| Objective:Bilateral common carotid artery ligation(2-VO)is a classic method to establish a model of vascular cognitive impairment.However,in pharmacological research,this method has the problem of "large differences in the range and degree of cognitive impairment in rats" in terms of technical means.This is in contrast to the clinically vascular cognitive impairment caused by ischemic stroke not only manifested as dementia mainly characterized by decline in learning and memory,but also as changes in other cognitive domains,including negative emotions such as communication disturbances and depression.One question matches.Therefore,we put forward the hypothesis that the way and degree of changes in the cognitive domain of rats are related to the degree of cerebral ischemia.After many person-time verifications in the early stage of the laboratory,it is found that the experimental operators have different ability to control the ligation tension,and the behavioral changes of the rats are quite different.It is speculated that the force during the ligation process may be an important reason for the degree of cerebral ischemia in rats.Therefore,this experiment is based on the metal strain gauge as the core component to design and prepare the vascular occlusion device,and establish the relationship between "control ligation tension-rat cerebral ischemia degreecognitive domain range and degree".And verify this hypothesis from the overall level,the level of tissues and organs,and the level of cells and molecules,aiming to perfect the basic research of the 2VO method to establish animal models of vascular cognitive impairment,and to achieve the technical rigor of the methodological process in pharmacological research.It is of certain significance to improve the uniformity and stability of pharmacological methodological research.Methods:(1)Based on the metal strain gauge as the core component,the vascular occlusion device was designed and prepared,and the intravenous infusion tube and the rat small intestine were selected for verification.(2)The quantified 2-VO method of vascular blocker was used to establish cerebral ischemia models of different degrees:100 male Wistar rats were randomly divided into SO group,2-VO group,0.5 N group,1.5 N group,and 2.5 N group.The cerebral blood flow was measured with a dual-channel laser Doppler cerebral blood flow meter before and after the model was established,14 d,28 d,and 42 d;and the HE staining method was used to observe the changes of brain tissue morphology on 42 d.(3)To verify the changes in cognitive domains of different degrees of cerebral ischemia from the overall level:Morris water maze was used to test the learning and memory ability of rats on 42 days;social experiments,open field experiments,sucrose preference experiments,and forced swimming were used on 28 days and 42 days.The experiment detects changes in social,anxiety-like and depression-like behaviors in rats.(4)To verify the effects of different degrees of cerebral ischemia on cognitive impairment at the cellular and molecular level:42 days to detect the activity of CHE,SOD,MDA,GSH-Px and CAT in brain tissue homogenate using enzyme activity detection kit;42 days Western blot was used to detect the expression of BDNF and TNF-α in brain tissue.Results:The applicable range of the vascular blocker is between 0.0-3.0 N.The cerebral ischemia model was established based on the vascular blocker combined with 2-VO method,and the death rate of rats was reduced from 55.0%to 11.1%.The results of cerebral blood flow measurement showed that compared with the SO group,the cerebral blood flow of the 2-VO group,0.5 N group,1.5 N group,and 2.5 N group decreased significantly after the model operation,14 d,28 d,and 42 d(p<0.01);Compared with the 2-VO group,the cerebral blood flow of the 0.5 N group increased significantly(p<0.01,p<0.05),and the cerebral blood flow of the 2.5 N group decreased significantly(p<0.01).Compared with the 0.5 N group,the cerebral blood flow of the 1.5 N group and the 2.5 N group was significantly decreased(p<0.01);compared with the 1.5 N group,the cerebral blood flow of the 2.5 N group was significantly decreased(p<0.01);tail cuffs were used There was no significant difference in blood pressure measured by tube method(p>0.05).The results of HE showed that cerebral ischemia rats had different degrees of neuronal pyknosis,some neurons were lost,and the distribution of cytoplasm and nucleus was unclear.The behavioral changes of rats with different levels of cerebral ischemia showed:(1)The results of Morris water maze showed that:when the model was established 42 days,compared with SO group,2-VO group,0.5 N group,1.5 N group,2.5 The escape latency and total distance of rats in group N increased significantly(p<0.01,p<0.05),and the number of platform crossings in groups 1.5 N and 2.5 N was significantly reduced.Compared with group 0.5 N,rats in group 1.5 N and 2.5 N The escape latency increased significantly(p<0.05).(2)The social results showed that compared with the SO group,the rats in the 2.5 N group showed significant differences in the duration and frequency of contact at 28 days after the model was established(p<0.05),which lasted to 42 days;There was a significant difference in the continuous contact time at 28 days and 42 days after the model was established(p<0.05).Compared with the 0.5 N group,the 2.5 N group had a significant reduction in the continuous contact time(p<0.05).(3)The results of the open field experiment showed that when the model was established on 28 d and 42 d,the standing times of the 1.5 N group and the 2.5 N group,the peripheral time of the rat,the number of times of entering the center and the number of squares,the total number of squares,the distance and speed of the movement There are significant differences(p<0.01,p<0.05).(4)The results of sucrose preference and forced swimming experiments showed that the consumption of sugar and water in the 1.5 N group and 2.5 N group was significantly reduced at 28 days and 42 days after the model was established(p<0.01,p<0.05).The detection results of the biochemical indicators of rat hippocampus tissue with different levels of cerebral ischemia showed that compared with the SO group,the activities of CHE,SOD,and GSH-Px in the 2-VO group,0.5 N group,1.5 N group,and 2.5 N group were significantly decreased(p<0.05),MDA activity was significantly increased(p<0.05),CAT activity did not change significantly(p>0.05);compared with 0.5 N group,1.5 N group,2.5 N group CHE,SOD,GSH-Px activity significantly decreased(p<0.05),MDA activity was significantly increased(p<0.05);CAT activity did not change significantly(p>0.05);compared with SO group,BDNF protein expression in 2-VO group,1.5 N group,and 2.5 N group Decrease(p<0.05).Compared with 0.5 N group,the expression of BDNF protein in 1.5 N group and 2.5 N group decreased(p<0.05).Compared with the SO group,the expression of TNF-α protein in the 2-VO group,1.5 N group,and 2.5 N group increased(p<0.05).Compared with the 0.5 N group,the TNF-α protein expression in the 1.5 N group and 2.5 N group was increased.Increased expression level(p<0.05).Conclusion:Within a certain range,the vascular blocker can achieve the purpose of quantifying the cerebral ischemia model by controlling the ligation force.There is a positive correlation between the degree of cerebral ischemia and the ligation force within a certain range,but after the ischemia reaches a certain degree,even if the ligation force increases,the degree of cerebral ischemia no longer changes,and the main manifestation is that the cognitive domain and biochemical indicators no longer change.The changes to the cognitive domain tend to become more singular.This experiment can indicate that in the basic research of cerebral ischemia-related pharmacology,the vascular blocker can achieve the purpose of quantifying the cerebral ischemia model.If the research of vascular dementia is carried out,the bilateral neck should be ligated with 1.5 N-2.5 N force.The common artery is sufficient;for post-stroke depression and post-stroke anxiety-related research,a force of about 1.5 N can be used for ligation. |
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