Changes in Distribution of Cystatin C, Apolipoprotein E and Ferritin in Rat Hypothalamus after Hypophysectomy

To clarify the mechanism underlying the process of degeneration of injured CNS neurons, we have immunohistochemically examined the distribution of cystatin C, apolipoprotein E, IgG, transferrin and ferritin in the hypophysectomized rat hypothalamus. Stainings for ferritin revealed that reactive microglial cells massed in the paraventricular and supraoptic nuclei 14 days after hypophysectomy, when the degeneration of vasopressin neuronal cell bodies was apparent. Cystatin C-positive magnocellular neurons first appeared at 4 days and the number of intensely-stained cells increased rapidly up to the 7th day of hypophysectomy, followed by a decrease thereafter. Most of such cystatin C-positive neurons were simultaneously stained with anti-vasopressin serum. Accumulation of apolipoprotein E in extra-cellular spaces was obvious in the both hypothalamic magnocellular nuclei at 7 days. Several apolipoprotein E-positive cells were localized in the supraoptic nucleus, although the number of apolipoprotein E-positive cells was much smaller than that of cystatin C-positive cells. The experiments performed with the transferrin and IgG antibodies showed undetectable levels of such molecules in and around the degenerating magnocellular neurons during whole experimental periods. These findings suggest the importance of cystatin C and apolipoprotein E in the process of degeneration and/or regeneration of magnocellular neurons after hypophysectomy.     

Expression of endothelial nitric oxide synthase in the ischemic penumbra: relationship to expression of neuronal nitric oxide synthase and vascular endothelial growth factor

Expressional patterns of the endothelial and neuronal forms of nitric oxide synthase (NOS) in cerebral ischemia were studied utilizing a permanent middle cerebral artery occlusion (PMCAO) model. Motor performance and infarct volumes were determined in the rats. Immunohistochemical staining for eNOS, nNOS and neurofilament were performed at 1, 2, 3, 5, 7 and 14 days after PMCAO. Vascular endothelial growth factor (VEGF) expression was determined by in-situ hybridization. PMCAO caused a reproducible cortical infarct with motor deficits in the rats. Double immunohistochemical stainings indicated that eNOS and nNOS were induced in ischemic neurons. Most stained neurons were positive for both NOS forms but some reacted with only one NOS antibody. nNOS expression peaked at 24-48 h after PMCAO, stained mainly the cytoplasm of core neurons, and disappeared after the 3rd day. eNOS expression increased until the 7th day, stained mainly the cytoplasm and membrane of penumbral cells and disappeared by the 14th day after PMCAO. VEGF expression was significantly induced in the penumbral zone in a similar distribution to eNOS. The anatomical and temporal pattern of VEGF and eNOS induction in the brain after permanent ischemia suggest that these mediators may play a role in protecting penumbral tissue from additional ischemic damage.     

Expression of neuron specific phosphatase, striatal enriched phosphatase (STEP) in reactive astrocytes after transient forebrain ischemia

We studied the distribution and change of striatal enriched phosphatase (STEP) in the gerbil hippocampus after transient forebrain ischemia. STEP was expressed in the perikarya and in neuronal processes; it was not detected in non-neuronal cells of control animals. After 5-min forebrain ischemia, STEP immunoreactivity (STEP-IR) was preserved for 2 days; it disappeared 4 and more days after ischemia with completion of delayed neuronal death (DND) in the CA1 subfield. Furthermore, only in the CA1 after ischemia, STEP was expressed in reactive astrocytes for 4 to 28 days, showing different patterns of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. After non-or less-than lethal ischemia, STEP expression in reactive astrocytes corresponded with the degree of neuronal degeneration. Immunoblot analysis of the CA1 subfield revealed the expression of three isoforms, STEP45, -56 and -61; their expression patterns changed with time after ischemia. These data suggest that neuronal STEP is preserved until cell degeneration after ischemia and that STEP is expressed in reactive astrocytes only after lethal ischemia, with different expression patterns for its isoforms. Of STEP45, -56 and -61, STEP61 was the most strongly expressed in the reactive astrocytes; both STEP45 and -61 were expressed in neurons and the expression of STEP56 was weak. STEP may play an important role not only in neurons but also in reactive astrocytes after ischemia, depending on neuronal degeneration.     

Pattern of neuronal loss in the rat hippocampus following experimental cardiac arrest-induced ischemia.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered 'relatively resistant'. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes.     

Calpain induces proteolysis of neuronal cytoskeleton in ischemic gerbil forebrain

We investigated the relationship between the activity of calcium-dependent protease (calpain) and the ischemic neuronal damage. We also investigated the mechanism of ischemic resistance in astrocytes. In gerbil, a 10-min forebrain ischemia was induced by occlusion of both common carotid arteries. The calpain-induced proteolysis of cytoskeleton (fodrin) was examined by immunohistochemistry. Immunolocalization of micro and m-calpain was also examined. Intact fodrin was observed both in neurons and astrocytes, but proteolyzed fodrin was not observed in normal brain. Fifteen minutes after ischemia, proteolysis of fodrin took place in putamen, parietal cortex and hippocampal CA1. The proteolysis extended to thalamus 4 h after ischemia after which the immunoreactivity faded down in all areas except hippocampus. On day 7, the proteolysis was still observed only in hippocampus. Neurons with the proteolysis of soma resulted in neuronal death. Throughout the experiment, the proteolysis was not observed in astrocytes. micro -Calpain was observed only in neurons but m-calpain was observed both in neurons and astrocytes. The ischemia induced only micro -calpain activation, which resulted in fodrin proteolysis of neurons with differential spatial distribution and temporal course. The proteolysis was developed rapidly and was completed within 24 h in all vulnerable regions except hippocampal CA1. The proteolysis preceded the neuronal death. The mechanism of the proteolysis seemed to be involved by Ca(2+) influx via glutamate receptor and rapid neuronal death seemed reasonable. The reason why neuronal death in CA1 evolved slowly was not clarified. In astrocytes, fodrin was not proteolyzed by m-calpain. The low Ca(2+)-sensitivity of m-calpain may be the reason of ischemic resistance in astrocytes.     

Complete cerebral ischemia with short-term survival in rat induced by cardiac arrest. II. Extracellular and intracellular accumulation of apolipoproteins E and J in the brain

The distribution of apolipoprotein E (apo E) and apolipoprotein J (apo J) was investigated immunocytochemically in rats at various time intervals after 10 min global cerebral ischemia (GCI) induced by cardiac arrest. Strong apo E and weaker apo J immunoreactivity was found extracellularly in multiple deposits located close to the microvessels. These deposits appeared 3 h after GCI and were present, but not in all the animals, at all time intervals studied post-GCI. In some rats, apo E immunoreactivity was also found in small necrotic foci. Widespread, neuronal apo E immunostaining appeared 6 h post-GCI. However, the strongest neuronal apo E immunoreactivity was found 7 days post-GCI in those neurons, most often observed in the CA1 hippocampal region, exhibiting signs of ischemic cell damage. These ischemically damaged neurons displayed weaker immunoreactivity to apo J, despite its increase in the response to GCI in the various brain regions examined. Our data show that mechanisms operating in ischemia are able to supply large amounts of apo E and apo J to the brain tissue and suggest involvement of both apo E and apo J in a complex series of events occurring in the ischemic brain. Perivascular deposits of apo E/apo J colocalized with amyloid beta protein precursor epitopes that have been disclosed by us previously in this model. Whether this phenomenon is limited to postischemic brain tissue, or can be encountered also in other pathological conditions will require further elaboration.     

The expression of transforming growth factor-beta1 (TGF-beta1) in hippocampal neurons: a temporary upregulated protein level after transient forebrain ischemia in the rat

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.     

Postischemic neuronal damage causes astroglial activation and increase in local cerebral glucose utilization of rat hippocampus

The purpose of the present study was to determine the consequences of postischemic neuronal damage on CMRglc. Forebrain ischemia of 10 min duration was induced in male Wistar rats. The extent of neuronal damage and the numbers of immunocytochemically detected astrocytes in the hippocampal CA1 subfield as well as CMRglc were determined 2, 5, 7, and 14 days after ischemia. CBF was additionally measured 7 days postischemia. CMRglc was decreased in cortical and thalamic structures up to 5 days postischemia, and was normalized again on day 7 after ischemia. In the hippocampal areas, CMRglc was decreased only on day 2 after ischemia, was normalized after 5 days, and increased in the stratum oriens and pyramidale of the CA1 subfield from postischemic day 7 onward. Neuronal damage was clearly demonstrable 5 days after ischemia and further increased up to day 7. The number of GFAP-reactive astrocytes increased markedly at day 7 postischemia. It is assumed that the activation of astrocytes is induced by neuronal damage, and that the astroglial metabolism is responsible for the increase in CMRglc of the CA1 subfield 7 days after ischemia. The decrease in CBF of the CA1 subfield 7 days after ischemia could be caused by a reduced density of perfused capillaries.     

Response of Hippocampal Neurons and Glial Cells to Alternating Magnetic Field in Gerbils Submitted to Global Cerebral Ischemia

The purpose of this study was to determine whether exposure to an extremely low-frequency magnetic field (ELF-MF, 50 Hz) affects the outcome of postischemic damage in the hippocampus of Mongolian gerbils. After 10-min bilateral carotid occlusion, the gerbils were continuously exposed to ELF-MF (average magnetic induction at the center of the cage was 0.5 mT) for 7 days. The impact of ELF-MF was estimated immediately (the 7th day after reperfusion) and 7 days after cessation of exposure (the 14th day after reperfusion) compared with ischemic gerbils without ELF-MF exposure. Applying stereological methods, histological evaluation of changes in the hippocampus was done for determining its volume, volume densities of degenerating neurons and astrocytes, as well as the number of microglial cells per unit area. ELF-MF per se did not induce any morphological changes, while 10-min global cerebral ischemia led to neuronal death, especially in CA1 region of the hippocampus, as expected. Ischemic gerbils exposed to ELF-MF had significantly a lower degree of cell loss in the examined structure and greater responses of astrocytes and microglial cells than postischemic gerbils without exposure on the seventh day after reperfusion (immediate effect of ELF-MF). Similar response was observed on the 14th day after reperfusion (delayed effect of ELF-MF); however, differences in measured parameters were low and insignificant. Applied ELF-MF has possible neuroprotective function in the hippocampus, as the most sensitive brain structure in the model of global cerebral ischemia, through reduction of neuronal death and activation of astrocytes and microglial cells.     

Immunocytochemical investigation of L-glutamic acid decarboxylase in the rat hippocampal formation: the influence of transient cerebral ischemia

The hippocampus is especially vulnerable to ischemic damage. Neurons in the CA3c region and dentate hilus demonstrate fast progressive damage while CA1 pyramidal cells demonstrate delayed neuronal damage. The delayed CA1 pyramidal cell loss could be caused by postischemic neuronal hyperactivity if hippocampal interneurons are lost after ischemia. Therefore we have counted the L-glutamic acid decarboxylase (GAD)-immunoreactive neurons in the hippocampus from control rats and rats surviving 4 or 11 days after 20 minutes of cerebral ischemia. All rats were injected intraventricularly with colchicine before they were killed. The hippocampal cell counts showed an increase in GAD-immunoreactive somata visualized on the fourth postischemic day. Eleven days after ischemia, the number of GAD-immunoreactive neurons visualized in the hippocampus CA1 and CA3c region decreased. GAD-immunoreactive baskets were visualized in the pyramidal cell layer and the granule cell layer in controls and 4 days after ischemia, but not in the CA1 and CA3c pyramidal cell layer 11 days after ischemia. We suggest the number of GAD-immunoreactive neurons visualized on the fourth postischemic day increases because somatal GAD accumulation increases and, therefore, ischemia may enhance GAD production. Our previous counts of CA1 interneurons 21 days after ischemia in toluidine-stained semithin sections demonstrated no interneuron loss. Therefore we suggest that the decreased number of CA1 and CA3c GAD-immunoreactive neurons visualized 11 days after ischemia is related to a decreased GAD production. It is possible at this stage after ischemia that the interneurons have decreased their GAD production because they have lost their input and/or target cells. We conclude that our counts of GAD-immunoreactive neurons visualized after ischemia express changes in the content of somatal GAD rather than the actual number of GAD-immunoreactive somata. Finally, we conclude that the delayed loss of CA1 pyramidal cells seen 4 days after ischemia is not preceded by loss of hippocampal GAD-immunoreactive neurons.     

Astrocyte targeted overexpression of Hsp72 or SOD2 reduces neuronal vulnerability to forebrain ischemia

Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well‐studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte‐specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter‐1 immunoreactivity at 5‐h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation. © 2010 Wiley‐Liss, Inc.     

Implication of "Down syndrome cell adhesion molecule" in the hippocampal neurogenesis of ischemic monkeys

Molecular signals regulating adult neurogenesis in primates are largely unknown. Here the authors used differential display to analyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and characterized its expression pattern at the protein level. In contrast with approximately threefold upregulation of Dscam gene on days 5 and 7, immunoblotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or betaIII-tubulin, while another is astrocytes positive for S100beta. Young astrocytes were in intimate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of primate hippocampus upregulated after ischemia.     

Ischemic neuronal injury in the rat hippocampus following transient forebrain ischemia: evaluation using in vivo microdialysis

Neuronal vulnerability to ischemia in the rat hippocampus was investigated by the measurement of high potassium evoked overflow of neurotransmitters using in vivo microdialysis. Changes in the extracellular level of amino acids caused by high potassium (100 mM) stimulation were measured on the 5th day after 20 min of forebrain ischemia, and the ratio of stimulated to basal levels or the peak concentration following the stimulation were correlated to neuronal activities. The responses to high potassium stimulation of glutamate and aspartate were reduced to 35-40% of the control values on the 5th day after 20 min ischemia, whereas the responses of gamma-aminobutyric acid (GABA) and taurine were not reduced on the 5th day after the ischemia. These results suggest that excitatory amino acid neurons (glutamatergic and aspartatergic) are more vulnerable than inhibitory amino acid neurons (GABAergic and taurinergic) in the hippocampus. Histologically, hippocampal CA1 pyramidal cells, which are believed to be glutamatergic or aspartatergic, demonstrated a marked neuronal necrosis on the 5th days after 20 min ischemia. Biochemical features revealed by high potassium stimulation may be an expression of 'delayed neuronal death' in the hippocampal CA1 area.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Immunohistochemical distribution of protein kinase C isozymes is differentially altered in ischemic gerbil hippocampus

We used monoclonal antibodies to examine the immunohistochemical distribution of the three major Ca(2+)-dependent protein kinase C (PKC) isozymes (I, II, and III) in ischemic gerbil hippocampus. Groups of four animals were sacrificed at 15 min, 4 h, 1 day, 2 days, 3 days, and 7 days after a 10-min episode of global forebrain ischemia. In control animals, PKC-I immunoreactivity was greater in CA1 neurons than in CA3-4. Terminal-like staining was not evident. PKC-II immunoreactivity was observed in all CA fields and in the outer molecular layer of the dentate gyrus. PKC-III staining was present in the CA fields, the inner molecular layer of the dentate gyrus and the subiculum. Dentate granule cells and mossy fibers were not stained with any of the PKC antibodies. Fifteen minutes and 4 h after ischemia, PCK-I, -II and -III immunoreactivity were all increased in CA1 neurons and PKC-III immunoreactivity alone was visualized in granule cells and mossy fibers. Staining patterns returned to baseline one day after ischemia. PKC-II and -III terminal-like staining were preserved in the stratum lacunosum-moleculare for 3 days and 2 days after ischemia respectively and then disappeared. The altered patterns of PKC staining in the hippocampus may reflect activation and/or down-regulation of PKC isozymes. Ca(2+)-dependent PKC isozymes may, therefore, potentially play a role in the pathogenesis of delayed ischemic neuronal death.     

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus following transient global ischemia

To investigate a potential role of ciliary neurotrophic factor (CNTF) in transient global ischemia, we have studied the postischemic regulatory changes in the expression of CNTF and its receptor, the ligand-binding alpha-subunit (CNTFRalpha). Immunoblot analysis demonstrated CNTF levels were slightly upregulated already during the first day after ischemia and then increased markedly by more than 10-fold until 2 weeks postischemia. Immunoreactivity for CNTF became detectable 1 day after ischemia and was localized in reactive astrocytes. The intensity of the immunolabeling was maximal in CA1 during the phase of neuronal cell death (days 3-7 postischemia) and in the deafferented inner molecular layer of the dentate gyrus. Upregulation of CNTF expression was less pronounced in CA3 and absent in the stratum lacunosum moleculare and the outer molecular layer of the dentate gyrus and thus did not simply correlate with astroliosis as represented by upregulation of glial fibrillary acidic protein (GFAP). As shown by in situ hybridization, expression of CNTFRalpha mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Following ischemia, reactive astrocytes, identified by double labeling with antibodies to GFAP, transiently expressed CNTFRalpha mRNA with a maximum around postischemic day 3. This astrocytic response was most pronounced in CA1 and in the hilar part of CA3. These results show that CNTF and its receptor are differentially regulated in activated astrocytes of the postischemic hippocampus, indicating that they are involved in the regulation of astrocytic responses and the neuronal reorganizations occurring after an ischemic insult.     

脑缺血再灌注后海马CA1区谷氨酸的表达变化及氟桂利嗪的影响

目的　观察暂短性脑缺血再灌注后沙土鼠海马区谷氨酸的表达变化以及氟桂利嗪干预的影响。方法　按照Kirino的方法 ,制作缺血再灌注模型。于缺血再灌注后 1、2、7天采取免疫组化方法检测谷氨酸表达 ,并于 7天在电镜和光镜下观察组织学变化。结果　缺血再灌注组 1天及 2天海马CA1区谷氨酸表达增高 (P <0 .0 1) ,7天恢复正常。光镜及电镜可见给药组存活的神经元数目明显多于缺血再灌注组 (P <0 .0 1)。结论　谷氨酸表达增高可能是鼠脑海马区迟发性神经元死亡的原因之一 ,氟桂利嗪可抑制谷氨酸的表达 ,对缺血的神经元起保护作用。     

Morphological investigation of the neuroprotective effects of graded hypothermia after diverse periods of global cerebral ischemia in gerbils

Hypothermia is known to be the most effective method to protect the neuronal damage induced by ischemia. In the present study, we investigated the histopathological consequences of hippocampal CA1 pyramidal neurons as well as the glial reactions in the hippocampus, after diverse periods of ischemic insult at graded intra-ischemic hypothermia ranging from 32 to 20°C. Gerbils were exposed to forebrain ischemia by clamping the bilateral common carotid arteries for 5–120 min depending upon the temperatures. The morphological study was performed 7 days after ischemia or sham-operation. Histopathological evaluation of delayed neuronal death (DND) was performed by Cresyl violet (CV) staining and MAP2 immunoreactivity. Glial reactions were examined by GFAP immunostaining and isolectin B4 histochemistry, corresponding to astrocytes and microglia, respectively. The forebrain ischemia at 32°C for 10 min and at 28°C for 20 min did not induce DND in the CA1 region. However, the ischemia at 32°C for 20 min and at 28°C for 30 min caused extensive degeneration of CA1 pyramidal neurons as observed in normothermic ischemic animals. Under the condition of deep hypothermia, the ischemia for 60 min at 24°C and for 120 min at 20°C which were the longest durations of each temperature within the limitation of the animal survival following 7 days, induced no DND in CA1 pyramidal neurons. The reactive changes of astrocytes were observed not only in ischemic animals with DND, but also in ischemic animals without DND. Computer image analysis showed that the area fraction of GFAP-positive structures in the CA1 region was significantly increased in both ischemic cases with and without DND compared with each sham group. In contrast, the distribution of activated microglia was much more restricted to the CA1 region and they were always accompanied by DND at 7 days postischemia. The present results demonstrate the remarkable neuroprotective effect of deep hypothermia that has been widely used in cardiovascular surgeries as the cerebroprotective strategy during total circulatory cessation. The findings also suggest that even under the condition of hypothermia, glial reactions may play an important role in neuronal survival and death after ischemia.