| Background and objectiveChronic cerebral hypoperfusion(CCH) is a kind of clinical frequent pathologic state.It leads to dizziness,cerebaria and insensible feeling,and mayby play an important role in a few diseases such as artherosclerosis, Alzheimer's disease(AD), vascular dementia(VD), Parkinson disease,et al.Especially,some research show that CCH switches on or maintaine-sthe process of AD probably in recent years.The machenism that CCH causes AD is not clear,but vasculat factor provides a direction to prevent and cure AD.So,nitric oxide(NO), a kind of vasodilator substance,maybe a target poit for prevention and treatment of AD.NO,which originates from nitric oxide synthase(NOS),is an important vasodilator substance and neurotransmitter in central nervous systerm.NO is the key messenger in long term potentiation(LTP) of synaptic transmission and playes an important role in learning and memory. It also takes part in onset and development of acut cerebral ischemia.NOS has three isoforms.NO which originates from endothelial NOS(eNOS) shows neuroprotection, but which originates from overexpression of neuronal NOS(nNOS) and inducible NOS(iNOS) shows cytotoxic effect.To explore medicine which can interfere in NOS maybe have latent clinical value.But in the pathologic process of dementia which caused by CCH,it is not known whether NO/NOS has dual effects which have been shown in the process of acut cerebral ischemia. Nimodipine,a kind of Ca2+ channel antagon,shows neuroprotection effect and can treat dementia.The specific mechanism is unknown.In this study,we make CCH rats model by 2VO,and treat CCH rats with nimodipine.We observe the change of cognitive ability of rats and the expression of NOS isoforms in CA1 region of hippocampus to explore the roles of NOS isoforms in the process of CCH and the possible neuroprotectin mechanism of nimodpine.This study will provide further experimental evidence of nimodipine.Materials and methods1. 30 healthy male Sprague-Dawley rats of 10～12 months old and 300～350g weight were randomly assigned into three groups:sham operation group, only ischemia group, post-ischemia treated group.2. Rat models of CCH were established by occluding and snipping of bilateral common carotid arteries via using 0# thread permanently in only ischemia group and post-ischemia treated group. Bilateral common carotid arteries were isolated but not ligated or snipped in sham operation group. All rats were raised in common condition after the operation. Blood pressure were messured befor and one time once a week after operation. The post-ischemia treated group swallowed nimodipine (1mg/kg) dissolved by SCMC, while the sham operation group and only ischemia group swallowed the same amount of SCMC twice a day for 60 days since 24h of post-operation. Cognitive ability of all rats was assessed in a Y model maze and shown with the times of electric attack after 60 days. And then, they were anesthetized and perfused with normal saline through heart. Subsequently, brains of them were removed and fixed in 4% polyformaldehyde. Finally, brains were embedded, sectioned and processed for HE staining and immunohistochemical staining.3. Observation indexes included blood pressure,cognitive ability, pathological change of brain tissue by HE staining,and expression of nNOS,iNOS and eNOS by immunohistochemistry.4. Statistic software SPSS10.0 were exploited to analysis data. Deviation and difference were compared using the ANOVA. The differential significance was judged by a=0.05. Results1. All the rats were in low spirits, reacted slowly and took food less than normal after operation. On the next day, sham operation group recovered, reacted more quickly and took food normally, but only ischemia groups didn't show manifest signs of rehabilitations. In the process befor they were killed, sham operation group reacted quickly but only ischemia group depressed and reacted clumsy.The appearance of post-ischemia treated group is better than only ischemia group but less than sham operation group.Blood pressure of three group were not shown difference befor and after operation(P>0.05). Blood pressure of post-ischemia treated group were not shown difference neither befor nor after nimodipine treatment(P>0.05).2. The results of cognitive ability: The times of electric attack in sham operation group was 35.50±5.10, in only ischemia group was 81.60±5.95 and in post-ischemia treated group 58.60±7.23. There was significant difference among the three groups(P < 0.01). These results indicate that congintive ability were injured by 2VO and nimodipine can alleviate cognitive impairment in rats with CCH.3. The results of pathological section with HE staining:Only a few degenerated and dead neurocytes were found, while most neurocytes were normal in morphology in sham operation group. The number of degenerated and dead neurocytes significantly increased in only ischemia group compared with sham operation group, and some white matter lesions were found in this group. The number of degenerated and dead neurocytes was less in post-ischemia treated group than that in only ischemia group.4. The results of immunohistochemistry for nNOS: The average gray scale of nNOS positive cells in CA1 region of hippocampus of rats brains in sham operation group was 57.50±10.11,in only ischemia group was 34.90±5.22 and in post-ischemia treated group 43.10±6.56. There was significant difference among the three groups(P < 0.01). These results indicate that expression of nNOS were descended by 2VO and nimodipine can improve it in rats with CCH.5. The results of immunohistochemistry for iNOS: The average gray scale of iNOS positive cells in CA1 region of hippocampus of rats brains in sham operation group was 10.40±3.50,in only ischemia group was 82.70±2.80 and in post-ischemia treated group 63.60±6.57. There was significant difference among the three groups(P < 0.01). These results indicate that expression of iNOS were improved by 2VO and nimodipine can inhibit it in rats with CCH.6. The results of immunohistochemistry for eNOS: The average gray scale of eNOS positive cells in CA1 region of hippocampus of rats brains in sham operation group was 62.40±4.20,in only ischemia group was 21.20±2.74 and in post-ischemia treated group 38.30±3.13. There was significant difference among the three groups(P < 0.01). These results indicate that expression of eNOS were descended by 2VO and nimodipine can improve it in rats with CCH.Conclusions1. In CA1 region of hippocampus in rats with CCH, the expression of nNOS and eNOS was descended while the expression of iNOS was improved due to CCH.This change correlated with cognitive impairment after CCH.2. Neurotoxicity of iNOS injured nNOS and eNOS neurocytes and led to the inhibition of LTP.Maybe it was one of machenisms that CCH caused cognitive impairment. This experiment did not support neurotoxicity effect of nNOS in CCH.3. Nimodipine can alleviate the damages of neurocytes and improve cognitive ability after CCH.This maybe due to that nimodipine can improve the expression of nNOS and eNOS and inhibite the expression of iNOS. |
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