Fluoro-Jade B histofluorescence staining detects dentate granule cell death after repeated five-minute transient global cerebral ischemia

Transient global cerebral ischemia (tGCI)-induced neuronal damage is variable according to its duration and degree. There are many studies on the damage or death of pyramidal cells of the hippocampus proper (CA1–3) in rodent models of tGCI. However, studies on the death of granule cells in the hippocampal dentate gyrus (DG) following tGCI have not yet been addressed. In this study, we examined the damage/death of granule cells in the gerbil DG at 5 days after various durations (5, 10, and 15 min) of single tGCI and repeated tGCI (two 5-min tGCI with 1-h interval) using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B (F-J B) histofluorescence staining. Neuronal death was observed only in the polymorphic layer in all single tGCI-operated groups. However, in the repeated tGCI-operated group, massive neuronal death was observed in the granule cell layer as well as in the polymorphic layer by using F-J B histofluorescence staining. In addition, microgliosis in the DG was significantly increased in the repeated tGCI-operated group compared to the 15-min tGCI-operated group. Taken together, these findings indicate that repeated brief tGCI causes granule cell death in the DG which could not occur by a longer duration of single tGCI.

     

Estrogen can act via estrogen receptor alpha and beta to protect hippocampal neurons against global ischemia-induced cell death

Estradiol at physiological concentrations intervenes in apoptotic death cascades and ameliorates neuronal death in experimental models of focal and global ischemia. The cellular targets that mediate estradiol protection of hippocampal neurons in global ischemia are, however, unclear. The present study examined the hypothesis that estradiol protects hippocampal neurons in ovariectomized rats via estrogen receptor (ER)alpha and/or beta. Estradiol (14 d pretreatment) afforded robust protection of CA1 neurons against global ischemia-induced death. The broad-spectrum ER antagonist ICI 182,780 (intracerebroventricularly, 0 and 12 h after ischemia) abolished estrogen protection, consistent with a role for ERs. To evaluate the potential roles of ERalpha vs. ERbeta in estrogen protection, we administered subtype-selective agonists for 14 d before and 7 d after ischemia. The ERalpha-selective agonist propyl pyrazole triol (PPT, 10 mg/kg) and ERbeta-selective agonist WAY 200070-3 (1 mg/kg) produced nearly complete protection of CA1 neurons in approximately 50% of the animals. PPT, but not WAY 200070-3, at doses used for protection, elicited lordosis, induced negative feedback inhibition of LH release, and reduced weight gain. These findings establish the efficacy of the PPT dose in neuroendocrine assays and specificity of WAY 200070-3 for ERbeta. We also examined the ability of estradiol and neuronal injury to regulate ERalpha and ERbeta expression. Both estradiol and global ischemia markedly increased ERalpha, but not ERbeta, protein in CA1. These data indicate that estradiol can act via ERalpha and ERbeta to protect CA1 neurons from global ischemia-induced death and that both estradiol and global ischemia modulate ERalpha expression in hippocampal CA1.     

Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia

The inducible isoform of the enzyme cyclooxygenase-2 (COX2) is an immediate early gene induced by synaptic activity in the brain. COX2 activity is an important mediator of inflammation, but it is not known whether COX2 activity is pathogenic in brain. To study the role of COX2 activity in ischemic injury in brain, expression of COX2 mRNA and protein and the effect of treatment with a COX2 inhibitor on neuronal survival in a rat model of global ischemia were determined. Expression of both COX2 mRNA and protein was increased after ischemia in CA1 hippocampal neurons before their death. There was increased survival of CA1 neurons in rats treated with the COX2-selective inhibitor SC58125 {1-[(4-methylsulfonyl) phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole} before or after global ischemia compared with vehicle controls. Furthermore, hippocampal prostaglandin E₂ concentrations 24 h after global ischemia were decreased in drug-treated animals compared with vehicle-treated controls. These results suggest that COX2 activity contributes to CA1 neuronal death after global ischemia.     

糖尿病大鼠脑缺血再灌注后细胞外信号调节激酶1/2表达与海马CA4区神经元凋亡

目的 探讨糖尿病大鼠脑缺血再灌注后神经元细胞外信号调节激酶(extracellularsignal-regulated kinase,ERK)1/2表达及其意义.方法 72只健康成年SD大鼠随机分假手术组、正常血糖脑缺血组和糖尿病腑缺血组,每组根据缺血再灌注不同时点分为缺血 15 min再灌注1 h、3 h,6 h亚组,每个亚组6只.采用链脲佐菌素诱导糖尿病,双血管闭塞联合放血法建立糖尿病大鼠全脑缺血模型.应用TUNEL和免疫组化方法观察海马CA4区神经元凋亡和磷酸化ERK1/2表达.结果 糖尿病脑缺血组在缺血 15 min、冉灌注1 h,3 h,6 h各时间点海马CA4区神经元凋亡发生率均明显高于正常血糖脑缺血组(P<0.05);糖尿病脑缺血组在各时间点磷酸化ERK1/2均有较高表达,在再灌注1 h和3 11时均明显高于正常血糖组(P<0.01).结论 ERK1/2可能参与了糖尿病加重脑缺血冉灌注后神经元损伤的机制.     

糖尿病大鼠脑缺血再灌注后细胞外信号调节激酶1/2表达与海马CA4区神经元凋亡

目的 探讨糖尿病大鼠脑缺血再灌注后神经元细胞外信号调节激酶(extracellularsignal-regulated kinase,ERK)1/2表达及其意义.方法 72只健康成年SD大鼠随机分假手术组、正常血糖脑缺血组和糖尿病腑缺血组,每组根据缺血再灌注不同时点分为缺血 15 min再灌注1 h、3 h,6 h亚组,每个亚组6只.采用链脲佐菌素诱导糖尿病,双血管闭塞联合放血法建立糖尿病大鼠全脑缺血模型.应用TUNEL和免疫组化方法观察海马CA4区神经元凋亡和磷酸化ERK1/2表达.结果 糖尿病脑缺血组在缺血 15 min、冉灌注1 h,3 h,6 h各时间点海马CA4区神经元凋亡发生率均明显高于正常血糖脑缺血组(P<0.05);糖尿病脑缺血组在各时间点磷酸化ERK1/2均有较高表达,在再灌注1 h和3 11时均明显高于正常血糖组(P<0.01).结论 ERK1/2可能参与了糖尿病加重脑缺血冉灌注后神经元损伤的机制.     

An immunohistochemical study of parvalbumin containing interneurons in the gerbil hippocampus after cerebral ischemia

We investigated postischemic changes of non-pyramidal neurons in the gerbil hippocampus 1 h - 7 days after 10 min of cerebral ischemia, with parvalbumin and microtubule-associated protein 2 (MAP2)-immunohistochemistry. Parvalbumin-immunoreactive interneurons in the hippocampus were unaffected up to 24 h after ischemia. A slight reduction of the immunoreactivity in neuronal processes was seen in the hippocampal CA1 sector 48 h after ischemia. Seven days after ischemia, a marked loss of parvalbumin-immunoreactive interneurons was observed in the hippocampal CA1 and CA3 sectors. Furthermore, reduced staining in the dentate granular and molecular layers was observed. MAP2-immunoreactive pyramidal neurons in the hippocampus were unchanged up to 48 h after ischemia. Seven days after ischemia, a severe loss of MAP2 immunoreactivity was found in the hippocampal CA1 and CA3 neurons and dentate hilar neurons. However, scattered CA1 neurons, most likely interneurons, preserved MAP2 immunoreactivity. The results demonstrate that transient cerebral ischemia can cause a loss of parvalbumin-immunoreactive interneurons in the hippocampus. Furthermore, some interneurons seem to lose parvalbumin synthesis. Although dentate granule cells are resistant to ischemia, considerable reductions of afferent input was suggested by parvalbumin staining.     

Blockade of calcium-permeable AMPA receptors protects hippocampal neurons against global ischemia-induced death

Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn(2+) in CA1 neurons just before the onset of histologically detectable cell death. Here we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca(2+)/Zn(2+)-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn(2+) and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn(2+) in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn(2+) and death of CA1 neurons, although a direct action at the time of the rise in Zn(2+) is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans.     

BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia

Mitochondria and cytochrome c release play a role in the death of neurons and glia after cerebral ischemia. In the present study, we investigated whether BID, a proapoptotic promoter of cytochrome c release and caspase 8 substrate, was expressed in brain, activated after an ischemic insult in vivo and in vitro, and contributed to ischemic cell death. We detected BID in the cytosol of mouse brain and primary cultured mouse neurons and demonstrated, by using recombinant caspase 8, that neuronal BID also is a caspase 8 substrate. After 2 h of oxygen/glucose deprivation, BID cleavage was detected in neurons concurrent with caspase 8 activation but before caspase 3 cleavage. Bid(-/-) neurons were resistant to death after oxygen/glucose deprivation, and caspase 3 cleavage was significantly reduced; however, caspase 8 cleavage did not differ from wild type. In vivo, BID was cleaved 4 h after transient middle cerebral artery occlusion. Infarct volumes and cytochrome c release also were less in Bid(-/-) mice (-67% and -41%, respectively) after mild focal ischemia. These findings suggest that BID and the mitochondrial-amplification pathway promoting caspase activation contributes importantly to neuronal cell death after ischemic insult.     

Involvement of caspase-2 long isoform in Fas-mediated cell death of human leukemic cells

Engagement of the plasma membrane receptor Fas can induce apoptosis of leukemic cells. Signaling through Fas requires the formation of a death-inducing signaling complex (DISC) that involves the cytoplasmic domain of Fas, the adaptor molecule FADD/MORT-1, and procaspase-8. The present study investigated whether another caspase, known as procaspase-2L, played a role in Fas-mediated cell death. A series of human leukemic variant cells was derived by stable transfection with a CASP2L antisense construct (CASP2L/AS). Specific down-regulation of procaspase-2L decreased the sensitivity of these cells to apoptosis induced by an agonistic anti-Fas antibody (Ab, clone CH11), as determined by studying DNA fragmentation, chromatin condensation, and externalization of phosphatidylserine on the plasma membrane. In leukemic cells transfected with an empty vector, anti-Fas Ab treatment activated caspase-8, decreased the expression of the BH3 domain-only protein Bid, triggered the release of cytochrome c from the mitochondria to the cytosol, and activated caspase-3. All these events could not be observed when CASP2L/AS cells were similarly treated with anti-Fas Abs. CASP2L/AS transfection did not inhibit the formation of the DISC and no direct interaction between procaspase-2L and either Fas or FADD or procaspase-8 was identified. Down-regulation of procaspase-2L inhibited anti-Fas Ab-mediated cleavage of c-FLIP (FLICE-inhibitory protein), a protein that interferes with the formation of a functional DISC. These results suggest that the long isoform of caspase-2 plays a role in the Fas-mediated pathway to cell death by contributing to caspase-8 activation at the DISC level.     

The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations

We have considered the extracellular serine protease thrombin and its receptor as endogenous mediators of neuronal protection against brain ischemia. Exposure of gerbils to prior mild ischemic insults, here two relatively short-lasting occlusions (2 min) of both common carotid arteries applied at 1-day intervals 2 days before a severe occlusion (6 min), caused a robust ischemic tolerance of hippocampal CA1 neurons. This resistance was impaired if the specific thrombin inhibitor hirudin was injected intracerebroventricularly before each short-lasting insult. Thus, efficient native neuroprotective mechanisms exist and endogenous thrombin seems to be involved therein. In vitro experiments using organotypic slice cultures of rat hippocampus revealed that thrombin can have protective but also deleterious effects on hippocampal CA1 neurons. Low concentrations of thrombin (50 pM, 0.01 unit/ml) or of a synthetic thrombin receptor agonist (10 microM) induced significant neuroprotection against experimental ischemia. In contrast, 50 nM (10 units/ml) thrombin decreased further the reduced neuronal survival that follows the deprivation of oxygen and glucose, and 500 nM even caused neuronal cell death by itself. Degenerative thrombin actions also might be relevant in vivo, because hirudin increased the number of surviving neurons when applied before a 6-min occlusion. Among the thrombin concentrations tested, 50 pM induced intracellular Ca(2+) spikes in fura-2-loaded CA1 neurons whereas higher concentrations caused a sustained Ca(2+) elevation. Thus, distinct Ca(2+) signals may define whether or not thrombin initiates protection. Taken together, in vivo and in vitro data suggest that thrombin can determine neuronal cell death or survival after brain ischemia.     

Ischemia-induced calpain activation causes eukaryotic (translation) initiation factor 4G1 (eIF4GI) degradation, protein synthesis inhibition, and neuronal death

Persistent protein synthesis inhibition (PSI) is a robust predictor of eventual neuronal death following cerebral ischemia. We thus tested the hypothesis that persistent PSI inhibition and neuronal death are causally linked. Neuronal viability strongly correlated with both protein synthesis and levels of eukaryotic (translation) initiation factor 4G1 (eIF4G1). We determined that in vitro ischemia activated calpain, which degraded eIF4G1. Overexpression of the calpain inhibitor calpastatin or eIF4G1 resulted in increased protein synthesis and increased neuronal viability compared with controls. The neuroprotective effect of eIF4G1 overexpression was due to restoration of cap-dependent protein synthesis, as well as protein synthesis-independent mechanisms, as inhibition of protein synthesis with cycloheximide did not completely prevent the protective effect of eIF4G1 overexpression. In contrast, shRNA-mediated silencing of eIF4G1 exacerbated ischemia-induced neuronal injury, suggesting eIF4G1 is necessary for maintenance of neuronal viability. Finally, calpain inhibition following global ischemia in vivo blocked decreases in eIF4G1, facilitated protein synthesis, and increased neuronal viability in ischemia-vulnerable hippocampal CA1 neurons. Collectively, these data demonstrate that calpain-mediated degradation of a translation initiation factor, eIF4G1, is a cause of both persistent PSI and neuronal death.     

Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.     

Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca(2+) influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca(2+)-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.     

Galanin acts as a neuroprotective factor to the hippocampus

The expression of the neuropeptide galanin is markedly up-regulated in many areas of the central and peripheral nervous system after injury. We have recently demonstrated that peripheral sensory neurons depend on galanin for neurite extension after injury, mediated by activation of the second galanin receptor subtype (GALR2). We therefore hypothesized that galanin might also act in a similar manner in the CNS, reducing cell death in hippocampal models of excitotoxicity. Here we report that galanin acts an endogenous neuroprotective factor to the hippocampus in a number of in vivo and in vitro models of injury. Kainate-induced hippocampal cell death was greater in both the CA1 and CA3 regions of galanin knockout animals than in WT controls. Similarly, exposure to glutamate or staurosporine induced significantly more neuronal cell death in galanin knockout organotypic and dispersed primary hippocampal cultures than in WT controls. Conversely, less cell death was observed in the hippocampus of galanin overexpressing transgenic animals after kainate injection and in organotypic cultures after exposure to staurosporine. Further, exogenous galanin or the previously described high-affinity GALR2 agonist, both reduced cell death when coadministered with glutamate or staurosporine in WT cultures. These results demonstrate that galanin acts an endogenous neuroprotective factor to the hippocampus and imply that a galanin agonist might have therapeutic uses in some forms of brain injury.