| Background and objectiveWhite matter lesions are very common in clinical Neurological diseases, in recent years , people have attached more importance to the study of white matter lesions. Studies show that chronic cerebral ischemia is the main cause of it. Inflammation, including oxide nitrogen and free radical has an important role in the process of white matter lesions after chronic cerebral ischemia. There are abundant of gliocyte in white matter, including astrocyte and microglia, which take part in the inflammatory reaction after chronic cerebral ischemia. So it is very important to investigate the effect of gliocyte and inflammation reaction on the white matter changes after chronic cerebral ischemia for searching effective measures to protect the white metter lesions. In the situation of chronic cerebral ischemia, the content of free oxidate increases and the activity of free oxidate's clearer-superoxidase dismutase (SOD) decreases, the increased free oxidate leads to more action of lipid peroxide, and its reactants malondialehyde (MDA) increases accordingly, all of which will cause the lesions of white matter. Reactive gliosis, demyelination and axonal damage are mainly signs of white matter lesions. Glial fibrillary acidic protein(GFAP) is a special signal protein of astrocyte, which will certainly increase in reactive gliosis.8-Amyloid precursor protein( P -APP) is transported by fast anterograde axonal transport, so the accumulation of this protein seems to be an indicator of disturbed axonal transport. Cyclooxygenase-2(COX-2) is the synthase in the synthesis of prostanoids from arachidonic acid, there are a lot of free radical be produced in this synthetical process. So COX-2 is a determinant factor of the cytotoxicity connected with inflammation following ischemic injury to the brain. Recently, cydooxygenase-2 mRNA level has been shown to be significantly increased within neurons and vascular cells after acute focal cerebral ischemia, the COX-2 inhibitor can reduce the concentration of PGE2 and the injury of neuron in the infarct. However, there are little study for the protective effect of COX-2 inhibitor on chronic cerebral ischemia and the relevent white matter lesions. In this study, we will use chronic cerebral ischemia animal model and investigate the changes of MDA and SOD, the situation of astrocyte, myelination and axone in the white matter to study the changes of white matter after chronic cerebral ischemia. We also use the COX-2 inhibitor nimesulide to treat the animals and investigate the neuroprotective effect of it.Materials and methods80 healthy Sprague-Dawley rats weighing 300~400g were used in this study. The animals were distributed into four groups randomly , Group A: sham operation group (n=20); group B: sham operation with nimesulide group (n=20), group C: operation group (n=20); group D: operation with nimesulide group (n=20). The models of chronic cerebral ischemia were established by permanent bilateral occlusion of the common carotid arteries (2VO) in groups C and D. Only isolated Bilateral common carotid arteries but not ligated them in groups A and B. The rats of groups B and D received a daily oral dose of 6mg/kg nimesulide (dissolved by 0.5% carboxymethylcellulose ) from 24h after the operation to 60 days, the rats of groups A and C received the same volume of vehicle( 0.5% carboxymethylcellulose ). In every group, the rats (n=10) used to detect MDA and SOD were decapitated and taken out brains. The coronal brain block thick 5mm from optic chiasm were triturated to make 10% brain tissue solution, and detected the level of MDA contents and SODactivities. The rest of rats in every group (n=10) decapitated and removed the brain. The coronal brain block thick 3mm from optic chiasm were embedded in paraffin for tissue sections. Brain sections (4 n m thick) were prepared to observe the histopatho-logical changes of brain by hematoxylin and eosin (HE) stain, the activation of astrocytes by immunohistochemistry for GFAP, the changes of myelination by luxol fast blue (LFB)stain and the damage of axonal by immunohistochemistry for 8-APP. All the datum were handled with SPSS10.0 statistic software. The difference of every two groups was compared with one-way analysis of variance and the least significant difference(LSD), significant level is a =0.05.Results1. The conditions of animal All the rats were depressed, reacted slowly, ate little after operation. The next day, sham groups recovered significantly, however the operation groups recovered until a week later.2. The results of HE stain There were a few degenerated and dead neurons be found in groups A and B. But more degenerated and dead neurons were found in group C, and we also found some white matter lesions in this group. Compared with group C, the degenerated and dead neurons were less in group D.3. The results of MDA and SOD The content of MDA( nmol/mg prot ) in brains of every group rats were 4.55 + 0.58,4.57 ± 0.62,9.61 + 0.70,6.72 + 0.49 respectively. The activity of SOD(y/mg ? prot) were 109.43 + 3.47, 110.20±4.84, 85.00±4.51, 98.33+3.25 respectively. The content of MDA were markedly elevated and the activities of SOD decreased in group C, all the changes were less in group D. The difference between every two groups is significant (P<0.05) except groups A and B in MDA and SOD.4.The results of immunohistochemical for GFAP The number of GFAP immunopositive cells in internal capsule in rats brain of every group were 25.20+ 2.82, 25.40+3.41, 39.50+4.22, 30.70+3.13 respectively, and in the corpus callosum were 17.10+2.60,17.30+2.00,26.00+2.91,20.10 + 2.77 respectively. The number of GFAP immunopositive cells were markly increased in group C in each part, all thechanges were less in group D. The difference between every two groups is significant (i><0.05) except A and B.5. The results of LFB stain According to Wakita H's standard, we juged the results to detect the damage of myelination. The grade in the corpus callosum in rats brain of every group were 0, 0,1.7 ±0.67, 0.6+0.70 respectively. The grade were markly increased in group C, the changes were less in group D. The difference between the two groups is significant (P<0.05).6. The results of immunohistochemical for P -APP According to Wakita H's standard, we juged the results to detect the damage of axone, the grade in the corpus callosum in rats brain of every group were 0, 0, 1.40+0.70, 0.70+0.67 respectively, The grade were markly increased in group C, the changes were less in group D. The difference between the two groups is significant (P<0.05).Conclusions1. We found the content of MDA were markedly elevated and the activities of SOD decreased in the brain tissue after chronic cerebral ischemia. Obvious proliferation and hypertrophy of GFAP immunopositive astrocytes also were observed in those brains, meanwhile, we also found demyelination and axonal damage in white matter after chronic cerebral ischemia.2. Nimesulide can reverse the elevation of MDA content and the reduction of SOD activity, and reduce the number of astrocytes after chronic cerebral ischemia. Moreover, it can alleviate the demyelination and axonal damage in white matter after chronic cerebral ischemia. But it had no effect on normal rats.3. The present study provides the experimental and theoretic evidences for the study of the mechanism of the white matter lesion after chronic cerebral ischemia, and the investigation of protection of the cyclooxygenase-2 inhibitor nimesulide to reduce the white matter damages induced by chronic cerebral ischemia.
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