Death Of CA1 Pyramidal Neurons Research Articles

Hyperthermia accelerates neuronal loss differently between the hippocampal CA1 and CA2/3 through different HIF‑1α expression after transient ischemia in gerbils.

The hippocampus has a different vulnerability to ischemia according to the subfields CA1 to CA3 (initials of cornu ammonis). It has been reported that body temperature changes during ischemia affect the degree of neuronal death following transient ischemia. Hypoxia-inducible factor 1α (HIF-1α) plays a key role in regulating cellular adaptation to low oxygen conditions. In the present study, we investigated the pattern of neuronal death (loss) in CA1 and CA2/3 following 5 min transient forebrain ischemia (TFI) under hyperthermia (39.5±0.2°C) and the relationship between neuronal death and changes in HIF-1α expression using western blot analysis and immunohistochemistry in gerbils. Normothermia or hyperthermia was induced for 30 min before and during the TFI, and neuronal death and HIF-1α expression were observed at 0, 3, 6 and 12 h, 1, 2 and 5 days after TFI. Under normothermia, TFI-induced neuronal death of CA1 pyramidal neurons occurred on day 5 after TFI, but CA2/3 pyramidal neurons did not die. In contrast, under hyperthermia, the death of CA1 and CA2/3 pyramidal neurons was observed on day 2 after TFI. Under normothermia, HIF-1α expression was significantly elevated in both CA1 and CA2/3 pyramidal neurons at 12 h and 1 day after TFI, and the increased HIF-1α immunoreactivity in CA1 was dramatically reduced from 2 days after TFI, but not in CA2/3 pyramidal neurons. Under hyperthermia, the basal expression of HIF-1α in the sham group was significantly higher in both CA1 and CA2/3 pyramidal neurons at 0 h after TFI than in the normothermia group. HIF-1 expression was continuously higher, peaked at 12 h after TFI, and then significantly decreased from 1 day after TFI. Overall, the present results indicate that resistance to ischemia in CA2/3 pyramidal neurons is closely associated with the persistence of increased expression of HIF-1α after ischemic insults and that hyperthermia-induced exacerbation of death of pyramidal neurons is closely related to decreased HIF-1α expression after ischemic insults.

Participation of Amyloid and Tau Protein in Post-Ischemic Neurodegeneration of the Hippocampus of a Nature Identical to Alzheimer's Disease.

Recent evidence suggests that amyloid and tau protein are of vital importance in post-ischemic death of CA1 pyramidal neurons of the hippocampus. In this review, we summarize protein alterations associated with Alzheimer’s disease and their gene expression (amyloid protein precursor and tau protein) after cerebral ischemia, as well as their roles in post-ischemic hippocampus neurodegeneration. In recent years, multiple studies aimed to elucidate the post-ischemic processes in the development of hippocampus neurodegeneration. Their findings have revealed the dysregulation of genes for amyloid protein precursor, β-secretase, presenilin 1 and 2, tau protein, autophagy, mitophagy, and apoptosis identical in nature to Alzheimer’s disease. Herein, we present the latest data showing that amyloid and tau protein associated with Alzheimer’s disease and their genes play a key role in post-ischemic neurodegeneration of the hippocampus with subsequent development of dementia. Therefore, understanding the underlying process for the development of post-ischemic CA1 area neurodegeneration in the hippocampus in conjunction with Alzheimer’s disease-related proteins and genes will provide the most important therapeutic development goals to date.

Altered Nurr1 protein expression in the hippocampal CA1 region following transient global cerebral ischemia.

Nuclear receptor related 1 protein (Nurr1), a member of the nuclear receptor 4 family of orphan nuclear receptors, has been reported to display anti-inflammatory properties. The present study investigated the alteration of Nurr1 immunoreactivity in the gerbil hippocampus proper following 5 min of transient global cerebral ischemia. In sham operated gerbils, Nurr1 immunoreactivity was observed in pyramidal neurons in all cornu ammonis 1–3 (CA1-3) subfields of the hippocampus proper. In ischemia-operated gerbils, Nurr1 immunoreactivity was altered in the CA1 subfield. Nurr1 immunoreactivity in CA1 pyramidal neurons gradually decreased until 2 days post-ischemia, and, at 4 days post-ischemia, Nurr1 immunoreactivity was concentrated in CA1 pyramidal neurons. Additionally, Nurr1 immunoreactivity was newly expressed in microglia in the CA1 subfield at 4 days post-ischemia. Conversely, in the CA2/3 subfield, time-dependent alteration of Nurr1 immunoreactivity was not identified at any time following ischemia. These results indicated that the alteration of Nurr1 expression in the CA1 subfield in the hippocampus may be associated with the death of CA1 pyramidal neurons.

Melatonin improves vascular cognitive impairment induced by ischemic stroke by remyelination via activation of ERK1/2 signaling and restoration of glutamatergic synapses in the gerbil hippocampus.

Vascular dementia affects cognition by damaging axons and myelin. Melatonin is pharmacologically associated with various neurological disorders. In this study, effects of melatonin on cognitive impairment and related mechanisms were investigated in an animal model of ischemic vascular dementia (IVD). Melatonin was intraperitoneally administered to adult gerbils after transient global cerebral ischemia (tGCI) for 25 days beginning 5 days after tGCI. Cognitive impairment was examined using a passive avoidance test and the Barnes maze test. To investigate mechanisms of restorative effects by melatonin, neuronal damage/death, myelin basic protein (MBP, a marker for myelin), Rip (a marker for oligodendrocyte), extracellular signal-regulated protein kinase1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2), and vesicular glutamate transporter (VGLUT)-1 (a glutamatergic synaptic marker) in the hippocampal Cornu Ammonis 1 area (CA1) were evaluated using immunohistochemistry. Melatonin treatment significantly improved tGCI-induced cognitive impairment. Death of CA1 pyramidal neurons after tGCI was not affected by melatonin treatment. However, melatonin treatment significantly increased MBP immunoreactivity and numbers of Rip-immunoreactive oligodendrocytes in the ischemic CA1. In addition, melatonin treatment significantly increased ERK1/2 and p-ERK1/2 immunoreactivities in oligodendrocytes in the ischemic CA1. Furthermore, melatonin treatment significantly increased VGLUT-1 immunoreactive structures in the ischemic CA1. These results indicate that long-term melatonin treatment after tGCI improves cognitive deficit via restoration of myelin, increase of oligodendrocytes which is closely related to the activation of ERK1/2 signaling, and increase of glutamatergic synapses in the ischemic brain area.

Transient Oxygen/Glucose Deprivation Causes a Delayed Loss of Mitochondria and Increases Spontaneous Calcium Signaling in Astrocytic Processes.

Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca(2+) channel blocker), two inhibitors of reversed Na(+)/Ca(2+) exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca(2+) indicator Lck-GCaMP-6S, we observed two types of Ca(2+) signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca(2+) through reversed Na(+)/Ca(2+) exchange triggers mitochondrial loss and dramatic increases in Ca(2+) signaling in astrocytic processes. Astrocytes, the most abundant cell type in the brain, are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission.

Neuroprotection by a novel NMDAR functional glycine site partial agonist, GLYX-13

GLYX-13 (threonine-proline-proline-threonine-amide) is an amidated di-pyrrolidine that acts as a functional partial agonist at the glycine site on N-methyl-D-aspartate glutamate receptors (NMDARs). GLYX-13 can both increase NMDAR conductance at NR2B-containing receptors, and reduce conductance of non-NR2B-containing receptors. Here, we report that GLYX-13 potently reduces delayed (24 h) death of CA1 pyramidal neurons produced by bilateral carotid occlusion in Mongolian gerbils, when administered up to 5 h post-ischemia. GLYX-13 also reduced delayed (24 h) neuronal death of CA1, CA3, and dentate gyrus principal neurons elicited by oxygen/glucose deprivation in in-vitro hippocampal organotypic slice cultures, when applied up to 2 h post-oxygen/glucose deprivation. The glycine site full agonist D-serine completely occluded neuroprotection, indicating that GLYX-13 acts by modulating activation of this site.

Ischemia-related changes in naive and mutant forms of ubiquitin and neuroprotective effects of ubiquitin in the hippocampus following experimental transient ischemic damage

Ubiquitin binds to short-lived proteins and denatured proteins produced by various forms of injury. The loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structure and function. The aim of the present study is to observe the chronological changes in ubiquitin naive form and its mutant form (ubiquitin +1) in the hippocampal CA1 region (CA1) after transient cerebral ischemia in gerbils. Delayed neuronal death in the CA1 was confirmed 4 days after ischemic insult with NeuN immunohistochemistry. Ubiquitin immunoreactivity and protein level in the CA1 were lowest at 12 h after ischemia/reperfusion; thereafter, they were increased with time. Ubiquitin +1 immunoreactivity and protein levels in the CA1 were slightly decreased at 3 h after ischemia/reperfusion, and they were significantly increased 1 day after ischemia/reperfusion. In addition, ubiquitin and ubiquitin +1 immunoreaction was expressed in astrocytes after delayed neuronal death in the ischemic CA1. To elucidate the protective effect of ubiquitin on ischemic damage, the animals were treated with ubiquitin (1.5 mg/kg body weight) intravenously via the femoral vein. Ubiquitin treatment significantly reduced ischemia-induced locomotor hyperactivity, neuronal death and reactive gliosis such as astrocytes and microglia. In addition, 5 days after ubiquitin treatment in the ischemic group, ubiquitin immunoreactivity was similar to that in the ubiquitin-treated sham group, however, ubiquitin +1 immunoreactivity was higher than that in the ubiquitin-treated sham group. These findings indicate that the depletion of ubiquitin and the accumulation of ubiquitin +1 in CA1 pyramidal neurons after transient cerebral ischemia may inhibit ubiquitin proteolytic pathway and this leads to delayed neuronal death of CA1 pyramidal neurons directly or indirectly after transient cerebral ischemia.

Differential regulation of CXCL12 and PACAP mRNA expression after focal and global ischemia

Pituitary adenylate cyclase activating peptide (PACAP) and the chemokine stromal cell-derived factor (SDF-1) have been implicated in neuroprotection, neurogenesis, and regeneration. Focal ischemia is associated with rapid upregulation of PACAP in perifocal neurons and delayed induction of SDF-1 in hypoxic/ischemic tissues, the latter process being involved in the recruitment of stem cells and inflammatory cells. Here, we studied mRNA patterns of PACAP, SDF-1 and the cognate receptors PAC1 and CXCR4 by in situ hybridization in the rat hippocampus after transient global ischemia, a rat model for programmed death of CA1 pyramidal neurons. Cell death in CA1 was not associated with local induction of PACAP and SDF-1 expression or recruitment of CXCR4-expressing infiltrates. However, there was a transient, almost complete loss of SDF-1 expression in microvessels in all hippocampal regions. Granule cells transiently showed a decrease of SDF-1 and an increase of PACAP expression. While PAC1 mRNA was moderately decreased throughout the hippocampus, CXCR4 expression was selectively increased in the subgranular layer. We propose that altered PACAP and SDF-1 gene expression in granule cells plays a role in regulated neurogenesis after global ischemia. The finding that programmed neuronal death after global ischemia was not associated with SDF-1 upregulation or recruitment of CXCR4-expressing cells is in sharp contrast to SDF-1/CXCR4-mediated infiltration of infarct tissue after focal ischemia. Hence, the different modes of neuronal death after focal and global ischemia are associated with distinct SDF-1 and PACAP gene regulation patterns and distinct reorganization mechanisms.

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.

Ischemic insults promote epigenetic reprogramming of μ opioid receptor expression in hippocampal neurons

Transient global ischemia is a neuronal insult that induces delayed, selective death of hippocampal CA1 pyramidal neurons. A mechanism underlying ischemia-induced cell death is activation of the gene silencing transcription factor REST (repressor element-1 silencing transcription factor)/NRSF (neuron-restrictive silencing factor) and REST-dependent suppression of the AMPA receptor subunit GluR2 in CA1 neurons destined to die. Here we show that REST regulates an additional gene target, OPRM1 (mu opioid receptor 1 or MOR-1). MORs are abundantly expressed by basket cells and other inhibitory interneurons of CA1. Global ischemia induces a marked decrease in MOR-1 mRNA and protein expression that is specific to the selectively vulnerable area CA1, as assessed by quantitative real-time RT-PCR, Western blotting, and ChIP. We further show that OPRM1 gene silencing is REST-dependent and occurs via epigenetic modifications. Ischemia promotes deacetylation of core histone proteins H3 and H4 and dimethylation of histone H3 at lysine-9 (H3-K9) over the MOR-1 promoter, an signature of epigenetic gene silencing. Acute knockdown of MOR-1 gene expression by administration of antisense oligodeoxynucleotides to hippocampal slices in vitro or injection of the MOR antagonist naloxone to rats in vivo affords protection against ischemia-induced death of CA1 pyramidal neurons. These findings implicate MORs in ischemia-induced death of CA1 pyramidal neurons and document epigenetic remodeling of expression of OPRM1 in CA1 inhibitory interneurons.

Overexpression of Ca 2+-permeable AMPA receptor promotes delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia

To examine the role of Ca 2+ entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca 2+-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca 2+-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca 2+-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.

Anti-ischemic and cognition-enhancing properties of NNC-711, a γ-aminobutyric acid reuptake inhibitor

NNC-711 [1-(2-((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride], a γ-aminobutyric acid (GABA) reuptake inhibitor with anticonvulsant activity, was investigated with respect to its cognition-enhancing and neuroprotective potency. In the rat, administration of NNC-711 immediately prior to training prevented amnesia for a passive avoidance task induced by the acetylcholine receptor antagonist scopolamine. NNC-711 was also effective in protecting against ischemia-induced death of CA1 pyramidal neurons in a model of bilateral common carotid artery occlusion in the gerbil. In addition to a neuroprotective activity, NNC-711 exhibited significant cognition-enhancing actions. Daily administration of NNC-711, immediately prior to a spatial learning task, significantly reduced escape latencies in the water maze paradigm in both mature (postnatal day 80) and aged (28 months) rats. All of the above actions exhibited a bell-shaped response with an optimal dose of 0.5–1.0 mg/kg. These investigations with NNC-711 and previous clinical observations on the structurally related anticonvulsant tiagabine confirm the potential of GABA reuptake inhibitors as anti-amnesia and cognition-enhancing agents.

NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus.

The effect of N-methyl-D-aspartate (NMDA) and 2-(aminomethyl)phenylacetic acid/kainate (AMPA/kainate) glutamate receptors on dentate cell proliferation and hippocampal synapsin-I induction was examined after global ischemia. Cell proliferation was assessed using BrdU labeling, and synaptic responses were assessed using synapsin-I expression. Systemic glutamate receptor antagonists (MK-801 and NBQX) increased BrdU-labeled cells in the dentate subgranular zone (SGZ) of control adult gerbils (30% to 90%, P < 0.05). After global ischemia (at 15 days after 10 minutes of ischemia), most CA1 pyramidal neurons died, whereas the numbers of BrdU-labeled cells in the SGZ increased dramatically (>1000%, P < 0.0001). Systemic injections of MK801 or NBQX, as well as intrahippocampal injections of either drug, when given at the time of ischemia completely blocked the birth of cells in the SGZ and the death of CA1 pyramidal neurons at 15 days after ischemia. Glutamate receptor antagonists had little effect on cell birth and death when administered 7 days after ischemia. The induction of synapsin-I protein in stratum moleculare of CA3 at 7 and 15 days after global ischemia was blocked by pretreatment with systemic or intrahippocampal MK-801 or NBQX. It is proposed that decreased dentate glutamate receptor activation--produced by glutamate receptor antagonists in normal animals and by chronic ischemic hippocampal injury--may trigger dentate neurogenesis and synaptogenesis. The synapsin-I induction in mossy fiber terminals most likely represents re-modeling of dentate granule cell neuron presynaptic elements in CA3 in response to the ischemia. The dentate neurogenesis and synaptogenesis that occur after ischemia may contribute to memory recovery after hippocampal injury caused by global ischemia.

Neurogenesis and Gliogenesis in the Postischemic Brain

Stem cells are found throughout the adult mammalian brain, including the subventricular zone (SVZ) adjacent to the lateral ventricles, and in the hippocampal dentate subgranular zone (SGZ). Cells born in the SVZ migrate to the olfactory bulb. Those born in the SGZ migrate into the granule cell layer. Following 5 or 10 min of global ischemia in the adult gerbil, there is a tenfold increase in the birth of new cells in the SGZ as assessed using bromo-deoxy-uridine incorporation. This begins at 7 days, peaks at 11 days, and decreases thereafter. Over the ensuing month, approximately one-fourth of the newborn cells disappear. Of the remaining cells, 60% migrate into the granule cell layer where two-thirds of these become NeuN, calbindin, and MAP-2 immunostained neurons. The remaining 40% of the cells migrate into the dentate hilus where one-fourth of these become glial fibrillary acidic protein-labeled astrocytes. Death of CA1 pyramidal neurons does not stimulate neurogenesis because ischemia-induced tolerance—which does not produce CA1 injury—also stimulated cell proliferation. It is proposed that ischemia-induced neurogenesis contributes to the recovery of function, specifically of anterograde and retrograde recent memory function that is lost following global ischemia in man.

Delayed death of CA1 pyramidal neurons in gerbil hippocampus after brief ischemia is regulated by lysosomal proteinases

Delayed death of CA1 pyramidal neurons in gerbil hippocampus after brief ischemia is regulated by lysosomal proteinases

Increased intracellular Ca 2+ concentration in the hippocampal CA1 area during global ischemia and reperfusion in the rat: a possible cause of delayed neuronal death

The crucial role of free cytosolic Ca 2+ in ischemic neuronal damage has been studied in recent years. In the present report, changes in the intracellular Ca 2+ concentration in the hippocampal CA1 area during transient global ischemia and reperfusion were measured using in vivo Ca 2+ fluorometry with fura-2 in the four-vessel occlusion and reperfusion model in halothane-anesthetized rats. Marked changes were seen during 10-min global ischemia, with the intracellular Ca 2+ concentration increasing gradually following application of the ischemic insult and rapidly about 2 min after the beginning of ischemia, and continuing to increase until reperfusion. On reperfusion, the intracellular Ca 2+ concentration began to decrease and returned to the pre-ischemic level within 15 min. Induction of severe global ischemia was confirmed by the complete suppression of synaptic activity and the decrease in hippocampal temperature in the CA1 area. After seven days, CA1 pyramidal cell loss was observed histopathologically in the same rats which had undergone measurement of the intracellular Ca 2+ concentration changes. In the present study, a temporal profile of the free cytosolic Ca 2+ dynamics during ischemic and early post-ischemic period was determined in vivo. The results demonstrate that the intracellular Ca 2+ concentration in the hippocampal CA1 area is transiently and markedly increased during a brief ischemia-inducing delayed neuronal death, implying that Ca 2+ overload during cerebral ischemia is a possible cause of the delayed cell death of CA1 pyramidal neurons.

Bcl-x1 Bax Interaction after Transient Global Ischemia

Five minutes of bilateral common carotid artery occlusion in the Mongolian gerbil results in a selective, delayed death of CA1 pyramidal neurons. Although Bcl-2 appears to protect a variety of cells from cell death, the precise role of this apoptosis-regulating protein is complicated. We used immunoblots to estimate levels of Bcl-2, Bcl-x(l), and Bax at various times after carotid occlusion. Rather than Bcl-2, Bcl-x(l) appears to be the predominant neuroprotective form of this family of proto-oncogenes in the gerbil hippocampus. After transient ischemia, Bcl-2 and Bcl-x(l) protein levels remained the same. However, Bax levels were dramatically increased at 6 hours after ischemia, compared with sham-operated animals, and were still elevated at 72 hours after ischemia. To monitor dimerization interactions among theses apoptosis-regulating molecules, immunoprecipitation studies were conducted. These studies demonstrated that Bcl-x(l) association with Bax increases after ischemia. Therefore, Bax may disrupt the more favorable Bcl-x(l) (Bcl-2) interactions necessary for normal neuronal functioning and thus promote transient ischemic death.

Transient global forebrain ischemia induces a prolonged expression of the caspase-3 mRNA in rat hippocampal CA1 pyramidal neurons.

The authors recently cloned a cDNA for an ICE/CED3-related cysteine protease from rat brain, which is closely related to human CPP32 (now designated caspase-3). In situ hybridization histochemistry revealed a profound developmental regulation of the caspase-3 transcript in rat brain, with relatively high levels of caspase-3 mRNA observed in neurons of the fetal and neonatal brain and low levels of mRNA in neurons of the adult brain. The authors report that transient forebrain ischemia, which results in a delayed apoptotic death of CA1 pyramidal neurons, results in prolonged expression of caspase-3 mRNA in these same pyramidal neurons. Up-regulation of caspase-3 mRNA in CA1 pyramidal neurons is prominent 24 hours after transient global ischemia, and expression is maintained at higher levels for at least 72 hours after ischemia. However, by 96 hours after ischemia, a marked decrease in caspase-3 mRNA expression is observed in CA1 pyramidal neurons, showing severe degenerative changes (e.g., nuclear condensation). By contrast, there is no change in the expression of a closely related member of caspase family, caspase-2, in CA1 pyramidal neurons after global ischemia. Instead, caspase-2 mRNA is induced in lamina layers of cerebral cortex 24 hours after the ischemia. A selective and prolonged induction of the caspase-3 gene in committed CA1 pyramidal neurons suggests that transcriptional activation of this caspase-3 gene may be involved in the apoptotic cell death cascade of CA1 neurons after transient global ischemia.

Involvement of calcineurin A alpha and A beta in neuronal death in a gerbil model of cerebral ischemia

Treatment with FK506, an inhibitor of Ca2+/calmodulin dependent phosphatase (calcineurin, CaN), within 1 hr after transient ischemia afforded protection from apoptotic death in CA1 pyramidal neurons. To investigate isoform-specific roles of CaN in the neuronal cell death, we localized CaN A alpha and CaN A beta in the gerbil hippocampus using isoform-specific antibodies. In control gerbils, immunoreactions of both isoforms were highly enriched in hippocampal CA1 pyramidal neurons. Four to seven days after the induced ischemia, immunoreactivities of both isoforms were markedly reduced in the CA1 pyramidal cell and lacunosum-molecular layers. The CaN A alpha immunoreactivity was increased in the CA1 radiatum and oriens layers, whereas that of CaN A beta was enhanced in reactive astrocytes in the CA1 region. These findings suggest that CaN A alpha is involved in sprouting of afferent fibers in CA1 and that CaN A beta is involved in the reaction of astrocytes such as assembly of glial fibril acidic protein.

Eliprodil, a non-competitive, NR2B-selective NMDA antagonist, protects pyramidal neurons in hippocampal slices from hypoxic/ischemic damage

The N-methyl- d-aspartate (NMDA) subtype of glutamate receptor is one pathway through which excessive influx of calcium has been suggested to trigger ischemia-induced delayed neuronal death. NMDA receptors are heterooligomeric complexes comprised of both NR1 and NR2A-D subunits, in various combinations. NR2B-containing NMDA complexes exhibit larger, more prolonged conductances than those lacking this subunit. We tested the ability of the non-competitive, NR2B-selective NMDA antagonist eliprodil to (a) protect synaptic transmission in in vitro hippocampal slices from hypoxia, and (b) reduce ischemic delayed neuronal death in hippocampal organotypic slice cultures. Eliprodil markedly improved the recovery of Schaffer collateral-CA1 excitatory postsynaptic potentials following a 15 min hypoxic insult, with an EC50 of approximately 0.5 μM. In contrast to this functional protection, eliprodil did not reduce delayed death of CA1 pyramidal neurons in organotypic hippocampal slice cultures treated with severe hypoxia plus hypoglycemia, though it did potently protect CA3 pyramidal neurons in the same cultures. These data indicate that NMDA receptors containing NR2B subunits may play a role in long-term recovery of hippocampal synaptic function following ischemia/hypoxia. Furthermore, the selective protection of CA3, but not CA1, pyramidal neurons suggests that NR2B-containing NMDA receptors may preferentially contribute to an excitotoxic component of ischemia-induced delayed neuronal death.