高盐饮食自发性高血压大鼠脑小静脉管壁基质改变观察

Observation on the changes of cerebral venules wall matrix in spontaneous hypertensive rat with high-salt diet

目的研究高盐饮食自发性高血压大鼠(SHR)中脑小静脉管壁基质的改变。方法10周龄雄性SHR及雄性WistarKyoto(WKY)大鼠各6只,给予SHR组10个月高盐饮食(1%氯化钠饮用水及4%高盐饲料),给予WKY组大鼠10个月正常饮用水及饲料。大鼠均饲养于复旦大学附属华山医院神经病学研究所,恒温22~24℃,恒湿(55±5)%,人工光照明暗各12h。通过苏木素-伊红(HE)染色观察小血管形态变化,通过α-平滑肌细胞肌动蛋白(α-SMA)鉴别小动脉及小静脉,免疫荧光检测脑小静脉Ⅰ型胶原蛋白(COLⅠ)、Ⅳ型胶原蛋白(COLⅣ)、纤连蛋白及层黏连蛋白(LN)表达情况。将脑组织划分为皮质、深部灰质、海马并对各脑区脑小静脉胶原沉积及相关细胞外基质表达情况进行比较。结果(1)HE染色可见高盐饮食SHR小动脉血管重构,符合小动脉硬化型脑小血管病的病理特点。(2)与WKY组比较,高盐饮食SHR组脑小静脉COLⅠ及LN沉积均明显增加(分别为:153±12比106±8,t=3.253,P=0.004;135±11比99±9,t=2.575,P=0.017);COLⅣ沉积有增加趋势,但差异无统计学意义(118±12比99±4,t=1.508,P=0.142);FN表达差异无统计学意义(104±4比101±3,t=0.664,P=0.512)。(3)与WKY组比较,高盐饮食SHR组皮质及深部灰质小静脉COLⅠ沉积明显增加(皮质:159±15比108±7,t=3.075,P=0.007;深部灰质:139±12比96±9,t=2.868,P=0.009),海马区小静脉COLⅠ沉积两组间差异无统计学意义(169±16比133±9,t=1.926,P=0.072);高盐饮食SHR组和WKY组各脑区的小静脉COLⅣ及纤连蛋白沉积差异均无统计学意义(均P>0.05);与WKY大鼠比较,高盐饮食SHR大鼠深部灰质小静脉LN表达显著增加(125±12比77±5,t=3.767,P=0.002),皮质和海马区小静脉LN表达差异均无统计学意义(皮质:138±18比109±7,t=1.460,P=0.174;海马区:153±16比133±13,t=0.960,P=0.359)。结论高盐饮食SHR脑小静脉管壁COLⅠ及LN沉积增加,COLⅠ沉积以皮质及深部灰质最为显著,LN沉积以深部灰质为主。

Objective To investigate the changes of wall matrix of the cerebral venules in the high-salt fed spontaneously hypertensive (SHR) rats.Methods Six SHR and six Wistar Kyoto (WKY) male rats at the age of 10 weeks were experimented.Rats in the SHR group were given high-salt diet (1% sodium chloride drinking water and 4% high-salt) for 10 months,and rats in the WKY group were given normal drinking water and fed for 10 months,The rats were all raised in the Institute of Neurology,Huashan Hospital,Fudan University.The feeding conditions were kept at a constant temperature of 22 - 24 ℃ and constant humidity of 55±5%,with artificial light and shade for 12 h each.Morphological changes of cerebral small vessels were observed by hematoxylin-eosin (HE) staining.Venules were differentiated from arterioles through the presence of α-smooth muscle actin.Immunofluorescence was performed to observe the expression of collagen Ⅰ(COLⅠ),collagen Ⅳ(COLⅣ),fibronectin (FN) and laminin (LN) in cerebral venules.The brain tissue was divided into cortex,deep gray matter and hippocampus regions.The collagen deposition and expression of related extracellular matrix of venules in these regions were compared.Results (1) HE staining showed vascular remodeling of arterioles in high-salt fed SHR,which was consistent with the pathological characteristics of arteriosclerotic cerebral small vessel disease.(2) Compared with the WKY group,the high-salt fed SHR group showed obviously increased cerebral venules COLⅠ deposition and LN expression (153±12 vs.106±8,t=3.253,P=0.004;135±11 vs.99±9,t=2.575,P =0.017);COLⅣ deposition had an increasing trend,but without statistically significant difference (118±12 vs.99±4,t=1.508,P=0.142 ).There was no significant difference in FN expression ( 104±4 vs.101±3,t= 0.664,P=0.512 ).(3) Compared with the WKY group,the SHR group had significantly increased deposition of COLⅠin cortex and deep gray matter venules (159±15 vs.108±7,t=3.075,P=0.007;139±12 vs.96±9,t= 2.868,P= 0.009);No statistically significant difference was found between the two groups in hippocampus region (169±16 vs.133±9,t=1.926,P= 0.072);No statistical significance was found between the high-salt diet SHR group and the WKY group in COLⅣ and fibronectin deposition of venules in these regions ( P >0.05);Compared with the WKY group,the LN expression of deep gray matter venules in the SHR group after high-salt diet was significantly increased (125±12 vs.77±5,t=3.767,P= 0.002),and there was no statistically significant difference in both cortical and hippocampal regions (138±18 vs.109±7,t=1.460,P=0.174;153±16 vs.133±13,t=0.960,P= 0.359).Conclusions The expression of COLⅠ and LN of cerebral venules wall was increased in the high-salt fed SHR.The COLⅠ deposition was most significant in cortex and deep gray matter,and the LN deposition was mainly in deep gray matter.