and (B) US-PZ and S-PZ. (Unpaired t-test/Mann-Whitney test). Standard error depicted. .. 108

Figure 33- Comparison of Sholls analysis of the total number of neurite intersections

xvii

between (A) US-PV and S-PV and (B) US-PZ and S-PZ. Bar indicates significance * p<0.05

(Unpaired t-test/Mann-Whitney test). Standard error depicted. ......................................... 108

Figure 34- Comparison of neuronal densities for PV substrates (A) and PZ substrates (B) **

p<0.01 (Unpaired t-test/Mann-Whitney test). Standard error depicted. ............................. 109

1

Chapter 1

Introduction

Spinal Cord Injury (SCI) is a complex problem, with acute and chronic scenarios. In chronic

injuries, pressure sores, thermoregulatory and respiratory problems are the most common

problems, being diseases of the respiratory system the primary cause of death after one year of

injury. In order to find the cure for SCI, contributions at different levels are necessary, ranging

from strategies for tissue regeneration, effective tools of assessment, good modeling of the injury,

which also includes targeting physiological systems with high priority. Biomedical engineering is

a relatively emerging field within the engineering disciplines and it is defined by the application

of the engineering principles to a biomedical problem. It entails mostly interdisciplinary work and

interfaces between many fields. It is in a context like spinal cord injury, were biomedical

engineering approaches are most useful. This thesis is a comprehensive study which addresses

and contributes to different parts of this problematic from several biomedical perspectives.

First, I address the two most problematic aspects in the context of spinal cord injury research:

injury delivery and the measurement of the tissues. The first goal of the thesis is to develop

effective tools to track functional improvement of regenerative therapies for spinal cord injury in

animals. That aim is addressed in two ways. One is by developing an injury model that effectively

mimics motor neuron denervation of the respiratory system and the other is by developing a

2

technique that studies function, which also is more sensitive than behavioral tests.

Histology is a useful method to show improvement at cellular level but cannot show restoration

of function, while behavioral tasks may not be appropriate for the system under study.

Functionality is the missing link in measurements of regenerative improvement. Regarding injury

site, high priority systems like the respiratory system have traditionally been neglected in

comparison with the locomotor system.

Electromyography is a common functional outcome but is mostly confined to qualitative studies

due to low resolution. Commercialy available softwares are partly accountable for the low

resolution of automatic electromyography (EMG) analysis observed in animals. Those softwares

are intended for humans and do not take into account particulars of animal electrophysiology. As

the first part of my work, I developed new software able to do automatic analysis of the EMG

signal of animals, which also identifies the most sensitive parameters. Custom-made software

also allows for implementation of signal processing techniques that might have been neglected

before and add to overall resolution. I have validated the technique and the model of injury with a

stem cell transplant.

In the second part of the thesis I propose as a regenerative strategy for the neuronal population, a

piezoelectric substrate that can deliver electrical stimulation. This substrate would be the

precursor of a piezoelectric platform for combinational therapies. I present here the in vitro

studies with those substrates. Futures in vivo studies will test the efficacy of those scaffolds with

the model of injury and the technique I described in the first part.

3

1.1. Hypothesis

My contribution to the field of spinal cord injury strategies can be summarized by the answer to

the following hypothesis.

1. A thoracic level bilateral contusion injury is a suitable model of denervation of the

intercostal muscles via damage of the central nervous system.

2. Interference Pattern Analysis is a sensitive tool to study denervation and reinnervation of

the intercostal muscles and can detect effectiveness of treatment for Spinal Cord Injury

and Spinal Muscular Atrophy Type I.

3. Piezoelectricity enhances branching in neurons with independence of the material and

cell type and vibration.

1.2. Contributions to the field

In the context of the first hypothesis, I developed an injury model to study effectiveness of a

transplant by tackling the respiratory system.

In the context of the second hypothesis the tasks I carry out are:

Create custom software to perform interference pattern analysis in animals

Use different signal processing techniques to improve resolution on analysis of the

electromyography signal

Determine which electrophysiological parameters have an appropriate resolution to track

reinnervation

Test the electrophysiology technique in a stem cell transplant and correlate results with

histology and behavior.

Finally, in the context of the third hypothesis, the different tasks performed are:

4

Develop methods for fabricating and characterizing biocompatible and biodegradable

piezoelectric polymer films as cellular growth substrates.

Develop platforms to stimulate and test PZ polymers in tissue culture applications.

Test effect of PZ stimulation and vibration on neurons

5

Chapter 2

Assessment of treatments in Spinal Cord Injury

2.1. Goal of this chapter

Spinal Cord Injury is a deleterious condition with strong physical, psychological and economical

costs for individuals and society. Proper assessment techniques are crucial in targeting therapies

with regenerative capabilities. In this chapter, I present the current assessment techniques and the

gap there is left in two fronts. One is assessment of functionality and the other is targeting the

respiratory system. I review some of the most common functional methods and I focus on the

reasons of deciding on electromyography.

2.2. Etiology of Spinal Cord Injury

Spinal Cord Injury (SCI) is damage of the spinal cord that results in loss of function, such as

mobility or feeling, or dysfunction of its normal activities. Usually is a result of a traumatic event

like car accidents, falls and gunshots, although tumors, neurodegenerative or demyelinative

diseases can be causes of spinal cord injury as well.

According to National Spinal Cord Injury Statistical Center (NSCISC) report of 2006, the

6

annual incidence of spinal cord injury in the U.S., not including those who died at the scene of the

accident, is approximately 11,000 new cases each year, adding to a total of around 200,000 cases

[1]. The lesion can damage myelinated fiber tracts, gray matter and sensorimotor neurons, as well

as cause loss of neurons.

The average age at injury is 38 years and the expectancy of life for that population is between

24.7 and 11.1 years, depending on the severity of the injury. Unlike the peripheral nervous system

(PNS), the central nervous system (CNS) does not regenerate spontaneously. Although there

might be a certain degree of recovery during the first months, most of the losses are permanent

after a while. This leaves a big population of relatively young people with difficult challenges to

face.

The consequences of a spinal cord injury vary widely depending on the level of the lesion, the

type of the injury and the time and type of intervention. The outcome is often a certain degree of

paralysis or sensory loss, complete or incomplete, of the parts of the body controlled by the

segments of the spinal cord below the injury site. The loss depends on the extension of the lesion

as well as the concrete pathways that were affected. Neuropathic pain is also a common

dysfunction on people affected by spinal cord injury. Other health issues for those affected with

SCI are related to the disuse and decrease of self- autonomy imposed by the injury, as pressure

sores and urinary infections. Not less important are the psychological burden put on the

individual and their families.

2.3. Considerations on priorities in the context of spinal cord injury

According to the Anderson study from 2004, the priorities for people with spinal cord injuries do

not match the efforts from the scientific and clinical communities [2]. Locomotor function does

7

not rank the highest in the concerns of those suffering with spinal cord injuries, and yet, around

75% of the studies on spinal cord injury between 2001 and 2007 are focused on that subject. A

further analysis on the focus of spinal cord injury studies in scientific journal shows an obvious

under-representation of the autonomic system as well as of the nature of pain. [3]

Altogether, it suggests that the world of the benches needs to approach the necessities of the

people they intend to help. It can be done directly through studies to extend our knowledge on the

way SCI affects those systems, by finding specific approaches to treat these conditions or

indirectly by developing injury models focused in them to evaluate the effectiveness of therapies.

2.4. Histology, molecular biology and genetic tests

The first and most direct answer to the effectiveness of a treatment are histological, molecular and

genetic tissue studies. In the absence of functional and behavioral recovery, analysis of the tissue

targets effective therapies that have not yet reached their full potential. In the presence of

functional recovery, those analyses are means to identify the mechanism, adjust it to the purpose

and so enhance the efficacy of the therapy.

Various tissue analyses can detect reduction of secondary damage in the spinal cord, directly by

studying the spinal tissue as seen in tissue sparing, secretion of growth factors, up or down

regulation of certain genes, nerve sprouting, nerve tracing and types of cells present in spinal cord

or indirectly by observing the effect that reduction has in the innervated muscle, as decrease in

muscular atrophy and alteration of muscular composition. Here follows a list of them and some of

the assays to obtain them:

a) Morphometry: Changes in size and shape of the tissue are useful to identify the epicenter

of the injury and correlate results with distance

8

b) Tissue Sparing: Staining against Neuronal Nuclei (NeuN) or cleaved caspase 3 allows

observation and quantification of neuronal survival. A concrete population can be

targeted using the appropriate antibody like choline acetyltransferase (ChAT) for motor

neurons

c) 5-HT Sprouting: Immunohistochemistry against the 5-hydroxytryptamine (5-HT)

receptors identify serotonergic projections allowing visualization of axonal sprouting in

ascending and descending pathways.

d) Nerve tracing: Labeling nerves with 5-bromodeoxyuridine (BrdU) and using horseradish

peroxidase (HRP) to observe innervation and interrupted pathways.

e) Growth Factor Secretion: Specific antibodies can be used against different growth factors

like Neurotrophin-3T (NT-3), Neurotrophin-4/5 (NT-4/5), Nerve Growth Factor (NGF)

or Vascular endothelial growth factor (VEGF)

f) Gene Expression: Polymerase Chain Reaction (PCR) analysis is done in extracted RNA

of homogenized tissue. Reverse transcription is performed with selected primers to

identify up-regulation or down-regulation of genes

g) Protein Expression: Enzyme-linked immunosorbent assay (ELISA) is used to quantify

expression levels of different proteins and determination of human protein levels.

h) Muscular fiber diameters and fiber types: Staining with hematoxylin and eosin (H&E) to

observe cellular nuclei (deep blue) and boundaries of myocytes (clear). Further

classification of fiber types can be done using Adenosine triphosphatase (ATPase) at

different PH levels.

2.5. Behavioral tests

To study the efficacy of treatment through recovery of function, there are several behavioral tests

available. Most of them are devoted to locomotor function, although some examine the sensory

9

system as well.

The Basso-Beattie-Bresnahan test (BBB) [4] was developed to assess improvement in hind-limb

function in rats after a spinal cord contusion injury at the thoracic level. Nowadays is the standard

method of assessment of locomotor function in rats not only for injuries beyond the thoracic level

but also for injuries different than contusion.

Other locomotor tests address the shortcomings or improve the resolution of the BBB test at

certain time-points in recovery. For instance, to account for inter-limb coordination and remove

the scorer intervention in the BBB test that sometimes is needed to stimulate rats to make a pass,

some alternative tests are the GridWalk from Barth et al. [5] and Kunkel et al [6]., the horizontal

ladder beam of Soblosky et al. [7], which counts missed steps, and the straight alley of Wong et

al.[8].

To obtain and analyze gait-related data during locomotion exists the CatWalk automatic gait

analysis [9], which uses thermally-impressed paw-prints to obtain parameters as stride length and

swing duration, pressure during locomotion and support of the base during the stride. To evaluate

forelimb function there are grip strength meters as described by et al. [10], for vertical exploration

there is the clear cylinder test of [11] et al and the food pellet reaching task of Metz and

Whishaw [12].

Return of sensation and also the presence of hyperalgesia or allodynia can be tested by applying

controlled mechanical stimuli like in the pin-prick test and the Von-Frey hair test [13], or by

observing the response to temperature applied by the hot plate test. Other sensory tests are the

visual cliff test, which evaluates the ability to see the drop-off edge of a horizontal surface, the

10

acoustic startled test which sets thresholds for audition as well as noxious auditory amplitude by

observing flinching in the animal.

2.6. Functional tests

In addition to histology and behavior, efficacy of therapies can be assessed by functional tests.

They are better quantifiable and have a higher resolution than behavioral tests, especially for low

levels of improvement. As an advantage over histological results, they are able to un-mask

physiological changes that do not translate into functional improvement.

Most functional tests have been developed around systems that lack a primarily voluntary input,

mainly because of the absence of behavioral tests for those systems. Not many standardized

techniques are offered to evaluate the function of the cardiovascular, respiratory, sexual and

gastrointestinal systems, the lower urinary tract and thermoregulation but it is necessary to point

out that there are fewer studies on sensation compared to locomotion, and even fewer studies deal

with recovery in the autonomic system. Hereafter, I present some functional tests. Reviewed from

[3].

In the cardiovascular context, we can study problems like hypotension, tachycardias and

arrhythmias by monitoring fluctuations of blood pressure and heart rates with time. The

measurement is done by implanting a cannula and connecting it to a pressure transducer.

Malfunction of the gastrointestinal (GI) after SCI, for instance in motility, can be studied with

spectrophotometry by dye recovery on the GI tract after oral marker ingestion. Electromyography

(EMG) is another tool to study external anal-sphincter (EAS) hyperreflexia. EMG can be used

also to asses ending of spinal shock which is characterized by recovery of autonomic function.

11

Manometry measures pressure changes inside the GI tract by inserting a catheter filled with fluid,

which is later attached to the recording probes. However, a more feasible way of continuously

measuring pressure changes in moving animals is by using strain gauges. On another line,

hydrogen breath tests measures the increase in hydrogen expiration which follows carbohydrate

administration and is used to track oro-cecal transit time.

Cystometric urodynamic analysis is the common method for assessing low urinary tract (LUT)

function and it can be performed by a catheter implanted into the bladder dome, which allows for

simultaneous EMG recording of the external urethral sphincter (EUS). Bladder volume is

proportional to severity of SCI and consequently, the volume of unire expressed at micturition is

a very useful output as well.

Sexual function is studied in animals by telemetric monitoring of pressure within the corpus

cavernosum (CC) and the corpus spongiosum (CSP). EMG of the perineral muscles during sexual

intercourse is one of complementary tests. Sexual arousal in cats might be studied by laser

doppler flowmetry, which records vaginal blood flow.

The effect that SCI has in thermoregulation can be tracked by measuring core temperature,

cutaneous temperature and blood flow. The latter gives information on the sympathetic

vasomotor pathways, which can be studied by micro-neurography as well.

Finally, in the context of the respiratory system, four tests yield useful information to track

recovery: phrenic nerve conduction, pneumotachometry, diaphragmatic electromyogrpahy and

plethysmography. A pneumotachometer measures the airflow and a plethysmograph changes in

air volume. They are both useful resources to study respiration in adult humans but they have

limitations when measuring from children and animals.

12

2.7. Significance of electrophysiological analysis of treatment

Histology and behavioral testing, mostly locomotive, pose the edges of a spectrum where there

are not many in-between outcome features and often neglects somatic or autonomic responses.

The functional output is the missing stone, better allowing to fine-tune therapies and identifying

where they are failing.

Electromyographic measurements are one of the most useful resources for therapeutic assesment

in terms of function, especially for systems with strong autonomic input. However, they have not

been used extensively as a validation tool of potential treatments. That is partly due to the

inherent variation of the technique which renders resolution too low to asses improvement with

statistical significance between injured and non-injured groups. Resolution of the

electrophysiology signal can improve with a combined approach. On one side, the use of

electrophysiological parameters most robust to variability, on the other, the use of signal

processing techniques to prepare the signal and finally creating software that can automatically

track the changes in

13

Chapter 3

Electromyography


This chapter presents the main concepts related to electromyography (EMG), the effect of injury

in the electrophysiological parameters and the specific electrical features of muscle activity in

rats. First, I introduced the concept of the Motor Unit Potential (MUP) and common vocabulary

related to intramuscular EMG. The chapter focuses in the different analysis comprised under the

label of Interference Pattern Analysis (IPA). Then, the effects that injury and reinnervation have

in the EMG signal are described. Finally, I have presented the specifics of the EMG signal of rats

as compared to humans, thus justifying the necessity of new software to automatically perform

IPA in animals. Chapter 4 will introduce EMGvet, the custom software I developed to perform

IPA in rats based in specific characteristics of their MUPs.

3.2. Electrophysiology of the skeletal muscle

Electromyography is a technique to measure the electrical activity in muscle fibers. A motor unit

is the basic structure of the skeletal system. It is constituted by a single motor neuron and all the

fibers it innervates.

14

The neuromuscular junction is the location where the motor neuron axon synapses with the

muscle. The muscle at that site is known as motor end plate and it has a very excitable membrane.

Depolarization of the motor end plate as a response to the acetylcholine neurotransmitter released

by the motor neuron is called end plate potential (EPP). When the EPP reaches threshold, an

action potential is triggered and each muscle fiber enervated by that motor neuron contracts

Spontaneous depolarizations of the end plate, which are caused by random release of

acetylcholine vesicles by the motor neuron, are called miniature en potentials (MEEP). They

hardly ever are able to trigger an action potential by themselves.

In the skeletal muscle of mammals, there are no inhibitory synapses. Thus, activation of the α

motor neuron results in the contraction of all the fibers it innervates, which are all of them of the

same type, whether fast-twitching or slow-twitching. The fibers innervated by a motor neuron are

usually scattered through the muscle. The set of motor units that innervates a concrete muscle is

known as the motor neuron pool for that muscle. Figure 1 is a diagram depicting that fact.

Figure 1.Schematic shows 2 motor unit pools and the patterns of innervation of each neuron in

different colors.

There are two main different types of electromyography: surface electromyography (sEMG) and

15

intramuscular electromyography, also known as needle EMG. Intramuscular recordings are taken

using a bipolar electrode or a concentric needle electrode. All recordings performed within the

purview of this thesis work are of the latter type. Intramuscular EMG avoids the problems of

attenuation, absorption and scattering associated with tissue. Because of those effects, the

electrical signal in sEMG does not permit a proper analysis on Motor Units.

The electrode measures all the electrical activity from the fibers within its detection range. A

single fiber action potential (SFAP) has a shape as the one depicted in Figure2. The farther the

electrode is from it, the lower in amplitude and longer in duration the SFAP is.

A B

Figure 2. Schematic of SFAP potentials recorded at short distance (A) and far distance (B).

Activation of a motor unit reflects contemporaneous activation of all the fibers in the motor unit

pool. Therefore, activation of a motor unit creates an electrical profile which depends on the

addition of each individual SFAP for that MUP and their relative distance to the recording site.

Such a profile is known as a motor unit potential (MUP) and the study of its features can be used

as a characterization as well as diagnostic tool of muscle activity.

16

Figure 3. Schematic representation of motor unit potential (MUP) generation and recording by

needle electrode.

Important features regarding MUP are listed in Table 1.

PARAMETER DESCRIPTION

Amplitude Maximum peak to-peak amplitude

Duration Time interval between the first deviation of the signal from baseline and

its last return

Area Area under the rectified waveform

Thickness Area/amplitude

Number of phases A phase is the portion of signal between deviation from baseline to its

return to baseline.

Rise time Slope of the main phase of the MUP

Number of turns A turn is a change in polarization of more than 100uV

Satellite potentials Linked low-amplitude waveform linked to the main MUP

Complexity. A MUP with more than 4 phases (polyphasic), 5 turns or satellite

potentials is a complex one

Jiggle Vertical instability of the MUP

Firing pattern Periodicity of MUPs

Recruitment Pattern Number of MUPS present for different levels of force

Table 1- MUP features

Some of these concepts have been depicted in Figure 4.

17

Figure 4. Main features of a MUP.

Electrical activity in the EMG signal comes not only from action potentials elicited by the same

motor neuron but also from action potentials elicited by different motor neurons. For low levels

of activity, it is easy to distinguish between different MUPs since they are not-synchronous and

their firing patterns are different. However, for high levels of muscle activity MUPs overlap

making it difficult to distinguish each one individually.

Some considerations on factors influencing MUP parameters are due at this point:

The amplitude of the electrode recording of the SFAP diminishes with distance to the power of 2.

Therefore, the MUP amplitude and shape is dominated by those fibers close to the electrode. On

the other hand, the fibers remote from the electrode will contribute more significantly to duration

of the MUP. In general, the higher the absolute number of muscle fibers in a motor unit results in

a longer MUP duration.

In addition to that it should be pointed out that higher levels of force are achieved by recruiting a

higher number of active motor units and increasing their individual firing frequencies. In normal

18

activity, low-threshold motor units are recruited first, and levels of contraction are increased by

increasing their firing frequency. That value reaches a plateau, but simultaneously, higher-

threshold motor units have started getting recruited to maintain an increase in contraction levels.

The frequency at which a motor neuron starts firing is called the onset frequency, and the

frequency at which the next motor unit is recruited is the recruitment frequency.

Analysis of all of these MUP features and deviation from their normalized values allow

identifying abnormal EMG signal caused by trauma or disease and diagnose a diverse range of

neuropathies and myopathies.

3.3. Automatic MUP Analysis

Analysis of MUP features in an automated manner was first presented by Stalberg in their Multi-

MUP software [14]. Multi-MUP analysis is a decomposition analysis of the EMG signal which

allows automatic identification of MUPs. The analysis is performed over epochs of 4.8s and

gathers 6 different MUPs per epoch. It uses a decomposition method based on template matching.

MUPs are signals meeting the criteria of starting with a negative peak which exceeds 30uV,

having a positive slope higher than 30uV/0.1ms and a separation between other potential MUPs

of more than 2.5ms. Each of those signals joins a pre-existing class if the difference is below a

certain value or starts a new class. At the end, the six classes with more hits are each averaged.

MUP parameters provided in a Multi-MUP analysis have been listed in Table 2, along with the

criteria used by the analysis [14]:

19

PARAMETER CRITERIA Zero threshold Zeroing below 20uV to erase noise fluctuations

Duration Time difference between the end and starting point of the MUP. Time

points are determined using a slope criterion of more than 30uV/0.1ms, a

negative peak exceeding 30uV and separation with adjacent MUPs of

more than 2.5ms, after zero-thresholding.

Amplitude Difference between maximum and minim peaks of the MUP

Rise time Time between the maximum negative and the maximum positive peak

Area Area of the MUP within duration time

Thickness Area/amplitude

Number of phases Each phase being a section of an MUP that falls between two baseline

crossings and reaches an absolute value of more than 20uV, after zero-

thresholding.

Table 2- MUP parameters obtained by a Multi-MUP Analysis and criteria used by it.

3.4. IP Analysis (IPA)

MUAP analysis is a useful analysis to understand the MUP activity and recruitment but can only

be performed at low levels of activity when the EMG signal is not very full and individual

MUAPs can be identified. After a certain amount of activity (in voluntary muscles that would be

above 10% of maximum voluntary contraction) the summation and overlap of MUAPs creates an

interference pattern, which introduces a lot of error in the automatic MUAP analysis. In such

cases, the interference Pattern Analysis (IPA) is a good alternative. The interference pattern

depends on the number of recruited MUs, size, duration, firing rates, time of recovery…etc.

The inspiratory burst associated with the electrical activity of the intercostal muscles firing during

the inspiration cycle can be analyzed by different techniques. The Turns/amplitude Analysis

(TAA) is the closest approximation to MUP analysis and yields certain parameters very

dependent on MUP features. (Reviewed from [15], [16]).

The whole burst can also be studied through integration, room mean square or envelope. All of

them provide gross features of burst amplitude, burst duration and breathing frequency.

20

Alternatively, information can be extracted from power spectral analysis. In the end, some of

these analyses are redundant and convey the same information through different paths.

3.1.4. Zero-crossings

The number of zero-crossings through the zero line can indicate degree of muscle activity. To

avoid background noise, it is established a threshold level so only crossings between those two

thresholds are counted. Usually it is established 25uV as the threshold.

3.2.4. Turns/amplitude analysis (TAA)

Willison developed a method to study the IPA based in measuring turns on the Interference

Pattern of the EMG [17]. A turn is a local maximum that changes direction by at least 100uV in

amplitude compared to the preceding and subsequent turns (other versions of the algorithm

establish a threshold of 50uV for the turns). A threshold of 30uV is set to cope with noise. The

number of turns is linearly related to number of zero-crossings, but has extra value as a diagnostic

parameter.

Three useful derivations of this concept are the number of turns per second (T/S) often referred

simply as turns, the mean amplitude of the turns (A/T), which is the average amplitude between

two turns and the segment, which is the time lapse between turns. These parameters, together

with the ratio between them (T/S: A/T) are useful diagnostic tool of disease.

In myopathies, T/S during a fixed time increases, and in neuropathies, T/S does not change while

A/T increases. The ratio T/S: A/T was increased in myopathies with respect to the normal case

and decreased in neuropathies. [18]

21

Usually, in interference pattern analysis, the parameters under consideration are the recruitment

of MUPs with increasing force and the amplitude, duration and complexity of the MUPs. Stalberg

et al developed three features, activity, upper centile amplitude and number of small segments

which are equivalent to those MUP features.

Activity is a quantification of the time with MUP activity, whether coming from individual or

superimposed MUPS. As the force of contraction increases, more MUPs are recruited and the IP

signal becomes fuller. Activity measures that feature and we can see how it increases linearly

with the number of MUAP discharges up to 80% of its theoretical value. Activity is the time per

second that IP signal is active. Actually, this parameter is used not only to assess the number of

motor units recruited but also the firing rate of the active units.

For the IP Analysis as developed by Nandedkar, those thresholds were derived from the biceps

brachii of healthy humans at 10%, 25%, 50% and 80% of maximum force. Histograms of the

segments amplitudes overlayed with scatter plots of the values of duration versus amplitudes

were used to set the mentioned thresholds by trial and error.

As a result, segments are said to be active if duration is less than 5ms for amplitudes greater than

2mV, or less than 3ms for amplitudes between 0.5mV and 2mV, or less than 0.5ms for

amplitudes less than 0.5mV

The number of small segments (NSS) measures the number of segments with amplitude of less

than 2mV from the segments defined as active. NSS gives information about the low-amplitude

high-frequency components of the IPA and it is a good indicator of polyphasicity. A high value of

NSS would indicate absence of large amplitude MUP even if the UCA index detects one large

22

amplitude of MUP. Therefore, it is important to take into account UCA along with NSS [18].

The Upper Centile Amplitude (UCA) defines the upper limit of the peak-to-peak amplitude of

the motor unit action potential contained in the epoch. UCA is measured by the largest amplitude

change between successive turns after excluding the 1% largest values. As the force of

contraction increases, so does the amplitude of the largest spikes in the IP.

The EMG Envelope Amplitude (ENAMP) reflects the amplitude of the largest MUAP in the IP.

That value is obtained by measuring the difference between the peak that has the fifth most

positve amplitude from the peak that has the fifth most negative amplitude from an epoch of

500ms. Ignoring up to the 4th strongest peak allows avoiding solitary peaks of large MUAPs when

the contraction is close to its recruitment threshold. [19]

UCA and the ENAMP have a similar meaning. Along with activity those parameters increase in

value with the force of contraction [19].

These parameters relate directly or indirectly to MUAP features. Similarly, they can be used as a

diagnosis tool. It has been seen that a neuropathy results in an increase in the UCA parameter,

while the NSS stays normal or decreases. On the other hand, a myopathy results in increased

NSS, while UCA stays normal or decreases.

3.3.4. Amplitude measurement of electromyographic burst

There are different ways of depicting the general features of the inspiratory burst. Each one has

its tradeoffs.

Methods like root mean square or integration of MUPs can sometimes yield an estimate muscle

23

force level, but they are not immune to artifacts. The linear envelope using low-pass filters (5-

10Hz cuttoff) of the full-rectified signal can detect onset of the burst, but suffers from the same

problem. A more robust estimate of muscle force can be provided by applying the moving

average of the signal. The results such obtained correlate with the levels of contraction of the

muscle [15].

Data regarding a mean average analysis are typically expressed as mean EMG amplitude,

duration of contraction, mean EMG-spiking activity (ESA) and durations of activity or

contractions.

3.4.4. Power Spectral Analysis

The general observation that in patients with neuropathies the power spectrum is displaced

toward higher frequencies while in patients with myopathy, it gets tracked toward lower

frequencies suggests a power spectral analysis of the EMG signal a potential diagnostic tool. The

total power of the burst has been studied as an indication of activity and polyphasia [15].

However, the diagnostic capabilities of the power spectrum are still under scrutiny Kopec and

Hausmamowa-Petrusewicz have stated PS as a better diagnostic tool for myopathies. When

compared to individual MUP, it was found that PS analysis can detect myopathies better than

neuropathies [20]. Similar conclusions were reached by Fuglsan-Frederiksen [21]. As of now, the

PS stays a complementary technique and it should be used for redundancy.

Usual outcomes of the power spectral analysis are the mean frequency, and power at frequencies

140Hz, 1400Hz, 2800 and 4200Hz.

24

3.5.4. Summary of IPA parameters

The most common parameters obtained in analysis of the IP signal are listed in Table 3.

They have been organized according to the type of analysis that provides them.

ANALYSIS PARAMETER DESCRIPTION

ZERO-

CROSSINGS

Zero-crossings per

second

Number of voltage crossings of the baseline per

second

TURNS-

AMPLITUDE

ANALYSIS

(TAA)

Turns per second

(T/S)

Number of changes in polarization of more than

100uV per second

Amplitude per Turn

(A/T)

Average amplitude between two turns

Ratio (T/S:A/T) Ratio between T/S and A/T

Activity Time per second that IP signal is active

Number small

segments (NSS)

Number of segments with amplitude of less than

2mV from the segments defined as active

Upper Centile

Amplitude (UCA)

Largest amplitude change between successive

turns after excluding the 1% largest values

EMG Envelope

Amplitude (ENAMP)

Difference between the 5th most positive peak

amplitude from the peak with the 5th most

negative amplitude from an epoch of 500ms

BURST

ANALYSIS( by

moving average,

root mean

square or

integration)

Amplitude Average amplitude of the electrical burst

Duration Time length above a certain % of amplitude of

burst as defined by criterion

Burst rate Frequency between burst

POWER

SPECTRUM

Total Power

Mean frequency

Power at a certain

frequency

Usually at 140Hz and 1400Hz.

Table 3- Parameters obtained from analysis of the IP signal.

There is usually a zero-thresholding of around 25uV before performing Zero-crossings and TA

analysis to avoid fluctuations due to noise.

3.5. Electrophysiological parameters after nerve injury

Analysis of the EMG signal may provide information about injury and disease of the

musculoskeletal system and the nervous system. Neuropathies like Spinal Muscular Atrophy or

25

traumatic events like contusion of the spinal cord affect the population of lower motor neurons

and produce a characteristic electrical profile after injury. Diseases like primary lateral sclerosis

which affect the upper motor neuron population have a much different electrical profile.

In this document, I will focus on how a discrete insult to the nervous system can affect on the

EMG signal. The insult will cause partial axonal/motor neuron loss; surviving axons will be

present. Sequellae following injury include the process of regeneration, which has ongoing effects

on the shape of MUAPs as well as firing rates, power spectral profile and the burst activity.

Hereafter I will summarize some of those effects.

Partial denervation is first characterized by silencing of the muscle. Spontaneous activity appears

in the denervated fibers between one to three weeks after injury. The time depends on the length

of remaining nerve in the muscle. Electrical instability of the muscle membrane typical in a fiber

which has lost its innervation is the most probable cause. Positive waves and fibrillation

potentials are the most common instances of spontaneous activity.

It is believed that between 4 to 12 weeks is necessary for enough reinnervation to occur to track

changes in the MUP. Early findings during reinnervation include an increase in fiber density,

amplitude of the MUP, duration of the MUP and polyophasia [22].

During the reinnervation process, denervated muscle fibers are re-innervared by adjacent motor

units by axonal sprouting. This leads to increases in the amplitude of the MUP as well as duration

increases as well, since there are more fibers innervated, and thus, more long distance SFAP

contributing to the overall MUP., causing an. Long distance fibers have an effect in duration that

is not irrelevant. Moreover, inmature synapses have slow conduction velocity which leads to

dispersion of the signal and results in polyphasic MUPs. Fiber density also increases.

26

As the reinnervating process advances, synapses mature. Hence, polyphasia disappears and

duration of the MUP decreases. Amplitude in the MUP tends to decrease slightly although it will

remain above the ones characteristics of non-injured muscle.

In terms of firing patterns, the onset frequency and the recruitment frequencies of a muscle with

significant axonal loss are increased as compared to the non-injured muscle. Therefore, for a

given level of force, there are fewer MUPs firing at higher rates than in the normal muscle.

The power spectrum of the EMG signal of patients with neuropathies is displaced toward higher

frequencies.

3.6. Electrophysiological differences between rats and humans

In previous sections, I presented automated softwares commercially available to perform MUAP

Analysis and IP Analysis. All of them used thresholds for automatic identification of MUPs that

were obtained from normalized data of human MUPs and trial and error during implementation of

the code.

MUPs for animals have different characteristics than those for humans. Studies in the rectus

abdominis of humans have reported MUP amplitudes of 373.8uV and duration of 9.9ms [23]. The

equivalent in rats shows values of 193.4 uV for amplitude and 5.2ms for duration [183]. Despite

accepting the difference in function that might arise from difference usage in those muscles

between rats and humans, MUPs of rats are smaller in amplitude. The scaling from humans to rats

is approximately 2:1 for amplitudes. Therefore, the thresholding applied before the MUP

Analysis needs to be adjusted for rats. In Chapter 5, it will be presented the method and the

27

results achieved for automatic detection of rat MUPs.

3.7. Summary

Muscle denervation by traumatic injury or disease, reinnervation and myelination are processes

that affect different factors of the EMG signal. Diagnosis of injury, injury type and the stage of

reinnervation can be identified in the injured muscle by study of MUP features and the

parameters of IPA. Some of these parameters are especially sensitive to these processes, which

allow them to be used as discriminatory tools to set apart controls from animals treated with

therapies that enhance or speed up recovery of function in the muscle

Commercial softwares for automatic MUP analysis are available to study the EMG signal via

IPA, but these softwares are intended for clinical studies and have been developed for humans.

MUPs features differ across species. For rats, MUPs have lower amplitudes than for humans.

Therefore, commercial available softwares for automatic MUP or IP analysis need to be adapted

for animals if the EMG signal is to be used as assessment tool of regenerative treatments.

28

Chapter 4

EMG Software Development


This Chapter presents EMGvet, the software I developed to overcome the lack of a program for

automatic Interference Pattern Analysis (IPA) of the Electromyographic (EMG) signal in rats. I

first introduce the necessity for developing such software, then I give some of the technical

details about its implementation, especially in terms of the algorithm to remove the ECG signal

and finally, I present the parameters I obtained, which allow performing automatic analysis of the

Interference Pattern (IP) in rats. I also present the methods that lead to these new thresholding

parameters. In Chapter 6, EMGvet has been used to analyze the EMG signal to show the

regenerative capabilities of a stem cell therapy by functional improvement of the respiratory

intercostal muscles as compared to controls. As a conclusion, EMGvet is a useful tool for

researchers to study electromyography in animals.

4.2. Need for Software modifications

Raw measurements of the EMG signal were recorded using the Dantec Keypoint® portable

system (Natus Medical Incorporated, San Carlos, CA) with standard filter settings (5-10Hz).

Keypoint®allows quantitative EMG recordings, single fiber EMG and nerve conduction studies.

29

Both intra-needle EMG and surface EMG are available. Moreover, Keypoint® provides

proprietary software which performs such common analysis as Motor Unit Potential Analysis

(MUPA), Interference Pattern Analysis (IPA), insertional activity and spontaneous activity.

Keypoint software is intended as a diagnosis tool in clinics and medical centers, therefore, MUPA

Analysis and IPA analysis have been coded as described by [14]. The method of Stalberg is an

automated analysis to detect and extract MUPS features from human EMG signals.

The Keypoint software required adaptation to the EMGs of rats. MUPs of animals are quite

different from those of human as seen in section 4.6. Indeed, the analysis of our data showed that

Keypoint software missed many MUPs that were obvious to a trained observer. Lost MUPs

cannot be modified manually without adding too much input from the researcher which,

eventually, compromises the bias in the study. To improve resolution of rat EMGs therefore it

was necessary to write custom routines.

For this thesis, I have developed EMGvet, a software for electromyographic analysis customized

across species. Typical electrophysiological parameters of IPA as activity, turns/second,

amplitude/turn, upper centile amplitude (UCA) and the number of small segments (NSS) have

also been translated to comprise the particularities of animals. Moreover, EMGvet performs

moving and ensemble averages on the EMG burst to obtain amplitude and duration, onset times

and frequency of respiration. It also performs power spectral analysis.

A compilation of all the specifics of the software is out of the scope of this document, but general

considerations on some basic issues are presented hereafter. The following sections present some

of the solutions I adopted in the development of EMGvet that differ from available commercial

30

EMG software or that were necessary tools to achieve them. The IPA parameters provided by

EMGvet are listed in Table 4.

ANALYSIS PARAMETER

ZERO-CROSSINGS Zero-crossings per second

TURNS-AMPLITUDE

ANALYSIS (TAA)

Turns per second (T/S)

Amplitude per Turn (A/T)

Ratio (T/S:A/T)

Activity

Number small segments (NSS)

EMG Envelope Amplitude (ENAMP)

BURST ANALYSIS( by moving

average)

Amplitude

Duration

Burst rate

POWER SPECTRUM Total Power

Mean frequency

Power at a 150,hz, 1500Hz, 3000Hz, and 6000Hz

Table 4-IPA parameters provided by EMGvet software.

4.3. Electrocardiogram removal

Intercostal recordings were taken from the right side of the ribcage to minimize the interference

of the electrocardiogram signal (ECG). Even so, ECG interference is still very strong and present

in the respiration bursts, altering the parameters under study. The first requirement to any analysis

on the EMG signal is the effective removal of the ECG signal.

The ECG signal shares with the respiration burst a similar power distribution in the frequency

domain. Bruce (p.281) [24] suggests a 2nd

Butterworth high-pass filter with a cutoff of 70Hz to

remove it. In our studies, the distortion of the respiration signal of interest thus filtered is too high

to be acceptable.

Similarities of both signals in the temporal domain makes difficult to set them apart using

temporal techniques. The ECG signal may have stronger, smaller or a similar amplitude that the

31

signal of interested. Therefore, there is no option to use that as a discriminative feature to set both

signals apart. It was not taken a reference signal of just the ECG signal to allow adaptive line

enhancement (ALE) filtering.

Independent component analysis (ICA) could not be performed since principal component

analysis (PCA) did not find two independent signals. Motor Unit Potentials and ECG have such a

similar shape that confounds most of the shape tracking approaches, including wavelet analysis

using the Ricker wavelet that other groups have used.

The final algorithm used in EMGvet is a template tracking algorithm. The ECG-template is

constructed from an average of ECG signals identified within the regions with absence of

inspiratory activity.

The ECG-removal-function starts with conditioning of the signal by filtering with a Butterworth

band-pass filter of 4th order, with cutoff frequencies at 30Hz and 500 Hz. Mean of the signal is

removed from the signal prior to rectification to reduce the noise. Then, moving average filtering

is applied to the rectified signal using a triangle window of 41ms.

A digital signal constituted by respiratory pulses (EMG and ECG interference) and ECG-only

pulses is obtained by static amplitude thresholding. Pulses are identified as ECG-only or

respiratory based on a static thresholding in duration. Both thresholds were obtained empirically.

A synthetic ECG is constructed taking five different templates of ECG-only pulses and averaging

them after aligning their peaks. The period of the ECG is identified and the algorithm can predict

the approximate position of the ECG within the respiratory burst. The exact position is identified

by auto-correlation, then, the template of ECG is removed from the inspiratory burst.

32

A B

Figure 5- EMG signal of the IIIrd intercostal muscle before (A) and after (B) being processed by

the cleansing algorythm. In (A) it was also included the digitalized signal, which is green for only

EKG, magenta for respiratory burst, and red for smoothed signal.

4.4. Automatic zero-crossings tracking

The number of zero-crossings through the zero line is used as indication of activity on the

muscle. To avoid background noise, it is established a threshold level so only crossings between

those two thresholds are counted. The usual value in clinical studies establishes 25uV as the

threshold. EMGvet uses a lower threshold of 15uV to adapt it to rat IPA.

4.5. Turns/amplitude analysis (TAA)

EMGvet uses 30uV of difference between turns. Figure shows an inspiratory burst and the

identified turns as asterisks.

33

Figure 6-Turns are depicted as black asterisk. Maximum are depicted in red dots for maximum

and green for minimum.in a time series (vector)

Thresholds that determine activity are very dependent on MUP features as well: As it was shown

in section 1.4, MUP features for animals, and rats in concrete, are different than in humans.

Therefore, in order to analize rat EMG signals using IP analysis, EMGvet changed the thresholds

obtained by Nandekahr for humans [25],[26], [19]. In order to do so, EMGvet uses the same

procedure of plotting histograms of segment amplitudes overlayed with the scatter plots of

amplitude of segments versus their duration. However, Nandekarh was able to gradually increase

EMG activity by asking their subjects to produce gradually greater amounts of force and set their

thresholds for different amounts of activity. Rats cannot be directed to do so. Instead, it was used

the signal of the intercostal muscles at different time points after injury to display different

amounts of activity and set thresholds in those different situations. Figure 7, Figure 8 and Figure

9 show the histograms for two animals along with the thresholds of the activity criteria.

As shown in Figure 7, after injury the MUPS are very small and there are not many of them and

activity is low.

34

A B

Figure 7- Histograms of the segment amplitudes (blue) are shown, overlayed with scatter plots

of the values of the duration versus the amplitude of the individual segments (red), and

threshold values for activity (black) for two different animals (A and B) 1 week after injury.

Figure 8 depicts the situation four weeks post injury. MUPS have larger amplitudes since

remaining MUPS are reinervating the denervated nearby muscles and more activity is observed.

A B



threshold values for activity (black) for two different animals (A and B) 4 weeks after injury.

Five weeks post-injury, MUPS decrease slightly in amplitude and diminish in duration as new

fibers get remyelinated as it is shown in Figure 9.

35

A B



threshold values for activity (black) for two different animals (A and B) 5 weeks after injury

As a result, segments in EMGvet are said to be active if duration is less than 10ms for amplitudes

greater than 100uV, or less than 7ms for amplitudes between 30uV and 100uV, or less than 3ms

for amplitudes less than 30uV. Based on these thresholding, the activity of a typical inspiratory

burst is presented in Figure 10.


presented in red while the general inspiratory burst in blue.

In EMGvet, the number of small segments (NSS) measures the number of segments with

amplitude of less than 100uV from the segments defined as active,

Upper Centile Amplitude (UCA) and EMG Envelope Amplitude (ENAMP) have been defined

36

similarly than in traditional. Figure shows the effects that it has in a burst of inspiratory activity.


presented in red while the general inspiratory burst in blue

UCA and the ENAMP have a similar meaning. Along with activity those parameters increase in

value with the force of contraction [27].

4.6. Analysis of the electromyographic burst

Duration and mean amplitude of the inspiratory burst, onset time and breathing rate are features

that can be studied by using integrating techniques of the EMG signal.

EMGvet adopts a moving average technique. The moving average is a good method to

determine levels of contraction. EMGvet performs the moving average on the EMG once been

cleaned from ECG artifact. Before doing the sliding-average, the signal is full-rectified and the

mean is extracted to reduce the noise. This concrete implementation uses a rectangular window of

41.6ms. There is a delay in the onset of burst by the same amount but it is acceptable.

Digitalization of the clean signal is done similarly to the ECG-removal function but using a

dynamic amplitude thresholding of 6% of the mean of the signal. Then, the ensemble average is

37

done on four inspiratory bursts to increase smoothing without adding delay.

Moreover, to diminish the fact that different insertions yield fairly different EMG amplitude

(depending on proximity of electrode to different motor unit pools in each insertion), it was

averaged the result of the three insertions. Before averaging, it was assessed the quality of the

signal, to prevent aberrant measures to be included. Finally, it is obtained the mean of the burst

ensemble and the duration of the burst ensemble.

A B

Figure 12- (A) raw and digitized EMG signals of the IIIrd intercostal muscle for several

inspirations before processing. Digitalized signal was also included, green for only EKG,

magenta for respiratory burst, and red for smoothed signal. (B) EMG during a single

inspiration after processing by the cleansing algorithm.

4.7. Summary

In order to discriminate between spontaneous recovery and effective treatment, this method

requires of appropriate software to perform Interference Pattern Analysis. Aptitude to the task is

defined by how accurately the automated software identifies IPA features. Commercial softwares

do not evaluate properly the IP of animals because they use thresholds for humans to track rat

MUPs. I developed EMGvet to address this necessity.

A summary of the new criteria set in EMGvet which allows automatically identify rat MUPs and

38

properly quantify the principal parameters of IPA is listed in Table 5. These values have been

compared to the commercial available software Keypoint.

PARAMETER Keypoint CRITERIA EMGvet CRITERIA

Zero thresholding 25uV 15uV

Zero-crossings 30uV 15uV

Turns 100uV 30uV

Activity d<5ms & amp> 2mV

d<3ms & 0.5mV<amp<2mV

d<0.5ms & amp<0.5mV

d<10ms & amp> 100uV

d<7ms & 30uV<amp<100uV

d<3ms amp<30uV

Number small segments d<3ms & 0.5mV<amp<2mV

d<0.5ms amp<0.5mV

d<7ms & 30uV<amp<100uV

d<3ms amp<30u

Moving Average

window

Information not available 41.6 ms

Moving Average

threshold for duration

Information not available 6% of total amplitude

Power at a certain

frequency

140Hz, 1400Hz, 2400Hz,

4800Hz

150Hz, 1500Hz, 3000Hz,

6000Hz

Table 5- Comparison of criteria for IPA parameters between Keypoint and EMGvet, where d is

duration of segments and amp is amplitude of segments.

Aptitude to the task requires also a system with enough resolution to set apart treated from control

groups, if there is indeed a functional improvement in the former group. Several signal processing

techniques are available to increase resolution in EMG analysis, mostly involving the removal of

interference and noise in the signal. EMGvet has been designed with stress in resolution and

different techniques like averaging across time (embedding) and recordings were used to achieve

that goal. Especial attention was paid to filtering and ECG removal.

As a summary, EMGvet is a necessary analysis for researchers studying the EMG signal of

animals. Animal studies using commercial available softwares might be erroneously tracking

MUPs and/or investing much time in manual correction. EMGvet will soon be provided as Open

Access software to the scientific community increasing the accuracy and efficiency in those

studies.

39

Chapter 5

Rat Model of Injury


Within the context of developing a model of injury and a technique to track functional

improvement of regenerative therapies for spinal cord injury, I decided on the electromyographic

signal (EMG) of the intercostal muscles as the most suitable method. Chapter 3 and Chapter 4

focused in the technique. I explained the usefulness of the EMG as a measurement of functional

improvement and the EMGvet software which was developed to analyze it with accuracy. In

Chapter 2, I presented the advantages of targeting the respiratory system as a model of injury.

This chapter describes the model of injury itself. First, I provide information on the anatomical

and functional background of the intercostal muscles as the rationale for the particulars of this

model. Later, I describe the level of injury, muscle of choice, location of recordings and

experimental protocol.

5.2. Physiology of the respiratory system

The main respiratory muscles are the diaphragm, intercostal muscles and abdominals. The

respiratory dynamics are mediated primarily by diaphragm contraction, which expands the

ribcage, diminishing the pressure inside of the lungs with respect to atmospheric pressure. As a

40

consequence, it forces entrance of air through the trachea into the lungs. Inspiration is facilitated

by the synchronized action of the external and internal intercostal muscles. Expiration is caused

by relaxation of the diaphragm which causes elastic retraction of the lung and tissues around the

thoracic cavity. Forced expiration, as it will be explained hereafter, is caused by interosseus

internal intercostals and assisted by other muscles in the thoracic and abdominal area. Reviewed

from [3].

Phrenic motor neurons located in the cervical spinal cord innervate the diaphragm, respiratory

intercostal muscles are controlled from the thoracic spinal cord and abdominal muscles are

innervated from the thoracolumbar spinal cord. The ventro-lateral medulla, modulates the activity

of these and other accessory muscle which facilitate respiration

The airways receive parasympathetic and sympathetic innervation too. Parasympathetic

neurons in the nucleus ambiguous innervate the trachea and bronchii, reducing airway

diameter upon activation. Their activation is excitatory via muscarinic cholinergic receptors.

On the other hand, sympathetic neurons at the 4th thoracic level (in rats and humans) project

to the paraventral ganglia activate their b-adrenergic receptors and as a result reduce

bronchial diameter they control.

The airways also have afferent innervation mostly through vagal mechanoreceptors, whose

cell bodies are in the nodose ganglia and whose central axons project to the nucleus of the

solitary tract in the brainstem. That link synchronizes respiration with the necessities of blood

flow.

41

5.3. Anatomy of intercostal muscles

Reviewed from The intercostal muscles are two layers of muscles laying in between pairs of ribs

and assisting the diaphragm to alter the ribcage dimensions in respiration. External intercostal

muscles attach at the tubercles of the ribs next to the vertebrae and extend dorsally up to the

costodronchal junction. Internal intercostal muscles extend dorsally from the sternocostal junction

in the sternum.

The two layers of muscles only super-impose laterally. Between the costodronchal junction and

the sternum, the external intercostal layer is replaced by the anterior intercostal membrane. Thus,

only the intercartilaginous internal intercostals are present in the ventral side of the thoracic cage.

This ventral portion of the internal intercostal muscles is also referred as paraesternals. Similarly,

internal intercostals do not reach the most dorsal part of the thoracic cage, close to the vertebrae.

External intercostal muscles at that point are mirrored by the levator costal. The parasternal

intercostal muscles are covered by the transversus thoracis. A deeper layer of internal intercostals

are the innermost intercostals which move together with the internal intercostals.

The muscle fibers of the internal and external intercostal muscles are skewed and run in opposite

directions. While the internal intercostal bundles run from rostral to caudal and dorsal to ventral,

the external intercostal muscles run from caudal to rostral and dorsal to ventral. The fibers of the

transversus thoracis are also set perpendicular to the internal intercostals.

Mechanically, because the bundles are skewed, the distance from the lower and upper insertion of

the muscle into the rib to the center of rotation of the rib is different, which translates into a

different torque magnitude for each. For the external intercostal muscles, the net torque produces

the ribs to upper and the lungs to inflate. The torque acting on the internal intercostal muscles is

42

smaller in the lower rib than in the upper one and the net effect is to lower the ribs and deflate the

lung. This generalization will be further extended on the following sections. This is a

generalization, which will be further extended in section.

5.4. Respiratory function of intercostal muscles

The traditional view sets that inspiration is produced mainly by contraction of the diaphragm and

the external intercostals, which elevates the ribs and the sternum, thus, expanding the transverse

dimensions of the thoracic cavity. During expiration, the diaphragm would relax, which would

decrease the dimensions of the thoracic cavity, driving the air out of the lungs. In forced

expiration, the internal intercostal muscles would contract to force the ribs down and drive air out

of the lungs. This is an oversimplification of the respiration process. It has been seen that specific

locations of both external and internal intercostals might have a role in inspiration and expiration.

It is suggested that its specific role depends on their mechanical advantage, even more, that the

electrical activity of the muscle is set according to its mechanical advantage [28].

In a canine model it was observed that contraction of the parasternal intercostal muscles by

electrical stimulation lifts the ribs up and increases lung volume, asserting the inspiratory role of

internal intercostals of specific locations in the dog [29]. Moreover, the inspiratory role of the

canine internal intercostal muscles decreases laterally and caudally at some point reversing its

role into expiratory [30], [31]. Therefore, the parasternal intercostal muscles at the 1st and 2

nd

interspaces have the strongest inspiratory function. In terms of nomenclature, throughout this

document the interscostal space between the 1st and 2

nd rib will be called 1

st intercostal , between

ribs 2nd

and 3rd

2nd

intercostal and the same for the 12th intercostal spaces of the rat.

The same trend has been seen in anesthetized or decerebrated cats, like any young cat usually is

43

[32]. Equivalently, the inspiratory effect of external intercostals decreases caudally and towards

the costochondral junction, up to the point of getting reversed into an expiratory function in the

lower and more ventral parts of the ribcage. Thus, both the external and parasternal intercostal

muscles in the upper spaces are active during inspiration and, in the lower spaces external and

internal intercostals tend to have an expiratory role.

Again, the same trend has been observed in humans [33]. A higher electrical activity in the upper

intercostal muscles has been measured not only qualitatively. The phasic inspiratory duration as a

percentage of total respiration time and the average discharging frequency of motor neurons (in

six subjects both decrease with the intercostal space in a rostrocaudal manner. All measurements

were taken close to the sternum

Troyer et al analized the mechanical advantage of different portions of the ribcage using an

extension of the mechanical model of Hamberger [34], and correlated those results with the role

in respiration of the internal and external intercostal muscles. Indeed, the topographic distribution

of mechanical advantage correlates with the topographic distribution of the respiratory effect,

both inspiratory and expiratory.

The net moment exerted by the muscle depends on the angular position around the rib, which for

the external intercostal muscle it is the strongest dorsally and is reversed in the ventral region.

The transition from expiratory to inspiratory effect depending on the position can be seen also for

internal intercostals [28].

In the dog, it has been seen that bundle of the para-sternal intercostal close to the sternum, which

have the greatest inspiratory mechanical advantage due to the angle of the bundle, contract the

most during inspiration than lateral bundles.

44

Histochemical study of the properties of intercostal muscles in terms of fiber type shows that

intercostal muscles routinely recruited in inspiration have significantly higher proportion of slow-

oxidative muscle fibers in the cat [35]. In the hamster parasternal intercostal muscles, it has been

observed a decrease in the slow oxidative fiber percentage and increase in the fast twitch fiber

percentage from rostral to caudal, which is in the same direction as inspiratory function decreases

[36].

5.5. Innervation of intercostal muscles

Trunk muscles in general are supplied by each spinal nerve deriving from its respective somite. In

concrete, innervation of intercostal muscles is segmentally distributed in the spinal cord [37],[38].

However, in humans it was found that intercostal nerves have communicating branches. There are

some conflicting results about the extent of innervation within the spinal cord of the motor

neurons for a concrete intercostal muscle Reviewed from [39].

In cats, it has been observed that segmented parts of the serratus dorsalis are supplied by a single

or double spinal segments, while intercostal muscles in adult rats and cats are supplied by each

segmental level. In neonatal rats, retrograde labeling located the soma of motors neurons

innervating external intercostal muscles within one or two segments [40] while electrophysiology

showed a strongest response to the stimulation of just one segmental level.

Therefore, injury of a segmental level of the spinal cord mostly denervates the corresponding

intercostal space, although it could have some effect in the innervation of adjacent intercostal

spaces. When the effect in intercostal innervation of spinal cord injury in an animal model,

special attention should be given to the fact that spinal cord segments are slightly decalated

45

rostrally with respect to their vertebral bodies.

5.6. Injury location and type

Under the light of the previous information it was decided as a suitable injury model a bilateral

contusion injury at the 3rd

thoracic level (T3). A thoracic injury affects the respiratory system and

yet the stress and surviving ratios for the animals are higher than in cervical injuries.

Regarding the intercostals, injury at the T3 level mostly denervates the 3rd

thoracic intercostals,

although it has also some effect in the 2nd

and 4th intercostals and it affects indirectly all the

intercostals in downstream pathways as well. Nevertheless, not being a complete injury like

transection, spares part of the function of the diaphragm and rest of intercostals. A contusion

injury also translates better than other models of injury, like hemisection, to the real scenario of

spinal cord injury in humans. According to the last etiology studies, contusion are the most

common type of spinal cord injury [1].

Electrophysiology of the intercostal muscles has been used to assess the functional improvement

that a potential beneficial treatment evokes on denervated intercostal muscles. Both internal and

external intercostals are affected by this type of injury. The parasternal intercostals are the muscle

of choice as site of recording.

Para-sternal intercostals allow for a minimally invasive setup of electromyographic recordings. In

a supine position, they offer easy access. Exposure of the parasternal intercostal only requires a

minimal incision of the ventral skin and tearing apart of the major pectoralis fibers. Altogether, it

has a minimal effect in pain and locomotion of the rat. Besides, it avoids the dorsal area where

the laminectomies were performed which would decrease the healing rate of those injuries and

46

increase distress in the animals. Moreover, by placing the rat in a supine position, there is no

restriction on its breathing due to body weight.

While more rostral intercostal muscles have stronger electromyographical signals, lesions at

higher levels in the spinal cord impose stronger disabilities in the animals. There is a trade-off to

reach. Ventral branches of the 1st and 2

nd thoracic nerves are part of the brachial plexus

controlling the forelimbs [41] and should be avoided to minimize the impact on the locomotor

system. At the T4 level there is an anatomical landmark in the form of an artery, which results in

death of the animal when torn. For practical reasons and to ensure survival, it is better to avoid

laminectomies at T4 vertebral body.

As a results of all those considerations, it has been decided T3 as the site of injury. It is important

to note that the function of internal intercostal muscles at the 3rd

intercostal space is solely

inspiratory and increases ventrally. Therefore, recordings were taken 5mm away from the

sternum at the midpoint between ribs.

5.7. Experimental Protocol

Twenty Sprague-Dawley female rats (200-275g of 6-8 weeks of age) were anesthetized by

injection of Xylazine (10 mg/kg, i.p.; Western Medical Supply, Los Angeles, CA) and Ketamine

(100 mg/kg). Animal losses are typically 10%. Because the laminectomy procedure for a T3

injury can result in severe blood loss due to critical arteries and veins being present within the

surgical area, we hypothesized that there would be more than 15% animal loss. Animals were

tagged at the time of surgery by tail markings as well as attachment of a clip with the animal

number to the ear. The dorsal area was shaved and disinfected with serial provodone and 70%

ethanol scrubs. A midline incision exposed the spinal column at the level of T1–T5, and the

47

paravertebral muscles were dissected bilaterally to visualize the transverse apophyses.

Laminectomy was performed at T3 and animals were subjected to a bilateral contusion injury

using the Infinite Horizons Device (Precision Systems, Kentucky, IL) with a 220Kdyne weight.

After injury, animals are kept in a heating pad for 12 hours and receive a solution of 5ml ringers,

0.02 ml Baytril (2.5 mg/kg/d; s.c.; Bayer, Shawnee Mission, KS), and 0.4ml of dilute

buprenorphine (0.025 mg/kg/d, s.c.; Western Medical Supply, Los Angeles, CA) subcutaneously

during 2 days post injury. Animals are bladder expressed twice a day and monitored for health

problems for a minimum of 3 days or as needed.

Five days after injury animals are divided into two groups of ten subjects comparable in terms of

injury behavioral outcome according to the Basso, Beattie and Bresnahan Locomotor Rating

Scale procedure, which was described in Chapter 2. One week post-injury, one of these groups is

exposed to the treatment whose effectiveness wants to be assessed while the other group is treated

to become their appropriate controls, whether that implies injecting a vehicle solution, implanting

an inert bioscaffold, etc...

EMG recordings from the 2nd

and 3rd

intercostal spaces were taken before injury to act as

baselines, one week post-injury before transplant, four weeks post-injury (three weeks after

transplant) and five weeks post-injury ( four weeks after transplant). Special care was taken while

recording to minimize the effect of noise and interference. Proper grounding and a Faraday cage

effectively removed most of the noise and the 60Hz interference.

Animals are placed in a supine position within the Faraday cage with the limbs immobilized to

avoid disturbance during the procedure. The animals need to be anesthetized but Xylazine should

be avoided in this protocol because it is a muscle relaxant. Ketamine is a sedative and it is not

48

acceptable for use as the primary anesthetic method in surgeries that require incision. Isofluorane

is a general anesthetic which potentiates the effect of muscle relaxants but its use at 2% was

found to have a slight muscle relaxant effect itself. The final approach was a combination of 60

mg/kg Ketamine and isoflurane at a lower dose of 1.0%.

Electrophysiological recordings are taken with a 30 gauge concentric needle with a standard

recoding surface (0.07mm2), placed 5 mm away from the sternum, since the electrical activity

decreases dorsally. At the same time, recordings were taken at the midpoint in between ribs and

approximately 3mm inside the muscle, ensuring by observation of the electromyographic signal

that the muscle was active during inspiration and that the electrode had not gone too far and

reached the transversus thoracis instead.

Recordings were taken from the right intercostal to minimize the effect of the electrocardiogram

(ECG) signal. A reference electrode was placed near the left side to minimize that presence

although signal filtering has been applied by software to eliminate the remainders

Three electrode insertions were performed approximately at the same spot, per animal per

session, and at least 20 seconds of recordings were taken from each one of them. Although these

recordings are minimally invasive, animals receive an injection of 1 ml of the analgesic solution

(5ml ringers, 0.02 ml Baytril and 0.4ml of dilute buprenorphine).

5.8. Summary

One of the goals of this thesis is the development of an injury model that targets denervation of

the respiratory system and assess efficacy of regenerating therapies with a functional test, being

49

EMG the specific functional technique here applied. In this Chapter, I have proposed the contused

spinal cord at the thoracic level 3 as a good model of injury to mimic denervation of the accessory

muscles of respiration due to injury or disease. I have also presented the recovery of the

intercostal muscle functionally as a good indicative of neuronal regeneration. At the same time,

not affecting the phrenic nerve or producing much locomotion or homeostasis deficits like

cervical injuries entail, the survival ratios of the animals and all the practical considerations

relating maintenance and recurrent EMG measurements are facilitated.

50

Chapter 6

Results on a stem cell repair therapy


In previous chapters, I presented my work on developing a rat-injury model and

electromyography (EMG) program to test efficacy of cures in conditions affecting the motor

neuron population. In this chapter, I validate the model and the technique by assessing the

efficacy of a stem cell therapy for the treatment of Spinal Muscular Atrophy (SMA) type I and

identify the parameters with enough resolution to use as a discriminatory tool between groups.

The MotorGraft (Motor Neuron Progenitors) transplantation into the injured spinal cord of rats

produced accelerated regeneration, as I documented using electrophysiological methods.

Histological and behavioral tests are also presented to correlate with the functional outcomes of

EMG. The results confirm that EMG is a useful tool to identify beneficial therapies where

behavioral tests lack resolution or target the wrong system and yet there is functional efficacy. I

performed the work here presented in the Hans Keirstead laboratory at University of California-

Irvine in collaboration with California Stem Cell, Inc.

This study represents one of the first successful uses of stem cells in restoring respiratory neurons

51

and the work here presented is part of the application to the Investigational New Drug (IND)

program of the Food and Drug Administration (FDA), as a preliminary requirement to enter

Phase 1 clinical trials for SMA Type I. If the application is cleared, it will become the second

clinical trial in the stem cell therapy field.

6.2. Background

Spinal Muscular Atrophy (SMA) type I is a genetic disease characterized by degeneration of

certain pools of motor neurons, including the anterior horn motor neurons of the spinal cord.

Symptoms presented by children with SMA type I are hypotonia and a progressive weakness of

voluntary muscles. The diagnosis of SMA Type I is usually made before 3 months of age. A

common scenario with life-threatening implications is the presence of abnormal respiration

patterns and respiratory insufficiency due to weakness of the chest muscles, including the

intercostal muscles.

Transplanted stem cells can influence the injured environment by acting as survival factors,

guidance molecules, or cues for proliferation and differentiation of endogenous stem and

progenitor cells [42] [43, 44] [45, 46]. Motorgraft© is a population of high purity motor neuron

progenitors derived from hESCs by California Stem Cell, Inc. which has been shown to produce

growth factors in vitro [47]. In this study, Motorgraft©, has been transplanted into rat thoracic

spinal cord according to the model presented in chapter 3 with the aim of sparing the motor

neurons that control the intercostal muscles. Two action mechanisms are considered: secretion of

growth factors to delay loss of motor neurons and replacement of dying motor neurons.

52

6.3. Methods

6.1.3. Spinal cord injury:

Injury was performed according to the experimental protocol described in Chapter 3.

6.2.3. Basso, Beattie, and Bresnahan (BBB) Locomotor Rating Scale Procedure:

Five days following spinal cord injuries, all animals were tested according to the BBB test in

order to place them in groups with similar injury profiles. The groups have been listed in Table 6.

Following transplantation, BBB was performed on animals from groups I and II once a week for

the duration of the study. Rats were scored independently by two observers in a blinded fashion.

Groups were compared at the end of the study using a mixed factorial ANOVA with differences

(p>0.05) used for assessment of statistical significance.

Group Condition Outcome Measures Sacrifice

points

n

I MotorGraft

Transplant

BBB, EMG, Tissue sparing analysis, 5HT

sprouting, growth factor

immunohistochemistry

1 month post

transplant 18

II Vehicle

Control

BBB, EMG, Tissue sparing analysis, 5HT

sprouting, growth factor

immunohistochemistry

1 month post

transplant 18

III MotorGraft

Transplant RNA extraction for PCR

14 days post

transplant 5

IV Vehicle

Control RNA extraction for PCR

14 days post

transplant 5

V MotorGraft

Transplant Protein extraction for ELISA

1 month post

transplant 10

VI Vehicle

Control Protein extraction for ELISA

1 month post

transplant 10

Total 66

Table 6- Animal groups, condition,outcome measuremes, sacrifice points and number of

subjects (n)

The result from groups III and IV for gene expression and groups V and VI for growth factor

expression analysis have not been included in this report.

53

6.3.3. Immunosuppression and Cell Transplantation:

MotorGraft was manufactured by California Stem Cell, Inc., using protocols similar to those

described in [47] and implanted 1 week post injury. Twenty four hours prior to transplantation,

animals began daily immunosuppression with Cyclosporin A (20 mg/kg/d, s.c.; Bedford

Laboratories, Bedford, OH). Animals also received antibiotic treatment of Baytril (2.5 mg/kg/d;

s.c.; Bayer, Shawnee Mission, KS) beginning one day prior to transplantation, and continued as

necessary thereafter.

Animals were anesthetized as above and the laminectomy site re-exposed. A total of 100,000

MotorGraft cells or Cell Transplant Solution (CTS) (vehicle control) was injected in a total of

four sites, 2 bilateral injections rostral and caudal to the injury site (0.5mm lateral, 1.2mm deep)

to target the ventral horn. Each injection was a total volume of 1 µL, at a dose of 25,000 cells/

µL.

6.4.3. Electromyography measurements:

Electromyography was performed in groups I and II according to the Experimental Protocol

described in Chapter 3.

6.5.3. Tissue Extraction:

Spinal cord injury epicenter segments of animals in groups III and IV were fresh extracted 14

days post transplantation. The spinal cord injury epicenter segments of animals in groups III and

IV were also fresh extracted 1 month post transplantation to extract protein for analysis with

ELISA. Animals in groups I and II were sacrificed by trans-cardiac perfusion with 4%

paraformaldehyde. The entire spinal cord was extracted and cryopreserved for sectioning. The

54

spinal cord was cut into 1 mm transverse segments and embedded into OCT compound. Sections

of the embedded spinal cord were then cut with a cryostat into 20µm sections and placed onto

slides. Ten slides from each animal were then used for 5-HT staining and anti-human nuclei

staining, NeuN and anti-human nuclei staining, and ChAT and anti-human nuclei staining.

6.6.3. MotorGraft Cell Survival:

Animals in groups I and II were used for MotorGraft cell survival analysis. Animals in group II

served as a negative control for the presence of human cells. Transverse sections of the spinal

cord were analyzed for presence of human cells using an antibody against human nuclei (1:200;

Abcam, Cambridge, MA). MotorGraft cells, as determined by human nuclei staining, were

expected to be located near the injection site.

6.7.3. Immunohistochemistry (IHC):

The spinal cords from animals in groups I and II were used for analysis of MotorGraft

engraftment, neuronal sparing, motor neuron sparing and serotonergic sprouting. Rat spinal cords

were cryosectioned into 20 m sections, and placed onto slides. Five slides per animal, spaced

200 m apart, from transplanted and non-transplanted groups were used for each analysis.

IHC staining was performed on slides using mouse anti-NeuN (neuronal nuclear protein, 1:200;

Millipore, Billerica, MA) primary antibody and rabbit anti-Ku80 (human nuclear protein, 1:200;

Abcam, Cambridge, MA) primary antibody for neuronal sparing analysis. For motor neuron

sparing analysis, IHC staining was performed on slides using mouse anti-ChAT (choline

acetyltransferase, 1:200; Millipore, Billerica, MA) primary antibody and rabbit anti-Ku80 (human

nuclear protein, 1:200; Abcam, Cambridge, MA) primary antibody. For serotonergic sprouting

analysis, IHC staining was performed on slides using mouse anti-5HT (serotonin, 1:200; Abcam,

55

Cambridge, MA) primary antibody and rabbit anti-Ku80 (human nuclear protein, 1:200; Abcam,

Cambridge, MA) primary antibody. Hoechst was used as a nuclear counter-stain. Following IHC

staining, the tissue was imaged using an Olympus AX-80 microscope. Images were taken at 200x

magnification of the dorsal and ventral horns using the MicroFire software. The transverse

section of the spinal cord was broken down into four quadrants; left and right dorsal horns, and

left and right ventral horns. These images represented more than 30% of the total grey matter.

Analysis of human nuclear staining was performed on all stained tissue sections. The presence of

human nuclear staining was recorded for each section, allowing for the identification of

transplanted animals in which human nuclei were and were not detected. The results are reported

as the number of transplanted animals with detectable MotorGraft cells.

Analysis of endogenous motor neuron sparing was conducted from images from the ventral

and dorsal horns, and represent sparing up to 2mm rostral and caudal of the injury epicenter. The

number of motor neurons from the quadrants (images) was quantified using ImageJ software

(U.S. National Institute of Health; Bethesda, Maryland). It should be noted that the sections

quantified did not contain human positive cells. If a cell double stained positive for both ChAT

and Ku80 then that area of tissue was not used in quantification. At least 3 of the 5 slides for the

animal had to be quantifiable to be included in the analysis. The counts for the slides for each

animal were averaged. Statistical analysis was performed using a Student’s T-test (2 tailed

distribution and two sample equal variance).

Analysis of serotonergic sprouting was conducted from images from the ventral and dorsal

horns, which were taken at the same exposure, imported into ImageJ software (U.S. National

Institute of Health; Bethesda, Maryland), and inverted to grayscale images with the same

threshold applied to all images. The density of the staining was determined by multiplying the

56

grey value in pixels by the area measured. The results interpret serotonergic sprouting 1mm

rostral and 1mm caudal to the injury epicenter. Transplanted and nontransplanted groups were

averaged, and statistical analysis was performed using a student’s t-test (2 tailed distribution and

two sample equal variance).

6.8.3. SCI Morphometry:

Animals in groups I and II were used for morphometric analysis of the injury site. Sections that

have been used for other histological analyses (MotorGraft cell survival and IHC) were used to

examine the gross morphometry of the spinal cord at the injury epicenter and surrounding tissues.

The area of intact tissue, including that of pathologic tissue, was traced on the images using the

Olympus MicroSuite B3SV software to determine the area and perimeter values.

6.4. Electrophysiological results

Histology analysis of the transplanted and vehicle groups proved a regenerative and protective

effect of the Motorgraft transplant in the host. Genetic assays also show up-regulation of neuro-

protective and anti-inflammatory genes, while protein assays show significant higher levels of

neuron growth factors in transplanted animals as compared to vehicles (Data shown in following

sections).

Different parameters of the Interference Pattern Analysis were scrutinized looking for resolution

that allowed using them as a discriminatory tool in identifying beneficial treatments which

correlate with histological improvement. I included in the scrutiny parameters obtained from

Zero-Crossing Analysis, Turns-Amplitude Analysis (TAA), Power Spectral Analysis (PSA) and

Burst Analysis.

57

Although results obtained by these analyses support their respective findings, the only parameters

that were able to show significant differences between groups were Zero-Crossing and Turns per

second (T/S). I conclude that Electromyography studies should focus in these parameters as their

main functional outcomes.

The number of turns per second (T/S), one of the parameters obtained from a Turns-Amplitude

Analysis (TAA), is known to increase as there is an increase in the percentage of polyphasic

potentials [48]. Polyphasic potentials are abnormal electrical configurations of a motor unit that is

typically observed after nerve injury and/or neuropathology [49]. An increase in turns per second

was observed in both MotorGraft and vehicle control animals 3 or 4 weeks after transplant, which

is to be expected as all animals received spinal cord injuries prior to transplantation (Figure 13).

A B

Figure 13- IP analysis of EMG signal from intercostal muscles 2 (a) and 3 (b) presented as turns

per second. Data expressed as mean and standard error. Student t-test with *p<0.05, **p<0.005.

The timing of these polyphasic potentials is consistent with the literature that states that the

earliest manifestation of axonal denervation can be detected 1-4 weeks after nerve injury [49].

Turns per second in the vehicle control group increased with time at both the second (Figure 13a)

and third (Figure 13b) intercostal muscles, and these exceeded that of the MotorGraft transplanted

58

group at 5 weeks post transplantation (p<0.05). These data suggest that MotorGraft

transplantation decreases polyphasia, or abnormal electrical configurations in a state of motor

neuron loss.

6.5. Behavioral results

The BBB locomotor scale is often used to group animals into treatment groups following SCI [50,

51]. Thus, the BBB scores 5 days post injury were used for placement of animals into groups

such that all groups had similar BBB scores at time of treatment. As shown in Figure 14 BBB

locomotor scores increased over the course of the study to 11 and 12 for the MotorGraft and

vehicle groups, respectively 27 days after treatment. These data demonstrate that transplantation

of MotorGraft at T3 did not affect lower motor pool recovery, as was expected due to T3 motor

neurons not being involved in hindlimb locomotion.

Figure 14-BBB locomotor scores for MotorGraft treated and vehicle control animals with a T3

SCI. Testing was performed at 5 days post injury prior to MotorGraft transplantation for

pretreatment comparison (5D Post-injury/Pre-MG). Testing was preformed again at 1 week, 2

weeks, 20 days, and 27 days after MotorGraft transplantation (1 Wk Post-MG, 2 Wks Post-MG,

20 D Post-MG, and 27 D Post-MG, respectively). The locomotor capability of MotorGraft and

vehicle control treated animals did not differ. Data are expressed as mean ± standard error.

6.6. Histological results

6.1.6. MotorGraft cells survive following transplantation

IHC staining for human nuclear protein to identify the presence of MotorGraft cells was

performed on spinal cords of all animals. Human nuclei were detected in all but two (2) of the

59

MotorGraft transplanted animals analyzed. Typically there were a lot of human nuclei present, as

opposed to just a few, within a section of analyzed spinal cord tissue (Figure 15a-f). These data

demonstrate that MotorGraft survives following transplantation into the spinal cord. ChAT

immunohistochemical staining was performed to determine if transplanted MotorGraft cells

express markers of mature motor neurons. ChAT staining localized with MotorGraft and

surrounding cells (Figure 15g-i).

Figure 15-MotorGraft cells survive and begin to express ChAT. a, d) Human nuclei (green) were

detected in the spinal cords of transplanted animals. b, c, e, f) Hoechst nuclear counter stain

(blue) (b and e) colocalizes with human nuclear staining (c and f). g, h, i) ChAT positive staining

(red) is localized with MotorGraft (human nuclei in green) and surrounding cells (Hoechst in

blue). Images taken at 200X magnification.

All analyses comparing MotorGraft transplanted to vehicle control groups did not include the two

animals where MotorGraft cells were not detected

6.2.6. MotorGraft Transplantation Spares Endogenous Neurons

The average number of endogenous neurons in the MotorGraft transplanted group, rostral to the

60

injury epicenter, was 38 neurons with a standard error of +/- 3. The average number of neurons in

the vehicle control group, rostral to injury epicenter, was 29 neurons with a standard error of +/-

2. Rostral to the injury epicenter the MotorGraft transplanted group showed a significant (p <

0.01) sparing of neurons compared to the vehicle control group, (p<0.01) (Figure 16a). The

difference in neuronal sparing is evident in representative pictures of NeuN staining (Figure 16e,

f). The average number of neurons in the MotorGraft transplanted group, caudal to the injury

epicenter, was 36 neurons with a standard error of +/- 4. The average number of neurons in the

vehicle control group, caudal to the injury epicenter, was 37 neurons with a standard error of +/-

3. Caudal to the injury epicenter the MotorGraft transplanted group did not demonstrate statistical

significance in neuronal sparing (Figure 16a). Therefore, caudal to the injury site neuronal

sparing was not affected by the transplantation of MotorGraft, whereas MotorGraft spared

neurons rostral to the injury epicenter.

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Figure 16-a) The average number of neurons in a quadrant of spinal cord cross sections from

vehicle control (ave= 29) and MotorGraft transplanted animals(ave=38). There were

significantly greater number of neurons in transplanted animals (ave=38). b) The average

number of motor neurons in a ventral horn of vehicle control (ave= 7) and MotorGraft

transplanted animals.(ave=12) rostral to the injury epicenter. c, d) Representative NeuN

staining (red) in transplanted (c) and vehicle control (d) spinal cord sections. e, f)

Representative ChAT staining (red) in transplanted (e) and vehicle control (f) spinal cord

sections. Images taken at 200X magnification

6.3.6. Transplantation Spares Endogenous Motor Neurons

The average number of endogenous ChAT positive cells in the MotorGraft transplanted and

vehicle control groups was compared to examine whether the neuronal sparing may have

included endogenous motor neurons. The average number of ChAT positive cells counted in a

single ventral horn of the MotorGraft transplanted group in sections that are rostral to the injury

epicenter was 12 cells with a standard error of +/- 2 cells (Figure 16b). The average number of

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ChAT positive cells counted in a single ventral horn of the vehicle control group in sections that

are rostral to the injury epicenter was 7 cells with a standard error of +/- 2 cells (Figure 16b). The

number of ChAT positive cells rostral to the injury epicenter in the MotorGraft transplanted

group was significantly (p<0.05) greater than that of the vehicle control group, demonstrating that

MotorGraft transplantation results in sparing of endogenous motor neurons (Figure 16b). The

difference in motor neuron sparing is evident in representative pictures of ChAT staining (Figure

4c, d). There was no significant difference in the number of ChAT positive cells caudal to the

injury site despite the MotorGraft transplanted group having 13 +/- 2 cells as compared to the

vehicle control group having 9 +/- 2 cells (Figure 16b). Although there was no significant

difference caudal to the injury site, this suggests that there was a trend in the data.

6.4.6. Morphometry

The area of intact spinal cord tissue, whether it be normal or pathologic, was examined.

Morphometric analysis of the spared intact spinal cord tissue resulted in no significant difference

between MotorGraft transplanted and vehicle control groups (data not shown). These data

implicate that although there is significant sparing of neurons and motor neurons in particular,

there is not an effect on the overall area of the intact spinal cord tissue. The lack of difference in

the area of intact tissue reveals that the comparison in the number of neurons and motor neurons

is justified as the area of tissue was comparable.

6.5.6. Serotonergic Sprouting

Serotonergic fiber sprouting analysis demonstrates that the MotorGraft transplanted group has

significantly greater sprouting as compared to the vehicle control group at 2mm rostral to the

injury epicenter (Figure 17a). Although there was not statistical significance at 1mm rostral, 1mm

caudal, and 2mm caudal, the data suggests that there may be a trend to support increased

63

serotonergic sprouting with MotorGraft transplantation. These data suggest that MotorGraft may

have a trophic effect on the local environment.

Furthermore, as serotonin is thought to be involved in recruitment of more motor neurons with

afferent input after SCI [52], these data suggest that the electrophysiological properties of motor

units would be improved, just as was observed with MotorGraft transplantation (see Figure 13).

A

B C

Figure 17- Comparison of serotonergic fiber sprouting (Stained by 5-HT) in vehicle control and

MotorGraft transplanted groups. MotorGraft transplantation resulted in increased integrated

density of 5-HT fibers (red) 2mm rostral (+2) to the injury epicenter. Analysis is at 2mm and

1mm rostral (+2 and +1, respectively) and 2mm and 1mm caudal (-2 and -1, respectively) to the

injury epicenter. Images taken at 200X magnification. Data is expressed as mean ± standard

error. *p<0.05.

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6.6.6. Gene Expression Analysis and Growth Factor Expression Analysis

Although not shown in this document, PCR analysis of transplanted animals detected a decrease

in host-expression of the pro-inflammatory and pro-apoptotic genes CD40, CRP, Gadd45,

Caspase 4, and TGFβ2 and increased expression of IL-10, which has anti-inflammatory functions.

There was also detected a significant increase in the growth factors NT-3 and NGF of the spinal

cord of animals as compared to vehicle animals, via ELISA assays, proving increased expression

of in vivo secretion of growth factors by the transplant.

6.7. Discussion

Reinnervation of a denervated muscle is characterized by an increase in the amplitude of the

signals. The surviving motor neurons sprout towards denervated muscle fibers, and their MUPs

increase both in amplitude and duration. Early findings are characterized by polyphasia due to

differences in conduction between newly innervated fibers and old ones. As time goes and the

connections in the end-plate mature, the duration of individual MUP decrease and so do the

amplitudes. MUP analysis or IP analysis indirectly can provide information about individual

MUPs.

I studied different parameters of the IPA to use as indication of re-innervation and overall

beneficial effects that correlated with histological improvement. I found that parameters related to

amplitude as mean amplitude per turn (A/T) and mean amplitude of the burst are too variable to

provide the necessary resolution to discriminate between groups, even after applying averaging

techniques across measurements and ensemble average across time. Power Spectral Analysis does

not show significance either. Turns per second from the Turns-Amplitude Analysis and Zero-

Crossings have found to be the most sensitive parameters to track regeneration.

65

At the end of the study, the transplanted group displayed lower turns per second and

zerocrossings. This data supports the hypothesis that a motor neural transplant enhances

reinnervation of the internal intercostals after a traumatic insult to the spinal cord. The results are

corroborated by the results from histology, gene expression analysis and growth factors

expression analysis.

6.8. Summary

Rats subjected to the model of injury described in section 5.6 were transplanted with MotorGraft

from California Stem Cell, Inc (Motor Neuron Progenitors) and were measured according to the

experimental protocol described in section 5.7 and section 6.3.

The transplant, as observed from histology, has significant beneficial effects in neuronal sparing

and sprouting. PCR analysis showed decreased expression of pro-inflammatory and pro-apoptotic

genes and increased expression of anti-inflammatory genes in transplanted animals, while ELISA

analysis detected increased levels of growth factors in vivo in the transplanted animals as

compared to vehicles. Altogether, it supports the beneficial role of the transplant in regeneration

and protection in the injured environment.

However, the behavioral BBB locomotor test does not detect a difference between groups,

probably because the injury does not target specifically locomotion. On the other hand, turns,

zerocrossings of the intercostal muscles were able to show accelerated regeneration. As a

summary, in the results shown in this Chapter supports the usefulness of the model of injury and

the techniques here developed as assessment tool of efficacy of therapies in the context of

denervation diseases or traumatic injuries to the spinal cord.

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Chapter 7

Effects of electrical and mechanical stimuli in

neurons


In previous chapters, I presented the development of a model of rat injury that mimics

denervation of the respiratory system due to disease or injury at the spinal cord level and

evaluates efficacy of regenerative treatments by analyzing the electromyography (EMG) signal. I

validated the technique and the model in Chapter 6 using a motor neuron progenitor transplant.

In the following chapters, I propose a new scaffold made of piezoelectric polymers as the

method to enhance neuronal plasticity via electrical stimulation. Piezoelectric polymers are an

ideal platform for combinational therapies in regeneration since they can combine electrical,

mechanical, topographical and chemical cues. This chapter starts by reviewing the effect that

different of these cues have in neuronal plasticity, focusing in the cues inherent to a piezoelectric

polymer (mechanical and electrical). Then, I review alternative means for delivery of electrical

fields to the site of injury and show the advantages of piezoelectric polymers over the others.

Finally, I present the significance and originality of my contribution.

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7.2. Strategies for Spinal Cord Injury

Contrary to previous knowledge, nowadays it is known that the CNS has an intrinsic capability

for regeneration, which is not successful because of lack of enough stimulation cues as well as the

presence of inhibitory cues. Enhancing the former and blocking the latter is the rationale behind

regeneration of damaged pathways.

The problem of neuronal repair in the context of spinal cord injury can be simplified to the

processes of growth (trophism), guidance towards the target (tropism) and establishment of

functional synapses at the end of that process

Several strategies have been implemented in order to promote such growth, and modulation of the

following factors may also act as guidance cues. These include:

a) Chemical growth factors

b) Substrate mechanical support

c) Stiffness

d) Mechanical stimuli

e) Electric stimuli

The combined effects of several growth enhancement factors, based on well-characterized

features of nerve cell behavior are extremely important. These mechanisms, once understood,

would lead in time to important new approaches to growth enhancement and regeneration of

spinal cord injuries. Despite efforts, none of the approaches have yield significant results.

Nowadays, there is a special interest in looking for combinational treatments where scaffolds,

neurotrophic growth factors, cells and different biomaterials are combined in search of

combinational if not synergistic effects ([53], [54], [55], [56], [57] and recent review articles:[58],

[59],[60]).

68

7.3. Chemical growth factors

Neurotrophins enhance cell survival and axonal growth in vivo but they require a permissive

substrate to promote such a plastic effect. NGF, BDNF, NT-3 stimulates dendritic branching and

axonal growth. Blocking of inhibitory molecules like nogo has a similar effect. These growth

factors might be supplied or secreted by cells, or doped into substrates with an engineered release

time. (Reviewed from [61])

7.4. Structural support

Scaffolds that enhance axonal growth in the SC are divided in two groups: Implants of cells into

the wounded area and cell-free bridges made with biocompatible materials. The first group

includes grafts with Schwann cells, olfactory ensheating glia, genetically manipulated fibroblasts

and stem cells, among others. Immuno-reaction is a problem for those drafts that use ensheathing

cells from another human or animals. For grafts where the own person cells are used, purification

and expansion of the cell population will take more time than the required to start an effective

treatment for an acute SCI. The problem is also the poor knowledge about these natural

mechanisms of regeneration, which makes them difficult to manipulate [62].

The last point lead to the use of substrates, engineered to contain the necessary properties to

promote regeneration. For some time it has been known that polymeric substrates can enhance

growth of neurons and muscle cells due to their structural properties [63], [64], [65], [66], [67].

In the context of biocompatible materials, polymers can be divided between non- biodegradable

and biodegradable. The concerns for these materials implantable materials are the degradability,

reabsorption and biocompatibility. Long-term exposure would promote scar formation and be

69

treated as a foreign body [61]. Undegradable substrates are easily manufactured, more

hypdrophobic and hence, less cellularly adhesive, and pose a higher risk of inflammation ([68],

[69], [70]). On the other hand, the manufacture design of biodegradable polymers is usually

challenging and poses constraints in volume and degradation rates to prevent a significant change

on the PH of the extracellular matrix [71]. In both groups there are FDA approved polymers to

use in human implants like polyacrylonitrile polyvinylchloride (PAN=PVC),

poly(tetrafluoroethylene) (PTFE), poly(glycolic acid) (PGA) and poly(lactide-co-caprolactone)

(PLCL).

Some examples of nerve conduits with non-degradable polymers include PAN=PVC channels

used to deliver Schwann cells or olfactory ensheathing glia, PTFE channels and poly(2-

hydroxyethyl methacrylate) (PHEMA) [68].

Among the common degradable materials that have been studied for use in nerve guidance

channels in the CNS there is the polymer family of poly(a-hydroxyacids,) which include synthetic

polymers and copolymers such as PGA,PLCL, poly(lactic acid) (PLA) and poly(lactic acid-co-

glycolic acid) (PLGA) and natural occurring polymers like collagen and chitosan. The last group

is abundant but the manufacturing of scaffolds is difficult, often because of insolubility in the

most common solvents. Chitosan has also a low mechanical strength.

At this point there is not one polymer with clear advantages respect to the others, and so there is

much search for new materials as well as modification of known substrates to enhance their

mechanical and chemical properties, like doping of chemical factors onto the substrates or

copolymerization to increase hydrophilicity. The use of hydrogel materials it is wide spreading

since they can expand to fill the entire wound, which makes them very suitable in the context of

SCI ( [72],[73]; [74]).

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7.5. Stiffness

The structural properties of substrates can enhance neuronal plasticity. Stiffness has been shown

to be a modulator of neuronal morphology. However, the effects of stiffness in neuronal

branching have produced conflicting results. Neurons grown on gels of the range of 300–3000 Pa

experience increased dendrite branching in stiffer gels [75]. While some studies support the same

increase in dendrite branching, others show decreased branching with stiffness. Cell density,

stiffness ranges, methods and cell age could account for the differences.

The mechanism under that effect seems to involve adhesivity of the substrate. Focal adhesion

kinases have been seen to change behavior on compliant substrates. The trans-membrane protein

tyrosine phosphatase a transduces mechanical forces via av/b3-integrin activation. Integrins have

been shown to play a role in mechano-sensing (Reviewed in [76]). Integrins are receptors of shear

stress in endothelial cells. Strength of integrin–cytoskeleton linkages is dependent on substrate

rigidity and fibrobast cultures grown on stiffer substrates overexpressed integrins.

Furthermore, anisotropy of the mechanical moduli can serve as a guidance cue as well. Stiffness

gradients have been shown to direct growth of axons, due to identified components in the cell

membrane that respond to mechanical traction. Dorsal root ganglia (DRG) from chick embryos

cultured in substrates with a gradient of stiffness between 60Pa and 365Pa grew neurites followed

the compliant side with a 300%, while neurons grown in isotropic substrates experienced a 20%

variation [77].

7.6. Mechanical stimuli

Although cells are mechano-sensitive entities, there are few studies on the effect of mechanical

71

stimuli on cell in terms of stress, whether stretch, compression or vibratory. Lately, studies on the

effects different mechanical stimuli have in a variety of cells as well as on mechanisms of

actuation have wide spread due to the advances of micro technology to control stimulation at the

cellular level while new optical approaches allow real time imaging of its effects. The field of

biomechanics is blossoming and, in the context of spinal cord injury, there is much need to

elucidate the response in growth that mechanical cues may have in cells of the Central Nervous

System (CNS).

The nature of the mechanical response of the cell depends much on the cell type, some of them

being intrinsically designed to respond to mechanical stimuli. It would be the case of many

sensory receptors, which are the sensory end-organs of primary neurons on the Peripheral

Nervous System (PNS). The responses of sensory receptors are not only specific to the type of

stimuli, which includes stretch, vibration and shear stress, but also to the spectral content and

amplitude of stimulation. Activation is generally mediated by mechanically gated ion channels.

Usually, opening of those channels depolarizes the cell membrane. If a threshold is reached, it

fires an action potential, although depolarization can also set the release of a neurotransmitter that

excites other cells, like in the case of cochlear hair cells.

Beyond sensory receptors, neurons have inherent mechano-sensitive properties themselves.

Controlled mechanical stimulation of sensory axons has shown specificity in their response to

static and vibration stimuli in the same manner their sensory receptors would, although end-

organs are required to fine-tune the threshold of activation [78]. The mechanism of mechano-

transduction seems to involve different ion channels depending on the type of stimuli. Rapidly

adapting currents are likely to have a strong contribution from [Na+] ion channels, while it is

suggested that slowly adapting currents are mediated by [Ca++

] ion channels [79]. In line with the

last study, [Ca++

] transients were affected by dynamic axial stretching [80].

72

If we focus on the cellular response to vibratory stresses alone, there are not many studies

dedicated to the neuronal population, apart from the study of sensory receptors and sensory

neurons. However, many studies address the effect of whole-body vibrations in different systems,

including the nervous system.

Although the actual stimuli experienced in the tissues depend on elastic matching and attenuation

factors within the body at different frequencies, the effects of whole-body vibration on the tissues

are still indicative of the response of a certain cell type to vibration. The frequency range to which

the human body is more sensitive is from 1 to 80Hz.

The interest on the impact that chronic vibration has on human body started back in the 1950s

within the context of work safety. The epidemiology of low back pain reveals a higher incidence

of spine disorders among professional drivers, higher even among those with longer driving

duties, which suggested vibration as a deleterious agent of spine health. The ISO 2631 standard,

from the International Standard Organization, was developed to evaluate the effect of human

exposure to whole-body vibration and to set thresholds delimiting injury [81].

Blurred vision, loss of balance, loss of concentration and other circulatory, digestive and neural

deficiencies are all examples of the deleterious effects of vibration in many systems[82]. On the

other hand, vibration in the 18-80Hz range has been shown to decrease low back pain in a manner

comparable to conventional physiotherapy [83] and [84].

We found other examples of the positive effects of vibration in the context of sports medicine,

where vibratory platforms seem to enhance physical performance. Whole-body vibration at 26 Hz

resulted in increased jumping height [85] and exercise in vibration plates at 40Hz increased

73

strength in naïve and non-naïve subjects more than resistance exercise alone [86].

Frequencies and amplitudes are the most relevant features when considering the positive or

negative effects of vibration, although axis of loading and duration of exposure are also

important.

Not many cellular studies exist at the neuronal level, but chronic vibration has been found to

affect rat neurons of the inferior vestibular nucleus (IVN) in a deleterious manner. Neurons

exposed for 5, 10 and 15 days, in 2h-sessions to vibration at 60Hz and 0.4mm in amplitude,

changed all their main features including an increase mean frequency of the background activity

and a more populated and chaotic firing pattern [82].

Another interesting finding on the effects of vibration in cells is the possible enhancement of

endocytosis. Rat cortical CryoCells increased antibody take upon vibration at 500um and 200Hz

for 3h, although some cell detachment was also observed[87].

Many studies have scrutinized the most deleterious frequencies involved in degeneration of the

intervertebral discs (IVDs), which correlates with low-back pain. Close to the resonant frequency

of the spine (4-5Hz), most degeneration of the IVD occurs although that is probably due to

mechanical energy transfer and not to vibratory frequency itself, while frequencies in the 20-

300Hz range seem to stimulate protein synthesis on IVDs and surrounding tissue, which is

regarded as beneficial contribution to IVD health.

Cultures of annulus cells loaded at 1.7MPa and 20Hz in sessions of 30 min for 9 days stimulated

protein synthesis and reduced protein degradation on the extracellular matrix while they did not at

0.3MPa and 1Hz[88]. IVDs subjected to axial vibrations on sessions of 10-60min, up-regulated

74

collagen type II (normally high) in the extracellular matrix at 80Hz, while at 8Hz decorin

(normally high) was up-regulated and at 40Hz was down-regulated. On the other hand, at 0.4-

0.5g, but not below 0.4g, collagen type I (normally low) was up-regulated, while versican

(normally high) was down-regulated, which, according to the authors, could suggest adverse

effects of vibration at those amplitudes [89].

Lately, there is much interest in the ability of vibration to build up muscle. Vertical stretch of

0.4mm amplitude on the range of 8-10Hz for 10/min day for 3 days has shown to enhance

number, length and average area of myotubes in C2C12 myoblasts [90]. On the other hand,

chondrocytes exposed to 1.4 g vibration at 400Hz arrested DNA synthesis [91]. Many studies

have demonstrated the positive effects of bodily vibration on muscle and bone maintenance [92-

95].

Although it would be important for the field of neuronal regeneration, there are few studies on

possible trophic effects of mechanical stimulation in the neural population. Disrupting

myelination produces in the stress–strain response of spinal cord tissue in uniaxial tension, which

suggests that both types of glia and myelin are important components of the structure–function

relationship in spinal cord tissue [96]. Stretch forces have been found to be a stimulatory cue in

axonal-growth. Axons can stretch up to 8mm/day and reach 10cm of elongation over 14days,

while getting a 35% increase in their calibers[97], but the impact of vibration on neuronal

plasticity remains a matter for study.

Recently, one study addressed the effect of nano-vibration, at 10 KHz, on neurite growth in PC12

cells, showing significant enhancement after several days. The mechanism behind this non-

physiological stimulation was attributed strictly to vibratory enhancement of nerve growth

factor[98], but still lacks an insight on the effects of vibration in a neuronal population.

75

As it has been review, vibration can induce changes in cells in terms of attachment, endocytosis,

cell viability, protein synthesis, firing patterns and plasticity. We could also consider [Na+] and

[Ca++

] mechano-sensitive channels as potential mediators in some of these phenomena.

Deleterious and beneficial effects of vibration over those features depend on the frequency,

amplitude and duration of stimulation.

As a first approach and admitting that thresholds might differ between cell types, amplitudes

above 1.4g seem to have deleterious effects on general viability of the cells and so would seem

for amplitudes between 0.4-0.5g but not below. Attachment is inversely correlated with amplitude

as well. Vibration on the frequency range of 20-300Hz is innocuous at worst and beneficial if we

account for protein synthesis, in concrete collagen presence in the extracellular matrix at 80Hz.

At very low frequencies (8-10Hz), vibration can play a trophic role.

As a summary, cells exposed to vibration should be ensured an amplitude and frequency range

that promotes well-being and maintenance of the culture. Not deleterious conclusions should be

inferred without more consideration, as even beneficial effects can be observed with the proper

settings.

7.7. Electrical Stimuli

Different cells are responsive to electrical fields in a manner that depends on the cell type.

Migration, proliferation and trophic responses in terms of dendritic branching and axonal growth

and guidance are a subject of study that has challenged the scientific community since the times

of Ramon y Cajal and Ingvar in 1920.

76

Endogenous electrical fields (EFs) are naturally occurring during the process of wound healing to

promote growth and protein absorption adsorption [99], [100]. Different studies have measured

the amplitude of the steady EF created in the injured tissue of amphibians and chick embryos.

Endogenous EFs generated upon injury are necessary for epithelium and corneal regeneration.

The amplitudes range between 40-50mV/mm in the cornea [101] and 100-150mV/mm in the skin

[102, 103], and some have been seen to persist for days. Reviewed from [104]and [105].

Additionally, migration of more than 15 cell types has been observed upon exposure of electrical

fields [106],[105],[107]. For example, endothelial cells have been observed to migrate towards

the cathode after exposure to EF above 100-200 mV/mm. It was also observed asymmetrical

distribution of filamentous actin in the cytoplasm, with a transient increase of 80% in the side of

the cathode [108]. Corneal epithelial cells under EF between 100–150 mV/mm for 16 hours

experienced asymmetric up-regulation of epidermic growth factor receptors, which is enhanced

by fibronectin and laminin [109]. EF’s of 120mV/mm also directed neuronal migration of

hippocampal neurons towards the cathode [110].

Direct electrical fields have an effect also in alignment. It has been shown that a direct electrical

field of 500mV/mm for 24 hours induces astrocyte alignment [111]. Electrodes providing an

extracellular voltage of 300-400uV/mm for 15 days post injury with a reverse polarity every 15

min to the injured spinal cord of dogs, has shown to change the density and orientation of

astrocytes[112].

Focusing in the neuronal context, it would seem reasonable that a cell type devoted to

transmission of electrical signals is susceptible to the effect of EFs. Indeed, neurons are

electrically sensitive. Electrical activity, either received directly from neurons or from electrodes,

can enhance dendritic growth and branching. The activity dependence of growth and branching of

77

dendritic arbors has also been noted [113]. Conversely, deprivation of synaptic input causes

dendritic shrinkage, and branch loss [114].

Neurons of the central nervous system (CNS) do not regenerate spontaneously. There is

increasing evidence that externally applied electric fields (EF’s) can induce neuronal growth and

guidance in the CNS. In vivo studies showed that regeneration of the lamprey spinal cord was

facilitated by a small steady current (10 µA, cathode at the distal end) across the lesion for 6 days

[115].

Axonal EF-mediated nerve regeneration in non-mammals has been reproduced in mammals [100,

116-127]. A weak, steady electric field ( 400 mV/mm) applied by electrodes implanted across

transected spinal neurons in guinea pigs induced axonal growth and penetration through the glial

scar within the lesion [128]. Follow-up studies showed that after 1 month of constant current

delivery, there was regeneration of the spinal cord, exhibited by growth cones, extensive

penetration and branching of axons within the lesion, as well as crossing of ascending axons into

the rostral segment [129].

Borgens and colleagues have studied EF-mediated responses of neurons and astrocytes in injured

guinea pig spinal cords [112, 129]. The same group used oscillating electrical fields with a

periodicity of 15 min for bidirectional regeneration in dogs suffering from spinal cord injury

[130] and in humans for Phase 1 clinical trials [131]. They based the periodicity setting in the

study of McCaig, which establishes a time-window during which cathode attraction is stimulated

but anodal retraction is avoided [132].

Different in vitro studies have proven the efficacy of EFs as low as 10mV/mm for directing

axonal growth and stimulating neurite outgrowth and directional branching. However, the effects

78

of polarity in guidance and branching between different studies are confusing and contradictory.

Cell type, charge of the substrate and if the process is axonal or dendritic seem to play a role in

the attraction or repulsion towards the cathode experienced by the neurites exposed to EFs [105].

Chick dorsal root ganglia (DRG) exposed above 40mV/mm showed higher growth rates towards

the cathode, [133], [134]. Spinal neurons of the Xenopus have faster growth rates towards

cathode and retraction from anode at 7-20mV/mm [135], increased sprouting towards the cathode

at 250mV/mm [136]and doubling of branching at 120-150mV/mm, with 80% of the branches

present in the cathodal side[137].

EF’s of 1000mV/mm applied to cultured spinal Xenopus neurons caused directional growth of

neurites toward the cathode and away from the anode if cultured in a negatively charged

substrate, but the opposite in a positively charged substrate, and the response was graded with

adhesivity. Thus, it seems that expression of adhesion molecules may interact with electrical

fields to modulate the guidance properties[105].

On the other hand, hippocampal neurons of rat embryos exposed to 28-219mV/mm had neurites

oriented perpendicular to the EF and a decreased number of neurites facing the cathode [125].

Another study with the same type of neurons, showed that dendrites are attracted to cathodes

while axons are not[138]. And rat PC12 cells growth was biased towards the anode [139] upon

exposure to EFs.

Finally, Zebrafish exposed to 100mV/mm were completely irresponsive to EFs[140] Therefore, it

seems that animal species might also have a role in the effect of neurons to direct electrical fields.

About the mechanism by which EFs exert their influence in neuronal plasticity, it has been found

79

that activation of [Na+]channels and voltage-gated [Ca

++] channels stimulates dendritic growth

and branching in various neurons, incuding cerebrocortical neurons [141, 142]. In the Xenoopus

spinal growth cones, attraction to the cathode seems to be mediated by the neurotrophin receptors

TrkB and Trkc, the nicotinic receptor nAChR, receptor tyrosine kinase,s phospholipase C,

different isoforms of kinase C , extracellullar Ca2++

and intracellular reserves of Ca2++

. Moreover,

cAMP could be a regulatory molecule modulating EF response along with Ca2++

[105].

Another study suggests the role that electrical fields have in protein adsorption and neurite

outgrowth. PC-12 cells grown on conductive films of polypyrrole were electrically stimulated

simultaneously to serum containing media and also pure fibronectin, which result higher levels of

fibronectin and protein absorption and also in longer neurite outgrowth, with respect to cells non-

electrically stimulated of electrically stimulated after protein exposure. This suggests that

absorption of fibronectin and immediate electrical stimulation are necessary for neurite outgrowth

[100].

Besides plasticity, steady state EFs have also an effect in neuronal excitability. EFs of less than

40mV/mmm have been seen to alter the threshold of action potentials evoked by orthodromic

stimulation in rat hippocampal slices [143].

There is much less study of neuronal plasticity by non-steady electrical fields. Electrical

stimulation at 20Hz for 1h of the completely transected sciatic nerve in the rat, showed higher

levels of re-innervation and a higher number of myelinated fibers in the distal nerve by histology

and electrophysiology [144]. Electrical stimulation at 20 Hz for 1 h during 7 days of the sciatic

nerve in vitro produced a 4 fold increase on the DRG neurite outgrowth. After a thoracic hemi-

laminectomy, stimulation at 20 Hz for 1 h during 7 days of the sciatic nerve increased axon

projections into the central lesion of the spinal cord. [145]. Electrical stimulation increased the

80

levels of intracellular cAMP, which makes it a candidate for the mediating mechanism under EF-

induced plasticity.

Because oscillating electric fields not have been studied much, there is little information on which

to select as appropriate stimulation frequencies for enhancement of neuroplasticity.

7.8. Electrically active polymers

While the above experiments were performed with metal electrodes, attractive alternatives are

being developed using quasi-permanent surface charge (electrets), polymers that generate electric

charge upon applied mechanical stress (piezoelectrics), and electrically conducting polymers.

The main advantage of a biocompatible polymer approach versus electrode implantation could be

effectiveness in stimulation delivery and minimization of infection risks. Many synthetic

materials are more biocompatible that metals, including inert metals. Some are even

biodegradable. They provide great flexibility of manufacture, which translates into localization of

EFs where they are most effective. Three-dimensional geometries can be used to deliver stimuli

to deep areas. Their mechanical properties better resemble the ones of the extracellular matrix.

They also can be easily doped with growth factors. Altogether, it makes electrically active

polymers the best platform for combinational therapies.

The most common conductive polymer is polypyrrole (PP). Films of PP have been used to study

neuronal plasticity [146, 147]. PC12 cells cultured on PP films and subjected to a steady field of

100mV for 2h showed a significant increase in neurite length [148]. Similar results were found

when PC12 cells were grown on glass coated with nano-particles of gold and subjected to pulsed

electrical fields [149]. Neural stem cells grew more neurites when their conductive polymer

81

matrix was electrically stimulated [147] Although PP is a non-degradable conducting polymer,

degradable alternative to PP have been studied. Pyrrole-thiophene oligomers have been linked

together with hydrolyzable esters [150] and blends of PP with chitosan or hyaluronic acid are

being investigated to create conducting materials with more biocompatible properties [151],

[152].

Electret materials and piezoelectric materials have the benefit in front of conductive polymers of

not requiring an external source for electrical stimulation. Examples of the effect of an electret is

found in the use of charged forms of poly(tetrafluoroethylene) (PTFE) which has resulted in a

higher number of myelinated axons upon injury with respect its non-charged form [153].

However, electrets have not caught much attention in the scientific community.

A potentially more powerful means of producing EF’s is via piezoelectric polymers. A

piezoelectric polymer generates a variation in the charge density of its surfaces in response to a

mechanical deformation. Thus, it can be used as an autonomous source of EF’s. Polymers with

piezoelectric (PZ) properties, such as polyvinylidene fluoride (PVFD), have been used to enhance

growth and myelination of peripheral nerve axons [154-156]. When grown on PZ-PVFD at

1200Hz for 72 hours, mouse neuroblastoma cells grew neurites at a rate 4 times faster than cells

growing on an ordinary, non-piezoelectric polymer [155]. The use of the piezoelectric copolymer

of PVDF, polyvinylidenefluoride-trifluoroethylene (PVDFTrFe), has also resulted in a higher

number of myelinated axons in the sciatic nerve after injury when compared to the

nonpiezoelectric form [99]. The generation of EF’s by the PZ materials is critical to their action,

since un-polarized, non-PZ polymers of identical composition do not enhance neurite outgrowth.

82

7.9. Significance

In this study, I tested the biocompatible piezoelectric PVFD for its effect on the outgrowth of

neurites in neurons of the central nervous system, and in specific, rat spinal cord neurons. Various

modifications of PVDF have been shown to provide suitable substrates for growth of neurons in

culture[157] but it has not been studied the effects that electricity delivered by a polymer has in

neurons of the central nervous system. Herein I show that spinal cells grown on stimulated

piezoelectric PVDF films generate significantly greater neurite branching compared to those

grown on non-stimulated and non-piezoelectric films. My studies include consideration of

different types of neurons (spinal cord neurons and cortical neurons) and different materials

(PVDF and polyvinylidene fluoride-trifluoroethylene, PVDF-TrFe). All of them show the same

trends, supporting the hypothesis that delivery of electrical fields via a piezoelectric polymer

enhances arborization in neurons of the central nervous system.

The novelty of this project is the development of a for spinal cord injury repair system that

integrates three methods in one platform: (1) the use of a polymer substrate and (2) the use of

electric fields (EF) (3) the use of vibration on cells.

83

Chapter 8

Piezoelectricity


In Chapter 8, I first introduce the principle of piezoelectricity and the basic vocabulary related to

the field. Then, I explain the different classes and the constitutive equations describing the

behavior of piezoelectric materials. Finally, I present three of the most useful piezoelectric

polymers with promising potential in the biomedical field due to its biocompatibility and/or

biodegradability and I explain the choice of materials which I use in the experiments presented in

Chapter 10.

8.2. History

The direct piezoelectric effect was discovered by Jacques and Pierre Curie in 1880. The Curie

brothers were able to measure charge produced in the surface of materials like tourmaline, zinc

blende, quartz, topaz or cane sugar once subjected to mechanical stress. The name comes from

the Greek word piezo, which means pressure. Thus, piezoelectricity is the ability of some

materials to generate an electric potential in response to an applied mechanical stress. The

converse effect is true as well, as Lippmann predicted in 1881. Piezoelectric materials create

strain as response to an applied electrical potential. For that reason, piezoelectric materials are

84

used both as sensors and actuators.

Piezoelectricity was only an experimental curiosity and mathematical challenge until World War

I, when most popular piezoelectric applications were conceived and developed. Crystal resonators

started being used as frequency stabilizers in oscillators. In 1917 Paul Langevin developed the

sonar, a submarine detector and communication system, which uses both the direct and reverse

piezoelectric effects. Microphones, accelerometers, bender element actuators, speakers, sound

pick-ups and signal filters were also inventions of those times. After 1940, piezoelectric

applications became more focused in actuators, like piezo-ignition systems and relays. Nowadays,

the need for micro-electromechanical systems (MEMS) that can perform both detection and

analysis on a chip has in piezoelectric materials its best ally.

8.3. Piezoelectric classes

The piezoelectric effect dwells on the crystal structure of the material and the ability for charge

separation which that crystal structure entails. In a piezoelectric crystal, the positive and negative

electric charges are separated forming an electric dipole. A dipole p

is defined as two equal and

opposite charges ±q separated by the vector of distance d

.

dqp

Dipoles are usually randomly oriented, so that the material is electrically neutral in macroscopic

terms. Some physical processes, like uniaxially or biaxilly drawing and poling, can align the

dipoles so macroscopically the material loses its electrical neutrality. Poling is a procedure by

which a strong electric field is applied across the material, usually at elevated temperatures,

forcing a remnant polarization at 0V/m.

From the total 32 crystal classes, the 21 that have a non-Centro symmetric crystal structure are

85

piezoelectric, of which 11 classes are neutral at 0 volts and 10 classes contain a remnant

polarization .

The polar group is also pyroelectric, meaning that an applied temperature gradient produces an

electrical field. Ferroelectrics are a subset within pyroelectric materials. In pure dielectric

materials, the polarization P

is linearly related to electric field E

by the susceptibility of the

medium e

and the permittivity of the free space o . In ferroelectric materials, the polarization

P

follows a hysteresis loop, which presents a remnant polarization at 0V/m and which can be

reversed upon exposure to a strong electrical field.

Under normal circumstances, even polarized materials do not display a net dipole moment. The

intrinsic dipole moment is neutralized by electric charge from the medium or from thermal

conduction that builds up on the surface of the material. When a mechanical stress is applied, the

symmetry of the polar crystal is disturbed and the variation of the dipole moment creates a charge

misbalance that generates a voltage across the material.

8.4. Constitutive equations for a piezoelectric material

Piezoelectric materials are electromechanical transducers. The constitutive equations describing

the mechanical and electrical behavior of the material must be extended to include the elastic

response to an applied electrical field and the electrical response to strain. Besides, piezoelectric

materials usually undergo processes like drawing and poling to render them piezoelectric

macroscopically. Those processes introduce asymmetries which break the isotropic configuration.

All of that must be taken into account in the equations of electro-mechanical coupling which

model the behavior of piezoelectric materials.

86

The electrical response of a generic anisotropic material is:

EP

1

EPED o

2

Where P

is the remnant polarization, E

is electric field, χ is the susceptibility matrix, D

is the

electric displacement, εo is the vacuum permittivity constant and ε is permittivity matrix.

For the development that comes afterwards, they can be described more conveniently in their

axial components:

jij

ji EP for i,j=1to3 3

j

jiji ED for i,j=1to3 4

Where 1

o

ij

ij 5

Hooke’s law for a generic material states that a mechanical stress produces a proportional strain.

Stress can be applied as compression, extension and shear, and thus, stress and strain are tensors.

TsS

6

Or alternatively,

iji

ij TsS for i,j=1 to 6, 7

Where S

is the strain tensor, T

is the stress tensor and s is the compliance matrix. The

compliance matrix s is symmetrical and so, only 21 of those 36 constants are independent.

Figure 18 depicts the nomenclature used. The stress for direction 1, 2 and 3 is tensile stress

applied in direction 1, 2 and 3 respectively, while 4,5 and 6 describe shear stress around direction

1 2 and 3 respectively. In that context, T4 would be shear stress around axis 1 and –T3 would be

87

compression stress on the axis 3.

Figure 18- Nomenclature of axis

In a piezoelectric material, a mechanical stress T

produces a polarization in the material that

contributes to the total displacement D

, along with the displacement created by the electrical

field E

itself. This contribution of T

is proportional by a factor of d, called the direct

piezoelectric constant. Equation 1.9 is the expression of the direct piezoelectric effect.

j

j

j

ijjiji TdED for i=1to3, j=1 to 6 8

Similarly, a piezoelectric material exposed to an electrical field E

generates a strain that adds to

the elastic response of the material to the stress T

. This contribution to the total strain S

is also

proportional to the constant d. Equation 1.10 models the inverse piezoelectric effect.

i

iiji

i

jij EdTsS for i=1to3, j=1 to 6 9

Or more compactly,

TdED

10

EdTsS t

11

A d tensor can be identified for each of the 20 classes of piezoelectric crystal structures. We

88

describe the d tensor by a matrix of 3 by 6, dij,, where i is the electrical field in the i direction

created by stress applied in the j direction. Due to symmetries, this matrix is symmetrical and

only 15 of its 18 coefficients are independent.

8.5. Piezoelectric polymers

A polymer is macromolecule consisting of repeating subunits or mers linked by covalent bonds,

which form chains that can arrange spatially in amorphous regions, crystalline regions or a mix of

both. The percentage and type of crystallinity depends not only of the material but also

temperature, pressure and time in cooling off.

Crystals of piezoelectric polymers are non-centro-symmetric and so they have a net dipole

charge. However, the crystalline regions are randomly oriented and overall, any polarization or

piezoelectric effect cancels out and the polymer is macroscopically neutral. Polymers can be

rendered macroscopically piezoelectric by drawing or poling the material.

Many polymers exhibit some piezoelectricity, like PVDF and several of its copolymers (PVDF-

TrFe), nylons (Nylon 7, Nylon 11), cellulose and polyureas (P(MDA/MDI), Polyurea 5, Polyurea

9). Their piezoelectric constants range from 20pC/N for d31 for PVDF at 20oC to 1.7 pC/N for

Polyurea 9 at 20oC, passing by -10pC/N for d36 for PLLA at 20

oC [158].

8.1.5. PVDF

Probably, the most commercially used piezoelectric polymer is Poly-vinylidene fluoride (PVDF).

PVDF is biocompatible aside of displaying one of the highest piezoelectric constant within the

polymeric group. The schematic of the PVDF molecule is shown in Figure 19.

89

Figure 19-PVDF Molecule

PVDF can crystalize in different conformations, namely α, β, ϒ and δ. The α-phase is obtained by

cooling off the melt at a normal rate of about 10oC/min. This gives a trans-gauche-trans-gauche

+

structure which makes the crystal non-polar. Slower cooling of the sample gives rise to the ϒ-

phase. Poling of the α-phase gives rise to δ-phase. None of these phases has significant polarity

since the fluor atoms at both sides of the carbon backbone which tend to cancel each other’s

polarity; therefore, none if these phases are piezoelectric.

The only piezoelectric form of PVDF, the β-phase, can be obtained by uniaxial stretching of the

α-phase at room temperature, which forces a reorientation of the bonds into an all-trans

configuration. In the β-phase, the molecules arrange in an orthorhombic lattice. Because all the

fluorine atoms are on the same side of the carbon backbone, there is net dipole moment. For

macroscopic results, is customary to pole as well.

The point group of the PVDF crystal in its β form is C2V (mm2). This group belongs to one of the

polar classes; therefore, PVDF is not only piezoelectric but pyroelectric and ferroelectric as well.

The piezoelectric constant pertaining to that group is

d=

000

00000

00000

333231

24

15

ddd

d

d

The axis nomenclature used in the piezoelectric matrix is the following. In order to display

90

piezoelectricity macroscopically, piezoelectric materials are often drawn and poled. Drawing

aligns the crystals in the stretching direction. That direction is named 1 by convention. The

direction of poling is usually perpendicular to the plane of the film and is named 3. Direction 2

would be the direction perpendicular to the other two

Figure 20- Axis nomenclature for the piezoelectric matrix

The d constant matrix of PVDF uniaxially stretched and poled is the same as the individual PVDF

crystal. Thus, the piezoelectric matrix for uniaxially stretched and poled PVDF has

piezoelectricity in both the shear and tensile axes.

8.2.5. PVDF-TrFe

Another piezoelectric polymer is the PVDF copolymer Polyvinylidene fluoride-trifluoroethylene

(PVDF-TrFe). PVDF-TrFe crystallizes directly into the all-trans configuration and because of

that, there is no need to stretch to align the crystals. Polarization is still needed in order to

synchronize all the crystal regions towards a known direction. The point group of PVDF-TrFe is

the same as for PVDF. As a result, the same type of d matrix applies.

8.3.5. PLLA

Poly-Lactic Acid (PLA) is also a piezoelectric polymer. PLA is biocompatible and biodegradable.

Because of these properties, it has been used extensively in sutures, scaffolds and in biomedical

devices for implantation. PLA is a chiral molecule and as such two conformations exist: Poly-L-

91

Lactic Acid (PLLA) and Poly-D-Lactic Acid (PDLA). The only form that can be rendered

piezoelectric is PLLA.

Figure 21- PLLA molecule. The asterisk indicates the chiral carbon atom.

Due to its chirality, PLLA arranges in a helix. There are two known crystal structures: the

pseudo-orthorhombic α-phase contains two chains in the unit cell, which arrange in a 10/3 helix

conformation, the β-phase is an orthorhombic unit cell and contains six chains, which have a 3/1

helix conformation.

Works by Fukada [158] and Tajitsu [159] show that uniaxially stretching PLLA films orients the

polymer chains and crystals, giving a piezoelectric constant responsive to shear. Processing

conditions have to be such that the crystals are formed in the α form, which is the piezoelectric

one. PLLA belongs to one of the non-polar groups and therefore is piezoelectric but not

pyroelectric.

The point group of PLLA crystal in its α-phase is D2 (222), but uniaxially stretched PLLA can be

identified with point group D∞ (422). Thus, the piezoelectric matrix for stretched PLLA shows

only shear piezoelectricity:

d=

36

36

00000

00000

000000

d

d

92

8.6. Summary

Piezoelectric polymers have smaller piezoelectric constants than other piezoelectric materials,

their versatility, wide temperature range of operation, elastic properties and behavioral properties

like biocompatibility and biodegradability make them an interesting alternative to their peers,

especially in the biomedical field.

In Chapter 8 I have presented three of the most important piezoelectric polymers, PVDF, PVDF-

TrFE and PLLA. PVDF and their copolymers are biocompatible and display a stronger

piezoelectric effect than PLLA. The advantage of PLLA dwells in its biodegradability. A

combinational platform based on piezoelectric biomaterials to provide electrical and mechanical

stimulatory cues for neuronal outgrowth would most benefit of the biodegradation capability of

PLLA.

In the experiments described in Chapter 10, I have used PVDF and PVDF-TrFE instead of PLLA

due to their higher piezoelectric constant. These experiments are intended to test the principle that

piezoelectric polymers are a practical mean to deliver electrical field and modulate neuronal

plasticity. I have proved their potential for combinational therapies and that further studies should

be devoted to fine tune their stimulation patterns to exploit neuronal regeneration cues.

93

Chapter 9

Experimental design and characterization of

piezoelectric materials


Chapter 8 presented poly-vinylidene fluoride (PVDF), the most popular piezoelectric polymer in

the market due to their high piezoelectric constant and good mechanical properties. For those

reasons, included biocompatibility, PVDF is the materials of choice for the substrates I used in

the experiments presented in Chapter 10. First, I describe the manufacture process of the PVDF

films I fabricated for these set of experiments. I designed the process to confer these materials

with very specific crystalline, mechanical and piezoelectric properties to optimize their suitability

in cell culture. I have also included the results of characterization for those films. Then, I present

the experimental set-up and the characterization of VIBES, the vibratory device which was

developed to vibrate cell-plates with autonomy within the incubator. Finally, I present the

analysis tools I used to study arborization and alignment.

94

9.2. Fabrication of piezoelectric films

Films of polyvinylidene fluoride were fabricated from pellets with a molecular weight, Mn. of

180,000 (Soltex, Houston, TX). Films were extruded with a Brabender Extruder® at 185ºC at a

rate of 10 rpm and extruder speed of 70 rpm. First, films were shaped according to the ASTM

International standard D638 to produce uniform strain during stretching. To convert films from

crystalline phase α (paraelectric) to phase β (ferroelectric), they were stretched (uniaxially

oriented) and some were then polarized [160]. Stretching ratios up to 3.5:1 were achieved using a

custom film stretcher at 50oC and a drawing speed of 8 μm/second. After stretching, films were

cooled in place at 25oC for 12 hours to allow relaxation. Final thickness ranged between 17μm

and 22μm. The ferroelectric films were then polarized at 150 MV/m for 1 hour using contact

electrodes, which re-oriented the crystals by ferroelectric switching of the molecular dipoles and

made them piezoelectric. For non-piezoelectric control samples, films were identically

manufactured but not polarized. For simplicity, stretched films that were polarized and therefore

piezoelectric are denoted as PZ, and those not polarized (and hence non-piezoelectric) are

denoted as PV.

9.3. Characterization of materials

9.1.3. Rheolograph

Gold electrodes, with offset tabs, were sputtered on opposite sides of PVDF films. The relative

dielectric constant, εr , young modulus , c31, and piezoelectric coefficients d31 and e31, were

measured with a Rheolograph Solid® (ToyoSeiki, Japan) at a frequency of 10 Hz with a static

load of 5g at room temperature to keep the films from buckling during the compressive part of the

measurement cycle. Values of those constants for drawn and polarized PVDF films (PZ) and are

listed in Table 7. Both real and imaginary parts were measured, with no significant contribution

of the imaginary part. All errors are standard errors.

95

R[Young

Modulus[

I[Young

Modulus] R[d31] I[d31] R[e31] I[e31] R[εr] I[εr ]

(GPa) (GPa) (pC/N) (pC/N) (mC/m2) (mC/m2)

3.26±0.07 0.13±0.00 22.60±1.23 -0.84±0.09 73.90±3.99 0.17±0.06 11.30±0.72 -0.25±0.03

Table 7-Rheolograph measurements for PZ films fabricated as described in methods

Drawn and non-polarized PVDF films (PV) exhibited topographic, elastic and dielectric

properties equivalent to the polarized PVDF films (PZ) but had approximately zero

piezoelectricity (d31~0, e31~0).

9.2.3. Digital Scanning Calorimetry

The thermal characteristics of the samples, such as glass transition temperature (Tg) or melting

temperature(Tm), from extruded PVDF films were obtained using a DSC 2910 (TA Instruments,

Texas). All DSC measurements were performed at a rate of 10 °C/min for the heating ramp and

under a dry nitrogen gas atmosphere. The Glass transition temperature (Tg) for this polymer is

around -140oC [161] and it is not shown in Figure 22. S70 acronym represents the films extruded

at a speed of 70rpm which were used in the experiments hereafter described.

Figure 22- DSC graphic showing the melting temperature of PVDF.

96

9.3.3. Fourier Transform Infrared Spectroscopy

The crystal form of the drawn films was studied by Fourier Transform Infrared Spectroscopy

(FTIR) using a Nicolet Magna 760 IR spectrometer and analyzed using Omnic software (Fisher

Scientific). The piezoelectric strain and stress coefficients d31 and e31, the axial elastic modulus,

c31, and dielectric constant, εr, were measured with a Rheolograph Solid® (ToyoSeiki, Japan) at a

frequency of 10 Hz with a static load of 5g at room temperature to keep the films from buckling

during the compressive part of the measurement cycle.

The effect of polarization on FTIR spectra for PVDF films is shown in Figure 23. These results

confirm the transition from the α phase (paraelectric) in extruded un-drawn films to β-phase

(ferroelectric) in drawn films. The characteristic peaks of the α-phase and β-phase have been

marked for convenience.

Figure 23- FTIR spectra of non-polarized PVDF (PV, top) and piezoelectric PVDF (PZ, bottom).

The characteristic peaks of the α-phase and β-phase have been marked for convenience.

97

9.4. Cell culture

9.1.4. Mixed Spinal Cord (SC) Cell Cultures

Spinal cords were dissected from embryonic day 16 rats, and cells were dissociated by

mechanical trituration and seeded in 24 well plates at a density of 50,000 cells/cm2. For cells

grown in PVDF-TrFe films the density used was 100,000 cells/cm2. Cells were cultured in

Neurobasal medium (Gibco) containing 2% B27 supplement (Gibco), 69µg/ml L-glutamine,

25µM beta-mercaptoethanol, and 1% penicillin/streptomycin. Cells were grown for 5 days at

standard incubation conditions [162]. After plating, 24 hours were allotted for cells to attach

before onset of stimulation.

9.5. Setup and Stimulation Protocol

Polymer films were cleaned and sterilized with 70% ethanol prior to use and placed into 24 well

culture dishes with cross-linked poly(dimethyl siloxane) (PDMS) (Sylgard, Dow Corning, MI) O-

ring anchors were placed on top of the films. The O-rings secured the films to the bottom of the

well as seen in Figure 24. Films were coated for 1 hour with 0.2 mg/ml Poly-D-Lysine (PDL) in

borate buffer at 37oC and washed three times with phosphate-buffered saline (PBS).

Figure 24- Set-up of the well plate for seeding.

Cells were tested using four sets of film conditions: unstimulated piezoelectric polymer (US-PZ),

stimulated piezoelectric polymer (S-PZ), unstimulated polymer (US-PV), and stimulated polymer

(S-PV). Cells were seeded and rested for 24 hours before starting mechanical stimulation.

98

Stimulated plates were subjected to vibration from VIBES (Figure 25). VIBES is a custom

vibrating platform as shown in Figure 24 that can be sterilized and placed in the incubator. It

provides a vertical vibration to plates for up to 42 hours before recharging, with selectable

frequency and amplitude. Un-stimulated plates were cultured in the incubator on top of PDMS

mats to isolate them from the vibration of the incubator, and VIBES was isolated itself with the

same type of mats. All films were placed with the dipole orientation up.

Figure 25- VIBES: Polycarbonate base for stimulation of well plate cell cultures.

The stimulation protocol consisted of a mean vibratory frequency at 50 Hz and fixed vibratory

amplitude of approximately 0.3g, to produce a varying electric field on the surfaces of the

polymer. Wells used in the experiment were arranged in a radial configuration around the center

of the plate. For a single experiment, each condition was run at least in duplicate wells. For

experiments with SC neurons grown on PVDF materials vibration was applied continuously for

96 hours.

Variations in vibration frequency and amplitude between wells were approximately 0.5% and

12%, respectively. A MSI® sensor was used to characterize the well plate by gluing it to the

bottom of one of the wells (Figure 26)

99

Figure 26- Mechanical characterization of well-plate stimulation system: contour plot of

frequency (left) and amplitude (right) across the well plate.

9.1.5. Electrical Field Measurement

The piezoelectric response was measured on films coated with gold electrodes sputtered onto

both surfaces. The variation in voltage between the surfaces of the film when vibrated at different

amplitudes was measured using a voltage amplifier and a Hewlett Packard digitizing

oscilloscope. Calculation of charge density such created was made using the dielectric properties

of the film.

Polarization magnitude, P

, can be estimated from the charge density on the surface of the

material, , according to equation (1), where d̂ is the unit vector in the direction of polarization,

in this case, the thickness of the polymer.

dP ˆ

12

The electric displacement, D

, can be described by (2), where o is the permittivity of vacuum

and E

is the electrical field.

PED o

, 13

Due to the boundary conditions, on the surface of the polymer, D

is constant in the direction

normal to the surface. The contribution to D

within the material thus is P

, and outside, the

contribution is E

. Thus,

100

o

PE

14

Therefore, vibratory oscillations modify the net polarization of the piezoelectric material in such a

way that EFs on the PZ film surface changes its magnitude in synchrony with them.

To correlate the electrical field sensed by the cells upon vibration with the volume and

frequencies of VIBES, one of the fabricated piezoelectric films with sputtered electrodes is placed

in the well in the same arrangement used for cell culture.

Knowing the voltage across electrodes as measured by the voltage amplifier and the dielectric

constant of the material, the following relation between volume of VIBES and electrical fields

was found. Calibration of VIBEs was made both in empty wells and in wells with deionized

water. Results in Figure 27 account for the latter.

Figure 27: Mechanical characterization of well plate stimulation at 80Hz, 50Hz and 20Hz.

Therefore, in the presented experiments, when vibrated at 30 points, VIBES at 50 Hz, the S-PV

film exhibited an oscillating electrical field of 50,000 mV/mm peak, corresponding to a voltage

difference between surfaces of 52 mV peak, or a polarization variation of 44.24pC/cm2 peak.

101

9.6. Immunocytochemistry

After 5 DIV (or 4DIV in the case of cortical neurons), neuronal cultures were fixed with 4% PBS

for 30 min at 37 °C. Neurons were permeabilized in blocking solution (5% normal goat serum,

0.02% sodium azide, and 0.1% Triton X-100 in PBS). Neurons were immunostained using a

1:500 dilution of anti-MAP2 (Chemicon, Temecula, CA, USA.) for 1–2 h at room temperature

and then incubated for 1–2 h with a secondary antibody conjugated to fluorophore (Jackson

ImmunoResearch, West Grove, PA, USA).

9.7. Cell analysis and imaging

9.1.7. Neuronal Cell Cultures

Neurons were imaged on an Olympus Optical IX50 microscope (Tokyo, Japan) with a Cooke

Sensicam CCD cooled camera and fluorescence imaging system and ImagePro software

(MediaCybernetics, Silver Spring). Images were inverted to black cells on a white background to

visualize dendrites with greater accuracy. For each well, an average of 15 neurons was analyzed.

Pictures were randomly selected by scanning the well from left to right and from top to bottom

until enough neurons had been acquired.

Analysis was done exclusively on isolated neurons, with sufficient space to grow. Excluded from

the analysis were those that were part of a cluster, binary system, or overlapped with another

neuron, and those with less than a 180o

spatial range. This method was intended to allow the

processes to reach their maximum expansive potential. Processes are designated generically as

neurites.

Division by order was performed using a centrifugal labeling scheme, where processes attached

to the soma have a number of 1 and the number increases at each branch point, as illustrated in

102

Figure 28.

Figure 28- Centrifugal labeling scheme. Processes attached to the soma (green circle) have an

order of 1. At each branch point the order is increased by one.

For neurite analysis, we used Bonfire software, developed in the Firestein laboratory at Rutgers

University [163]. Bonfire is a semi-automatized software program that assists in neurite

branching analysis by interfacing between the plugin NeuronJ [164] of Image J (NIH, Bethesda,

MD, USA) and Neuron Studio [165]. Bonfire uses MATLAB scripts to calculate different metrics

that characterize neurite arborization. It computes number of branch points, terminal points, and

number of processes, in total and broken down by order. It also performs Sholl analysis, with the

possibility of breaking the curve down into branch order as well.

Sholl analysis is a method used to quantify the morphology of dendritic fields. It is performed by

counting the number of neurites that cross each circle from a set of successive concentric circles

drawn around the centroid of the soma of the neuron. We performed Sholl analysis with an inner

ring at 9.3 μm and at ring intervals of 6 μm from the first ring.

Neuronal density was done by counting images at 20X and using the corrective factor 7

cells/mm2. At least 5 images per well were averaged as described in [166].

103

9.8. Statistical analysis

9.1.8. Neuronal Cell Cultures

Statistics were calculated using GraphPad Instat Software (San Diego, CA, USA). The non-

parametric test of Mann–Whitney was performed followed by the appropriate post hoc test. [167],

[168], [169],[170].

9.9. Summary

This chapter has reported the materials and methods for the experiments I describe in Chapter 10.

It has been included both the design, fabrication and characterization I made of the materials, the

design and characterization of the stimulation, the methods of culture for neurons and the

analyses performed in the data to study arborization, elongation and alignment.

104

Chapter 10

Growth of cells on piezoelectric polymer films


In Chapter 10, I report the effects of piezoelectricity and vibration in the branching features of rat

spinal cord neurons in terms of number of branch points, terminal points, number of processes

and length. Morphology of the neurite arbor is shown by Sholl analysis. I also show the

distribution by order of the process for all these metrics. Then, I present the effect that

piezoelectricity and vibration have on cell density. In all these experiments, the appropriate

controls allowed studying vibration, topography and piezoelectricity independently. Finally, I

discuss the implications of all these results in terms of the potential mechanisms of electrical

sensory.

10.2. Effects of piezoelectric polyvinylidene fluoride in spinal cord

neurons

The effect that vibration of a piezoelectric polymer as polyvinylidene fluoride (PVDF) has in

neuronal branching has been tested using vibrated polarized PVFD (S-PZ), non-vibrated and

polarized PVDF (US-PZ), vibrated non-polarized PVDF (S-PV) and non-vibrated non-polarized

PV (S-PV).

105

Stretched and polarized PVDF displays piezoelectricity upon mechanical deformation. Hence, S-

PZ displays piezoelectricity, US- PZ does not. But vibration itself may affect cells and that needs

to be accounted for. While US-PZ controls for the effects that polarized PZ can have in initial

cell-attachment, S- PV controls for the effects of solely vibration on the cells.

Hereafter I reported the effects of piezoelectricity and vibration on spinal cord neurons. PVDF

was obtained from extrusion, stretched to a ratio of 3.5:1 and polarized at 150MV/m to induce

piezoelectricity, with a piezoelectric constant of d31~19pC/N. Rat spinal cord neurons E16 were

plated at a density of 50,000 cells/cm2 density on films coated for 1h in Poly-D-lysine (PDL).

Stimulation of the plates started after 24 hours of seeding and produced and stress of 0.3g and an

electric field of 50,000mV/mm. Stimulation was delivered continuously for 4 days and cultures

were fixed and stained with MAP2 at 5 DIV.

A B

C D

Figure 29- Spinal cord cultures immunostained with a MAP2 antibody after 5 DIV in the four

conditions: (A) US-PV, (B) S-PV, (C) US-PZ and (D) S-PZ. Scale bar=30 µm.

Representative images of immune-stained neurons for each of the four conditions are shown in

106

Figure 29.

10.1.2. Branching, terminal points and process number per cell

Arborization of neurite fields were quantified according to 3 metrics: (1) number of branch

points, (2) terminal points, and (3) number of processes. Overall results are presented in Figure

30, where the left graph shows the effect of vibration on PV and the right graph shows the effect

of vibration on PZ.

As seen in Figure 30A, vibration of the non-PZ films reduced all of the arborization measures

substantially (25-50%) but not significantly. When applied to PZ films (Figure 30B), vibration

increased all 3 measures: branch points, terminal points, and processes (p<0.001). These data

support the positive effect of piezoelectric growth stimulation of neurites, independent of strictly

mechanical cues, such as structural film properties and vibration

A B

Figure 30- Comparison of branching features between (A) US-PV and S-PV and (B) US-PZ and

S-PZ. *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-Whitney test). Standard error

depicted.

The results of PV and PZ have been split in two graphs because of possible differences in coating

due to polarization. PZ films were placed with the net dipole facing up. This might have an effect

in the coating of PZ films with the positively charged PDL, which would not be present in the

107

coating of PV films.

10.2.2. Average number of processes per cells split by order

If divided by order, branching in the piezoelectric polymers is significantly bigger. Analysis of

arborization, in term of number of neurites, as a function of branching order is shown in Figure

31. Firstly, vibration did not significantly affect neurite number, at any order, as seen in Figure

31A, although trends were similar to that in Figure 30A. Vibration of PZ films, however,

enhanced branching at all order levels, as seen in Figure 31B. The neurons grown on stimulated

PZ has significantly more primary, secondary, and higher order processes than those grown on

non-stimulated PZ.

A B


and (B) US-PZ and S-PZ. * p<0.05, *** p<0.001, **** p<0.0001 (Unpaired t-test/Mann-Whitney

test). Standard error depicted.

10.3.2. Average length of process per cell by order

Vibration had no significant effect on the length of neurites found in neurons grown on either PV

or PZ materials as shown in Figure 32.

A B

108


and (B) US-PZ and S-PZ. (Unpaired t-test/Mann-Whitney test). Standard error depicted.

10.4.2. Sholl Analysis

The morphology of the arbor field is studied by Sholl analysis. As seen in (Figure 33A, vibration

did not affect intersections of neurons grown on PV films. Neurons grown on the vibrated PZ

film, in contrast, (Figure 33B), exhibited around 85% more intersections. These results again

support the stimulatory effect of piezoelectric activity on neurite outgrowth and branching.

A B

Figure 33- Comparison of Sholls analysis of the total number of neurite intersections between

(A) US-PV and S-PV and (B) US-PZ and S-PZ. Bar indicates significance * p<0.05 (Unpaired t-

test/Mann-Whitney test). Standard error depicted.

109

10.5.2. Neuronal density

Figure 34 shows the effect that vibration and piezoelectricity have in neuronal density after four

days of stimulation. There is a significant increase in neuronal density between US-PZ and S-PZ

but not in the PV materials, suggesting that piezoelectricity might increase the viability of cells,

reduce apoptosis or enhance the expression of integrins and that effect is independent of

vibration.

A B

Figure 34- Comparison of neuronal densities for PV substrates (A) and PZ substrates (B) **

p<0.01 (Unpaired t-test/Mann-Whitney test). Standard error depicted.

10.3. Discussion

Rat spinal neurons were subjected to alternating electrical fields applied directly from their

culture substrate for a total of 4 days, the polarization variation being of 44.24pC/cm2 at its peak.

In order to establish an independent effect of piezoelectric activity on neurite growth, it is

necessary to rule out confounding variables that may affect growth. These include both structural

features of the substrate and the mechanical vibration. It is well known that the first variable

directly influences neurite growth since neurons are exceedingly sensitive to substrate

morphology at the nano-to micro scale [171-173]. The surface of PZ film is fibrillar, with

uniaxially oriented nanofibers on the order of 10 nm, produced during the stretching process as

characterized by [174]. This type of surface morphology provides a permissive substrate for cell

growth and provides cues for growing neurons [175].

110

To eliminate the structural cues as a systematic variable, I restricted the analysis to substrates that

were fabricated identically, coming from the same sample of stretched and polarized PVDF and

differing only in their treatment in the incubator: vibrated or not. These two substrates, labeled

US-PZ and S-PZ in Figure 30B, Figure 31B, and Figure 33B, yielded neurons with significantly

different morphological attributes. Arborization, as measured by branch points, more than

doubled, and the number of terminal processes and intersections increased by approximately 80%

in neurons grown on the S-PZ substrate as compared to those grown on the US-PZ.

To identify whether vibration played an independent role in regulating neuritogenesis, I tested its

effects on neurons grown on non-piezoelectric films. The PZ effect couples a mechanical input to

an electrical output. To establish a baseline effect of mechanical vibration on neuronal plasticity, I

measured the morphological attributes of neurons under the isolated influence of mechanical

vibration, i.e. on polymers not induced with PZ activity via polarization. Results from these two

conditions, labeled S-PV and US-PV, are seen in Figure 30A, Figure 31A, and Figure 33A. As

seen in Figure 30A, vibration does not increase growth but rather reduces all 3 metrics, although

this effect does not reach a statistical significance at the P<0.05 level. The results here presented,

not only point toward the possible detrimental role of vibrations on stimulating growth but also

suggest that the EF produced by the 50 Hz piezoelectric effect may overcome its negative effects

on neurite growth. S-PZ substrates did not show a significant decrease due to vibration.

Contextualization of this result is difficult, as few studies have investigated effects of vibration on

cultured cells. One study on the effect of nano-vibration, at 10 KHz, on neurite growth in PC12

cells showed significant enhancement after several days. The mechanism behind this non-

physiological stimulation was attributed strictly to vibratory enhancement of nerve growth

factor[98]. However, the different range of frequency and cell type may account for the

111

discrepancies Altogether, my results support the idea that EF’s produced by vibrating PZ films

enhance neurite branching at all order levels in neurons on spinal cord neurons

Factors that may affect arborization include cell density, neuronal density, growth factor

concentration, glutamate concentration, substrate stiffness and calcium concetration. Some of

them work in combination and/or have mechanistic feedback loops (reviewed from [75]). Figure

34B shows that neuronal densities increased significantly (more than 100%) in S-PZ substrates as

compared to those grown on US-PZ. However, there is a non-significant decrease in neuronal

density between US-PV and S-PV as shown in Figure 34A. Those findings suggest a possible

role of piezoelectricity in the activation of cell adhesion proteins.

Mechanical properties of substrates have been reported to activate the integrin population [176],

[177]. Integrins show increase expression in neurons grown in stiffer substrates and integrin

expression has been to cause increased neurite branching [178]. Whether the branching observed

in these experiments as a result of piezoelectricity is due to integrin over-expression partly or

entirely remains to see.

One likely mediator of the EF-induced neurite branching are increases in internal calcium,

[Ca++

]I. The crucial role of [Ca++

]I in cellular movement associated with neurite growth is well

known [179], and its moment-to-moment level within microdomains is a primary indicator of cell

growth [180]. Turning direction of growth cones correlates well with local [Ca++

]I as shown by

the action of chemotactic agents, such as netrin, that increase Ca++

selectively within the turning

side of the growth cone. Direct evidence that [Ca++

]i , and specifically voltage-gated Ca

++

channels, play a major roles in EF galvanotropism was shown in mouse neuroblastoma cells,

whose neurite extension and growth cone elongation toward the cathode correlated directly with

cathode-directed elevation in Ca++

levels and depolarization [181]. Calcium dynamics also

112

regulate the activation of integrins, suggesting another potential mechanism of neuronal

outgrowth via Ca++

dynamics. Another likely mediator of neurite growth is cAMP, as suggested

in a study where outgrowth from both DRGs in vitro and spinal sensory nerves in vivo was

enhanced by electrical stimulation at 20 Hz [145].

10.4. Summary

The results here presented show that PZ stimulation of central neurons (spinal cord) promotes

their outgrowth in culture. Using image analysis, I showed that EFs from PZ promote branching

of neurites, increasing their number at all order levels. The total range of the neurite field

increases also upon exposure to piezoelectricity, although the average process length of the

processes is not affected by it. These phenomena are independent of vibration and topography of

the substrates. The observation of increased neuronal density upon piezoelectricity exposure

provides some mechanistic insight of the phenomena into the possible role of integrins

The results from these experiments also show that mild vibrations at 50Hz to neurons of the

central nervous system do not have a significant effect in outgrowth, although the trends suggest

that vibration might decrease branching. Vibrations at 50Hz have not shown to have any effect in

neurite length, total extension of the neurite field or neuronal density.

113

Chapter 11

Conclusion

I have developed: (1) an injury model that allows assessment of treatments for injuries and

diseases that lead to muscle denervation, and (2) a novel treatment paradigm based on

piezoelectric polymers. This injury model is appropriate for spinal cord injuries produced by

contusion and for motor neuron diseases affecting lower motor neurons like spinal muscular

atrophy, amyotrophic lateral sclerosis, progressive muscular atrophy, pseudobulbar palsy and

myasthenia gravis. The injury model I proposed is a bilateral contusion at the thoracic level which

denervates the intercostal muscles. The use as model of injury of such a relevant system like

respiration, encourages the study of problems related to the autonomic and somatic systems in the

context of spinal cord injury. Although I have used a contusion injury, as it is the most relevant

scenario, the model could be easily extended to hemisection with the additional advantage that

the same animal could be used as their own controls.

The technique of assesment of function involves injurying the spinal cord and studying the

electromyographic signal of the denervated intercostal muscle with time. I found that the most

sensitive parameters to identify functional improvement are the turns/second and zero/seconds of

the Interference Pattern Analysis. The signal was studied with EMGvet, a new software I

developed to account for specifics of the electromyographic signal of rats.

114

Many in vivo studies have demonstrated the power of electrode stimulation to enhance neurite

growth and promote axonal regeneration. Oscillating electrical fields delivered by electrodes have

achieved clinical success with spinal cord regeneration [182], and thus, development of new,

more versatile electrical interfaces is an important task. In vitro studies have described the effect

of electrical fields in mammalian neurons but have focused mostly in the peripheral nervous

system. Herein I have shown that electrical stimulation can be delivered directly to cells via a

piezoelectric polymer. The use of polymer substrates for applying electrical charge in vivo may

have advantages over electrodes because polymers can also provide structural and chemical

growth enhancement while delivering electrical fields intimately within the cellular environment.

Piezoelectric polymers represent a versatile platform by which to promote neuronal growth, since

they not only can deliver EFs efficiently, they can be doped with chemical growth factors, and

arranged as scaffolds, providing a complete complement of physical and chemical growth

enhancers.

Herein, I have presented a multidisciplinary approach to the problem of Spinal Cord Injury. I

have provided researchers of the field with a refined set of tools that include software, techniques

and models to test more effectively future therapies. I have also studied the effect in neuronal

branching of piezoelectricity and I have presented the potential of piezoelectric biomaterials for

combinational therapies leading to regeneration of the central nervous system. Importantly, the

work here presented establishes a foundation on which future work in SCI research can be

performed

115

Chapter 12

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