Ultrastructural investigation of the CA1 region of the hippocampus after transient cerebral ischemia in gerbils

Summary Ultrastructural damage leading to delayed neuronal death was investigated in the mid-CA1 region of the hippocampus from the stratum (str.) moleculare to oriens after transient bilateral forebrain ischemia in Mongolian gerbils. After ischemia for 5 min without recirculation, mild swelling of the peripheral part of the apical and basal dendrites was already apparent in the str. moleculare and str. oriens. Mitochondria in the dendrites were also swollen in the same area. During recirculation for 12 h to 3 days, swelling of the dendritic cytoplasm persisted with formation of microvacuoles, but swelling of mitochondria receded. Microvacuolation and loss of microtubules were also observed in the proximal part of the dendrites during this period, and swelling and disruption of internal cristae were observed in mitochondria after recirculation for 3 days. The dendrites became severely degenerated after recirculation for 4 days. In the pyramidal cell bodies, no abnormality was observed at the end of ischemia for 5 min, but disaggregation of polyribosomes and swelling of the endoplasmic reticulum were observed 12 h after recirculation. Proliferation of the endoplasmic reticulum in parallel arrays occurred after recirculation for 1 day and persisted. Severe degeneration of the pyramidal cell bodies was obvious after recirculation for 4 days. The findings observed in the present investigation suggested that the neuronal structure most vulnerable to ischemia was the peripheral part of the dendrites and postischemic neuronal damage occurred early in this part of the dendrites.Supported by the grant NS-06663 from the National Institutes of Health, U.S. Public Health Service     

Temporal changes in extracellular acetylcholine and CA1 pyramidal cells in gerbil hippocampus following transient cerebral ischemia

Temporal changes in cholinergic functions following transient cerebral ischemia (10 min) were studied in the hippocampus of awake unrestrained gerbils using in vivo microdialysis. These data were compared with the results for temporal change in the area of each CA1 cell soma, measured with a microcomputer imaging device. KCl-induced release of acetylcholine (ACh) tended to be lower within 1 day after recirculation, and was significantly lower on the 4th, 7th and 14th days. Atropine-induced release of ACh gradually decreased over the test period. In histological estimation, no differences were observed within the 1st day, but a significant decrease of the area of CA1 cell soma was observed from the 4th to 14th days. Moreover, ischemia over 2 min decreased KCl- and atropine-induced ACh release on the 14th day without significant changes of hippocampal CA1 pyramidal cell. From these results, it is clear that ischemia produced dysfunction of hippocampal cholinergic neurons, and that dysfunction of the hippocampal cholinergic system following transient ischemia precedes pyramidal cell damage in the hippocampal CA1 subfield.     

The immunoreactivity and activity of adenylate cyclase type I are changed in the hippocampal CA1 region after transient forebrain ischemia in gerbils

Adenylate cyclase (AC) has a specific sensitivity to Ca²⁺/calmodulin. AC-I, one of the mediator of learning and memory, plays an important role in signal transduction underlying learning and memory function. In the present study, we found ischemia-related changes of AC-I in the hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, AC-I immunoreactive neurons were detected in pyramidal and non-pyramidal cells in the hippocampus proper. AC-I immunoreactivity was significantly increased at 3 h in the CA1 region after ischemic insult. Thereafter, AC-I immunoreactivity was gradually decreased. Four days after ischemic insult, AC-I-immunoreactive CA1 pyramidal cells in the stratum pyramidale were very few due to delayed neuronal death. The results of Western blot analysis showed that changes of AC-I protein contents were similar to immunohistochemical data after ischemic insult. Gpp(NH)p-dependent AC-I activity in hippocampal CA1 region was not changed in all groups, while Ca²⁺/calmodulin-dependent AC-I activity in hippocampal CA1 region was significantly decreased 24 h after ischemia–reperfusion. These results suggest that the decrease of AC-I activity may be associated with impairment of neurodevelopment and neuroplasticity including learning and memory although the AC-I immunoreactivity was maintained 24 h postischemic group compared to that of the sham-operated group.     

Temporal alterations in voltage gated Ca2+ channel immunoreactivities in the gerbil hippocampus following ischemic insults

In the present study, temporal changes of voltage-gated Ca²⁺ channel (VGCC) immunoreactivities were evaluated in the gerbil hippocampus following ischemia. P/Q-type VGCC immunoreactivity was elevated in the hippocampus in the 3 h post-ischemic group. In the 30 min post-ischemic group, N-type VGCC immunoreactivity began to increase only in the CA1 region. L-type (1C) VGCC immunoreactivity was significantly increased in the 12 h post-ischemic group. L-type (1D) VGCC immunoreactivity began to increase in the CA1 region in the 30 min post-ischemic group and peaked in the 12 h post-ischemic group. These findings suggest that the altered VGCC immunoreactivities following ischemia may play an important role in the ischemic neuronal injury.     

脑缺血再灌注后海马CA1区谷氨酸的表达变化及氟桂利嗪的影响

目的　观察暂短性脑缺血再灌注后沙土鼠海马区谷氨酸的表达变化以及氟桂利嗪干预的影响。方法　按照Kirino的方法 ,制作缺血再灌注模型。于缺血再灌注后 1、2、7天采取免疫组化方法检测谷氨酸表达 ,并于 7天在电镜和光镜下观察组织学变化。结果　缺血再灌注组 1天及 2天海马CA1区谷氨酸表达增高 (P <0 .0 1) ,7天恢复正常。光镜及电镜可见给药组存活的神经元数目明显多于缺血再灌注组 (P <0 .0 1)。结论　谷氨酸表达增高可能是鼠脑海马区迟发性神经元死亡的原因之一 ,氟桂利嗪可抑制谷氨酸的表达 ,对缺血的神经元起保护作用。     

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.     

An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia

Changes in MAP2 and clathrin immunoreactivity were studied in gerbil hippocampus after transient cerebral ischemia. MAP2 immuno-reactivity decreased significantly by 1 h in the subiculum-CA1 and CA2 areas which correspond to reactive change, while no decrease was observed in CA1 until day 4. Before the initiation of delayed neuronal death, MAP2 immunoreactivity was not changed in CA1. On the other hand clathrin immunoreactivity increased in the pyramidal cell layer of CA1 by 3 h after ischemia and remained high for 2 days. Clathrin immunoreactivity in the pyramidal cell layer of CA1 diminished after delayed neuronal death. The transient change of clathrin was noted especially in CA1 in the period prior to delayed neuronal death. These results imply an abnormal change in clathrin turnover after ischemia, which may participate in the pathogenesis of delayed neuronal death.     

Effect of transient forebrain ischemia on superoxide dismutases in gerbil hippocampus

Substantial generation of oxygen-derived free radicals has been implicated in pathophysiology of ischemic brain damage. Immunoreactive mitochondrial manganese and cytosolic copper-zinc superoxide dismutases, initial and essential enzymes to scavenge superoxide radical anions, increased in the gerbil hippocampal neurons after transient forebrain ischemia. Neuronal cells responded to oxidative stress in ischemia and induced the protective mechanism to increase superoxide dismutases.     

Transport of fragmented DNA in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia

Transport of fragmented DNA in apical dendrites of the CA1 pyramidal neurons of gerbil hippocampus is observed in the apoptotic process following transient forebrain ischemia. The time-course of specific DNA fragmentation was examined after the ischemic insult by in situ nick-end-labeling method and fluorescence detection technique by DAPI. Although the role of the fragmented DNA movement is unclear, the transport mechanism of fragmented DNA is still active in the late phase of apoptotic process.     

Chemical preconditioning with 3-nitropropionic acid: lack of induction of neuronal tolerance in gerbil hippocampus subjected to transient forebrain ischemia

Chemical preconditioning using the mitochondrial toxin, 3-nitropropionic acid (3-NP) has been reported to induce neuroprotection against subsequent global ischemia. To investigate the underlying mechanisms, Mongolian gerbils were pretreated with either vehicle or 3-NP at the dose of 3 or 10 mg/kg, intraperitoneal, 3 days prior to a 5-min bilateral carotid artery occlusion followed by either 48 h or 7 days of blood recirculation. Neuronal damage was assessed by a cresyl violet/fuchsin acid staining. Induction of heat shock protein 72 (HSP72) and manganese superoxide dismutase (MnSOD) expression was evaluated by Western blotting. Astroglial and microglial activation was detected by immunohistochemistry (glial fibrillary acid protein) and by histochemistry (isolectin B4 staining), respectively. Present data show that the hippocampal neuronal damage induced by ischemia were of similar extent between the vehicle- and 3-NP-treated gerbils, whatever the dose tested, indicating that 3-NP did not induce tolerance to transient forebrain ischemia under our experimental conditions. The lack of difference in the post-ischemic level of HSP72 and MnSOD protein expression and in the intensity of astroglial and microglial activation represents further indirect indications of the absence of 3-NP preconditioning effect. In conclusion, although chemical preconditioning with 3-NP is a well-established phenomenon at least in vitro and in models of focal ischemia, the relevance of 3-NP as a preconditioning molecule towards global brain ischemia remains an open question.     

Time interval after ischaemic preconditioning affects neuroprotection and gliosis in the gerbil hippocampal CA1 region induced by transient cerebral ischaemia

Objectives: Ischaemic preconditioning (IPC) can increase ischaemic tolerance of the central nervous system (CNS) to a subsequent longer or lethal period of transient ischaemia. In this study, we examined neuroprotective effects of time intervals after IPC against ischaemic insult in the hippocampus.

Methods: Animals were randomly assigned to six groups; sham-operated-group, ischaemia-operated-group, and three IPC (12?hours, 1- and 2-day intervals after IPC) plus ischaemia-groups (IPC-12?hour, 1 and 2-day interval-ischaemia-operated-groups). For neuroprotection, we carried out cresyl violet (CV) staining neuronal nuclei (NeuN) immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, we examined gliosis using immunohistochemistry for GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia).

Results: A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischaemia-operated-group and IPC-12?hours interval-ischaemia-operated-groups. In the IPC-1?day interval-ischaemia-operated-group, CA1 pyramidal neurons were well protected from ischaemic insult; the neuroprotective effect in the IPC-2?day interval-ischaemia-operated-group was less than that in the IPC-1?day interval-ischaemia-operated-group. On the other hand, we observed changes in glial cells (astrocytes and microglia) in the CA1 of all groups. The distribution pattern of glial cells only in the IPC-1?day interval-ischaemia-operated-group was similar to that in the sham-group.

Conclusion: In brief, our findings indicate that 1?day after IPC displays a mighty neuroprotection and shows an inhibition of glial activation in the CA1 induced by transient ischaemic insult.     

Na+/Ca2+ exchanger 1 alters in pyramidal cells and expresses in astrocytes of the gerbil hippocampal CA1 region after ischemia

Alterations of immunoreactivity and protein contents of Na(+)/Ca(2+) exchanger 1 (NCX1) were observed in the gerbil hippocampus proper after 5 min of transient forebrain ischemia. NCX1 immunoreactivity was significantly changed in the hippocampal CA1 region, but not in the CA2/3 region after ischemia/reperfusion. In the sham-operated group, NCX1 immunoreactivity was mainly detected in CA1 pyramidal cells. However, 30 min after ischemia/reperfusion, NCX1 immunoreactivity was significantly decreased and then increased at 1 day after ischemia. At this time, NCX1 immunoreactivity in CA1 pyramidal cells was similar to that of the sham-operated group. At 3 days after ischemia, NCX1 immunoreactivity was significantly reduced in the CA1 region compared to that of the sham-operated group and NCX1 immunoreactivity was significantly increased again 4 days after ischemia. Thereafter, NCX1 immunoreactivity was decreased time-dependently in ischemia groups. Between 15 min and 6 h post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum of the CA1 region. From 3 days post-ischemia, NCX1 immunoreactivity was expressed in astrocytes in the strata oriens and radiatum. Ischemia-induced changes in NCX1 protein contents in the hippocampus proper concurred with immunohistochemical data post-ischemia. Our results suggest that changes in NCX1 in CA1 pyramidal cells and astrocytes after ischemia are associated with intracellular Na(+) concentrations and that NCX1 may induce an intracellular calcium overload, which may be related to neuronal death.     

Relationship between magnitude of hypothermia during ischemia and preventive effect against post-ischemic DNA fragmentation in the gerbil hippocampus

Protective effect of hypothermia against DNA fragmentation in hippocampal CA1 field after transient forebrain ischemia in gerbils was evaluated by changing the magnitude of hypothermia. Inhibition of DNA fragmentation was proportional to the magnitude of hypothermia. The result indicates that, in terms of susceptibility to ischemia, hippocampal CA1 neurons are sensitive to a relatively small decrement of temperature, with temperatures ≤35°C being critical for the prevention of apoptotic process following transient forebrain ischemia.     

Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil

Synapsin-I is a vesicular phosphoprotein, which regulates neurotransmitter release, neurite development, and maturation of synaptic contacts during normal development and following various brain lesions in adulthood. In the present study, we have examined by immunohistochemistry possible modifications in the expression of synapsin-I in the hippocampus of Mongolian gerbils after transient forebrain ischemia. The animals were subjected to 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and were examined at different time-points post-ischemia. Transient forebrain ischemia produces cell death of the majority of CA1 pyramidal neurons of the hippocampus and polymorphic hilar neurons of the dentate gyrus. This is followed by reactive changes, including synaptic reorganization and modifications in the expression of synaptic proteins, which provide the molecular bases of synaptic plasticity. Transient decrease of synapsin-I immunoreactivity was observed in the inner zone of the molecular layer of the dentate gyrus, thus suggesting denervation and posterior reinervation in this area. In addition, a strong increase in synapsin-I immunoreactivity was observed in the hilus of the dentate gyrus and in the mossy fiber layer of the hippocampus at 2, 4 and 7 days after ischemia. Parallel increases in synaptophysin immunoreactivity were not observed, thus suggesting a selective induction of synapsin-I after ischemia. The present results indicate that synapsin-I participates in the reactive response of granule cells to transient forebrain ischemia in the hippocampus of the gerbil, and suggest a role for this protein in the plastic adaptations of the hippocampus following injury.     

Effects of fluoxetine on ischemic cells and expressions in BDNF and some antioxidants in the gerbil hippocampal CA1 region induced by transient ischemia

Fluoxetine, a selective serotonin reuptake inhibitor, alters several physiological processes, for example, elevating intracellular cAMP level, in the hippocampus. We examined the effect of fluoxetine on ischemia-induced neuronal death, the expression of brain-derived neurotrophic factor (BDNF) and changes in some antioxidative enzymes in the hippocampal CA1 region induced by transient ischemia. In addition, we also studied the effect of fluoxetine on locomotor activity in gerbils after ischemia/reperfusion. Animals were administered with various doses of fluoxetine (10, 20, and 40 mg/kg, i.p.) once daily for 3 days before the ischemic surgery. The treatment of 10 mg/kg and 20 mg/kg fluoxetine did not show significant neuroprotective effects on CA1 pyramidal cells 4 days after ischemia/reperfusion, while the treatment with 40 mg/kg fluoxetine in ischemic animals showed about 77% neuronal survival rate compared to the control group. The treatment of 40 mg/kg fluoxetine in ischemic animals enhanced significantly BDNF, catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase-1 (SOD1) immunoreactivity in the CA1 region compared to those in the saline-treated group 4 days after ischemia/reperfusion. In addition, the treatment of fluoxetine (10, 20, 40 mg/kg) significantly inhibited post-ischemic hyperactivity. In brief, treatment with fluoxetine protects neuronal damage after transient ischemia, and the neuroprotective effect of fluoxetine in an ischemic animal model may be related with the up-regulation of BDNF, CAT, GPX, and SOD1 expression.     

Elevation of Na+-K+ ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia

In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na(+)-K(+) ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na(+)-K(+) ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia-reperfusion, Na(+)-K(+) ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na(+)-K(+) ATPase was not altered. Interestingly, the density of Na(+)-K(+) ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na(+)-K(+) ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na(+)-K(+) ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult.     

DNA fragmentation in the CA2 sector of gerbil hippocampus following transient forebrain ischemia

It has been reported that following transient forebrain ischemia in the gerbil, "delayed neuronal death" and "reactive change" occur in hippocampal CA1 and CA2 sectors, respectively. In the present study, using the gerbil transient forebrain ischemia model, we examined brain sections after various recirculation periods and demonstrated, employing the in situ nick-end labeling (TUNEL) method, a nuclear DNA fragmentation in the damaged CA2 neurons.     

Nitric oxide synthase inhibitor reduces delayed neuronal death in gerbil hippocampal CA1 neurons after transient global ischemia without reduction of brain temperature or extracellular glutamate concentration

We planned a study to determine whether or not the mechanism of nitric oxide (NO) neurotoxicity involves the elevation of extracellular glutamate or changes of brain temperature in the pathogenesis of delayed neuronal death of gerbil hippocampal CA1 neurons following 5-min transient forebrain ischemia. Intraventricular injection of 5 μl of 5.0 mg/ml N^ω-nitro-l-arginine (LNNA) significantly preserved neuronal density in the central part of the CAI region examined 7 days after 5-min ischemia [188.5 ± 8.5/mm: 90.0% of the 209.5 ± 11.1 /mm density in the sham-operated controls vs. 16.7 ± 6.4/mm in those injected with artificial cerebrospinal fluid (CSF) only]. There was no difference between these two groups in hippocampal temperature before, during or after 5-min ischemia. The glutamate concentration ([Glu.]) during 5-min ischemia measured by a microdialysis technique was similar in the two groups (peak [Glu.] = 2.76 ± 0.62 pmol/μl dialysate in the artificial CSF group and = 2.93 ± 0.64 pmol/μ1 dialysate in the LNNA group). It was found that the neuronal toxicity of NO does not involve hyperthermia or the increase of extracellular glutamate concentration in the hippocampal CA1 region during 5-min ischemia.     

Expression of Bax and Bcl-2 protein in the gerbil hippocampus following transient forebrain ischemia and its modification by phencyclidine

Abstract

To determine the effect of phencyclidine (a noncompetitive NMDA receptor antagonist) on expression of Bax and Bcl-2 (apoptosis-regulating proteins) in gerbil hippocampus after transient forebrain ischemia, brain sections were immunohistochemically evaluated 48, 72, 96 hand 7 days following ischemia. In ischemic control animals, the expression of Bax in CA 7 neurons was increased with time and peaked at 72 h, then disappeared at 96 h. In the phencyclidine (5 mg kg^-1 , intraperitoneally)-treated animals, the intensity of Bax expression at 72 h was weaker than that of ischemic control animals. Furthermore, at 96 h, Bax expression was still observed in CA1 neurons. No expression of Bcl-2 in the CA1 neurons was detected in either control or phencyclidine-treated animals. From these results, it is possible that NMDA receptor antagonists exert their preventive effect against delayed neuronal death through inhibition of Bax protein expression, although the precise relationship between the function of Bax protein and delayed neuronal death is still unclear. [Neural Res 1997; 19: 629-633]     

短暂陛全脑缺血/再灌注损伤致大鼠海马组织BNIP3表达水平的改变

目的 观察全脑缺血/再灌注损伤后大鼠海马组织Bc12/腺病毒E1819kD相互作用蛋白3(BNIP3)表达水平的改变. 方法 取10只成年雄性Wistar大鼠,采用随机数字表法分为假手术组和全脑缺血/再灌注72h组,每组5只.通过尼氏染色检测缺血模型是否引起海马神经元死亡.另取14只成年雄性Wistar大鼠采用随机数字表法分为假手术组(4只)、全脑缺血/再灌注1h组(5只)、全脑缺血/再灌注6h组(5只).在全脑缺血/再灌注后,于对应时间点取三组大鼠海马组织制备蛋白样品.western blot检测各时间点BNIP3表达水平的改变. 结果 (1)与假手术组比较,全脑缺血/再灌注72 h组中海马CAI区神经元形态不规则.细胞皱缩.胞核碎裂消失,表明海马神经元死亡.(2)大鼠海马组织BNIP3单体表达水平在全脑缺血/再灌注后上调,与假手术组相比.全脑缺血/再灌注1 h组(2.543±0.473)与全脑缺血/再灌注6 h组(2.942±0.777)的海马组织BNIP3单体蛋白表达水平都升高,差异有统计学意义(P<0.05).但全脑缺血/再灌注1 h组与全脑缺血/再灌注6 h组间比较差异无统计学意义(P>0.05).BNIP3双聚体在各时间点表达水平差异无统计学意义(P>0.05). 结论 全脑缺血/再灌注损伤可以引起大鼠海马组织BNIP3单体表达水平上调.