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.     

FK506 ameliorates the discrimination learning impairment due to preventing the rarefaction of white matter induced by chronic cerebral hypoperfusion in rats

We examined the effects of the immunosuppressant tacrolimus (FK506) on the discrimination learning impairment induced by chronic cerebral hypoperfusion in rats. Chronic cerebral hypoperfusion was prepared by permanent ligation of bilateral common carotid arteries for male Wistar rats aged 9 weeks. FK506 (0.05 mg/kg, s.c.) recovered the learning impairment and also prevented the rarefaction of white matter and striatal neuronal cell damage. Our findings suggest that FK506 ameliorates the learning impairment mainly due to preventing neuropathological alterations.     

Mild hypothermia enhances the neuroprotective effects of FK506 and expands its therapeutic window following transient focal ischemia in rats

FK506 (tacrolimus), an immunosuppressant, reportedly reduces ischemic brain injury following transient middle cerebral artery occlusion (MCAO) in rats. The authors previously reported that the therapeutic window of FK506 in this model is more than 1 h, but less than 2 h. The aim of the present study is to determine whether mild hypothermia (35 degrees C) enhances the neuroprotective effects of FK506 and expands its therapeutic window. Sprague-Dawley rats were subjected to 2 h MCAO followed by 24 h reperfusion. Animals were randomly divided into four groups: (I) vehicle-treated normothermic group; (II) FK506-treated normothermic group; (III) vehicle-treated hypothermic group; (IV) FK506-treated hypothermic group. Animals received a single injection of FK506 (0.3 mg/kg) or vehicle intravenously at 2 h after ischemic induction. During ischemia, temporal muscle and rectal temperatures were maintained at 37 degrees C in the normothermic animals and at 35 degrees C in the hypothermic animals. Infarct volumes and neurological performance were evaluated at 24 h after reperfusion. The combination of FK506 and mild hypothermia significantly reduced infarct volume (cortex, -61%; striatum, -31%) and edema volume (cortex, -57%; striatum, -41%), while mild hypothermia or FK506 alone failed to improve ischemic brain damage. Furthermore, this combination also provided for the best functional outcome. These results demonstrate that the combination of FK506 and mild hypothermia significantly reduces ischemic brain damage following transient MCAO in rats, and expands the therapeutic window for FK506. This therapy may be a new approach for treatment of acute stroke.     

Chronic cerebral hypoperfusion induces white matter lesions and loss of oligodendroglia with DNA fragmentation in the rat

Cerebrovascular white matter lesions represent an age-related neurodegenerative condition that appears as a hyperintense signal on magnetic resonance images. These lesions are frequently observed in aging, hypertension and cerebrovascular disease, and are responsible for cognitive decline and gait disorders in the elderly population. In humans, cerebrovascular white matter lesions are accompanied by apoptosis of oligodendroglia, and have been thought to be caused by chronic cerebral ischemia. In the present study, we tested whether chronic cerebral hypoperfusion induces white matter lesions and apoptosis of oligodendroglia in the rat. Doppler flow meter analysis revealed an immediate reduction of cerebral blood flow ranging from 30% to 40% of that before operation; this remained at 52–64% between 7 and 30 days after operation. Transferrin-immunoreactive oligodendroglia decreased in number and the myelin became degenerated in the medial corpus callosum at 7 days and thereafter. Using the TUNEL method, the number of cells showing DNA fragmentation increased three- to eightfold between 3 and 30 days post-surgery compared to sham-operated animals. Double labeling with TUNEL and immunohistochemistry for markers of either astroglia or oligodendroglia showed that DNA fragmentation occurred in both of these glia. Messenger RNA for caspase-3 increased approximately twofold versus the sham-operated rats between 1 and 30 days post-surgery. Immunohistochemistry revealed up-regulation of caspase-3 in the oligodendroglia of the white matter, and also in the astroglia and neurons of the gray matter. Molecules involved in apoptotic signaling such as TNF- and Bax were also up-regulated in glial cells. These results indicate that chronic cerebral hypoperfusion induces white matter degeneration in association with DNA fragmentation in oligodendroglia.     

Epidermal growth factor receptor in proliferating reactive glia following transient focal ischemia in the rat brain

Severe transient focal cerebral ischemia causes brain infarction with a strong glial reaction. We have studied whether postischemic reactive glial cells express epidermal growth factor receptor (EGFR) following middle cerebral artery occlusion in the rat. We have also looked for signs of proliferating activity, as EGFR is known to be involved in cell growth and proliferation in certain non-neural cells. EGFR was studied using three different antibodies which were found to stain for a tyrosine-phosphorylated protein (p170) corresponding to the membrane-anchored EGFR. Neurons of the control brain were strongly immunoreactive to EGFR, but a decrease of EGFR-immunoreactivity was seen in the ipsilateral brain side from 24 h postischemia due to neuronal loss. However, the presence of abundant glial cells strongly immunoreactive to EGFR became apparent in this area from 4 days postischemia onward. The use of microglial (lectin or OX-42) and astroglial (GFAP) markers showed that these postischemic EGFR-stained cells were reactive microglia/macrophages and astroglia. The subcellular localization of EGFR in reactive microglia/macrophages was compatible with the network of the Golgi apparatus, as revealed with an antibody against a peripheral membrane-bound protein of the Golgi. The presence of abundant proliferating cells in the ischemic brain was detected from 4 days postischemia with an antibody against proliferating cell nuclear antigen. Proliferating reactive microglia/macrophages were abundant within the infarcted brain side, whereas proliferating astrocytes were found mainly in the immediate periphery of the infarct limiting the necrotic area from the undamaged tissue. These proliferating cells were immunoreactive to EGFR. The results show the presence of EGFR in postischemic reactive glial cells and suggest that EGFR-dependent pathways mediate signal transduction in reactive glia following transient focal cerebral ischemia. GLIA 23:120–129, 1998. © 1998 Wiley-Liss, Inc.     

Cytopathological alterations and therapeutic approaches in Binswanger's disease

Binswanger's disease (BD) is a condition characterized by prominent brain atrophy with ventricular dilatation, diffuse white matter (WM) lesions and a scattering of lacunar infarcts. BD patients have dementia, and have vascular risk factors, focal cerebrovascular deficits and evidence of subcortical cerebral dysfunction. From our clinical studies, the most effective prophylaxis against the development of BD is to manage the hypertension, especially a high nocturnal blood pressure, in the early stage patients showing only a scattering of lacunes and/or mild WM lesions. The pathogenesis of BD is likely to be chronic cerebral ischemia due to hypertensive small artery disease with capillary collagenosis, which causes the multiple lacunes and the alterations in the glia and axons. In addition, arterial hypertension and a subsequent dysfunction of the blood–brain barrier (BBB) may cause the WM lesions. A compromised BBB will permit the entry of serum components, immunoglobulins, complements and fibrinogen into the perivascular neural parenchyma. These substances may subsequently activate both astro- and microglia and thus damage the myelin structures. Experimentally, immunosuppressants, cyclosporin A and FK 506 suppressed both the glial activation and WM changes after chronic cerebral hypoperfusion. The pro-thrombotic state of the microcirculation in BD patients may also contribute to local inflammation and the BBB dysfunction, because thrombin and prostanoids are in-volved in various tissue reactions including brain edema and glial activation. Therefore, novel therapeutic ap-proaches using the administration of anti-thrombin and cyclo-oxygenase-2 inhibitors as well as immunosup-pressants may be useful for preventing the progression of BD.     

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.     

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.     

Neuroprotective effect of immunosuppressant FK506 in transient focal ischemia in rats: Therapeutic time window for FK506 in transient focal ischemia

Abstract

Tacrolimus (FK506), an immunosuppressant currently used in clinic, is known to have neuroprotective properties. However, effects in focal ischemia are shown only in a endothelin induced middle cerebral artery (MCA) occlusion model or with filament technique at a relatively high dose. We have previously shown that FK506 had significant protective effects at a low dose of 0.3mg kg^-1 when administered immediately after ischemia. In this study, we explored the therapeutic time window of FK506 at this low dose, in a transient focal ischemia model using filament technique. Male Sprague-Dawley rats were subjected to 2 h MCA occlusion and subsequent reperfusion. They received FK506 or vehicle (0.3 mg kg^-1) i.v. at 30, 60 or 120 min after induction of ischemia, and were decapitated 24 h after ischemia. FK506 injected at 30 and 60 min significantly reduced cortical infarction volume (FK506 vs. vehicle; 30 min: 95 ± 33 mm³ vs. 170 ± 62 mm³, p < 0.05; 60 min: 93 ± 45 mm³, vs. 168 ± 35 mm³, p < 0.05, respectively). FK506 was ineffective when given at 120 min after ischemia. FK506 had no effect on edema formation, nor on the infarct volume in striatum. The therapeutic time window for this low dose of FK506 given i.v. is between 60 and 120 min in this model. [Neurol Res 2001; 23: 755-760]     

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.     

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.     

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.     

Effects of FK506 on the translocation of protein kinase C and CaM kinase II in the gerbil hippocampal CA1 neurons

To explore biochemical basis for cerebroprotective effect of immunosuppressant FK506, we studied changes in subcellular distribution of protein kinase C gamma (PKC gamma) as well as calcium/calmodulin-dependent protein kinase II (CaMKII) after ischemia. Male Mongolian gerbils were subjected to 5 min forebrain ischemia. FK506 (1 or 3 mg kg-1) was administered at 1 min after recirculation, which was confirmed to be cerebroprotective by histological examination at seven days after ischemia. At the designated time points (before ischemia, 5 min ischemia, 1 and 24 h recovery), heads were frozen and samples were taken from CA1 subfield of hippocampus. Western blot analysis was carried out. Persistent translocations of PKC gamma and CaMKII to synaptosomal P2 fraction were observed in vehicle-treated group. FK506 significantly decreased levels of PKC gamma and CaMKII in P2 fraction at 24 h of recovery. The present results suggest FK506 downregulates translocated PKC gamma and CaMKII, which may contribute to its survival promoting effect after cerebral ischemia.     

慢性间断缺氧对老年期大鼠脑白质的影响

目的研究慢性间断缺氧对老年期大鼠脑白质的影响。方法建立老年期SD大鼠慢性间断缺氧模型,免疫组化检测大鼠脑室旁白质髓鞘碱性蛋白（myelin basic protein,MBP）、神经微丝H＋L（neutofilament—H＋L,NF-H＋L）、胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）表达并行图像分析;电镜下观察大鼠脑内髓鞘、轴突的超微结构。结果慢性间断缺氧可使老年期大鼠脑白质MBP、NF-H＋L表达减少及GFAP表达增加（P均〈0．05）,MBP与NF-H＋L呈高度正相关（R^2=0．908,P〈0．01）。电镜观察与空白对照组比较,缺氧组大鼠脑内可见较多的髓鞘脱失、轴突受损。结论慢性间断缺氧可对老年期大鼠脑白质产生不良影响,表现为髓鞘脱失、轴突变性以及神经胶质增生。     

A case of FK 506-induced leukoencephalopathy]

We reported a 15-year-old boy with an acute myelomonocytic leukemia and FK 506-induced leukoencephalopathy. He was received FK 506 for graft versus host disease occurred after peripheral blood stem cell transplantation. He, four weeks later, had generalized seizures and consciousness disturbance. The serum level of FK 506 was high (27.5 ng/ml). His brain MRI showed abnormal high intensity areas in the frontal and parietal white matter lesions on T2-weighted images. Neuropathological studies revealed the destruction of myelin sheeths and axons in the cerebral white matter corresponded with abnormal lesions on MRI. There were calcification and mineralization in the small vessel walls of the cortex and white matter. Osteopontin immunoreactivity was detected in the endothelial cells of small vessels. These findings suggest that the vascular damage was involved in the FK 506-induced leukoencephalopathy.     

Insulin-like growth factor (IGF)-1 suppresses oligodendrocyte caspase-3 activation and increases glial proliferation after ischemia in near-term fetal sheep.

Insulin-like growth factor (IGF-1) markedly increases myelination and glial numbers in white matter after ischemia in near-term fetal sheep; however, it is unclear whether this is due to reduced cell loss or increased secondary proliferation. Brain injury was induced in near-term fetal sheep by 30 minutes of bilateral carotid artery occlusion. Ninety minutes after the occlusion, fetuses were given, intracerebroventricularly, either a single dose of IGF-1 (either 3 or 30 micro g), or 3 micro g followed by 3 micro g over 24 hours (3 + 3 micro g). White matter was assessed 4 days after reperfusion. Three micrograms, but not 30 micro g of IGF-1 prevented loss of oligodendrocytes and myelin basic protein density (P < 0.001) compared to the vehicle-treated ischemia controls. No additional effect was observed in the 3 + 3 micro g group. IGF-1 treatment was associated with reduced caspase-3 activation and increased glial proliferation in a similar dose-dependent manner. Caspase-3 was only expressed in oligodendrocytes that showed apoptotic morphology. Proliferating cell nuclear antigen co-localized with both oligodendrocytes and astrocytes and microglia. Thus, increased oligodendrocyte numbers after IGF-1 treatment is partly due to suppression of apoptosis, and partly to increased proliferation. In contrast, the increase in reactive glia was related only to proliferation. Speculatively, reactive glia may partly mediate IGF-1 white matter protection.     

Neuroprotective FK506 does not alter in vivo nitric oxide production during ischemia and early reperfusion in rats.

BACKGROUND AND PURPOSE: Previous studies have demonstrated that the immunosuppressant FK506 provides neuroprotection in experimental brain injury and suggest that this action may be mediated by suppression of neuronal nitric oxide synthase activation that occurs after ischemic depolarization. We sought to determine whether FK506 reduces histological injury after middle cerebral artery occlusion (MCAO) in the rat and whether the neuroprotective effect is mediated via suppression of in vivo nitric oxide (NO) production during ischemia or early reperfusion. METHODS: Under controlled conditions of normoxia, normocarbia, and normothermia, halothane-anesthetized male Wistar rats were subjected to 2 hours of MCAO by the intraluminal occlusion technique in a blinded, randomized experimental trial. Ipsilateral parietal cortical laser-Doppler flowmetry was monitored throughout ischemia. Animals were randomly assigned to 4 pretreatment groups: intravenous FK506 0.3 mg/kg or 1. 0 mg/kg, vehicle (cremaphor), or an equivalent volume of saline administered 30 minutes before MCAO. Infarction volume was assessed by a triphenyltetrazolium chloride staining at 22 hours of reperfusion. In separate experiments, microdialysis probes were placed bilaterally into the striatum. Rats were perfused with artificial cerebrospinal fluid containing 3 micromol/L [14C]- L-arginine for 3 hours and then subjected to 2 hours of right MCAO. Intravenous 0.3 mg/kg FK506 or cremaphor was given 30 minutes before right MCAO. Right-left differences between [14C]-L-citrulline in the effluent were assumed to reflect differences in NO production. RESULTS: All values are mean+/-SE. FK506 at 0.3 mg/kg reduced infarction volume in cortex: 40+/-12 mm3 compared with saline (109+/-15 mm3) and cremaphor vehicle (148+/-23) (P<0.05). Striatal infarction was also reduced by low-dose FK506: 16+/-4 mm3 versus 36+/-4 mm3 and 34+/-4 mm3 in saline and vehicle groups, respectively (P<0.05). High-dose treatment reduced infarction volume in cortex (61+/-14 mm3, P<0.05 from saline and vehicle groups) and in striatum (22+/-5 mm3, P<0.05 from saline and vehicle groups). [14C]-L-citrulline recovery via microdialysis was markedly enhanced in ischemic compared with nonischemic striatum. However, ischemia-evoked [14C]-L-citrulline recovery was not different in FK506-treated rats compared with vehicle-treated animals. CONCLUSIONS: These data demonstrate that FK506 provides robust neuroprotection against transient focal cerebral ischemia in the rat. The mechanism of protection in vivo is not through attenuation of ischemia-evoked NO production during MCAO and early reperfusion.