Physiological and morphological heterogeneity of dentate gyrus-hilus interneurons in the gerbil hippocampus in vivo

A variety of morphological types of dentate gyrus/hilus interneurons have been described, but little is known about their corresponding physiological characteristics. To address this issue, intracellular responses to current injection and perforant path stimulation were obtained from putative dentate interneurons in anaesthetized adult gerbils. Our sample of interneurons showed heterogeneity in their intrinsic physiological characteristics and spike thresholds to perforant path stimulation, suggesting the existence of distinct physiologically-defined classes. 'Fast-spiking' interneurons had a low threshold to perforant path stimulation, whereas 'slow-spiking' interneurons responded with predominantly inhibitory potentials. In several cases, cells were intracellularly labelled with biocytin for visualization. Interneurons with different physiological traits had distinct morphological features. These results confirm that, as in hippocampus proper, morphologically identifiable interneurons in the dentate hilus show electrophysiological features that are likely to reflect functionally specific roles in informational processing.     

Early NMDA receptor blockade impairs defensive behavior and increases cell proliferation in the dentate gyrus of developing rats.

These studies were conducted to determine whether (a) early N-methyl-{d}-aspartate (NMDA) receptor blockade impairs defensive behavior and (b) a relationship exists between defensive behavior and the production of granule cells in the dentate gyrus. Rat pups were treated with different doses of the NMDA receptor antagonist CGP 43487 on postnatal day (P) 5, and their behavior was observed following exposure to an unfamiliar adult male rat, a potential predator, on P13, P20, and P30. A dose-dependent impairment in freezing behavior was observed in rat pups treated with NMDA receptor antagonist on P13, P20, but not P30. Moreover, a dose-dependent increase in the number of –3H-thymidine-labeled cells in the dentate gyrus was detected following CGP 43487 treatment, suggesting that an inverse relationship exists between cell proliferation and freezing behavior in rat pups following NMDA receptor blockade. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Network properties of the dentate gyrus in epileptic rats with hilar neuron loss and granule cell axon reorganization

Neuron loss in the hilus of the dentate gyrus and granule cell axon reorganization have been proposed as etiologic factors in human temporal lobe epilepsy. To explore these possible epileptogenic mechanisms, electrophysiological and anatomic methods were used to examine the dentate gyrus network in adult rats that had been treated systemically with kainic acid. All kainate-treated rats, but no age-matched vehicle-treated controls, were observed to have spontaneous recurrent motor seizures beginning weeks to months after exposure to kainate. Epileptic kainate-treated rats and control animals were anesthetized for field potential recording from the dentate gyrus in vivo. Epileptic kainate-treated rats displayed spontaneous positivities ("dentate electroencephalographic spikes") with larger amplitude and higher frequency than those in control animals. After electrophysiological recording, rats were perfused and their hippocampi were processed for Nissl and Timm staining. Epileptic kainate-treated rats displayed significant hilar neuron loss and granule cell axon reorganization. It has been hypothesized that hilar neuron loss reduces lateral inhibition in the dentate gyrus, thereby decreasing seizure threshold. To assess lateral inhibition, simultaneous recordings were obtained from the dentate gyrus in different hippocampal lamellae, separated by 1 mm. The perforant path was stimulated with paired-pulse paradigms, and population spike amplitudes were measured. Responses were obtained from one lamella while a recording electrode in a distant lamella leaked saline or the gamma-aminobutyric acid-A receptor antagonist bicuculline. Epileptic kainate-treated and control rats both showed significantly more paired-pulse inhibition when a lateral lamella was hyperexcitable. To assess seizure threshold in the dentate gyrus, two techniques were used. Measurement of stimulus threshold for evoking maximal dentate activation revealed significantly higher thresholds in epileptic kainate-treated rats compared with controls. In contrast, epileptic kainate-treated rats were more likely than controls to discharge spontaneous bursts of population spikes and to display stimulus-triggered afterdischarges when a focal region of the dentate gyrus was disinhibited with bicuculline. These spontaneous bursts and afterdischarges were confined to the disinhibited region and did not spread to other septotemporal levels of the dentate gyrus. Epileptic kainate-treated rats that displayed spontaneous bursts and/or afterdischarges had significantly larger percentages of Timm staining in the granule cell and molecular layers than epileptic kainate-treated rats that failed to show spontaneous bursts or afterdischarges. In summary, this study reveals functional abnormalities in the dentate gyri of epileptic kainate-treated rats; however, lateral inhibition persists, suggesting that vulnerable hilar neurons are not necessary for generating lateral inhibition in the dentate gyrus.     

Cyclic AMP-dependent protein kinase enhances hippocampal dentate granule cell GABAA receptor currents

1. The effects of activation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), intracellular application of PKA and inhibition of endogenous PKA by protein kinase inhibitory peptide (PKIP) on hippocampal dentate granule cell gamma-aminobuturic acid A (GABAA) receptor (GABAR) currents were characterized. 2. GABAR currents evoked by repeated application of GABA (30 or 100 microM) were enhanced by application of 1 mM norepinephrine (52 +/- 26%; mean +/- SE; n = 11) and of 500 mM 8-bromo cAMP (15 +/- 2%, n = 7). 3. GABA concentration response curves were obtained from six dentate granule cells before and after application of 500 microM 8-bromo cAMP. The maximal current was increased significantly by 89 +/- 36%, but the mean EC50 was not significantly changed (68 +/- 42 microM vs. 25 +/- 10 microM). 4. The GABA concentration response relationship was studied in a group of 7 granule cells recorded with pipettes containing PKIP and 2 mM ATP and compared with another group of 12 cells recorded with 2 mM ATP in the pipette. When currents were recorded with intracellular PKIP, the mean EC50 for GABA was no different (43 +/- 9 microM vs. 45 +/- 16 microM); however, the maximal current obtained was smaller, (961 +/- 102 pA vs. 658 +/- 104 pA). 5. Concentration response data were obtained from four granule cells using recording pipettes containing the cPKA and an ATP regeneration system and compared with seven cells recorded with the ATP regeneration system. With cPKA, the maximal GABAR current was significantly larger (1,224 +/- 132 pA vs. 718 +/- 56 pA), but the EC50 for GABA was not significantly altered (21 +/- 2.0 microM vs. 79 +/- 25 microM).     

The paired-pulse index: a measure of hippocampal dentate granule cell modulation

This study was undertaken to assess whether the paired-pulse index (PPI) is an effective measure of the modulation of dentate granule cell excitability during normal development. Paired-pulse stimulations of the perforant path were, therefore, used to construct a PPI for 15-, 30-, and 90-day old, freely moving male rats. Significant age-dependent differences in the PPI were obtained. Fifteen-day old rats showed significantly less inhibition at short interpulse intervals [interpulse interval (IPI): 20 to 30 msec), a lack of facilitation at intermediate IPIs (50 to 150 msec), and significantly less inhibition at longer IPIs (300 to 1,000 msec) than adults.     

Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress

Although granule cells continue to be added to the dentate gyrus of adult rats and tree shrews, this phenomenon has not been demonstrated in the dentate gyrus of adult primates. To determine whether neurons are produced in the dentate gyrus of adult primates, adult marmoset monkeys (Callithrix jacchus) were injected with BrdU and perfused 2 hr or 3 weeks later. BrdU is a thymidine analog that is incorporated into proliferating cells during S phase. A substantial number of cells in the dentate gyrus of adult monkeys incorporated BrdU and approximately 80% of these cells had morphological characteristics of granule neurons and expressed a neuronal marker by the 3-week time point. Previous studies suggest that the proliferation of granule cell precursors in the adult dentate gyrus can be inhibited by stress in rats and tree shrews. To test whether an aversive experience has a similar effect on cell proliferation in the primate brain, adult marmoset monkeys were exposed to a resident-intruder model of stress. After 1 hr in this condition, the intruder monkeys were injected with BrdU and perfused 2 hr later. The number of proliferating cells in the dentate gyrus of the intruder monkeys was compared with that of unstressed control monkeys. We found that a single exposure to this stressful experience resulted in a significant reduction in the number of these proliferating cells. Our results suggest that neurons are produced in the dentate gyrus of adult monkeys and that the rate of precursor cell proliferation can be affected by a stressful experience.     

The effect of hippocampal dentate granule cell lesions upon the limbic seizure model of rats

The purpose of this study was to determine the role of dentate granule cells upon limbic seizure of Wistar rat caused by unilateral intra-amygdaloid administration of kainic acid (KA). Stereotactic surgery was performed in Wistar rats and stainless steel injection chemitorode was inserted in the left amygdala. Left dentate granule cells lesion were induced by microinjection of colchicine. The rats obtained recovery period for 7 days, postoperatively. The rats were divided into two groups. One group were used for observation of symptoms and electroencephalographic findings during the limbic seizure for 6 hours after the KA injection. Another group was processed for measuring local cerebral glucose utilization (LCGU) during limbic seizure status. The histological study demonstrated a selective loss of dentate granule cells in the left hippocampus 7 days after the colchicine injection. After the KA injection, initiation of the spike discharge was significantly retarded not only in the hippocampus (from 6.01 min. to 37.25 min.) but also in the amygdala (from 2.96 min. to 10.8 min.). Progression, frequency and intensity of the KA induced seizures were also inhibited by the colchicine-induced dentate granule cells lesion. During limbic seizure status, LCGU obtained by 14C-deoxyglucose autoradiography were significantly decreased not only in the hippocampus but also in the amygdala on the site of KA injection. These data suggest that hippocampal dentate granule cells play an important role on initiation and progression of the KA induced limbic seizure. The result suggested that there was an acceleration mechanism of the limbic seizure between amygdala and hippocampus.     

An association between granule cell density in the dentate gyrus and two-way avoidance conditioning in the house mouse.

In 2 experiments, 102 hybrid male mice (C57BL/6J and C58/J stock) with genetically associated variations in the number and density of granule cells in the dentate gyrus were tested for open-field activity, spatial maze learning, and 2-way avoidance conditioning. The number of granule cells was not associated with any behavior. Only avoidance conditioning was related to granule cell density, which had a negative correlation with performance on the shuttlebox task. This result was replicated in 2 genetically different stocks of mice. Density of the more caudal portion of the dentate was associated with early stages of avoidance learning, whereas the more rostral portion was associated with later stages. Results are discussed in relation to theories of functional dissociation within the hippocampus. (38 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Dopamine and the regulation of cell proliferation in gerbil (Meriones unguiculatus) pyloric mucosa

The epithelium of the digestive system mucosa consists of a highly dynamic cell population. The conditions under which mitotic activity in the gastrointestinal epithelium is regulated is as yet poorly understood. Nevertheless, it is assumed that some biogenic amines might be involved. Having demonstrated that dopaminergic cells occur in the stomach of gerbils (Meriones unguiculatus), in the present study we examined the influence of dopamine antagonist haloperidol on the proliferation of epithelial cells in the mucosa of the stomach. Proliferating cells were detected immunocytochemically and quantified after in-vivo labeling with 5-bromo-2'-desoxyuridine in both haloperidol- and saline-treated animals. The results show that acute doses of haloperidol significantly increases the proliferation rate in the pyloric mucosa, suggesting that dopamine plays a probable modulatory role in the regulation of mitotic activity. These findings are discussed with regard to the role of paraneurons in regulating epithelial mitosis.     

Morphology and electrophysiology of dentate granule cells in the rhesus monkey: comparison with the rat

The morphologic and electrophysiologic properties of dentate granule cells in the young adult rhesus monkey (Macaca mulatta) were examined for the first time with whole-cell patch clamp recordings and intracellular biocytin filling in in vitro hippocampal slice preparations. Data from monkeys were compared with data generated in an identical manner from adult Sprague-Dawley rats. Intracellularly filled monkey and rat granule cells were identical in numerous morphologic parameters, including area of somata, total dendritic length, dendritic spread, segment number and length, and branching pattern. The single statistically significant difference in morphology was the vertical extent of the dendritic tree (distance from soma to fissure), which was 20% greater in the monkey. The passive membrane properties (resting membrane potential, input resistance, and membrane time constant) measured under current clamp conditions were virtually identical. The thresholds and amplitudes of action potentials were the same, but significant differences were seen in the kinetics of single action potentials. Monkey granule cell action potentials were significantly longer in duration (with slower rise and fall times) than action potentials in rat cells. These differences were likely due to a much smaller fast after hyperpolarization in the monkey as compared with the rat cells. Thus, with the exception of action potential properties, the principal finding of this study is that there is significant conservation of both form and function in dentate granule cells in these two species, despite the enormous phylogenetic separation. This suggests that granule cell properties may be extremely stable across diverse mammalian species.     

The calretinin-containing mossy cells survive excitotoxic insult in the gerbil dentate gyrus. Comparison of excitotoxicity-induced neuropathological changes in the gerbil and rat

BACKGROUND: The locoregional failure rate remains high in advanced cervical carcinoma. Chemotherapy (CT) was added to radiotherapy (RT) in order to increase disease control and to improve 5-year survival. METHODS: CT + RT included cisplatin administered 100mg/m2, d.1 plus 5-fluorouracil 1000 mg/m2 D.1 to 5, ci (120 hrs), q every 3rd week for 3 cycles, followed by RT. RT included external beam irradiation 64.8 Gy in 1.8 Gy fractions, five days a week, by 4-field box technique. The median follow-up was 46 months. Ninety-four patients were evaluable for survival, 47 in the CT + RT group and 47 in the RT group. Ninety-two patients were evaluable for response. Known prognostic factors were equally distributed between the two groups. RESULTS: Of the 43 patients evaluable before RT, 31 (72%) achieved a partial or complete response after CT alone. After RT, 52 patients attained a complete response, 25 in the CT + RT group and 27 in the RT-group. Sixty-three patients developed distant metastases or local relapse, 30 in the CT + RT group and 33 in the RT group. In the CT + RT group 6 of the 9 patients with metastases also had local progression at relapse, in the RT group, 7 of 17 patients. The survival rates for the two groups are not statistically different. Thirty-seven patients are alive, 29 have no evidence of disease. Fifty-seven have died, 29 in the CT + RT group and 28 in the RT group. Fifty-four deaths were related to cancer, and 3 to therapy. CONCLUSIONS: Sequential CT and RT did not improve the survival, local control, or metastasis rate compared with RT alone.     

Effect of transient cerebral ischaemia on acetylcholine release in the gerbil hippocampus

To clarify the relationship between presynaptic cholinergic dysfunction and postsynaptic cell death in the hippocampus, extracellular levels of acetylcholine (ACh) were assayed and CA1 pyramidal cells were histologically investigated in gerbils which had undergone 2, 5 and 10 min ischaemia. It was found that the KCl- and atropine-induced release of ACh, an index of the functioning cholinergic system at the presynaptic terminals, was significantly lower in the ischaemic groups than in control groups. The hippocampal CA1 pyramidal cell area of the 5 and 10 min ischaemic animals was also significantly decreased, but the 2 min ischaemia caused no cell damage. These findings indicate that the presynaptic terminals of the cholinergic neurone are vulnerable to ischaemic insult and that cholinergic dysfunction precedes postsynaptic CA1 pyramidal cell death in the hippocampus.     

Enhanced bursts of IPSCs in dentate granule cells in mice with regionally inhibited long-term potentiation

Until recently, most studies on the synaptic-cellular basis of learning and memory concentrated on the activity-dependent changes occurring in principal cells such as hippocampal pyramidal cells and dentate granule cells. However, the ability of the inhibitory interneurons to regulate synaptic plasticity remains less understood. This study tested the hypothesis that the gamma-aminobutyric-acid (GABA)-mediated inhibitory neurotransmission is enhanced in mice that show no detectable long-term potentiation in the dentate gyrus in the absence of the GABAA receptor antagonist bicuculline. Patch clamp recordings were made from dentate granule cells in brain slices from wild-type and Thy-1 knockout (KO) mice. The frequency, amplitude and kinetics of miniature inhibitory postsynaptic currents (mIPSCs, generated by the action potential-independent release of GABA) was not different between animals. However, bursts of spontaneous IPSCs (sIPSCs, generated by both action potential-independent and -dependent GABA release) in KO mice were associated with larger synaptic charge transfers and increased durations. When pairs of IPSCs were evoked at varying intervals, the amplitude of the second response with respect to the first was significantly larger in KO animals. These results further support the concept that enhancement of interneuronal functions in cortical structures can have profound effects on the activity-dependent synaptic plasticity observed in principal cells.     

Physiological identification and analysis of dentate granule cell responses to stimulation of the medial and lateral perforant pathways in the rat

Stimulation of the dorsomedial or ventrolateral perforant pathways resulted in quantitatively different extracellularly recorded EPSPs in the fascia dentata of the rat. The two potentials differed in latency to peak and in width at half amplitude in a manner consistent with the different locus of termination of the two pathways on the granule cell dendrites. Both potentials were able to follow brief stimulus trains of 100 Hz, which suggests that they are monosynaptic. Medially elicited responses had their peak negativity approximately 100 to 180 micrometer deeper in the molecular layer than laterally elicited responses. Stimulation at short intervals along a dorsomedial to ventrolateral track in the angular bundle yielded a step function rather than a continuum of EPSP peak latency and half-width, in agreement with Hjorth-Simonsen's ('72) evidence for the separateness of the two pathways. Both pathways were able to induce granule cell discharge. Laterally elicited spikes, however, were delayed. Stimulation at intermediate locations frequently elicited double spikes from the granule cell population. Population spikes elicited by either pathway were inhibited for as long as 100 msec after a single discharge. Both pathways showed facilitation with double stimuli at short intervals, and both showed post-tetanic potentiation lasting at least 30 minutes. Under conditions where it could be shown that the two pathways at least partially converged onto the same granule cells, the response of one pathway did not increase when long lasting potentiation was induced on the other.     

Mitochondrial redox change in gerbil hippocampus before and after transient ischemia

Athletes who need high endurance capacity often use training at moderately high altitude (1500-3000 m) to improve oxygen delivery and utilization because of a hypoxia-induced increase of the red blood cell volume and adaptations at the muscular level. As maximal heart rates decrease at high altitude and plasma lactate levels for a given workload change during prolonged exposure to high altitude, it can be difficult to control and adapt the intensity and duration of the work-outs. Furthermore, maximal performance capacity decreases and therefore training intensity at high altitude is usually reduced compared to training at sea level. To avoid these disadvantages at high altitude a concept of living at moderately high altitude and training at lower elevations, termed "live high-train low" evolved, opposing the conventional concept of "live high-train high". A third option using a hypobaric chamber ("live low-train low") is hardly used anymore for training athletes. Studies on the effects of conventional high-altitude training for the improvement of athletic performance often lack a rigorous controlled design and yield controversial results. Regarding the new concept of "live high-train low" there is only one controlled study on college athletes and it shows a minor advantage of this new approach compared to conventional high-altitude training. However, training concepts are especially important for elite competitive athletes, and controlled studies with such individuals are very difficult to perform. Therefore, it appears that today we cannot answer the question of whether altitude-specific physiologic factors or non-altitude-related benefits of training camps account for the success of individual athletes.     

Forebrain ischemia in the gerbil increases lambda opiate binding in hippocampal mossy fibers

Transient forebrain ischemia was produced in gerbils by short-term occlusion of the common carotid arteries under halothane anesthesia. Histological analysis of brains 7 days post-ischemia demonstrated characteristic destruction of CA1 pyramidal cells. lambda Opiate binding (measured with [3H]naloxone in the presence of 300 nM diprenorphine) at 7 days post-ischemia was significantly increased in the stratum lucidum of the hippocampus (the mossy fiber layer), but not in any other region measured, including other hippocampal regions, cortex, amygdala, caudate putamen, thalamus, and hypothalamus. The increase in mossy fiber lambda binding was slow to develop (no increase detected up to 48 h post-ischemia), and long-lasting (binding remained elevated at 32 days post-ischemia). While MK-801 significantly inhibited CA1 pyramidal cell destruction when administered 20 min prior to ischemia, the increase in mossy fiber lambda binding was still evident. None of seven different opioid agonists and antagonists examined had an effect on either the pyramidal cell damage or increased mossy fiber lambda binding seen 7 days after ischemia.     

Glutamate currents in morphologically identified human dentate granule cells in temporal lobe epilepsy

Glutamate-receptor-mediated synaptic transmission was studied in morphologically identified hippocampal dentate granule cells (DGCs; n = 31) with the use of whole cell patch-clamp recording and intracellular injection of biocytin or Lucifer yellow in slices prepared from surgically removed medial temporal lobe specimens of epileptic patients (14 specimens from 14 patients). In the current-clamp recording, low-frequency stimulation of the perforant path generated depolarizing postsynaptic potentials that consisted of excitatory postsynaptic potentials and phase-inverted inhibitory postsynaptic potentials mediated by the gamma-aminobutyric acid-A (GABA(A)) receptor at a resting membrane potential of -62.7 +/- 2.0 (SE) mV. In the voltage-clamp recording, two glutamate conductances, a fast alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitatory postsynaptic current (EPSC; AMPA EPSC) and a slowly developing N-methyl-D-aspartate (NMDA)-receptor-mediated EPSC (NMDA EPSC), were isolated in the presence of a GABA(A) receptor antagonist. NMDA EPSCs showed a voltage-dependent increase in conductance with depolarization by exhibiting an N-shaped current-voltage relationship. The slope conductance of the NMDA EPSC ranged from 1.1 to 9.4 nS in 31 DGCs, reaching up to twice the size of the AMPA conductance. This widely varying size of the NMDA conductance resulted in the generation of double-peaked EPSCs and a nonlinear increase of the slope conductance of up to 37.5 nS with positive membrane potentials, which resembled "paroxysmal currents," in a subpopulation of the neurons. In contrast, AMPA EPSCs, which were isolated in the presence of an NMDA receptor antagonist (2-amino-5-phosphonovaleric acid), showed voltage-independent linear changes in the current-voltage relationship and were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The AMPA conductance showed little variance, regardless of the size of the NMDA conductance of a given neuron. The average AMPA slope conductance was 5.28 +/- 0.65 (SE) nS in 31 human DGCs. This value was similar to AMPA EPSC conductances in normal rat DGCs (5.35 +/- 0.52 nS, mean +/- SE; n = 55). Dendritic morphology and spine density were quantified in the individual DGCs to assess epileptic pathology. Dendritic spine density showed an inverse correlation (r2 = 0.705) with a slower rise time and a longer half-width of the excitatory postsynaptic potentials mediated by the NMDA receptor. It is concluded that both AMPA and NMDA EPSCs contribute to human DGC synaptic transmission in epileptic hippocampus. However, a wide range of changes in the slope conductance of the NMDA EPSCs suggests that the NMDA-receptor-mediated conductance could be altered in human epileptic DGCs. These changes may influence the generation of chronic subthreshold epileptogenic synaptic activity and give rise to pathological excitation leading to epileptic seizures and dendritic pathology.     

Effects of repeated cerebral ischemia on extracellular amino acid concentrations measured with intracerebral microdialysis in the gerbil hippocampus

BACKGROUND AND PURPOSE: To clarify the role of elevated extracellular amino acid concentrations during ischemia on the cumulative neuronal damage after repeated cerebral ischemic insults, using a microdialysis technique we measured concentrations of the amino acids glutamate, glutamine, glycine, taurine, and gamma-aminobutyric acid in the gerbil hippocampus over three 2-minute forebrain ischemic insults induced at 1-hour intervals. METHODS: Under light anesthesia, the bilateral common carotid arteries were occluded with aneurysm clips at 1-hour intervals. Samples were collected by microdialysis at 10-minute intervals, and the amino acid concentrations were determined using a high-performance liquid chromatography system. RESULTS: During and immediately after the first ischemic insult, concentrations of glutamate, glycine, and taurine, but not glutamine, increased significantly. Glutamate and taurine concentrations rose again during the second and third ischemic insults, but the increases were smaller than those during the first insult. By contrast, glutamine concentrations increased slightly but significantly during the second and third ischemic insults. The extracellular concentration of gamma-aminobutyric acid before the ischemic insults was below the level of detectability but increased markedly during each ischemic insult, with similar declines in the amounts released during later insults. Concentrations of all amino acids returned to baseline after 10 minutes of reperfusion and remained at baseline until the subsequent ischemic insult was induced. CONCLUSIONS: It is well established that glutamate released during ischemia plays a crucial role in ischemia-induced neuronal death. However, the present results indicate that cumulative neuronal damage following sublethal ischemic insults is not caused by an exaggerated release of excitatory amino acids during subsequent ischemic insults but strongly suggest that increased intracellular reactions leading to cell death play a major role.