| Vascular cognitive impairment(VCI) is an acquired intelligence damage syndrome caused by all kinds of cerebrovascular disease with learning and memory dysfunction. The late stage of VCI is called vascular dementia(VD). At present, the VD has become the second type of dementia after Alzheimer’s disease in the worldwide. after. However, so far, the pathogenesis of VD is not fully clear and lack of effective treatments. Studies have shown that chronic cerebral hypoperfusion(CCH) injury plays an important role in the pathogenesis of VD process.Thus it has become one of the hot research topics of modern medicine to explore the pathogenesis of VD induced by chronic cerebral hypoperfusion damage, and to seek effective prevention and treatment drugs of dementia.Emerging studies have shown that autophagy and apoptosis of neurons play an important role in the pathogenesis of VD induced by chronic cerebral hypoperfusion damage. So the regulation of autophagy and apoptosis in neurons is very important. Phosphoinositide 3 kinase/protein kinase B(PI3K/Akt) signal pathway is a classic antiapoptotic and survival promotion pathway. The activation of the PI3K/Akt pathway plays an important role in hypoxic ischemia neurons injury. In recent years, research has shown that PI3K/Akt pathway and its related downstream signal factor-mammalian target of rapamycin(m TOR) has a regulatory effect on the activation of autophagy. Oxiracetam(ORC) is a new type of nootropic drugs of pyrrolidone, which chemical name is 4-hydroxy-2-oxo-1-pyrrolidineacetamide. Animal experiments have shown that the ORC can go through the blood brain barrier, selectively effect on hippocampus, cortex and striatum to improve the brain’s ability to use oxygen and glucose and promote the functional recovery of nerve cells. ORC drug itself has no direct blood vessel activity, also have no central stimulants, but can be by enhancing protein kinase C(protein kinases C, PKC) activity, promoting synaptic plasticity, promote brain long cheng(long term potentiation, LTP) the production of a variety of ways, such as on the improvement of the learning and memory functions play a lasting role in promoting. However, the ORC for chronic cerebral ischemia dementia rats nerve protective effect is not very clear.Based on the above research background, the study established rat VD model with chronic cerebral hypoperfusion induced by BCCAO) to observe and investigate(1)the effect of BCCAO on cognitive function in rats and hippocampus morphology change,(2) the protective effect of ORC on nerve cell at different time and different, and weather the protective effect was related to the activation of PI3K/Akt signal pathway?(3) the effect of ORC on the expression of regulation autophagy and apoptosis related proteins in hippocampal neurons and the mechanism of ORC to improve cognitive dysfunction.Part one The establishment of the vascular dementia rat model and the effects of ORCObjective: To observe the behavioral changes and hippocampal pathology changes 4 weeks and 8 weeks after BCCAO in SD rat, and to observe if ORC can improve learning and memory impairment induced by cerebral chronic cerebral hypoperfusion.Methods: 3-month-old male Sprague–Dawley rats weighing 250-300 g were randomly divided into four groups: Sham group, vehicle group, ORC low-dose group(ORC-L), ORC high-dose group(ORC-H). Each group was investigated for 4 and 8 weeks postoperative(subgroup, n = 10). Permanent bilateral common carotid artery occlusion was used to establish VD model of rats. From the next day of the operation, sham group and vehicle group were provided with an equal volume of Na Cl, while ORC-L and ORC-H were treated with 100mg/kg and 200mg/kg respectively through oral treatment.Results: In 4-week groups, general statistical results show that there were statistically difference in the training days(F=166.03, P<166.03), the comparison between groups(F=51.132, P<51.132), and the interaction of training days and group(F=3.515, P<0.01) were. In the place navigation test, the escape latency of all the rats was gradually shortened with training. Compared with the sham group, the escape latency of rats in VD vehicle group was significantly prolonged(P<0.01).Compared with model group, from the beginning of the second day, the escape latency of ORC-L group rats was shorten, in the third and fifth days were the most significant difference(P< 0.05). While the escape latency of ORC-H group were shorten in day 3 to day 5 with obvious statistical significance(P<0.05, P<0.01, P<0.05). In the five days of training, there was statistically difference of escape latency between ORC-H group and ORC-L group, especially in the fourth and fifth day(P < 0.05). In 8-week groups, the general statistical results show that the training days(F=119.222, P<119.222), the comparison between groups(F=13.376, P <13.376), and the interaction of training days and group(F=2.011, P<0.05) were statistically difference. Compared with the sham group, the escape latency of rats in VD vehicle group was significantly prolonged(P<0.01).Compared with the sham group, the escape latency of model group rats were significantly prolong in the five training days with statistical significance(P<0.01 in day1, P<0.05 in day 2, P<0.01 in day3 to day5), suggesting the effective VD model was made in the experiment. In the spatial probe test, There was significant difference in the ratio of time that rats spent in the target quadrant among four groups. Compared with the sham group, the vehicle group spent less time in the target quadrant, with statistic significance(P<0.01). While the deficits were significantly improved with treatment of ORC(P<0.01).These ORC treatment effects were highly significant in both learning and memory functions. The peak value was observed at 8 weeks in 4 and 8 weeks of observation period.Conclusion: BCCAO rat model is a useful animal model for studying cognition impairment and pathological changes associated with VD, which to some extent is similar to the onset of clinical VD process and is an ideal animal model of VD.ORC can effectively improve the cognitive functions of VD rats, which exerts a neuroprotective effect in the learning and memory abilities.Part two ORC with different doses and treated time improves cognitive function and pathological damage in VD ratsObjective: To further evaluate the protective effects of different doses and treated time of ORC on pathological damage after BCCAO tested by HE staining.Methods: After behavioural evaluation, pathological changes of hippocampal CA1 region were examed by HE staining.Results: The pyramidal cells in the hippocampal CA1 areas of the sham group arranged densely and orderly. The size of cells was normal and the morphology was integrity. The nucleolus and cytoplasm were clear. Compared with the sham group, there was significantly loss in neurons in vehicle group. The arrangement of pyramidal neurons was loosely and irregularly. Some neurons turned karyopyknosis, and cytoplasm hyperchromatic. After the intervention of ORC, the morphological changes described above had improved. The loss of neurons in CA1 area of hippocampus reduced, but there still exits a little amount of karyopyknosis. Rats in the vehicle group 4 weeks after BCCAO suffered the most injury with only a small amount of neuron survival. In the ORC low dose and high dose treatment group, neuron loss was reduction, there was still a small nuclear pyknosis.Conclusion: Chronic cerebral hypoperfusion damage after BCCAO caused learning and memory disorders in rats, at the same time can cause pathological changes in hippocampal neurons, especially in 4 week vehicle group. After ORC intervention, the cognitive function of rat was improved, pathological changesof the hippocampus neuron damage has also been improved in a certain degree. The effect of high dose group is better than the lower dose treatment groups.Part three The influence of ORC in PI3K/Akt pathway in VD ratsObjective: To observe the Akt, p-Akt, m TOR, p-m TOR protein expression in hippocampus of VD rats and the effects of ORC.Methods: The expressions of Akt, p-Akt, m TOR and p-m TOR in hippocampus were studied by measure of Western Blot at 4 weeks and 8 weeks after BCCAO operation.Results: Compared with the vehicle group rats, ORC-L and ORC-H group rats showed a strong expression of p-Akt(P<0.01).ORC induced expression of p-m TOR Ser2448 in the hippocampus of vehicle rats. There is no marked change in total Akt and m TOR among 4 groups(P>0.05).However, p-m TOR Ser2448 were significantly increased in the VD model rats compared with those in the sham group(P<0.01). Moreover, ORC-L and ORC-H group rats showed a strong expression of p-m TOR compared with the vehicle group rat(P<0.01, P<0.01).There was the same trend of the expressions of Akt, p-Akt, m TOR and p-m TOR in 4 weeks and 8 weeks after BCCAO operation.Conclusion: The chronic cerebral hypoperfusion damage caused hippocampal neuron damage, the application of ORC allviated hippocampal neurons injury and improved cognitive function in rats, which the effects may be mediated by the activation of PI3K/Akt signal transduction pathway.Part four The influence of ORC on the expression of apoptosis and autophagy related proteins in vascular dementia ratsObjective: To observe the apoptosis related proteins Bcl- 2 / Bax ratio and autophagy related proteins LC3II/I ratio, Beclin 1, p62 protein expression in hippocampus of VD rats, further to observe the intervention effect of ORC on neuron apoptosis and autophagy.Methods: The expressions of Bcl-2 / Bax ratio, LC3II/I ratio, Beclin 1 and p62 protein in hippocampus were studied by measure of Western Blot at 4 weeks and 8 weeks after BCCAO operation.Results: C ompared with control group, Bcl-2/Bax ratio in VD model group rats was decreased with statistical significance(P<0.01). Compared with vehicle group, the ratio was increased after ORC treatment with statistically significant(P<0.05). There was no statistical difference between low dose and high dose group(P>0.05). Compared with the sham group, LC3-II/I ratio increased, Beclin 1 protein expression level increased significantly, p62 protein expression decreased significantly in the vehicle group(P<0.05, P<0.01, P<0.01). Compared with vehicle group, LC3-II/I ratio decreased, p62 protein expression was significantly increased in ORC treatment group. The expression changes in high dose treatment group rats was obvious than rats in low dose treatment group(P<0.05); Beclin 1 protein expression was decreased without statistically significant(P>0.05). There was the same trend of the expressions of Bcl-2/Bax, LC3II/I,Beclin 1 and p62 in 4 weeks and 8 weeks after BCCAO operation..Conclusion: C hronic cerebral hypoperfusion damage induced cell apoptosis and autophagy excessive activation, which resulted in hippocampus neuron degeneration and death. Application of ORC can play a role of neuron protection, which may be acted by adjusting the neuron apoptosis and autophagy level. |
