1.2.5 组织学检查及RGC存活指数计算 分别于造模后第14和28天采用速眠新Ⅱ注射液(0.6 mL/kg)腹腔注射麻醉后,按我们以往的方法[7],在角膜缘12∶00位缝线作标记并摘取眼球固定于10%甲醛液中,眼球摘除后再注射过量麻药处死。眼球固定24 h后沿12∶00至6∶00经线并沿视神经中央对剖开眼球,弃去晶状体和玻璃体,保留眼球壁。常规脱水、包埋、连续切片(3张)、苏木素伊红染色、封片。在光学显微镜下,从12∶00至6∶00锯齿缘(经视盘中央),计数整个经线上RGC核的数目,以其左眼作对照计算RGC的存活指数[8],每眼计数3张,取其平均值。计算公式:存活指数=(右眼垂直经线RGC数目/左眼垂直经线RGC数目)×100%。
1.2.6 PERG记录方法 按BenShlomo等[9]报道的方法记录PERG。采用日本Tomey系统,软件为EP1000 Vision 1.2.3,刺激方式为棋盘格,刷新频率为65 Hz,通频带设为5~100 Hz,平均叠加次数30~60次,取样频率为2 000 Hz,刺激间隔时间为338 ms。豚鼠在自然光适应状态下,用速眠新Ⅱ注射液麻醉后固定于一个可以三维移动的动物实验台上(自制)。记录电极为环形氯化银电极,滴加14 mg/mL甲基纤维素后固定于右眼角膜缘处,不锈钢针制作的参考电极和地极,分别置于被检眼同侧颞侧皮下和背部。豚鼠的角膜缘与荧光屏平行,角膜顶点与刺激野中心距离为20 cm,然后于明适应状态下记录。每眼记录3次以上,取其平均值。按照国际临床视觉电生理学会(International Society for Clinical Electrophysiology of Vision, ISCEV)推荐的方法从P50的波峰至N95的波谷测量N95振幅[10]。
1.3 统计学方法 所有数据采用x±s表示。采用SAS 6.12统计软件包进行单因素方差分析,如差异有统计学意义,则进行各组均数间两两比较(StudentNewmanKeuls test, SNK);RGC数目和P50波振幅相关性检测采用直线相关分析。检验水准α=0.05。
2 结果
2.1 EGb 761对RGC凋亡的影响 凋亡细胞的细胞核在3通道激发光下呈绿色荧光着染,所有细胞核在5通道激发光下呈红色荧光着染,在8通道激发光下凋亡细胞的细胞核呈黄绿色或黄色荧光着染。正常对照组和假手术组视网膜神经节细胞层均未见黄色荧光着染的RGC。模型组和生理盐水组均见有较多强荧光着染的RGC。EGb 761组未见荧光着染的RGC。见图1。
2.2 光学显微镜观察 豚鼠视网膜可见清晰的10层组织学结构,但全层无血管样结构。正常对照组神经纤维层较厚,RGC呈立方形,排列紧密,胞浆丰富,核饱满,染色较均匀,而胶质细胞胞体较小,核深染。假手术组除RGC略为稀疏外,与正常对照组无明显差异。视神经横断后28 d,模型组和生理盐水组RGC的数目明显减少,神经纤维层明显变薄且RGC稀少,胞体变小。EGb 761组RGC胞体与正常大小无明显区别,但数目略少。见图2。
2.3 EGb 761对RGC存活指数的影响 正常豚鼠视网膜垂直经线上RGC的数目为(318±13)个。视神经横断后14 d时,模型组与生理盐水组RGC存活指数显著低于正常对照组和假手术组(P<0.05);EGb 761组虽比正常对照组和假手术组低(P<0.05),但显著高于模型组和生理盐水组(P<0.05)。视神经横断后28 d时,模型组与生理盐水组RGC存活指数进一步降低,显著低于正常对照组和假手术组(P<0.05);EGb 761组RGC存活指数也有所降低,虽比正常对照组和假手术组低(P<0.05),但显著高于模型组和生理盐水组(P<0.05)。见图3。
图1 TUNEL检测EGb 761对RGC凋亡的影响(荧光显微镜,×400)(略)
Figure 1 Effects of EGb 761 on RGC apoptosis checked by TUNEL (Fluorescein microscopy, ×400)
GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; TUNEL: DeadEnd fluorometric terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatebiotin nick endlabeling; PI: Propidium iodide. A: Normal control group, no TUNEL positive RGCs; B: Shamoperated group, no TUNELpositive RGCs in retina; C: Untreated group, a few TUNEL positive RGCs (white arrows) presented in retina; D: Normal saline group, a few TUNELpositive RGCs (white arrows) presented in retina; E: EGb 761 group, no TUNELpositive RGCs presented in retina.
图2 HE染色观察EGb 761对视网膜组织结构的影响 (光学显微镜,×400)(略)
Figure 2 Effects of EGb 761 on retinal structure observed by HE staining (Light microscopy, ×400)
GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer. A: Normal control group. There was no vascular structure in retina, and cubic RGCs arrayed regularly and the nerve fibers layer (NFL) of retina was thick. B: Shamoperated group. The number of RGCs was close to that in normal control group. C: Untreated group. The number of RGCs and the thickness of NFL were obviously less than those in normal control group. D: Normal saline group. RGCs were rare and NFL was very thin. E: EGb 761 group. The number of RGCs and the NFL were slightly less than those in normal control group. All cells white arrows pointed at were RGCs.
图3 视神经横断后14和28 d各组RGC存活指数(略)
Figure 3 Survival index of RGC in each group 14 and 28 days after optic nerve transection
A: Normal control group; B: Shamoperated group; C: Untreated group; D: Normal saline group; E: EGb 761 group. Data were represented as x±s, n=6. *P<0.05, vs normal control group; △P<0.05, vs shamoperated group; ▲P<0.05, vs untreated group; □P<0.05, vs normal saline group.
2.4 EGb 761对PERG N95振幅的影响 视神经横断14 d时模型组和生理盐水组N95振幅下降,显著低于正常对照组、假手术组和EGb 761组(P<0.05);EGb 761组无显著变化,与正常对照组和假手术组相近(P>0.05)。视神经横断28 d时模型组和生理盐水组N95振幅进一步下降,显著低于正常对照组、假手术组和EGb 761组(P<0.05);与正常对照组和假手术组比较,EGb 761组无显著变化(P>0.05)。见图4和图5。
2.5 N95振幅与RGC存活指数的相关性 对N95振幅和RGC的存活指数进行相关分析,结果为N95振幅与RGC的存活指数呈正相关关系,相关系数r为0.858 68(P=0.001 5)。见图6。
图4 视神经横断后14和28 d各组N95 振幅的变化(略)
Figure 4 Changes of N95 amplitude in each group 14 and 28 days after optic nerve transection
A: Normal control group; B: Shamoperated group; C: Untreated group; D: Normal saline group; E: EGb 761 group. Data were represented as x±s, n=6. *P<0.05, vs normal control group; △P<0.05, vs shamoperated group; ▲P<0.05, vs untreated group; □P<0.05, vs normal saline group.
图5 视神经横断后28 d各组PERG波形(略)
Figure 5 PERG wave form in each group 28 days after optic nerve transection
N35: Negative wave 35; P50: Positive wave 50; N95: Negative wave 95. A: Normal control group; B: Shamoperated group; C: Untreated group; D: Normal saline group; E: EGb 761 group.
图6 N95振幅与RGC存活指数的相关关系(略)
Figure 6 Correlation of N95 amplitude with survival index of RGC
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