Protective Effects Of Lithium Chloride Treatment On Repeated Cerebral Ischemia–reperfusion Injury In Mice

Vascular cognitive impairment(VCI) is caused by a variety of cerebrovascular disease with learning and memory dysfunction as the main symptoms of intelligent syndrome. With the elderly population increasing and aging speeding up, the incidence of cerebrovascular disease has increased year by year. It seriously influences the life quality for the elderly, and has a trend for the young. Except limb dysfunction caused by cerebrovascular disease, it also causes cognitive dysfunction and mood disorders. So it is important to take early positive and effective prevention and treatment to reduce the burden of patient’s themselves and their family.Cerebral repeated ischemia-reperfusion(IR) injury plays an important role in the pathogenesis of VCI. Repeated IR mice model has been considered as a useful animal model for studying cognition impairment associated with VCI. Lithium is a renowned pharmacological treatment for mood disorders. Recently lithium has also been recognised as a neuroprotective agent for managing neurodegenerative diseases and cerebrovascular damage. However, the exact mechanism for lithium treating learning and memory is not clear.Studies show that hippocampus is not only the main brain region associated with learning and memory, but also the ischemia vulnerable area. Protein kinase B(Akt)/glycogen synthase kinase-3β(GSK-3β) signal pathway is the major member of neurotrophin-dependent signaling pathways and has a major function in learning and memory in the hippocampus. Repeated IR results in a large number of reactive oxygen species generation and causes a lipid peroxidation damage. Reducing oxidative stress damage timely and effectively may be one of feasible methods for the prevention and treatment of VCI. Brain-derived neurotrophic factor(BDNF) is the most abundant neurotrophin in the brain and absence of neurotrophins promotes neuronal death. BDNF also exerts an anti-apoptotic effect by stimulating the production of the Bcl-2 family proteins to play neuroprotective effects. One of the most well known transcriptional regulators of BDNF gene expression is the cyclic AMP responsive element binding protein(CREB). The activation of CREB through its phosphorylation on Serine-133(p-CREB) has been implicated in synaptic plasticity, long term memory formation and consolidation.Based of these, the VCI model was made by repeated bilateral common carotid artery occlusion(BCCAO) for 20 min repeated thrice with an interval of 10 min. Pretreatment and treatment with 2 mmol/kg or 5mmol/Kg Li Cl were administrated to observe:(1) whether it can change the learning and memory function and hippocampus morphology in mice by adjusting Akt and GSK-3β protein expression;(2)whether it can reduce oxidative stress by adjusting MDA content, SOD, GSH-PX and CAT activities;(3)whether it can depress apoptosis and activate BDNF/CREB signaling pathway by adjusting Bcl-2, Bax, BDNF and CREB gene or protein levels. Part Ⅰ Protective effects of lithium chloride treatment on repeatedcerebral ischemia-reperfusion injury in mice by activating Akt/GSK-3β signaling pathwayObjective: To establish a VCI mice model induced by cerebral repeated ischemia-reperfusion and to determine whether Akt and GSK-3β activities are the essential components in the repeated IR injury after 2 weeks, 4 weeks and 6 weeks. And if so, whether 2mmol/Kg lithium postoperative enhances or counteracts this action to play the protective effects on the hippocampus of mice.Methods: A total of 144 C57Bl/6 male mice were randomly distributed into four groups:(1) Na Cl-treated sham group(with sham operation, physiological saline administered, n = 36);(2) Na Cl-treated model group(repeated IR, physiological saline administered, n = 36);(3) Li Cl-treated sham group(with sham operation, Li Cl administered, n = 36); and(4) Li Cl-treated model group(repeated IR, Li Cl administered, n = 36). Each group was investigated for 2, 4 and 6 weeks postoperative(subgroup, n = 12). The model groups were suffered from repeat bilateral common carotid artery occlusion(BCCAO) for 20 min thrice with an interval of 10 min. The Li Cl-treated mice were intraperitoneally administered with 2 mmol/kg Li Cl daily postoperative. The next day after the final injection,all the survival mice were evaluated learning and memory abilities by the water maze test for four consecutive days. The swimming time and error count on the third and fourth days were recorded as the learning and memory score. Shortly after behavioural evaluation, hematoxylin-eosin(HE) staining and TUNEL assay were used for pathological and apoptotic examination. Akt and GSK-3β protein expression were analyzed by Western blot.Results: The mortality rates were 16.67% and 11.11% in the Na Cl- and Li Cl-treated model groups. The numbers of deaths at 2, 4, and 6 weeks postoperative were listed as 2, 1, and 3(Na Cl-treated model group) and 1, 1, and 2(Li Cl-treated model group), respectively. No mortality was observed in the sham groups.Cognitive abnormalities were observed before and after the surgical operation. No difference was observed in learning and memory function among each group preoperative(0 week)(P>0.05). The swimming time and error count in learning and memory function both increased in model groups compared with those in sham groups(P<0.05). Li Cl treatment decreased the prolonged swimming time and reduced the increased error count compared with Na Cl-treated groups(P<0.05). These lithium treatment effects were highly significant in both learning and memory functions. The peak value was observed at 4 weeks in 2, 4 and 6 weeks of observation period.HE staining demonstrated many tightly arranged and ordered pyramidal cells in the hippocampal areas of the Na Cl- and Li Cl-treated sham groups. The nucleus were circinal and large with a clear nucleolus, and TUNEL-positive cells were difficult to detect. By contrast, the 4 and 6 week Na Cl-treated model groups showed fewer pyramidal cells and were loosely arranged in the hippocampal area. The nuclei also showed pyknosis, were deeply stained, and with no clear nucleolus seen. The TUNEL-positive cells were abundant as compared with sham group(P<0.05). Most damage was observed 4 weeks postoperative, which showed only several neurons alive in some areas. However, the animals treated with Li Cl showed less cell reduction and pyknosis. The number of apoptotic cells decreased, especially at 4 and 6 weeks(P<0.05).Western blot assay showed that the phosphor-Akt Ser473 and phosphor-GSK-3β Ser9 protein levels were higher in the Li Cl-treated group than in the Na Cl-treated group(P<0.05). The model animals exhibited to some extent higher phosphor-Akt Ser473 and phosphor-GSK-3β Ser9 than those of the sham group. However, the levels of these changes were much lower than those of the Li Cl-treated group. The expressions of the two proteins varied at different time points postoperative, and the highest amount was observed at 4 weeks postoperative.Conclusion: Repeated IR mice model is a useful animal model for studying cognition impairment and pathological changes associated with VCI. 2 mmol/kg Li Cl exerts a neuroprotective effect in the learning and memory functions by improving pathological changes, reducing apoptotic cell count and increasing phosphorylation of Akt and GSK-3β. Part II Lithium chloride with different doses and treated time improves cognitive function and pathological damage in VCI miceObjective: To further evaluate the protective effects of different doses and treated time of lithium chloride on cognitive function and pathological damage tested by Morris water maze and Nissl staining.Methods: Sixty mice were randomly distributed into 6 groups:(1) sham group;(2) vehicle group;(3) pre-Li Cl-low model group(pre-Li-L);(4) Li Cl-low model group(Li-L);(5) pre-Li Cl-high model group(pre-Li-H);(6) Li Cl-high model group(Li-H). Each group was investigated for 4 weeks postoperative(n = 10). The model groups were suffered from repeated BCCAO for 20 min repeated thrice with an interval of 10 min as in Part I. The pre-Li mice were injected intraperitoneally with 2 mmol/kg(low concentration) or 5mmol/Kg(high concentration) Li Cl daily pre-operative for 7 days and the Li mice were injected intraperitoneally with 2 mmol/kg or 5mmol/Kg Li Cl daily postoperative for 28 days. The next day after the final injection,all the survival mice were evaluated learning and memory abilities by Morris water maze test for six consecutive days.(1) Place navigation test: each mouse received six trails per day for five consecutive days. The time to find the submerged platform(escape latency) was recorded in each trial as the learning score.(2) Spatial probe test: The next day after the place navigation test, the mice were tested on a spatial probe trial in which the platform was removed, and the mice were placed in the opposite quadrant and allowed to swim freely for 60 s. The time percentage of rats spent in the target quadrant where the platform had been located and the count crossed the platform was recorded as memory score. After behavioural evaluation, pathological changes and normal neuron count of hippocampal CA1 region were examed by Nissl staining.Results: On day 29 after repeated IR, a total of 56 mice were evaluated spatial learning and memory abilities by the Morris water maze test. All mice showed a progressive decline in the escape latency with training every day. Further analysis by SNK test revealed that beginning on day 2, mice in vehicle group took significantly longer time to find the platform(P<0.05)compared with sham group. Mice in Li-L, pre-Li-H and Li-H group showed shorter mean latencies compared with vehicle group(P<0.05)on day 3. Over the next two days, mice treated with pre-Li and Li(2mmol/kg and 5mmol/kg) showed significant shorter escape latencies compared with vehicle group(P<0.05), meanwhile, 2mmol/kg Li Cl resulted in the shortest escape latency than the other three groups. Although it was not statistically significant, administration of pre-Li 5mmol/kg resulted in shorter escape latencies than pre-Li 2mmol/kg group. But administration of Li 5mmol/kg group resulted in longer escape latencies than Li 2mmol/kg group which was opposite with expectation. In the probe trial without the platform, mice in vehicle group stayed in the target quadrant for significantly less time and less count crossed the platform than sham group(P<0.05). Compared with vehicle group, mice treated with pre-Li and Li(2mmol/kg and 5mmol/kg) evidently increased the ratio of time spent in the target quadrant and more count crossed the platform(P<0.05).Nissl staining demonstrated that the pyramidal neurons in the CA1 region of hippocampus in sham group were tightly ranked in order, and the neurons were clear and moderate in size with normal microstructure after repeated IR. In vehicle group, obvious pathological changes were exhibited with loosely arranged neurons and neuronal shrinkage, loss and light color staining, and the normal neuron count was significantly decreased(P<0.05). Administration of either pre-Li or Li, especially Li 2mmol/kg per day, evidently reversed the morphologic changes and up-regulated the survival neuron count(P<0.05).Conclusion: Repeated IR insulted impaired spatial learning and memory abilities in Morris water maze, accompanied by less normal neuron count. 2mmol/Kg and 5mmol/Kg pre-Li and Li treatment significantly improved the impaired spatial learning and memory function and increased the normal neuron count which was best in 2mmol/Kg Li group. Part III Lithium chloride with different doses and treated time reduces oxidative stress damage in VCI miceObjective: To evaluate the dynamic MDA content, SOD activity, GSH-PX activity and CAT activity changes after repeated IR and explore whether 2mmol/Kg or 5mmol/Kg pretreatment or post treatment with Li Cl would correct the abnormal expression to reduce oxidative stress damage after repeated IR insult.Methods: First stage: twenty-four mice were randomly distributed into 6 groups:(1) sham group;(2)1 d group after repeated IR;(3) 3 d group after repeated IR;(4) 7 d group after repeated IR;(5) 14 d group after repeated IR;(6) 28 d group after repeated IR. Each group has 4 mice. Second stage: grouping and model preparation were same as in Part III. MDA content, SOD activity, GSH-PX activity and CAT activity were evaluated according to the manufacturer’s instructions in different groups after repeated IR and groups with 2mmol/Kg or 5mmol/Kg pretreatment or post treatment with Li Cl.Results: Compared with the sham group, MDA content increased continuously from 1 day to 28 days, with the peak value at 28 days(P<0.05). SOD activity, GSH-PX activity and CAT activity were significantly lower than the sham group(P<0.05), with the peak value at 7 days after repeated IR. 2mmol/kg and 5mmol/kg pre-Li and Li can significantly reduce the MDA content(P<0.05)and increase SOD and GSH-PX activity(P<0.05), which was best in 2mmol/kg Li Cl treat group. CAT activity was also improved in some extent, but only the 2mmol/kg Li Cl treat group reached the statistical difference(P<0.05).Conclusion: Repeated IR insulted oxidative stress damage from 1 day to 28 days, leading to more reactive oxygen species generation and inhibited antioxidant enzyme activities. 2mmol/kg and 5 mmol/kg pre-Li and Li treatment can play the neuroprotective effect by improving the variety of antioxidant enzyme activities and reducing reactive oxygen species content.Part IV Lithium chloride with different doses and treated time ameliorates cognitive deficits through depressing apoptosis and activating BDNF/CREB signaling pathway in VCI miceObjective: To evaluate the dynamic expression of Bcl-2, Bax and BDNF protein after IR and explore whether 2mmol/Kg and 5mmol/Kg pretreatment and post treatment with Li Cl would depress apoptosis and increase the key molecular of BDNF and p-CREB protein, mediating neuronal survival after repeated IR insult.Methods: Grouping and model preparation were carried out in accordance with Part III. Western blot assay was used for evaluating the dynamic expression of Bcl-2, Bax and BDNF protein after repeated IR. Real Time-q PCR and Western blot were used for detecting Bcl-2, Bax, BDNF, CREB and p-CREB levels after 2mmol/Kg and 5mmol/Kg pre-Li and Li.Results: Western blot assay showed that compared with the sham group, the ratio of Bcl-2/Bax in VCI group was reduced(P<0.05)and the peak value was on IR 7d. The Bcl-2 protein expression was not significant difference among all groups(P>0.05). The insult led to a sustained increase in the BDNF expression compared with the sham group, but it was not significant(P>0.05).Western blot analysis also demonstrated that 5mmol/kg pre-Li Cl significantly increased repeated IR-stimulated reduce of Bcl-2/Bax ratio(35% upregulation)(P<0.05). The effects of 2mmol/kg pre-Li and Li on Bcl-2/Bax ratio were increased by 9% and 17%, respectively, not as effective as 5 mmol/kg pre-Li Cl. However, the Bcl-2 protein levels were not different in each group(P>0.05).Real Time-PCR showed that BDNF m RNA levels was significantly reduced in the hippocampus of VCI mice compared to sham group(P<0.05). However, compared with vehicle group, BDNF m RNA levels extremely increased in group treated with pre-Li and Li(P<0.05), which was highest in Li 2mmol/kg group among the four treatment groups. We next investigated the effects of Li Cl on BDNF, CREB and p-CREB protein expression. 2mmol/kg Li Cl significantly increased the BDNF expression compared with the vehicle group(P<0.05). The effect of 2mmol/kg pre-Li, 5mmol/kg pre-Li Cl and Li on BDNF expression also increased by 35%, 32% and 32%, respectively. 5mmol/kg pre-Li Cl also led to a significantly increase in the expression of p-CREB/ CREB(33% upregulation)(P<0.05). None of these treatments altered the CREB protein expression among all groups(P<0.05).Conclusion: Repeated IR stimulated apoptosis and upregulated BDNF expression with a self-protection from 1 day to 28 days. 2mmol/kg and 5 mmol/kg pre-Li and Li treatment have the ability in depressing apoptosis and increasing hippocampal BDNF and p-CREB expression to play the neuroprotective resulting after IR.