缺氧诱导因子-1α在慢性脑缺血大鼠海马中的表达

目的　观察缺氧诱导因子 - 1α在慢性脑缺血大鼠海马中的表达 ,并探讨其表达的意义。方法　采用永久性双侧颈总动脉结扎术 ( 2 VO)制备大鼠慢性脑缺血的动物模型 ,运用 Western印迹和免疫组化检测 HIF- 1α的表达。结果　 Western印迹显示 2 VO1d后 HIF- 1α的表达即上调 ,2周后进一步升高 ,保持这一水平直至第 6周。免疫组化见海马各区均有免疫阳性细胞分布。结论　 HIF- 1α在慢性脑缺血大鼠海马中的表达上调 ,可能参与了慢性脑缺血的代偿机制。     

Allograft-inflammatory factor-1 in rat experimental autoimmune encephalomyelitis,neuritis, and uveitis: Expression by activated macrophages and microglial cells

Allograft inflammatory factor-1 (AIF-1) is a Ca²⁺binding peptide expressed predominantly by activated monocytes. In order to investigate the role of AIF-1 in autoimmune lesions of the rat nervous system, we have used a synthetic gene to express AIF-1 in E.coli and have produced monoclonal antibodies against AIF-1. AIF-1 was localized to monocytes/macrophages with rather selective staining of a minor rat monocyte subpopulation of lymphoid tissue. We then investigated expression of AIF-1 in experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), and uveitis (EAU). Within the local inflammatory lesions, infiltrating macrophages are prominently stained. In the diseased brain, AIF-1-positive microglial cells are not only found in the direct vicinity of the infiltrate, but widespread activation is seen in the parenchyma. This is the first demonstration that AIF-1 is present in autoimmune lesions. Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance. Thus, AIF-1 might be a valuable marker to dissect the local monocyte heterogeneity in autoimmune disease. GLIA 24:244–251, 1998. © 1998 Wiley-Liss, Inc.     

Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat

Cerebral ischemia induces microglial and astroglial activation, which may play a crucial role in the development of ischemic neuronal damage. In this study, we examined the role of cell cycle proteins in glial proliferation in the hippocampus following 10min of global cerebral ischemia in the rat. Proliferating cells were identified with immunostaining for proliferating cell nuclear antigen (PCNA), and glial cells were visualized with immunostaining for microglial response factor-1 (microglia/macrophages) and glial fibrillary acidic protein (astrocytes). Expression of cyclin D1 and cyclin-dependent kinase-4 was also examined with double label immunohistochemistry. Proliferating cells in the CA1 region after ischemia consisted of microglia and much fewer astrocytes. Microglial activation and proliferation (7.6-fold increase in number after 7 days) were preceded by an increase in PCNA-positive microglia; 83% of microglia were PCNA-positive after 2 days. Astrocytes increased by 1.8-fold after 7 days, and only 6% of astrocytes became PCNA-positive by day 7. Cyclin D1 and cyclin-dependent kinase-4 immunoreactivity appeared in these glial cells in parallel with the expression of PCNA. The findings suggest that the accumulation of brain macrophages elicited by transient cerebral ischemia is caused predominantly by activation and proliferation of resident microglia through the upregulation of cell cycle proteins.     

Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: The purpose of this study was (1) to examine the contribution of microglia and macrophages with their interleukin-1beta production and (2) to assess the vulnerability and response of oligodendrocytes in cerebral infarction. METHODS: Male Wistar rats were subjected to permanent occlusion of the left middle cerebral artery. Expansion of ischemic infarction and response of oligodendrocytes were investigated together with accumulation of inflammatory cells, production of interleukin-1beta, and disruption of the blood-brain barrier. Apoptotic cell death was inferred from fragmented DNA and the expression of proapoptotic Bax protein. RESULTS: During expansion of infarction, amoeboid microglia and extravasation of serum albumin were observed not only in the infarcted area but also in the adjacent surviving area, whereas macrophages accumulated along the boundary and granulocytes migrated into the center of the infarction. Both amoeboid microglia and macrophages produced interleukin-1beta, an inflammatory cytokine, during an early ischemic period. Furthermore, macrophages within the infarcted tissue expressed Bax protein and subsequently showed fragmented nuclear DNA. Oligodendrocytes were detected in the infarcted area even after 24 hours following middle cerebral artery occlusion, but they subsequently developed fragmented DNA. A week after onset of ischemia, oligodendrocytes were found to be accumulated in the intact area bordered with the infarct together with reactive astrocytes. CONCLUSIONS: Our results suggest the importance of amoeboid microglia, macrophages, and their interleukin-1beta production in gradual expansion of cerebral infarction. Resident oligodendrocytes may be resistant to ischemic insults, and oligodendrocytes accumulated at the border of the infarction may participate in tissue repair after cerebral infarction.     

Selective accumulation of cyclooxygenase-1-expressing microglial cells/macrophages in lesions of human focal cerebral ischemia

Cyclooxygenases (COX; prostaglandin endoperoxide H synthases) are key enzymes in the conversion of arachidonic acid into prostanoids which mediate inflammation, immunomodulation, mitogenesis, ovulation, fewer, apoptosis and blood flow. Here, we report COX-1 expression following focal cerebral infarctions (FCI). In healthy control brains, COX-1 was localized by immunohistochemistry to a few endothelial cells, single neurons and rare, evenly distributed brain microglia/macrophages. In infarctioned brains, COX-1+ cells accumulated highly significantly (P < 0.0001) in peri-infarctional areas and in the developing necrotic core early after infarction. Here, cell numbers remained persistently elevated up to several months post infarction. Further, clusters of COX-1+ cells were located in perivascular regions related to the Virchow-Robin space. Double-labeling experiments confirmed co-expression of COX-1 by CD68+ microglia/macrophages. Co-expression of the activation antigens HLA-DR, -DP, -DQ (MHC class II) or the macrophage inhibitor factor-related protein MRP-8 (S100A8) by most COX-1+ microglia/macrophages was only seen early after infarction. Thus, COX-1 appeared to be expressed in microglial cells regardless of their activation state. However, the prolonged accumulation of COX-1+ microglia/macrophages restricted to peri-infarctional areas enduring the acute post-ischemic inflammatory response points to a role of COX-1 in tissue remodeling or in the pathophysiology of secondary injury. We have identified localized, accumulated COX-1 expression as a potential pharmacological target following FCI. Therefore we suggest that therapeutic approaches based on selective COX-2 blocking might not be sufficient for suppressing the local synthesis of prostanoids.     

Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas

Allograft inflammatory factor-1 (AIF-1) is a Ca²⁺-binding peptide that constitutes a potential modulator of macrophage activation and function during the immune response of the brain. Peptides termed microglia response factor-1 or ionized calcium-binding adaptor molecule-1 have been reported to be identical with AIF-1. We have investigated the expression of AIF-1 in the rat C6 glioblastoma and 9L gliosarcoma tumor models and additionally assessed AIF-1 expression in a diverse range of human astrocytomas by immunohistochemistry. AIF-1 was expressed by activated microglial cells and a subset of infiltrating macrophages in areas of infiltrative tumor growth and in compact tumor areas in both rat and human gliomas. Double-labeling experiments in rats and humans characterized the nature and the functional status of AIF-1⁺ cells. AIF-1 expression was detected in cells expressing major histocompatibility complex class II molecules and in a subset of activated macrophages/microglial cells. All MRP-8⁺ cells coexpressed AIF-1. In humans, there was a strong correlation of AIF-1-expressing activated macrophages/microglial cells with tumor malignancy (P < 0.0001). These results suggest that AIF-1 defines a distinct subset of tumor-associated activated macrophages/ microglial cells. Received: 5 October 1998 / Revised: 15 June 1999, 6 August 1999, 28 February 2000 / Accepted: 28 February 2000     

Pyruvate dehydrogenase activity in the rat cerebral cortex following cerebral ischemia

The effect of cerebral ischemia on the activity of pyruvate dehydrogenase (PDH) enzyme complex (PDHC) was investigated in homogenates of frozen rat cerebral cortex following 15 min of bilateral common carotid occlusion ischemia and following 15 min, 60 min, and 6 h of recirculation after 15 min of ischemia. In frozen cortical tissue from the same animals, the levels of labile phosphate compounds, glucose, glycogen, lactate, and pyruvate was determined. In cortex from control animals, the rate of [1(-14)C]pyruvate decarboxylation was 9.6 +/- 0.5 nmol CO2/(min-mg protein) or 40% of the total PDHC activity. This fraction increased to 89% at the end of 15 min of ischemia. At 15 min of recirculation following 15 min of ischemia, the PDHC activity decreased to 50% of control levels and was depressed for up to 6 h post ischemia. This decrease in activity was not due to a decrease in total PDHC activity. Apart from a reduction in ATP levels, the acute changes in the levels of energy metabolites were essentially normalized at 6 h of recovery. Dichloroacetate (DCA), an inhibitor of PDH kinase, given to rats at 250 mg/kg i.p. four times over 2 h, significantly decreased blood glucose levels from 7.4 +/- 0.6 to 5.1 +/- 0.3 mmol/L and fully activated PDHC. In animals in which the plasma glucose level was maintained at control levels of 8.3 +/- 0.5 mumol/g by intravenous infusion of glucose, the active portion of PDHC increased to 95 +/- 4%. In contrast, the depressed PDHC activity at 15 min following ischemia was not affected by the DCA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The microglial reaction in the rat hippocampus following global ischemia: immuno-electron microscopy

Summary Transient arrest of the cerebral circulation leads to neuronal cell death in selectively vulnerable regions of the central nervous system. It has recently been shown at the light microscopical level that neuronal necrosis is accompanied by a rapid microglial reaction in ischemia (Gehrmann et al. (1992) J. Cereb. Blood Flow Metab. 12:257–269). In the present study we have examined the postischemic microglial reaction in the dorsal rat hippocampus at the ultrastructural level using immuno-electron microscopy. Global ischemia was produced by 30 min of four-vessel occlusion and the microglial reaction then studied after 8, 24 and 72 h. In sham-operated controls microglial cells were not phagocytic; they were randomly distributed throughout the neuropil and occasionally made contacts with other structures such as dendrites in CA1. Ultrastructural signs of activation were observed from 1 day postlesion onward. Reactive microglial cells were consistently seen to phagocytose degenerating neurons particularly in the CA1 stratum pyramidale and in the CA4 sector. They were sometimes interposed between two morphologically distinct types of CA1 neurons, i.e., dark (degenerating) and pale (surviving) types of neurons. Phagocytic microglial cells also became positive for major histocompatibility complex (MHC) class II antigens at these locations from 1 day after ischemia onward. Furthermore, activated microglial cells were frequent along degenerating dendrites in the stratum radiatum of CA1. After survival times of up to 72 h microglial cells, but not astrocytes, were occasionally observed to undergo mitosis. In addition to their random distribution across the neuropil, microglial cells were frequently observed in a perivascular position under normal conditions. These perivascular microglial cells rapidly expressed MHC class II antigens, extended broad cellular processes and showed signs of phagocytic activity from 1 day onward. These results demonstrate that upon ischemic injury microglial cells proliferate and are rapidly recruited to the site of injury. By virtue of their pronounced cytotoxic potential, microglial cells could be further involved in mediating tissue destruction in ischemia, thus constituting the main immuneffector cell population in this pathological state.     

The microglial reaction in the rat dorsal hippocampus following transient forebrain ischemia.

We have examined the distribution and time course of the microglial reaction in the rat dorsal hippocampus after 25-min transient forebrain ischemia (four-vessel occlusion model). Microglial cells were visualized in brain sections using lectin staining with the Griffonia simplicifolia B4-isolectin following intervals of reperfusion ranging from 20 min to 4 weeks. Increased staining of microglial cells was detected in the dentate hilus and area CA1 as early as 20 min after reperfusion. These same regions demonstrated more intense microglial staining after 24 h. The strongest microglial reaction was observed 4-6 days after reperfusion when reactive microglia were abundant throughout all laminae of CA1 and the dentate hilus. Following longer reperfusion intervals, the microglial reaction became less intense; however, it remained above normal levels until the end of the fourth week. At all time points examined, microglial reactivity in the CA3 pyramidal and dentate granule cell layers was considerably lower than that observed in CA1 and dentate hilus. Our results are consistent with the known serial pathological changes associated with cerebral ischemia, but, in addition, show that the examination of the microglial reaction provides an extremely sensitive indicator of subtle and morphologically nonapparent neuronal damage during the early stages of injury.     

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.     

Brain glutamine synthetase increases following cerebral ischemia in the rat.

Changes in astrocyte glutamine synthetase (GS) in postischemic rat brain were evaluated and correlated with regional neuronal vulnerability or resistance to ischemia. Rats subjected to 20 or 30 min of cerebral ischemia were allowed to survive for 3 or 24 h after ischemia; normal animals served as controls. Resultant neuronal necrosis was severe in the striatum by 24 h and in the CA1 region of the hippocampus at 72 h; neurons in paramedian cortex and CA3 region of the hippocampus were not permanently damaged. Glutamine synthetase (GS) immunocytochemistry was performed on vibratome sections of paraformaldehyde-fixed brains and enzyme activity was assayed in frozen samples of cerebral cortex, striatum and hippocampus. At 3 and 24 h after ischemia, GS immunoreactivity increased and was secondary to enlargement of GS-positive cell bodies and processes as well as to increased numbers of GS-positive astrocytes. Enzyme activity also increased in cortex, striatum and hippocampus at 3 and 24 h (P less than or equal to 0.03). This study shows that increase in astrocyte GS occurs rapidly after ischemia, and prior studies indicate that this increase occurs in parallel with proliferative changes in astrocyte organelles. The results also suggest that astrocyte metabolism of glutamate increases after ischemia. The increased capacity for glutamine synthetase may be important in normalizing extracellular glutamate following ischemia and protecting brain from the neurotoxic effects of this excitatory amino acid.     

Selenoprotein S expression in the rat brain following focal cerebral ischemia

Recent studies on cerebral ischemic stroke have demonstrated the importance of the inflammatory response. Ongoing inflammatory insults have been implicated as a secondary mechanism underlying neuronal injury induced by ischemia, and anti-inflammatory strategies have gained considerable interest. Selenoprotein S (SelS), which is an endoplasmic reticulum resident protein, is known to promote cell survival by regulating inflammation. Moreover, SelS has been shown to be responsive to ischemia in cultured astrocytes. A Finnish report revealed that a variation in the SelS gene locus is associated with a higher predisposition to ischemic stroke in humans, suggesting a crucial role for SelS in protection against brain ischemia. However, the time-course of SelS expression following cerebral ischemia in vivo remains unknown. In the present study, we show, for the first time, differential SelS expression from 3 h to 7 days after reperfusion in rats with transient focal cerebral ischemia induced by a 1-h middle cerebral artery occlusion. We found that the SelS protein level decreased in the ischemic core 3–7 days after reperfusion. Furthermore, SelS expression was upregulated in the ischemic penumbra adjacent to the ischemic core 3–7 days after reperfusion and is matched by reactive astrogliosis. Thus, we propose that the upregulation of Sels represents a reaction of astrocytes against inflammatory stimuli, and the findings of this study open a new chapter in the research of the interrelationships between SelS and cerebral ischemic stroke.     

Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia

The present study was undertaken to correlate calcium accumulation with the development of neuronal necrosis following transient ischemia. After 10 min of forebrain ischemia in the rat--a period that leads to reproducible damage of CA1 pyramidal cells--determination of calcium concentration and evaluation of morphological signs of cell body necrosis in the dorsal hippocampus were performed at various recirculation times. Tissue calcium concentration was not different from control at the end of ischemic period and did not change after 3, 6, 12, or 24 h of recirculation. However, after 48 h, calcium content increased significantly, with a further increase being seen after 72 h. At early recovery periods, only scattered necrotic neurons were observed. After 48 h, only 2 of 12 hemispheres showed more than 25 necrotic cells per section. More conspicuous neuronal death was observed after 72 h. The results thus demonstrate that net accumulation of calcium in regio superior of the hippocampus precedes marked necrosis of CA1 pyramidal cells. The results suggest that one primary event in the delayed death of these cells is membrane dysfunction with increased calcium cycling.     

Comparison of C1q-receptors on rat microglia and peritoneal macrophages.

A comparison of the expression and ligand specificity of the C1q (first complement component) receptor on rat microglia and peritoneal macrophages was made. This revealed that radiolabelled C1q was competed from the peritoneal macrophages with intact C1q, and additively displaced by calf-skin collagen and purified C1q globular heads, suggesting the presence of at least two receptors. This was in contrast to microglia, where radiolabelled C1q was displaced with intact C1q and to a modest degree with collagen, but not with globular heads. Taken together, this implies that under these conditions, peritoneal macrophages and microglia both express a C1q receptor which binds to the collagen-like region, and that peritoneal macrophages additionally express a molecule which binds to the globular head of C1q. Analysis of the ligand bound by these cells reflected the differences observed in the competitive binding experiments, with the novel identification of naturally-occurring peptides from the globular head of C1q bound to the peritoneal macrophages, but not the microglia.     

The microglia/macrophage response in the neonatal rat facial nucleus following axotomy

Microglia represent a population of brain macrophage precursor cells which are intrinsic to the CNS parenchyma. Transection of the facial nerve in the newborn rat causes death of the affected motor neurons which is accompanied by massive activation of local microglia. Many of these cells develop into macrophages as can be shown by immunocytochemistry for OX-42 and ED1. Using the new polyclonal microglial marker ionized calcium binding adapter molecule 1, iba1, in combination with immunocytochemical double-labeling for the proliferating cell nuclear antigen (PCNA), or [³H]thymidine autoradiography, and confocal microscopy, qualitative as well as quantitative differences can be demonstrated between the newborn and the adult axotomized rat facial nucleus. While microglial cells are the only cell population which responds to axotomy by cell division in the adult facial nucleus, GFAP positive reactive astrocytes can be shown to undergo mitosis following axotomy in the newborn rat. Furthermore, ED1 immunoreactivity, early expression of MHC class II molecules and morphological transformation of microglia into macrophages can only be observed under conditions of neuronal degeneration, i.e., in the neonatal rat facial nucleus. Thus, the combination of cellular markers described here should be useful for studies employing the neonatal rat facial nucleus as an in vivo assay system to test the efficacy of neurotrophic factors.     

Changes of hypoxia-inducible factor-1 signaling and the effect of cilostazol in chronic cerebral ischemia

Hypoxiainducible factor1 and its specific target gene heme oxygenase1, are involved in acute cerebral ischemia. However, very few studies have examined in detail the changes in the hy poxiainducible factor1/heme oxygenase1 signaling pathway in chronic cerebral ischemia. In this study, a rat model of chronic cerebral ischemia was established by permanent bilateral common carotid artery occlusion, and these rats were treated with intragastric cilostazol （30 mg/kg） for 9 weeks. Morris water maze results showed that cognitive impairment gradually worsened as the cerebral ischemia proceeded. Immunohistochemistry, semiquantitative PCR and western blot analysis showed that hypoxiainducible factorla and heme oxygenase1 expression levels in creased after chronic cerebral ischemia, with hypoxiainducible factorla expression peaking at 3 weeks and heme oxygenase1 expression peaking at 6 weeks. These results suggest that the elevated levels of hypoxiainducible factorla may upregulate heine oxygenase1 expression fol lowing chronic cerebral ischemia and that the hypoxiainducible factor1/heme oxygenase1 sig naling pathway is involved in the development of cognitive impairment induced by chronic cerebral ischemia. Cilostazol treatment alleviated the cognitive impairment in rats with chronic cerebral ischemia, decreased hypoxiainducible factorla and heme oxygenase1 expression levels, and reduced apoptosis in the frontal cortex. These findings demonstrate that cilostazol can protect against cognitive impairment induced by chronic cerebral ischemic injury through an antiapoptotic mechanism.