Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus

To analyze the role of specific genes and proteins in neuronal signaling cascades following global cerebral ischemia, it would be useful to have a reproducible model of global cerebral ischemia in mice that potentially allows the investigation of mice with specific genomic mutations. We first report on the development of a model of reversible cardiocirculatory arrest in mice and the consequences of such an insult to neuronal degeneration and expression of immediate early genes (IEG) in the hippocampus. Cardiocirculatory arrest of 5 min duration was induced via ventricular fibrillation in mechanically ventilated NMRI mice. After successful cardiopulmonary resuscitation (CPR), animals were allowed to reperfuse spontaneously for 3 h (n=7) and 7 days (n=7). TUNEL staining revealed a selective degeneration of a subset of neurons in the hippocampal CA1 sector at 7 days. About 30% of all TUNEL-positive nuclei showed condensed chromatin and apoptotic bodies. Immunohistochemical studies of IEG expression performed at 3 h exhibited a marked induction of c-Fos, c-Jun, and Krox-24 protein in all sectors of the hippocampus, peaking in vulnerable CA1 pyramidal neurons and in dentate gyrus. In contrast, sham-operated animals (n=3) did not reveal neuronal degeneration or increased IEG expression in the hippocampus when compared with untreated control animals (n=3). In conclusion, we present a new model of global cerebral ischemia and reperfusion in mice with the use of complete cardiocirculatory arrest and subsequent CPR. Following 5 min of ischemia, a subset of CA1 pyramidal neurons was TUNEL-positive at 7 days. The expression of IEG was observed in all sectors of the hippocampus, including selectively vulnerable CA1 pyramidal neurons. This appears to be a good model which should be useful in evaluating the role of various genes in transgenic and knockout mice following global ischemia.     

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。     

大鼠脑缺血再灌注后星形胶质细胞反应性变化差异与海马CA1区的迟发性神经元死亡

BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion.
OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion.
DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007.
MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia.
MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL.
RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）.
CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.     

亚低温对大鼠全脑缺血再灌注损伤后海马CA1区c-Jun蛋白表达的影响

目的研究亚低温对大鼠全脑缺血再灌注损伤后海马CA1区神经元的保护作用,并探讨其可能的机制。方法采用四血管阻断法建立大鼠全脑缺血模型。SD大鼠,随机分为假手术组(SH组)、常温组(IR组)和亚低温组(HIR组)。各组在全脑缺血15min后分别再灌注6h、12h、1d、3d,采用苏木素-伊红(HE)染色观察各时间点海马CA1区细胞形态学变化和TUNEL法检测海马CA1区神经元凋亡,免疫印迹检测c-Jun蛋白表达。结果(1)HE染色结果 IR组和HIR组于全脑缺血再灌注后6h,HE染色未见明显改变,IR组缺血再灌注1d时CA1区出现严重改变,3d时损伤最严重,出现细胞数目减少,细胞胞体缩小、胞核固缩深染,损伤严重,排列紊乱,核膜不清,核仁消失。而HIR组海马存活的锥体细胞数较之IR组12h、1d、3d时间点均明显增加(P<0.05)。(2)TUNEL标记IR组于缺血再灌注后6h在海马CA1区阳性细胞开始增多,缺血再灌注1 d时阳性细胞数最多。而HIR组各时间点阳性细胞数均较IR组明显减少(P<0.01)。(3)免疫印迹结果全脑缺血再灌注后6h c-Jun蛋白在IR组海马CA1区表达开始增加,12h达高峰,持续到3d;HIR组在各时间点的表达均弱于IR组(P<0.01)。结论亚低温通过减少海马CA1区c-Jun的表达,抑制海马CA1区神经元的凋亡,可能是亚低温脑保护作用的机制之一。     

Expression of the RNA-binding protein TIAR is increased in neurons after ischemic cerebral injury

T-cell restricted intracellular antigen-related protein (TIAR) is an RNA recognition motif-type RNA-binding protein that has been implicated in the apoptotic death of T-lymphocytes and retinal pigment epithelial cells. Western blots prepared with a monoclonal antibody against TIAR showed expression in normal rat hippocampus, and induction by 15 min of global cerebral ischemia. This increased expression was evident at 8 hr after ischemia and maximal at 24 hr, whereas expression at 72 hr was reduced below basal levels. Expression of TIAR protein was also increased in parietal cortex 6 and 24 hr after 90 min of focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, as well as in cultured cortical neurons and astroglia after exposure to hypoxia in vitro. Immunocytochemistry showed that increased expression of TIAR occurred mainly in the CA1 sector of hippocampus 24 hr after global ischemia, and in cortical and striatal neurons 24 hr after 20 or 90 min of focal ischemia. Double-labeling studies showed that TIAR protein expression was co-localized with DNA damage in neuronal cells. The findings suggest that TIAR may be involved in neuronal cell death after cerebral ischemic injury.     

Pretreated quercetin protects gerbil hippocampal CA1 pyramidal neurons from transient cerebral ischemic injury by increasing the expression of antioxidant enzymes

Quercetin(QE; 3,5,7,3′,4′-pentahydroxyflavone), a well-known flavonoid, has been shown to prevent against neurodegenerative disorders and ischemic insults. However, few studies are reported regarding the neuroprotective mechanisms of QE after ischemic insults. Therefore, in this study, we investigated the effects of QE on ischemic injury and the expression of antioxidant enzymes in the hippocampal CA1 region of gerbils subjected to 5 minutes of transient cerebral ischemia. QE was pre-treated once daily for 15 days before ischemia. Pretreatment with QE protected hippocampal CA1 pyramidal neurons from ischemic injury, which was confirmed by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, pretreatment with QE significantly increased the expression levels of endogenous antioxidant enzymes Cu/Zn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in the hippocampal CA1 pyramidal neurons of animals with ischemic injury. These findings demonstrate that pretreated QE displayed strong neuroprotective effects against transient cerebral ischemia by increasing the expression of antioxidant enzymes.     

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.     

Neuronal damage and calcium accumulation following transient cerebral ischemia in the rat

The purpose of this study was to examine the distribution of neuronal damage following transient cerebral ischemia in the rat model of four-vessel occlusion utilizing light microscopy as well as⁴⁵Ca-autoradiography. Transient ischemia was induced for 30 min. The animals were allowed to survive for 7 d after ischemia. In the animals subjected to ischemia, the most frequently and seriously damaged areas were the paramedian region of hippocampus, the hippocampal CA1 sector, and the dorsolateral part of striatum, followed by the inferior colliculus, the substantia nigra, the frontal cortex, and the thalamus, which were moderate damaged. Furthermore, the cerebellar Purkinje neurons, the hippocampal CA4 sector, the medial geniculate body, and the hippocampal CA3 sector were slightly affected.⁴⁵Ca-autoradiographyic study also revealed calcium accumulation in the identical sites of ischemic neuronal damage, except for the frontal cortex. Regional cerebral blood flow during 10 min of ischemia was severely decreased in selectively vulnerable areas. The blood flow in the medial geniculate body, the substantia nigra, the inferior colliculus, and the cerebellum was less pronounced than that in the selectively vulnerable areas. The present study demonstrates that transient cerebral ischemia can produce significant neuronal damage not only in the selectively vulnerable regions, but also in the brainstem.     

Global ischemia downregulates the function of metabotropic glutamate receptor subtype 5 in hippocampal CA1 pyramidal neurons

Within the hippocampus, electrophysiological and immunohistochemical studies showed that metabotropic glutamate receptor subtype 5 (mGluR5) is the major postsynaptic mGluR expressed in CA1 pyramidal neurons. To better understand the role of mGluR5 in ischemia-induced neuronal death, whole-cell patch-clamp recordings using hippocampal slices were performed to investigate the functional change of mGluR5 in CA1 pyramidal neurons following transient global ischemia. Our results indicated that 6 to 24 h after global ischemia, mGluR5-induced cationic currents and mGluR5-mediated enhancement of NMDA-evoked currents in CA1 pyramidal neurons were significantly reduced. Further TaqMan real-time quantitative RT-PCR assay showed that mGluR5 mRNA expression in hippocampal CA1 region or single CA1 pyramidal neurons was significantly downregulated following ischemic insults. The present study suggests that transient global ischemia downregulates mGluR5 function of CA1 pyramidal neurons by decreasing mGluR5 mRNA and that the resulting reduced mGluR5-mediated excitotoxicity could contribute to the survival of CA1 pyramidal neurons after ischemic insult.     

Induction of NADPH-diaphorase activity in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1–7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.     

Keratinocyte growth factor prevents ischemia-induced delayed neuronal death in the hippocampal CA1 field of the gerbil brain

Fibroblast growth factors (FGFs) are polypeptides with various biological activities in vivo and in vitro, and their receptors are expressed in the widespread and specific neuronal populations of the brain. In this study, we asked whether keratinocyte growth factor (KGF), one of the FGF superfamily, would express in the brain, and have neuroprotective against ischemic brain injury. In situ hybridization analysis revealed that intense silver grains for KGF mRNA are observed in the neuronal cells of the cerebral cortex, hippocampus and amygdala in gerbil brain. Continuous cerebroventricular infusion of KGF (20 microg) for a 7 day period to gerbils starting 2 days before temporary right carotid artery occlusion (20 min) resulted in a higher survival rate than seen in vehicle-treated ischemic animals. Subsequent histological examinations showed that KGF effectively prevented delayed neuronal death of the hippocampal CA1 region. In situ detection of DNA fragmentation (TUNEL staining) revealed that ischemic animals infused with KGF contained fewer TUNEL-positive neurons in the hippocampal CA1 field than those infused with vehicle alone at the forth and seventh day after ischemia. KGF-treated brain showed over-expression of KGF mRNA in the neuronal cells of the cerebral cortex, hippocampus only in the right hemisphere, which was the side of carotid artery occlusion, 8-10 h after ischemia. These findings suggest that KGF has a protective effect against ischemic hippocampal neuronal damage in vivo, which may provide a new therapeutic strategy in the survival and reconstruction of neurons in response to cerebral injury.     

Induction of 27-kDa heat shock protein following cerebral ischemia in a rat model of ischemic tolerance

Preconditioning the brain with sublethal cerebral ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). The purpose of this study is to investigate the role of low-molecular weight stress proteins, 27-kDa heat shock protein (HSP27) and αB crystallin, in ischemic tolerance. We measured the content of these proteins with enzyme immunoassay in the rat hippocampus and cerebral cortex following 6 min of ischemia with and without preconditioning with 3 min of ischemia and 3 days of reperfusion. We also visualized the localization of HSP27 immunohistochemically in comparison with that of HSP70. A 3-min period of ischemia caused a 2.4-fold increase in HSP27 content in the hippocampus after 3 days. Immunohistochemical localization of HSP27 was found in glial cells in all subregions of the hippocampus, whereas HSP70 immunostaining was seen only in CA1 pyramidal neurons. HSP27 content in the hippocampus decreased 2 h after 6 min of ischemia. HSP27 content progressively increased in the unpreconditioned hippocampus after 1 and 3 days, but returned to preischemic levels in the preconditioned hippocampus. HSP27 and HSP70 immunostaining was seen in CA1 pyramidal neurons after 1 day both with and without preconditioning. After 3 and 7 days, an intense HSP27 staining was observed in reactive glial cells in the CA1 without preconditioning, whereas the staining decreased in the preconditioned hippocampus. HSP70 staining was seen only in neurons at these time points. We observed no significant changes in HSP27 content in the cerebral cortex although neurons in the third and fifth layers were immunostained after 1 and 3 days. We observed no alterations in αB crystallin content after ischemia both in the hippocampus and the cortex. The present study demonstrated that cerebral ischemia induces HSP27 expression but not αB crystallin. Both HSP27 and HSP70 induction had a good temporal correlation with the induction of ischemic tolerance. However, different sites of action were suggested because the localization and cell types of HSP27 induction were quite different from those of HSP70 induction. The result suggests that it is unlikely that HSP27 is directly involved in the protection afforded by ischemic preconditioning.     

Naftidrofuryl protects neurons against ischemic damage

The effects of naftidrofuryl on postischemic neuronal damage and on local cerebral blood flow (LCBF) were examined in a rat model of forebrain ischemia (occlusion of carotid arteries and hypotension). Ischemia was induced for 10 min. LCBF was measured after 2 and 10 min of recirculation. A histological evaluation of cell loss in the hippocampal areas was performed 7 days after ischemia. Naftidrofuryl (10 mg/kg) was administered intraperitoneally 15 min before ischemia. The drug reduced the percentage of necrotic neurons in the CA1 and CA4 sector of the hippocampus, while the LCBF of these hippocampal sections was not significantly altered. Thus, naftidrofuryl is suggested to protect hippocampal neurons against ischemic damage mainly by a direct effect on brain parenchyma.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.     

Apoptosis associated genes are induced in gerbil hippocampus following global ischemia

Numerous studies have demonstrated evidence of DNA nick end-labeling and DNA laddering following cerebral ischemia. To determine whether genes directly implicated in apoptosis were induced by ischemia, the expression of bcl-2, bcl-x and ICE mRNAs were examined using oligonucleotide probes. Northern blots demonstrated induction of bcl-2 mRNA and bcl-x mRNAs in hippocampus 24 and 72 h following 5 min of global ischemia. In situ hybridization demonstrated induction of bcl-2 and bcl-x mRNAs in CAl pyramidal neurons of hippocampus at 24 h following ischemia which decreased by 72 h. ICE-like mRNA was induced in non-neuronal cells in the CAl region at 72 h following global ischemia. The data show that genes implicated in either protecting against or promoting programmed cell death in other systems are induced following cerebral ischemia. It is hypochesized that CAl neuronal cell death could be accounted for by the failure of the ischemic cells to make protective proteins that protect the cells from an ischemic induced apoptotic-like cell death.     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

Chronological alterations of neurofilament 150 immunoreactivity in the gerbil hippocampus and dentate gyrus after transient forebrain ischemia

In this study, we observed the chronological alterations of neurofilament 150 (NF-150) immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. NF-150 immunoreactivity in the sham-operated group was mainly detected in mossy fibers and in the hilar region of the dentate gyrus. NF-150 immunoreactivity and protein contents of NF-150 and RT 97 (polyphosphorylation epitopes of neurofilament) were significantly decreased at 15 min after ischemic insult. Between 30 min and 12 h after ischemic insult, NF-150 immunoreactivity and protein content were significantly increased as compared with the sham-operated group. Thereafter, NF-150 immunoreactivity and protein content started to decrease. At 12 h after ischemic insult, unlike dentate gyrus, NF-150 immunoreactivity increased in pyramidal cells of the CA1 region. Thereafter, NF-150 immunoreactivity in the CA1 region started to decrease, and 4 days after ischemic insult, NF-150 immunoreactivity nearly was similar to that of the sham-operated group. These biphasic patterns of NF-150 immunoreactivity in the hippocampus and dentate gyrus are reverse correlated with that of the intracellular calcium influx. For calcium detection in the CA1 region, we also conducted alizarin red staining. Alizarin red positive neurons were detected in some neurons at 15-30 min after ischemic insult. At 12 h after ischemia, alizarin red positive neurons were decreased. Thereafter, alizarin red positive neurons started to decrease, but alizarin positive neurons were significantly increased in dying neurons 4 days after ischemia. These results suggest that ischemia-related changes of NF-150 expression may be caused by the calcium following transient forebrain ischemia.     

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.     

Delayed neuronal death and damage of GDNF family receptors in CA1 following focal cerebral ischemia

Delayed neuronal death (DND) of pyramidal neurons in the CA1 and CA3 regions of the hippocampus has been extensively studied following global brain ischemia, whereas only little is known about DND in this highly vulnerable brain region after focal brain ischemia. In the present study, the distribution and time course of hippocampal neuronal apoptosis were studied following transient middle cerebral artery occlusion (MCAO) in rats 1, 3, 7, 14, and 30 days after the insult. In 60% of the animals, more than 90% of CA1 pyramidal neurons showed strong nick-end labeling (TUNEL) staining at day 3 with fragmentation and marginalization of the nuclei in approximately 40% of these cells. The number of TUNEL-positive cells decreased within the next days, but 30 days after MCAO, some apoptotic neurons were still present. Analysis of the expression of the glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha1, GFRalpha2, and GFRalpha3 using triple immunofluorescence and confocal laser scanning microscopy revealed that in all animals showing marked hippocampal DND, the neuronal staining for GFRalpha1, GFRalpha3, and GDNF decreased prior to the onset of TUNEL staining in CA1. After 7 days, some apoptotic neurons still expressed GFRalpha3, whereas only few showed GFRalpha1 immunoreactivity, indicating that GFRalpha1 may be beneficial for the survival of hippocampal neurons. The data suggest that reduced expression of GDNF and impairment of GFRalpha1/3 may contribute to hippocampal DND after focal brain ischemia.