Joseph C. LaManna, PhD, Duke University 1975 - US grants

Cerebral Resuscitation following cardiac arrest is the focus of many intense controversies. The role of the impaired reperfusion phenomenon in the development of continuing cerebral damage after total cerebral ischemia (TCI) remains unclear. This cerebral vasospasm seen post-TCI has been shown in this laboratory to be similar in both dogs and primates. Using a double occlusion balloon model for TCI developed in this laboratory and radiolabelled microspheres for sequential measurement of regional cerebral blood flow (RCBF), we have evaluated the effect of various pharmacological interventions on both RCBF after TCI and on chronic neurological outcome (nitroprusside, thiopental, nifedipine, indomethacin and 1-Benzyl Imidazole). Over the next 3 years, we plan to conduct multi-disciplinary studies on the basis mechanisms of cerebral damage following TCI, employing new techniques to monitor cerebral oxidative metabolism (reflectance spectrophotometry), glucose metabolism (3-deoxyglucose autoradiography), neurotransmitter stores/enzymatic activities, and RCBF (microspheres). Pharmacological tools will be used to "dissect" the mechanism of continuing cerebral damage (thromboxane synethetase inhibitors, calcium ionophore antagonists, prostacyclin, adenyl cyclase stimulation, opiate antagonists) and hopefully provide a rational basis for future clinical protocols for cerebral resuscitation.

The specific aims of this project represent an attempt to quantitatively determine the inter-relationships among neuronal work, oxygen delivery and metabolism, and glucose delivery and metabolism in the rat brain during and after complete, reversible cerebral ischemia. Accomplishment of the aims will provide a basis for the description of the mechanisms which couple metabolism to function and how they are altered during and after stroke preventing full functional recovery. These goals will be reached through the use of reliable techniques and methodologies combining electrophysiological recording of brain activity, extracellular potassium and hydrogen ion activity, and tissue oxygen tension with optical monitoring of mitochondrial metabolism and intracellular pH by reflection spectrophotometry in situ under well controlled physiological and pathophysiological conditions. The data recorded from in vivo experiments will be compared directly with neurochemical correlates describing the metabolic state of the brain at significant times in the tissue response to stroke. The results from these proposed experiments will be directly applicable to the problem of brain dysfunction and recovery of function due to reversible or irreversible cell damage accompanying the imbalance between function and metabolism due to the pathophysiological stress which occurs during stroke. The uniqueness of this approach lies in the integration of various techniques which allow concurrent measurements of electrical brain activity, tissue oxygen content, cytochrome oxidase, potassium ion, hydrogen ion, and blood flow, volume and hemoglobin oxygenation; applied to the intact cerebral cortex in a manner which allows continuous determination in each rat during a variety of experimentally controlled conditions. This will allow specific analysis of the threat to continued normal brain function brought about by the failure of compensatory mechanisms which exist as a consequence of the total reliance of the brain on energy produced by oxygen metabolism.

The long-term goal of this project is to determine the role of tissue acid-base and metabolic derangements in the temporal and spatial progression of focal stroke. Focal models of ischemia are generally more relevant to human stroke process than are global models, since most human strokes involve the occlusion of a single process than are global models, since most human strokes involve the occlusion of a single vessel. However, a number of problems arise in focal stroke that have limited its usefulness as an experimental model: 1) the spatial configuration of the stroke is not reproducible and the affected regions vary between animals, and 2) the insult elicited by the occlusion is not restricted to the infarct but extends to adjacent regions which, unlike the ischemic focus, are partially perfused. The fate of these partially perfused peri-focal areas, their location, and their response to the insult will determine the eventual scope of infarcted tissue, and, therefore, the severity of the stroke. The necessary first step is to locate and define the condition of these compartments. This has been achieved by the intravenous injection of the diffusible dye, neutral red. The advantages of the neutral red technique are: 1) it visually defines infrazones of stroke, 2) it is compatible with metabolic microanalysis of the tissue, and 3) it is simple and inexpensive compared to other methods for indicating tissue perfusion. The distribution of neutral red delineates three major visible regions without ambiguity. The relationship between stain intensity and tissue perfusion will be evaluated in conjunction with iodoantipyrine metabolic and histological studies. The viability of the various regions will be assessed by evaluating certain metabolic and pH parameters at various stages of recovery in rat brain frozen in situ, sectioned on a cryostat, lyophilized and dissected in reference to the neutral red staining pattern. Energy balance in the areas of altered perfusion will be determined by the microquantitative measurement of ATP, P- creatine, glucose, glycogen, and lactate; and functional integrity assessed from the levels of GABA, glutamate, and cyclic nucleotides. The affected region will also be labeled with neutral red to determine the intracellular pH by spectral analysis, and for in vivo studies on potassium and hydrogen ion concentration using ion-selective extracellular microelectrodes. This use of neutral red permits, for the first time, a multidisciplinary approach to identify the spatial and temporal concomitants of focal stroke. This information collectively will serve as a basis for developing and evaluating new therapeutic regimens.

Stroke is a leading cause of death and disability. At the present time, there are no effective therapeutic methods that can be used once a stroke has occurred that have been shown to ameliorate the damaging effects of the stroke. In addition, there are no predict eventual survival or residual neurological deficit. In order to rectify this situation it is not sufficient to simply screen potential pharmacologic agents, regardless of theoretical justification. What is needed is to study and understand the major cytotoxic cellular mechanisms that are initiated during and shortly after stroke. This project is designed to investigate the possibility that one of the cytotoxic mechanism includes the activation of the sodium/proton exchange transporter during the early period after reperfusion following stroke, resulting in an intracellular alkaline shift and producing and increased metabolic stress due to the volume changes caused by the accompanying intracellular influx of water. Our primary specific aim is to determine the time cause of the changes in brain intracellular pH after 10 minutes of reversible total cerebral ischemia in the rat. Cerebral intracellular pH will be determined by spectrophotometric measurements of the vital dye and pH indicator, neutral red, in vivo and in tissue frozen in situ. The method allows the simultaneous microregional determination of other metabolically significant substances in the the same tissue. Thus, we will be able to study the cellular reactions which result in acidification during ischemia, and those which are responsible for recovery from intracellular acidosis. A secondary aim involves the study of the cellular mechanisms controlling hydrogen ion homeostasis in ischemia in a somewhat more simple preparation, without the additional variable of blood flow. These mechanisms will be studied in the isolated brain slice preparation in vitro where the time course and reversibility of the effects can be determined in each slice. These studies are also designed to begin the attempt at separating the relative contribution of the different cell types (neurons, glial, endothelial cells) to observed phenomena. The fruits of these studies will be related back to the intact animal through experimental protocols designed to examine the possible influences of the hydrogen ion/volume regulation mechanisms on the active control of cerebral capillary function especially that part devoted to matching capillary recruitment to cerebral metabolic demand.

Initially, more than half of the cardiac arrests that occur can be resuscitated successfully, but long-term survival and recovery of neurologic function are limited by metabolic failure in the central nervous system. In addition, there are no physiological variables, which can be measured soon after resuscitation, that can be used to predict long-term morbidity and mortality. It continues to be the long-term goal of this project to investigate the metabolic pathophysiological response to cardiac arrest and resuscitation in rat model in order to 1) discover measurable variables which predict survival and neurologic outcome, and 2) provide theoretical underpinning for development of treatment strategies to improve overall brain resuscitation rates and decrease neuronal loss of function. Intracellular pH has been thought to play a central role in brain damage in ischemia, due to both primary acidification during ischemia and delayed swelling accompanying alkalinization during reperfusion. This hypothesis will be directly tested in the proposed experimental protocols both in the in vitro hippocampal slice preparation and by microregional measurement of cerebral blood flow by autoradiographic methods, by gross regional determinations of tissue wet weight to dry weight ratios, and by microregional measurements of intracellular pH through the application of color film histophotometry of the pH indicator dye neutral red in rat brain frozen in situ after cardiac arrest and resuscitation. Potential treatment strategies intended to reverse the loss of the brain adenylate pool by adenosine administration; to counteract the osmotically driven cellular edema by hyperosmotic solutions; and, to decrease cell water that accumulates through the activity of the amiloride-sensitive Na+/H+ exchanger by administration of specific inhibitors will be applied in the early hours after resuscitation and provide a focus for monitoring and manipulating the critical metabolic variables that may be responsible for cerebral death and delayed specific neuronal loss.

Adaptation to continued moderate hypoxia in the rat brain includes structural and metabolic changes. The most striking evidence of this hypoxia induced metabolic and vascular plasticity is capillary angiogenesis characterized by increased capillary density. This project proposes to study the time course of angiogenesis in response to continued and intermittent hypoxia, and return to normoxia. The appearance and disappearance of capillaries will be documented in the standard adult Wistar rat model and compared to the response of obese/hypertensive, lean/hypertensive, and streptozotocin-induced chronic hyperglycemic rats. Functional consequences of vascular re-structuring will be examined through measurements of blood flow and mean transit time and blood volume under resting conditions and in response to challenges of acute hypoxia, acute hypercapnia and acetazolamide. Brain energy metabolites and intracellular Ph will be measured to characterize the metabolic adaptations in these models. Dynamic metabolic responses to hypoxia in adapted and non-adapted rats will be studied by H and 31P-NMR spectroscopy. These studies will help to define the boundaries of the structural and metabolic changes that occur in response to continued hypoxic exposures. Neuronal function is coupled tightly to both blood flow and oxygen metabolism. This tight coupling directly suggests mechanisms which could explain the cognitive impairments commonly associated in humans with sleep-disordered breathing. Certainly, acute moderate hypoxemia might impair neuronal function. Incomplete or unsuccessful adaptation to continued hypoxia could also explain functional impairment. Finally, even successful adaptation to continued hypoxemia might result in cognitive impairment because of the functional consequences of the structural adaptations. These considerations provide a rationale for obtaining evidence of similar metabolic and vascular plasticity in human subjects that have continuous hypoxemic episodes. The extent of metabolic and vascular adaptation to chronic hypoxia will be tested in human patients using positron emission tomography for measuring blood flow, blood volume, oxygen extraction fraction and glucose consumption in sleep apneic patients. Additional protocols for studying patients using 1H and 31P spectroscopy will be pursued based on the animal spectroscopy studies. It is our long-term goal to provide a basic understanding of the physiological and pathophysiological responses triggered by hypoxic exposure. This information would be used to diagnose and quantify hypoxic exposure in human subjects using currently available PET and MRI- MRS methods; and would also provide tools for determining and need for, and evaluating the success of therapeutic interventions.

DESCRIPTION (Applicant's abstract): While severe acidosis exacerbates brain injury due to global cerebral ischemia, moderate acidosis appears to promote recovery during reperfusion. This application proposes to test 3 mechanistic hypotheses related to the effects of ischemic and postischemic tissue acidosis and alkalosis on animal survival and neural cell death in a rat model of cardiac arrest and resuscitation. The first hypothesis is that the primary cause of death within the first 6 hr of reperfusion is metabolic and vascular failure as a result of cytotoxic edema initiated by activation of the Na exchanger in the brainstem nuclei that control cardiovascular and respiratory function. The second hypothesis is that death within a few days after resuscitation is metabolic and a result of vascular failure in the brainstem due to vasogenic edema caused by blood-brain barrier leakiness through factors such as VEGF induced by oxidative stress. The third hypothesis is that delayed neuronal death in selectively vulnerable areas such as the hippocampal CA1 region is promoted by acidosis during and immediately following cardiac arrest. The general experimental approach to testing these hypotheses will be to define the regional intracellular pH changes in the brainstem and hippocampus of rats in response to cardiac arrest and resuscitation and to correlate these changes to structural and functional indicators of metabolic stress and energy balance. The project will utilize animals that are either normoglycemic and normocapnic, hyperglycemic and normocapnic, or normoglycemic and hypercapnic. Some animals will also be treated with Na+/H+ transport inhibitors to provide further support that this transporter promotes postischemic intracellular alkalosis and worsens neurological outcome.

[unreadable] DESCRIPTION (provided by applicant): Prolonged mild hypoxia initiates a sequence of vascular and metabolic adaptations in the rat brain. Angiogenesis and the resultant increased capillary density that occurs over a 3 week time course is an important aspect of the adaptation process. The long term goals of these investigations are to understand and to be able to manipulate the molecular mechanisms responsible for microvascular remodeling in the brain. These mechanisms that allow the neurovascular unit to adapt to environmental challenges are also likely to be involved in the vascular remodeling that occurs with learning, and which may be diminished with age. These processes can have an important contribution to the pathophysiology of ischemia and other metabolic and oxidative stresses. There appear to be 2 major pathways responsible for brain angiogenesis: a hypoxia-inducible factor-1 (HIF-1a) dependent upregulation of vascular endothelial growth factor (VEGF), and a HIF-1a independent upregulation of angiopoietin-2 (Ang-2). The first specific aim of this proposal is to determine if Ang-2 expression occurs by a mechanism involving prostaglandin E2 (PGE2) production after upregulation of cyclooxygenase-2 (COX-2) activity in mice exposed to hypoxia. When hypoxic adapted animals are returned to normoxia (recovery), the subsequent capillary regression is apparently due to upregulation of Ang-2 in the absence of VEGF inducing endothelial cell apoptosis. The second aim of this proposal is to use a binary mouse molecular genetics approach to explore the role of Ang-2 and VEGF in angiogenesis and capillary regression. The working hypothesis of these investigations is that capillary structure is greatly affected by the balance between VEGF and Ang-2. When Ang-2 is increased in the presence of VEGF then angiogenesis will occur; if Ang-2 is elevated in the absence of VEGF then the endothelial cells will undergo apoptosis. The third aim will explore the consequences of the impaired response of HIF-1a to hypoxia and to determine if angiogenesis is thereby inhibited in aged rats; and, if the Ang-2 response is still robust, to determine whether capillary regression might be induced instead. An attempt will be made to induce HIF-1 accumulation in these older rats, through tilorone treatment to restore microvascular plasticity. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A significant number of individuals die soon after a successful cardiopulmonary resuscitation form cardiac arrest, and of those that survive a significant fraction have residual neurological deficit. Most of the morbidity and mortality after initially successful resuscitation from cardiac arrest can be assigned to the immediate and delayed effects of reperfusion injury in the central nervous system. With aging, there is an increased incidence of cardiac arrest and subsequent death or residual neurological deficit secondary to reperfusion injury. This outcome is probably due to an increase in free radical production and decrease in defense mechanisms that exacerbates reperfusion injury in the aging. Nevertheless, the poor ability of the brain to recover from such reversible ischemic events remains, in all ages, unpromising and is most likely due to free radical damage and unstabilized energy metabolism. This project represents a new collaboration between investigators specializing in the area of reperfusion injury. We propose to investigate new treatment strategies aimed at using alternate energy substrates such as, pyruvate and ketones in combination with antioxidant type drugs, such as, melatonin, N-t-Butyl-a-Phenyl-nitrone, adenosine and methylisobutyl amiloride, as therapies for improving recovery from cardiac arrest. Novel esters will also be tested for their neuroprotective properties in brain against reperfusion injury. These compounds are unique in that they are metabolized to physiological substrates, such as, pyruvate, glycerol and N-acetylcyteine, and have shown to decrease the effects of reperfusion injury in other organ systems. An animal model of cardiac arrest and resuscitation, adult (3 mos) and aged (18 mos) rats will be used to test the efficacy of these new treatment strategies. One set of experimental protocols measures delayed loss of hippocampal CA1 neurons 4 and 30 days after cardiac arrest and resuscitation and survival rates. Another set of experimental protocols will elucidate specific mechanism(s) by which the selected energy substrates and agents and are effective.

DESCRIPTION (provided by applicant): Neurodegeneration associated with oxidative stress limits recovery from stroke and other pathophysiological challenges. There are many physiological factors that play a role in recovery from oxidative stress and survival such as those related to stabilization of energy metabolism and vascular integrity. For example, prolonged mild hypoxia initiates a sequence of vascular and metabolic adaptations in brain. Angiogenesis and the resultant increased capillary density is a fundamental aspect of the hypoxic adaptation process. In the aged brain, however, there is an apparent deficiency in glucose metabolism and lack of hypoxic response. Cerebral blood flow (CBF) and metabolic rate for glucose (CMRglu) as well as the coupling between CBF and CMRglu are known to decline as a function of age, suggesting dysfunction of the neurovascular unit. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates adaptive responses to the lack of oxygen in mammalian cells and has multiple functions related to cellular homeostasis and survival, such as the upregulation of VEGF and EPO, known neuroprotective molecules. In the aged brain, the HIF-1 response to hypoxia is severely attenuated. The inability to stabilize HIF-1 is consistent with the deleterious outcomes with even mild hypoxia suffered by the aged brain that the young adult brain can overcome. We have recently shown accumulation of HIF-1a (without hypoxic stimulation) and increased vascular density in young mature rat brain in rats made ketotic through a ketogenic diet. The increased blood ketones were accompanied by up-regulation of the primary substrate transporters for glucose (GLUT1) and ketones (monocarboxylates;MCT1) at the blood-brain barrier. We intend to determine if similar responses can be elicited in the ketotic, aged rat brain. Ketone bodies are alternate energy substrates to glucose and are signaling molecules that stabilize glucose metabolism by relieving metabolic blocks. We will investigate if ketosis (via a ketogenic diet) in the aged rat results in a similar angiogenic response as the younger adult brain and test if there is improved adaptation response to hypoxia. The common mechanism by which ketones act is most likely related through the stabilization of glucose metabolism and citric acid cycle intermediates resulting in increased brain succinate levels. Increased succinate levels result in inhibition of prolyl-hydroxylase (PHD), a key enzyme of the HIF-1a degradation pathway. Inhibition of PHD will result in greater accumulation of HIF-1a and should result in better tolerance to hypoxia as measured by molecular, structural and behavioral responses. PUBLIC HEALTH RELEVANCE: Neurodegeneration as a result of stroke and other pathophysiological challenges related to aging remains to be explored. We propose to investigate if ketosis, induced by ketogenic diet (low carbohydrate, high fat) results in improved adaptation response to hypoxia in aged rat brain. The ability to alter brain metabolism and produce neuroprotection by dietary adjustments would be a significant new strategy for stroke prevention and recovery.

DESCRIPTION (provided by applicant): The uptake and metabolism of ketone bodies in brain have been of interest to researchers and clinicians for decades but how they affect the coupling of blood flow and glucose metabolism, especially during chronic ketotic conditions, remains unclear. Although glucose is considered the primary fuel for brain, ketones supplement brain metabolism, especially under conditions of glucose sparing, such as fasting, starvation or high fat-low carbohydrate diet. The enzymes for ketone metabolism and the monocarboxylate transporters at the blood brain barrier are known to increase with fasting or ketogenic diet. It is known that cerebral ischemia, such as stroke (induced by cardiac arrest and resuscitation), results in altered glucose metabolism, the reduction of intracellular energy metabolites such as ATP, ADP and phosphocreatine and the accumulation of metabolic intermediates, such as lactate and adenosine. Degree of recovery of neurologic function following stroke (oxidative stress) is limited by the ability of the central nervous system to recover from an ischemic event. Based on our experiences we have developed the working hypothesis that ketones are effective against pathology associated with oxidative stress and/or altered glucose metabolism. The rationale is ketosis stabilizes glucose metabolism through the normalization of redox (lactate/pyruvate ratio) in brain. One potential mechanism is that ketone body metabolism differs from glucose such that when oxidized, acetyl-CoA units enter the Krebs-TCA cycle at the level of citrate bypassing glycolysis (the step after pyruvate dehydrogenase complex which is often a metabolic block following oxidative stress) and through feed-back regulation is known to down regulate glycolytic rates at the level of citrate, phosphofructokinase or hexokinase. Another proposed mechanism may be that ketosis facilitates anaplerosis (replenishment of the Krebs-TCA cycle intermediates) after oxidative challenges, a mechanism for neuroprotection. To investigate the effects of ketosis on cerebral metabolic rate for glucose (CMRglu) imaging modalities, such as PET analysis, and an in vivo rat model of ketosis will be used to determine if ketosis improves CMRglu and metabolic outcome following cardiac arrest and resuscitation. PUBLIC HEALTH RELEVANCE: Fasting, prolonged starvation or consumption of high fat-low carbohydrate diet (ketogenic diet) is known to result in ketosis and has been used for the treatment of intractable epilepsy, especially where the seizures are caused by insufficient glucose transport into the brain. Based on animal studies, ketosis has been suggested to possess neuroprotective properties against neurodegenerative diseases such as Alzheimer's, Parkinson's and recovery from focal stroke. However, there has been little progress in developing therapies that optimize ketosis as an approach to the medical treatment of neurodegenerative diseases, and, therefore, we would like to investigate the effects of ketosis on brain metabolism of glucose (CMRglu) using image (PET) analysis in ketotic rat and to determine if there is improved outcome following cardiac arrest and resuscitation.

DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Brain Energy Metabolism and Blood Flow as part of the Gordon Research Conference series to be held in Andover, NH, August 22 - 27, 2010. The broad and long term goal of the conference is to increase understanding of the fundamental mechanisms enabling and supporting normal brain function from the uninterrupted supply of nutrients, particularly glucose, and oxygen with an emphasis on the failure of these mechanisms which leads to neurodegeneration. The biennial Gordon research conference is devoted to the presentations of the results of frontline research on brain metabolism, cell signaling, cell-cell interactions and vascular regulation in the normal and injured brain. Therefore, the conference is a unique opportunity for senior and junior researchers alike to exchange state-of- the-art advances in methodology and concepts, as well as to outline promising avenues for collaborative research. The specific aims of this meeting will be to convene 23 speakers that represent critical areas of neuroenergetics research with a total of 135 participants for a five day conference in a relatively isolated setting. The program will have a keynote address and eight sessions that broadly address current issues of brain blood flow and metabolism as vital to the normal mammalian nervous system and as bases for functional imaging. In addition, two evening poster sessions will permit all participants to contribute to these topics. The significance of this application is that the Gordon Research Conference is a critical component of the yearly series of conferences that propel research in the international community of neuroenergetics researchers. The health relatedness of this application is that dramatic progress has been made in the last decade in the fields of molecular biology, biophysics, and genetics of brain. The progress impacts on our understanding of brain energy metabolism, neural organization, cell signaling, and vascular regulation. In addition, new technologies have emerged to measure blood flow and metabolism with high spatial and temporal resolution. The stage is set to use this methodological progress to address fundamental issues related to the organization of brain function, blood flow and metabolic activity. We anticipate that progress in this field will drive new discoveries in the experimental and clinical neurosciences and affect diagnosis and treatment of stroke and other neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: This conference is a unique opportunity to bring together senior and junior investigators to exchange state-of- the-art advances in methodology and concepts and to outline promising avenues for collaborative research in brain energy metabolism and blood flow. Progress in this field will drive new discoveries in the experimental and clinical neurosciences and affect diagnosis and treatment of stroke and other neurodegenerative disorders.

DESCRIPTION (provided by applicant): The goal of the Neurodegeneration Training Program (NTP) is to provide rigorous pre-doctoral training in various aspects of neurodegeneration and mechanisms of diseases involving neurodegeneration. An outstanding pool of trainees will combine with world-class faculty to investigate a wide range of neurodegeneration-related topics spanning research areas such as protein structure, cell and molecular biology and in vitro and in vivo models of disease. The NTP spans departments, schools and institutions to include multiple departments at Case Western Reserve University, particularly the School of Medicine, and its affiliated institutions, University Hospitals Case Medical Center (UHCMC), The Louis Stokes Cleveland Veteran's Administration Medical Center (VAMC), and the Cleveland Clinic Foundation (CCF, including the Lerner Research Institute). All of these institutions are within walking distance of each other and this rich training environment enjoys very active basic science and clinical activities and state of the art resources to enrich the training of pre- doctoal trainees as they engage in basic and/or translational research in the field of neurodegeneration. Training in the NTP involves NTP course work, formal and informal seminars, an annual retreat, and a research experience resulting in scholarly publications. A unique component of this training program is the inclusion of a required 1 semester, half day per week, mentored experience in a neurodegenerative disease clinic. The combination of didactic and experiential training opportunities afforded by the NTP will provide trainees a solid foundation for a future career in the scientific inquiry of neurodegeneration.