| Background and objectiveChronic cerebral ischemia (CCI) is a common ischemic cerebrovascular disease, which is closely associated with chronic hypoperfusion injury of brain due to various reasons. It has been confirmed that white matter is more easily to be damaged by chronic cerebral hypoperfusion than neurons.There are abundant nerve fibers and neuroglial cells in white matter. Glial cells known as oligodendrocytes, myelin-forming cells in CNS, are damaged by CCI and decreased in number. The impairment of oligodendrocytes results in loss of myelin. More importantly, myelinoclasis is a serious interference of neural transmission that can lead to dysfunction of learning and cognitive. Glial cells known as astrocytes enlarge and proliferate to form a scar and produce inhibitory molecules that inhibit regeneration of a damaged or severed axon. Meanwhile, ultra-reactionary astrogliosis may injure neurons and neuroglial cells by producing some neurovirulent substances.Glial cells have a powerful ability to undergo cell plerosis and regeneration after an injury, while neurons not. All about these show great potentialities to treatment of diseases presenting as white matter injured, and bring points that are more valuable for studies. The Ginkgo biloba extract (EGb761), a standardized extract, is a complex mixture of ingredients including 24% flavonoid and 6% terpenoid, etc, with a uniquely broad spectrum of pharmacological activities. Nowadays, so many studies have supported the potential usefulness of EGb761 in neuronic protective. However, the studies about the effect of EGb761 on the injured neuroglial cells in cerebral ischemia is still lacking and its association with neuroglial cells secondary injury remains to be proved.Consequently, the model of CCI was established in Sprague Dawley rats by occluding and snipping of bilateral common carotid arteries permanently (2VO). Then, the testing technique of ethology and the histopathologic and immunohistochemical techniques were employed to observe and evaluate the changes in learning ability, neurons, myelin sheath and glial cells in CCI rats. It woud help us to estimate and investigate the potential neuroprotective effects of EGb761 on cognitive disfunction and changes of glial cells and myelin sheath, which might provide theoretical basis and work out new countermeasures in neuroprotection in CCI.Materials and Methods1 Experimental animals and grouping:Thirty healthy male Sprague Dawley rats (body weight:220±30g) were employed in the study, which were randomly divided into three groups including sham-operated group (Group A), ischemia group (Group B) and EGb761 group (Group C). There were ten rats in each group.2 Model establishment:Chronic cerebral ischemia model was established by occluding and snipping of bilateral common carotid arteries permanently in ischemia group and EGb761 group. Bilateral common carotid arteries were isolated but not ligated or snipping in sham-operated group. Since 24-hour of post-operation, the rats of EGb761 group swallowed Tanakan (40mg, Tanakan; IPSEN Laboratories, Paris, France) dissolved by distilled water(5mg/ml,0.5ml/100g), while the sham-operated group and ischemia group swallowed the same amount of distilled water once a day for 12 weeks.3 Specimen collection:The ability of learning and memory of all rats was tested with Y-maze at 12 weeks after operation. And then, they were anesthetized and perfused with physiological saline and 4% paraformaldehyde through heart. Subsequently, the brain-tissues of all rats were took out and fixed. Finally, the brains were embedded in paraffin, sectioned and processed for hematoxylin and eosin (HE) staining, ponceau 2R-brilliant green double myelin staining and immunohistochemical staining.4 Observation indexes:(1) The ability of learning and memory of all rats was detected with Y-maze in the noiseless darkroom. (2) HE staining was performed to observe pathological changes of brain tissue under light microscope. (3) Changes of myelin in corpus callosum were observed by ponceau 2R-brilliant green double staining. (4) Oligodendrocytes and astrocytes were symbolized by their specific antibodies,2'3'-cyclic nucleotide3'-phosphode-esterase (CNPase) and glialfibrillary acidic protein (GFAP). The expressions of them in hippocampus, corpus callosum and cortex were examined by immunohistochemistry and the positive cells of CNPase and GFAP were counted.5 Statistic analysis:The experimental data were expressed by mean±standard deviation (χ±s) and the data were handled with SPSS16.0 statistic software. Significance level is judged byα=0.05.Results1 The conditions of animals:All rats showed spiritless in varying degrees, unresponsive, sluggish, suffering from lack of appetite and low ability of self-cleaning after the operation of 2VO. Just one day after operation, the behavior of group A was obviously ameliorated. After the treatment, the behavior of group C was more ameliorated than group B's.2 The ability of learning:The learning record is the number of times that rats could run to the exit passageway according to the signal lamp, when they were stimulated by electric stimulation. The times of electric attacks in sham-operated group were 33.30±1.16; the times in ischemia group were 68.30±2.06; the times in EGb761 group were 46.90±1.45. There was significant difference between sham-operated group and ischemia group in the ability of learning (P<0.01). The difference between ischemia group and EGb761 group was also significant (P<0.01).3 The results of pathological section with HE staining:Mostly, the neurons were dense and lined tidily and cells were in normal shape in sham-operated group. There were only a few degenerated and necrotic neurons to be found. In ischemia group, the neurons were distributed irregularly. A number of neurous died and were loss. Cells were abnormal in shape. There was a blank area around the cell. The staining of nucleus pyknosis was intensive. However, from the structure of the neurons to the number of neurous, EGb761 group were more significantly ameliorated than ischemia group's.4 The results of myelin stain with ponceau 2R-brilliant green double staining: Changes of myelin in corpus callosum were observed according to Wakita H's standard, in order that we could judge the degree of myelin lesions. The grade in sham-operated group was 0.052±0.025; the grade in ischemia group was 2.808±0.176; and the grade in EGb761 group was 1.66±0.271. Compared with sham-operated group, the grade in ischemia group and EGb761 group were remarkably increased: And the difference was significant (P<0.01). The grade in EGb761 group was more decreased than that in ischemia group. The difference was also significant (P<0.01).5 The results of immunohistochemisty for CNPase:The positive cells of oligodendrocytes in the cortex, corpus callosum and hippocampus were counted in each group. In the cortex:group A 9.70±1.34; group B 4.80±1.03; group C 7.10±1.10. In the corpus callosum:group A 27.80±1.93; group B 18.20±1.75; group C 23.80±1.32. In the hippocampus:group A 7.70±0.95; group B 3.90±0.88; group C 5.70±0.82. According to the data, the CNPase positive cells of group B were the lowest among all three groups. The positive cells of group B and group C were lower than that in group A, and the differences were all significant among every group (P<0.01).6 The results of immunohistochemisty for GFAP:The positive cells of astrocytes in the cortex, corpus callosum and hippocampus were counted in each group. In the cortex:group A 12.90±1.66; group B 28.20±1.87; group C 19.40±1.43. In the corpus callosum:group A 17.01±1.49; group B 28.50±1.58; group C 22.00±1.49. In the hippocampus:group A 10.50±2.55; group B 24.30±2.46; group C 16.60±1.51. Compared with the GFAP positive cells of group A, both of group B and group C were significantly increased (P<0.01). The positive cells of group B were more increased than these of group C, and there was significant difference. Conclusions1. Chronic cerebral ischemia (CCI) leads to reduction in numbers of oligodendrocytes, myelinoclasis, and excess hyperplasia of astrocytes and closely related dysfunction of learning and cognitive.2. EGb761 plays the role that could reduce the ischemic reaction of oligodendrocytes and the reactive hyperplasia of astrocytes and improve the related myelinoclasis and cognitive dysfunction.3. EGb761 can protect neurons from injury of chronic cerebral ischemia as well as have some protective effects on gliacyte.
|
PDF Full Text Request