Melatonin administration protects CA1 hippocampal neurons after transient forebrain ischemia in rats

Melatonin administered at the beginning of cerebral reperfusion protected CA1 neurons against 10, 20 and 30 min of transient forebrain ischemia. Intraperitoneal injections of saline or melatonin (10 mg/kg) were given after 0, 2 and 6 h, or 1, 2 and 6 h of cerebral reperfusion, or 30 min prior to ischemia. One week later, quantitative histological analysis demonstrated that CA1 neuronal density was significantly increased in the melatonin groups that were treated at 0, 2, 6 h compared to the saline-treated controls. Ischemic protection of CA1 was lost in the animals in which the melatonin treatment was delayed by 1 h, or given 30 min prior to the ischemia.     

Delayed transient ischemic attacks kill some CA1 neurons previously salvaged with postischemic hypothermia: neuroprotection undone

Delayed hypothermia reduces ischemic hippocampal CA1 injury. However, there are residual structural and functional abnormalities. Therefore, we studied whether these apparently vulnerable rescued neurons are susceptible to secondary insults. All gerbils were subjected to normothermic forebrain ischemia (ISC, 5 min) or SHAM operation. Gerbils were treated with mild hypothermia (HYPO; 33 degrees C for 24 h+35 degrees C for 24 h) beginning 12 h after surgery, or they remained normothermic (NORMO). Then 5 and 6 days following ISC/SHAM operation gerbils received sublethal transient ischemic attacks (TIA, 1.5 min) or sham (SH) surgeries. Behavioral testing was done and animals survived for 30 days for quantification of medial, middle and lateral CA1 sector cell death. The SHAM groups were not significantly different. The ISC+NORMO+SH group lost 87.3% (of SHAM) of medial CA1 neurons, which was not significantly exacerbated in the ISC+NORMO+TIA group (91.1%, P=0.633). However, the ISC+HYPO+TIA group (58.8% loss) had significantly more cell death than the ISC+HYPO+SH group (42.8%; P=0.035), although CA1 protection was still better than in ISC+NORMO groups (P<0.001). Trends were similar in middle and lateral CA1, but the deleterious effects of TIAs were not statistically significant. Behavioral testing did not distinguish groups with or without TIA, but did reveal deficits in ISC+NORMO groups and protection in ISC+HYPO groups. These data, like previous ultrastructural findings, show that while most hypothermia-rescued CA1 neurons are healthy, some are susceptible. Perhaps other neuroprotectants, especially weaker ones, might be undone by delayed insults (e.g. TIA, fever).     

Delayed neuronal death in hippocampal CA1 pyramidal neurons after forebrain ischemia in hyperglycemic gerbils: amelioration by indomethacin

Hyperglycemia worsens ischemic-induced neuronal damage. Many reports argue the delayed neuronal cell death (DND) after forebrain ischemia in gerbils is due to apoptosis. We examined the effects of hyperglycemia and indomethacin on DND after forebrain ischemia in gerbils. Complete occlusion of both common carotid arteries was performed for 3.5 min followed by declamping and reperfusion. Blood glucose levels were maintained at 25-30 mmol/1 for 24 h after reperfusion in the hyperglycemic groups. We examined morphological changes consistent with DND using Nissel-stained sections and DNA fragmentation using TUNEL staining, at 12, 24, 36, 48, 60, 72, 84, 96, 108, 120 h, and 7 days after reperfusion. DND was noted 96-120 h after ischemia in normoglycemic group. Hyperglycemia enhanced the development of DND at an earlier stage (48-84 h after ischemia). TUNEL positive neurons were detected 72-108 h after reperfusion in normoglycemic group, but very few TUNEL positive neurons were detected in hyperglycemic group at 36-48 h. Indomethacin reduced the number of TUNEL-positive cells in normoglycemia and completely inhibited the appearance of TUNEL-positive cells under hyperglycemia. The number of viable neurons at 7 days after ischemia was markedly higher in indomethacin-treated groups than vehicle-treated group. Our results indicate that hyperglycemia worsens DND after forebrain ischemia in gerbils but such process is not associated with DNA fragmentation. Our results also showed that indomethacin provides a neuroprotective effect in normo- and hyperglycemic conditions.     

Nitrotyrosine immunoreactivity in gerbil hippocampal CA1 region after transient forebrain ischemia

We examined whether or not nitration of tyrosine residues takes place in the gerbil hippocampal CA1 region after transient forebrain ischemia. The nitration of tyrosine residues to produce nitrotyrosine is a footprint of peroxynitrite, a reaction product of nitric oxide (NO) with superoxide. Nitrotyrosine immunoreactivity had been detected in the CA1 region from the early stage in a reperfused brain at 30 min after transient ischemia until DNA fragmentation and neuronal death appeared at 4 days after transient ischemia. In electron microscopy, we detected, prominently, nitrotyrosine immunoreactivity after transient ischemia in the cytoplasm of the CA1 neurons. Therefore, it is considered that the nitration of tyrosine residues by peroxynitrite may be closely related to apoptosis after transient ischemia.     

Gamma-vinyl GABA prevents hippocampal and substantia nigra reticulata damage in repetitive transient forebrain ischemia

GABAergic inhibitory mechanisms may offer protection to neurons after global ischemia. We tested the effects of γ-vinyl GABA, a GABA-transaminase inhibitor, via continuous infusion in the third ventricle (Alza pumps) in a gerbil model of repetitive forebrain ischemia. We used two episodes of 3 min duration with a ’reperfusion’ interval of 1 h between the insults. Histological analysis was done with silver staining 5 days after the insult. Our results show that there is significant protection of the hippocampus CA1 region and substantia nigra reticulata in treated animals compared to controls. An increase in GABA levels, decrease in glutamate, or mild hypothermia, may be potential mechanisms for this protection. GABAergic agents may prove useful agents in repetitive ischemia.     

Recovery of protein synthesis in tolerance-induced hippocampal CA1 neurons after transient forebrain ischemia

Protein synthesis at various recirculation times after 5-min transient forebrain ischemia was evaluated in gerbil hippocampal CA1 pyramidal neurons that had acquired tolerance to delayed-type ischemic injury. Evaluation was performed by observing polyribosomes under electron microscopy, and by [¹⁴C] leucine autoradiography. Hippocampal CA1 pyramidal neurons in the gerbils acquired stable and reproducible tolerance to delayed-type ischemic injury subsequent to a 5-min ischemia by pretreatment that consisted of loading two 2-min ischemic periods at a 1-day interval, followed by 48 h of recirculation. During the early phase following the 5-min ischemia, polyribosomal disaggregation, loss of dendritic microtubules, and significant suppression of radiolabeled leucine incorporation were observed in the tolerance-induced CA1 neurons as well as in the non-tolerance-induced neurons. While these findings persisted in the non-tolerance-induced neurons throughout the duration of the experiment, most of the tolerance-induced neurons demonstrated reaggregation of cytosomal ribosomes, increase in the number of dendritic microtubules, and restoration of impaired amino acid incorporation 24 h after the ischemia. These findings suggest that revovery of protein synthesis during the early post ischemic phase is essential for CA1 neuron survival after ischemic injury.Supported by the Ehime Health Foundation. This study was carried out in compliance with the Guidelines for Animal Experimentation at Ehime Univesity School of Medicine, Ehime, Japan     

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.     

Selective proteasomal dysfunction in the hippocampal CA1 region after transient forebrain ischemia.

Delayed neuronal death in the hippocampal CA1 region after transient forebrain ischemia may share its underlying mechanism with neurodegeneration and other modes of neuronal death. The precise mechanism, however, remains unknown. In the postischemic hippocampus, conjugated ubiquitin accumulates and free ubiquitin is depleted, suggesting impaired proteasome function. The authors measured regional proteasome activity after transient forebrain ischemia in male Mongolian gerbils. At 30 minutes after ischemia, proteasome activity was 40% of normal in the frontal cortex and hippocampus. After 2 hours of reperfusion, it had returned to normal levels in the frontal cortex, CA3 region, and dentate gyrus, but remained low for up to 48 hours in the CA1 region. Thus, the 26S proteasome was globally impaired in the forebrain during transient ischemia and failed to recover only in the CA1 region after reperfusion. The authors also measured 20S and 26S proteasome activities directly after decapitation ischemia (at 5 and 20 minutes) by fractionating the extracts with glycerol gradient centrifugation. Without adenosine triphosphate (ATP), only 20S proteasome activity was detected in extracts from both the hippocampus and frontal cortex. When the extracts were incubated with ATP in an ATP-regenerating system, 26S proteasome activity recovered almost fully in the frontal cortex but only partially in the hippocampus. Thus, after transient forebrain ischemia, ATP-dependent reassociation of the 20S catalytic and PA700 regulatory subunits to form the active 26S proteasome is severely and specifically impaired in the hippocampus. The irreversible loss of proteasome function underlies the delayed neuronal death induced by transient forebrain ischemia in the hippocampal CA1 region.     

Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils

In the present study, we investigated chronological changes of galanin (GAL), well known as the potassium channel opener, immunoreactivity and GAL protein level in the hippocampus of the gerbil at the various times after 5 min transient forebrain ischemia. In the sham-operated group, weak GAL immunoreactivity was found in non-pyramidal cells. At 12 h after ischemia-reperfusion, the number of GAL-immunoreactive neurons and GAL immunoreactivity were significantly increased in the hippocampus compared to 3 h after ischemic insult, especially in the hippocampal CA1 region. Thereafter the number of GAL-immunoreactive neurons and GAL immunoreactivity decrease time-dependently in the hippocampus. Four days after transient ischemia, GAL immunoreactivity was low as compared with the sham-operated group. At this time point after ischemic insult, GAL immunoreactivity was shown in microglia in the CA1 region because delayed neuronal death happened in the CA1 pyramidal cells. The result of Western blot showed the pattern of GAL expression similar to that of immunohistochemical data. These results suggest that the early increase of GAL in the CA1 pyramidal cells may be associated with the reduction of the excitotoxic damage, that long-lasting enhanced expression of endogenous GAL at 12 h-2 days after ischemia may be associated with efflux of potassium ion into the extracellular space, and that GAL expression in microglia 4 days after ischemia may be associated with reduction of ischemic damage.     

Laser doppler flowmetry in CA1 sector of hippocampus and cortex after transient forebrain ischemia in gerbils.

BACKGROUND AND PURPOSE: Local differences in the hemodynamic response to transient ischemia could be involved in the development of selective vulnerability. These differences were studied in vulnerable and nonvulnerable regions of the brain. METHODS: Five gerbils were subjected to 10 minutes of bilateral forebrain ischemia, and cerebral blood flow was measured continuously in the frontal cortex and CA1 sector of the hippocampus using laser Doppler flowmetry. Carotid artery pressure was recorded simultaneously with a pressure transducer. RESULTS: After induction of ischemia, blood flow in the cortex and CA1 sector decreased to 11.8% and 18.0% of the baseline value, respectively. After release of the vascular occlusion, blood flow in the cortex returned to the preischemic level at 7.5 minutes (recovery time), reached the hyperemic peak (123.8%) at 12.4 minutes (peak latency), and again decreased to the preischemic level at 27.2 minutes. In the CA1 sector, blood flow returned to the preischemic level at 2.1 minutes, reached the hyperemic peak (122.2%) at 5.7 minutes, and decreased again to the preischemic level at 21.3 minutes. In both the cortex and CA1 sector, recovery time and peak latency correlated inversely with the amount of residual blood flow during ischemia. Histologically, cortical neurons were not injured but only 14.6% of CA1 neurons survived 1 week after ischemia. CONCLUSIONS: CA1 neurons were selectively injured despite the milder percentage decrease of blood flow during ischemia and the more prompt recovery of flow after ischemia. These findings stress the importance of intrinsic rather than hemodynamic factors in the pathogenesis of selective vulnerability of CA1 neurons after transient bilateral forebrain ischemia.     

小鼠脑缺血后氧化DNA损伤的早期修复与神经保护的相关性研究

目的建立原位检测C57BL／6小鼠脑缺血所致AP位点和3＇-PO4型氧化DNA损伤的方法。同时采用TUNEL测定神经细胞凋亡，探讨两者的相关性，并观察nNOS在此过程中的作用。方法制作小鼠前脑缺血／再灌注模型（FbIR）：夹闭双侧颈总动脉90min后恢复血流，按实验设计的再灌注不同时间点处死动物后制作脑组织冰冻切片。用大肠杆菌核苷酸外切酶Ⅲ敏感位点法（EXOSS）检测AP位点和3’-PO4末端型两种氧化DNA损伤；采用TUNEL技术观察细胞凋亡，并检测特异性nNOS抑制剂3BR7NI对此氧化DNA损伤及细胞凋亡的影响。结果与假手术对照组相比，EXOSS阳性率在再灌注15min时至少增高20倍（P〈0．01），并在此后的30min内持续增高；EXOSS阳性主要出现在神经元的细胞核内；细胞死亡在再灌注24h组能检测到，并主要发生在神经元细胞内；在一定时间内大脑皮质区神经元细胞内的氧化DNA损伤和细胞凋亡均能被特异性nNOS抑制剂3-bromo-7-nitroindazole（3BR7NI，30mg／kg，腹腔内注射）所抑制。结论EXOSS是一种原位检测AP位点和3’-PO4末端型氧化DNA损伤的有效方法，该损伤在一定时间内可以得到修复，nNOS在这一过程中起着重要作用。     

Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils

Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct. To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils. MK administration at the dose of 0.5-2 microg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult. The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult. MK administration at 2 h after occlusion did not ameliorate the neuronal death. These findings suggested that the therapeutic time window was narrow. The two to four times repeated administration of 2 microg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone. These findings suggested that the therapeutic time window was relatively narrow. The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.     

The therapeutic window of hypothermic neuroprotection in experimental ischemic neuropathy: protection in ischemic phase and potential deterioration in later reperfusion phase

Hypothermia will neuroprotect peripheral nerve from ischemia-reperfusion (IR) injury, but the therapeutic window of hypothermic neuroprotection has not been defined. Unilateral IR injury was produced by the ligation and release of nooses tied around supplying arteries to the right sciatic-tibial nerve of the rat. Using this model, 114 rats were divided into 12 groups according to the delay (0, 1, 3, and 4 h) and the depth of hypothermia (28, 32, and 35 degrees C). All rats were subjected to 3 h ischemia and 7 days reperfusion followed by behavioral, electrophysiological, and pathological evaluations. We demonstrated significant hypothermic neuroprotection with both deep (28 degrees C) and mild (32 degrees C) hypothermia initiated during ischemia (0 and 1 h delay), but not hypothermia initiated during reperfusion (3 and 4 h delay) in both behavioral and electrophysiological evaluations. In addition, the pathologically significant differences were observed between deep hypothermia (28 degrees C) and normothermia (35 degrees C) initiated during ischemia. We conclude that the therapeutic window of hypothermic neuroprotection is optimal during the intraischemic period and that mild and deep hypothermia provide neuroprotection. Prolonged delay of hypothermic treatment results in worsening of IR injury.     

Caffeinol confers cortical but not subcortical neuroprotection after transient focal cerebral ischemia in rats

The combination of low-dose ethanol and caffeine (caffeinol) protects cortical areas of the brain from damage produced by distal focal ischemia in rats. There are no data, however, as to whether caffeinol influences injury in subcortical brain regions. Rats were anesthetized with halothane and subjected to 2 h of MCAo by poly-l-lysine-coated intraluminal suture. Caffeinol [a combination of ethanol, 0.33 g/kg, and caffeine, 10 mg/kg (n=5)] or vehicle (0.9% NaCl; n=7) was administered by i.v. infusion over a 2.5-h period beginning 15 min after reperfusion. Neurological status was evaluated daily, and histopathology was quantified at 3 days. Caffeinol therapy significantly improved the neurological score, reduced the total infarct volume (by 52%) and cortical infarct areas at multiple coronal levels, but subcortical infarction and brain swelling were not affected.     

Neuronal damage,glial response and cerebral metabolism after hypothermic forebrain ischemia in the rat

Summary We investigated the effect of 30°C whole body hypothermia on neuronal injury, astroglial reactivity and intracellular pH in rats subjected to 15 min of forebrain ischemia. Experimental groups included: (1) normothermic ischemia (n=8), ischemia induced under 37°C body temperature, (2) hypothermic ischemia (n=6), ischemia induced under 30°C body temperature. Cerebral intracellular pH was measured using in vivo ³¹P NMR spectroscopy over 7 days. Neuronal injury and astrocytic reactivity were evaluated using hematoxylin and eosin staining, and immunoreactivity to glial fibrillary acidic protein, respectively. Normothermic animals revealed significant alkalosis (P<0.01) at 48 h after ischemia compared to the pre-ischemic value. No significant intracellular pH change was detected after ischemia in the hypothermic group. Ischemic neuronal injury was prevented in the hypothermic animals, compared to the severe neuronal injury found in the normothermic animals (P<0.01). The marked astrocytosis of normothermic animals was significantly inhibited in the hypothermic animals (P<0.01). Our data indicate, that hypothermia significantly inhibits neuronal injury as well as post-ischemic alkalois and astrocytosis, induced by 15 min of forebrain ischemia in the rat.Supported by NINDS grants NS23393 and NS29463     

Asymmetric damage of the CA1 sector of Ammon's horn after short-term forebrain ischemia in Mongolian gerbils.

The studies were carried out on 98 three-month-old Mongolian gerbils, submitted to short-term (5 or 7.5 min) forebrain ischemia induced by bilateral ligation of common carotid artery. After 5-day survival animals were sacrificed by transcardiac perfusion with 10% formaldehyde. Paraffin brain sections were stained with cresyl violet and according to Klüver-Barrera method. Pickworth benzidine method was also applied to evaluate hippocampal vascular network. Varying susceptibility of individual animals to the ischemic incident was found. This was expressed by differences in the intensity and extent of structural lesions of CA1 pyramidal neurons. No abnormalities were found in 41.7% of animals, total neuronal loss in the CA1 sector was observed in 33.3% of cases, while the partial neuronal loss appeared in the remaining 25% of animals. Asymmetric distribution of the neuronal changes observed in 18.4% of cases was a very striking feature. Differences of the angioarchitectonics of CA1 sector as compared with neighbouring parts of Ammon's horn were found. In the pyramidal cell layer very scarce fragments of the blood vessels were present. In adjacent cortical layers (stratum oriens and stratum radiatum) relatively dense capillary network was characterized by appearance of specific vascular loops and tangles localized on the border of stratum pyramidale. It is supposed that particular spatial arrangement of the vascular network in pyramidal layer of CA1 sector, favouring appearance of local hemodynamic and rheologic abnormalities after temporary brain ischemia, may play an essential pathogenic role in both, selective vulnerability of this neuronal population and individual variations in the intensity and distribution of the neuronal changes.     

Indefatigable CA1 sector neuroprotection with mild hypothermia induced 6 hours after severe forebrain ischemia in rats.

Considerable controversy exists about whether postischemic hypothermia can permanently salvage hippocampal CA1 neurons or just postpone injury. Studies of very brief cooling in rat have found transient benefit, whereas experiments in gerbil using protracted hypothermia report lasting protection. This discrepancy might be because of the greater efficacy of longer cooling or it might, for example, represent an important species difference. In the present study, a 48-hour period of mild hypothermia was induced starting 6 hours after 10 minutes of severe four-vessel occlusion ischemia in rats. Untreated normothermic ischemia resulted in total CA1 cell loss (99%), whereas delayed hypothermia treatment reduced neuronal loss to 14% at a 28-day survival. In unregulated rats, brain temperature spontaneously fell during ischemia, and stayed subnormal for an extended period after ischemia. This mild cooling resulted in more variable and less severe CA1 injury (75%). Finally, vertebral artery cauterization under halothane anesthesia caused an approximately 2 degrees C drop in brain temperature for 1 hour, but prevention of this hypothermia did not significantly affect CA1 damage. In summary, protracted postischemic hypothermia provided robust and long-term CA1 protection in rat. These results encourage the clinical assessment of prolonged hypothermia and its use as a model to understand ischemic CA1 injury.     

Enhancement of GABA neurotransmission after cerebral ischemia in the rat reduces loss of hippocampal CA1 pyramidal cells

Increased excitation may be involved in the development of delayed CA1 pyramidal cell death in hippocampus after global cerebral ischemia. Therefore we investigated the possible neuroprotective effect of the GABA uptake inhibitor, R-(-)-1-(4,4-(3-methyl-2-thienyl)-3-butenyl)-3-piperidine carboxylic acid (No-328), in a rat cerebral ischemia model of delayed CA1 pyramidal cell death. No-328 in doses of 36 mg/kg given 30 min before, and 1, 24, 48 and 72 h after ischemia significantly reduced the CA1 neuron loss. Doses of 50 mg/kg of No-328 given immediately before, 24 h and 48 h after ischemia, also reduced the CA1 neuron loss significantly. Furthermore, we demonstrated that postischemic treatment with diazepam (4 x 15 mg/kg) significantly reduced the CA1 neuron loss. However, postischemic treatment with several doses (5 x 12 mg/kg) of the GABA analog, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), offered no CA1 neuron protection when given alone, but when administrated together with diazepam (4 x 15 mg/kg) it significantly reduced the CA1 neuron loss. We conclude that enhancement of postischemic GABA neurotransmission, during the first 2-3 days after ischemia, may reduce the ischemic CA1 damage through a continuous increase in hippocampal GABA extracellular levels (No-328), or through an increase in sensitivity to GABA neurotransmission (diazepam).     

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.