灯盏细辛对NMDA所致大鼠视网膜神经元损伤的保护作用

目的:已有一些临床试验和基础研究显示灯盏细辛(GerigeronBreviscapus(vant)Hand-Maz2EBHM),对青光眼患者及动物模型有神经保护的作用,本研究探讨灯盏细辛是否对NMDA导致的大鼠RGCL神经元兴奋毒性有保护作用。方法:60只健康SD大鼠随机分为4组,其中6只为正常对照组(A组),其余54只随机分为3组,分别为B组(EBHM组),C组(生理盐水+NMDA组),D组(EBHM+NMDA组),每组各18只大鼠。C、D两组大鼠右眼玻璃体内注射NMDA10nmol/2μL制成视网膜损伤模型,左眼玻璃体内注射相同剂量PBS液作为自身对照。B组及D组均在NMDA注射前7d起按150mg/(kg·d)日剂量予60g/LEBHM腹腔内注射,C组予生理盐水0.5mL腹腔内注射。在NMDA处理后4,7,14d处死动物剥取视网膜作全层铺片行RGCL神经元计数分析。结果:正常对照组双眼RGCL神经元计数比较差异无显著性意义(P=0.200)。NMDA干预后4,7,14dEBHM组RGCL神经元计数,双眼与正常对照组比较差异无显著性意义(P>0.05)。生理盐水+NMDA及EBHM+NMDA组实验眼在以上各时段RGCL神经元计数与正常组比较差异均有非常显著性意义(P<0.001),左眼与正常对照组比较差异无显著性意义(P>0.05)。实验眼14d时RGCL神经元计数EBHM+NMDA组高于生理盐水+NMDA组,二者比较差异有显著性(P=0.044),但仍低于正常对照组(P<0.05)。结论:单独使用EBHM对正常大鼠RGCL神经元计数无影响,EBHM可对NMDA所致大鼠RGCL神经毒性提供部分保护作用。     

银杏叶提取物对大鼠慢性高眼压视网膜损伤的保护作用

目的:探讨银杏叶提取物(EGb)对实验性高眼压大鼠视网膜神经节细胞层(RGCL)神经元损伤的保护作用.方法:取健康SD大鼠60只,正常对照组和正常银杏叶治疗组各6只,其余48只采用烧烙法,烙闭大鼠左眼2条浅层巩膜静脉,制作大鼠持续性高眼压模型,从中选出眼压稳定在实验要求水平的大鼠30只,随机分为生理盐水组,治疗A组(每日EGb 50mg/kg)、治疗B组(每日EGb100mg/kg)、治疗C组(每日 EGb 150mg/kg)、治疗D组(每日EGb 200mg/kg),治疗时间为1mo,处死大鼠后做视网膜全层铺片,对 RGCL 神经元做特异性染色后行神经元计数分析来评估神经元的情况.结果:正常对照组双眼RGCL神经元计数比较无显著性差异(P＞0.05),正常对照组与正常 EGb 治疗组 RGCL神经元计数比较无显著性差异(P＞0.05),各浓度EGb治疗组实验眼与正常对照组RGCL计数比较均有显著性差异(P＞0.05),各浓度EGb治疗组实验眼与其自身对照眼RGCL神经元计数均有显著性差异(P＞0.05),各浓度EGb治疗组实验眼与生理盐水组实验眼RGCL神经元计数均有显著性差异(P＞0.05),治疗A,B,C,D 4组实验眼RGCL神经元计数两两比较均有显著性差异(P＜0.05).结论:单独使用EGb对正常大鼠RGCL神经元计数无影响,EGb可对持续慢性高眼压导致的大鼠RGCL神经元损伤提供部分保护作用,且在一定范围内保护作用大小与用药剂量呈正相关.     

N-甲基-D-天门冬氨酸对大鼠视网膜神经节细胞层神经元的损伤作用

目的 探讨N—甲基—D—天冬氨酸(NMDA)对大鼠视网膜神经节细胞层(RGCL)神经元的毒性作用。方法 通过大鼠玻璃体内注入不同浓度的NMDA，观察注射前后不同时间视网膜神经节细胞层的神经元计数。结果 大鼠RGCL神经元计数随NMDA浓度增加及时间延长而逐渐减少。其中以大神经元对NMDA兴奋毒性最敏感。结论 NMDA可导致RGCL神经元损伤，并显示以大神经元最先受损，即与青光跟性视神经损伤相似，此模型可应用于研究青光跟性视神经损伤机制。     

重组人促红细胞生成素对慢性高眼压致大鼠视网膜神经元损伤的保护作用

目的:探讨玻璃体内注射重组人促红细胞生成素(recombinant human erythropoietin,rHuEPO)对慢性高眼压所致的大鼠视网膜神经元损伤的保护作用及其可能的作用机制。方法:健康SD大鼠30只随机分为5组,每组6只,分别为损伤组I,损伤组Ⅱ、DMSO组、rHuEPO组和rHuEPO+Wortmannin(WM)组。各组均以右眼为实验眼,损伤组Ⅱ大鼠激光处理1d后处死,其余各组激光处理8wk后处死。损伤组用532nm激光烧灼大鼠角巩膜缘浅层静脉,阻断房水回流的方法建立大鼠慢性高眼压模型,采用Tono-pen眼压计测量眼压。DMSO组、rHuEPO组和rHuEPO+WM组大鼠均在激光处理后1d开始分别向玻璃体内注射DM-SO、rHuEPO和rHuEPO+WM,1次/wk。正常对照组选取损伤组左眼作为对照眼。用免疫组织化学法和TUNEL染色检测观察各组实验结果。结果:与正常对照组比较,各组视网膜神经节细胞层(retinal ganglion cell layer,RGCL)中促红细胞生成素受体(erythropoirtin receptor,EPOR)表达增加,差异具有统计学意义(P<0.05)。与正常对照组比较,各组RGCL中蛋白激酶B(AKT)表达无明显改变,差异无统计学意义,损伤组I和DMSO组RGCL中磷酸化蛋白激酶B(PAKT)表达也无明显改变,但rHuEPO组RGCL中PAKT表达增加(P<0.05)。与损伤组I和DMSO组比较,rHuEPO组RGCL中凋亡神经细胞数目减少(P<0.05)。与rHuEPO组比较,rHuEPO+WM组RGCL中AKT表达无明显变化,但PA-KT表达减少(P<0.05),凋亡神经细胞数目增加(P<0.05)。结论:rHuEPO对慢性高眼压所致的大鼠视网膜神经元损伤有保护作用,且rHuEPO通过PI-3K/AKT信号途径抑制神经细胞凋亡而发挥其视神经保护作用。     

灯盏细辛抑制外源性VEGF诱导的大鼠早期视网膜血管病变

目的:探讨灯盏细辛对外源性VEGF诱导的大鼠早期视网膜血管病变的作用,并阐明其对视网膜内核层单个毛细血管管腔面积、内皮细胞面积和视网膜电图振荡电位的影响。方法:大鼠48只随机分为正常对照组、VEGF组、VEGF+EBHM组和VEGF+NS组。VEGF+EBHM组每天ip灯盏细辛浸膏溶液。首次注射VEGF后2wk行荧光素眼底血管造影、视网膜电图振荡电位和组织学检查。结果:注射VEGF实验眼,约10d后表现为眼底大血管呈结节样扩张,周围视网膜水肿。VEGF+EBHM组较VEGF组、VEGF+NS组轻。实验眼在视盘、视盘周围、后极部视网膜大血管出现高荧光,荧光素从曲张区中央开始消退。VEGF+EBHM组实验眼后极部血管局限性高荧光较VEGF组、VEGF+NS组弱、显示的范围小。VEGF组、VEGF+NS组实验眼视网膜内核层毛细血管管腔缩窄,血管内皮细胞肥大,与正常对照组比较,差异有显著性(P<0.05)。VEGF+EBHM组毛细血管管腔面积无减少,血管内皮细胞无肥大,与VEGF组比较,差异有显著性(P<0.05)。与正常对照组比较,差异无显著性(P>0.05)。VEGF组,VEGF+NS组实验眼视网膜电图振荡电位总波幅降低,与正常对照组比较,差异有显著性(P<0.05)。VEGF+EBHM组视网膜振荡电位总波幅没有变化,与VEGF组比较差异有显著性(P<0.05)。与正常对照组比较,差异无显著性(P>0.05)。结论:EBHM对VEGF诱导的大鼠早期视网膜血管病变有抑制作用。能抑制rrVEGF164诱导的大鼠早期视网膜内核层毛细血管管腔变窄、视网膜血管内皮细胞肥大。能抑制rrVEGF164诱导的大鼠早期视网膜电图振荡电位总波幅的降低。     

色素上皮源性因子对缺血-再灌注视网膜神经节细胞的保护作用

目的:研究色素上皮源性因子(pigment epithelium derived factor,PEDF)对高眼压诱导的大鼠视网膜缺血-再灌注后视网膜神经节细胞的保护作用.方法:经眼角膜进行前房平衡盐水(BSS)灌注,维持眼内压110 mmHg,以阻止视网膜正常血液灌注.60 min后取出灌注针头,恢复视网膜正常血流,从而建立大鼠视网膜缺血-再灌注模型.实验分为正常非缺血组和视网膜缺血-再灌注组,后者又分为生理盐水注射对照组和PEDF注射实验组,再灌注模型建立后立即向实验组大鼠玻璃体腔内注射0.2 g/L PEDF 2 μL.实验对照组用同样方法注射等量生理盐水.分别于注射后2 d和7 d进行眼球摘除,对视网膜进行光学显微镜形态学观察和Fas原位杂交免疫学分析,探讨PEDF对缺血-再灌注视网膜神经节细胞的保护作用.结果:缺血-再灌注2 d时生理盐水注射组和PEDF注射组视网膜神经节细胞明显少于正常对照组(P＜0.01)和(P＜0.05),PEDF注射组视网膜神经节细胞数较生理盐水注射组明显较多,相比有显著性差异(P＜0.05);视网膜神经节细胞计数再灌注7 d后结果与2 d时类似.再灌注2 d生理盐水注射组Fas阳性染色细胞比PEDF注射组明显较多(P＜0.05),生理盐水组比PEDF注射组阳性细胞百分率明显较高(P＜O.01);再灌注7 d时两组Fas阳性细胞计数无明显差异.结论:视网膜缺血-再灌注后即刻行玻璃体腔内PEDF注射可以改善视网膜神经节细胞的损伤并有一定保护作用.     

结膜下和玻璃体内注射重组人PEDF对氧诱导大鼠RNV的作用

目的:观察结膜下和玻璃体注射重组人色素上皮衍生因子(pigment epithelium-derived factor,PEDF)两种注射方式对氧诱导大鼠视网膜新生血管(retinal neovascularization,RNV)的作用。方法:新生大鼠2只进行左眼结膜下注射PEDF,Westernblot检测视网膜PEDF的表达。氧诱导SD新生大鼠建立类似早产儿视网膜病变动物模型。新生鼠48只随机分为6组(A:空气对照组,B:高氧对照组,C:高氧+玻璃体注射PEDF2μg组,D:高氧+结膜下注射PEDF2μg组,E:高氧+结膜下注射PEDF4μg组,F:高氧+结膜下注射PEDF8μg组)。当新生大鼠脱离氧时,C,D,E和F组大鼠左眼用不同剂量和注药次数进行注射PEDF,ADP酶视网膜血管染色观察视网膜血管形态,石蜡切片计数突破视网膜内界膜的新生血管内皮细胞核数目。结果:结膜下注射重组人PEDF,可检测到视网膜PEDF蛋白表达。视网膜铺片结果显示:A组视网膜血管发育正常;B组视网膜大量的新生血管生成;C组新生血管明显减少;D,E,F组新生血管稍减少。组织病理学检测突破视网膜内界膜的新生血管内皮细胞计数可见:A组视网膜内界膜平滑,偶见突破的视网膜内皮细胞。B组明显高于A组,差异有统计学意义(P<0.05)。C组明显低于B组(P<0.05),D,E,F组与C组差异有统计学意义(P<0.05)。结论:巩膜和脉络膜-色素上皮层对PEDF是有渗透性的,可以跨越结膜下组织到达视网膜,但与玻璃体注射组相比,抑制新生血管作用有显著减弱;玻璃体注射可有效抑制氧诱导大鼠RNV。     

视网膜对形觉剥夺性近视鸡巩膜MMP-2的调控

目的建立鸡形觉剥夺性动物模型,通过观察用庆大霉素破坏小鸡视网膜后,后极部巩膜基质金属蛋白酶Ⅱ(matrix metalloproteinase-2,MMP-2)的变化,探讨视网膜在形觉剥夺性近视(formdeprivation myopia,FDM)发生、发展中的影响作用。方法48只白色来亨鸡(鸡龄2d,SPF级)分为A、B、C、D4组,每组12只,其中A组、B组在第2d即行双眼半透明眼罩遮盖同时行右眼玻璃体内1次注射庆大霉素400μg;C组仅右眼玻璃体内1次注射庆大霉素400μg不予遮盖,左眼不作处理为自身对照;D组不作任何处理,为正常对照组。在第3周末,对全部小鸡行检影验光测屈光度后,将A、C2组鸡处死,迅速摘除双眼球,测量其前后径。并随机取出2只送病理组织切片;B组摘除双眼眼罩后继续饲养3周,在第6周末对B、D2组行检影验光后将其处死,摘除双眼球测量眼球前后径。用7mm直径环钻钻取后极部巩膜组织,研磨离心后取上清液作为标本,对所有标本采用ELISA(即酶联免疫吸附)试剂盒测定MMP-2活性浓度。所有数据经过EXCEL和SPSS统计软件进行处理。结果第3周末,A、B两组双眼屈光度之间均具有显著性差异(P<0·001),C组和D组双眼屈光度间无显著性差异(P=0·088,0·920),且A、B2组与C组双眼屈光度比较均分别具有显著性差异(P<0·001);A组双眼眼轴测量值具有显著性差异(P<0.01),C组双眼眼轴未见显著性差异(P>0.05),A组与C组比较时,右眼眼轴无显著性差异(P>0.05),而左眼间差异具有显著性(P<0.001);第6周末,B组去遮盖后双眼屈光度及眼轴测量值之间均无显著性差异(P>0.05),且与D组比较差异亦无显著性意义(P>0.05),但B组去遮盖前后双眼屈光度之间具有明显显著性差异(P<0.001)。ELISA结果显示,除了A组双眼后级部巩膜MMP-2含量自身对照及与其他各组比较均有显著性差异外(P<0.001),另外3组之间差异均无显著性意义(P>0.05)。结论视网膜色素上皮层在形觉剥夺性近视的发生发展过程中起着非常重要的作用,破坏视网膜色素上皮层能一定程度的减轻近视的发展程度。     

活化的巨噬细胞促进视神经损伤后视网膜中生长相关蛋白-43的表达

目的评估活化巨噬细胞对于视神经损伤后视网膜神经节细胞中生长相关蛋白43(GAP43)表达的影响。方法成年Wistar大鼠按夹伤后存活时间不同(1、3、7d)分为3组,每只大鼠右眼接受玻璃体内注射酵母多糖(实验组),左眼接受玻璃体内注射PBS(对照组)。采用免疫荧光技术检测视网膜神经节细胞中GAP43和ED1的表达。结果对照组中未见ED1阳性巨噬细胞,实验组中视网膜内表面见ED1阳性巨噬细胞,C组GAP43较多(189.26±26.16),与B组(65.29±21.32)相比有显著差异,而对照组中无GAP43的表达。结论玻璃体内注射酵母多糖可激活巨噬细胞,从而促进视神经损伤后视网膜神经节细胞中GAP43的表达。     

白蒺藜皂苷对慢性高眼压兔视网膜SOD活性和MDA含量的影响

目的:观察白蒺藜皂苷对慢性高眼压兔SOD活性和MDA含量的影响,探讨其对兔慢性高眼压模型视网膜氧化损伤的抑制作用。方法:将24只健康新西兰白兔随机分为正常对照组(A组),高眼压模型空白组(B组),高眼压模型灯盏细辛(erigeron brevicapas hand mass,EBHM)治疗组(C组),高眼压模型蒺藜皂苷(gross saponins of tribulus terrestris,GSTT)治疗组(D组);以20g/L甲基纤维素前房注射建立兔慢性高眼压模型,EBHM治疗组实验兔每日耳缘静脉推注EBHM注射液4.5mg/kg,GSTT治疗组实验兔每日耳缘静脉推注GSTT针剂5mg/kg,连续用药4wk并每日测眼压,于第28d摘取眼球检测兔视网膜组织中超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(maleic dialdehyde,MDA)含量。结果:兔眼造成青光眼模型后,其眼压在观察期内维持在32～39mmHg;高眼压模型空白组与正常对照组、EBHM治疗组、GSTT组比较,视网膜中MDA和SOD的含量差异有统计学意义(P<0.05),EBHM治疗组、GSTT组之间数值差异无统计学意义(P>0.05),EBHM治疗组、GSTT组与正常对照组比较视网膜中MDA的含量仍略高(P<0.05),SOD的含量略降低(P<0.05)。结论:白蒺藜皂苷能有效提高慢性高眼压下兔视网膜中SOD活性,降低MDA含量,从而对持续性高眼压视网膜氧化应激具有保护作用。     

Neuroprotective effect of Erigeron Breviscapus (vant) Hand -Mazz on NMDA -induced retinal neuron injury in rats

To investigate whether Erigeron Breviscapus (vant) Hand-Mazz (EBHM) has neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced neuron death in retinal ganglion cell layer (RGCL). · METHODS: Sixty healthy SD rats were randomly divided into four groups. 6 animals were normal control group (group A). The others were divided as group B (EBHM group), group C (normal saline+NMDA group) and group D(EBHM + NMDA group), each group had 18 rats. 10nmol NMDA was intravitreally injected to induce partial damage of the neurons in RGCL in the right eyes of Groups C and D. Same volume PBS was intravitreally injected into the left eyes as self-control. Groups B and D were pretreated intraperitoneally with 6g/L EBHM solution at a dose of 150mg/kg body weight/day seven days before and after NMDA treatment. Group C were administrated intraperitoneally with 9g/L normal saline at the same time of EBHM injection. Rats were sacrificed at 4,7,14 day after NMDA treatment. Flat whole retinas were stained with 5g/L cresyl violet and neuron counting in RGCL from both eyes were observed. Each subgroup had 6 rats. · RESULTS: There was no significant difference of neuron counting in RGCL between the right eye and the left eye in group A. There was no significant difference between normal control group and EBHM group either in the right eyes or in the left eyes at 4, 7 and 14 day respectively after intravitreal injection of 10nmol NMDA in group C and group D. (P = 0.636, P =0.193). Neuron counting of RGCL in group C and D was significantly decreased in the NMDA- treated eyes 4, 7 and 14 days after intravitreal injection (P < 0.001). There was no significant difference between self-control eyes group and normal control group. However, neuron counting was significantly higher in the EBHM+NMDA group than normal saline +NMDA group 14 days after intravitreal injection (P = 0.044), but lower than normal control group (P <0.05). · CONCLUSION: EBHM has no effect on neuron counting of RGCL when administered alone in normal rats. The results indicate that EBHM plays a partial protective role in NMDA-induced neuron loss in RGCL in rats.     

N -methyl- D -aspartate (NMDA) induced apoptosis in adult rabbit retinas

Previously we showed that apoptosis is involved in N -methyl- D -aspartate (NMDA) induced excitotoxicity in adult rat retinas. Since rabbits have a higher endogenous level of glutamate in the retina and very different retinal structures, it is not clear if apoptosis is similarly involved in adult rabbit retinas after intravitreal injection of NMDA. In this study, we used ultrastructural features, TdT-mediated biotin-dUTP nick end labeling (TUNEL) and two caspase inhibitors to examine whether apoptosis is involved in NMDA-induced excitotoxicity in adult rabbit retinas. At 18 hr after an intravitreal injection of 400 nmoles NMDA, typical apoptotic features in degenerative cells in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) were noted by electron microscopy. TUNEL positive nuclei were detected in these layers as early as 4 hr showing maximal numbers at 18 hr. At 7 days, significant loss of nuclei from the RGCL was noted at the visual streak, the superior and the inferior retinas. These losses were abolished by simultaneous administration of MK-801 and ameliorated by YVAD, a caspase-1 inhibitor, but not by IETD, a caspase-8 inhibitor. These results indicated that, similar to adult rat retinas, apoptosis is involved in NMDA receptor-mediated excitotoxicity in rabbit retinas and that specific caspases may play important roles.     

灯盏细辛注射液对鼠实验性高眼压视神经轴浆运输的影响

探讨灯盏细辛注射液是否对急性实验性高眼压大鼠视神经浆运输阻滞有促进恢复作用。方法健康ＳＤ大鼠３０只,右眼制作成急性高眼压模型后,随机分成３组。Ａ组６只鼠,做经左侧上丘逆行辣根过氧化物酶（ｈｏｒｓｅ ｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ,ＨＲＰ）标记,并行视网膜神经节细胞（ｒｅｔｉｎａｌ ｇａｎｇｉｏｎ ｃｅｌｌｓ,ＲＧＣｓ）计数。Ｂ组１２只鼠,再分灯盏细辛腹腔注射治疗组与对照组,每组各６只鼠,高眼压     

腺病毒介导的色素上皮衍生因子抑制大鼠脉络膜新生血管的研究

目的 探讨腺病毒介导的色素上皮衍生因子(AdPEDF)对大鼠脉络膜新生血管(CNV)的抑制作用.方法 实验研究.选取6～8周雌性BN大鼠68只(136只眼),CNV模型建立后,采用单纯随机抽样方法将68只大鼠随机分为5组,空白对照组(N组)4只大鼠(8只眼),其余64只大鼠(128只眼)作为实验组.根据给药方式不同将实验组随机分为玻璃体腔注射实验组(A组)、玻璃体腔注射对照组(B组)、球周注射实验组(C组)、球周注射对照组(D组).A组玻璃体腔注射AdPEDF1 μl;B组玻璃体腔注射腺病毒载体注射液(AdNull)1 μl;C组球周注射AdPEDF 1 μl;D组球周注射AdNull 1 μl.于给药后3、7、14及28 d行组织病理、TUNEL染色、荧光素眼底血管造影(FFA)检查、及CNV中央最大厚度测量.应用SPSS 11.5统计学软件对光凝斑荧光素渗漏行秩和检验;对CNV发生率行X2检验;对CNV中央最大厚度行单因素与析因方差分析.结果 A组(54.7%)和C组(56.3%)给药后比给药前荧光素渗漏减轻(t=2.75,3.15;P＜0.01).给药后7d,A组(57.3%)、C组(57.8%)CNV数量减少;B、D组CNV呈显著纤维血管增殖.给药后A、C组CNV中央最大厚度分别为(44.51±0.53)和(44.37±0.48)μm,较N组减小(F=7.57,8.85;P＜0.01),并且随时间延长而减小(F=4.31,5.25;P＜0.05).给药后3 d A组CNV中央最大厚度为(46.35±0.62)μm,比C组(44.90±0.44)μm大(F=3.55,P＜0.05);给药后14及28 d,A组CNV中央最大厚度比C组减少(F=6.54,P＜0.01;F=4.41,P＜0.05).给药后A、C组CNV内皮细胞出现部分TUNEL阳性细胞.术后并发症为白内障(5只眼).结论 AdPEDF对BN大鼠CNV有抑制作用,治疗后7 d起效,14 d抑制作用最强,可持续至28 d.玻璃体腔注射比球周注射起效慢,但抑制作用强.     

玻璃体腔重复注射抗血管内皮生长因子单克隆抗体bevacizumab对糖尿病大鼠视网膜的毒性观察

目的 观察玻璃体腔重复注射抗血管内皮生长因子(VEGF)单克隆抗体bevacizumab(商品名Avastin)对糖尿病大鼠视网膜的毒性作用.方法 40只健康成年雄性Sprague-Dawley大鼠随机分为正常组(A组)和糖尿病组,分别为10、30只.糖尿病组采用链脲佐菌霉素(STZ)尾静脉注射方法制作糖尿病动物模型.随机选取10只作为糖尿病视网膜病变(DR)组(B组),不作任何处理;其余20只大鼠左眼为实验组(C组),玻璃体腔注射25 mg/ml的bevacizumab 3 μ1,共注射3次,每次间隔10 d;右眼为实验对照组(D组),不给予任何处理.末次注射后20 d,采用闪光视网膜电图(F-ERG)对各组大鼠行视网膜功能检测;溴化乙锭(EB)染色视网膜铺片,荧光显微镜观察各组大鼠视网膜血管变化情况;苏木精-伊红(HE)染色,光学显微镜观察大鼠视网膜形态学变化;免疫组织化学染色法观察大鼠视网膜各层Thy-1及VEGF阳性表达情况.结果 F-ERG检测显示,A、B、C、D 4组暗适应a、b波潜伏期,暗适应b波振幅及振荡电位(Ops)总振幅比较,差异均有统计学意义(F=33.165,36.162,19.955,23.243;P值均=0.000);A、B、C、D4组暗适应a波振幅比较,差异无统计学意义(F=0.097,P=0.961).荧光显微镜观察发现,A组大鼠视网膜血管走行良好,B组大鼠视网膜血管走行纡曲、扩张,C组大鼠视网膜血管走形规则、变细,D组大鼠视网膜可见微血管瘤.光学显微镜观察发现,A组大鼠视网膜层次结构整齐分明,B组大鼠视网膜各层细胞结构排列紊乱,C组大鼠视网膜各层细胞排列整齐,D组大鼠视网膜各层细胞排列不整齐.免疫组织化学染色发现,Thy-1阳性表达主要位于神经节细胞层(GCL),极少数位于内丛状层、内核层.VEGF阳性表达主要定位于GCL,少量位于神经纤维层、内核层及视网膜色素上皮层.结论 玻璃体腔重复注射bevacizumab对糖尿病大鼠视网膜有一定毒性作用.

Abstract：

Objective To observe the retinal toxicity of repeated intravitreal injection with bevacizumab(Avastin)in diabetic rats.Methods Forty male Sprague Dawley(SD)rats were randomly divided into normal group(Group A,10 rats)and diabetes mellitus group(30 rats).The rats in diabetes mellitus group were induced with streptozotocin injection for diabetic retinopathy model.And then randomly divided into diabetic retinopathy(DR)group(Group B,10 rats),the rats were not intervened;the left eyes of the other 20 rats were intravitreal injected with bevaeizumab 3 μ1(25 mg/m1)for 3 times as experimental group(Group C);the right eyes of the 20 rats were not intervened as experimental control group(Group D),20 days after last intravitreal injection,retinal function was measured by Flicker Electroretinogram (F-ERG);retinal vascular pattern was determined by fluorescence microscopy of ethidium bromide(EB)stained retinal flat mounts;retinal morphological changes were determined by light microscope on hematoxylin-eosin (HE) stained sections;Thy-1 and VEGF expression was measured by immunohistochemistry staining.Results F-ERG showed that-the differences of a-and b-waves-the b-wave amplitude and the Ops-wave amplitude in the implicit time between group A,B,C and D were significant (F=33.165,36.162,19.955,23.243;P=0.000);the differences of a-wave amplitude between group A,B,C and D was not significant(F=0.097,P=0v961).Retinal blood vessel pattern was normalin Group A;retinal vascular vessels were tortuous and irregularly expanded in Group B:retinal vascular vessels of Group C were regular and thinner than Group A;microaneurysm were showed in Group D.Light microscope displayed that the layers of the rat retina of Group A were regular,the retinal architectures of Group B were irregular,the retinal layers were regular in Group C,the retinal layers were irregular in Group D.Immunohistochemistry staining discovered that Thy-1 and VEGF were mainly expressed in ganglion cell layer(GCL).Conclusion Repeated intravitreal injection of bevacizumab iS toxic tO retina of diabetes mellitus rats.     

糖尿病大鼠药物性玻璃体松解术的研究

目的 研究在糖尿病大鼠中能否采用药物性玻璃体松解术诱导完全性玻璃体后脱离(PVD).方法 实验研究.成年SD大鼠用链脲佐菌素(STZ)腹腔注射诱发糖尿病,4周后用视觉电生理、视网膜血管铺片、透射电镜观明确视网膜病变的发生.发病4周后将糖尿病大鼠分为4组:A组10只,右眼玻璃体内注射透明质酸酶5 U;B组10只,右眼玻璃体腔内注射纤溶酶0.5 U;C组10只,右眼玻璃体腔内注射纤溶酶0.5 U+透明质酸酶5 U;D组10只,右眼玻璃体注射BSS 2 μl.注射后一周内观察眼部一般情况并检查视觉电生理变化(F-ERG).一周时取各组大鼠视网膜做扫描电镜检查和组织切片,观察视网膜组织结构及玻璃体后脱离的发生情况.对数据采用t检验进行统计学分析.结果 糖尿病大鼠发病4周时已经有视觉电生理的改变,血管铺片显示周细胞的减少(t=6.1,P<0.01);Ops振幅降低、峰时延迟(t=2.8,3.4;P<0.05);透射电镜显示毛细血管周细胞水肿变性.玻璃体腔注射药物一周后,通过扫描电镜观察A组和D组无PVD发生(0/10);B组发生部分性PVD 6/10,完全性PVD 4/10;C组发生完全性PVD 10/10.视觉电生理检查各组与注射前相比差异均无统计学意义(P>0.05),组织切片检查未显示视网膜组织结构的异常改变.结论 STZ诱导糖尿病4周大鼠视网膜已经出现了病理改变,在其玻璃体腔中注射0.5 U的纤溶酶加5 U的透明质酸酶能够稳定地诱发出完全性PVD,而单独使用纤溶酶仅能诱发出部分性PVD,透明质酸酶不能诱发出PVD.各组未见药物对视网膜的结构和功能造成明显的毒性反应.