Ischemic preconditioning: a long term survival study using behavioural and histological endpoints

In this study we sought to determine if ischemic preconditioning provided long term behavioral and histological protection. A second goal was to see if ischemic preconditioning conveys its protective effect on CA1 neurons by altering post-ischemic brain temperature. While preconditioning episodes of short duration ischemia (i.e. 1.5 min) provided significant histological protection of CA1 pyramidal cells against a subsequent severe ischemic insult (i.e. 5 min), this did not result in complete behavioural protection. Preconditioned ischemic animals initially displayed habituation deficits in an open field test that were comparable to untreated ischemic gerbils. A significant decline in CA1 preservation in preconditioned animals was observed when survival time was extended from 10 (81% protection) to 30 (53% protection) days. In addition, protection was not observed in the subiculum and CA2 sector of the hippocampus where consistent damage was observed in 21/22 gerbils. Ischemic preconditioning did not markedly affect post-ischemic brain temperature suggesting that the observed protection was not due to a reduction in temperature during or after the severe ischemic insult. The lack of functional protection within the first 10 days after ischemia, along with the decline of cellular preservation over time, suggests that this paradigm may not provide permanent protection.     

Competing processes of cell death and recovery of function following ischemic preconditioning

The goal of the present study was to determine the neuroprotective efficacy of ischemic preconditioning using behavioral, electrophysiological and histological endpoints at various time points up to 90 days postischemia. Gerbils were exposed to a brief, non-injurious episode of forebrain ischemia (1.5 min) on each of 2 consecutive days. Three days following this preconditioning procedure, the animals received a 5 min occlusion. Other animals underwent sham surgery or a 5 min occlusion without preconditioning. Ischemic preconditioning appeared to provide striking histological protection at both rostral (80% and 67% of sham) and posterior levels of hippocampus (94% and 78% of sham) at 3 and 10 days survival, respectively. However, in spite of the near normal number of CA1 neurons, animals displayed marked impairments in an open field test of habituation as well as reduced dendritic field potentials in the CA1 area. Additionally, in ischemic animals the basal and apical dendritic regions of CA1 were nearly devoid of the cytoskeletal protein microtubule associated protein 2 (MAP2). Staining levels of MAP2 in preconditioned and sham animals were similar. With increasing survival time, open field behavior as well as CA1 field potential amplitude recovered. Nonetheless, CA1 cell death in ischemic preconditioned animals continued over the 90-day survival period (P<0.05, vs. sham levels). Ischemic preconditioning provides a significant degree of neuroprotection characterized by a complex interplay of protracted cell death and neuroplasticity (recovery of function). These competing processes are best elucidated using a combination of functional and histological endpoints as well as multiple and extended survival times (i.e., greater than 7–10 days).     

Cerebral ischemic preconditioning induces lasting effects on CA1 neuronal survival, prevents memory impairments but not ischemia-induced hyperactivity

In ischemic preconditioning, prior exposure to a short 3-min global ischemia provides substantial protection against the deleterious effects of a subsequent prolonged ischemic insult in rats. The objective of the present study was to determine if the neuronal protection induced by ischemic preconditioning influence functional recovery following a 6-min ischemic insult in rats. Animals received either sham-operation, a 3-min ischemia, a preconditioning 3-min global ischemia followed 3 days later by a 6-min global ischemia or a single 6-min global ischemia. Open field habituation, memory performance in the 8-arm radial maze and object recognition were assessed at different intervals following ischemia. Our findings revealed that preconditioning reversed ischemia-induced spatial memory deficits in the 8-arm radial maze, as suggested by significant reduction of working memory errors in preconditioned as compared to ischemic animals. Preconditioning also attenuated ischemia-induced object recognition deficits at short-term intervals. Nonetheless, preconditioning failed to alter ischemia-induced hyperactivity as demonstrated by enhanced behavioral activity in the open field in both preconditioned and ischemic animals compared to 3-min ischemic and sham-operated rats. CA1 cell counts revealed significant neuronal sparing in preconditioned animals that was observed 6-month following reperfusion. Together, these findings suggest that neuronal survival in preconditioned rats is associated with significant improvements of hippocampal-dependent memory functions and, further support that ischemia-induced hyperactivity may not solely depend on selective neuronal damage to hippocampal neurons.     

缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系

目的 探讨缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系。方法 实验动物被随机分为手术组、缺血组、预缺血组、预缺血后再缺血组。阴断沙土鼠双侧颈总动脉造成前脑缺血模型。采用细胞特异性抗原胶质纤维酸性蛋白（GFAP）免疫组化法标记星形胶质细胞。结果 预缺血后1－7天,海马CA1区GFAP阳性的星形胶质细胞数轻度增加,至28天时增生非常显著（P＜0．01）。预缺血后1－7天再缺血,海马CA1区存活正常神经元数逐渐下降,预缺血后28天再缺血又显著增加（P＜0．01）。结论 缺血预处理后,神经元可出现迟发性缺血耐受,反应性星形胶质细胞增生可能起了重要作用。     

缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系初步探讨

目的动态观察缺血预处理后大鼠大脑皮层和海马CA1区神经元凋亡与Fas蛋白表达变化情况,初步探讨缺血预处理后Fas蛋白表达与迟发性神经元凋亡的关系。方法四血管阻断法复制全脑缺血模型,动物随机分为非缺血对照组、预处理对照组、缺血预处理组和缺血组。采用尼氏和TUNEL染色法观察皮层及海马CA1区神经元存活数和凋亡细胞数,免疫组化方法检测Fas蛋白在缺血预处理后表达变化情况。结果缺血组缺血6h在皮质及海马CA1区Fas阳性表达细胞计数升高,12h达高峰;缺血预处理组缺血12h阳性细胞计数升高,24h达高峰。缺血组缺血6h出现凋亡细胞,48h凋亡细胞数达到高峰;缺血预处理组凋亡细胞数较缺血组明显减少。缺血组缺血7d神经元数明显减少,12周时神经元大量减少;缺血预处理组缺血7d时神经元数无明显变化,但12周时神经元同样大量减少。结论全脑缺血可能通过诱导Fas蛋白的表达增多,启动细胞凋亡,导致缺血后神经元凋亡的发生;缺血预处理虽可延缓缺血后神经元的凋亡,但无法提供真正的长时期的神经元保护作用,其有限的保护作用可能是通过延缓Fas蛋白的表达而减缓了神经元凋亡的进程。     

Sevoflurane-induced preconditioning protects against cerebral ischemic neuronal damage in rats

In the present study, we tested the ability of sevoflurane to induce early and late preconditioning against ischemic neuronal injury using an in vivo model of global cerebral ischemia in the rat. Seven-minute global ischemia was induced by cardiac arrest, followed by resuscitation and recovery for 7 days. Hippocampal slices were then prepared and the amplitude of extracellularly recorded, orthodromically evoked, CA1 population spikes (neuronal function) was quantified. Rats were preconditioned for 30 min with 1.0 minimum alveolar concentration (MAC) of sevoflurane once or on 4 consecutive days, 15 min (single exposure, early) or 24 h (four exposures, late preconditoning) prior to cardiac arrest. After early or late preconditioning, sevoflurane reduced ischemic neuronal damage from 43 +/- 3% [sham rats, (mean +/- SEM)] to 30 +/- 3% and 35 +/- 4%, respectively. Histopathology demonstrated a preserved morphology of the CA1 region of preconditioned rats, whereas pyknosis was present in control and sham-treated rats. Sevoflurane-induced preconditioning confers neuroprotection during an early as well as late time window.     

3-硝基丙酸诱导沙士鼠脑缺血耐受时海马促红细胞生成素的表达

目的:探讨促红细胞生成素(erythropoietin,Epo)在小剂量3-硝基丙酸(3-nitropropionic acid,3-NPA)预处理诱导脑缺血耐受鼠脑海马的表达变化,揭示3-NPA预处理的机制。方法:将沙土鼠腹腔注射5mg/kg3-NPA后3天,阻断沙土鼠双侧预总动脉3.5min造成前脑缺血模型,通过尼氏染色和免疫组化观察海马锥体细胞损伤和Epo的表达变化。结果:对照组海马CA_1区已失去正常结构,锥体细胞大部分丧失,存活神经元计数显著低于3-NPA预处理组。对照组脑缺血再灌注6h,海马可检测到脑源性Epo的表达,再灌注12h,脑源性Epo表达达到较高水平,以后随时间延长逐渐下调。3-NPA预处理组海马Epo的表达在脑缺血再灌注后6h和12h高于对照组,1d、3d、7d与对照组比较无显著性差异。结论:3-NPA预处理可以诱导脑缺血耐受,在3-NPA预处理后脑缺血再灌注早期Epo的表达增强,提示Epo可能是3-NPA预处理形成的机制之一。     

Dynamic changes in CA1 dendritic spines associated with ischemic tolerance

Hippocampal CA1 neurons are particularly vulnerable to 5-10 min durations of global ischemia. These cells can develop tolerance to ischemia through prior exposure to brief episodes of ischemia (ischemic preconditioning, IP). Dendritic spines are implicated in various forms of neuroplasticity including memory and recovery of function. Here we characterized the changes in hippocampal CA1 dendritic spines during the development of ischemic tolerance and the subsequent postischemic recovery period. Gerbils received 5 min, bilateral carotid artery occlusions preceded by two 1.5 min occlusions each of which were 24 h apart (tolerance groups). Spine densities were calculated from CA1 apical and basilar dendrites in tolerant animals that survived 3 (IP3), 10 (IP10) or 30 (IP30) days as well as sham-operated animals and those that received only the two preconditioning episodes (PO). Habituation to a novel open-field was assessed 3, 7, 10 and 30 days after ischemia to gauge CA1 functional integrity. Dendritic spines were quantified from Golgi-Cox stained sections of the CA1 subfield. IP10, IP30 and PO animals had significantly higher CA1 basilar and apical spine densities than all other groups. Tolerant animals initially displayed open-field habituation impairments at a time when spine densities were reduced. Behavioral impairments gradually subsided over time in coincidence with an increase in CA1 spine densities. These findings suggest that dendritic spines may play a role in recovery of function associated with ischemic tolerance and stroke.     

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.     

Short-term ischemia usually used for ischemic preconditioning causes loss of dendritic integrity after long-term survival in the gerbil hippocampus

Ischemic preconditioning has been established as a powerful experimental neuroprotective strategy, both after global and focal cerebral ischemia. Little is known, however, about the structural and functional long-term outcome. Therefore, our present study was designed to check for potential subtle alterations in the hippocampus after long-term survival. Gerbils were subjected either to short-term ischemia of 2.5 min duration usually used for ischemic preconditioning (n=8) or to sham operation (n=6) and allowed to survive for 6 weeks. Hippocampi with neuronal densities comparable to those of sham-operated control animals were analyzed for dendritic marker proteins MAP2, MAP1B and synaptopodin, respectively. Although MAP2 immunoreactivity was widely unchanged in all hippocampal subfields, both MAP1B and synaptopodin protein expression was decreased to about 80% to 90% of sham controls. A significant reduction, however, was only seen for synaptopodin immunoreactivity in stratum oriens and pyramidale of hippocampal CA1 subfield. In conclusion, our data indicate a dissociation of neuronal survival and dendritic integrity 6 weeks after short-term ischemia usually used for ischemic preconditioning. Analysis of postischemic synaptopodin protein expression is the most sensitive method to detect subtle dendritic changes compared to the classical dendritic marker molecules MAP2 and MAP1B.     

Diazepam delays the death of hippocampal CA1 neurons following global ischemia

Although diazepam provides limited long term neuroprotection, it may be useful for expanding the therapeutic time window after stroke by delaying neuronal death. However, it is not known to what extent diazepam maintains normal cellular structure and function in the first few days after ischemia. We used histological, immunohistochemical and electrophysiological endpoints to address this question. Gerbils underwent 5 min of global ischemia followed by 10 mg/kg diazepam (D) given 30 and 90 min later. Other animals were subjected to sham surgery, normothermic ischemia (I) or ischemia at 32 °C (Hypo). Postischemic brain temperature was regulated at  37 °C for 24 h. Gerbils in the D and I groups were sacrificed 1, 2 and 3 days after ischemia. Sham and Hypo gerbils were sacrificed on day 3. CA1 cell counts, MAP2 staining and CA1 field potentials were performed at each survival time. Hypothermia prevented CA1 necrosis, preserved MAP2 integrity and maintained CA1 field potential amplitude. Ischemic gerbils showed a significant reduction in these 3 outcome measures by day 3. Diazepam-treated gerbils exhibited near normal levels of CA1 neurons and MAP2 staining. Most importantly, CA1 field potentials were similar to sham values and significantly preserved relative to non-treated ischemic gerbils. Diazepam maintains near normal structural and functional integrity up to 3 days after a global ischemic insult. As such, this drug may be useful for extending the therapeutic time window after cardiac arrest, stroke and related disorders.     

Protection against ischemic brain damage using propentofylline in gerbils

We studied the xanthine derivative propentofylline (HWA 285) to determine its protection against ischemic brain damage when administered before and after ischemia. Transient forebrain ischemia was produced in 81 Mongolian gerbils by occluding both carotid arteries. The necessary surgery was performed under anesthesia with intraperitoneal pentobarbital/chloral hydrate 2 days before occlusion. We tested the parameters delayed selective hippocampal nerve cell damage, generation of seizures, and survival. Determination of the dose-response relation revealed the optimal dose of HWA 285 to be 10 mg/kg i.p. The effect of the drug on delayed selective nerve cell damage in the hippocampus was assessed by measuring the intensity of Nissl staining in the CA1 area by means of densitometry 4 days after a 10-minute occlusion. Gerbils treated with HWA 285 revealed significant protection of the CA1 neurons even when the drug was applied 1 hour after the end of the occlusion. In contrast to HWA 285, pentobarbital provided no detectable protection of the CA1 neurons in our experimental model when administered 1 hour after occlusion, suggesting different mechanisms of action. After a 15-minute occlusion, all six untreated gerbils developed convulsions and died within 2 days. Chronic (daily) treatment of nine gerbils with HWA 285 prevented the generation of convulsions in eight and allowed seven to survive for greater than 12 days.     

Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures

The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12-14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193+/-0.015 in control (sham) (n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108+/-0.018 (n=5) in CA1 (*P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190+/-0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event.     

Neuroprotective effects of ischemic preconditioning on hippocampal CA1 pyramidal neurons through maintaining calbindin D28k immunoreactivity following subsequent transient cerebral ischemia

Ischemic preconditioning elicited by a non-fatal brief occlusion of blood flow has been applied for an experimental therapeutic strategy against a subsequent fatal ischemic insult. In this study, we investi-gated the neuroprotective effects of ischemic preconditioning (2-minute transient cerebral ischemia) on calbindin D28k immunoreactivity in the gerbil hippocampal CA1 area following a subsequent fatal tran-sient ischemic insult (5-minute transient cerebral ischemia). A large number of pyramidal neurons in the hippocampal CA1 area died 4 days after 5-minute transient cerebral ischemia. Ischemic preconditioning reduced the death of pyramidal neurons in the hippocampal CA1 area. Calbindin D28k immunoreactivity was greatly attenuated at 2 days after 5-minute transient cerebral ischemia and it was hardly detected at 5 days post-ischemia. Ischemic preconditioning maintained calbindin D28k immunoreactivity after transient cerebral ischemia. These findings suggest that ischemic preconditioning can attenuate transient cerebral ischemia-caused damage to the pyramidal neurons in the hippocampal CA1 area through maintaining cal-bindin D28k immunoreactivity.     

Comparative study on Cu,Zn-SOD immunoreactivity and protein levels in the adult and aged hippocampal CA1 region after ischemia-reperfusion

In the present study, we investigated chronological changes in Cu,Zn-superoxide dismutase (SOD1) immunoreactivity and its protein levels in the hippocampal CA1 region of adult and aged gerbils after transient forebrain ischemia to compare ischemia-related changes in SOD1 in adult and aged gerbils. Delayed neuronal death in the CA1 region at 4 days after ischemic insult was prominent in adult gerbils compared to that in aged gerbils. In sham-operated gerbils, SOD1 immunoreactivity and protein level in the aged group were significantly higher than that in the adult group. At 12 h after ischemia-reperfusion, SOD1 immunoreactivity and protein level were increased in both the groups. At 1 day after ischemia, SOD1 immunoreactivity and protein level in the adult group were significantly increased: the SOD1 immunoreactivity was increased in non-pyramidal cells as well as pyramidal cells. At this time after ischemia, SOD1 immunoreactivity and protein level in the aged group were decreased: the immunoreactivity was decreased significantly in pyramidal cells. At 4 days after ischemia, SOD1 immunoreactivity was detected only in non-pyramidal cells of the CA1 region in both the groups. These results suggest that SOD1 in the gerbil hippocampal CA1 region is higher in sham-aged group than that in sham adult one, and that different changes in SOD1 in CA1 pyramidal cells after ischemia in adult and aged gerbils may indicate different processes in delayed neuronal death with time after ischemic insult.     

Protective preconditioning by transient global ischemia in the rat: components of delayed injury progression and lasting protection distinguished by comparisons of depolarization thresholds for cell loss at long survival times.

Robust ischemic preconditioning has been shown in rodent brain, but there are concerns regarding the persistence of neuron protection. This issue was examined in rat hippocampus following 4-vessel occlusion (4-VO) ischemia, using DC shifts characteristic of ischemic depolarization to reproducibly define insult severity. Preconditioning ischemia producing 2 to 3.5 mins depolarization was followed at intervals of 2, 5, or 7 days by test insults of varied duration, after which CA1 counts were obtained at 1, 2, 4, or 12 weeks. Neuron loss in naive animals increased with depolarization time longer than 4 mins regardless of postischemic survival interval. Preconditioning 2, 5, or 7 days before test insults prolonged the injury threshold evaluated at 1 week survival to 15, 9, or 6 mins, respectively, showing robust protection and a rapid decay of the protected state. However, by 2 weeks survival after preconditioning at a 2-day interval, the injury threshold dramatically regressed from 15 to 9 mins. Thereafter protection remained relatively stable through 1 month, but slight progression of neuron injury was evident at 3 months. Inflammatory responses were seen in both naive and preconditioned hippocampi throughout this interval, appropriate to the extent of neuron injury. These studies show distinct components of transient and lasting protection after ischemic preconditioning. Finally, it was found that ischemic depolarization was delayed by approximately 1 min in optimally preconditioned rat hippocampus, in contrast to previous results in the gerbil, identifying one specific mechanism by which insult severity is reduced in this model.     

Ischemic tolerance preserves propagation of membrane depolarization

The functional integrity of the synaptic connections within the hippocampus in gerbils that had acquired ischemic tolerance was investigated. The propagation of membrane depolarization across the hippocampus in response to electrical stimulation of CA1 was monitored with the use of a high speed optical recording technique. In comparison to control slices, propagation was significantly depressed and depolarization was shortened in slices from gerbils subjected to 5 min of ischemia. Hippocampal slices from gerbils who were preconditioned with prior sublethal ischemia demonstrated only a slight reduction in propagation. The duration of depolarization was longer than that of ischemia group. These findings suggest that ischemia induces a functional disturbance of synaptic transmission and membrane depolarization. Ischemic preconditioning significantly reduced the extent of this functional disturbance.     

Extracellular signal-regulated kinase and c-Jun N-terminal protein kinase in ischemic tolerance.

The alterations and involvement of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK) activation were examined in the hippocampal CA1 region in a rat model of global brain ischemic tolerance. Western blotting study showed that ERK activation (diphosphorylation) level was decreased (3.75-, 0.56-, and 0.23-fold vs sham control) and JNK activation level was increased (3.82-, 4.63-, and 5.30-fold vs sham control) 3 days after more severe ischemic insults (6 min, 8 min, and 10 min of ischemia, respectively). These alterations were significantly prevented by pretreatment with preconditioning ischemia, which also provided neuronal protection against ischemic injury. Inhibition of ERK activation after preconditioning ischemia by PD98059, a specific ERK kinase inhibitor, significantly prevented the inhibitory effects of preconditioning ischemia on both JNK activation and ischemic injury. The results suggest that ERK activation after preconditioning ischemia may result in the prevention of JNK activation and thus be involved in the protective responses in ischemic tolerance in hippocampal CA1 region.     

Aging blunts ischemic-preconditioning-induced neuroprotection following transient global ischemia in rats

The present study examines the hypothesis that aging defined by the 50% survival age compromises neuroprotection afforded by ischemic preconditioning (IPC). Sixty-four male F344 rats aged 4- and 24-months, respectively, were subjected to IPC, (3-min ischemia) or sham-surgery followed by 10-min (full) ischemia or sham-surgery 2 days later. There were 4 groups at each age: sham-surgery-sham-surgery (SS), preconditioning-sham-surgery (PS), preconditioning-ischemia (PI) and sham-surgery-ischemia (SI) groups. Assessments of histology and immunoreactivities of N-methyl-D-aspartic acid receptor 1 (NMDAr1) and caspase-3 active peptide (C3AP) in the hippocampal CA1 region were performed 8 days after full ischemia. The CA1 "living cell ratio" was greater in the aged SI group than in the young SI group (32+/-6% vs. 17+/-5%, p<0.05), whereas the degree of protection against full ischemia afforded by IPC was reduced in the aged compared with the young (53+/-17% vs. 241+/-25%, P<0.0001). The basal level of NMDAr1 immunofluorescence was significantly higher in young animals, while the numbers of C3AP-positive cells were greater in all three aged ischemic groups as compared to respective young groups (p<0.01, p=0.055 and p<0.05). A fourth method of assessing cell damage using Fluoro Jade C labeled degenerating neurons that were also intensively eosinophilic. Counts of Fluoro Jade C-positive cells were higher in the young SI group than in the aged SI group (P<0.05), suggesting that mechanisms of ischemic cell death may change with aging. In conclusion, aging alters mechanisms of ischemic cell death in CA1 neurons and ischemic tolerance mechanisms are blunted by aging.     

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.