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
作者：解正高, 吴星伟, 庄朝荣, 陈放, 王正, 王雅坤, 华欣
【摘要】 目的: 探讨腹腔注射银杏叶提取物（Ginkgo biboba extract，EGb 761）对豚鼠视神经横断伤后视网膜神经节细胞（retinal ganglion cell, RGC）形态及功能的保护作用。方法: 75只白化豚鼠随机等分为正常对照组、假手术组、模型组、生理盐水组和EGb 761组。分离暴露豚鼠的右眼视神经并于球后1.0 mm处进行横断造模，正常对照组不作任何处理，假手术组仅分离暴露视神经。生理盐水组和EGb 761组分别于实验开始前1周每日腹腔注射1次相应体积生理盐水和EGb 761（100 mg/kg），术后继续给药4周。术后第4天，各组随机处死3只豚鼠，采用脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase?mediated deoxyuridine triphosphate?biotin nick end?labeling, TUNEL)法检测RGC凋亡；分别于术后第14和28天进行图形视网膜电图（pattern electoretinogram, PERG）检查；摘取眼球作组织病检查并计数视网膜垂直经线RGC数目。结果: 术后第4天，正常对照组、假手术组和EGb 761组均未见TUNEL阳性RGC，模型组和生理盐水组均见有TUNEL阳性RGC。术后第14和28天，模型组和生理盐水组RGC数目均少于正常对照组和假手术组（P<0.05），EGb 761组少于正常对照组和假手术组（P<0.05），但显著多于模型组和生理盐水组（P<0.05）；术后第14和28天，EGb 761组PERG的N95振幅较模型组和生理盐水组高（P<0.05），与模型组和假手术组相近（P>0.05）。RGC数目与N95振幅呈正相关（r=0.859, P=0.001 5）。结论: 腹腔注射银杏叶提取物EGb 761能抑制豚鼠视神经横断后的RGC凋亡，对RGC的结构和功能具有保护作用。
【关键词】 银杏叶提取物; 视神经损伤; 视网膜神经节细胞; 图形视网膜电图; 神经保护; 豚鼠
Objective: To investigate the effects of Ginkgo biloba extract (EGb 761) on the morphology and function of retinal ganglion cells (RGC) in guinea pigs with optic nerve transection.
Methods: Seventy?five albino guinea pigs were randomly divided into five groups: normal control group, sham?operated group, untreated group, normal saline group and EGb 761 group. No operation was performed in the normal control group. Optic nerve was merely exposed in the sham?operated group, but transected at 1.0 mm from posterior pole of the eye ball in the untreated, normal saline and EGb 761 groups. Guinea pigs in the EGb 761 group or the normal saline group received daily intraperitoneal injection of EGb 761 (100 mg/kg) or corresponding volume of normal saline from 7 days before experiment to 28 days after experiment. Three guinea pigs in each group were sacrificed for apoptosis assay (TUNEL method) of RGC. Pattern electoretinograms (PERGs) were recorded 14 and 28 days after transection, respectively. At the end of the examination, six guinea pigs were killed for histological examination and RGC count.
Results: No TUNEL?positive cells were observed in the normal control, sham?operated and EGb 761 groups, but there were TUNEL?positive cells in the untreated group and the normal saline group. The numbers of RGCs in the untreated and normal saline groups were less than those in the normal control and sham?operated groups at 14 days or 28 days (P<0.05). Although the number of RGCs in the EGb 761 group was less than those in the normal control and sham?operated groups (P<0.05), it was more than those in the untreated and normal saline groups (P<0.05). N95 amplitude in EGb 761 group was higher than those in the untreated and normal saline groups (P<0.05) and close to those in the normal control and sham?operated groups (P>0.05) at 14 days or 28 days. The number of RGCs was positive correlated to N95 amplitude (r=0.859, P=0.001 5).
Conclusion: EGb 761 can inhibit the apoptosis of RGCs in guinea pigs after optic nerve transection, thus protect the morphology and function of RGCs.
Keywords: Ginkgo biloba extract; optic nerve lesion; retinal ganglion cell; pattern electroretinogram; neuroprotective effect; guinea pigs
青光眼是一种威胁视神经视觉功能，主要与眼压升高有关的眼病，也是目前我国第二大致盲眼病。虽然青光眼的具体发病机制尚不清楚，但目前一致认为视网膜神经节细胞（retinal ganglion cell, RGC）凋亡为青光眼神经损害的中心环节。研究证明银杏叶提取物（Ginkgo biloba extract，EGb 761）具有抗氧化［1］、抗缺血［2］、保护线粒体［3］和抑制一氧化氮合酶活性［4］等广泛的生物学效应。因此，我们采用横断豚鼠球后视神经的方法建立RGC损伤模型观察EGb 761对RGC结构的保护作用，并采用图形视网膜电图（pattern electroretinogram, PERG）评价EGb 761对RGC功能的保护作用。
1 材料和方法
1.1 实验材料
1.1.1 实验动物 75只雌性白化豚鼠，体质量190～210 g，购于院上海实验动物中心，许可证号为SCXK（沪）2007?005。
1.1.2 实验药物及试剂 EGb 761，17.5 mg/支（5 mL），批号为9941209C，德国威玛舒培博士大药厂产品；速眠新Ⅱ注射液，又名846合剂，1 mg/mL，军事医学科学院军事兽医研究所产品，批号为(2004)005013；陆醒宁注射液，1 mg/mL，军事医学科学院军事兽医研究所产品，批号为(2006)006011；甲基纤维素，上海建华精细生物制品有限公司产品；脱氧核苷酸转移酶介导的dUTP缺口末端标记（terminal deoxynucleotidyl transferase?mediated deoxyuridine triphosphate?biotin nick end?labeling, TUNEL）检测试剂盒，美国Promega公司产品；碘化丙啶（propidium iodide，PI），美国Sigma公司产品。
1.1.3 实验设备 手术显微镜（SM?2000J，上海轶德医疗设备有限公司）；PERG检查仪(EP1000，日本Tomey公司)；荧光显微镜（Axioplan 2，德国Zeiss公司）；光学显微镜（Olympus BX50，日本Olympus公司）。
1.2 实验方法
1.2.1 实验动物分组 豚鼠使用严格遵循视觉与眼科研究协会（Association for Research in Vision and Ophthalmology, ARVO）有关视觉和眼科研究动物使用条款。所有豚鼠均在12 h明/12 h暗，温度为22～25 ℃，湿度为55%～60%的环境中饲养。采用机随机数字法将75只豚鼠分为5组：正常对照组、假手术组、模型组、模型+生理盐水组（生理盐水组）、模型+EGb 761组（EGb 761组），每组15只。每只鼠的右眼为实验眼，左眼为用于计算RGC存活指数的对照眼。
1.2.2 视神经横断方法 按照Nakazawa等［5］报道的方法进行视神经横断，建立豚鼠视神经横断模型。臀部注射速眠新Ⅱ注射液0.6 mL/kg麻醉后，在手术显微镜下剪开外眦角；自12∶00处沿角膜缘逆时针呈180°打开球结膜并向后钝性分离暴露视神经，钝性分离视神经鞘表面的血管后于球后1.0 mm处横断球后视神经。分别缝合球结膜和外眦角，涂金霉素眼膏。手术结束后立即在臀部注射陆醒宁（1.2 mL/kg）催醒。术中尽可能钝性分离血管以不影响眼球的血液供应，同时尽可能减少对视神经的牵拉。
1.2.3 各组处理方法 正常对照组：不给任何处理；假手术组：按造模方法暴露视神经，但不横断；模型组：按上述视神经横断方法横断视神经；生理盐水组：于实验前7 d腹腔注射生理盐水，每日1次，体积参照EGb 761的剂量，视神经横断后继续注射28 d；EGb 761组：于实验前7 d腹腔注射EGb 761，剂量为100 mg/kg［6］，每日1次，持续至视神经横断后继续注射28 d。视神经横断后第4天，各组随机处死3只豚鼠，摘取眼球作石蜡切片，进行RGC凋亡的TUNEL标记检测。分别于视神经横断后14 d和28 d进行PERG检查，检查后每组处死6只豚鼠作视网膜组织病理学检查，并作RGC计数。
1.2.4 TUNEL检测RGC凋亡 按TUNEL检测试剂盒说明书提供的方法进行石蜡切片和常规脱蜡。用蛋白酶K孵育后滴加TUNEL反应混合液，最后用PI衬染细胞核。经上述处理后，在荧光显微镜下进行观察并采用AxioVision软件进行同步图像采集，视网膜组织中凋亡的RGC细胞核呈黄色荧光着色。
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Ben?Shlomo等［9］报道的方法记录PERG。采用日本Tomey系统，软件为EP?1000 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 Soci?ety 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（Student?Newman?Keuls 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 transferase?mediated deoxyuridine triphosphate?biotin nick end?labeling; PI: Propidium iodide. A: Normal control group, no TUNEL positive RGCs; B: Sham?operated group, no TUNEL?positive RGCs in retina; C: Untreated group, a few TUNEL positive RGCs (white arrows) presented in retina; D: Normal saline group, a few TUNEL?positive RGCs (white arrows) presented in retina; E: EGb 761 group, no TUNEL?positive 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: Sham?operated 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: Sham?operated 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 sham?operated 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: Sham?operated 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 sham?operated 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: Sham?operated 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］。Ben?Shlomo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。
【】
1 Xie Z, Wu X, Gong Y, Song Y, Qiu Q, Li C. Intra?peritoneal injection of Ginkgo biloba extract enhances antioxidation ability of retina and protects photoreceptors after light?induced retinal damage in rats. Curr Eye Res. 2007; 32(5): 471?479.
2 Chung HS, Harris A, Kristinsson JK, Ciulla TA, Kagemann C, Ritch R. Ginkgo biloba extract increases ocular blood flow velocity. J Ocul Pharmacol Ther. 1999; 15(3): 233?240.
3 Chandrasekaran K, Mehrabian Z, Spinnewyn B, Chinopoulos C, Drieu K, Fiskum G. Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity?induced neuronal death. Pharmacopsychiatry. 2003; 36(Suppl 1): S89?S94.
4 Marcocci L, Packer L, Droy?Lefaix MT, Sekaki A, Gardès?Albert M. Antioxidant action of Ginkgo biloba extract EGb 761. Methods Enzymol. 1994; 234: 462?475.
5 Nakazawa T, Tamai M, Mori N. Brain?derived neurotrophic factor prevents axotomized retinal ganglion cell death through MAPK and PI3K signaling pathways. Invest Ophthalmol Vis Sci. 2002; 43(10): 3319?3326.
6 Ranchon I, Gorrand JM, Cluzel J, Droy?Lefaix MT, Doly M. Functional protection of photoreceptor from light?induced damage by dimethylthiourea and Ginkgo biloba extract. Invest Ophthalmol Vis Sci. 1999; 40(6): 1191?1199.
7 Gong YY, Song Y, Xie ZG, Wu XW. Protective effect of Huangban Granule against light?induced retinal damage in rats. J Chin Integr Med. 2008; 6(11): 1159?1163. Chinese with abstract in English.
宫媛媛, 宋毅, 解正高, 吴星伟. 中药黄斑颗粒对大鼠视网膜光损伤的防护作用. 中西医结合学报. 2008; 6(11): 1159?1163.
8 Li Y, Schlamp CL, Nickell RW. Experimental induction of retinal ganglion cell death in adult mice. Invest Ophthalmol Vis Sci. 1999; 40(5): 1004?1008.
9 Ben?Shlomo G, Bakalash S, Lambrou GN, Latour E, Dawson WW, Schwartz M, Ofri R. Pattern electroretinogram in a rat model of ocular hypertension: functional evidence for early detection of inner retinal damage. Exp Eye Res. 2005; 81(3): 340?349.
10 Bach M, Hawlina M, Holder GE, Marmor MF, Meigen T, Vaegan, Mivake Y. Standard for pattern electroretinography. International Society for Clinical Electrophysiology of Vision. Doc Ophthalmol. 2000; 101(1): 11?18.
11 Hirooka K, Tokuda M, Miyamoto O, Itano T, Baba T, Shiraga F. The Ginkgo biloba extract (EGb 761) provides a neuroprotective effect on retinal ganglion cells in a rat model of chronic glaucoma. Curr Eye Res. 2004; 28(3): 153?157.
12 Holder GE. Significance of abnormal pattern electroretinography in anterior visual pathway dysfunction. Br J Ophthalmol. 1987; 71(3): 166?171.
13 Viswanathan S, Frishman LJ, Robson JG. The uniform field and pattern ERG in macaques with experimental glaucoma. Invest Ophthalmol Vis Sci. 2000; 41(9): 2797?2810.
14 Mittag TW, Danias J, Pohorenec G, Yuan HM, Burakgazi E, Chalmers?Redman R, Podos SM, Tatton WG. Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model. Invest Ophthalmol Vis Sci. 2000; 41(11): 3451?3459.
15 Maffei L, Fiorentini A. Electroretinographic responses to alternating gratings before and after section of the optic nerve. Science. 1981; 211(4485): 953?955.
16 Maffei L, Fiorentini A, Bisti S, Holl?nder H. Pattern ERG in the monkey after section of the optic nerve. Exp. Brain Res. 1985; 59(2): 423?425.
17 Holder GE. Pattern electroretinography (PERG) and an integrated approach to visual pathway diagnosis. Prog Retin Eye Res. 2001; 20(4): 531?561.
18 Berardi N, Domenici L, Gravina A, Maffei L. Pattern ERG in rats following section of the optic nerve. Exp Brain Res. 1990; 79(3): 539?546.
19 Berninger T, Schuurmans RP. Spatial tuning of the pattern ERG across temporal frequency. Doc Ophthalmol. 1985; 61(1): 17?25.
20 Hood DC, Xu L, Thienprasiddhi P, Greenstein VC, Odel JG, Grippo TM, Liebmann JM, Ritch R. The pattern electroretinogram in glaucoma patients with confirmed visual field deficits. Invest Ophthalmol Vis Sci. 2005; 46(7): 2411?2418.
