Expression of S100 protein and protective effect of arundic acid on the rat brain in chronic cerebral hypoperfusion

S100 protein is expressed primarily by astroglia in the brain, and accumulates in and around the ischemic lesions. Arundic acid, a novel astroglia-modulating agent, is neuroprotective in acute cerebral infarction, whereas the protective effects remain unknown during chronic cerebral hypoperfusion. Rats undergoing chronic cerebral hypoperfusion were subjected to a bilateral ligation of the common carotid arteries, and were allowed to survive for 3, 7 and 14 days. The animals received a daily intraperitoneal injection of 5.0, 10.0 or 20.0 mg/kg of arundic acid, or vehicle, for 14 days. Alternatively, other groups of rats received a delayed intraperitoneal injection of 20.0 mg/kg of arundic acid or vehicle, which started from 1, 3 or 7 days after ligation and continued to 14 days. The degree of white matter (WM) lesions and the numerical density of S100 protein-immunoreactive astroglia were estimated. In the WM of rats with vehicle injections, the number of S100 protein-immunoreactive astroglia increased significantly after chronic cerebral hypoperfusion as compared to the sham-operation. A dosage of 10.0 and 20.0 mg/kg of arundic acid suppressed the numerical increase in S100 protein-immunoreactive astroglia and the WM lesions. These pathological changes were suppressed with delayed treatment up to 7 days in terms of astroglial activation, and up to 3 days in terms of the WM lesions. The protective effects of arundic acid against WM lesions were demonstrated in a dose-dependent manner, and even after postischemic treatments. These results suggest the potential usefulness of arundic acid in the treatment of cerebrovascular WM lesions.     

The role of immunologic reactions in the pathogenesis in Binswanger's disease; a clue to therapeutic approach]

We examined the alterations of glial cells in the brains with Binswanger's disease. In comparison to the brains with lacunar cerebral infarction and those from non-neurological controls, oligodendroglia was decreased in number, and microglia was increased and activated in the white matter lesions in Binswanger's disease. Astroglia occasionally showed a regressive change, termed clasmatodendrosis. In rodents, similar white matter lesions could be induced after chronic cerebral hypoperfusion along with a numerical decrease of oligodendroglia, an increase of astroglia and activation of microglia in the corresponding white matter. These white matter lesions were suppressed by injection of immunosuppressant, cyclosporin A or FK506, with a suppression of microglial activation. These findings indicate that the lesion development in the white matter may be mediated by an immunologic reaction. Unfortunately, these drugs may be inappropriate for a practical use in terms of their side effects. Alternatively, we tried the use of anti-thrombin drug, argatroban. In acute neurological exacerbation of Binswanger's disease, markers for coagulation-fibrinolysis pathway were elevated significantly, and this activation was suppressed along with a recovery of neurological symptoms such as parkinsonism and pseudobulbar palsy. Thrombin contributes to a microcirculatory disturbance and may also enhance an inflammatory response through the action of thrombin receptor.     

Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brains angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.     

Neuroprotection by cilostazol, a phosphodiesterase type 3 inhibitor, against apoptotic white matter changes in rat after chronic cerebral hypoperfusion

In the present study, we elucidated effect of cilostazol to prevent the occurrence of vacuolation and rarefaction of the white matter in association with apoptosis induced by bilateral occlusion of common carotid arteries in the male Wistar rats. Rats orally received vehicle (DMSO) or 60 mg kg(-1) day(-1) (orally) cilostazol for 3, 7, 14 or 30 days. In the vehicle group, increased vacuolation and rarefactions in the white matter were accompanied by extensive activation of both microglial and astroglial cells with suppression of oligodendrocytes in association with increased TNF-alpha production, caspase-3 immunoreactivity and TUNEL-positive cells in the white matter including optic tract. Post-treatment with cilostazol (60 mg kg(-1) day(-1)) strongly suppressed not only elevated activation of astroglia and microglia but also diminished oligodendrocytes following chronic cerebral hypoperfusion. In conclusion, cilostazol (60 mg kg(-1) day(-1), orally) significantly reduced the apoptotic cell death in association with decreased TNF-alpha production and caspase-3-positive cells in the white matter of rat brains subjected to bilateral occlusion of common carotid arteries, consequently ameliorating vacuoles and rarefaction changes in the white matter.     

Cumulative white matter changes in the gerbil brain under chronic cerebral hypoperfusion

Summary An animal model of chronic brain hypoperfusion has been developed by applying coiled clips to the bilateral carotid artery of Mongorian gerbils. The brain tissue damage was neuropathologically studied after 1, 4, 8, and 12 weeks of hypoperfusion. The hippocampus, basal ganglia, and cerebral cortex of the chronically hypoperfused gerbil showed lesions with various severity which are probably due to ischemic episodes. In the cerebral white matter, however, two types of lesions were observed; one similar to those in the gray matter, and the other observed only in the white matter after more than an 8-week duration of brain hypoperfusion. The lesion specific to the white matter showed rarefaction and gliosis without locally associated ischemic changes. This type of the white matter lesion was never found in the gerbil brain before 8 weeks and, significantly, increased in number and size by 12 weeks post operation. The accumulation of the white matter lesions is characteristic in the gerbil with chronic hypoperfusion. The observed white matter-specific lesion resembles the histological changes in aged brain with cerebrovascular diseases.     

Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.     

A cyclooxygenase-2 inhibitor attenuates white matter damage in chronic cerebral ischemia.

The effects of nimesulide, a cyclooxygenase-2 inhibitor, were examined during chronic cerebral hypoperfusion. After bilateral ligation of the common carotid arteries in 30 rats, 21 received dosages of 2 or 5 mg/kg nimesulide daily and nine received vehicle daily for 14 days. The serum was then analyzed biochemically, and pathological changes were estimated in the white matter by the emergence of major histocompatibility complex (MHC) antigen-immunoreactive activated microglia and white matter lesions. In the vehicle-treated animals, activated microglia and white matter lesions were observed. Following treatment with either 2 or 5mg/kg nimesulide, the magnitude of these changes was reduced (p < 0.001) without significant side effects. These results indicate a potential use for cyclooxygenase-2 inhibitors in cerebrovascular disease.     

TUNEL-positive staining in white and grey matter after fatal head injury in man.

Paraffin sections from the hippocampus, the cingulate gyrus and the insula of 18 head-injured patients who survived between 5 hours and 10 days, and 18 age-matched controls, were stained by the terminal deoxynucleotidyl transferase mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL) technique for evidence of in situ DNA fragmentation. Additional staining techniques (HE, combined LFB/CV and immunohistochemistry for GFAP and CD68) were used to characterize any lesions and their time course. Only the occasional TUNEL+ cell per area was seen in the control brains. TUNEL+ cells were identified in both grey and white matter of the head-injured material and their numbers peaked between 24 and 48 hours and were still present at 10 days. Within the hippocampus, fewer TUNEL+ cells were seen in grey (between 3-5 per area) than in the white matter, (up to 51+ per area) whereas in the cingulate gyrus and in the insula, the number of TUNEL+ cells was always greater in the cortex (between 11-20 per area) than in white matter (6-10 per area). In the grey matter, most TUNEL+ cells had the morphology of necrosis. However, the histological appearances of some of the neurons (2-3%), and of oligodendroglia and macrophages in white matter (about 5%) were those of apoptosis.     

氧化应激在慢性缺血性脑白质损伤中的作用

目的 阐明氧化应激是否参与大鼠慢性脑缺血所致的脑白质损伤.方法 健康雄性Wistar大鼠按照完全随机数字表法分为假手术组,持久性双侧颈总动脉结扎3 d组、7 d组、3周组及6周组,每组6只.应用大鼠双侧颈总动脉结扎制备慢性脑缺血模型,检测大鼠脑白质内超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、谷胱甘肽(GSH)含量以及脂质过氧化产物丙二醛(MDA)和4-羟基壬烯醛(4-HNE)加合物的变化.结果 与假手术组比较,慢性脑缺血大鼠脑白质内MDA含量在手术后3周明显增加,手术后6周进一步增高,差异有统计学意义(P<0.05).手术后3d至6周,慢性脑缺血大鼠脑白质内4-HNE蛋白加合物逐渐增高,与假手术组比较有差异有统计学意K(P<0.05).SOD活性在手术后3周和6周才明显降低,与假手术组比较差异有统计学意义(P<0.05).此外,慢性脑缺血大鼠脑白质内GSH含量在手术后7d即开始降低,而在手术后3周及6周则进一步下降,与假手术组比较差异有统计学意义(P<0.05).结论 慢性脑缺血导致大鼠脑白质氧化性损伤增加,抗氧化防御能力降低:氧化性损伤的增加和抗氧化防御能力的降低与慢性脑缺血所致的脑白质损伤密切相关.     

Chronic cerebral hypoperfusion induces MMP-2 but not MMP-9 expression in the microglia and vascular endothelium of white matter.

White matter lesions are closely associated with cognitive impairment and motor dysfunction in the aged. To explore the pathophysiology of these lesions, the authors examined the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the white matter in a rat model of chronic cerebral hypoperfusion. After bilateral clipping of the common carotid arteries, myelin staining revealed demyelinating changes in the optic tract and the corpus callosum on day 7. Zymographic analyses indicated an increase in the level of MMP-2, but not MMP-9, after the hypoperfusion. Immunohistochemical analyses revealed the presence (most abundantly on day 3) of MMP-2-expressing activated microglia in the optic tract and corpus callosum. In contrast, the capillary endothelial cells expressed MMP-2 later. IgM-immunoreactive glial cells were absent in the sham-operated animals, but were present in the hypoperfused animals by day 3, reflecting the disrupted blood-brain barrier. These findings suggest that the main sources of the elevated MMP-2 were the microglia and the endothelium, and that these cells may contribute to the remodeling of the white matter myelin and microvascular beds in chronic cerebral hypoperfusion.     

大鼠局灶性脑缺血再灌流后Caspase-3激活与神经元凋亡的关系

目的探讨Caspase-3与局灶性脑缺血后神经元凋亡的关系。方法局灶性脑缺血再灌流大鼠模型,按再灌流时间不同随机分为5组。用半定量RT-PCR观察了脑缺血后不同时程Caspase-3mRNA表达及四肽荧光底物法检测Caspase-3蛋白酶活性;用TUNEL方法检测不同时程细胞凋亡。结果脑缺血2h再灌流2h组Caspase-3mRNA水干即已升高(P＜0.05)),蛋白酶活性轻度升高,再灌流6h后两者均明显升高(P＜0.05);脑缺血再灌流后不同时程细胞凋亡具有与Caspase-3相似的变化趋势。结论提示脑缺血后细胞凋亡的发生与Caspase-3的激活有关。     

Cerebral white matter contains PDGF-responsive precursors to O2A cells

Cells dissociated from the cerebral white matter of immature rats were maintained in monolayer culture. Treatment with platelet-derived growth factor (PDGF) caused a large increase in the numbers of "O2A" oligodendroglial precursor cells (which bind the monoclonal antibody A2B5) and subsequently in the numbers of galactocerebroside (galC)-positive oligodendroglia. A2B5-negative "pre-O2A cells" in cerebral white matter cultures in which O2A cells and oligodendroglia had been killed by antibody-dependent complement-mediated cytolysis were induced by PDGF to proliferate and to differentiate into O2A cells and subsequently into oligodendroglia and type 2 astroglia. The most mature pre-O2A phenotype in these cultures was a small, round, process-bearing cell which expressed vimentin but not glial fibrillary acidic protein or galC. Cells of this phenotype were not observed upon PDGF treatment of immature rat optic nerve monolayer cultures from which O2A cells and oligodendrocytes had been depleted, and PDGF also failed to elicit the accumulation of O2A cells and oligodendroglia in such cultures.     

Leukoencephalopathy in diffuse hemorrhagic cerebral amyloid angiopathy

We have studied 12 patients with diffuse hemorrhagic cerebral amyloid angiopathy clinically and at postmortem examination. The brains in 8 patients had diffuse bilateral loss of myelin in the hemispheric white matter sparing the U fibers, corpus callosum, and internal capsules. The periventricular areas were predominantly affected. Microscopic examination of the white matter showed an association with subacute or chronic edematous lesions: spongiosis, swollen oligodendroglia, widening of the perivascular spaces with edema fluid or siderophages, hyalinization of the blood vessel walls, incomplete myelin loss, and astrocytic gliosis. Three of 8 autopsied patients had undergone computed tomographic examination, which showed bilateral hypodensity of the hemispheric white matter. The brains of 4 patients with illnesses of shorter duration showed only discrete but similar lesions in the centrum semiovale. These white matter changes are similar to those observed in Binswanger's subcortical encephalopathy. We suggest that a common mechanism of hypoperfusion of the distal white matter causes the leukoencephalopathy.     

Astrogliosis in EAE spinal cord: derivation from radial glia, and relationships to oligodendroglia

A prominent feature of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is the accumulation of enlarged, multipolar glial fibrillary acidic protein (GFAP) and brain lipid binding protein (BLBP) immunoreactive astroglia within and at the margins of the inflammatory demyelinative lesions. Whether this astrogliosis is due to both astroglial hyperplasia and hypertrophy or solely to astroglial hypertrophy is controversial. We now report that coincident with the first appearance of inflammation and clinical deficits in mice with myelin oligodendrocyte glycoprotein peptide (MOG peptide)-induced EAE, the radially oriented, bipolar, GFAP, and BLBP positive cells (adult radial glia) present in normal spinal cord white matter undergo mitosis and phenotypic transformation to hypertrophic astroglia. To facilitate visualization of relationships between these hypertrophic astroglia and dying and regenerating oligodendroglia, we used mice that express enhanced green fluorescent protein (EGFP) in cells of the oligodendroglial lineage. During the first week after onset of illness, markedly swollen EGFP+ cells without processes were seen within lesions, whereas EGFP+ cells that expressed immunoreactive cleaved caspase-3 were uncommon. These observations support the hypothesis that necrosis contributes to oligodendroglial loss early in the course of EAE. Later in the illness, EGFP+ cells accumulated amongst hypertrophic astroglia at the margins of the lesions, while the lesions themselves remained depleted of oligodendroglia, suggesting that migration of oligodendroglial lineage cells into the lesions was retarded by the intense perilesional gliosis.     

Dose-dependent, protective effect of FK506 against white matter changes in the rat brain after chronic cerebral ischemia

Neuroprotective effects of immunosuppressive agents have been shown in cerebral ischemia. To investigate the role of immunosuppressive agents in chronic cerebral ischemia and to design a drug protocol with safe therapeutic windows, we examined the effects of FK506, a potent immunosuppressive agent, on chronic cerebral ischemia. Both common carotid arteries were ligated in 73 male Wistar rats. Fifty-eight of these rats received a chronic injection of FK506 (0.2, 0.5, 1.0 mg/kg) and the remaining 15 received a vehicle solution injection. Microglia/macrophage was investigated with immunohistochemistry for leukocyte common antigen and major histocompatibility complex, and astroglia was examined with glial fibrillary acidic protein as markers. White matter rarefaction and the number of immunopositive glial cells were assessed from 7 to 30 days after the ligation. In the vehicle-treated animals, there was persistent and extensive activation of the microglia/macrophages and astroglia in the white matter, including the optic nerve, optic tract, corpus callosum, internal capsule, anterior commissure and traversing fiber bundles of the caudoputamen. In the FK506-treated rats, the number of activated microglia/macrophages was significantly reduced in a dose-dependent manner (p<0.01) as compared to the vehicle-treated rats. Rarefaction of the white matter was also inhibited by FK506 in a dose-dependent manner (p<0.01). Thus, a clinically-relevant dosage of FK506 attenuated both glial activation and white matter changes in chronic cerebral ischemia in the rat. These results indicate a potential use for FK506 in cerebrovascular diseases.     

MCP-1-mediated activation of microglia promotes white matter lesions and cognitive deficits by chronic cerebral hypoperfusion in mice

Microglia activation played a vital role in the pathogenesis of white matter lesions (WMLs) by chronic cerebral hypoperfusion. In addition, hypoxia induced up-regulated expression of MCP-1, promotes the activation of microglia. However, the role of MCP-1-mediated microglia activation in chronic cerebral ischemia is still unknown. To explore that, chronic cerebral hypoperfusion model was established by permanent stenosis of bilateral common carotid artery in mice. The activation of microglia and the related signal pathway p38MAPK/PKC in white matter, and working memory of mice were observed. We found that stenosis of common carotid arteries could induce MCP-1-mediated activation of microglia through p38MAPK/PKC pathway and white matter lesions. Taken together, our findings represent a novel mechanism of MCP-1 involved in activation of microglia and provide a novel therapeutical strategy for chronic cerebral hypoperfusion.     

Phosphodiesterase III inhibition promotes differentiation and survival of oligodendrocyte progenitors and enhances regeneration of ischemic white matter lesions in the adult mammalian brain

Vascular dementia is caused by blockage of blood supply to the brain, which causes ischemia and subsequent lesions primarily in the white matter, a key characteristic of the disease. In this study, we used a chronic cerebral hypoperfusion rat model to show that the regeneration of white matter damaged by hypoperfusion is enhanced by inhibiting phosphodiesterase III. A rat model of chronic cerebral hypoperfusion was prepared by bilateral common carotid artery ligation. Performance at the Morris water-maze task, immunohistochemistry for bromodeoxyuridine, as well as serial neuronal and glial markers were analyzed until 28 days after hypoperfusion. There was a significant increase in the number of oligodendrocyte progenitor cells in the brains of patients with vascular dementia as well as in rats with cerebral hypoperfusion. The oligodendrocyte progenitor cells subsequently underwent cell death and the number of oligodendrocytes decreased. In the rat model, treatment with a phosphodiesterase III inhibitor prevented cell death, markedly increased the mature oligodendrocytes, and promoted restoration of white matter and recovery of cognitive decline. These effects were cancelled by using protein kinase A/C inhibitor in the phosphodiesterase III inhibitor group. The results of our study indicate that the mammalian brain white matter tissue has the capacity to regenerate after ischemic injury.     

FK506 treatment inhibits caspase-3 activation and promotes oligodendroglial survival following traumatic spinal cord injury

The focus of this study is to examine the ability of FK506, an immunosuppressant that inhibits calcineurin activation, to limit caspase-3 activation in oligodendroglia following spinal cord injury (SCI). To better establish a role for calcineurin and caspase-3 activation in oligodendroglia following SCI, rats received a contusion injury to the spinal cord followed by treatment with FK506 or rapamycin (another immunosuppressant with no detectable inhibitory action on calcineurin activation). Animals were then sacrificed at 8 days postinjury and spinal cord tissue was processed using immunofluorescence histochemistry to examine cellular caspase-3 activation in ventral and dorsal white matter. In all treatment groups, numerous oligodendroglia were found to express the activated form of caspase-3 in regions proximal and distal to the injury epicenter. However, our findings suggest that treatment with FK506, but not rapamycin reduces the number of oligodendroglia expressing activated caspase-3 and increases the number of surviving oligodendroglia in dorsal white matter. These results provide initial evidence that agents that reduce the actions of calcineurin and subsequent caspase-3 activation may prove beneficial in the treatment of traumatic SCI.     

TUNEL-positive staining of surface contusions after fatal head injury in man

In frontal lobe contusions obtained post mortem from 18 patients who survived between 6 h and 10 days after head injury, DNA fragmentation associated with either apoptotic and/or necrotic cell death was identified by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL) histochemical technique. Additional histological techniques were also used to identify regional and temporal patterns of tissue damage. TUNEL-positive cells were present in both the grey and white matter of the contusion, where they peaked in number between 25 and 48 h, and were still identifiable at 10 days post injury. Fewer TUNEL-positive cells were observed in grey than in white matter; and most TUNEL-positive neurons in the grey matter demonstrated the morphological features of necrosis. However, the morphology of some TUNEL-stained neurons, and of TUNEL-stained oligodendroglia and macrophages in white matter was suggestive of apoptosis. Apoptosis was not seen in age- and sex-matched controls, none of whom had died from intracranial pathology or had pre-existing neurological disease. These findings suggest that multiple cell types in frontal lobe contusions exhibit DNA fragmentation and that both necrosis and apoptosis are likely to contribute to post-traumatic pathology. These findings provide further evidence that the observations made in animal models of traumatic brain injury have fidelity with clinical head injury. Received: 25 November 1999 / Revised: 3 February 2000 / Accepted: 9 February 2000     

Blood-brain barrier disruption in white matter lesions in a rat model of chronic cerebral hypoperfusion.

Blood-brain barrier damage has been implicated in the pathogenesis of cerebrovascular white matter lesions. This type of lesion is responsible for cognitive impairment in the elderly and can be induced by permanent ligation of the bilateral common carotid arteries in the rat. Because it is unclear whether the blood-brain barrier is impaired, we examined whether vascular permeability to horseradish peroxidase is altered using this model. According to light microscopic results, the reaction product of horseradish peroxidase was most intensely localized to the paramedian part of the corpus callosum in the brain, occurring to a small degree at 3 hours, day 1, markedly on day 3, but reduced on days 7 and 14. By electron microscopic study of the same area, the reaction product of horseradish peroxidase was localized to the plasmalemmal vesicles in the endothelial cells 3 hours after ligation, but appeared in the cytoplasm on days 1 and 3, suggesting a diffuse leakage of horseradish peroxidase. In addition, the reaction product was dispersed into the cytoplasm of glial cells in the perivascular regions on day 3. The luminal surface of the endothelial cell cytoplasm appeared irregular on day 7, suggesting a conformational change of the endothelial cells. Collagen fibrils proliferated in the thickened basal lamina and mitochondria degenerated in the pericyte on days 7 and 14. Perivascular glial endfeet were swollen throughout the survival period. In sham-operated rats, the reaction product of horseradish peroxidase was not observed at any time interval, except in vesicular structures. These findings indicate that chronic cerebral hypoperfusion induces blood-brain barrier damage with subsequent morphologic changes of the vascular structures in the corpus callosum. An extravasation of macromolecules, such as proteases and immunoglobulins, may contribute to the pathogenesis of white matter lesions.