| objective:Chronic cerebral hypoperfusion is one of important risk factors in cognitive impairment.However,its pathogenesis has not been elucidated nowadays.This study dynamically observed the characteristics of cognitive impairment in the experimental rats with chronic cerebral hypoperfusion,furthermore,we quantitatively analyze of the coverage of the astrocytic endfeet around microvascular in the hippocampal and the expression of astrocytic endfeet AQP4 and microvascular basement membrane CollagenⅣto investigate the mechanism of gliovascular interface and neurovascular unit damaged in the chronic cerebral hypoperfusion induced cognitive impairment in rat.Methods:According to the random number table method,96 healthy adult male SD rats were randomly divided into Sham operation control group（abbreviation:control group,n=44）and chronic cerebral hypoperfusion group（abbreviation:CCH group,n=52）,and then each group were randomly divided into 1 week,1 month,3 months,6 months four subgroups（each subgroups of CCH group was 13,each subgroups of control group was 11）.Experimental rats with chronic cerebral hypoperfusion group used bilateral common carotid artery occlusion to induce chronic cerebral hypoperfusion model,and the control group rats except not bilateral common carotid arteries ligation,other operations were consistent with the chronic cerebral hypoperfusion group.After the model was established successfully at1 week,1 month,3 months,6 months,we started to conduct behavioral testing to evaluate the rat’s autonomic exploration behavior responses and the ability of object recognition and spatial learning and memory of cognitive functions by open field test,object recognition test,Morris water maze test.Then we conducted the histopathological examination of the brain.We used the immunofluorescence double staining to label the astrocytic endfeet of AQP4and microvascular basement membrane CollagenⅣand colocalization analyze the coverage of the astrocytic endfeet around microvascular,the expression of astrocytic endfeet AQP4 and microvascular basement membrane CollagenⅣin 1 week,1 month,3 months,6 months.Furthermore,we used the projection electron microscope to observe the changes of ultrastructure of astrocytic endfeet,microvascular basement membrane,gliovascular interface and neurovascular unit.Results:（1）The result of behavioral test:In the open field test,compared with control group,from 1 week to 6 months autonomic exploration behavior responses of CCH model group（from 1 week to 6months the control group vs.CCH model group:2401.87±191.41vs.12813.07±237.95,2628.79±366.78 vs.1728.57±299.64,2539.14±348.67 vs.2192.07±360.64,2642.44±341.75 vs.2202.33±459.07）was reduced significantly（1 week P<0.001,1 month P<0.001,3 months P=0.042,6 months P=0.026）;In the object recognition test,compared with control group,from 1week to 6 months the object recognition of CCH model group（from 1 week to6 months the control group vs.CCH model group 0.72±0.07 vs.0.64±0.09,0.87±0.12 vs.0.70±0.15,0.81±0.20 vs.0.64±0.10,0.78±0.16 vs.0.61±0.07）was reduced significantly（1 week P=0.041,1 month P=0.015,3 months P=0.025,6 months P=0.009）too;In the place navigation test of Morris water maze,compared with control group,the escape latency of CCH model group was prolonged significantly and the situated learning of CCH model group was decline significantly(1 week:F（1,18）=20.61,P<0.001,1 month F（1,18）=11.82,P=0.003,3 months F（1,18）=32.48,P<0.001,6 months F（1,18）=19.56,P<0.001);In the spatial probe test of Morris water maze,compared with control group,the times of crossing platform in CCH model group（from 1 week to 6 months the control group vs.CCH model group:3.80±1.75 vs.1.30±0.48,2.8±1.14vs.1.4±1.51,4.20±1.32vs.2.40±1.65,3.50±1.90vs.1.30±1.34）was reduced significantly（1 week P<0.001,1 month P=0.031,3 months P=0.015,6 months P=0.008）;In the working memory test of Morris water maze,compared with control group,the escape latency of CCH model group was prolonged significantly and the working memory of CCH model group was decline significantly(1 week F（1,18）=29.29,P<0.001,1 month F（1,18）=25.29,P<0.001,3 months F（1,18）=22.34,P<0.001,6 months F（1,18）=38.36,P<0.001).（2）The result of double-label immunofluorescence:Compared with control group,colocalization analyzing the coverage of the astrocytic endfeet around microvascular of CCH model group（from 1 week to 6 months the control group vs.CCH model group:1 week 93%±0.61%vs.91.64%±1.13%,1 month93.37%±1.83%vs.90.7%±1.03%,3 months 91.57%±0.83%vs.88.69%±0.81%,6months90.83%±1.33%vs.88.70%±1.63%）wasdecreased significantly（1week P=0.016,1 month P=0.006,3 months P<0.001,6 months P=0.020）.The coverage of the astrocytic endfeet around microvascular began to decrease in the 1week until 3 month,in 3 month it was lowest.Although it began to increase in the 6 month and it was lower than the normal level prominently(F（3,24）=10.85,P<0.001).The the expression of perivascular astrocytic endfeet AQP4 of CCH group（from 1 week to 6 months the control group vs.CCH model group:2.33±1.11 vs.0.76±0.33,1.90±0.80 vs.1.12±0.42,2.01±0.50 vs.0.96±0.49,2.42±1.27 vs.1.71±0.99）was decreased significantly from 1 week to 3 month,but it was not significantly in 6 months（1week P=0.004,1 month P=0.042,3 months P=0.002,6 months P=0.268）.All the CCH group was compared in different time-point,The the expression of perivascular astrocytic endfeet AQP4 began to decrease in the 1week until 3months,and it was lowest in 3 months,and it began to increase in the 6months(F（3,24）=3.11,P=0.044);The the expression of microvascular basement membrane CollagenⅣdecreased of CCH group（from 1 week to 6 months the control group vs.CCH model group:6.73±0.97 vs.4.76±0.88,6.82±2.50vs.4.23±1.02,6.57±2.28 vs.3.64±1.24,5.44±1.05vs.5.44±1.28）was decreased significantly from 1 week to 3 month,but it was not significantly in 6 month（1week P=0.002,1 month P=0.026,3 months P=0.012,6 months P=0.996）.All the CCH group was compared in different time-point,The the expression of microvascular basement membrane CollagenⅣbegan to decrease in the1week until 3 months,and it was lowest in 3 months,it increased in the 6months(F（3,24）=3.24,P=0.040);（3）the result of the ultrastructure in the projection electron microscope:Compared with control group,the ultrastructure revealed that the hippocampal astrocytic endfeet were swollen and destroyed,which were detached from the basement membrane of the microvessel,the gliovascular interface and the neurovascular unit was damaged in the chronic cerebral hypoperfusion group from 1 week to 6 months.Conclusion:Chronic cerebral hypoperfusion induced autonomic exploration behavior responses,the ability of object recognition and learning and memory cognitive function impaired with experimental rats sustainably.At the same time,and it caused the abnormal expression of AQP4 in astrocytic endfeet persistently,and the coverage of astrocytic endfeet around microvessel in the brain tissue decreased persistently,and the gliovascular interface and the neurovascular unit damaged persistently.Furthermore,the ultrastructural revealed that astrocytic endfeet swollen and destroyed,and detached from the basement membrane of the microvessel.It Showed that perivascular astrocytic endfeet of AQP4 changes may be one of important reasons for the gliovascular interface and the neurovascular unit damage.And it may be one of the important pathogenesis of cognitive impaired induced by chronic cerebral hypoperfusion. |
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