逆行灌注白藜芦醇对猪脊髓缺血再灌注损伤的神经保护作用

Protective effect of retrograde venous perfusion with cold saline and resveratrol on spinal cord ischemiareperfusion injury in swines

目的 探讨逆行灌注白藜芦醇（resveratrol,Res）对猪脊髓缺血再灌注（ischemia-reperfusion,I/R）损伤的神经保护作用.方法 将18只西藏小型猪按“随机数字表法”随机分为3组：手术对照组（I/R组,n=6）;冷液（cold saline,CL）逆灌组（CL组,n=6）;Res逆灌组（CL＋Res组,n=6）.I/R组：常温阻断降主动脉70 min;CL组：阻断前游离出副半奇静脉,阻断降主动脉70 min,同时经副半奇静脉逆行灌注低温（4℃）9 g/L氯化钠溶液,灌注流速为16.65 mL/min;CL＋Res组：阻断降主动脉70 min,同时经副半奇静脉逆行灌注含Res 10 mg/kg的低温（4℃）9 g/L氯化钠溶液.于术后6、24、48 h行神经功能评分,术后48 h取L2～L4段脊髓,免疫组化法和Western Blot检测Bax及Bcl-2蛋白表达变化.结果 18只西藏小型猪全部存活.术后6、24、48 h各组行为学评分均降低,I/R组低于CL＋Res组、CL组,CL＋Res组高于CL组,差异有统计学意义（F=26.341、18.553、9.637,P＜0.05）.将主动脉阻断后各组实验动物的鼻咽温度和椎管温度均有下降,I/R组、CL＋Res组和CL组分别在阻断70 min和再灌注20 min时降至最低.再灌注后,I/R组的鼻咽温度和椎管温度逐渐回升,至再灌注60 min时与阻断前相近（P=0.293、0.115）;而CL组和CL＋Res组回升不明显,在再灌注60 min时仍明显低于阻断前（P＜0.01）.Western Blot检测结果与免疫组化结果一致：Bcl-2蛋白表达水平在CL＋Res组与CL组均高于I/R组,差异有统计学意义（P＜0.05）;且CL＋Res组高于CL组,差异有统计学意义（P＜0.05）.Bax蛋白表达水平在CL＋Res组与CL组均低于I/R组,差异有统计学意义（P＜0.05）;且CL＋Res组低于CL组,差异有统计学意义（P＜0.05）.结论 经猪副半奇静脉逆行灌注低温9 g/L氯化钠溶液及Res对脊髓I/R损伤有保护作用,其机制可能是通过下调Bax及上调Bcl-2蛋白的表达,抑制细胞凋亡而发挥作用.

Objectives To observe the protective effect of retrograde venous perfusion with cold saline and resveratrol （Res） on spinal cord ischemia-reperfusion （I/R） injury in a porcine model of abdominal aortic cross-clamping.Methods Totally 18 swines were randomly assigned to three groups,6 in each：I/R injury group （I/R group）,retrograde perfusion with cold saline group （CL group） and retrograde perfusion with cold saline and Res group （CL＋Res group）.At the time points of 6,24,48 hours after reperfusion,behavior scores were respectively given.The expressions of Bax and Bcl-2 proteins were detected at 48 hours after reperfusion by immunohistochemistry and Western Blot analysis in spinal cord tissue.Results All the 18 swines survived.Behavior scores of the three groups decreased at all the time points.Behavior scores of CL＋Res and CL group were significantly higher than that of I/R group and CL＋Res group was higher than CL group （F=26.341,18.553,9.637; P〈0.05）.Nasopharyngeal temperature and spinal cord temperature of the three groups descended after aorta was obstruction,and they were the lowest at 70 min after obstruction and 20 min after reperfusion.Nasopharyngeal temperature and spinal cord temperature of I/R group upswinged after reperfusion,and by the time of 60 min after reperfusion,they were almost the same as those before obstruction （P=0.293,0.115）.However,they did not obviously upswing in CL group and CL＋Res group,and by the time of 60 min after reperfusion,they were still lower than those before obstruction （P〈0.01）.Immunohistochemistry and Western Blot studies both revealed that expressions of Bcl-2 protein in CL＋Res and CL groups were significantly higher than that in I/R group （P〈0.05）,and CL＋Res group was significantly higher than CL group （P〈0.05）.Expressions of Bax protein in CL＋Res and CL group were obviously lower than that in I/R group （P〈0.05）,and CL＋Res group was significantly lower than CL group （P〈0.05）.Conclusions Retrograde venous perfusion with cold saline and Res may protect the spinal cord from I/R injury through inhibiting cell apoptosis.