大鼠视网膜缺血-再灌注损伤后P38丝裂原活化蛋白激酶和半胱氨酸天冬氨酸蛋白酶表达的变化及尼莫地平对二者的影响

Changes of expression of the P38 MAPK and caspase-3 in rat retinal ischemia-reperfusion model and the influence of Nimodipine

目的探讨L型钙通道阻滞剂尼莫地平对大鼠视网膜缺血再灌注损伤的保护作用及其对细胞信号转导通路的影响。方法Wistar大白鼠95只,随机分为4组。A组正常(空白)对照组5只,B组视网膜缺血再灌注组(实验对照组)30只,C组视网膜缺血再灌注+尼莫地平组(实验组)30只,D组低压灌注组(假手术组)30只,以前房灌注升高眼压的方法制备大鼠视网膜缺血再灌注模型,分别于再灌注发生后2、6、12、24、72、168h各处死5只大鼠,石蜡包埋后切片。以原位杂交方法检测各时间点视网膜P38丝裂原活化蛋白激酶类(P38MAPK)mRNA表达情况,以免疫组化法检测视网膜半胱氨酸天冬氨酸蛋白酶3(caspase3)表达情况。结果于Metamorph软件上处理,取平均吸光度值作统计分析。结果视网膜缺血再灌注损伤后,P38MAPK和caspase3表达增强。视网膜P38MAPKmRNA原位杂交信号位于节细胞层和内核层的细胞核内,正常视网膜只有少量表达。P38于B组缺血再灌注后,6h表达即明显增加,至12h达到顶峰,持续至24h,72h后逐渐下降。C组使用尼莫地平后,P38表达趋势与B组相同,但表达水平下降。caspase3表达情况与P38相似。2h开始见内核层细胞核散在阳性,6h后节细胞层内核层细胞核阳性数增加,至24h达到顶峰。C组caspase3表达趋势同B组,阳性细胞数及着染深度均小于B组。对平均吸光度值行统计学分析表明:P38MAPKmRNA表达,在6、12、24、72h,B组与A、C组,C组与A、D组间差异有统计学意义。caspase3表达,除0h、168h这两个时间点外,其他各时间点A、D组与B组,A、D组与C组,B组与C组间差异均有统计学意义。A组与D组间差异无统计学意义。结论P38MAPK和caspase3均参与了视网膜缺血再灌注损伤中视网膜神经细胞信号的转导,尼莫地平通过下调P38MAPK和caspase3的表达而实现对视网膜的保护作用。

Objective To investigate the protective effects of L-type calcium blocker Nimodipine on retinal ischemia-reperfusion of rat and its affect on cell signal transduction system. Method Ninety-five Wistar rats were divided into 4 groups randomly. Group A was normal （blank） control group and included 5 rats; Group B was retinal ischemia-reperfusion （experimental control） group which had 30 rats; Group C was retinal ischemia-reperfusion plus Nimodipine （experimental） group that included 30 rats; and group D was low-pressure irrigation （pseudo-operation） group that had 30 rats. The rat retinal ischemia-reperfusion model was prepared by elevate the pressure of anterior chamber to 110 mm Hg. Five rats were executed at 2, 6, 12, 24 and 72 hours after reperfusion. The specimens were embedded in paraffin, the expression of P38 MAPK and caspase-3 in the retina was evaluated by in situ hybridization and immunohistochemical studies on every time point, respectively. The results were analyzed by Metamorph software, take the average optical density （A） to perform statistical analyses. Results After retinal ischemia-reperfusion, the expression of P38 MAPK mRNA and caspase-3 were increased. The in situ hybridization signal of P38 was located in retinal ganglion cell layer and inner nuclear layer, there was only little expression in normal retina. In group B, P38 expression increased 6 hours after reperfusion, reached its peak after 12 hours, continued to 24 hour, decreased gradually after 72 hours. In group C, after Nimodipine medication, the expression trend of P38 was similar to group B, but the expression level was lower. Statistical analyze using average optical density as parameter indicated that the difference of expression of P38 MAPK at 6, 12 , 24 and 72 hours was statistical significant （ P 〈 0.05） between group B and C, group B and A, group C and A and D. The expression of caspase-3 was similar to that of P38. Two hours after reperfusion, there were scattering positive nucleus in inner nuclear layer; 6 hours later, positive nucleus were located in ganglion cell layer and inner nuclear layer, and reached its peak after 24 hours. In group C, the expression trend of caspase-3 was similar to that of group B, but had less positive cells and showed lighter staining. Conclusion Both P38 MAPK and caspase-3 participate in signal transduction of retinal neurons during retinal ischemia-reperfusion. Nimodipine can protect the retina by means of down-regulate P38 MAPK and caspase-3 expression. （China J Ophthalmol , 2006, 42 ： 435-442 ）