Shedding Light on Mitochondrial Function In VivoShedding Light on Mitochondrial Function In Vivo::From Experimental animals to clinical ApplicationsFrom Experimental animals to clinical Applications

October 4th 2012

The Mina & Everard Goodman Faculty of Life-SciencesThe Mina & Everard Goodman Faculty of Life-Sciences andand

The Leslie & Susan Gonda Multidisciplinary Brain Research CenterThe Leslie & Susan Gonda Multidisciplinary Brain Research Center

Bar-Ilan University, Ramat-Gan, 52900, IsraelBar-Ilan University, Ramat-Gan, 52900, Israel

ESCTAIC 23rd Congress 2012

Timisoara, Romania

Email: mayevskya@gmail.comEmail: mayevskya@gmail.com

Bar Ilan University Ramat Gan ISRAELBar Ilan University Ramat Gan ISRAEL

Short History –Monitoring of Mitochondrial function and Tissue Energy Metabolism .

“There is no instance in which it can be proven that an organ increases its activity under physiological conditions, without also increasing in its call for oxygen, and- in no organ excited by any form of stimulation can it be shown that positive work is done without the blood supply having to respond to a call for oxygen”.

Barcroft J. The Respiratory Function of the Blood.

Cambridge Univ. Press, Cambridge, 1914

HbO 2

Microcirculatory Hemoglobin Oxygenation

(Tissue Oximeter)

Venule

O2

O2

O2

O2

2

O2

O2

O2

O2

O2

O2

O2

O2

Major artery

Arteriole

Capillary

Tissue Blood Flow (Laser Doppler Flowmetry)

MicrovascularArterioles & capillaries

O2

Systemic HemoglobinOxygenation

(Pulse Oximeter)

Large Artery A

B

C

D

O2

ETC-OXPHOS

ADP+Pi

ATP

H2O

AcC

oA

CO2H+

NAD+ NADH

TCA

GlycolysisGlucosePyruvate

Lactate

Mitochondrial NADH

(Fluorometry)

NADHNADH

100

20-30

1

95

0

50

100

150

AlveoliArterial Blood

AIR Tissue Intramitochondrial

Oxy

gen

Par

tial P

ress

ure

(mm

Hg)

160N2

O2

The partial pressure of oxygen inside the mitochondria is less than 1 mmHg

ATP Production in the Cell ATP Production in the Cell

Valid assessment of the metabolic – oxygenation state at the Valid assessment of the metabolic – oxygenation state at the tissue / cellular leveltissue / cellular level

Real-time – continuous measurementReal-time – continuous measurement

Measures sensitive to metabolic deviations as well as to Measures sensitive to metabolic deviations as well as to corrective interventionscorrective interventions

Clinical Unmet NeedsClinical Unmet Needs

ICU: A Complex Setting ICU: A Complex Setting in Search of Valid in Search of Valid

Diagnostic & Treatment Diagnostic & Treatment Support ModalitiesSupport Modalities

88

100

20-30

1

95

0

50

100

150

Alveoli Arterial Blood

AIR Tissue Intramitochondrial

Oxy

gen

Par

tial P

ress

ure

(mm

Hg)

160N2

O2End Tidal

CO2 Heart Rate &ECG

Cardiac Output

Systemic Blood Pressure

Systemic Saturation(Pulse Oximetry)

CritiViewMicrocirculation blood flow and

oxygenationNADH redox state

In 1857, Albert von Kölliker described what he called “granules” in the cells of muscles.

The discovery of mitochondria in general came in 1886 when Richard Altman, a cytologist, identified the organelles and dubbed them “bioblasts.”.

Carl Benda, in 1898, coined the term mitochondria from Greek thread, mitos, and granule, chondros.

Discovery of the Mitochondrion

There is no real single answer regarding who discovered mitochondria. The process of discovery and identification was a gradual one that has spanned the last 150 years.

The use of light in studying Mitochondrial function was introduced by my Post-Doc Mentor and teacher

Prof. Britton Chance more than 50 years ago .

The created light is helping us to shed new light into the darkness of Mitochondrial Functions

Mitochondrial Function and NADH fluorescence measurementsMitochondrial Function and NADH fluorescence measurements

The definition of mitochondrial metabolic state in 1955, by Chance and Williams, opened up a new era in spectroscopic measurements of respiratory chain enzyme’s redox state In Vitro as well as In Vivo.

NADH Oxidation-NADH Oxidation-Reduction StateReduction State is the is the

best parameter for best parameter for evaluating Mitochondrial evaluating Mitochondrial

Function In VivoFunction In Vivo

Chance et al in 1973 concluded that “For a system in a steady state, NADH is at the extreme low potential end of the chain, and this may be the oxygen indicator of choice in isolated

mitochondria and tissues as well.”

Chance, B., Oshino, N., Sugano, T., Mayevsky, A., 1973. Basic principles of tissue oxygen determination from mitochondrial signals. In: Internat. Symposium on

Oxygen Transport to Tissue, Adv. Exp. Med. Biol. Vol.37A, pp.277-292. Plenum Pub Corp, New York ,

Why NADH ???

NADH Oxidation-Reduction StateNADH Oxidation-Reduction State is is

the best parameter for evaluating the best parameter for evaluating

Mitochondrial Function In VivoMitochondrial Function In Vivo

Lubbers, D.W. 1995. Optical sensors for clinical monitoring. Acta Anaesth. Scand. Suppl. 39, 37-54.

Lubbers in 1995 concluded that “the most important intrinsic

luminescence indicator is NADH, an enzyme of which the reaction is

connected with tissue respiration and energy metabolism”

Why NADH ???

Am. J. Physiol. Cell Physiol. 292: C615-C640 )2007( .

A. NADH - The Mitochondrion “Flag”

B. Absorption Spectra of NAD+ and NADH

C. NADH Fluorescence spectra

nm

y = 0.0191x + 0.3529

R2 = 0.9945

y = 0.0195x + 0.4522

R2 = 0.9918

0

1

2

3

4

5

6

7

0 100 200 300 400NADH)mM(

Cri

tiV

iew

)V

olt

s(

set#1

set#2

NADH Calibration in SolutionNADH Calibration in Solution

In Vivo Monitoring of NADH redox stae using Optical FibersIn Vivo Monitoring of NADH redox stae using Optical Fibers

A

Head Holder

Micromanipulator

Probe Holder

Surgical ToolsB

1 2

3

C

The first Fiber optic based Time-Sharing Fluorometer/Reflectometer The first Fiber optic based Time-Sharing Fluorometer/Reflectometer

Mayevsky and Chance 1972

Anoxia Ischemia

RefRef

FluFlu

CFCF

ECoGRightECoGRight

100%

100%

100%

100mV

B

A

Metrazol

A

B

C

Metrazol

KCl NaCl

Dog Heart- Open Chest

Fiber Optic Holder

B

b

a

c

C

A

A

B

kidney

Testis

Small Intestine

Liver

Heart

Urethra

Spinal cord

Animal

ClinicalClinicalPigs

Ischemia

NE

NE

Ischemia

NE

HypercapniaPapaverine

Ischemia

N2

NEHemorrhage AAA

ICU

Bypass

Pacing

Hypopnea

Ischemia

Drugs )Ach, NE,

vasoactive(

Compression

Ischemia

Oxygen deficiencyIschemia

NO

Drugs

TBI

HyperbariaHBO

Clinical

Activation

CO

Hemorrhage

Hypothermia

Aging

Sepsis

Epilepsy

SD

Mannitol

ICP elevation

Retraction

Anoxia

Hypoxia

Hypercapnia

Nimodipine

Ethanol

Anesthetics

UncouplerDuring operation

ICU

Brain

Multiparametric Monitoring Multiparametric Monitoring of Tissue Energy Metabolismof Tissue Energy Metabolism

WHYWHY? ?

Limitations in NADH monitoring in Vivo:

1. Relative Measurements and not calibrated yet in absolute units.

2. The NADH fluorescence signal must be corrected , in blood perfused organs, for hemodynamic artifacts.

3. In order to interpret the NADH signal it is necessary to monitor the microcirculatory events as well.

Monitoring of NADH and Tissue Blood Flow

TissueNADH

Tissue Blood Flow

min.Normal

NormalNormal

Brain

Hyperoxia

Ischemia

Hypocapnia

Hypoxia

CO Exposure

Spreading Depression

Hypercapnia

Monitoring of 2 Parameters

min. Tissue Blood FlowNormal

Normal

Brain

Hyperoxia

Hypocapnia

Ischemia Hypoxia

Heart pacing

Tissue activation

Hypercapnia

Tissue Blood Flow Alone

min.

TissueNADH

NormalNormal

Brain

Hyperoxia

Ischemia

Hypocapnia

Hypoxia

Heart pacing

Tissue activation

Hypercapnia

Tissue NADH Alone

NA

DH

Red

ox S

tate

Cerebral Blood Flow

0 100

100Normal

BrainHyperoxia

IschemiaVasopasm

Death

Hypoxia

CO low

SD

Hypercapnia

200

200

CO high

Seizures

Anes.SD+Ischemia

Hypocapnia

SD=Spreading Depression; CO=Carbon Monoxide; Anes.=Anesthesia

“The more parameters you monitor...The better you can differentiate between

states”

Multiparametric Monitoring of Multiparametric Monitoring of Tissue Vitality in Vital and Tissue Vitality in Vital and

Less-Vital organs:Less-Vital organs:

Experimental Animal ResultsExperimental Animal Results

33

Blood flowRedistribution

Blood flowRedistribution

CardiacArrest

CardiacArrest

Body Emergency Metabolic State )BEMS(

Body Emergency Metabolic State )BEMS(

TraumaTraumaHypoxiaHypoxiaPrenatal

HypoxemiaPrenatal

HypoxemiaSepsisSepsisShockShock

Activation of the Sympathetic Nervous System

Activation of the Sympathetic Nervous System

Secretion of Adrenaline intoblood stream

Secretion of Adrenaline intoblood stream

Less Vital Organs Skin

MuscleG-I tractUrogenital

Less Vital Organs Skin

MuscleG-I tractUrogenital

Decreased Tissue Perfusion

Decreased Tissue Perfusion

MitochondrialDysfunction

MitochondrialDysfunction

Energy FailureEnergy Failure

Highly VitalProtected Organs

BrainHeart

Adrenal Glands

Highly VitalProtected Organs

BrainHeart

Adrenal Glands

Increase tissue Perfusion

Increase tissue Perfusion

Better O2 Supplyto mitochondria

Better O2 Supplyto mitochondria

Energy productionPreservation

Energy productionPreservation

Body Blood Flow Redistribution Under Body Blood Flow Redistribution Under

Emergency Metabolic StatesEmergency Metabolic States

Multi-Site Multi-Parametric Multi-Site Multi-Parametric systemsystem

Med Sci monit, 2007: BR211-219

Effects of Hypoxia Effects of Hypoxia (12% O(12% O22))

Brain

Intestine

Intestinal (red) and Brain (blue) responses to

Epinephrine 10 µg/kg

0

100

200

*

0

100

200 * *

0

100

200

300

400

* *

0

100

200

-2 -1 0 1 2 3 4

Time )min(

* *

EPINEPHRINE 10µ

NA

DH

(%

)TB

F (%

)M

AP (

mm

Hg)

Brain

Intestine

Mitochondria In-Vitro* Brain In-Vivo**

NA

DH

.. Seizures

Anaesthesia

~100

99

53

~0

NADH%

RespirationRate

Limiting Substance

2

4

3

5

State#

Slow

Slow

Fast

0

Substrate

Oxygen

Resp. Chain

ADP

Min.

..

Metabolic state

Max.

B l

o o

d

F l

o w

Max.

Min.Death

Ischemia

Hypoxia HBO

S.D.

HypoxiaHBO

Normoxia

HBO - Hyperbaric OxygenationS.D. - Spreading Depression

*According to Chance & Williams 1955 **According to Mayevsky 1984

Critical Clinical Situations Requiring Critical Clinical Situations Requiring

Measurement of Tissue and Body Measurement of Tissue and Body

VitalityVitality

Real time Monitoring of Vitality parameters in PatientsReal time Monitoring of Vitality parameters in Patients

System Oriented Monitoring

Specific Organ Oriented Monitoring

Systemic General Parameters

Systemic EarlyWarning Parameters

Heart Rate

Blood Pressure

End Tidal CO2

HbO2 Sat.

Blood pO2, pCO2, pH

Core Temperature

Non Vital Organs

Parameters

Skin

Muscle

GI Tract

Bladder

Urethra

pO2 , pCO2 , pH

HbO2

TBF

CritiView

Brain

Heart

Transplanted Organs

Skin and Muscle Flap

Limb Vascular Surgery

In the OR

In ICU

Aneurysm

Retraction

Bypass Grafting

A

B2

B

B1

Monitoring of Specific Organ Monitoring of Specific Organ

Vitality in PatientsVitality in Patients

Floating Probe in NeurosurgeryFloating Probe in Neurosurgery

Brain Tissue Probe in NeurosurgeryBrain Tissue Probe in Neurosurgery

Decrease of ICP by Suction of CSFDecrease of ICP by Suction of CSF

Real time Monitoring of Vitality parameters in PatientsReal time Monitoring of Vitality parameters in Patients

System Oriented Monitoring

Specific Organ Oriented Monitoring

Systemic General Parameters

Systemic EarlyWarning Parameters

Heart Rate

Blood Pressure

End Tidal CO2

HbO2 Sat.

Blood pO2, pCO2, pH

Core Temperature

Non Vital Organs

Parameters

Skin

Muscle

GI Tract

Bladder

Urethra

pO2 , pCO2 , pH

HbO2

TBF

CritiView

Brain

Heart

Transplanted Organs

Skin and Muscle Flap

Limb Vascular Surgery

In the OR

In ICU

Aneurysm

Retraction

Bypass Grafting

A

B2

B

B1

Clinical BackgroundClinical Background In emergency metabolic states In emergency metabolic states –– the body protects the the body protects the

vital organs (heart, brain) by diverting blood flow from vital organs (heart, brain) by diverting blood flow from less-vital organs (skin, muscles, intestine, urethra etc.)less-vital organs (skin, muscles, intestine, urethra etc.)

Potential disturbances in tissue:Potential disturbances in tissue: Blood flow: insufficient amount of blood.Blood flow: insufficient amount of blood. Blood oxygenation: insufficient amount of oxygen Blood oxygenation: insufficient amount of oxygen

in RBCin RBC Cellular (mitochondrial) function: impaired oxygen Cellular (mitochondrial) function: impaired oxygen

utilization and production of energyutilization and production of energy

Global monitoring parameters (BP, CVP, CO, etc.)Global monitoring parameters (BP, CVP, CO, etc.) Not sensitive enough to changes at the tissue level.Not sensitive enough to changes at the tissue level.

Intensive care: key principles Intensive care: key principles Early detection of evolving conditionsEarly detection of evolving conditions Optimal adjustment of therapeutic meansOptimal adjustment of therapeutic means

Monitoring of Patients in Monitoring of Patients in

Operative Rooms and Operative Rooms and

ICUsICUs..

100

20-30

1

95

0

50

100

150

Alveoli Arterial Blood

AIR Tissue Intramitochondrial

Oxy

gen

Par

tial P

ress

ure

(mm

Hg)

160N2

O2End Tidal

CO2 Heart Rate &ECG

Cardiac Output

Systemic Blood Pressure

Systemic Saturation(Pulse Oximetry)

CritiViewMicrocirculation blood flow and

oxygenationNADH redox state

In-vivo tissue spectroscopy In-vivo tissue spectroscopy by the CritiViewby the CritiView

Tissue Blood Flow

Doppler (Frequency) Shift

NADH

Mitochondrial Function

Spectroscopy

NADH

Tissue Reflectance

Back Scattered Light

B.V=Blood volume

Time )Sec(

Various Light Sources

Blood Oxygenation

450 500 550 600 650

Spectroscopy

HbO2

Wavelength )nm(

Fiber Optic Probes

Light Source Unit Single Floor

LED 470nm

LED 375nm

LED 530nm

DL 785nmPhotodiode

785nm

Photodiode375nm

DTU

Doppler

Oximetry

NADH Fluor

Refl

Output Optical

Connector

Input Optical

Connector

Photodiode

PMT 450, 470, 530nm

Doppler

Oximetry

Oximetry

NADH+Refl

IntensityMonitoring

PD

AA BB

CC

TBF

R375

R470

R530

Flu

NADH

HbO2

N2

2 min

Time )min(R+LOccl

2 min

KCl NaCl

Time )min(

TBF

R375

R470

R530

Flu

NADH

HbO2

AA

BB

CritiView is an adjunctive monitor which provides:CritiView is an adjunctive monitor which provides:(a) Sensitive identification / warning of the body(a) Sensitive identification / warning of the body’’s s critical metabolic imbalances or tissue vitality; critical metabolic imbalances or tissue vitality;

( ( bb ) )End point of resuscitationEnd point of resuscitation

These early warning changes are expected to be These early warning changes are expected to be in advance of current critical care monitors in advance of current critical care monitors capabilities to detect physiological changes at the capabilities to detect physiological changes at the tissue level.tissue level.

Thus allowing the clinician to make more refined Thus allowing the clinician to make more refined corrective medical actions.corrective medical actions.

Critiview’s First Application: Operating rooms and ICUsCritiview’s First Application: Operating rooms and ICUs

52

Cross section

of catheter

Tip of the Fiber optic Probe

developed by CritiSense

The fiber-optic sensor for measuring Urethral wall

vitality, is embedded in a disposable 3-way Foley

catheter.

The 3-Way Foley catheter in the human

bladderThe “smart” Foley Catheter

The CritiView Device, Probes and Clinical ApplicationsThe CritiView Device, Probes and Clinical Applications

5454

CritiView is an adjunctive monitor which CritiView is an adjunctive monitor which providesprovides::

((aa ) )Sensitive identification / warning of the body Sensitive identification / warning of the body critical metabolic imbalances or tissue vitalitycritical metabolic imbalances or tissue vitality;;

((bb ) )Indication of the end point of resuscitationIndication of the end point of resuscitation..

These early warning changes are These early warning changes are detected in advance of current critical detected in advance of current critical care monitoring capabilities for care monitoring capabilities for detecting physiological changes at the detecting physiological changes at the tissue leveltissue level..

This allows the clinician to take more This allows the clinician to take more refined corrective medical actionsrefined corrective medical actions..

CritiView Primary Applications: CritiView Primary Applications: Operating rooms and ICUsOperating rooms and ICUs

Abdominal Aorta AneurysmectomyAbdominal Aorta Aneurysmectomy

Patient MS686Patient MS686

Clamping Declamping

10 min

Clamping of the abdominal aorta led to dramatic decrease of TBF and large increase in NADH. After declamping of the aorta TBF returned to normal levels while NADH became more oxidized may be due to improved mitochondrial function. This response proves that when blood supply to the Urethral wall is blocked, the mitochondrial NADH was accumulated to its maximal level.

1 - 10 min before clamp

2- 10 min after clamp

3- Mid point of clamp period

4- 10 min before declamp

5- 10 min after declamp

6- Last 5 min off monitoring

p≤0.05*

p≤0.01**

p≤0.001***

AAA operations_Mean±SEM)N=5(

0

20

40

60

80

100

120

140

160

180

1 2 3 4 5 6

Stage of operation

TB

F/N

AD

H R

elat

ive

Val

ues

TBF

NADH

***** **

*

Patient #2: Tamponade, Monitored in the Cardiac ICU After A Bypass Grafting Procedure

The left side of the figure shows that during the initial 3 hours of monitoring the NADH levels were very stable (+ 20% changes). During the next 40 minutes of monitoring (right side) a clear mitochondrial dysfunction was recorded (100% increase in NADH). During this period a cardiac tamponade was developed and the patient was returned to the OR to stop the bleeding.

The hemodynamic parameters (Heart Rate and Blood Pressure) shown in boxes below the figure were not clearly correlated to the development of the tamponade.

116 80/50

150 97/73

132 83/54

135 76/50

127 97/51

132 82/51

142 81/54

HR

BP Systolic/Diastolic

112 142/79113 115/78 122 94/52

106 146/87

Blood Flow

NADH

Patient DD932 - 4.2.07Patient DD932 - 4.2.07

38min

CABG

In this patient the hemodynamic and mitochondrial responses started very early in the operation procedure.

Chest open

Pump on

Chest closure

Pump off

GS942 22 JAN 2007- 15H40MGS942 22 JAN 2007- 15H40M

22min

In this patient clear responses to the procedure were recorded. At 16:49, the pump ON condition led to a large decrease in TBF as well as a large increase in NADH. The signals returned toward the initial values although base line was not reached )monitoring period ends at 18:14(

Patient DS785 – 8.1.07Patient DS785 – 8.1.07

A

B

Severe bleeding and cooling the

patient

Pump on Pump off )flow to head only(

Pump Pump onon

Cooling

A. During the severe systemic bleeding )under normothermia ~37°C( significant decrease in TBF was correlated to the increase in NADH.

B. Under severe hypothermia )17-18°C(, the pump-off shift led to a significant decrease in TBF while NADH remained quite stable )possible decrease(, indicating possible protection of mitochondrial function by the hypothermia.

37min

1 - 5 min Baseline

2 -5 min after washing

3 -10 min after chest opening

4 -10 min after HLM on

5 -Mid point of HLM

p≤0.05*

p≤0.01**

p≤0.001***

Bypass Operations_ Mean ±SEM )N=11(

0

20

40

60

80

100

120

140

160

180

200

1 2 3 4 5 6 7 8

Stage Of Operation

TBF/

NADH

Rel

ativ

e Va

lues

TBF

NADH*******

*

*** ** ** **

*

N=3

6- 10 min before HLM off

7- 10 min after HLM off

8- Last 5 min off monitoring

6363

Thank YouThank You

www.critisense.com