大鼠局灶性脑缺血再灌注后不同时间E-选择素、P-选择素和细胞间粘附分子-1的表达

目的观察大鼠脑缺血再灌注(I/R)后不同时间P-选择素(P-selectin)、E-选择素(E-selectin)和细胞间粘附分子-1(ICAM-1)的表达及中性粒细胞浸润脑组织的情况,探讨粘附分子在脑缺血再灌注损伤中的作用。方法采用Zea-Longa线栓法,建立大鼠局灶性脑缺血模型。缺血1 h后拔出栓线进行再灌注,假手术组除不插线外其余手术操作同模型组。分别于再灌注后4、8、12、24、48 h,将动物麻醉下处死,快速取出脑组织,采用HE染色的方法,观察缺血再灌注不同时间脑组织形态学变化;采用免疫组化方法观察再灌注不同时间脑组织中P-selectin、E-selectin和ICAM-1的阳性表达数及表达部位;采用免疫荧光双标法,观察P-selectin、E-selectin和ICAM-1的表达及其定位;采用流式细胞术定量检测P-selectin、E-selectin和ICAM-1的表达;采用生化法测定缺血侧脑组织中髓过氧化物酶(MPO)的活性,以反映白细胞浸润的情况。结果缺血再灌注后,缺血侧脑组织发生明显的病理学形态改变,并且随着再灌注时间的延长,病理学形态改变逐渐加重。缺血再灌注后P-selectin、E-selectin、ICAM-1共表达于血管内皮细胞,与假手术组〔P-selectin:(4.99±0.08)channel;E-selectin:(4.17±0.13)channel;ICAM-1:(4.17±0.13)channel〕相比,I/R 4 h〔(5.46±0.09)channel;(4.60±0.14)channel;(4.56±0.12)chan-nel〕、I/R 8 h〔(5.87±0.24)channel;(5.08±0.14)chan-nel;(5.41±0.22)channel〕、I/R 12 h〔(6.48±0.18)chan-nel;(5.72±0.18)channel;(5.66±0.16)channel〕、I/R 24 h〔(7.16±0.11)channel;(6.09±0.09)channel;(5.61±0.09)channel〕及I/R 48 h〔(5.82±0.28)channel;(5.37±0.25)channel;(5.27±0.16)channel〕各组均升高(P<0.01),且表达峰值出现在缺血再灌注后24 h左右。在缺血再灌注后4~24 h大鼠脑组织中P-selectin(r=0.975,P<0.01)、E-selectin(r=0.977,P<0.01)和ICAM-1(r=0.749,P<0.01)的表达增加具有时间依赖性。脑缺血再灌注后MPO活性明显升高,I/R 4 h组(0.107±0.015)U.g-1、I/R8 h组(0.202±0.010)U.g-1、I/R 12 h组(0.242±0.010)U.g-1、I/R 24 h组(0.273±0.006)U.g-1和I/R 48h组(0.294±0.006)U.g-1与假手术组(0.043±0.008)U.g-1相比,差异均具有显著性(P<0.01),其峰值出现在缺血再灌注后48 h左右。在缺血再灌注后4~48 h,MPO活性增高具有时间依赖性(r=0.982,P<0.01)。结论大鼠局灶性脑缺血再灌注后24 h粘附分子E-selectin、P-selectin和ICAM-1表达增加最明显,而MPO活性在脑缺血再灌注后48h增加最明显,E-selectin、P-selectin和ICAM-1上调共同参与炎症反应,介导白细胞浸润,引起缺血再灌注性脑损伤。

The E-selectin, P-selectin and ICAM-1 expression at different time points after focal brain ischemic-reperfusion in rats

Aim To study the expression of P-selectin,E-selectin and ICAM-1 and the migration of leukocyte at different time points after focal brain ischemia-reperfusion,and explore the role of cell adhesion molecules in cerebral ischemia-reperfusion damage.Methods The model of focal cerebral ischemia-reperfusion was established with the occluding suture as described by Longa.Sham operation was performed without inserting the suture.Rats were decapitated under anesthesia at 4,8,12,24,and 48 h after ischemia-reperfusion.Brains were immediately removed and samples were handled for following application.Morphological changes of the brain tissue were observed through hematoxylin-eosin staining.The positive expressions of P-selectin,E-selectin,and ICAM-1 were observed and located using immunohistochemistry and immunofluorescent histochemistry technique,respectively.The expressions of P-selectin, E-selectin and ICAM-1 in infracted cortex were measured quantitively with Flow cytometry.The activities of myeloperoxidase in the infracted hemispheres represented the migration of leukocytes were detected through biochemical method.Results The positive cells of P-selectin,E-selectin,and ICAM-1 expressed at the same position of microvessels in the infracted hemisphere were significantly increased after ischemia-reperfusion.Compared with the sham operation group(P-selectin:(4.99±0.08) channel,E-selectin:(4.17±0.13) channel,ICAM-1:(4.17±0.13) channel),the expressions of P-selectin,E-selectin,and ICAM-1 at I/R 4 h((5.46±0.09) channel,(4.60±0.14) channel,(4.56±0.12) channel),I/R 8 h((5.87±0.24) channel,(5.08±0.14) channel,(5.41±0.22) channel),I/R 12 h((6.48±0.18) channel,(5.72±0.18) channel,(5.66±0.16) channel),I/R 24 h((7.16±0.11) channel,(6.09±0.09) channel,(5.61±0.09) channel), and I/R 48 h((5.82±0.28) channel,(5.37±0.25) channel,(5.27±0.16) channel) were increased(P<0.01)and reached peak at 24 h or so after ischemia-reperfusion,which were time-dependent(r=0.975,0.977,0.749,respectively,P<0.01) during 4～24 h of ischemia-reperfusion.Activities of MPO were raised significantly(P<0.01) at I/R 4 h(0.107±0.015) U·g~(-1),I/R 8 h(0.202±0.010) U·g~(-1),I/R 12 h(0.242±0.010) U·g~(-1),I/R 24 h(0.273±0.006) U·g~(-1),and I/R 48 h(0.294±0.006) U·g~(-1),compared with the sham operation (0.043±0.008) U·g~(-1),which were time-dependent(r=0.982,P<0.01)during 4～48 h of ischemia-reperfusion.Conclusion The expressions of E-selectin,P-selectin,and ICAM-1 have the most increases at I/R 24 h,while activities of MPO attain the most increases at I/R 48 h.Polymorphonuclear leukocytes infiltration is correlated to upregulation of E-selectin,P-selectin,or ICAM-1 expresions.Upregulation of E-selectin,P-selectin,or ICAM-1 expressions is earlier than leukocyte infiltration,suggesting that upregulation of E-selectin,P-selectin,and ICAM-1 expressions could promote leukocyte infiltration and involve in inflammatory response after cerebral ischemia-reperfusion.