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.     

大鼠脑缺血再灌注后星形胶质细胞反应性变化差异与海马CA1区的迟发性神经元死亡

BACKGROUND： Blood supply to the hippocampus is not provided by the middle cerebral artery. However, previous studies have shown that delayed neuronal death in the hippocampus may occur following focal cerebral ischemia induced by middle cerebral artery occlusion.
OBJECTIVE： To observe the relationship between reactive changes in hippocampal astrocytes and delayed neuronal death in the hippocampal CA1 region following middle cerebral artery occlusion.
DESIGN, TIME AND SETTING： The immunohistochemical, randomized, controlled animal study was performed at the Laboratory of Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from July to November 2007.
MATERIALS： Rabbit anti-glial fibrillary acidic protein （GFAP） （Neomarkers, USA）, goat anti-rabbit IgG （Sigma, USA） and ApoAlert apoptosis detection kit （Biosciences Clontech, USA） were used in this study. METHODS： A total of 42 healthy adult male Wistar rats, aged 3–5 months, were randomly divided into a sham operation group （n = 6） and a cerebral ischemia/reperfusion group （n = 36）. In the cerebral ischemia/reperfusion group, cerebral ischemia/reperfusion models were created by middle cerebral artery occlusion. In the sham operation group, the thread was only inserted into the initial region of the internal carotid artery, and middle cerebral artery occlusion was not induced. Rats in the cerebral ischemia/reperfusion group were assigned to a delayed neuronal death （＋） subgroup and a delayed neuronal death （–） subgroup, according to the occurrence of delayed neuronal death in the ischemic side of the hippocampal CA1 region following cerebral ischemia.
MAIN OUTCOME MEASURES： Delayed neuronal death in the hippocampal CA1 region was measured by Nissl staining. GFAP expression and delayed neuronal death changes were measured in the rat hippocampal CA1 region at the ischemic hemisphere by double staining for GFAP and TUNEL.
RESULTS： After 3 days of ischemia/reperfusion, astrocytes with abnormal morphology were detected in the rat hippocampal CA1 region in the delayed neuronal death （＋） subgroup. No significant difference in GFAP expression was found in the rat hippocampal CA1 region at the ischemic hemisphere in the sham operation group, delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup （P 〉 0.05）. After 7 days of ischemia/reperfusion, many GFAP-positive cells, which possessed a large cell body and an increased number of processes, were activated in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression in the hippocampal CA1 region was greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.01）. Moreover, GFAP expression was significantly greater in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.01）. After 30 days of ischemia/reperfusion, GFAP-positive cells were present in scar-like structures in the rat hippocampal CA1 region at the ischemic hemisphere. GFAP expression was significantly greater in the delayed neuronal death （＋） subgroup and delayed neuronal death （–） subgroup compared with the sham operation group （P 〈 0.05）. GFAP expression was significantly lower in the delayed neuronal death （–） subgroup than in the delayed neuronal death （＋） subgroup （P 〈 0.05）. The delayed neuronal death rates were 42% （5/12）, 33% （4/12） and 33% （4/12） at 3, 7 and 30 days, respectively, followingischemia/reperfusion. No significant differences were detected at various time points （χ2 = 0.341, P 〉 0.05）.
CONCLUSION： The activation of astrocytes was poor in the hippocampal CA1 region during the early stages of ischemia, which is an important reason for delayed neuronal death. Glial scar formation aggravated delayed neuronal death during the advanced ischemic stage.     

Neuroprotective effects of willed-movement therapy in a rat model of cerebral ischemia/reperfusion

The present study established rat models of middle cerebral artery ischemia/reperfusion using the thread method.Rats performed willed-movement(climbing a ladder or wall in a box) when induced by food and water.Results revealed that Longa scores of neurological deficits significantly de-creased in the willed-movement group at 15 days after reperfusion,while expression of glial fibrillary acidic protein,neurotrophic factor-3,and growth-associated protein-43 significantly increased at 7 and 15 days after reper...     

Down-regulation of astroglial proteins in the rat cerebellum after portacaval anastomosis

The effect of short-term portacaval anastomosis (PCA) on the expression of specific astroglial markers [glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS)] in the rat cerebellum was examined to determine the influences of PCA on astroglial cells. The results suggest that PCA directly interferes with astroglial cytoskeleton, as indicated by the irregular distribution and reduced expression of GFAP observed after 1 month. PCA also decreased GS immunoreactivity in the Bergmann glial processes of the molecular layer, as well as in astrocytes of the granule cell layer. It might also modulate glutamatergic nervous activity as GS expression was reduced in 1 month post-PCA brains. Moreover, the GFAP and GS levels in PCA-exposed rats were lower than in control rats. This might contribute to the appearance of encephalopathy by increasing extracellular glutamate and/or ammonia concentrations. These results show that short-term PCA interferes with astroglial protein expression, with both GFAP and GS levels falling in astroglial cells.     

甲基强的松龙对伽玛刀照射后胶质细胞Cx43和GFAP表达的影响

目的 研究甲基强的松龙（MP）对伽玛刀照射后胶质细胞缝隙连接蛋白43（Cx43）和胶质纤维酸蛋白（GFAP）表达的影响。方法体外培养原代星形胶质细胞,经伽玛刀照射（边缘剂量32Gy）并培养36h后随机分为1μg／ml、5μg／ml、10μg／ml、20μg／ml、30μg／ml、40μg/ml组及实验对照组,各组进一步随机分为48h、72h、96h、120h4个亚组。将各组胶质细胞与相应剂量MP共培养,于48、72、96、120h各时间点采用免疫组化方法检测GFAP及Cx43的表达。结果伽玛刀照射后,胶质细胞Cx43表达降低,GFAp表达增高。添加MP后,20μg／ml、30μg／ml组Cx43呈时间依赖性上调;GFAP表达虽呈时间依赖性上升,但其峰值低于实验对照组,峰值出现时间亦晚于实验对照组。结论MP可上凋伽玛刀照射后胶质细胞Cx43的表达,抑制GFAP表达的上升趋势。     

Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfusion

White matter lesions are thought to result from chronic cerebral ischemia and constitute a core pathology of subcortical vascular dementia. This rarefaction has been known to be associated with microglial activation. We investigated whether minocycline, a microglial inhibitor, attenuates the white matter damage induced by chronic cerebral hypoperfusion that is used as a model of vascular dementia. Male Wistar rats were subjected to bilateral, permanent occlusion of the common carotid arteries (BCCAO) to induce chronic cerebral hypoperfusion. Minocycline or saline was injected daily for 2 weeks after BCCAO. In the corpus callosum and the optic tract, white matter damage observed with Klüver-Barrera staining was significantly attenuated in the minocycline-treated group compared to saline-treated controls. In control rats, immunoreactivities of major basic protein (MBP), Ox-42 as a microglial marker, and matrix metalloproteinase (MMP)-2 were increased in the corpus callosum. Minocycline significantly reduced these changes. Co-expression of Ox-42 and MMP-2 was confirmed by double immunofluorescence histochemistry. Our results suggest that chronic treatment with minocycline could be protective against at least some ischemic white matter damage, and its mechanism may be related to suppressing microglial activation.     

Astroglial reaction in the gray matter lumbar segments after midthoracic transection of the adult rat spinal cord

Previous studies of cordotomized rats revealed a glial reaction in the gray matter of the spinal cord at sites remote from the lesion, and the present study was done to explore this phenomenon further. Seventy-five young adult female rats were cordotomized and 10 hemicordotomized, both operations at T5. Between 1 and 28 days postoperatively, histologic sections of thoracic and lumbar segments stained by phosphotungstic acid hematoxylin (PTAH, pH 2.37), by periodic acid Schiffdimedon (PAS-D) or by an immunocytochemical method for glial fibrillar acidic protein (GFAP) revealed histological changes as follows: PTAH staining showed that astroglia in thoracic and lumbar regions of the cordotomized rats possessed a swollen, pink-staining cytoplasm and enlarged, thick, dark blue-staining fibrous processes. This response, first noted within 4 days, had intensified by 7 days and was maximal at 14 to 17 days postoperatively. By 28 days, the reaction had diminished but was still readily detectable. The more specific GFAP staining procedure confirmed that the reactive cells were astrocytes and demonstrated that their fibrillar density had increased. The PAS-D reaction revealed glycogen accumulation in glia of the lumbar gray matter within 2 days; this response intensified by 4 days and diminished to normal by 14 days. This reaction was largely concentrated in the perivascular end feet of astroglia, but also appeared in conjunction with perineuronal astroglia. The site of glial reactivity included both dorsal and ventral horns and was particularly noticed in the gray matter surrounding the central canal. In the hemicordotomized rats, the thoracic and lumbar glia response was much more pronounced ipsilaterally than contralaterally. These results support the interpretation that an astroglial response, involving hypertrophy, fibrillogenesis, and glycogen accumulation, occurs in response to degenerating nerve fibers caudal to sites of spinal cord injury.     

Acquisition of glial fibrillary acidic protein-containing fibres by astroglial cells in primary culture is orchestrated by a communicable factor

In a previous study we discovered that primary cultures initiated from the whole brain of 21-day foetal rats contained astroblasts that concertedly acquired glial fibrillary acidic protein (GFAP) fibres. The mechanism of this burst of cytoskeletal differentiation could not be investigated in these cultures because it occurred too quickly (completed within 2 h). We report that cultures initiated from the region of the third ventricle display an extended burst of GFAP acquisition whose rate could be markedly reduced by medium changing. Temporary medium deprivation or the addition of cytosine arabinoside to the growth medium had no effect. Our findings suggest that an as yet uncharacterised communicable factor is involved in the orchestration of cytoskeletal differentiation in culture. This factor may be responsible for synchronising the appearance of GFAP-positive cells in the periventricular regions of the foetal brain.     

Glial activation and white matter changes in the rat brain induced by chronic cerebral hypoperfusion: an immunohistochemical study

Activation of glial cells and white matter changes (rarefaction of the white matter) induced in the rat brain by permanent bilateral occlusion of the commom carotid arteries were immunohistochemically investigated up to 90 days. One day after ligation of the arteries, expression of the major histocompatibility complex (MHC) class I antigen in microglia increased in the white matter including the optic nerve, optic tract, corpus callosum, internal capsule, anterior commissure and traversing fiber bundles of the caudoputamen. After 3 days of occlusion, MHC class I antigen was still elevated and in addition MHC class II antigen and leukocyte common antigen were up-regulated in the microglia in these same regions. Astroglia, labeled with glial fibrillary acidic protein, increased in number in these regions after 7 days of occlusion. A few lymphocytes, labeled with CD4 or CD8 antibodies, were scattered in the neural parenchyma 1 h after occlusion. Activation of glial cells and infiltration of lymphocytes persisted after 90 days of occlusion in the white matter and the retinofugal pathway. However, cellular activation and infiltration in microinfarcts of the gray matter was less extensive and was substantially diminished 30 days after occlusion. The white matter changes were most intense in the optic nerve and optic tract, moderate in the medial part of the corpus callosum, internal capsule and anterior commissure, and slight in the fiber bundles of the caudoputamen. These results indicated that chronic cerebral hypoperfusion induced glial activation preferentially in the white matter. This activation seemed to be an early indicator of the subsequent changes in the white matter.     

BMP4 expression in the developing rat retina

We investigated the expression of bone morphogenetic protein-4 (BMP4) in the developing retina. At E19, we found very intense BMP4 immunoreactivity (IR) in the nerve fiber layer. At P1, the inner plexiform layer exhibited very strong BMP4-IR. Thereafter, abundant BMP4 expression was kept to the adult period. These results suggest that BMP4 plays pivotal roles in the retina not only in the early embryonic period but also in the late embryonic and postnatal periods, and even in the adult.     

Disruption of the blood-brain barrier and neuronal cell death in cingulate cortex,dentate gyrus,thalamus, and hypothalamus in a rat model of Gulf-War syndrome

We investigated the effects of a combined exposure to restraint stress and low doses of chemicals pyridostigmine bromide (PB), N, N-diethyl-m-toluamide (DEET), and permethrin in adult male rats, a model of Gulf-War syndrome. Animals were exposed daily to one of the following for 28 days: (i) a combination of stress and chemicals (PB, 1.3 mg/kg/day; DEET, 40 mg/kg/day; and permethrin, 0.13 mg/kg/day); (ii) stress and vehicle; (iii) chemicals alone; and (iv) vehicle alone. All animals were evaluated for: (i) the disruption of the blood-brain barrier (BBB) using intravenous horseradish peroxidase (HRP) injections and endothelial barrier antigen (EBA) immunostaining; (ii) neuronal cell death using H&E staining, silver staining, and glial fibrillary acidic protein (GFAP) immunostaining; and (iii) acetylcholinesterase (AChE) activity and m2-muscarinic acetylcholine receptors (m2-AChR). Animals subjected to stress and chemicals exhibited both disruption of the BBB and neuronal cell death in the cingulate cortex, the dentate gyrus, the thalamus, and the hypothalamus. Other regions of the brain, although they demonstrated some neuronal cell death, did not exhibit disruption of the BBB. The neuropathological changes in the above four brain regions were highly conspicuous and revealed by a large number of HRP-positive neurons (21-40% of total neurons), a decreased EBA immunostaining (42-51% reduction), a decreased number of surviving neurons (27-40% reduction), the presence of dying neurons (4-10% of total neurons), and an increased GFAP immunostaining (45-51% increase). These changes were also associated with decreased forebrain AChE activity and m2-AchR (19-25% reduction). In contrast, in animals exposed to stress and vehicle or chemicals alone, the above indices were mostly comparable to that of animals exposed to vehicle alone. Thus, a combined exposure to stress and low doses of PB, DEET, and permethrin leads to significant brain injury. The various neurological symptoms reported by Gulf-War veterans could be linked to this kind of brain injury incurred during the war.     

Skilled reaching training promotes astroglial changes and facilitated sensorimotor recovery after collagenase-induced intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is the most devastating type of stroke and a leading cause of disability and mortality worldwide. Although rehabilitation improves recovery after ICH the cellular mechanisms involved are poorly understood. We decided to examine if skilled (SK) and unskilled (US) training after sham or intracerebral hemorrhage (ICH) surgeries would induce GFAP+ astrocytic changes and whether these modifications can be associated with functional improvement. A 4-week course of motor training, involving either skilled and unskilled training began seven days after surgery; sensorimotor recovery was evaluated using Staircase, ladder walk and cylinder tests. Histological and morphometric analyses were used to assess GFAP+ cell bilaterally in forelimb sensorimotor cortex and dorsolateral striatum. All behavioral tests showed that ICH-SK rats experienced a greater degree of recovery when compared to ICH no task or ICH-US groups; no behavioral differences were found among all sham groups. Astrocytic density was increased in all analyzed structures for ICH no task, ICH-SK and ICH-US rats. Morphological analysis revealed an increased number of primary processes in ipsilateral (to lesion) sensorimotor cortex for all ICH groups. Present results also revealed that both ICH and SK induced an increased length of GFAP+ primary process; there was a further increase in length processes for ICH-SK group in sensorimotor cortex and ipsilateral striatum. We suggest that skilled reaching is an effective intervention to promote astrocytic plasticity and recovery after ICH.     

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.     

Concerted and predictable acquisition of glial fibrillary acidic protein filaments by a cohort of astroblasts in culture

Primary cultures of the 21 day foetal rat brain contain a cohort of astroblasts that concertedly acquire glial fibrillary acidic protein (GFAP) filaments between the 16th and 18th hour after plating. This burst of cytoskeletal differentiation is not observed in cultures initiated from the 18 day foetal brain and is not effected by the addition of cytosine arabinoside, an inhibitor of glial cell proliferation.     

Possible involvement of 5alpha-reduced neurosteroids in adrenergic and serotonergic stimulation of GFAP gene expression in rat C6 glioma cells

Influence of adrenergic and serotonergic stimulation on glial fibrillary acidic protein (GFAP) gene expression in rat C6 glioma cells was first examined as an in vitro model experiment for investigating the neuronal regulation of glial cell differentiation. Stimulation of these cells with isoproterenol and serotonin elevated GFAP mRNA levels followed by an increase in its protein contents, thus suggesting that both adrenergic and serotonergic stimulation might induce the differentiation of the glioma cells. In addition, progesterone and its 5alpha-reduced metabolite dihydroprogesterone also elevated GFAP mRNA levels in rat C6 glioma cells, consistent with their stimulatory actions on GFAP gene expression observed in rat astrocytes. Further studies showed that the elevation of GFAP mRNA levels induced by isoproterenol and serotonin as well as progesterone was abolished by pretreatment of the glioma cells with finasteride, an inhibitor of 5alpha-reduced steroid production. Moreover, the stimulatory actions of isoproterenol and serotonin on GFAP gene expression were inhibited by pretreatment with a GABA(A) receptor antagonist bicuculline and a progesterone receptor antagonist RU486. These findings suggest that both adrenergic and serotonergic stimulation may indirectly activate GFAP gene expression probably through the production of 5alpha-reduced steroid metabolites in rat C6 glioma cells, proposing the possibility that 5alpha-reduced neurosteroids may play a potential role in the neuronal regulation of glial cell differentiation.     

白果内酯抑制持续性脑栓塞大鼠水通道蛋白1，4及胶质纤维酸性蛋白的表达

实验结果发现白果内酯预处理可以降低永久性大脑中动脉闭塞大鼠脑组织含水量和梗死面积;下调水通道蛋白1,4 mRNA在水肿脑组织中的表达,继而抑制其合成,特别是在缺血的早期阶段（8 h）;抑制胶质纤维酸性蛋白的表达,减轻反应性胶质增生。说明白果内酯可通过抑制水通道蛋白的表达减轻脑水肿。     

Expression of glial fibrillary acidic protein in developing rat brain after intrauterine infection

In order to investigate the neuropathological effects on the developing rat brain after intrauterine infection, identification of GFAP was observed. Escherichia coli (E. coli) was inoculated into uterine horn of pregnant rats when gestation was 70% complete (15 days) and the control group was inoculated with normal saline. Immunohistochemistry was used for evaluation of GFAP expression in pup brains at postnatal day 1 (P1), P3, P7, P14 and P21, and RT‐PCR was used to analyze GFAP mRNA, interleukin‐1β mRNA (IL‐1β mRNA) and tumor necrosis factor‐α mRNA (TNF‐α mRNA) expression in pup brains at P1, P3 and P7. At P1 and P3. GFAP was expressed very scarcely in periventricular white matter but not in other brain regions between the two groups. Compared with the control group, at P7 GFAP expression of the E. coli‐treated pups was remarkably increased in periventricular white matter and hippocampus. The E. coli‐treated pups at P14 showed a marked increase of GFAP expression in periventricular white matter, corpus callosum and cortex. However, no significant difference in levels of GFAP expression in any brain regions were found at P21 between the two groups. GFAP mRNA expression of the E. coli‐treated pups was higher than the control at P1 and P3, but there was no significant difference between the two groups at P7. IL‐1β mRNA and TNF‐α mRNA expressions of the E. coli‐treated pups were higher than the control at P1 but there was no significant difference between the two groups at P3 and P7. These present results suggest that intrauterine infection could increase GFAP expression in the pup brain and indicate that intrauterine infection might damage the developing white matter and IL‐1β, TNF‐α might be a mechanism mediating between the two events.