Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil

Prompt dendritic damage has been observed in the hippocampus of the gerbil brain after transient cerebral ischemia. In the present study, we studied the frontoparietal cortex of the gerbil brain electron microscopically after brief bilateral carotid occlusion to assess the vulnerability of dendritic processes. After ischemia for 5 min, there was swelling of the periphery of dendrites accompanied by swelling of mitochondria, cytoplasmic vacuolation and disintegration of microtubules in layer I, which spread to layer III after ischemia for 20 min. After reperfusion for 3-24 h following ischemia for 20 min, swelling in the periphery of dendrites and of mitochondria inside receded but vacuole formation and disintegration of microtubules propagated proximally. In neuronal perikarya, polyribosomal disaggregation was observed after ischemia for 20 min and persisted thereafter, while fragmentation of rough endoplasmic reticulum (ER) and microvacuolation occurred after reperfusion for 3 h. Electron-dense clumping of neuronal perikarya was observed after reperfusion for 6 h particularly in layers III and Vb, which increased in number for up to 72 h. The observed progressive damage in dendrites may be common to neurons vulnerable to cerebral ischemia and may significantly contribute to development of delayed neuronal death.     

Immunoelectron microscopic investigation of creatine kinase BB-isoenzyme after cerebral ischemia in gerbils

Alteration of creatine kinase BB-isoenzyme (CK-BB) was investigated in the vulnerable CA1 region of the hippocampus of ischemic and postischemic gerbil brains using immunoelectron microscopy. CK-BB existed in the neuronal perikarya, dendrites and axons as well as in astroglias in the normal gerbil brain. Immunocytochemical reaction products were associated with microtubules and polyribosomes. Propagation of ischemic and postischemic damage with disintegration of microtubules was observed in the dendro-somatic direction in neurons, which progressed in parallel with dispersion and loss of the immunocytochemical reaction for CK-BB in the dendroplasm. After reperfuson for longer than 24 h, CK-BB was also observed in the extracellular space. The present result supported the notion that loss of the immunohistochemical reaction for CK-BB which has been observed by light microscopy after cerebral ischemia, was at least partly due to dispersion of this enzyme caused by disintegration the mirotubules and extracellular leakage of this enzyme, although other processes, including degradation of CK-BB per se, were also possible. The loss of CK-BB from the neuronal structure may delay the recovery from ischemic damage and may eventually lead to neuronal death.The present investigation was supported by the grant NS-06663 from the National Institute of Health, U.S. Public Health Services     

Immunoelectron microscopic study of tubulin and microtubule-associated proteins after transient cerebral ischemia in gerbils

Summary Differential vulnerability of microtubule components to cerebral ischemia has been reported previously. We investigated the disintegration of microtubules using immunoelectron microscopy for -tubulin and microtubule-associated protein 1A and 2 (MAP1A and 2). Mongolian gerbils were subjected to bilateral carotid occlusion for 10 to 30 min and reperfusion for up to 72h following ischemia for 10 min. After ischemia for 10 min, some dendrites in the stratum moleculare of the subiculum-CA1 region lost immunoreaction products for -tubulin and MAPs. Loss of the reaction products spread to the medial CA1 region during progressive ischemia for 30 min. In some dendrites, electron-dense precipitates for MAPs were dispersed in the dendritic cytoplasm with little reaction product on microtubules and without alteration of the reaction for -tubulin. After recirculation, loss of electron-dense precipitates for -tubulin and MAPs, as well as disintegration of microtubules, propagated further to the medial CA1 region and to the proximal dendrites. The present study demonstrated prompt disintegration of microtubules with rapid disappearance of the reaction for MAPs which seemed to be caused by detachment of MAPs from the microtubule cores.     

Ultrastructural investigation of the CA1 region of the hippocampus after transient cerebral ischemia in gerbils

Summary Ultrastructural damage leading to delayed neuronal death was investigated in the mid-CA1 region of the hippocampus from the stratum (str.) moleculare to oriens after transient bilateral forebrain ischemia in Mongolian gerbils. After ischemia for 5 min without recirculation, mild swelling of the peripheral part of the apical and basal dendrites was already apparent in the str. moleculare and str. oriens. Mitochondria in the dendrites were also swollen in the same area. During recirculation for 12 h to 3 days, swelling of the dendritic cytoplasm persisted with formation of microvacuoles, but swelling of mitochondria receded. Microvacuolation and loss of microtubules were also observed in the proximal part of the dendrites during this period, and swelling and disruption of internal cristae were observed in mitochondria after recirculation for 3 days. The dendrites became severely degenerated after recirculation for 4 days. In the pyramidal cell bodies, no abnormality was observed at the end of ischemia for 5 min, but disaggregation of polyribosomes and swelling of the endoplasmic reticulum were observed 12 h after recirculation. Proliferation of the endoplasmic reticulum in parallel arrays occurred after recirculation for 1 day and persisted. Severe degeneration of the pyramidal cell bodies was obvious after recirculation for 4 days. The findings observed in the present investigation suggested that the neuronal structure most vulnerable to ischemia was the peripheral part of the dendrites and postischemic neuronal damage occurred early in this part of the dendrites.Supported by the grant NS-06663 from the National Institutes of Health, U.S. Public Health Service     

Temporal profile of serum albumin extravasation following cerebral ischemia in a newly established reproducible gerbil model for vasogenic brain edema: a combined immunohistochemical and dye tracer analysis

Summary We investigated the temporal profile of the extravasation of serum albumin in a reproducible gerbil model of unilateral cerebral ischemia, using immunohistochemical and dye-tracer techniques to evaluate albumin accumulation and the occurrence of active extravasation, respectively. After 30 min of cerebral ischemia and subsequent reperfusion, immunostaining for albumin became visible in the lateral part of the thalamus during the first 3 h, and then expanded to other brain regions up to 24 h. At both 24 h and 3 days after reperfusion, massive extravasation of albumin was noted in the whole ischemic hemisphere, and this had decreased again by 7 days after reperfusion. The extent and the degree of albumin immunopositivity were almost the same in all animals examined at each period after reperfusion. The extravasation of Evans blue, which was allowed to circulate for 30 min before death, was limited to the lateral part of the thalamus during the first 6 h of reperfusion. In the circumscribed area of massive albumin extravasation, many neurons were immunopositive for albumin; most of these neurons appeared to be intact and also showed immunostaining for microtubule-associated protein 2. The current investigation clearly demonstrated that (1) albumin extravasation was produced with reliable reproducibility in this model, (2) the lateral part of the thalamus was the region most vulnerable to ischemic blood-brain barrier damage, and (3) many apparently intact neurons in the ischemic region were positive for albumin.Supported in part by a Grant-in-aid (01570485) from the Ministry of Education, Science and Culture and by a research grant for cardiovascular diseases (2A-2) from the Ministry of Health and Welfarc in Japan     

Sequential alteration of [H]rolipram binding in gerbil brain after transient cerebral ischemia

The time course of rolipram (Ca²⁺/calmodulin independent cyclic adenosine monophosphate inhibitor) binding sites changes following gerbil transient forebrain ischemia was determined using receptor autoradiography. Gerbils subjected to 10-min ischemia revealed a significant reduction in rolipram binding in most selectively vulnerable regions early in the recirculation (1–5 h). Marked reduction in the rolipram binding was seen in the selectively vulnerable areas 48 h or 7 days after ischemia. Thereafter, the rolipram binding in the hippocampal CA1 and CA3 sectors, which were most vulnerable to ischemia, was severely reduced up to 1 month after recirculation. In contrast, the reduction of the rolipram binding activity in other regions recovered to sham-operated level or showed a slight recovery. Interestingly, the dentate gyrus, which was resistant to ischemia, also exhibited a significant reduction of the rolipram binding activity up to I month after ischemia. Eight months after ischemia, the hippocampal CA 1 and CA3 sectors showed severe shrinkage and marked reduction in the rolipram binding. Other regions exhibited no significant reduction in the rolipram binding except for a slight reduction in the thalamus. These results demonstrate that transient cerebral ischemia causes severe reduction in rolipram binding sites in selectively vulnerable areas, and this reduction precedes the neuronal cell loss. These findings may reflect the alteration of an intracellular phosphodiesterase activity after ischemia.     

白细胞在实验性脑缺血中作用及中药川芎嗪和丹皮酚对其影响的研究

目的　研究沙鼠脑缺血后再灌注期周围血白细胞 (WBC)、脑实质中小胶质细胞和WBC数的变化规律及中药川芎嗪、丹皮酚对其的影响。　　方法　建立沙鼠脑缺血及再灌注模型 ,取周围血行WBC计数 ,脑组织切片在光镜下行WBC及小胶质细胞计数。　　结果　周围血WBC数在再灌注 4h升高 ,至 1 2h达到高峰 ,以中性粒细胞 (NC)增多为主 ;脑实质中小胶质细胞于再灌注 4h达到高峰 ,WBC在再灌注 2 4h增多最明显 ;川芎嗪、丹皮酚均可降低脑缺血再灌注后增加的周围血WBC数 ,亦能使脑实质中小胶质细胞和WBC数明显减少 ,　　结论　白细胞、小胶质细胞在脑缺血时明显增多 ,丹皮酚、川芎嗪能减少增多的白细胞和小胶质细胞。     

The highly selective cyclooxygenase-2 inhibitor DFU is neuroprotective when given several hours after transient cerebral ischemia in gerbils

Several studies suggest that cyclooxygenase-2 contributes to the delayed progression of ischemic brain damage. In this study we examined whether the highly selective cyclooxygenase-2 inhibitor DFU reduces neuronal damage when administered several hours after 5 min of transient forebrain ischemia in gerbils. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity and by histopathological evaluation of the extent of CA1 hippocampal pyramidal cell injury 7 days after ischemia. DFU treatment (10 mg/kg, p.o.) significantly reduced hippocampal neuronal damage even if the treatment is delayed until 12 h after ischemia. These results suggest that selective cyclooxygenase-2 inhibitors may be a valuable therapeutic strategy for ischemic brain injury.     

Three‐dimensional structural changes in cerebral microvessels after transient focal cerebral ischemia in rats: Scanning electron microscopic study of corrosion casts

Pathological changes of cerebral microvessels in transient ischemia were investigated by scanning electron microscopy of vascular corrosion casts. Wistar rats were treated with middle cerebral artery (MCA) occlusion for 30 min, 1 h, 3 h, 4 h, 5 h or 7 h and subsequent reperfusion for 2 h. The ultrastructures of the cast were observed and computer‐aided montage micrographs were obtained for visualization of the whole microvasculature in the ischemic brain hemisphere. Avascular areas representing ischemic areas were detected in the frontotemporal cortex and caudate putamen in the groups from 30 min to 5 h occlusion. Extravasation of the resin, which probably corresponded to the leakage of plasma or hemorrhage, was seen as spheroidal, conglomerative, large massive and worm‐like types. The spheroidal type, which probably indicated a small leakage or minor hemorrhage, began to appear in the 30‐min occlusion group. The conglomerative type, which probably indicated a larger leakage or moderate hemorrhage, appeared in the 3‐ to 5‐h occlusion groups. The large massive and worm‐like types, which probably indicated a significant hemorrhage, appeared in the 4‐ and 5‐h occlusion groups. The number of these extravasations increased significantly in the 4‐h occlusion group. Arterioles near the avascular area frequently showed vasospastic appearances, such as corrugations, fusiform indentations of endothelial nuclei, continuous circulatory constrictions and severe narrowing with interrupted branches. Arteriolar vasospasm possibly caused prolonged hypoperfusion even if reperfusion was achieved. The capillaries had a thin stringy appearance in the 4‐ and 5‐h occlusion groups. These changes seemed to relate closely with increased intracranial pressure by brain edema or hemorrhage. The present study suggested that the risk of brain edema or hemorrhagic infarction increased beyond 3 h of MCA occlusion, and vasospasm of the arterioles might participate in stroke pathophysiology.     

Ornithine decarboxylase in reversible cerebral ischemia: an immunohistochemical study

Summary Anesthetized Mongolian gerbils were subjected to 5-min ischemia and 8 h of recirculation. Vibratiom sections were taken for studying changes in ornithine decarboxylase (ODC) immunoreactivity using an antiserum to ODC, and tissue samples were taken for measuring ODC activity. After 5-min ischemia and 8-h recirculation ODC activity increased 11.5-, 5.9-, and 7.9-fold in the cerebral cortex, striatum and hippocampus, respectively (P0.05 to 0.01). In the cortex, striatum and hippocampus of control animals immunoreactivity was low but clearly above the detection limit. The reaction was confined to neurons. After 5-min ischemia and 8-h recirculation a sharp increase in immunoreactivity was observed confined to neurons, indicating that the postischemic activation of polyamine metabolism is a neuronal response to ischemia. The immunoreactivity was markedly increased in the perinuclear cytoplasm and the dendrites. In the striatum the density of neurons exhibiting a sharp increase in immunoreactivity was more pronounced in the lateral than in the ventral part. In the hippocampus a strong reaction was present in all subfields but the CA1 subfield was particularly affected. The present study demonstrates for the first time that biosynthesis of a protein is markedly activated during the first 24 h of recirculation after 5-min cerebral ischemia of gerbils even in the vulnerable CA1 subfield, in which the overall protein synthesis is sharply reduced at the same time. Studying polyamine metabolism after ischemia may, thus, provide new information about the basic molecular mechanisms responsible for the altered gene expression after metabolic stress.Supported by the Deutsche Forschungsgemeinschaft, Grant Pa 266/3-2, and by grants provided by the National Research Council for Natural Sciences, Academy of Finland and the Cancer Society of Finland.     

沙土鼠脑缺血和再灌流期间羟自由基的变化及氯胺酮对其影响

目的 研究脑缺血和再灌注期间的羟自由基变化及氯胺酮对其影响。方法 制作沙土鼠前脑缺血再灌注模型,脑缺血１０分钟,再灌流６０分钟,分为手术组、缺血组、血再灌流组和氯胺酮组。应用高效液相色谱测定纹状体和海马羟自由基（ＯＨ）三磷酸腺苷（ＡＴＰ）和多巴胺（ＤＡ）含量。结果 海马二羟基苯（２,３－ＤＨＢＡ）一缺血组和缺血再灌流组均明显高于假手术组;缺血再灌注组２,３－ＤＨＢＡ的含量高于缺血组;脑缺血和再灌注     

Differential vulnerability of microtubule components in cerebral ischemia

Summary Differential vulnerability of the major components of microtubules was examined in ischemic gerbil brains by a light microscopic, immunohisto-chemical method using monoclonal antibodies for microtubule-associated protein (MAP) 1A and MAP2, polyclonal antibody for MAP1 and 2 as well as monoclonal antibody for -tubulin. Progressive cerebral ischemia during unilateral carotid occlusion for 5, 15 and 120 min and reperfusion for 3, 12 and 48 h following bilateral carotid occlusion for 10 min were studied. Ischemic lesions in the subiculum-CA1 region were visualized by all antibodies after ischemia for 5 min but the antibody for -tubulin was less sensitive. The antibody for -tubulin was also less sensitive than antibodies for MAPs for detection of early postischemic lesions. Differential sensitivity was also observed in the cerebral cortex and other brain regions. Microtubules in myelinated axons were more stable than those in dendrites. The observed loss of immunohistochemical reactivities for MAPs and -tubulin may have been caused by activation of calcium-dependent proteolytic enzymes such as calpains. The discrepancy between MAPs and -tubulin could be due to differences in affinities or topographic distributions of these proteins within microtubules.Supported by the grant NS-06663 from the National Institutes of Health, U.S. Public Health Service     

大鼠短暂局灶性脑缺血再灌注后核转录因子-kB的表达

目的　研究核转录因子 - k B(NF- k B)在局灶性脑缺血再灌注中的动态表达规律及其作用。方法　采用线栓法建立大鼠局灶性脑缺血再灌注模型。应用细胞免疫组织化学法分析 NF- k B的移位 ,采用 Western- blot法检测脑组织中核 NF- k B的表达量。结果　局灶性脑缺血再灌注后 NF- k B明显从细胞浆移位于细胞核 ,核 NF- k B的表达量显著增加 (P<0 .0 1)。结论　局灶性脑缺血再灌注能够引起 NF- k B的表达增加 ,进一步产生炎症和免疫反应 ,从而参与了脑缺血再灌注损伤的发病机制     

缺血预处理对沙土鼠脑缺血再灌注后线粒体功能的影响

目的 观察缺血预处理对沙土鼠脑缺血及再灌注后脑组织线粒体功能的影响。方法 用沙土鼠双侧颈总动脉结扎制成全脑缺血模型（缺血２０分钟,再灌注３０分钟）。动物随机分为预缺血组、模型组和假手术组。预缺血组给予两次（各２分钟）缺血预处理（间隔４８小时）。用氧电极法测定呼吸功能和呼吸链的氧化酶（ＮＡＤＨ氧化酶、琥珀酸氧化酶、细胞色素Ｃ氧化酶）活性。结果 缺血模型组动物的呼吸控制率、磷氧比及氧化磷酸化效率均较假     

大鼠短暂性脑缺血后葡萄糖转运子3表达变化

目的 研究大鼠短暂性脑缺血葡萄糖转运子3（GLUT3）转录水平表达规律。方法 用插线法建立大鼠短暂性脑缺血模型。剥取缺血半暗带及中心区皮质组织,采用半定量逆转录-聚合酶链式反应（RT-PCR）,测定不同再灌注时间GLUT3mRNA水平的变化。结果 再灌注3h缺血半暗区GLUT3mRNA3h升高,48h达到高峰,再灌注1周后仍高于正常。结论 再灌注后,缺血半暗带GLUT3的表达明显上调,有可能是机体抗损伤性反应。     

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。     

Electron microscopic study of the gerbil dentate gyrus after transient forebrain ischemia

Summary Silver impregnation performed 1–2 days after transient forebrain ischemia in the Mongolian gerbil demonstrated terminal-like granular deposits in the outer two-thirds of the hippocampal dentate molecular layer (perforant path terminal zone), even though neither the cell bodies of origin of the perforant path nor the dentate granule cells were destroyed. Electron microscopic studies of the dentate gyrus were performed in an effort to discover the identity of these degenerating structures. Electron microscopy revealed that the granular silver deposits corresponded to electron-dense profiles. Many of these were degenerating boutons and some were degenerating postsynaptic dendritic fragments, but most of them could not be identified with certainty. Electron-dense profiles were less numerous than expected from the density of granular silver deposits. These structures were probably the degenerating axons, axon terminals and dendrites of CA4 neurons. The granular silver deposits and electron-dense boutons observed in the inner third of the dentate molecular layer 5 days after transient ischemia can probably be explained by the ischemia-induced degeneration of CA4 mossy cells, which give rise to the dentate associational-commissural projection. Finally, most mossy fiber boutons in area CA4 and some boutons in the molecular layer appeared watery and enlarged on postischemia days 1 and 2. Mossy fiber boutons with this ultrastructural appearance have previously been observed in seizure-prone animals and in animals undergoing convulsant-induced seizures. Although no postischemic seizures occur under the conditions of this study, these findings support the idea that excitatory pathways become hyperactive after transient ischemia.Supported by NIH Stroke Center grant NS 06233     

An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia

Changes in MAP2 and clathrin immunoreactivity were studied in gerbil hippocampus after transient cerebral ischemia. MAP2 immuno-reactivity decreased significantly by 1 h in the subiculum-CA1 and CA2 areas which correspond to reactive change, while no decrease was observed in CA1 until day 4. Before the initiation of delayed neuronal death, MAP2 immunoreactivity was not changed in CA1. On the other hand clathrin immunoreactivity increased in the pyramidal cell layer of CA1 by 3 h after ischemia and remained high for 2 days. Clathrin immunoreactivity in the pyramidal cell layer of CA1 diminished after delayed neuronal death. The transient change of clathrin was noted especially in CA1 in the period prior to delayed neuronal death. These results imply an abnormal change in clathrin turnover after ischemia, which may participate in the pathogenesis of delayed neuronal death.     

Induced hypertension treatment to improve cerebral ischemic injury after transient forebrain ischemia

The effect of induced hypertension treatment on cerebral ischemia is still controversial. We investigated the preferred blood pressure manipulation level and pressor agent required to reduce cerebral ischemic injury following transient forebrain ischemia induced by bilateral occlusion of the common carotid arteries in anesthetized gerbils. Following 60-min cerebral ischemia, we evaluated the preferred blood pressure manipulation level and pressor agent required to treat cerebral ischemic injury after reperfusion by examining the effects of different levels of mean arterial blood pressure (MABP), increased with phenylephrine or angiotensin II or decreased by blood withdrawal, on cerebral blood flow (CBF), survival ratio, cerebral edema, and brain energy metabolism following transient forebrain ischemia in gerbils. Mild phenylephrine-induced hypertension treatment (21+/-4 mmHg) during post-cerebral ischemia-reperfusion improved the survival ratio and reduced cerebral edema, which was also associated with an increase in local CBF and a recovery of brain energy metabolism. However, intense phenylephrine-induced hypertension, angiotensin II-induced hypertension, or hypotension worsen the survival rate and produced extra cerebral edema, that were also associated with deterioration of brain energy metabolism. These results demonstrate that a mild induced hypertension with phenylephrine (21+/-4 mmHg above the baseline level) results in reduction of the cerebral edema and improves the survival ratio and brain energy metabolism. Furthermore, angiotensin II may have neurotoxic effect to use as the pressor agent for induced hypertension after cerebral ischemia.     

Hyperbaric oxygen reduces basal lamina degradation after transient focal cerebral ischemia in rats

Hyperbaric oxygen (HBO) has been shown to preserve the integrity of the blood-brain barrier after cerebral ischemia. However, the underlying molecular mechanisms are currently unknown. We examined the effect of HBO on postischemic expression of the basal laminar component laminin-5 and on plasma matrix metalloproteinase-9 (MMP) levels. Wistar rats underwent occlusion of the middle cerebral artery (MCAO) for 2 h. With a delay of 45 min after filament introduction, animals breathed either 100% O2 at 1.0 atmosphere absolute (ata; NBO) or at 3.0 ata (HBO) for 1 h in an HBO chamber. Laminin-5 expression was quantified on immunohistochemical sections after 24 h of reperfusion. Plasma MMP-9 levels were measured using gelatin zymography before MCAO as well as 0, 6 and 24 h after reperfusion. Immunohistochemistry 24 h after ischemia revealed a decrease of vascular laminin-5 staining in the ischemic striatum to 43 +/- 26% of the contralateral hemisphere in the NBO group which was significantly attenuated to 73 +/- 31% in the HBO group. Densitometric analysis of zymography bands yielded significantly larger plasma MMP-9 levels in the NBO group compared to the HBO group 24 h after ischemia. In conclusion, HBO therapy attenuates ischemic degradation of cerebral microvascular laminin-5 and blocks postischemic plasma MMP-9 upregulation.