| Vascular dementia is a general term for dementia syndrome caused by series of cerebral vascular factors leading to brain damage, the diagnosis must have the following three or more impaired mental activities such as language, memory, visual spatial skills, emotion, personality, computing power and abstract sense.Common clinical vascular dementia include: multi-infarct dementia, dementia with massive cerebral infarction, subcortical arteriosclerotic encephalopathy (Binswanger disease), dementia due to the special position infarction and hemorrhagic dementia. With the aging of society and the continuous changing models of living, VD incidence has increased annually, becoming the second largest type of dementia preceded only by Alzheimer's disease (Alzheimer's disease, AD).VD is the common diseases of the Department of Neurology,which not only seriously affect the health and quality of life of the majority of patients, but also brought a heavy burden to society and the family . In recent years, VD appears a younger trend, attracting more attentions of the majority of research and clinical workers. At present the pathogenesis of VD is not yet fully clear, nor having precise and effective control methods, so probing the pathogenesis of VD and developing effective treatment programs have becoming a focus of medicine.Learning and memory is the advanced brain neurophysiological activities. Learning is the acquisition of experience and skills, memory is preserving and reproducing of experience and skills. Learning and memory are two different neuro-biological processes, which not only interrelated but also cooperated with each other, so that organisms adapt to the changing external environment. Many studies show that the structure of the hippocampus has great significance in learning and formating and maintaning of memory. Memory is divided into transient memory, short-term memory, long-term memory and permanent memory. Longer memories may be related with brain protein synthesis, whereas the permanent memory may be related with the establishment of new synapses. The unique structure and neurophysiological function of the hippocampus may be involved in the synthesis of this protein and the establishment of the synapse. The body's neurotransmitters and humoral factors involved in the process of learning and memory, Such as the cholinergic system, excitatory amino acids glutamate and its receptor, NO and other calmodulin, which are widely confirmed by basic and clinical science, but its mechanism is too complicated to be fully cleared.Butylphthalide (dl-3n-butylphthalide, NBP), is a class of new drugs developed by the Chinese Academy of Medical Sciences and the National Drug Research Institute of Peking Union Medical University, which we have independent intellectual property rights.NBP is of racemic yellow oily liquid firstly separated, purificated and synthesised from natural plant celery seed, having good anti-ischemic efficacy. Butylphthalide's mechanism against cerebral ischemia is complexity, which is a multi-gene, multi-target and multi-link process. NBP play a pharmacodynamics by reducing cerebral edema, improving cerebral energy metabolism, improving micro-circulation ischemic brain areas, inhibiting neuronal apoptosis, anti-thrombosis, anti-platelet aggregation, inhibiting of glutamate release, decreasing the intracellular calcium concentration, inhibiting free radical and improving antioxidant enzyme activities. Current studies about Butylphthalide are focused on cerebral ischemia field, lacking of relevant studies on VD. MMPs, especially MMP-2 and MMP-9's important roles in the pathogenesis of VD have attracted more and more attentions of scientific researchers.We have reasons to believe that MMPs may be one of butylphthalide's target for the treatment of VD. This study probe on Butylphthalide's therapeutic and protection mechanism to VD rats, and then to provide a scientific basis for VD's clinical treatment.Objective:To study butylphthalide's effect on learning and memory and hippocampal MMP-2, MMP-9 expression in rats with vascular dementia, studing on the therapeutic mechanism of butylphthalide to chronic cerebral ischemia.Methods :160 healthy male Wister rats aged of 12-16 weeks, weighing about 250-300g, were randomly divided into sham operation group (n = 40), model group (n = 40), high-dose butylphthalide group (n = 40) (a dose of 6mg/kg), low dose butylphthalide group (n = 40) (a dose of 2mg/kg). Vascular dementia model is made by the 2-Vo method. Bilateral carotid arteries are isolated from rats, sham operation group only isolated without ligation.In model, high-dose butylphthalide group and low dose butylphthalide group rats bilateral carotid artery are permanent ligatured.Large dose butylphthalide group and low dose butylphthalide group from the date of surgery is given the appropriate dose of butylphthalide intraperitioneal injection, the model group is given the same amount of saline and sham group were not administered. After administration of the 1d, 3d, 5d, 7d, 14d and 30d, at each time point three rats in each group were randomly selected,brains were removed after cardiac perfusion, which are coronal cut after the optic chiasm at 1mm and 4mm to remove the cerebellum and brain stem, taking the middle of the hippocampus, then to fixation, dehydration, dipping wax, embedded into paraffin sections, immunohistochemistry staining and obseving the expression of MMP-2 and MMP-9. After the 30 day's administration the learning and memory ability of the remaining rats in each group is tested by platform avoidance test and Morris water maze test .HE staining is used to obseve hippocampal pathology. Datas are presented as mean±standard deviation (x±s) and analysised by ANOVA analysis using the statistical software of SPSS 11.0, LSD-t test was used for each pairwise comparison between groups, P < 0.05 was statistically significant.Results:1. The general situation of experimental animalsThe rats were hair untidy, activity reducing, apathetic, less Eating and drinking after operation. Comparing to the postoperative anesthesia recovery time and physical recovery time, sham operation group was significantly earlier than the model group.High-dose butylphthalide group and low dose butylphthalide group, while high-dose butylphthalide group and low-dose butylphthalide group was significantly earlier than the model group. Sham control group need 1 days to restore the spirit,while butylphthalide group need about 2-3 days and model group need about 5-7 days'of recovery.2. Platform avoidance test and Morris water maze testThe rats are tested 31 days after operation by platform avoidance test and Morris water maze test as a study performance,and 32 days after operation as a memory performance. In platform we use avoidence test escape latency of rats and the number of errors as the academic performance,while latency and error frequency as the memory performance; In Morris water maze test we use avoid latency and error frequency as the academic performance,while staying in the target quadrant time as the memory performance..2.1 Platform test results showed that: In the academic test,compared with model group, high-dose butylphthalide group and low dose butylphthalide group have significantly shorter escape latency respectively (85.23±12.71 ,56.18±11.36,57.96±11.48)s and significantly less number of errors respectively (4.78±0.79,2.08±0.41,2.09±0.45); In the memory test,compared with model group, high-dose butylphthalide group and low dose butylphthalide group have significantly prolonged latency respectively (72.84±10.26 , 152.27±11.70 ,153.19±11.58) s and decreased number of errors respectively (1.86±0.13,0.53±0.05,0.55±0.06) .2.2 Morris water maze test results show that: In the academic test,compared with model group, high-dose butylphthalide group and low dose butylphthalide group have significantly shorter escape latency respectively (80.71±8.27,43.25±5.29 , 44.74±5.82)s and decreased number of errors respectively (37.53±7.69,14.76±5.01,15.68±5.32); In the memory test, compared with model group, high-dose butylphthalide group and low dose butylphthalide group have significantly longer the former platform quadrant time respectively (18.67±5.39,32.35±4.27,31.19±4.78)s.3. HE staining in hippocampus of ratsSham operation group: neuronal degeneration and necrosis and cell swelling was not obvious, the nucleus is large and round, cells are tightly packed, which have clear boundaries with the surrounding neurons; model group: neuronal degeneration and necrosis and cell edema are obvious, cells are nuclear pyknosis, which have significant edema boundaries with the surrounding neurons; Butylphthalide treatment groups: The level of apoptosis and degeneration in neuron is between the sham operation group and model groups, the structure between neurons is clear, edema boundaries with the surrounding neurons isn't obvious and damaged neurons were significantly reduced compared with the model. neuronal pathological damage in high dose butylphthalide group is ligter than the low dose butylphthalide group.4. MMP-2, MMP-9 immunohistochemical stainingThe rats were respectively narcosized in the 1d, 3d, 5d, 7d, 14d and 30d by 10% chloral hydrate,whose brains were removed, fixed by 4% paraformaldehyde perfusion, embedded in paraffin and sected coronaly,. SP immunohistochemical staining method is used to observe the MMP-2, MMP-9 expression status. Image acquisition system and image analysis software was used to analyze the images. Compared with model group, high-dose butylphthalide group and low dose butylphthalide group the expression of MMP-2 and MMP-9 at all time points was significantly reduced, p<0.05, statistic have significance, indicating that the butylphthalide can reduced the expression of MMP-2 and MMP-9.Conclusion:1. Butylphthalide dementia can improve learning and memory abilities of VD rats in platform avoidance test and Morris water maze test .2.MMP-2 and MMP-9 expression was significantly increased in hippocampal neurons of chronic cerebral ischemia rats.3. Butylphthalide can significantly reduce hippocampal neurons'expression of MMP-2 and MMP-9.4. Butylphthalide reducing expression of MMP-2 and MMP-9 may be involving for NBP's treatment for VD. |
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