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银杏叶提取物对豚鼠视神经横断伤后视网膜神经节细胞结构及功能的保护作用 2
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
3 讨论
EGb 761主要含有黄酮苷(24%)和萜类内酯(6%)两种成分。黄酮类化合物主要有槲皮素、山奈黄素、异鼠李素。萜类内酯除银杏内酯外还有银杏苦内酯A、B、C和J。该制剂可抗脂质过氧化,对缺血和(或)缺氧损伤、机械损伤的神经元具有保护作用。它能减轻谷氨酸毒性,抑制血小板凝集,对抗凋亡,还能使眼动脉血流量增加。本研究结果证明EGb 761能抑制视神经横断后RGC凋亡,对RGC有保护作用,该结果与Hirooka等[11]的研究结果一致。
PERG是客观评价RGC功能的方法之一,其波形主要特征为一个正向的P50波和一个向下的N95波。根据各种疾病PERG的不同表现,Holder[12]认为两个波的起源不同。药理学分析也证明,N95波可被河豚毒素完全阻断,N95波依赖于RGC产生的动作电位。尽管P50波的确切起源还不清楚,并不能被河豚毒素阻断,但在青光眼患者和猴青光眼模型中P50波振幅均有所下降,因此推测它可能起源于RGC的胞体和(或)RGC的远端[13]。以往评价慢性高眼压引起的RGC数目减少常常采用闪光ERG[14],然而闪光ERG只代表视网膜外层电反应,PERG才真正代表视网膜内层电反应[15, 16]。迄今,尽管对PERG的确切起源尚未完全清楚,但起源于视网膜内层是一致公认的,主要是RGC[17]。Berardi等[18]证实横断大鼠视神经后可引起RGC逆行性变性,导致PERG反应消失,而闪光ERG不受影响。目前PERG已被应用于临床评价高眼压症和青光眼患者的视功能,并认为青光眼对PERG的N95的影响比P50大[19, 20]。BenShlomo等[9]首次应用PERG评价大鼠高眼压后RGC功能,并研究N95振幅和RGC数目的相关性,结果发现两者呈正相关关系。我们的研究也证明豚鼠视神经横断后,随着RGC数目的减少N95振幅逐渐降低,且两者呈正相关关系(r=0.858 68,P=0.001 5),表明PERG的N95振幅可评价RGC数目丧失的程度。
总之,腹腔注射EGb 761可抑制视神经横断后RGC凋亡,从而保护RGC的结构和功能。PERG是评价视神经横断后RGC丧失程度的有效指标之一。
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