Metabolic mechanisms of anoxia tolerance in the turtle brain.
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Adenosine Triphosphate↗
Biomedical subjects
T J Sick
Publications and source records attributed to T J Sick.
Rapid preconditioning protects rats against ischemic neuronal damage after 3 but not 7 days of reperfusion following global cerebral ischemia.
Earlier studies indicated that sublethal ischemic insults separated by many hours may "precondition" and, thereby, protect tissues from subsequent insults. In Wistar rats, we examined the hypothesis tht ischemic preconditioning (IPC) can improve histopathological outcome even if the "conditioning" and "test" ischemic insults are separated by only 30 min. Normothermic (36.5-37 degrees C) global cerebral ischemia was produced by bilateral carotid artery ligation after lowering mean systemic blood pressure. The conditioning ischemic insult lasted 2 min and was associated with a time sufficient to provoke "anoxic depolarization" (AD) (i.e., the abrupt maximal increase in extracellular potassium ion activity). After 30 min of reperfusion, 10-min test ischemia was produced, and histopathology was assessed 3 and 7 days later. After 3 days of reperfusion, neuroprotection was most robust in the left lateral, middle and medial subsections of the hippocampal CA1 subfield and in the cortex, where protection was 91, 76, 70 and 86%, respectively. IPC also protected the right lateral, middle and medial subsections of the hippocampal CA1 region. These data demonstrate that neuroprotection against acute neuronal injury can be achieved by conditioning insults followed by only short (30 min) periods of reperfusion. However, neuroprotection almost disappeared when reperfusion was continued for 7 days. When test ischemia was decreased to 7 min, a clear trend of neuroprotection by IPC was observed. These data suggest that subsequent rescue of neuronal populations could be achieved with better understanding of the neuroprotective mechanisms involved in this rapid IPC model.
Animals↗
Mitochondrial hyperoxidation after cerebral anoxia/ischemia. Epiphenomenon or precursor to residual damage?
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Animals↗
Aging impairs regulation of intracellular pH in rat hippocampal slices.
We investigated whether aging changed H+ homeostasis in hippocampal slices bathed in HEPES buffer. Intracellular pH in hippocampal slices from rats aged 26-27 months (7.06 +/- 0.02) was significantly lower compared with that in slices from rats aged 6-7 months (7.16 +/- 0.04). Age did not influence extracellular ph. Age-related reductions in intracellular pH may reflect altered pH regulation that potentially affects brain function and could contribute to the increased vulnerability of the aged brain to metabolic stress.
Aging↗
Anoxic preconditioning in hippocampal slices: role of adenosine.
Prior studies have shown that sublethal anoxic/ischemic insults may "precondition" and thereby protect tissues such as heart and brain from subsequent insults. In hippocampal slices, we examined two hypotheses: (i) that anoxic preconditioning improves the ability of slices to recover synaptic activity following a second anoxic insult and (ii) that anoxic preconditioning involves adenosine receptors. Hippocampal slices were preconditioned by short periods of anoxia prolonged only until the onset of anoxic depolarization. The slices were then reoxygenated for 30 min, after which a second ("test") anoxic insult was induced. Amplitudes of evoked potentials recovered significantly better after "test" anoxic insults in preconditioned slices. In control slices, transient superfusion with adenosine or an adenosine A1 receptor agonist (2-chloroadenosine) 30 min prior to "test" anoxia markedly improved evoked potential recovery. Administration of 8-cyclopentyl-1,3-dipropylxanthine, an A1 receptor antagonist, blocked the protection afforded by preconditioning. These data support the hypothesis that adenosine, probably by its activation of A1 receptors, is involved in the neuroprotection afforded by anoxic preconditioning in hippocampal slices. Preconditioning insults may have a significant clinical impact, since certain surgical procedures may require, or produce, multiple periods of brain ischemia.
Adenosine↗
Mitochondrial hyperoxidation signals residual intracellular dysfunction after global ischemia in rat neocortex.
Reperfusion after global ischemia (10-60 min in duration) in rat neocortex most commonly provoked transient hyperoxidation of mitochondrial electron carriers, tissue hyperoxygenation, and CBF hyperemia. These responses were normally accompanied by recovery of K+ homeostasis and EEG spike activity. Goals of this research were to understand putative relationships among these postreperfusion events with special emphasis on determining whether mitochondrial hyperoxidation results from intracellular changes that may modulate residual damage. The amplitude of postischemic mitochondrial hyperoxidation (PIMHo) did not increase when CBF increased above an apparent threshold during reperfusion, and tissue hyperoxygenation was not required for PIMHo to occur or to continue. These findings suggest that PIMHo is not merely a response to increased CBF and tissue hyperoxygenation; rather, PIMHo is modulated, at least in part, by residual intracellular derangements that limit mitochondrial electron transport. This suggestion was supported by observations that NAD became hyperoxidized after reoxygenation in anoxic hippocampal slices. Also, PIMHo occurred and subsequently resolved in many animals, but K+o never was cleared fully to baseline and/or EEG spike activity never was evident. One suggestion is that PIMHo signals or initiates residual intracellular derangements that in turn impair electrical and metabolic recovery of cerebral neurons after ischemia; an alternative suggestion is that PIMHo and tissue hyperoxygenation are not the sole factors modulating the immediate restoration of electrical activity after ischemia. Present data also support the following: Decreased oxygen consumption, despite adequate oxygen delivery, likely contributes to tissue hyperoxygenation after ischemia; and mitochondrial hyperoxidation is modulated by a limitation in the supply of electrons to the mitochondrial respiratory chain.
Animals↗
Adenosine, a "retaliatory" metabolite, promotes anoxia tolerance in turtle brain.
Contrary to what is found in most vertebrates, the brains of certain turtle species maintain ATP levels and ion homeostasis and survive prolonged anoxia. The hypothesis tested here is that the release of adenosine and its binding to A1 receptors are essential for this anoxic tolerance. Studies were conducted in the isolated turtle cerebellum, which did release adenosine to the extracellular space during anoxia. When adenosine receptor antagonists [theophylline, 8-cyclopentyltheophylline (CPT), or 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)] were added to the superfusate under control conditions, they had no effect on extracellular potassium ion activity ([K+]o). During anoxia, however, these antagonists provoked maximal efflux of K+ (anoxic depolarization). Anoxic depolarization occurred earlier during anoxia with theophylline (a nonspecific adenosine receptor antagonist) than with CPT or DPCPX, which specifically block A1 receptors. Therefore, adenosine release and effects mediated by A1 receptors are essential to anoxia tolerance in turtle brain.
Adenosine↗
Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices.
Hippocampal slices exposed to brief anoxia combined with elevated glucose exhibit greater postanoxic recovery of synaptic transmission. Glucose may have improved recovery of synaptic transmission by enhancing the production of metabolic energy during and after anoxia. This enhancement should provide more ATP for energy-requiring ion transport processes, and lead (1) to a delayed onset of complete depolarization of CA1 pyramidal cells during anoxia (anoxic depolarization) and (2) to greater ion transport activity following anoxia. A delay in anoxic depolarization would protect neurons from damage if the duration of anoxic depolarization was shortened. Greater postanoxic ion transport would allow the re-establishment of ion gradients supportive of neuronal and synaptic excitability. The effects of glucose and anoxia on ion homeostasis and synaptic transmission were examined in rat hippocampal slices exposed to different glucose concentrations (5-20 mM). The duration of anoxic depolarization was held constant so that postanoxic damage related to this duration was controlled. We found that K+ transport and recovery of synaptic transmission after anoxia in hippocampal slices improved as glucose concentration increased. Also, anoxic depolarization was delayed as glucose concentration increased. Thus, added glucose may improve postanoxic recovery of synaptic transmission by better supporting ion transport.
Animals↗
Anoxic survival of the isolated cerebellum of the turtle Pseudemis scripta elegans.
Intact turtle brain provides a useful model for the study of anoxia and potential survival strategies, since this tissue maintains transmembrane ion gradients and ATP levels during prolonged anoxia and recovers functional activity afterwards. Since isolated tissues offer experimental advantages, the present study sought to determine effects of anoxia on the isolated turtle cerebellum and to define relationships between anoxia survival and glucose supply. In normoxia, the extracellular potassium ([K+]o) activity and evoked potentials were maintained with 5 mM glucose, but 20 mM glucose was required to maintain adenosine triphosphate (ATP) levels and prevent significant increases in [K+]o during anoxia. Inhibition of glycolysis by iodoacetic acid (IAA) during anoxia provoked large increases in [K+]o at all glucose levels. These results demonstrate the usefulness of the isolated turtle cerebellum for studies of anoxic survival since this tissue can maintain ATP levels and [K+]o during prolonged anoxia with 20 mM glucose in the artificial cerebrospinal fluid medium. They also suggest the presence of a Pasteur effect at least during the transition to a hypometabolic state.
Adenosine Triphosphate↗
Glycolysis, oxidative metabolism, and brain potassium ion clearance.
Studies were directed toward defining relationships between brain ion transport, glycolysis, and oxidative phosphorylation. This was done by examining the relative sensitivity to hypoxemia and to iodoacetate (IAA)-induced inhibition of glycolysis in rats anesthetized with pentobarbital. Both insults had minimal effects on K+o baseline. In response to neuronal activation, IAA increased the time required for K+o clearance from maximal values to half-recovery of baseline. Hypoxemia slowed the later phase of K+o clearance, when K+o was approaching "resting" levels. Hypoxemia produced greater declines in high-energy intermediates than did IAA, which indicated that the IAA effect was not due to a greater overall insult to metabolism and suggested a direct link between ATP produced by glycolysis and ion transport activity. These data demonstrate that K+o clearance requires energy from glycolysis and oxidative phosphorylation for different phases of the recovery process and that inhibition specific to glycolysis or oxidative phosphorylation may be temporally resolved within a single stimulus.
Animals↗
Glycolytic and oxidative metabolic contributions to potassium ion transport in rat cerebral cortex.
Putative roles of glycolytic and oxidative metabolism in the removal of potassium ion from the extracellular space were examined in rat cerebral cortex. In response to direct electrical stimulation of the cerebral surface, the activity of extracellular potassium ion (Ko+) transiently increased. Inhibition of glycolysis with iodoacetate prolonged the time required for dissipation of the elevated Ko+. This slowing was most evident in the early period after stimulation, when Ko+ was relatively high. Levels of high-energy intermediates were unchanged by iodoacetate. In contrast, severe hypoxemia was without effect during the early phase of K+ removal but hypoxemia slowed the later restoration of the Ko+ baseline. These data demonstrate that the rapid removal of potassium ion from the extracellular space following intense neuronal activity is aided by the Embden-Myerhoff metabolic pathways and perhaps by direct coupling of ATP produced by glycolysis. We suggest that removal of potassium ion from the brain extracellular space depends on two ATP pools, one derived from oxidative phosphorylation, the other from glycolysis. The glycolytic ATP pool may be most involved in the early and rapid phase of potassium clearance; the oxidative ATP pool may be more associated with the second and slower phase of Ko+ clearance, and with the maintenance of the Ko+ baseline under 'resting' conditions.
Adenosine Triphosphate↗
Downregulation of sodium channels during anoxia: a putative survival strategy of turtle brain.
In contrast to mammalian brain, which exhibits rapid degeneration during anoxia, the brains of certain species of turtles show an extraordinary capacity to survive prolonged anoxia. The decrease in energy expenditure shown by the anoxic turtle brain is likely to be a key factor for anoxic survival. The "channel arrest" hypothesis proposes that ion channels, which regulate brain electrical activity in normoxia, may be altered during anoxia in the turtle brain as a mechanism to spare energy. Goals of present research were to test this hypothesis and to determine whether down-regulation of sodium channels is a possible explanation for spike threshold shifts seen during anoxia in isolated turtle cerebellum. We report here that anoxia induced a significant (42%) decline in voltage-gated sodium channel density as determined by studies of the binding of a sodium channel ligand, [3H]brevetoxin. This study demonstrates that sodium channel densities in brain may be regulated by tissue oxygenation or by physiological events associated with anoxia. Moreover, it also suggests that downregulation of sodium channels may be a basis for changes in action potential thresholds, the electrical depression and energy conservation that provide the unique anoxic tolerance of turtle brain.
Animals↗
Membrane and synaptic activity during anoxia in the isolated turtle cerebellum.
Electrical depression in the turtle brain during anoxia has been suggested as a strategy for sparing energy and promoting the extraordinary survival capacity of this tissue. The present study was aimed toward defining the changes in membrane properties during anoxia that may underlie such electrical depression. Studies were conducted in isolated cerebellum from turtle brain. Anoxia caused transmembrane potentials (TMP) to become relatively less polarized in most Purkinje cells. In no cell, however, was TMP depolarized to levels associated with the complete loss of transmembrane ion gradients produced by tissue superfusion with iodoacetate during anoxia. Sodium (and likely calcium) spike thresholds were increased, postsynaptic responses from the major afferent input pathways to Purkinje cells were depressed, and input resistance decreased significantly during anoxia. These changes likely contribute to the sparing of energy needed for ion transport and perhaps other functions that may be directly related to cell survival.
Action Potentials↗
EEG suppression and anoxic depolarization: influences on cerebral oxygenation during ischemia.
Cerebral ischemia provokes sequential changes that include EEG suppression, anoxic depolarization (AD) with maximal increases in extracellular potassium ion activity (K+o), and anoxia with maximal decreases in tissue oxygen tension (tPO2) and increases in the reduction/oxidation (redox) ratios of the mitochondrial electron transport carriers. Studies were directed toward relationships among these events during cerebral ischemia ("four-vessel occlusion model") in pentobarbital anesthetized rats. Results demonstrate that EEG suppression and anoxic depolarization do not occur as a simple function of progressive oxygen decline during cerebral ischemia. Rates of K+ elevation, tPO2 decline, and cytochrome a,a3 reduction were decreased in the immediate period following EEG suppression. Latency to EEG suppression was inversely correlated with latency to maximal cytochrome reduction. In contrast, AD was associated with increased rates of tPO2 decline and cytochrome a,a3 reduction. Latency to AD was related to latency of subsequent maximal cytochrome a,a3 reduction. These data suggest that EEG suppression spares oxygen while AD accelerates the progression to energy failure by accelerating the decline in oxygen stores in brain following global ischemia.
Animals↗
Age-related modifications of potassium homeostasis and synaptic transmission during and after anoxia in rat hippocampal slices.
Age-related changes in the capacity of the brain to survive short anoxic episodes were studied in stratum pyramidale (region CA1) of hippocampal slices from control (6-7 months) and aged (26-27 months) rats. Our primary interest was in how aging affected the ability of slices to maintain or to recover extracellular potassium ion (K+o) homeostasis and orthodromically-stimulated field potentials during and after anoxia. During anoxia, K+o homeostasis was lost faster in slices from aged rats. Following anoxia, K+o homeostasis recovered more slowly, and synaptic transmission recovered less completely, in aged slices. These studies provide what is believed to be the first demonstration that aging diminishes the capacity of brain tissue to maintain K+o during anoxia and to recover K+o homeostasis and synaptic transmission following anoxia, and support suggestions that the aged brain is more vulnerable to anoxia.
Action Potentials↗
Extracellular pH and suppression of electrical activity during anoxia in turtle and rat brain.
To evaluate the contribution of extracellular H+ activity toward depression of brain electrical activity during anoxia, extracellular pH (pHe) and field potentials were measured in turtle and rat olfactory bulbs with ion-selective microelectrodes. This study tests the hypothesis that unique regulation of pHe contributes to the remarkable tolerance of turtle brain to prolonged anoxia. Hypercapnea (20% CO2 ventilation) depressed olfactory bulb evoked potentials 25-30% in both rat and turtle. During anoxia, evoked potentials were completely abolished within 1 min in rat olfactory bulb but decreased to only 40% of control after 4 h in the turtle despite similar changes in brain pHe. Anoxia-induced acidification of turtle brain was exacerbated by hypercapnea and was attenuated by hypocapnea or by hypocapnea plus intravenous infusion of sodium bicarbonate. However, these manipulations of pHe during anoxia in turtle brain had little effect on depression of evoked potentials. We conclude that energy failure, rather than extracellular acidification, is the major contributor toward suppression of electrical activity in mammalian brain and that preservation of energy balance, rather than unique pH regulation, is responsible for protection of turtle brain during anoxia.
Action Potentials↗
MPP+-induced pathophysiology demonstrates advantages of neurotoxicology studies in brain slices.
Since MPTP and its metabolite MPP+ produce nigrostriatal lesions and symptoms similar to Parkinson's disease, recent studies have aimed toward defining their selectivity and neurotoxic mechanisms. In mitochondria in vitro, MPP+ blocked electron transport and decreased oxygen consumption. However, these effects were not selective to striatal mitochondria or even to mitochondria from brain, they required concentrations of MPP+ much greater than those found in vivo, and physiological actions could not be related to intramitochondrial changes. Lower doses of MPP+ did produce highly selective degeneration of dopaminergic (DA) neurons in cell cultures. We report here that MPP+ provoked large (80%) oxidations of cytochrome b and large K+o increments (approximately 30 mM) in rat striatal slices. These effects were slowed by mazindol, which inhibits DA uptake, and were markedly attenuated in rat hippocampal slices which have little DA input. Since DA terminals comprise only 2-4% of the striatal mass, the large MPP+-induced changes suggest that while MPP+ neurotoxicity in brain requires the presence of functioning DA terminals, effects are not confined to these terminals. Such studies illustrate the complexity of MPP+ neurotoxicity and demonstrate the importance of investigations in models such as brain slices with an extracellular space and intracellular relationships as in intact brain.
1-Methyl-4-phenylpyridinium↗
Manipulating the intracellular environment of hippocampal slices: pH and high-energy phosphates.
The intracellular energetic environment of rat hippocampal slices was manipulated by bolstering ATP levels following the addition of adenosine to the incubation medium, or by manipulating intracellular pH. Addition of 8 mM adenosine to the incubation medium increased total tissue adenylate and ATP content, but did not prolong electrical function during anoxia. Further, it resulted in long-lasting alterations in normoxic evoked responses. Intracellular pH (pHi) was changed by manipulating the bicarbonate/CO2 ratio of the incubation medium, or by adding amiloride, a hydrogen/sodium antiport blocker. Estimates of intracellular pH using the creatine kinase equilibrium agree with those obtained by Neutral red scanning spectrophotometry in control conditions. However, only Neutral red indicated an acidification with amiloride treatment, while the creatine kinase equilibrium was preferentially affected by hypercapnia, suggesting the presence of at least two pH compartments in hippocampal brain slices. These manipulations cannot be carried out easily in vivo, and provide a means of determining the importance of metabolic changes on neural function during anoxia.
Action Potentials↗