Failure and function of intracellular pH regulation in acute hypoxic-ischemic injury of astrocytes

Astrocytes can die rapidly following ischemic and traumatic injury to the CNS. Brain acid-base status has featured prominently in theories of acute astrocyte injury. Failure of astrocyte pH regulation can lead to cell loss under conditions of severe acidosis. By contrast, the function of astrocyte pH regulatory mechanisms appears to be necessary for acute cell death following the simulation of transient ischemia and reperfusion. Severe lactic acidosis, and the failure of astrocytes to regulate intracellular pH (pH(i)) have been emphasized in brain ischemia under hyperglycemic conditions. Direct measurements of astrocyte pH(i) after cardiac arrest demonstrated a mean pH(i) of 5.3 in hyperglycemic rats. In addition, both in vivo and in vitro studies of astrocytes have shown similar pH levels to be cytotoxic. Whereas astrocytes exposed to hypoxia alone may require 12-24 h to die, acidosis has been found to exacerbate and speed hypoxic loss of these cells. Recently, astrocyte cultures were exposed to hypoxic, acidic media in which the large ionic perturbations characteristic of brain ischemia were simulated. Upon return to normal saline ("reperfusion"), the majority of cells died. This injury was dependent on external Ca2+ and was prevented by inhibition of reversed Na(+)-Ca2+ exchange, blockade of Na(+)-H+ exchange, or by low pH of the reperfusion saline. These data suggested that cytotoxic elevation of [Ca2+]i occurred during reperfusion due to a sequence of activated Na(+)-H+ exchange, cytosolic Na+ loading, and resultant reversal of Na(+)-Ca2+ exchange. The significance of this reperfusion model to ischemic astrocyte injury in vivo is discussed.     

Cell death in primary cultures of mouse neurons and astrocytes during exposure to and ‘recovery’ from hypoxia, substrate deprivation and simulated ischemia

Effects of hypoxia, substrate deprivationand simulated ischemia (combined hypoxia and substrate deprivation) on cell survival during the insult itself and during a 24 h ‘recovery’ period were studied in primary cultures of mouse astrocytes and in cerebral cortical neuronal-astrocytic co-cultures. Cell death was determined by release of the cytosolic high molecular enzyme lactate dehydrogenase (LDH) as well as morphologically (retention of staining with rhodamine 123 and lack of staining with propidium iodide as an indicator of live cells). Glutamate concentrations were measured in the incubation media at the end of the metabolic insults. Astrocytes were very resistant to hypoxia, but less so to simulated ischemia; under both conditions the glutamate concentrations in the media remained low. Cerebral cortical neurons were almost equally susceptible to damage by hypoxia and by stimulated ischemia, although hypoxia had a faster deleterious effects on some of the neurons and simulated ischemia during a long-term insult (9 h) killed all neurons, whereas a non-negligible neuronal subpopulation survived 9 h of hypoxia. Neuronal cell death after long-term hypoxia (but not after simulated ischemia) was correlated with high concentrations of glutamate in the incubation media. After certain insults, most notably relatively short lasting simulated ischemia (3 h) in neurons (which caused no increased cell death during the insult), there was a large release of LDH during the ‘recovery’ period.     

Evaluation of preconditioning treatments to protect near-pure cortical neuronal cultures from in vitro ischemia induced acute and delayed neuronal death

We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen--glucose deprivation, hypoxia, hypothermia and TNFalpha preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22--24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen--glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFalpha and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.     

NAD+ as a metabolic link between DNA damage and cell death

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The mechanisms by which PARP-1 activation leads to cell death are not understood fully. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation was induced by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), using medium in which glucose was the only exogenous energy substrate. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD+ restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation. These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood-brain barrier.     

Rapid astrocyte death induced by transient hypoxia, acidosis, and extracellular ion shifts

Death of astrocytes requires hours to days in injury models that use hypoxia, acidosis, or calcium paradox protocols. These methods do not incorporate the shifts in extracellular K(+), Na(+), Cl(-), and Ca(2+) that accompany acute brain insults. We studied astrocyte survival after exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), with respective [Ca(2+)], [K(+)], [Na(+)], [Cl(-)], and [HCO(-)(3)] of 0.13, 65, 51, 75, and 13 mM (15% CO(2)/85% N(2), pH 6.6). Intracellular pH (pH(i)) was monitored with the fluorescent dye BCECF. Cell death was indicated by a steep fall in the pH-insensitive, 440-nm-induced fluorescence (F440) and was confirmed by propidium iodide staining. After 15-40-min HAIR exposure, reperfusion with standard Ringer caused death of most cultured (and acutely dissociated) astrocytes within 20 min. Cell death was not prevented if low Ca(2+) was maintained during reperfusion. Survival fell with increased HAIR duration, elevated temperature, or absence of external glucose. Comparable durations of hypoxia, acidosis, or ion shifts alone did not lead to acute cell death, while modest loss was noted when acidosis was paired with either hypoxia or ion shifts. Severe cell loss required the triad of hypoxia, acidosis, and ion shifts. Intracellular pH was significantly higher in HAIR media, compared with solutions of low pH alone or with low pH plus hypoxia. These results indicate that astrocytes can be killed rapidly by changes in the extracellular microenvironment that occur in settings of traumatic and ischemic brain injury.     

Interstitial pO2 in ischemic penumbra and core are differentially affected following transient focal cerebral ischemia in rats.

Stroke causes heterogeneous changes in tissue oxygenation, with a region of decreased blood flow, the penumbra, surrounding a severely damaged ischemic core. Treatment of acute ischemic stroke aims to save this penumbra before its irreversible damage by continued ischemia. However, effective treatment remains elusive due to incomplete understanding of processes leading to penumbral death. While oxygenation is central in ischemic neuronal death, it is unclear exactly what actual changes occur in interstitial oxygen tension (pO2) in ischemic regions during stroke, particularly the penumbra. Using the unique capability of in vivo electron paramagnetic resonance (EPR) oximetry to measure localized interstitial pO2, we measured both absolute values, and temporal changes of pO2 in ischemic penumbra and core during ischemia and reperfusion in a rat model. Ischemia rapidly decreased interstitial pO2 to 32% +/- 7.6% and 4% +/- 0.6% of pre-ischemic values in penumbra and core, respectively 1 hour after ischemia. Importantly, whilst reperfusion restored core pO2 close to its pre-ischemic value, penumbral pO2 only partially recovered. Hyperoxic treatment significantly increased penumbral pO2 during ischemia, but not in the core, and also increased penumbral pO2 during reperfusion. These divergent, important changes in pO2 in penumbra and core were explained by combined differences in cellular oxygen consumption rates and microcirculation conditions. We therefore demonstrate that interstitial pO2 in penumbra and core is differentially affected during ischemia and reperfusion, providing new insights to the pathophysiology of stroke. The results support normobaric hyperoxia as a potential early intervention to save penumbral tissue in acute ischemic stroke.     

Brain acidosis, cerebral blood flow, capillary bed density, and mitochondrial function in the ischemic penumbra

Within the ischemic penumbra, there is a heterogeneous development of cortical intracellular acidosis that is associated with selective neuronal injury. This experiment, which used a rabbit model of moderate focal cerebral ischemia, examined the time course for changes in intracellular brain pH, cortical blood flow, capillary bed density, and mitochondrial function in the ischemic penumbra. After cortical annotation of regions of intracellular acidosis in the ischemic penumbra, the animals underwent transcardiac carbon black perfusion for measurement of capillary bed density. Analysis of variance and Pearson's correlation coefficients were used to determine the relationship between capillary bed density, brain intracellular pH, mitochondrial function, and cortical blood flow. Thirty minutes after the onset of ischemia, cortical blood flow declined from 46+/-2 to 22+/-1 mL/100gm/min (P<.01) in all groups. The overall cortical intracellular brain pH measured 6.78+/-.01 compared with a preischemic value of 6.98+/-.01 (P<.05). Within this moderately ischemic cortex, there were small regions (1,000 to 45,000 mum(2)) of increased acidosis, meauring 6.68+/-.01, not associated with focal changes in cortical blood flow, occurring within 15 minutes of ischemia and persisting throughout the ischemic period. Capillary bed density progressively declined with ongoing ischemia occurring after the development of acidosis. For example, capillary bed density in preischemic controls was 338+/-6/mm(2), whereas after 1 hour of ischemia, it measured 147+/-12/mm(2), at 3 hours 97+/-23/mm(2), and at 6 hours 92+/-16/mm(2). Mitochondrial function was reduced coinciding with the decrease in capillary bed density. These data support the hypothesis that cortical acidosis in the ischemic penumbra facilitates the development of perfusion defects that subsequently lead to mitochondrial dysfunction.     

Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats

Focal ischemia in the cerebral cortex results in acute and delayed cell death in the ischemic cortex and non-ischemic thalamus. We examined the hypothesis that neurons in ischemic and non-ischemic regions died from different mechanisms; specifically, we tested whether a mixed form of cell death containing both necrotic and apoptotic changes could be identified in individual cells.Focal barrel cortex ischemia in rats was induced by occlusion of small branches of the middle cerebral artery (MCA) corresponding to the barrel cortex, local blood flow was measured by quantitative autoradiography. Cell death was visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and caspase-3 staining 1 to 10 days after the ischemia. Electron microscopy was used for ultrastructural examination. Cell death occurred in the ipsilateral cortex 24 h after ischemia, followed by selective neuronal death in the ventrobasal (VB) thalamus 3 days later. TUNEL positive neurons were found in these two regions, but with striking morphological differences, designated as type I and type II TUNEL positive cells. The type I TUNEL positive cells in the ischemic cortex underwent necrotic changes. The type II TUNEL positive cells in the thalamus and the cortex penumbra region represented a hybrid death, featured by concurrent apoptotic and necrotic alterations in individual cells, including marked caspase-3 activation, nuclear condensation/fragmentation, but with swollen cytoplasm, damaged organelles and deteriorated membranes. Cell death in the thalamus and the cortex penumbra were attenuated by delayed administration of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-FMK). Our data suggest that TUNEL staining should be evaluated with morphological changes, the hybrid death but not typical apoptosis occurs in the penumbra region and non-ischemic thalamus after cerebral ischemia.     

Evolution of brain infarction after transient focal cerebral ischemia in mice.

The evolution of brain infarction after transient focal cerebral ischemia was studied in mice using multiparametric imaging techniques. One-hour focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Cerebral protein synthesis (CPS) and the regional tissue content of adenosine triphosphate (ATP) were measured after recirculation times from 0 hours to 3 days. The observed changes were correlated with the expression of the mRNAs of hsp-70, c-fos, and junB, as well as the distribution of DNA double-strand breaks, visualized by TUNEL. At the end of 1 hour of ischemia, protein synthesis was suppressed in a larger tissue volume than ATP in accordance with the biochemical differentiation between core and penumbra. Hsp70 mRNA was selectively expressed in the cortical penumbra, whereas c-fos and junB mRNAs were increased both in the lateral part of the penumbra and in the ipsilateral cingulate cortex with normal metabolism. During reperfusion after withdrawal of the intraluminal filament, suppression of CPS persisted except in the most peripheral parts of the middle cerebral artery territory, in which it recovered between 6 hours and 3 days. ATP, in contrast, returned to normal levels within 1 hour but secondarily deteriorated from 3 hours on until, between 1 and 3 days, the ATP-depleted area merged with that of suppressed protein synthesis leading to delayed brain infarction. Hsp70 mRNA, but not c-fos and junB, was strongly expressed during reperfusion, peaking at 3 hours after reperfusion. TUNEL-positive cells were detected from 3 hours on, mainly in areas with secondary ATP depletion. These results stress the importance of an early recovery of CPS for the prevention of ischemic injury and suggest that TUNEL is an unspecific response of delayed brain infarction.     

Viability thresholds and the penumbra of focal ischemia

The classic concept of the viability thresholds of ischemia differentiates between two critical flow rates, the threshold of electrical failure and the threshold of membrane failure. These thresholds mark the upper and lower flow limits of the ischemic penumbra which is thought ot suffer only functional but not structural injury. Recent studies of the functional and metabolic disturbances suggest a more complex pattern of thresholds. At declining flow rates, protein synthesis is inhibited at first (at a threshold of about 0.55 ml/gm/min), followed by a stimulation of anaerobic glycolysis (at 0.35 ml/gm/min), the release of neurotransmitters and the beginning disturbance of energy metabolism (at about 0.20 ml/min), and finally the anoxic depolariztion (<0.15 ml/gm/min). The penumbra, as defined by the classic flow thresholds, does not remain viable for extended periods. Since viability of the tissue requires maintenance of energy-dependent metabolic processes, penumbra is redefined as a region of constrained blood supply in which the energy metabolism is preserved. Imaging of the penumbra by combining autoradiographic cerebral blood flow measurements with bioluminescent images of adenosine triphosphate (ATP) demonstrates a gradual expansion of the infarct core (in which ATP is depleted) into the penumbra until, after a few hours, the penumbra has disappeared. It is suggested that the limited survival of the penumbra is due to periinfarct depolarizations, which result in repeated episodes of tissue hypoxia, because the increased metabolic workload is not coupled to an adequate increase of collateral blood supply. This explains pharmacological suppression of periinfarct depolarizations lowering the threshold of metabolic disturbances and reducing the volume of the ischemic infarct.     

Brain tissue acidosis and changes of energy metabolism in mild incomplete ischemia--topographical study

Regional changes of brain tissue pH and its correlation to energy metabolism were studied in various degrees of incomplete ischemia for 5 and 60 min in the unilateral common carotid occlusion of normally fed mongolian gerbils. The degree of ischemia was evaluated by the severity of neurological deficits following 60 min of occlusion, and animals were divided into three groups: symptomatic, borderline, and asymptomatic. Changes of NADH and ATP distribution corresponded well to the degree of ischemia. On the other hand, acidosis developed more clearly and extended in wider areas than the changes of NADH and ATP distribution. These changes were already seen at 5 min of occlusion. From the results of this experiment, it was suspected that acidosis in mild incomplete ischemia was due to stimulated anaerobic glycolysis that might supplement NADH oxidation and ATP yields. Further, acidosis without energy failure was considered not to be detrimental to neuronal cells.     

CHOP plays a pivotal role in the astrocyte death induced by oxygen and glucose deprivation

Ischemia has different consequences on the survival of astrocytes and neurons. Thus, astrocytes show a remarkable resistance to short periods of ischemia that are well known to cause neuronal death. We have used a cell culture model of stroke, oxygen, and glucose deprivation (OGD), to clarify the mechanisms responsible for the exclusive resistance of astrocytes to ischemia. The expression of genes implicated in both ischemia-induced astrocyte death and post-ischemic survival was analysed by the RNA differential display technique. Our study revealed that the expression of the CEBP homologous protein (CHOP)-coding gene is promptly an intensely upregulated following astrocyte oxygen and glucose deprivation. CHOP mRNA induction was accompanied by the activation of other genes (grp78, grp95) that, alike CHOP, are involved in the endoplasmic reticulum (ER) stress response. In addition, drugs that cause ER calcium depletion or protein N-glycosylation inhibition mimicked the effects of OGD on astrocyte survival, further supporting the involvement of ER in the astrocyte responses to OGD. Our experiments also demonstrated that upregulation of CHOP during the ER stress response is required for ischemia to cause astrocyte death. Not only the levels of CHOP mRNA and protein correlate perfectly with the degree of OGD-triggered cell injury, but also astrocyte death induced by OGD is significantly overcome by CHOP antisense oligonucleotide treatment. Nevertheless, we observed that astrocytes undergo apoptosis only when CHOP is permanently upregulated, and not when CHOP increases are transient. Finally, we found that the extent of CHOP induction is determined by the length of the ischemic stimulus. Taken together, our results indicate that permanent upregulation of CHOP is decisive for the induction of astrocyte death by OGD.     

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, ³H-glutamate and ⁸⁶Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive ⁸⁶Rb uptake. Concomitant transient increases (2–3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4–24 h of hypoxia, 4 h of glucose deprivation, and 2–4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply. © 1997 Wiley-Liss Inc.     

Tricarboxylic acid cycle substrates prevent PARP-mediated death of neurons and astrocytes.

The DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP1), contributes to cell death during ischemia/reperfusion when extensively activated by DNA damage. The cell death resulting from PARP1 activation is linked to NAD+ depletion and energy failure, but the intervening steps are not well understood. Because glycolysis requires cytosolic NAD+, the authors tested whether PARP1 activation impairs glycolytic flux and whether substrates that bypass glycolysis can rescue cells after PARP1 activation. PARP1 was activated in mouse cortical astrocyte and astrocyte-neuron cocultures with the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Studies using the 2-deoxyglucose method confirmed that glycolytic flux was reduced by more than 90% in MNNG-treated cultures. The addition of 5 mmol/L of alpha-ketoglutarate, 5 mmol/L pyruvate, or other mitochondrial substrates to the cultures after MNNG treatment reduced cell death from approximately 70% to near basal levels, while PARP inhibitors and excess glucose had negligible effects. The mitochondrial substrates significantly reduced cell death, with delivery delayed up to 2 hours after MNNG washout. The findings suggest that impaired glycolytic flux is an important factor contributing to PARP1-mediated cell death. Delivery of alternative substrates may be a promising strategy for delayed treatment of PARP1-mediated cell death in ischemia and other disorders.     

The ischemic penumbra: identification, evolution and treatment concepts

The concept of the ischemic penumbra is an important one for both basic investigators of cerebral ischemia and for clinicians who treat stroke patients. The ischemic penumbra has been defined in a variety of ways, but the most clinically relevant definition is that portion of the ischemic territory that is still potentially salvageable, if an appropriate treatment is given. Currently, three main challenges persist for those interested in the ischemic penumbra concept: how can this ischemic region be most accurately identified in stroke patients, what mechanisms of ischemic cell death are most important for progression from penumbra towards irreversible injury and what therapeutic modalities are most likely to impede the development of infarction? Much important information regarding each of these topics has become available recently and will be the focus of this paper.     

Therapeutic time window in the penumbra during permanent focal ischemia in rats: changes of free fatty acids and glycerophospholipids

To better define a therapeutic time window for reducing the extent of damage in ischemic penumbra, the time courses of changes in the glycerophospholipid and free fatty acid (FFA) levels were determined in the rat cerebral cortex following induction of the permanent focal ischemia. Focal ischemia induced a biphasic increase in FFA levels in the cerebral cortex, which had been recognized as the ischemic penumbra during the early stages after permanent occlusion of the middle cerebral artery (MCA). The first increase in FFA levels, in which the polyunsaturated fatty acid (PUFA) contained a large number of arachidonic acid (C20:4) molecules, began at 30 min and reached a peak at 1 h, followed by transient return to each sham level 2-6 h after the onset of MCA occlusion. Thereafter, the delayed increase in FFA levels, showing more increases of docosahexaenoic acid (C22:6) molecules than the C20:4 in PUFA compositions, occurred at 24 h. In contrast, the levels of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) decreased rapidly at 30 min of ischemia and returned transiently to each sham level at 1-6 h. The levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), including polyphosphoinositides (PIPs), began to decrease significantly during the late stages, i.e., 24 h after induction of ischemia. These results suggest that the time-dependent changes in FFA and PIPs levels during the early stages of ischemia (until 6 h after induction) might be an important determinant of the subsequent neuronal death in the ischemic penumbra and that the breakdown of glycerophospholipids in the later stages after the induction of focal ischemia was associated with the development of infarction in the cerebral cortex.     

Brain-derived neurotrophic factor reduces cortical cell death by ischemia after middle cerebral artery occlusion in the rat

Stroke is the major cause of adult brain dysfunction. In an experimental approach to evaluate the possible beneficial effects of administration of neurotrophic factors in stroke, we have used a model of distal middle cerebral artery (MCA) occlusion in adult rats. In this model, we found: (1) a permanent reduction of brain-derived neurotrophic factor (BDNF) and its full-length receptor, TrkB, in the infarcted core; (2) a transient increase in BDNF immunoreactivity in the internal region of the border of the infarct (penumbra area) at 12 h after MCA occlusion; (3) increased truncated TrkB immunoreactivity in astrocytes surrounding the area of the infarction; and (4) increased full-length TrkB immunoreactivity in scattered neurons, distant from the infarct, in ipsilateral and contralateral cortices at 24 and 48 h after MCA occlusion. We next studied the regulation of TrkB expression by BDNF, after ischemia, and its neuroprotective effects in vivo. In control non-ischemic rats, grafting of mock- or BDNF-transfected fibroblasts (F3A-MT or F3N-BDNF cell lines, respectively) in the medial part of the somatosensory cortex increased truncated TrkB immunoreactivity in neighboring astrocytes. Grafting alone also increased full-length TrkB in the vicinity of the mock graft (at 24 and 48 h) and the BDNF-grafted graft (at 4 days). Interestingly, ischemic animals grafted with the mock-transfected cell line did not show any further regulation of TrkB receptors. However, ischemic animals grafted with the BDNF cell line showed an up-regulation of full-length TrkB expression in neurons located in the internal border of the infarct. Analysis of nuclear DNA fragmentation in situ, combined with microtubule-associated protein 2 immunohistochemistry, revealed that most cells dying in the borders of the infarct (penumbra area) at 48 h following MCA occlusion were neurons. No differences in the infarct size were found between MCA occluded, mock-transfected MCA-occluded, and BDNF-transfected MCA-occluded rats. Moreover, cell death was similar in nongrafted and mock-grafted rats subjected to MCA occlusion. However, the number of cells with nuclear DNA breaks was significantly reduced in the penumbra area close to the BDNF graft in ischemic rats. Thus, our results show that BDNF specifically up-regulates its full-length TrkB receptor in cortical neurons of the penumbra area and prevents their death in an in vivo model of focal ischemia.     

Optogenetic translocation of protons out of penumbral neurons is protective in a rodent model of focal cerebral ischemia

BackgroundIntracellular acidosis in the ischemic penumbra can contribute to further cell death, effectively enlarging the infarct core. Restoring the acid-base balance may enhance tissue survivability after cerebral ischemia.ObjectiveThis study investigated whether translocating protons out of penumbral neurons could mitigate tissue acidification and induce neuroprotection in a rodent model of acute cerebral ischemia.MethodsWe modulated the penumbral neurons via a light-driven pump to translocate protons out (i.e., archaerhodopsin/ArchT group) or into (i.e., channelrhodopsin-2/ChR2 group) neurons after focal cerebral ischemia in rats. Intracellular pH values were imaged via neutral red (NR) fluorescence and cerebral blood flow (CBF) was monitored through laser speckle contrast imaging (LSCI). Global CBF responses to electrical stimulation of the hindlimbs were obtained 24 h and 48 h after ischemia to assess neurological function. Behavioral and histological outcomes were evaluated 48 h after ischemia. A control group without gene modification was included.ResultsThe reduction of relative pH (R_pH), the amplitude of negative peak of hypoemic response (R_NP) and the hemispheric lateralization index (LI) in ArchT group were significantly less than those of the ChR2 or control group. Moreover, R_pH was strongly correlated with R_NP (r = 0.60) and LI (r_24h = 0.80, r_48h = 0.59). In addition, behavioral and histological results supported a neuroprotective effect of countering neuronal acidosis in penumbra through optogenetic stimulation.Conclusion(s)These results indicate that countering intracellular acidosis by optogenetically translocating protons out of penumbral neurons during the acute ischemic stage could induce protection after ischemic brain injury.     

Hyperglycemic versus normoglycemic stroke: topography of brain metabolites, intracellular pH, and infarct size.

Hyperglycemia aggravates brain pathologic outcome following middle cerebral artery (MCA) occlusion in cats. We presently determined if hyperglycemia during occlusion leads to high lactic acid accumulations in the ischemic MCA territory. We measured brain metabolite concentrations in 14 MCA territory sites at 0.5 and 4 h following occlusion in hyper- (20 mM) and normoglycemic (5 mM) cats and correlated these results with previous brain pathologic findings. Hyper- versus normoglycemia during MCA occlusion resulted in significantly higher lactate concentrations in the ischemic territory and more numerous loci with lactates greater than 17 mumol/g. At 0.5 h of occlusion, ATP levels were lower in normoglycemic cats, while at 4 h, ATP was similarly reduced (40%) in both glycemia groups. At 4 h, PCr was more reduced in hyperglycemics secondary to a greater brain tissue acidosis. Carbohydrate substrates at 0.5 h were more markedly depleted in normoglycemics, likely limiting lactate accumulation (34.3% versus only 5.0% of sites in hyperglycemics with glucose less than 0.5 mumol/g). Although lactate was markedly elevated at both 0.5 and 4 h in hyperglycemic ischemic territories, clip release at 4 versus 0.5 h yields a significantly poorer brain pathologic outcome. Correspondingly, intracellular pH, calculated from the creatine kinase equilibrium, was more markedly depressed at 4 than at 0.5 h of occlusion, demonstrating a time-dependent dissociation between tissue lactate and hydrogen ion accumulations. The present findings show that following MCA occlusion (a) hyperglycemia increases the magnitude and topographic extent of marked tissue lactic acidosis, (b) infarct size following 0.5 h of clip release correlates more closely with tissue acidosis than with lactate concentrations, (c) ischemic tissue ATP concentrations correlate poorly with infarct size, (d) normoglycemia limits lactate accumulation during focal ischemia because tissue glucose is depleted, and (e) early during ischemia, tissue buffering or antiport mechanisms may prevent marked increases in intracellular hydrogen ion activity.     

Activation of CysLT receptors induces astrocyte proliferation and death after oxygen-glucose deprivation

We recently found that 5-lipoxygenase (5-LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT(1) and CysLT(2) receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5-LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen-glucose deprivation (OGD) followed by recovery to induce ischemic-like injury in the cultured rat astrocytes. We found that 1-h OGD did not injure astrocytes (sub-lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4-h OGD moderately injured the cells (moderate OGD) and led to death 24-72 h after recovery. Inhibition of phospholipase A(2) and 5-LOX attenuated both the proliferation and death. Sub-lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5-LOX inhibitors. Sub-lethal OGD increased the expressions of CysLT(1) receptor mRNA and protein, while moderate OGD induced the expression of CysLT(2) receptor mRNA. Exogenously applied leukotriene D(4) (LTD(4)) induced astrocyte proliferation at 1-10 nM and astrocyte death at 100-1,000 nM. The CysLT(1) receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT(2) receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD(4) (100 nM) increased the expression of CysLT(2) receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT(1) receptor-mediated proliferation and CysLT(2) receptor-mediated death.