Induction of tolerance to ischemia: Alterations in second-messenger systems in the gerbil hippocampus

Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu), [3H]cyclic adenosine monophosphate (cAMP) and [3H]rolipram, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [3H]IP3 binding (p < 0.01 vs. control) and 15-25% reductions in [3H]forskolin (p < 0.01 vs. control), [3H]cAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CA1 pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [3H]IP3 and [3H]rolipram binding in CA1 showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively, in the early reperfusion period. This downregulation of the IP3 system may play a role in the protection against cell death.     

Preserved neurotransmitter receptor binding following ischemia in preconditioned gerbil brain

Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine, [3H]muscimol, and [3H]PN200-110, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [3H]QNB binding in the CA1 subfield. Three-minute ischemia destroyed the majority of CA1 pyramidal cells and caused, in CA1, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA1 neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-110 binding transiently decreased in the early reperfusion period, suggesting down-regulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death.     

Ischemic tolerance to memory impairment associated with hippocampal neuronal damage after transient cerebral ischemia in rats

When rats were trained preoperatively with a three-panel runway task and were then exposed to 10-min ischemia by the method of 4-vessel occlusion, they showed no increase in the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points), having normal retention of memory performance learned before the ischemic insult. Next, we investigated the abilities of ischemic rats to acquire the three-panel runway task and to learn a subsequent reversal task, where the correct panel-gate locations were changed. Rats with 5-min ischemia exhibited performance as good as that of control rats, but rats exposed to 10- and 20-min ischemia showed more errors than control rats during 10 acquisition sessions and 5 subsequent reversal sessions, each of which (consisting of 6 trials) was given once a day. Marked neuronal degeneration was observed in the hippocampal CA1 sector from the rats with 10- and 20-min ischemia. Exposure to sublethal 5-min ischemia followed by 10-min ischemia at a 2-h interval had no effect on either the memory impairment during acquisition and reversal tests or the hippocampal CA1 damage. When rats were exposed to 5-min ischemia 2 days before lethal 10-min ischemia, they showed acquisition and subsequent reversal learning as good as that of control rats. Preconditioning with sublethal 5-min ischemia followed by 2 days of reperfusion also prevented the neuronal destruction of the hippocampal CA1 sector induced by 10-min ischemia. These findings suggest that postischemic hippocampal CA1 neuronal damage does not affect retention of spatial memory acquired before ischemia, but produces a significant impairment of acquisition and subsequent reversal learning. The present results also demonstrate that preconditioning with sublethal ischemia can develop tolerance to subsequent lethal ischemia to prevent the learning impairment related to the hippocampal CA1 neuronal damage.     

大鼠脑缺血再灌流后海马氨基酸水平变化与神经元损害的关系探讨

目的探讨脑缺血再灌流后海马氨基酸递质变化与神经元损害的关系。方法建立大鼠前脑缺血再灌流模型,测定海马ＣＡ１区和ＣＡ３／齿状回区游离氨基酸含量,观察阻断隔－海马通路对海马神经元损害和氨基酸水平的影响。结果（１）海马结构中仅ＣＡ１区神经元明显损害,但ＣＡ１区和ＣＡ３／齿状回区的Ｇｌｕ、Ａｓｐ和ＧＡＢＡ含量无差异。（２）阻断隔－海马通路可明显减轻海马神经元损害,但对海马氨基酸水平变化无影响。结论脑缺血再灌流后,氨基酸递质水平的异常变化不是海马ＣＡ１区神经元选择性易损的唯一决定因素,隔－海马通路末梢释放的神经递质也参与海马神经元损害过程。     

Postischemic administration of idazoxan, an alpha-2 adrenergic receptor antagonist, decreases neuronal damage in the rat brain

The effect of an alpha-2 receptor antagonist, idazoxan, on ischemic neuronal damage in the hippocampus and neocortex was studied in rats following 10 min of forebrain ischemia. Idazoxan was given 0.1 mg/kg i.v. immediately after recirculation, followed by 48 h of continuous infusion at a rate of 10 micrograms/kg/min. A histopathological examination of the CA1 region of the dorsal hippocampus and neocortex from each hemisphere was made on paraffin-embedded sections following 7 days of survival. In ischemic animals receiving an infusion of saline, 71% of the neurons in the hippocampal CA1 region were degenerated. In contrast, in the idazoxan-treated animals only 31% of the neurons were irreversibly damaged (p less than 0.01). We conclude that postischemic administration of the alpha-2 antagonist idazoxan protects neurons against damage following cerebral ischemia. Rapid postischemic administration of alpha-2 adrenergic receptor antagonists could be an effective treatment after stroke and cardiac arrest.     

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia

Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damages in the brain. In the present study, ischemia-induced changes in GLP-1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP-1R agonist exendin-4 (EX-4) against ischemic damage was studied. GLP-1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP-1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP-1R protein level was increased again at 10 days after I/R, and GLP-1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX-4 treatment attenuated ischemia-induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose-dependent manner. EX-4 treatment also induced the elevation of GLP-1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP-1R is altered in the ischemic region after an ischemic insult and that EX-4 protects against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglial activation against transient cerebral ischemic damage.     

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.     

Moderate hypothermia mitigates neuronal damage in the rat brain when initiated several hours following transient cerebral ischemia

Intraischemic moderate hypothermia generally protects the brain against ischemic cell death, while hypothermia instigated several hours into the reperfusion phase is considered to be less effective. Here we report the effect of hypothermia (32.5°–33.5°C) of 5-h duration, initiated at 2, 6, 12, 24 and 36 h into the recirculation phase following 10 min of transient cerebral ischemia, on ischemic neuronal injury in the hippocampus and striatum of the rat. Hypothermia induced at 2 h, and 6 h postischemia reduces neuronal damage in the entire hippocampal CA1 region by approximately 50%. In the lateral CA1 region hypothermia induced at 12 h postischemia, significantly mitigates necrosis. When initiated at 2 h postischemia, but not later, protection was also observed in the striatum. Hypothermia induced 24 and 36 h postischemia was ineffective. A period of hypothermia of 5 h, initiated 2 h postischemia, was required for marked neuronal protection in the CA1 region, while 3.5-h hypothermia decreased neuronal damage by approximately 10% and 30 min hypothermia was ineffective. The clinical implications of the data are that extended period of hypothermia initiated long into the recovery phase following ischemia may prove beneficial. Hypothermia protects brain regions displaying rapid as well as delayed neuronal damage, and a minimal time of hypothermia is required for effective neuronal protection. Also, strict temperature control for up to 24 h postischemia may be required for proper assessment of the efficacy of cerebro-protective drugs.Supported by the Swedish Medical Research Council (grant no. 08644), The Medical Faculty at Lund University, The Segerfalk Foundation, The Crafoord Foundation, Åke Wibergs Foundation, and the CNPq (Brazilian Council for Development of Science and Technology)     

Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects

We examined the response of the gerbil brain to secondary ischemic insult following pretreatment with brief ischemia at intervals of 5 min, 1 and 6 h, 1, 2, 4, 7 and 14 days. Two minutes of bilateral carotid artery occlusion produced no histopathological brain damage, whereas 3 min of occlusion caused a moderate to severe reduction in the number of hippocampal CA1 pyramidal cells. Two-minute occlusion followed by 3-min occlusion at 5-min, 1- and 6-h intervals resulted in almost complete destruction of CA1 neurons. Additional neuronal damage was observed in the striatum at a 1-h interval and in the thalamus and the neocortex at 1- and 6-h intervals. The neuronal damage was most severe at a 1-h interval. Two-minute ischemia followed by 3-min ischemia at intervals of 1, 2, 4 and 7 days, however, caused a marked protective effect, and the hippocampal CA1 neurons were preserved. The protective effect was not observed at a 14-day interval and following pretreatment with 1-min ischemia. Thus, pretreatment with brief ischemia leads to complex responses of the brain to secondary ischemic insult; cumulative damage at intervals of 1-6 h and protective effects at intervals of 1-7 days.     

Effect of hyperthermia on calbindin-D 28k immunoreactivity in the hippocampal formation following transient global cerebral ischemia in gerbils

Calbindin D-28K(CB), a Ca2+-binding protein, maintains Ca2+ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We investigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei(Neu N) and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia(39.5 ± 0.2°C) was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neuronal damage/death in the pyramidal layer of CA1–3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreactivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immunoreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults.     

Repeated focal cerebral ischemia in gerbils is associated with development of infarction.

We induced repeated focal cerebral ischemia in gerbils. Single 5-min ischemia produced neuronal damage limited to the ipsilateral CA1 and CA4 hippocampus. Two 5-min ischemic insults spaced at a 1-h interval caused selective neuronal damage to the CA1, CA3 and CA4 hippocampus, striatum, neocortex, and thalamus. Three 5-min ischemic insults at 1-h intervals produced infarction. Thus, repeated focal ischemia produced cumulative brain damage by conversion of sublethal damage into selective neuronal damage and of the neuronal damage into infarction.     

Rapid decline in protein kinase Cgamma levels in the synaptosomal fraction of rat hippocampus after ischemic preconditioning.

Neurons can be preconditioned against ischemic damage by a brief sublethal period of ischemia, applied several days before the second insult. Here we report on changes in the distribution and the levels of protein kinase Cgamma (PKCgamma) in nonconditioned and preconditioned rat hippocampal CA1 and neocortex regions after a 9 min ischemic episode induced by two-vessel occlusion ischemia. At the end of the second ischemia we found significantly lower levels of PKCgamma in the CA1 region but not neocortex of preconditioned brains than in non-conditioned brains. Protein kinase Cgamma levels in both CA1 and neocortex decrease simultaneously in the cytosolic fractions. We conclude that PKCgamma is translocated to cell membranes during ischemia and is rapidly removed or degraded during the second otherwise lethal ischemic insult in preconditioned brains. The data suggest that ischemic preconditioning enhances downregulation of cell signaling mediated by PKCgamma and may thereby provide neuroprotection.     

Vinconate prevents ischemic neuronal damage in the rat hippocampus

The neuroprotective effect of vinconate, a novel vinca alkaloid derivative, was examined in a rat model of forebrain ischemia induced by 4-vessel occlusion. Hippocampal cell loss was quantified histologically 3 days after 10 or 15 min of ischemia. Intraperitoneal application of vinconate (25 and 50 mg/kg) 10 min before and immediately after 10 min of ischemia significantly reduced the neuronal cell loss in the CA1 sector of the hippocampus. Protective effect of vinconate against 15 min of ischemia was reduced, but there was still significant protection at the higher dose. Autoradiography using 14C-vinconate showed that the drug easily penetrates the blood-brain barrier and distributes in the hippocampus. The result suggests that vinconate prevents ischemic neuronal damage by direct action on the hippocampal CA1 neurons.     

沙鼠脑缺血耐受的组织学变化及HSP在其中的作用

目的 :观察脑缺血耐受时的组织学变化及 HSP在其中的作用。方法 :通过 HE染色观察脑缺血耐受时的组织学变化 ,并通过免疫组化染色 ,了解 HSP70及 HSP2 7在其中的作用。结果 :一次性 5分钟缺血后 7天海马 CA1区神经元大多坏死 ,若在缺血前给予 2分钟的缺血预处理 ,该区神经元大多保留 ,表现出明显的保护作用。只给一次性 5分钟缺血 ,海马 CA1区神经元无 HSP70染色。若在缺血前给予预处理 ,海马 CA1区神经元可见明显 HSP70染色。而HSP2 7主要在胶质细胞表达 ,海马区的神经元未见其表达。结论 :缺血前给予预处理对以后的缺血有保护作用 ;在缺血耐受过程中 ,HSP70表达出一定的保护作用。     

Indole‐3‐propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus

Tryptophan‐derived indole compounds have been widely investigated as antioxidants and as free‐radical scavengers. Indole‐3‐propionic acid (IPA), one of these compounds, is a deamination product of tryptophan. In the present study, we used Mongolian gerbils to investigate IPA's neuroprotective effects against ischemic damage and its antioxidative effects in the hippocampal CA1 region (CA1) after 5 min of transient forebrain ischemia. The repeated oral administration of IPA (10 mg/kg) for 15 days before ischemic surgery protected neurons from ischemic damage. In this group, the percentage of cresyl violet–positive neurons in the CA1 was 56.8% compared with that in the sham group. In the vehicle‐treated group, glial fibrillary acidic protein (GFAP)‐, S‐100‐, and vimentin‐immunoreactive astrocytes and ionized calcium‐binding adapter molecule 1 (Iba‐1)– and isolectin B4 (IB4)–immunoreactive microglia were activated 4 days after ischemia/reperfusion, whereas in the IPA‐treated ischemic group, GFAP, S‐100, Iba‐1, and IB4, but not vimentin, immunoreactivity was distinctly lower than that in the vehicle‐treated ischemic groups. The administration of IPA significantly decreased the level of 4‐hydroxy‐2‐nonenal, a marker of lipid peroxidation, in ischemic hippocampal homogenates compared with that in the vehicle‐treated ischemic groups at various times after ischemia/reperfusion. In addition, immunostaining for 8‐hydroxy‐2′‐deoxyguanosine showed DNA damage in pyramidal neurons in the ischemic CA1 was significantly lower in the IPA‐treated ischemic groups than in the vehicle‐treated ischemic groups. These results suggest that IPA protects neurons from ischemia‐induced neuronal damage by reducing DNA damage and lipid peroxidation. © 2009 Wiley‐Liss, Inc.     

Neuronal damage and calcium accumulation following repeated brief cerebral ischemia in the gerbil

We investigated the distribution of neuronal damage following brief cerebral transient ischemia and repeated ischemia at 1-h intervals in the gerbil, using light microscopy and 45Ca autoradiography as a marker for detection of ischemic damage. The animals were allowed to survive for 7 days after ischemia induced by bilateral carotid artery occlusion. Following 2-min ischemia, neuronal damage determined by abnormal calcium accumulation was not observed in the forebrain regions. Following 3-min ischemia, however, abnormal calcium accumulation was recognized only in the hippocampal CA1 sector and part of the striatum. Two 2-min ischemic insults caused extensive abnormal calcium accumulation in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the substantia nigra and the inferior colliculus. The ischemic insults were more severe than that of a single 3-min ischemia. However, three 1-min ischemic insults caused abnormal calcium accumulation only in the striatum. On the other hand, three 2-min ischemic insults caused severe abnormal calcium accumulation in the brain. The abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus. Gerbils subjected to three 3-min ischemic insults revealed most severe abnormal calcium accumulation. Marked calcium accumulation was seen not only in the above sites, but also spread in the neocortex, the septum and the hippocampal CA3 sector. Morphological study after transient or repeated ischemia indicated that the distribution and frequency of the neuronal damage was found in the sites corresponding to most of the regions of abnormal calcium accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protective effects of peroxisome proliferator‐activated receptors γ coactivator‐1α against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia

Peroxisome proliferator‐activated receptors γ coactivator‐1α (PGC‐1α) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC‐1α in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC‐1α signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague‐Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4–48 hr after TGI. In addition, knock‐down of PGC‐1α expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC‐1α is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress. © 2009 Wiley‐Liss, Inc.     

Protective effects of serotonin reuptake inhibitors, citalopram and clomipramine, against hippocampal CA1 neuronal damage following transient ischemia in the gerbil

To clarify the role of serotonin in cerebral ischemia, we examined the effects of selective serotonin reuptake inhibitors, citalopram and clomipramine, on ischemic neuronal damage in the gerbil. Pretreatment with citalopram (40 mg/kg i.p.) and clomipramine (20 mg/kg i.p.) protected against neuronal destruction of hippocampal CA1 pyramidal cells following 5 min of forebrain ischemia. Furthermore, microdialysis assays showed that a striking increase in extracellular excitatory amino acid levels during ischemia was significantly inhibited by pretreatment with citalopram and clomipramine. However, citalopram (40 mg/kg i.p.) did not alter the extracellular amino acid concentrations in normal gerbils. Thus, serotonin reuptake inhibitors have a protective effect against ischemic neuronal damage. Furthermore, the present result suggests that the protective effect is mediated through prevention of the accumulation of extracellular excitatory amino acids during and after ischemia.     

Regulation of ischemic hippocampal damage in the gerbil: adrenalectomy alters the rate of CA1 cell disappearance

Adrenalectomy protects hippocampal pyramidal cells from transient ischemia in rats. We hypothesized that this effect of adrenalectomy could be generalized to the gerbil. We determined the effect of glucocorticoid manipulation on hippocampal CA1 cell death following transient forebrain ischemia in the gerbil. Adrenalectomy diminished hippocampal damage when performed immediately following transient forebrain ischemia, as in the rat, while glucocorticoid administration resulted in an increase in CA1 pyramidal cell damage. Furthermore adrenalectomy 24 h after the ischemic injury diminished hippocampal damage to roughly the same extent as immediate adrenalectomy. However, if gerbils were examined at longer survival periods after ischemia, the difference in hippocampal damage between adrenalectomized and sham-adrenalectomized was lost. These findings suggest that glucocorticoids influence the rate of hippocampal pyramidal cell disappearance following ischemia. Manipulation of glucocorticoids could be an important adjunct to therapy for preventing ischemic brain damage.