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Shedding Light on Mitochondrial Function In Vivo:

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.

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Shedding Light on Mitochondrial Function In Vivo:

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  1. 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 Email: mayevskya@gmail.com ESCTAIC 23rd Congress 2012 Timisoara, Romania October 4th 2012

  2. Bar Ilan University Ramat Gan ISRAEL

  3. 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

  4. Major artery A Large Artery D NADH Mitochondrial NADH (Fluorometry) Systemic Hemoglobin Oxygenation ( Pulse Oximeter) Arteriole NAD+ NADH O2 AcCoA TCA O2 O2 Microcirculatory Hemoglobin Oxygenation (Tissue Oximeter) O 2 B O 2 H+ CO2 O2 H2O Capillary O HbO2 2 O2 ETC-OXPHOS O2 O2 2 ATP ADP+Pi O2 O2 C O2 Microvascular Arterioles & capillaries O Pyruvate Glucose 2 Glycolysis Lactate Venule Tissue Blood Flow (Laser Doppler Flowmetry)

  5. 150 160 O2 N2 100 50 20-30 1 0 Arterial Blood Intramitochondrial Tissue AIR Alveoli Oxygen Gradients from AIR to Mitochondria Oxygen Partial Pressure (mmHg) 100 95 The partial pressure of oxygen inside the mitochondria is less than 1 mmHg

  6. ATP Production in the Cell

  7. Clinical Unmet Needs • Valid assessment of the metabolic – oxygenation state at the tissue / cellular level • Real-time – continuous measurement • Measures sensitive to metabolic deviations as well as to corrective interventions ICU: A Complex Setting in Search of Valid Diagnostic & Treatment Support Modalities

  8. 150 160 O2 N2 100 100 95 50 20-30 1 0 Arterial Blood Intramitochondrial Tissue AIR Alveoli Oxygen Gradients from AIR to Mitochondria End Tidal CO2 Oxygen Partial Pressure (mmHg) Heart Rate &ECG Cardiac Output Systemic Blood Pressure Systemic Saturation (Pulse Oximetry) CritiView Microcirculation blood flow and oxygenation NADH redox state

  9. 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. 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.

  10. 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.

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

  12. Mitochondrial 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.

  13. 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 State is the best parameter for evaluating Mitochondrial Function In Vivo

  14. 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” Lubbers, D.W. 1995. Optical sensors for clinical monitoring. Acta Anaesth. Scand. Suppl. 39, 37-54. Why NADH ??? NADH Oxidation-Reduction State is the best parameter for evaluating Mitochondrial Function In Vivo

  15. nm A. NADH - The Mitochondrion “Flag” C. NADH Fluorescence spectra B. Absorption Spectra of NAD+ and NADH Am. J. Physiol. Cell Physiol. 292: C615-C640 (2007).

  16. NADH Calibration in Solution

  17. In Vivo Monitoring of NADH redox stae using Optical Fibers

  18. C B 1 Probe Holder 3 A Head Holder 2 Micromanipulator Surgical Tools

  19. The first Fiber optic based Time-Sharing Fluorometer/Reflectometer Mayevsky and Chance 1972

  20. Anoxia Ischemia 100% Ref 100% Flu 100% CF ECoG Right 100mV A B

  21. A NaCl KCl B Metrazol C Metrazol

  22. Dog Heart- Open Chest A B C Fiber Optic Holder a c b

  23. A B

  24. Anoxia Hypoxia Hypercapnia Nimodipine Ethanol Anesthetics Uncoupler NADH During operation ICU Mannitol ICP elevation Retraction Brain Epilepsy SD Ischemia NE Compression Ischemia NE Pacing Hypopnea Ischemia Drugs(Ach, NE, vasoactive) N2 NE Ischemia NE Hypercapnia AAA ICU Bypass Hemorrhage Papaverine Ischemia NO Sepsis CO Oxygen deficiency Ischemia Drugs Hemorrhage Clinical Hyperbaria HBO TBI Aging Hypothermia Activation Liver Spinal cord Small Intestine Heart kidney Urethra Testis Animal Clinical Pigs Clinical

  25. Multiparametric Monitoring of Tissue Energy Metabolism WHY ?

  26. 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.

  27. Monitoring of NADH and Tissue Blood Flow

  28. Hypoxia Ischemia CO Exposure Hypocapnia Normal Brain Hypercapnia Hyperoxia Spreading Depression Monitoring of 2 Parameters Tissue NADH Normal min. Tissue Blood Flow Normal

  29. Hypoxia Heart pacing Ischemia Hypocapnia Normal Hyperoxia Brain Hypercapnia Tissue activation min. Tissue Blood Flow Normal Tissue Blood Flow Alone

  30. Tissue NADH Hypoxia Ischemia Heart pacing Hypocapnia Normal Normal Brain Hypercapnia Hyperoxia Tissue activation Tissue NADH Alone min.

  31. Normal Brain “The more parameters you monitor... The better you can differentiate between states” 200 Death Hypoxia Ischemia Vasopasm SD+ Ischemia NADH Redox State Anes. CO high Hypocapnia CO low 100 Hypercapnia Hyperoxia Seizures SD 0 100 200 Cerebral Blood Flow SD=Spreading Depression; CO=Carbon Monoxide; Anes.=Anesthesia

  32. Multiparametric Monitoring of Tissue Vitality in Vital and Less-Vital organs: Experimental Animal Results

  33. Decreased Tissue Perfusion Increase tissue Perfusion Mitochondrial Dysfunction Better O2 Supply to mitochondria Less Vital Organs Skin Muscle G-I tract Urogenital Highly Vital Protected Organs Brain Heart Adrenal Glands Energy Failure Energy production Preservation Body Blood Flow Redistribution Under Emergency Metabolic States Shock Sepsis Prenatal Hypoxemia Hypoxia Cardiac Arrest Trauma Body Emergency Metabolic State (BEMS) Activation of the Sympathetic Nervous System Secretion of Adrenaline into blood stream Blood flow Redistribution 33

  34. Multi-Site Multi-Parametric system

  35. Effects of Hypoxia (12% O2) Brain Intestine Med Sci monit, 2007: BR211-219

  36. NADH (%) TBF (%) MAP (mmHg) Intestinal (red) and Brain (blue) responses to Epinephrine 10 µg/kg Intestine Brain

  37. * According to Chance & Williams 1955 HBO - Hyperbaric Oxygenation S.D. - Spreading Depression ** According to Mayevsky 1984

  38. Critical Clinical Situations Requiring Measurement of Tissue and Body Vitality

  39. Real time Monitoring of Vitality parameters in Patients System Oriented Monitoring Specific Organ Oriented Monitoring A B Aneurysm In the OR Retraction Brain Systemic General Parameters Systemic Early Warning Parameters B1 B2 In ICU Bypass Grafting Heart Heart Rate Non Vital Organs Parameters Transplanted Organs Blood Pressure Skin pO2 , pCO2 , pH Skin and Muscle Flap End Tidal CO2 Muscle HbO2 HbO2 Sat. Limb Vascular Surgery GI Tract TBF Blood pO2, pCO2, pH Urethra CritiView Bladder Core Temperature

  40. Monitoring of Specific Organ Vitality in Patients

  41. Floating Probe in Neurosurgery

  42. Brain Tissue Probe in Neurosurgery

  43. Decrease of ICP by Suction of CSF

  44. Real time Monitoring of Vitality parameters in Patients System Oriented Monitoring Specific Organ Oriented Monitoring A B Aneurysm In the OR Retraction Brain B1 B2 Systemic General Parameters Systemic Early Warning Parameters In ICU Bypass Grafting Heart Heart Rate Non Vital Organs Parameters Transplanted Organs Blood Pressure Skin pO2 , pCO2 , pH Skin and Muscle Flap End Tidal CO2 Muscle HbO2 HbO2 Sat. Limb Vascular Surgery GI Tract TBF Blood pO2, pCO2, pH Urethra CritiView Bladder Core Temperature

  45. Clinical Background • In emergency metabolic states – the body protects the vital organs (heart, brain) by diverting blood flow from less-vital organs (skin, muscles, intestine, urethra etc.) • Potential disturbances in tissue: • Blood flow: insufficient amount of blood. • Blood oxygenation: insufficient amount of oxygen in RBC • Cellular (mitochondrial) function: impaired oxygen utilization and production of energy • Global monitoring parameters (BP, CVP, CO, etc.) • Not sensitive enough to changes at the tissue level. • Intensive care: key principles • Early detection of evolving conditions • Optimal adjustment of therapeutic means

  46. Monitoring of Patients in Operative Rooms and ICUs.

  47. 150 160 O2 N2 100 50 20-30 1 0 Arterial Blood Intramitochondrial Tissue AIR Alveoli Oxygen Gradients from AIR to Mitochondria End Tidal CO2 Oxygen Partial Pressure (mmHg) Heart Rate &ECG Cardiac Output 100 95 Systemic Blood Pressure Systemic Saturation (Pulse Oximetry) CritiView Microcirculation blood flow and oxygenation NADH redox state

  48. Tissue Blood Flow Mitochondrial Function Doppler (Frequency) Shift Spectroscopy NADH NADH Blood Oxygenation Tissue Reflectance Spectroscopy Back Scattered Light HbO2 B.V=Blood volume Time (Sec) Wavelength (nm) In-vivo tissue spectroscopy by the CritiView Fiber Optic Probes Various Light Sources

  49. DTU A B Photodiode 785nm Doppler PD DL 785nm Doppler Oximetry LED 530nm PMT 450, 470, 530nm NADH Fluor Oximetry LED 470nm Oximetry NADH+Refl LED 375nm Photodiode375nm Intensity Monitoring Refl Photodiode Output Optical Connector Input Optical Connector C Light Source Unit Single Floor

  50. A TBF R375 Flu NADH 2 min R470 B R530 HbO2 TBF Time (min) R+LOccl N2 R375 Flu NADH 2 min R470 R530 HbO2 Time (min) KCl NaCl

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