shedding light on mitochondrial function in vivo:
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Shedding Light on Mitochondrial Function In Vivo: From Experimental animals to clinical Applications. The Mina & Everard Goodman Faculty of Life-Sciences and The Leslie & Susan Gonda Multidisciplinary Brain Research Center Bar- Ilan University, Ramat- Gan , 52900, Israel. - PowerPoint PPT PresentationTRANSCRIPT
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: [email protected]: [email protected]
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
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