Efrat Barbiro-Michaely

Use of NADH fluorescence to determine mitochondrial function in vivo.

Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathological states, the real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. In this paper we are summarizing the following items: (1) presenting the solid scientific ground underlying nicotine amide adenine dinucleotide (NADH) NADH fluorescence measurements based on published materials. (2) Presenting NADH fluorescence monitoring and its physiological significance. (3) Providing the reader with basic information on the methodologies of the fluorometers reflectometers. (4) Clarifying various factors affecting the monitored signals, including artifacts. (5) Presenting the potential use of monitoring mitochondrial function in vivo for the evaluation of drug development. The large numbers of publications by different groups testify to the valuable information gathered in various experimental conditions. The monitoring of NADH levels in the tissue provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. Although NADH signals are not calibrated in absolute units, their trend monitoring is important for the interpretation of physiological or pathological situations. To better understand the tissue function, the multiparametric approach has been developed where NADH serves as the key parameter to be monitored.

Shedding light on mitochondrial function by real time monitoring of NADH fluorescence: I. Basic methodology and animal studies.

Normal mitochondrial function in the process of metabolic energy production is a key factor in maintaining cellular activities. Many pathological conditions in animals, as well as in patients, are directly or indirectly related to dysfunction of the mitochondria. Monitoring the mitochondrial activity by measuring the autofluorescence of NADH has been the most practical approach since the 1950s. This review presents the principles and technological aspects, as well as typical results, accumulated in our laboratory since the early 1970s. We were able to apply the fiber-optic-based NADH fluorometry to many organs monitored in vivo under various pathophysiological conditions in animals. These studies were the basis for the development of clinical monitoring devices as presented in accompanying article. The encouraging experimental results in animals stimulated us to apply the same technology in patients after technological adaptations as described in the accompanying article. Our medical device was approved for clinical use by the FDA.

Real-time Monitoring of Mitochondrial Nadh and Microcirculatory Blood Flow in the Spinal Cord

Study Design. We developed a real-time, in vivo monitoring system for the evaluation of spinal cord viability in rats during spinal cord ischemia. Objective. The aim of the present study was to apply a real-time multiparametric monitoring system in a rat spinal cord model exposed to ischemia or mechanical compression. Summary of Background Data. The evaluation of spinal cord integrity during spine surgeries is highly important, as it enhances the potential to prevent secondary irreversible damage to the spinal cord tissue. Mitochondrial NADH redox state is the most sensitive parameter for tissue oxygenation state and, together with microcirculatory blood flow, can estimate the metabolic status of the spinal cord tissue. Methods. We applied the Tissue Vitality Monitoring System (TVMS) that includes optical fibers for the simultaneous monitoring of the spinal cord blood flow (SCBF) using laser Doppler flowmetry, and the mitochondrial NADH fluorescence using the fluorometric technique. Additionally, systemic arterial blood pressure was measured. Two models involving the interruption of the spinal blood flow were tested: the occlusion of the abdominal aorta (ischemia) and spine mechanical compression. Results. The results clearly demonstrated the link between the level of ischemia and the viability state of the spinal tissue. When SCBF decreased, in both experimental models, mitochondrial NADH was elevated, while reperfusion was associated with NADH oxidation. Nevertheless, during the recovery phase, even though SCBF significantly increased (became hyperemic), no further oxidation of NADH was observed. Conclusion. The monitoring of the mitochondrial function together with SCBF by the TVMS reflects the viability of the spinal cord tissue and, together with the conventional monitoring techniques, may help to evaluate the spine conditions, especially under surgical procedures involving the deterioration of the spinal cord blood supply.

Real-time multi-site multi-parametric monitoring of rat brain subjected to traumatic brain injury

Abstract Introduction: Traumatic brain injury (TBI) is one of the major causes of death in the world, with at least ten million serious traumatic brain injuries occurring annually; nevertheless, the pathophysiologic events taking place immediately after the injury are not yet fully known. Objective: To study the effects of TBI on brain hemodynamic, metabolic and ionic homeostasis using the multi-parametric monitoring system. This system enables real-time monitoring of cerebral blood flow (CBF), mitochondrial NADH redox state, extracellular levels of K+, H+, DC potential, ECoG and ICP. Methods: In order to find the best brain location for the monitoring device in relation to the fluid percussion injury site, we used the multi-site multi-parametric monitoring system. Two groups of rats were connected to four monitoring probes at four different locations near the injury site, two in each hemisphere. We monitored CBF, NADH redox state, tissue reflectance and DC steady potential in each of the four sites. Results: Under anoxia, the initial CBF decrease was followed by an increase, NADH level increased, the reflectance decreased and dc potential showed a biphasic response, in all 4 locations. However, following fluid percussion injury, there was a significant variability in the responses in each of the 4 monitored locations. Conclusion: The advantage of the multi-parametric-monitoring approach for enhanced understanding of the injured brain was indicated. Moreover, we showed that contralateral monitoring of the injured brain gives good indication for the events taking place following fluid percussion brain injury

Multiorgan Monitoring of Hemodynamic and Mitochondrial Responses to Anoxia and Cardiac Arrest in the Rat

All tissues in the body are dependent upon the continuous supply of energy to perform their various functions. Glucose and oxygen are brought to the tissue through the bloodstream to fulfill this demand. The basic need of glucose as an energy substrate and oxygen for respiration is common to all tissues. However, the different organs in the body have various levels of demand for these substances. For this reason, the body distributes its resources differently to different organs.

Shedding light on mitochondrial function by real time monitoring of NADH fluorescence: II: human studies

Monitoring the mitochondrial function, alone or together with microcirculatory blood flow, volume and hemoglobin oxygenation in patients, is very rare. The integrity of microcirculation and mitochondrial activity is a key factor in keeping normal cellular activities. Many pathological conditions in patients are directly or indirectly related to dysfunction of the mitochondria. Evaluation of mitochondrial activity by measuring the autofluorescence of NADH has been the most practical approach since the 1950s. This review, which accompanies part I, presents the principles and technological aspects of various devices used in order to monitor mitochondrial NADH redox state and tissue viability in patients. In part I, the detailed technological aspects of NADH monitoring were described. Typical results accumulated in our studies since the mid-1990s are presented as well. We were able to apply the fiber optic based NADH fluorometry to several organs monitored in vivo in patients under various pathophysiological conditions.

Nitrite-induced improved blood circulation associated with an increase in a pool of RBC-NO with no bioactivity.

The reduction of nitrite by RBCs producing NO can play a role in regulating vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of RBC-NO. The nitrite infusion reversed the increase in MAP and decrease in CBF produced by L-NAME inhibition of e-NOS. At the same time there was a dramatic increase in RBC-NO. Correlations of RBC-NO for individual rats support a role for the regulation of vascular tone by this pool of NO. Furthermore, data obtained prior to treatment with L-NAME or nitrite are consistent with a contribution of RBC reduced nitrite in regulating vascular tone even under normal conditions. The role of the RBC in delivering NO to the vasculature was explained by the accumulation of a pool of bioactive NO in the RBC when nitrite is reduced by deoxygenated hemoglobin chains. A comparison of R and T state hemoglobin demonstrated a potential mechanism for the release of this NO in the T-state present at reduced oxygen pressures when blood enters the microcirculation. Coupled with enhanced hemoglobin binding to the membrane under these conditions the NO can be released to the vasculature.

Renal Viability Evaluated by the Multiprobe Assembly: A Unique Tool for the Assessment of Renal Ischemic Injury

Background: One of the major causes of transplanted organs’ dysfunction is ischemia-reperfusion injury, where mitochondrial dysfunction is the primary contributor to cell damage. Mitochondrial NADH fluorescence reliably describes intracellular oxygen deficiency and mitochondrial function. Therefore, its monitoring at the tissue level, together with other physiological parameters, can serve to evaluate tissue vitality. Methods: The multiprobe assembly (MPA) enabled the assessment of renal blood flow (RBF) using laser Doppler flowmetry, mitochondrial NADH redox state using the fluorometric technique, and ionic homeostasis using specific mini-electrodes (K+ and H+). The MPA was utilized in two rat groups in which ischemia was induced for a period of 25–30 min (group 1) or for 60 min (group 2), and RBF and NADH were also monitored in a group of rats that underwent a complete kidney ischemia 24 h before the monitoring – a well-known model of acute renal failure. Results: During ischemia, the RBF was completely abolished, NADH and extracellular potassium levels increased, and extracellular pH decreased. Immediately after the reperfusion, full recovery was observed; however, in the rats undergoing 60-min ischemia followed by 24-hour reperfusion, the tissue hemodynamic and mitochondrial functions were significantly impaired. Conclusion: This study demonstrates the advantage of using the MPA for real-time evaluation of kidney physiological state, which may serve as a practical instrument for the evaluation of graft viability during transplantation procedures.

Mitochondrial function and cerebral blood flow variable responses to middle cerebral artery occlusion.

Middle cerebral artery occlusion (MCAO), which leads to focal cerebral ischemia, serves as an experimental model for brain stroke. There is a large variation in protocols and techniques using the MCAO model, which may affect the outcomes seen in different studies. The current work presents and compares the diverse responses in mitochondrial NADH and cerebral blood flow (CBF) following focal ischemia induced by the MCAO technique. Ninety-six Wistar rats underwent focal cerebral ischemia by MCAO, and monitored in the core and the penumbra using a unique Multi-Site-Multi-Parametric (MSMP) system, which measures mitochondrial NADH using the fluorometric technique, and CBF using laser Doppler flowmetry (LDF). Following MCAO, 58% of the experiments yielded expected responses, namely a decrease in CBF and an increase in NADH. However, 42% of the experiments showed six other profiles of responses, in which CBF, NADH and tissue reflectance (Ref) responded differently. These profiles included: ischemia without reperfusion, death following reperfusion, minor responses in parameters during ischemia, CBF elevation in the penumbra following MCAO, spontaneous early reperfusion and late reperfusion. These results demonstrate that MCAO is a complex model, which may lead to different responses other than the common expected outcomes, i.e. mitochondrial damage and reduced blood flow in both core and penumbra. The MSMP monitoring system may serve as an important tool in early diagnosis of successful focal cerebral ischemia, reducing the percentage of unsuccessful experiments.

In vivo multiparametric monitoring of brain functions under intracranial hypertension following mannitol administration

Abstract Objective: Over the last 20 years, mannitol has replaced other osmotic diuretics. Its beneficial effects on intracranial pressure (ICP), cerebral perfusion pressure (CPP), cerebral blood flow (CBF) and brain metabolism are widely accepted. In the present study, we tested the effect of mannitol injection on brain hemodynamic, metabolic, ionic and electrical state in rats exposed to intracranial hypertension. Methods: The parameters monitored simultaneously included ICP, CBF using the laser Doppler flowmetry, mitochondrial NADH redox state by the fluorometric technique, extracellular K+ and H+ levels, DC potential, ECoG, blood pressure and calculated CPP. ICP was elevated to 30 mmHg for 30 minutes and mannitol was injected 15 minutes post-ICP elevation. Results: Our results showed that mannitol decreased ICP, and improved the levels of MAP, CPP and CBF. Moreover, mannitol completely prevented mortality following intracranial hypertension in rats. Conclusion: It seems that the multiparametric monitoring approach, used in intracranial hypertension models, is an important tool for brain functional state evaluation.