1989 — 1993 |
Chesler, Mitchell |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Intracellular Ph Homeostasis in Glia
The broad objective of this investigation is to elucidate the mechanisms which govern the intracellular pH of glial cells under resting, stimulated, and hypoxic conditions. The study of the relationship between brain electrical activity and glial intracellular pH will add new insights into the process of neuronal-glial signalling. In addition, characterization of the glial pH response to sustained electrical stimulation will provide information relevant to glial behavior during seizure activity. The investigation of glial pH regulation during tissue hypoxia will directly address current issues in ischemic brain pathophysiology. The project will have four specific aims. (1) Glial acid transport systems will be identified and their role in the regulation of glial intracellular pH will be determined. (2) The mechanisms underlying the modulation of glial intracellular pH during neuronal activity will be elucidated. (3) Activity- dependent extracellular pH transients will be related to concomitant glial intracellular pH shifts. (4) The response of glial intracellular pH to tissue hypoxia will be studied, and its relationship to glucose availability determined. The guinea pig olfactory cortical slice preparation will be used as an in vitro model system. The intracellular pH of cortical glial cells will be measured directly during continuous recording with double- barreled, pH-sensitive microelectrodes. Glial intracellular pH will be manipulated by acid-loading, stimulation of the lateral olfactory tract, and by exposure to oxygen-free media. Experiments will focus on the ionic-dependence and pharmacologic sensitivity of the pH modulatory and regulatory mechanisms.
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1993 — 1996 |
Chesler, Mitchell |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dynamics of Ph Regulation in Normal and Ischemic Brain
The regulation of pH is critical for the normal operation of the brain, as numerous channels and enzymes are sensitive to small shifts in hydrogen ion concentration. Acid base status can be particularly important in brain injury, where it can play a determinant role in the manifestation of brain damage and the recovery of function. Normal and pathological electrical activity have been shown to cause large changes in pH, of sufficient magnitude to influence brain physiology. These extracellular and intracellular pH shifts arise from specialized mechanisms in both neurons and glia, and therefore display marked regional heterogeneity, and stereotyped developmental patterns. During brain ischemia, these acid base fluxes can become extremely large, and may therefore influence the processes which lead to secondary tissue injury. The objective of this study is to elucidate the mechanisms which govern the dynamic behavior of pH in mature and developing brain, and to determine the functional relevance of the acid-base shifts under normal and ischemic conditions. The broad aim will be addressed by studying pH dynamics at the systems, regional and cellular levels. Accordingly, experiments will be conducted using anesthetized rats, brain slices, and isolated single cells. Pathophysiological studies on whole animals will be performed during focal ischemia, with emphasis on the infarct rim, a region known to undergo severe electrophysiological disturbances. Unprecedented resolution of acid base status will be provided by new microelectrode techniques, allowing the first real-time determination of pH, bicarbonate, and carbon dioxide in the extracellular space of the brain. These studies will provide new insights into the functional and developmental role pH, and will add critical detail to our understanding of how hydrogen ions affect outcome and recovery, following, stroke, perinatal hypoxia, and cardiac arrest.
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1996 — 2000 |
Chesler, Mitchell |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Acid-Base Homeostasis in Brain Injury @ New York University School of Medicine
Disturbances in acid-base homeostasis have long been implicated in the pathophysiology of ischemic and traumatic brain injury. The deleterious effects of acidosis have received particular attention. Numerous studies indicate that excessive production of lactic acid is particularly harmful to astrocytes. Although the basis of this susceptibility is not understood, studies of astrocyte pH regulation suggest that acid transport mechanisms of these cells are implicated in these pathophysiological responses. Of particular interest are voltage-dependent acid secretory mechanisms which are activated by glial depolarization. A principal aim of this proposal is to elucidate the role of these astrocytic regulatory processes in acute injuries to the CNS. Astrocyte pH regulation will be investigated with pH microelectrodes, optical recording methods and whole cell patch clamp techniques. The intracellular pH studies will be performed at the level of the whole animal, brain slice and isolated cell, capitalizing on the unique experimental advantages offered by each preparation. Neurons, by contrast, can be protected by acidosis in the injury setting, since external hydrogen ions block NMDA receptor-mediated activity. However, spreading depression and others forms of excessive excitory synaptic activity, are associated with extracellular alkalinization, capable of relieving the H+ block of the NMDA receptor. The magnitude of these pH shifts depends upon the speed of buffering, which is governed by the extracellular activity of carbonic anhydrase. These studies will determine the role of this enzyme in ischemic and traumatic injury to the CNS. Experiments will employ recently-developed microelectrode techniques, which will permit the first real-time determination of pH, bicarbonate and carbon dioxide in injured brain. The microelectrode studies will be conducted in anesthetized rats, in models of cardiac arrest (complete cortical ischemia), stroke (middle cerebral artery occlusion), and spinal cord injury. These experiments will provide a complete description of extracellular acid base status in these afflictions. In conjunction with these measurements, we will determine whether the kinetics of H+ buffering can be enhanced by treatment with buffers or by modulation of carbonic anhydrase activity. Experiments will be extended to the study of spreading depression, and hypoxia, in order to determine the role of extracellular buffering in the manifestation of these pathologies. These studies capitalize upon recent conceptual and technological developments in the study of brain pH regulation. The broad goal of this work is to extend our progress in basic research to the pathophysiological setting. Our efforts will provide insights and understanding into how elemental regulatory processes affect injuries arising from cardiac arrest, stroke, and CNS trauma.
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1997 — 2000 |
Chesler, Mitchell |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathophysiology of Brain Ph Regulation @ New York University School of Medicine
DESCRIPTION: (Adapted from Applicant's Abstract): The acid-base status of the brain has been shown to play a critical role in the manifestation of ischemic brain injury. Although modest reductions in the interstitial pH can mitigate insults, severe acidosis is associated with a significantly worse outcome. Astrocytes are thought to play an active role in the regulation of brain pH, and therefore the survival of these cells may be a determinant factor in the development of ischemic injuries. However, the role of astrocytes in ischemic acid-base disturbances is not understood. Under normal conditions, the intracellular pH of these cells undergoes rapid shifts in response to neuronal activity. This unique behavior has been attributed to a voltage-sensitive transport system that imports sodium and bicarbonate ions when the cells are depolarized. As a result, the pH of astrocyte cytoplasm rapidly increases while the interstitial compartment is acidified. In view of the voltage dependence of this transporter, it is likely to be an important contributor to the acid base status of cerebral ischemia, where membrane depolarization is extreme. This proposal will address the role of astrocyte Ph regulatory mechanisms in the context of cerebral ischemia. Studies will begin with the first complete description of interstitial acid base status in ischemic cortex, using recently developed methods for the simultaneous measurement of pH, carbon dioxide, bicarbonate and carbonate. These data will aid in the design of subsequent mechanistic studies, where factors faced by astrocytes in cerebral ischemia will be examined separately. Here, microspectrofluorometric and ion-selective microelectrode techniques will be combined in an in vitro investigation of astrocyte pathophysiology. Conclusions derived from in vitro studies will be compared against direct intracellular measurements obtained in vivo from ischemic rat cortex. These studies will elucidate the factors that govern brain Ph in cerebral ischemia. The results will therefore contribute to the understanding of the mechanisms of ischemic brain injury and the development of management strategies in the context of cardiac arrest and stroke.
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1998 — 2000 |
Chesler, Mitchell |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Center For Cns Injury Studies @ New York University School of Medicine
This application proposes four projects and core facilities to investigate mechanisms of CNS injury. Project 1 Experimental Therapy of Spinal Cord Injury. We recently discovered that cyclosporin A (CsA) reduces responses, releases vasodilatory and inflammatory substances, and modulates neurotransmitter receptors and release. We propose to study the role of calcineurin in spinal cord injury. The experiments will compare the effects of CsA, FK506 (another calcineurin inhibitor), alone and in combination with rapamycin (an antagonist of K506 (another calcineurin inhibitor), alone and in combination with rapamycin (an antagonist of FK506 inhibition of calcineurin), on spinal cord ionic shifts, lesion volumes, motor recovery, blood flow, and serotonin-,GABA- and glutamate-induced spinal excitability and injury. Project 2. Acid-base homeostasis in brain injury. The mechanisms and consequences of pH changes in injured tissues are not well-understood. We will measure pH, PCO2, HCO3, and CO32 in ischemic brains and injured spinal cords, to determine the role of buffer capacity, lactoacidosis, and carbonic anhydrase in the pH changes. We propose that pH shifts modulate NMDA receptors and that interstitial buffering influences the rate and magnitude of spreading depression and ischemic injury in brain slices. Using on-selective microelectrodes, voltage clamp, and spectrophotometric methods, we will test the hypothesis that voltage-dependent Na:HCO3 co-transport governs H+, Na+, and HCO3- distribution across astrocytic membranes in normo- and hyperglycemic ischemia. Project 3. Ascorbate and glutathione in CNS Injury. We hypothesize that ascorbate (Asc) and glutathione (GSH) loss contributes to brain cell vulnerability to oxidative damage. Extracellular Asc will be measured in hypoxic brain slices and in ischemic cerebral cortex after middle cerebral artery occlusion. Intracellular Asc will be estimated from extracellular volumes and total tissue levels. Preliminary studies suggest that Asc and GSH are localized in neurons and astrocytes respectively, that both Asc and GSH decline with age and are lower in females. We will confirm these results, determine whether hypoxia and age compromises Asc homeostasis, assess protective effects of Asc and GSH in hypoxic brain slices, identify mechanisms of Asc and GSH loss in ischemia, and correlate tissue damage with Asc loss in male and females. Project 4. Water compartmentalization in Ischemia. The objective of this project is to understand how water shifts between extra- and intracellular compartments in ischemic brain tissues. Water shifts will be assessed with extracellular tracers, ion-elective microelectrodes, and weighing-drying method. We will manipulate extracellular ionic levels and block ionic transport and glutamate receptors. Water movements will be investigated in slices from aged animals. The role of the Na:HCO3 cotransport will be assessed. Light scattering and Na-K changes will be compared with wet-dry weight and tracer measurements.
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1998 — 2000 |
Chesler, Mitchell |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dynamics of Ph Regulation in Brain @ New York University School of Medicine
DESCRIPTION The regulation of pH is critical for the normal operation of the brain, as numerous channels and enzymes are sensitive to small shifts in hydrogen ion concentration. Acid base status can be particularly important in brain injury, where it can play a determinant role in the manifestation of brain damage and the recovery of function. Normal electrical activity also results in large changes in interstitial and intracellular pH, of sufficient speed and magnitude to influence function. These pH shifts arise from specialized mechanisms in neurons and glia, and therefore display marked regional heterogeneity and stereotyped patterns. The objective of this study is to determine the mechanisms which govern the dynamic behavior of pH in the central nervous system and to elucidate the role of these processes in modulating neural function. This broad aim will be addressed by investigating the dynamics of activity-dependent pH shifts at both the cellular and regional level. The role of carbonic anhydrase in governing the interstitial buffering capacity will be a principal focus. These experiments on brain slices will utilize using new technologies that permit temporal resolution of pH shifts in the millisecond range. Combining this capability with established patch clamp methods will allow analysis of the pH change that directly modulate excitatory synaptic currents. At the level of the isolated cell, attention will be turned to the specific acid transport mechanisms of neurons and astrocytes. Neuronal studies will address the relationship between calcium influx and the generation of interstitial alkaline shifts. Experiments on astrocytes will focus on the role of these cells as regulators of interstitial pH. The investigation will benefit from the combination of novel fluorescence and ion-selective microelectrode methods, capable of resolving real time changes in pH, bicarbonate, and carbon dioxide tension. These studies will provide an understanding of the functional role of hydrogen ions and add insights into the pathophysiology of pH regulation following stroke and cardiac arrest.
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2003 — 2018 |
Chesler, Mitchell |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Dynamics of Ph Regulation in the Brain @ New York University School of Medicine
DESCRIPTION (provided by applicant): In the brain, normal and pathological electrical activity gives rise to rapid changes in extracellular and intracellular pH. This modulation of pH can feedback to influence neural activity and can impact the response to brain hypoxia and ischemia. It is known that the intracellular pH of glial cells increases in response to membrane depolarization, due to sodium driven, electrogenic, entry of bicarbonate. This influx simultaneously acts to acidify the extracellular fluid. By contrast, depolarization of neurons slowly acidifies the cytosol, a response associated with entry of calcium. Preliminary studies on hippocampal neurons indicate that such neural responses represent a balance between a calcium dependent acidification, and a nearly equivalent alkalinizing mechanism that is triggered by depolarization. These results suggest that neurons regulate their pH preemptively, utilizing one or more transporters responsive to membrane potential. Unlike the transport mechanism of glia, preliminary data indicate that neurons alkalinize in response to depolarization by one mechanism that requires chloride ions, and a separate process that is chloride-independent. Elucidation of these two mechanisms occupies the first two aims of this proposal. The third aim seeks to clarify their functional relevance in response to acidosis, repetitive firing, and hypoxic-ischemic insults. The last two aims concern the role of neurons in rapid regulation of extracellular pH. The fourth aim focuses on an extracellular carbonic anhydrase (type 14) localized to neurons in the hippocampus, and posits that this enzyme regulates pH in the fluid around synapses. The last aim focuses again on role of chloride dependent bicarbonate transport, but from the extracellular perspective. The project will employ tissue from mice with gene deletions of specific bicarbonate transporters. Studies will be conducted with intracellular pH imaging and whole cell recording techniques, complemented by quantitative polymerase chain reaction protocols to quantify transporter expression. Results of these experiments offer potentially groundbreaking insights into how pH is regulated in the CNS, and now that regulation impacts normal physiology, and the response to conditions such as hypoxia, cardiac arrest, stroke and traumatic brain injury. PUBLIC HEALTH RELEVANCE: This project will focus on the mechanisms used by nerve cells to regulate internal (cytoplasmic) acid base balance, during both normal and abnormal electrical activity. Elucidation of these mechanisms is critical to understanding how the brain maintains an internal microenvironment conducive to proper function. Moreover, this information should provide important new insights into how nerve cells respond to conditions such as cardiac arrest, stroke and traumatic brain injury.
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