复方丹参对脑缺血再灌注后大鼠海马和齿状回神经细胞凋亡及Bcl-2 mRNA表达的影响

目的:探讨中药复方丹参对大鼠脑缺血再灌注后海马和齿状回神经细胞凋亡及Bcl-2 mRNA表达的影响。方法:采用大脑中动脉内栓线法建立大鼠大脑中动脉缺血再灌注模型,应用原位细胞凋亡检测和原位杂交技术检测大鼠海马和齿状回神经细胞凋亡和Bcl-2 mRNA的表达并做图像分析。结果:与假手术对照组比较,缺血再灌注组凋亡神经细胞主要位于缺血侧海马CA1、CA3区,齿状回凋亡细胞较少。3个区神经细胞Bcl-2mRNA的表达在缺血再灌注2 h后升高,随时间的延长逐渐增强。复方丹参组神经细胞Bcl-2 mRNA的表达明显强于缺血再灌组,而凋亡神经细胞数明显较低。结论:复方丹参可通过上调神经细胞Bcl-2 mRNA的表达,抑制神经细胞凋亡,从而减轻缺血再灌注对大鼠海马和齿状回的损伤。     

Hypothermia differentially increases extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun terminal kinase activation in the hippocampus during reperfusion after asphyxial cardiac arrest

Mitogen-activated protein kinases are signal transduction mediators that have been implicated in cell survival and cell death. This study characterized the activation of pathways in the hippocampus during reperfusion after global cerebral ischemia, as well as the influence of a regimen of hypothermia that reduces ischemic cell death in the hippocampus. Circulatory arrest was induced in rats by 8 min of asphyxia. Relative levels of phosphorylated and total extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were measured in the hippocampus after 6, 12 or 24h of reperfusion using immunoblotting. Asphyxia induced a progressive increase in phosphorylated extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase, but no change in phosphorylated p38 mitogen-activated protein kinase. Induction of mild hypothermia (33 degrees C) during reperfusion increased extracellular signal-regulated kinase phosphorylation and produced a smaller increase in stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation at 24h. Hypothermia did not alter extracellular signal-regulated kinase activation in rats not subjected to ischemia. Extracellular signal-regulated kinase activation was associated with an increase in phosphorylation of the mitogen-activated protein kinase kinase 1/2, and was inhibited by administration of the specific mitogen-activated protein kinase kinase 1/2 inhibitor SL327. Immunohistochemical staining showed an increase in active extracellular signal-regulated kinase in the CA1, CA2, CA3 and dentate gyrus regions of the hippocampus after ischemia and reperfusion. In contrast, active stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity was most intense in the CA3 and dentate gyrus regions.These data demonstrate that both extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase pathways are activated during the first 24h of reperfusion after global cerebral ischemia, and that hypothermia increases the activation of extracellular signal-regulated kinase relative to stress-activated protein kinase/c-Jun N-terminal kinase. Thus, an increase in extracellular signal-regulated kinase activation may be associated with improved neuronal survival after ischemic injury.     

Ischemia-related changes of adrenocorticotropic hormone immunoreactivity and its protective effect in the gerbil hippocampus after transient forebrain ischemia

In the present study, the temporal and spatial alterations of adrenocorticotropic hormone (ACTH) immunoreactivity in the gerbil hippocampus after 5 min transient forebrain ischemia were investigated as followed up 7 days after ischemic insult, and the effects of ACTH after ischemic insult were also investigated 4 days after ischemic insult. The ectopic expression of ACTH (1-24 fragments) immunoreactive neurons in the cornus ammonis 1 (CA1) region of hippocampus and hilar region of the dentate gyrus 1 day after the ischemic insult was observed. Judging from the double immunofluorescence study, these neurons contain GABA. Four days after ischemic insult, the ACTH immunoreactivity was localized in CA1 pyramidal cells and glia near the stratum pyramidale, which normally do not express ACTH. In addition, in the saline-treated groups, the percentage of the detected Cresyl Violet positive neurons was 11.2% compared with the sham-operated group 4 and 7 days after ischemic insult. In these groups, the OX-42 immunoreactive microglia were detected in the strata pyramidale, oriens and radiatum. However, in the Org2766 (analog of ACTH)-treated group, 57.8% neurons compared with the sham-operated group were stained with Cresyl Violet 4 and 7 days after ischemic insult. In these groups, the OX-42 immunoreactive microglia were significantly reduced in the stratum pyramidale. These results suggest that transient forebrain ischemia may provoke selective ectopic and enhanced expression of ACTH in the hippocampus, and further suggest that ACTH plays an important role in reducing the ischemic damage.     

Heme oxgenase-1 is expressed in viable astrocytes and microglia but in degenerating pyramidal neurons in the kainate-lesioned rat hippocampus

The present study aimed to elucidate the distribution of heme oxygenase-1 (HO-1) in the hippocampus after intracerebroventricular injections of kainate. Very little or no staining of HO-1 was observed in the normal CA1, whilst moderate staining of dentate hilar neurons was observed in the dentate gyrus, in the normal hippocampus. At postinjection day 1, a slight increase in immunoreactivity in the neuropil of the lesioned CA fields and a marked increase in HO-1 immunoreactivity in glial cells of the stratum lacunosum moleculare of CA fields and the stratum moleculare of the dentate gyrus was observed. Electron microscopy showed that the glial cells had features of viable astrocytes. At postinjection day 3, glial cells in the dentate gyrus continued to express HO-1, whilst pyramidal neurons in the degenerating CA fields started to express intense HO-1 immunoreactivity in their cell bodies. At postinjection weeks 1–3, HO-1 was observed in glial cells in the center of the lesion, but also in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, whilst the neurons contained discontinuous cell membranes and nuclear outlines, and had features of degenerating neurons. Intense immunoreactivity was observed in the cytoplasm of the degenerating neurons. The density of staining was greater than that observed in astrocytes or microglia. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than 15-fold) were associated with significant oxygen toxicity. These and the present observations suggest a destructive effect of increased expression of HO-1 in neurons, and possible novel therapeutic approaches involving overexpression of HO-1 must therefore be approached with caution. Electronic Publication     

Early-activated microglia play a role in transient forebrain ischemia-induced neural precursor proliferation in the dentate gyrus of mice

Although it has been well established that ischemic insults promote cell proliferation in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), the mechanisms by which this occurs remain unclear. The present study demonstrates that early-activated microglia in the hilus of the DG play an important role in ischemia-induced cell proliferation. Transient forebrain ischemia induced by 20 min of bilateral common carotid artery occlusion (BCCAO) significantly increased cell proliferation in the SGZ of the DG beginning 4 days post-reperfusion. Moreover, BCCAO increased microglial activation in the hilus of the DG from 1 day post-reperfusion and in the CA1 layer from 4 days post-reperfusion. An injection of minocycline (10 or 100 nmol in 0.5 μl) into the DG immediately after reperfusion decreased microglial activation in the hilus of the DG 1 day post-reperfusion, but only a high dose of minocycline (100 nmol) significantly decreased microglial activation in the CA1 layer. Both high and low doses of minocycline significantly decreased the number of BrdU-positive cells at 7 days post-reperfusion. These results suggest that early-activated microglia in the hilus of the DG take part in the cell proliferation induced by transient forebrain ischemia.     

孕酮对沙土鼠脑缺血再灌海马小白蛋白免疫反应的影响

目的:观察孕酮对沙土鼠脑缺血再灌海马小白蛋白免疫反应的影响。方法:以双侧颈总动脉夹闭法制作脑缺血再灌注模型,免疫细胞化学方法显示海马小白蛋白免疫反应的变化。结果:脑缺血再灌后1～7 d,海马小白蛋白阳性神经元增多;第7天背侧海马CA1区锥体细胞层呈现小白蛋白阳性致密条带。脑缺血再灌孕酮处理后小白蛋白阳性神经元进一步增加,可见许多串珠状小白蛋白阳性突起,未见海马CA1区锥体细胞层小白蛋白阳性致密条带结构。结论:孕酮可能通过对小白蛋白的表达调控发挥对脑缺血再灌损伤的部分神经保护作用。     

缺血后大鼠海马NR1 mRNA的表达及其与细胞凋亡的关系

目的：研究短暂性前脑缺血后大鼠海马NR1 mRNA的表达及其与细胞凋亡的关系。方法：以四血管阻断法建立脑缺血动物模型，采用原位杂交、TUNEL染色和图像分析等技术。结果：(1)在CA1区和CA3区，NR1 mRNA的表达于缺血后2h上升，24h达高峰，然后下降，但CA3区幅度明显较小；在齿状回，缺血后0．5～72h，表达无显著性变化，缺血后7d才显著降低。(2)TUNEL阳性细胞主要位于CAl区，于缺血后24h出现，至72h达高峰，然后有所减少。结论：大鼠短暂性前脑缺血后，NR1 mRNA的表达和细胞凋亡在海马各区存在显著性差异；提示缺血后NR1 mRNA的表达与海马的选择性易损性和缺血性细胞凋亡之间可能存在着某种联系。     

Differential regulation of cyclooxygenase-2 in the rat hippocampus after cerebral ischemia and ischemic tolerance

We investigated the temporal changes and cellular localization of cyclooxygenase-2 (COX-2) in the rat hippocampus during the induction of acquired ischemic tolerance by sublethal ischemia, and compared these changes with those occurring following transient forebrain ischemia. Adult male Sprague Dawley rats were subjected to either 10 min of lethal global ischemia with or without 3 min of sublethal ischemic preconditioning, or 3 min of ischemia only. A short (3 min) cerebral ischemia as well as lethal ischemia with preconditioning substantially and significantly upregulated COX-2 expression in dentate granule cells, as confirmed by immunoblot analysis. This became evident by 4 h, peaked at 1-3 days, and returned to the basal level around 7 days. COX-2 expression was also increased in CA2 and CA3 neurons, although with weaker staining intensity, but in CA1 neurons very weak immunoreactivity was transiently observed. In the ischemic hippocampus, however, in agreement with previous reports, COX-2 expression was induced strongly in vulnerable CA1 and hilar neurons as well as in resistant CA3 and dentate granule cells. These data demonstrated that COX-2 expression is upregulated in neuronal subpopulations destined to survive, i.e., in CA3 and dentate granule cells after ischemia and ischemia-tolerance induction, as well as in ischemia-vulnerable neurons, i.e., in CA1 neurons after lethal ischemia, suggesting that hippocampal neuronal subpopulations have differential sensitivity to COX-2 upregulation.     

Activation of Src kinase in platelet-derived growth factor-B-dependent tubular regeneration after acute ischemic renal injury

We previously reported that the platelet-derived growth factor B-chain (PDGF-B)/PDGF receptor (PDGFR) axis is involved in tubular regeneration after ischemia/reperfusion injury of the kidney. In the present study, we examined the activation of Src tyrosine kinase, a crucially important signaling molecule for PDGFR, and assessed the role of Src in PDGF-B-dependent renal tubular regeneration afterischemia/reperfusion injury. Immunoblot using clone 28, a monoclonal antibody specific for the active form of Src kinases, demonstrated increased active Src expression in the injured rat kidney 6 hours after reperfusion with peak activation at 12 hours. In vitro kinase assay confirmed increased Src activity that concurred with PDGFR-beta activation as detected by the increment of receptor-phosphorylated tyrosine. Immunohistochemistry using clone 28 demonstrated that active Src was preferentially expressed in the S3 segment of the proximal tubule in reperfused kidney, where it is not normally expressed. This enhanced expression of active Src was co-localized with the increased PDGFR expression in the tubular cells that were undergoing cell proliferation cycle. Trapidil administration suppressed Src and PDGFR-beta activation in the reperfused kidney and resulted in deteriorated renal function. These findings suggest that active Src participates in PDGF-B-dependent regeneration of tubular cells from acute ischemic injury.     

Mineralocorticoid and glucocorticoid receptor expressions in astrocytes and microglia in the gerbil hippocampal CA1 region after ischemic insult

In the present study, we observed expression and changes of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the gerbil hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia. In blood, corticosterone levels were increased biphasically at 30 min and 12 h after ischemia/reperfusion, and thereafter its levels were decreased. In the sham-operated group, MR and GR immunoreactivities were weakly detected in the CA1 region. By 3 days after ischemia, MR and GR were not significantly altered in the CA1 region: at 12 h after ischemia, GR was expressed in a few neurons in the CA1 region, whereas MR was not expressed in any neurons after ischemic insult. From 4 days after ischemia, MR and GR immunoreactivities were detected in astrocytes and microglia in the CA1 region, and at 7 days after ischemia, MR and GR immunoreactivities peaked in the hippocampal CA1 region. At this time, 55% of astrocytes and 30% of microglia showed MR immunoreactivity, and 20% of astrocytes and 40% of microglia showed GR immunoreactivity. Western blot analyses showed that the pattern of changes in MR and GR protein levels was similar to the immunohistochemical changes observed after transient forebrain ischemia. From 4 days after ischemia, MR and GR protein levels were increased time-dependently after ischemia. In conclusion, enhanced MR and GR expressions in astrocytes and microglia were detected in the hippocampal CA1 region 4-7 days after ischemia/reperfusion. At this time, GR immunoreactivity was abundant in microglia, whereas MR immunoreactivity was prominent in astrocytes. The specific distribution of corticosteroid receptors in the astrocytes and microglia may be associated with the differences of MR and GR functions against ischemic damage.     

BDNF Up-Regulates TrkB Protein and Prevents the Death of CA1 Neurons Following Transient Forebrain Ischemia

The neurotrophin family of growth factors, which includes Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT4/5) bind and activate specific tyrosine kinase (Trk) receptors to promote cell survival and growth of different cell populations. For these reasons, growing attention has been paid to the use of neurotrophins as therapeutic agents in neurodegeneration, and to the regulation of the expression of their specific receptors by the ligands. BDNF expression, as revealed by immunohistochemistry, is found in the pre-subiculum, CA1, CA3, and dentate gyrus of the hippocampus. Strong TrkB immunoreactivity is present in most CA3 neurons but only in scattered neurons of the CA1 area. Weak TrkB immunoreactivity is found in the granule cell layer of the dentate gyrus. Unilateral grafting of BDNF-transfected fibroblasts into the hippocampus resulted in a marked increase in the intensity of the immunoreaction and in the number of TrkB-immunoreactive neurons in the granule cell layer of the dentate gyrus, pre-subiculum and CA1 area in the vicinity of the graft. No similar effects were produced after the injection of control mock-transfected fibroblasts. Delayed cell death in the CA1 area was produced following 5 min of forebrain ischemia in the gerbil. The majority of living cells in the CA1 area at the fourth day were BDNF/TrkB immunoreactive. Unilateral grafting of control mock-transfected or BDNF fibroblasts two days before ischemia resulted in a moderate non-specific protection of TrkB-negative, but not TrkB-positive cells, in the CA1 area of the grafted side. This finding is in line with a vascular and glial reaction, as revealed, by immunohistochemistry using astroglial and microglial cell markers. This astroglial response was higher in the grafted side than in the contralateral side in ischemic gerbils, but no differences were seen between BDNF-producing and non-BDNF-producing grafts. However, grafting of BDNF-producing fibroblasts two days before ischemia significantly and specifically prevented nerve cells from dying in the CA1 area of the ipsilateral hippocampus. Cell survival was associated with increased TrkB immunoreactivity as the majority of living cells were TrkB immunoreactive. Thus, our results show that BDNF is able to up-regulate the expression of TrkB in control and pathological states, and that BDNF prevention of neuronal death following transient forebrain ischemia is associated with increased expression of its specific receptor.     

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.     

Effects of transient cerebral ischemia on glial fibrillary acidic protein phosphorylation and immunocontent in rat hippocampus.

Transient global cerebral ischemia induced in rats by four-vessel occlusion for 20 min produced an increase in the immunocontent of glial fibrillary acidic protein and a protein phosphorylation response that was different in the CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (one, four, seven, 14 and 30 days) and observed that the immunocontent and in vitro rate of phosphorylation of glial fibrillary acidic protein in the CA1 region was significantly increased at all intervals after the ischemic insult, indicating that the astrocytic response was maintained for at least 30 days. After reperfusion for 14 days a significant increase in the ratio "in vitro phosphorylation rate/immunocontent" in the CA1 region was observed when compared to control values, to other intervals and to the dentate gyrus, suggesting a hyperphosphorylation of this intermediate filament protein at this interval. In the dentate gyrus, an area less vulnerable to the insult, labelling and immunocontent of glial fibrillary acidic protein were equally increased from four days of reperfusion and the increase remained significant until 30 days, confirming that neuronal death is not the only determining factor for gliosis to occur. In control sham-operated animals, neither the CA1 region nor the dentate gyrus showed significant increases in labelling or immunocontent. Changes in the phosphorylation of glial fibrillary acidic protein may be essential for the plastic response of astrocytes to neuronal damage, as neurons and astrocytes can act as functional units involved in homeostasis, plasticity and neurotransmission.     

Rapamycin保护大鼠缺血性脑损伤和特定脑区神经新生有关

目的:探讨缺血前给予自噬诱导剂对脑缺血损伤的保护效应及可能机制。方法:双侧颈总动脉结扎建立全脑缺血模型,再灌注后24 h,评价动物神经行为功能,连续脑切片,采用Nissl染色定量检测皮层及海马CA1区细胞密度;通过免疫荧光技术检测Caspase-3阳性神经元,计数皮层及海马CA1凋亡细胞数量;激光共聚焦显微镜观察并计数海马齿状回颗粒下层内呈Ki67阳性、GFAP(胶质纤维酸性蛋白)阴性(Ki67+/GFAP-)细胞的数量。结果:Rapamycin术前预处理可以改善缺血导致的神经功能缺陷(P<0.05)。Nissl染色结果表明Ra-pamincy术前1 h给药可以减轻缺血导致的皮层(P<0.01)及海马CA1区(P<0.01)细胞丢失。同时,Rapamycin术前给药也显著减少了缺血导致的皮层及海马CA1区内Caspase-3阳性细胞的数量,组间比较有显著性差异(P<0.05)。Rapamycin术前1 h给药增加了海马齿状回内Ki67+/GFAP-细胞的数量,和缺血组比较差异有显著性(P<0.05)。结论:在全脑缺血模型上,通过自噬活化途径的缺血预处理可以保护缺血性脑损伤,这一作用和Rapamycin减少凋亡、增加新生神经细胞的数量有关。     

Extracellular signal-regulated kinase involved in NGF/VEGF-induced neuroprotective effect

Compelling evidence has shown that extracellular signal-regulated kinase (ERK) is widely expressed in many tissues, including the brain. In the present work, we investigated the temporospatial alterations of ERK1 immunoreactivity in hippocampus and perifocal cortex, and the expression involved in NGF/VEGF-induced neuroprotective effect. We demonstrated that ERK1 expression was first increased in hippocampal CA3/DG 1 h after reperfusion, then it was also increased 6 h after reperfusion in other brain regions, with a peak at day 1-3, and then gradually decreased to basal level at day 14. The expression of caspase-3 was strongly increased 1 h after reperfusion, with peak demonstrated at 3d. NGF/VEGF significantly inhibited the expression of ERK1 and caspase-3. These results suggest that ERK1 signaling pathway may be involved in neuronal cell death and NGF/VEGF-induced neuroprotective effect and there appeared an association between ERK and caspase-3. Inhibition of the ERK signaling pathway might therefore provide an efficient way to prevent neuronal cell death after ischemic cerebral injuries.     

The effect of an AMPA antagonist (NBQX) on postischemic neuron loss and protein synthesis in the rat brain

Two groups of rats were subjected to 12 min of global cerebral ischemia and 6 days recirculation using the four-vessel occlusion model with hypotension and then treated with the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxa-linedione (Honoré et al. 1988). One group was used for routine and quantitative histology and immunostaining for glial fibrillary acidic protein (GFAP). The second group was subjected to autoradiographic studies of regional cerebral protein synthesis, with special emphasis on the hippocampus, the frontal cortex, and the thalamus. It was found that neuroprotective treatment with NBQX normalized cerebral protein synthesis rate (CPSR) in all investigated regions 6 days after ischemia. In untreated ischemic animals CPSR was normalized in all regions except for the CA3 and thalamus, where it had increased by 29% and 41%, respectively. Treatment of controls with NBQX had no effect on CPSR after 6 days. The histological investigations revealed that NBQX did not protect vulnerable cells in the dentate hilus and the reticular thalamic nucleus (RTN). In these regions reactive astrocytosis visualized by GFAP immunostaining was equally pronounced in both ischemic and NBQX-treated animals, and most neurons in the RTN were eosinophilic. The 80–100% pyramidal neuron loss in CA1 was accompanied by a high degree of reactive astrocytosis, whereas the NBQX-treated animals showed no signs of astrocytosis in this region. The ischemic CA1 pyramidal layer was also massively invaded by microglia. Together these observations strongly suggest that the quantitatively normal protein synthesis in this region after ischemia must be attributed to these glial cell populations.