The Changes Of Autophagy Related Proteins Expression In Hippocampus Of Vascular Cognitive Impairment Mice And The Intervention Effects Of Lithium Chloride

Objective: Vascular cognitive impairment(VCI) is caused by a variety of cerebrovascular disease with learning and memory dysfunction as the main symptoms of intelligent syndrome. It is a chronic progressive disease, which could eventually develop to irreversible. Along with population aging, cognitive impairment has imposed a heavy burden to individuals, families and society. And so it is urgent to looking for a kind of effective drug treatment. Considerable studies characterize repeated cerebral ischemia-reperfusion injury is one of the leading causes of VCI. Thus, exploring the mechanism by which repeated cerebral ischemia-reperfusion injury leads to VCI and seeking the effective drugs for VCI have become one of the focuses of the medical profession. Repeated cerebral ischemia, in particular subsequent reperfusion, can induce mitochondrial damage and result in producing a large amount of reactive oxygen radicals, which can produce amounts of dysfunctional proteins and damaged organelles. Hence, autophagy is promoted, which leads to autophagic cell death. It has been demonstrated that the activation of glycogen synthase kinase-3β/mammalian target of rapamycin( GSK3β/m TOR) signaling pathway is very important for neurons to resist the excessive activation of autophagy. The experimental studies have confirmed that lithium chloride can alleviate cognitive impairment induced by neurodegenerative diseases and acute brain injury. Despite the numerous elucidations on the impact of lithium chloride on cognition improvement, little is known regarding the benefit of lithium chloride that is associated with inhibition of autophagy through activating GSK3β/m TOR signaling pathway. In our previous studies, we observed that lithium chloride administration at the dose of 2 mmol/kg in mice subjected to repeated cerebral ischemia can inhibit the activity of GSK-3β. Based on the above research, the current study was to investigate the protective effect of lithium chloride on spatial cognitive impairment induced by repeated cerebral ischemia in mice and by futher exploring whether the therapeutic benefit of lithium chloride was associated with inhibiting autophagy through activating m TOR pathway.Methods: Male C57BL/6 mice weighting 24-26 g, aged 10-12 weeks, were subjected to cerebral ischemia for 20 min via bilateral common carotid artery(BCCA) occlusion, and this was repeated once after 10 min. The pattern was repeated three times. Experiment 1: C57BL/6 mice were randomly divided into sham, 1d, 3d, 7d, 14 d and 28 d groups after operation. The time course expressions of p-m TOR, microtubule-associated protein light chain 3(LC3)-Ⅱ/Ⅰ and Beclin1 in the hippocampus were evaluated by western blot. Experiment 2: C57BL/6 mice were randomly divided into 4 groups: Sham group, Vehicle group, Pre-Li group, Li group. Pre-Li group was injected intraperitoneally 2mmol/kg before operation for 7d, and Li group was injected intraperitoneally 2mmol/kg after operation for 14 d. Sham and vehicle group were injected intraperitoneally equal volume physiological saline.The Morris water maze test was conducted at day 15-20, to investigate the protective effect of lithium chloride on spatial cognitive impairment induced by repeated cerebral ischemia in mice. After the behavioral test, nissl staining and transmission electron microscopy(TEM) were used to observe the pathological changes in the hippocampus. Western blot were applied to detect m TOR, p-m TOR, Beclin1 and LC3-Ⅱ/Ⅰ expressions in hippocampus tissue.Results:1 Spatial cognitive ability of Morris water maze test: All the mice showed a progressive decline in the escape latency with training. The mice in the vehicle group exhibited significant prolonged escape latencies during the 5-day training period compared with the sham group(P<0.01). After administration with lithium chloride, these two groups showed shorter escape latencies compared with the vehicle group, with statistic significance(P<0.01). During the probe trial, there was difference in the percentage of time that mice spent in the target quadrant among all groups. The vehicle group spent significantly less time in the target quadrant compared with the sham group(P<0.01). Compared with the vehicle group, mice in Pre-Li group evidently increased the time spent in the target quadrant(P<0.01). 2 Nissl staining observation: Nissl staining demonstrated that the pyramidal neurons in the CA1 region of the hippocampus in the sham group were tightly ranked in order, and the neurons were clear and moderate in size with normal microstructure. By contrast, the vehicle group showed fewer pyramidal neurons with loosely arranged and neuronal shrinkage. Administration of lithium reversed the morphologic changes. 3 Ultrastructural alterations of CA1 in hippocampus: A complete morphology of neuron could be seen from TEM image of pyramidal neuron in the sham group CA1 region. Nuclear envelope was integral and chromatin is distributed evenly, and the organelles were abundant including mitochondria with the ridge clearly visible, rough endoplasmic reticulum and Golgi apparatus. Whereas mitochondrial vacuolization, pyknotic nuclei,chromatin margination, and detachment of ribosomes from rough endoplasmic reticulum could be seen in model group. However, the TEM images of Pre-Li and Li groups seemed better, the vacuolization of mitochondrial and detachment of ribosomes from rough endoplasmic reticulum were significantly alleviated. 4 Western blot results: 4.1 The dynamic expressions of m TOR, p-m TOR, Beclin1 and LC3Ⅱ/Ⅰ in hippocampus of mice after repeated cerebral ischemia: There was no marked difference in total m TOR expression among the six groups(P>0.05). But the expression of p-m TOR was up-regulated compared with the sham group(P<0.01). The expression of Beclin1 and the ratio of LC3Ⅱ/Ⅰand were up-regulated compared with the sham group, beginning at 1d and peaking at 14 d after repeated cerebral ischemia(P<0.01). 4.2 Lithium regulated the expression of p-m TOR,Beclin1 and the ratio of LC3Ⅱ/Ⅰ in hippocampus: The expression of p-m TOR was significantly increased in Pre-Li and Li groups compared with the vehicle group(P<0.01). However, there was no significant difference in total m TOR expression among the four groups(P>0.05). The ratio of LC3Ⅱ/Ⅰand the expression of Beclin1 were reduced in Pre-Li and Li groups compared with the vehicle group(P<0.01).Conclusions: The successful model establiased by repeated cerebral ischemia-reperfusion can be used to evaluate whether lithium is effective for spatial cognition. The present study indicates that autophagy related mechanisms may be involved in the process of VCI induced by repeated cerebral ischemia-reperfusion. The protective effects of lithium against cognitive impairment induced by repeated cerebral ischemia-reperfusion in mice are at least partly mediated by down-regulation the ratio of LC3Ⅱ/Ⅰ and the expression of Beclin1 through activating the m TOR pathway.