Effects Of Cyclosporin A On Blood-brain Barrier After Ischemia-Reperfusion In Mice

Objective :Through building the ischemia-reperfusion model on mice, intraperitoneal inject different doses of cyclosporine A, to observe the mice nerve function score, infarcts area, brain water content and the change of Evans blue leakage amount,Using Western Blot method analyze the changing trends of Cyp A, Akt, NF- k B, MMP9, Claudin- 5 and so on,to discuss the effects and mechanism of cyclosporine A on Blood-Brain Barrier after Ischemia-Reperfusion in mice, providing experiment basis for better treatment of cerebral Ischemia. Methods: 1. To copy the focal ischemia-reperfusion(I/R) MCAO model on right side of the brain in mice by nylon thread bolt method;2. The C57 and apoe-/-mice respectively equal amounts and randomly divided into: Sham group;Ischemia-reperfusion(I/R) group;Vehicle group;Small dose(10 mg/kg) of Cs A group;High doses(20 mg/kg) of Cs A group. On 1d, 3d, 5d, 7d different time points after operation and using different way to put to death respectively. 3.To evaluate the mice brain ischemia reperfusion model is successful or not after operation, and grade the nerve function score and the corner test on different time points; Using the 2% 2,3,5-chlorinated triphenyl four azole nitrogen(TTC) staining to detect the infarcts area of mice; 5. Using the dry wet weight method to detect the brain water content of mice in different group on different time points; 6. Intravenous injection of Evans blue, by measuring the optical densityvalue(OD value) and leakage amount to analyze the permeability ofblood brain barrier. 7.Using the Western Blot method to analyze thechange trends of Cyp A, Akt, NF- k B, MMP9, Claudin-5, etc. Results:1.The neural function assessment: Sham group mice had no postoperativeneurological dysfunction, other groups of mice showed different degreesof neurological dysfunction at different time points, compared with thecontrol group were statistically differences(P<0.01);High doses of Cs Agroup and small dose of Cs A group were statistically difference at eachpoint; 2.The infarction area: compared with I/R group, the brain infarctsarea both decreased in small dose group and high dose group significantly(P<0.01), the Vehicle group had no significant change, there was nostatistically significant difference(P>0.05), and high-dose group infarctsarea compared with low-dose group were decreased(P<0.05).infarctsarea of each Apoe-/- groups compared with C57 group had statisticaldifference(P<0.05); 3.The brain water content: the brain water content ofC57 and apoe-/- mice groups compared with Sham groups weresignificantly increased, the difference was statistically significant(P<0.01). Compared with I/R group the Vehicle group has no obviousdifference(P>0.05), but high-dose group and small dose group decreasedsignificantly compared with I/R group(P<0.05), and the brain tissuewater content of high-dose group was less than smaller doses group(P<0.05). The brain water content of Apoe-/- mice were higher than the C57 mice group in corresponding, with statistical difference(P<0.05). 4.Evans blue: the Evans blue leakage and OD value of C57 and apoe-/-mice groups compared with Sham group were significantly increased, with statistical difference(P<0.01). The Evans blue leakage and OD value of Vehicle group compared with I/R group has no obvious difference, there was no statistical difference(P>0.05),but the high-dose group and small-dose group compared with I/R group decreased significantly(P<0.01), and the high-dose group was less than small-dose group(P<0.05).the Evans blue leakage and OD value of Apoe-/- mice group were higher than C57 mice group in corresponding, with statistical difference(P<0.05). 5.Related protein expression: compared with Sham group, the expression of p-Akt, Cyclophilin A(Cyp A), NK-k B, MMP-9 in the rest groups were different degree incresased(P<0.05), the expression level of Claudin-5 was lower(P<0.05). Among them, compared with I/R group, the expression level of p-Akt, Cyclophilin A(Cyp A), NK-κB, MMP-9 in Cs A intervention group was reduced(P<0.05),and the expression of high-dose group reduced compared with small dose group(P<0.05), while the Claudin-5 expression level raised(P<0.05), and the expression of high-dose group raised compared with small dose group(P<0.05), while the Vehicle group compared with I/R had no statistical difference(P>0.05). Conclusion: 1.The middle cerebral artery area is the predilection parts of the human brain infarction.we choose line-lock method to make the right middle cerebral artery occlusion(MCAO) model in mice, the infarction area is stable, no invasive and accurate control of ischemia reperfusion time, and its pathological physiology change process similar to the human body, therefore, the model is ideal for the study of cerebral ischemia reperfusion. 2.The right middle cerebral artery occlusion(MCAO) model in mice after treatment with Cs A, the neural function was recovered, infarction area was reduced, and cerebral edema was relieved, the permeability of blood brain barrier was decreased and the inflammatory exudation was reduced, which have the effect of brain protection; 3.Proving that Cs A could through inhibit Akt/NF-κB passageway to promoting the Claudin-5 expression, protecting the integrity of the structure and function of blood brain barrier(BBB), reducing brain edema and exudation, thus reducing the secondary brain injury of MCAO model on mice.