The Changes Of Mitochondrial Dynamics In The Hippocampus Of Vascular Dementia Mice And The Intervention Effects Of L-3-n-Butylphthalide

Objectives: With the growth of population and the increase of lifeexpectancy, the incidence of cerebrovascular disease is still ascending year byyear. Consequently, the incidence of vascular dementia (VD) in the world isalso found in a continued upward trend. Since the highest incidence of strokein China, the number of VD patients is rising sharply. There are more socialburden, economic burden and family burden from VD patients, so muchconcerns have been paid on the medical profession of VD.The functions of learning and memory are closely related to hippocampalneuron. Hippocampal neuron is extraordinarily sensitive to ischemia andhypoxia. In the process of ischemic damage, the balance of mitochondrialdynamics may be disturbed in hippocampal neurons, which possibly interferewith energy metabolism and accelerate cell death through a variety ofchannels. Mitochondrial dynamics refers to the mitochondria in a dynamicprocess of continually fission and fusion. In such process, outer membranefusion is regulated mainly by mitofusin1(Mfn1) and mitofusin2（Mfn2）,while mitochondrial fission protein1(Fis1) involves in the fission. It isreported that the imbalance of mitochondrial dynamics plays an important rolein a variety of nervous system diseases, but its role in the pathogenesis of VDis not yet clear. In the present study, expression changes of thesemitochondrial fusion and fission proteins will be detected in the VD mousehippocampus, the effcets of l-3-n-butylphathlide (l-NBP) on mitochondrialdynamics will be observed as well.What we do might make a little effort toexplore the pathogenesis and medications of vascular dementia.Methods: C57BL/6mice (22-26g) of clean grade, aged10-12weeks,were subjected to20min ischemia and10min reperfusion by ligating the bilateral common carotid arteries for three repeats to establish VD mice model.The mice were divided into five groups: sham control group, VD model group,pretreated l-NBP group, low-dose treatment group (l-NBP30mg/kg) andhigh-dose treatment group (l-NBP60mg/kg). Mice in the pretreated groupwere administrated before operation for7days, the treatment groups wereadministered for28days after operation, while the model group were givencorn oil as drug-solvent control.(1) During the days from29thday to35thday,mice in each group were tested by Morris water maze to record the function oflearning and memory;(2) After behavioral tests, HE staining was used toobserve hippocampal histopathological changes;(3) RT-PCR was applicatedto detect the changes of Mfn1/2and Fis1mRNA levels of hippocampus tissue,while the expressions of Mfn1/2and Fis1protein were tested by Western-blot.Result:1Morris water maze test1.1The orientation navigating test during the29thday to34thday to evaluatethe function of learning:Compared with sham group, the eacape latency was significantlyprolonged in VD model group (P <0.05). Compared with VD model group,the eacape latency was significantly shortered in pretreated group andhigh-dose treatment group (P<0.05), while there was no significant differencein the low-dose treatment group.1.2The spatial searching test in the35thday to evaluate the function ofmemory:Compared with sham group, mice in VD model group spent more time inthe platform quadrant (P<0.05). Compared with VD model group, mice inboth pretreated group and high-dose treatment group spent more time in theplatform quadrant (P<0.05), while there was no significant difference in thelow-dose treatment group.2HE staining was used in mice hippocampus in each groupIn sham group, the outline of hippocampal pyramidal cells was neat,intact with clear nucleolus and plump nucleus. In other groups, a segment loss of pyramidal cells could be seen in varing degrees. The most significant lossof pyramidal cells was found in VD model group, followed by low-dosetreatment group.Compared with model group, less lesion of pyramidal cellsappreared in pretreated group and high-dose treatment group.3The levels of Mfn1, Mfn2and Fis1mRNA by PCR3.1The changes of Mfn1/2mRNA levelCompared with the sham group, there were significant increases ofMfn1/2mRNA levels in model group (P<0.05). Compared with the modelgroup, Mfn1/2mRNA levels were markedly reduced in pretreated group andhigh-dose treatment group (P<0.05), while Mfn1/2mRNA levels in low-dosetreatment group remained high.3.2The change of Fis1mRNA levelCompared with the sham group, there was a significant decrease of Fis1mRNA level in model group (P<0.05). Compared with the model group, Fis1mRNA level was markedly increased in pretreated group and high-dosetreatment group (P<0.05), while Fis1mRNA level in low-dose treatmentgroup was still low.4The expressions of Mfn1, Mfn2and Fis1protein by Western-blot4.1The expressions of Mfn1/2proteinsCompared with the sham group, there were significant increases ofexpressions of Mfn1/2proteins in model group (P <0.05). Compared with themodel group, expressions of Mfn1/2proteins were markedly reduced inpretreated group and high-dose treatment group (P<0.05), while Mfn1/2protein in low-dose treatment was still in a high expression levels.4.2The expression of Fis1proteinCompared with the sham group, there was a significant decrease of theexpression of Fis1protein in model group (P<0.05). Compared with the modelgroup, expression of Fis1protein were markedly increased in pretreated groupand high-dose treatment group (P<0.05), while Fis1protein in low-dosetreatment was still in a low expression level. Conclusion:1In the present study, a preferable VD mice model was established. Theimpairments of learning and memory functions were tested in Morris watermaze and obvious damages in hippocampal tissue were found by HE straining,which suggested that this model might successfully imitate the cognitiveimpairment of VD patients in clinical works and was feasible to study VD.2In VD mice, the expressions of Mfn1/2were increased while theexpression of Fis1was reduced significantly, accompanied with the loss ofhippocampal neurons. It was suggested the previous balance of mitochondrialdynamics might be disturbed after repeated cerebral ischemia and reperfusion,which could be associated with the damage of hippocampal neuron.3The functions of learning and memory in some mice and the imbalanceof mitochondrial dynamics could be improved to some degree with theintervention of l-NBP, which suggested its neuroprotective effects onregulating mitochondrial dynamics.