CNTF和Ad-BDNF对视神经夹伤后视网膜神经节细胞存活的影响

目的:观察大鼠视神经夹伤后玻璃体腔内注射睫状神经营养因子(CNTF)和腺病毒介导脑源性神经营养因子(Ad-BDNF)对视神经损伤后视网膜神经节细胞(RGC)存活的影响。方法:制作大鼠视神经定量夹伤模型,玻璃体腔内注射CNTF和Ad-BDNF,经上丘荧光金(FG)逆行标记RGC,计数视网膜铺片上的RGC并行统计学分析。结果:正常SD大鼠视网膜上RGC密度为2155±265个/mm2(n=12),视神经夹伤后RGC在1~2wk内下降速率最快,到3,4wk时RGC细胞数量虽仍有减少但下降速度已经明显减慢。CNTF组在视神经夹伤后1wk时视网膜RGC数显著高于对照组,但2~4wk的结果和对照组比较差异不明显。Ad-BDNF组视神经夹伤后1~4wk视网膜RGC数均显著高于对照组。结论:CNTF治疗组玻璃体腔内一次性注射CNTF可以在损伤早期2wk内为损伤的RGC提供神经营养因子,减少RGC的早期死亡。Ad-BDNF治疗组的这种保护作用可以持续到损伤后4wk,能够为RGC提供长时间地营养支持,但这种作用比较局限,可能与单一营养因子作用有关。     

腺病毒介导CNTF基因眼内转移对视神经损伤大鼠F-VEP的影响

目的观察腺病毒(adenovirus,Ad)介导的睫状神经营养因子(ciliaryn eurotrophic factor,CNTF)对视神经钳夹伤大鼠闪光视觉诱发电位(flash visual evoked potential,F-VEP)的影响。方法采用钳夹视神经法制作大鼠视神经损伤模型,夹伤后分别向各组大鼠伤眼内单次注射Ad-CNTF、PBS、Ad-LacZ、CNTF,分别于伤前和伤后1d、14d、28d检测F-VEP的P1波振幅和峰潜时变化。结果伤后1d,各组P1波振幅大幅下降,峰潜时明显延长。伤后14d,各组P1波峰潜时恢复正常,并维持至28d;Ad-CNTF组P1波振幅(10.79μV±2.38μV)与伤后1d(2.97μV±1.21μV)相比,有部分恢复,且好于各对照组。伤后28d,Ad-CNTF组P1波振幅(14.26μV±2.55μV)比各对照组恢复好,差异非常显著(P<0.01);并且好于本组14d时,差异非常显著(P<0.01)。结论视神经损伤后伤眼内单次注射Ad-CNTF可促进受损视神经传导功能的恢复,并可持续到伤后28d。     

视网膜下移植睫状神经营养因子编码基因修饰的细胞保护SD大鼠视神经横断伤后视网膜节细胞变性

目的探讨移植表达睫状神经营养因子(CNTF)的细胞对SD大鼠视神经横断伤后视网膜节细胞的保护作用。方法通过脂质体将CNTF表达质粒转移至人胚肺成纤维细胞,建立稳定、高水平表达CNTF的细胞株。采用双侧背外侧膝状体及上丘核团注射3%荧光金逆行标记视网膜节细胞。将标记后的大鼠分为两组,于标记后7d手术切断眶内段视神经其中一组左眼不做手术作为正常对照组,右眼切断视神经作为手术对照组;另一组双眼均手术切断视神经,左眼注射PBS作为治疗对照组,右眼视网膜下移植表达CNTF的细胞作为实验组。术后5、14、17、21及28d取出眼球,铺片后荧光显微镜观察并计数视网膜内存活的节细胞。结果手术切断眶内段视神经后2周,视网膜内节细胞数减少6744%,视网膜下移植表达CNTF的细胞后第5、17、21d视网膜内存活的节细胞数明显多于治疗对照组(P<005)。结论视网膜下移植高水平表达CNTF的细胞对视网膜节细胞有保护作用。     

人脐血干细胞玻璃体腔移植对大鼠外伤性视神经损伤的保护作用

目的观察人脐血干细胞(human umbilical cord blood stem cells,h UCBSC)移植对视神经部分受损SD大鼠视网膜神经节细胞(retinal ganglion cells,RGC)的保护作用。方法将48只健康成年SD大鼠随机分为2组,2组SD大鼠暴露视神经,应用40 g夹持力的视神经夹在大鼠眼球后2 mm处夹视神经30 s造成部分视神经损伤模型,均损伤左眼。A组:伤后1周玻璃体腔注射脐血干细胞载体PBS,24只。B组:伤后1周玻璃体腔注射h UCBSC,24只;2组均于注射后7 d、14 d、21 d、28 d处死动物。处死前7 d双上丘注射50 g·L-1荧光金逆行标记双眼RGC。然后按时间先后处死大鼠分离视网膜置于荧光显微镜下,计数2组RGC并计算RGC标识率。结果 A、B两组视神经损伤眼RGC计数均低于未损伤眼(均为P<0.05),且随时间延长两组RGC标识率均呈下降趋势(均为P<0.05),但B组注射后7 d、14 d、21 d、28 d RGC标识率(77.52±6.33)%、(74.12±8.23)%、(64.78±5.21)%、(59.93±9.00)%下降幅度明显较A组(75.68±8.74)%、(68.21±10.59)%、(56.24±9.34)%、(48.91±8.81)%平缓。同时间段B组RGC标识率均高于A组,且差异均有统计学意义(均为P<0.05)。结论玻璃体腔注射h UCBSC可减缓外伤性视神经损伤大鼠视网膜中RGC的凋亡,对RGC具有一定的保护作用。     

雪旺细胞源营养神经活性物质对大鼠视网膜节细胞损伤的作用

目的 观察雪旺细胞(Schwann cells,SC)源营养神经活性物质 (SC derived neurotrophic activity,SCNA)对Sprague-Dawly(SD)大鼠视神经损伤后视网膜节细胞(retinal ganglion cells,RGC)存活的影响。 方法 体外培养日龄3～5 d SD乳鼠SC,收集无血清条件培养液,经超滤浓缩后制成冻干粉。SD大鼠分为正常对照组,视神经夹伤对照组,视神经夹伤溶剂对照组,视神经夹伤SCNA 治疗组,每组20只眼。荧光金逆行标记RGC后7 d,除正常对照组外均行球后视神经夹伤,SCNA治疗组将100 ng SCNA注入大鼠玻璃体腔内。分别于视神经夹伤后第5、7、14、21、28 d 将动物灌注固定,做全视网膜铺片,行RGC计数。 结果 视神经夹伤后第7 d RGC开始减少,14 d时降至正常对照的70.2%,28 d时降至40.5%。SCNA治疗组7 d时RGC数开始减少,但14、21、28 d RGC数均明显多于视神经夹伤对照组及视神经夹伤溶剂对照组(P＜0.01)。 结论 在视神经夹伤后眼内注射SCNA能减少RGC的死亡对RGC损伤有保护作用。（中华眼底病杂志,2000,16：1-70）     

腺病毒介导睫状神经营养因子在大鼠视网膜中的表达定位

目的观察携带睫状神经营养因子(ciliary neurotrophic factor,CNTF)基因的腺病毒(adenovirus,Ad)载体在正常大鼠视网膜中的表达定位。方法正常成年大鼠40只,随机分为处理组(PBS组、Ad-LacZ组、Ad-CNTF组)及正常对照组,向处理组大鼠眼内分别注射相应溶液,各处理组均分为注射后7d、14d、28d共3个时相点。在相应时相点取大鼠眼球,行冰冻切片,进行免疫组织化学染色。结果Ad-CNTF组各时相点视网膜与其他对照组相比,CNTF阳性染色明显增强,分布更广泛,并且可一直持续到注射后28d。结论Ad-CNTF在正常大鼠眼内注射后,CNTF在视网膜的表达明显增加,且表达时限可达28d。     

脑源性神经营养因子对大鼠视网膜节细胞损伤的保护作用

目的：观察脑源性神经营养因子（brain-derived neurotrophic factor,BDNF)对Sprague-Dawly(SD)大鼠视神经损伤后视网膜节细胞（retinal ganglin cells,RGC)存活的作用。方法：将SD大鼠分为正常对照组,夹伤对照组,溶剂对照组,BDNF治疗组4组,每组20只眼。荧光金逆行标记RGC后7天,除正常组外行球后视神经夹伤,将100ng BDNF注入BDNF治疗组大鼠玻璃体腔内,分别于5,7,14,21,28天行RGC计数。结果：视神经夹伤后第7天RGC开始减少,14天时降至正常对照的70．2％,28天时降至40．5％。与正常组相比BDNF治疗组14天时RGC数开始减少,但7、14、21、28天RGC数均明显多于夹伤组（P＜0．01）。结论：在视神经夹伤后眼内注射BDNF能减少RGC的死亡,对RGC损伤有保护作用。     

利用荧光金逆行示踪技术评价视神经不完全损伤后视网膜神经节细胞的存活率

目的　利用荧光金逆行示踪技术评价正常及视神经不完全损伤后的视网膜神经节细胞 (retinalganglioncells,RGCs)的数目。 方法　正常成年LongEvans大鼠 2 0只 ,体重(2 70± 2 0 ) g ,雌雄不限。按对照组、损伤后 7d、14d、2 1d分组 ,每组 5只大鼠。在球后视神经钳夹伤前 7d行荧光金逆行标记。 7d后损伤组大鼠用反向血管夹于左眼球后 2mm处夹视神经 10s。对照组大鼠只暴露视神经不行钳夹 ,分别于各时间点将大鼠用 4 %多聚甲醛灌注固定后 ,行全视网膜铺片 ,3h内在荧光显微镜下观察。在每张视网膜的上、下、鼻、颞侧距视盘 1/ 6、1/ 2、5 / 6半径处共拍摄 12张荧光照片。对照片上标记的RGCs进行计数 ,求平均值 ,计算损伤后各时间点剩余的RGCs与正常视网膜中RGCs的百分比。结果　视网膜铺片的RGCs边界清晰 ,并可见明显的细胞突起 ,血管走行区未见节细胞分布。正常组每张铺片的平均节细胞数为 (2 0 31± 2 87)个·mm-2 ,损伤后 7d的RGCs存活率为 71% ,14d存活率为 5 1% ,2 1d存活率为 35 %。结论　荧光金逆行标记是评价视神经损伤后RGCs存活率可靠并且有效的方法。     

表没食子儿茶素没食子酸酯对大鼠视神经钳夹伤后视网膜神经节细胞的保护作用

　　目的　观察绿茶提取物表没食子儿茶素没食子酸酯(EGCG)对大鼠视神经钳夹伤视网膜神经节细胞（RGC）是否具有保护作用。方法　72只Wistar大鼠随机分为正常对照组（A组）、假手术+EGCG组（B组）、视神经钳夹+生理盐水组（C组）、视神经钳夹+EGCG组（D组）等4组,每组各18只。B、D组在视神经钳夹或假手术前2 d起给予腹腔注射EGCG  25 mg/（kg·d）,直至手术后2 d,共5 d;随后改为口服2 mg/（kg·d）。C组以生理盐水替代EGCG。每次每组取6只大鼠,采用3%荧光金经上丘逆行标记RGC方法,比较各组视神经钳夹伤后7、14、28 d RGC的存活数量;采用免疫组织化学染色及蛋白免疫印迹方法检测各组视神经组织神经丝蛋白（NF-L）的表达。结果　视神经钳夹伤后7 d,C、D组RGC存活数量分别为（943.61±85.06）、（1 134.45±117.85） 个/mm2;14 d时分别为（812.76±172.07）、（1 021.67±94.02） 个/mm2;28 d时分别为（766.94±171.45）、（1 009.72±126.40）个/mm2。各时间点D组RGC存活数量均显著高于C组（t=3.216,2.609,2.792;P=0.009,0.026,0.019）。各时间点A、B组间RGC存活数量差异无统计学意义（t=0.749,0.403,0.254;P值均＞0.05）;视神经钳夹后7、14、28 d,D组视神经组织NFL表达均高于C组（t=9.847,5.731,2.868;P=0.001,0.005,0.045）。结论　EGCG对大鼠视神经钳夹伤后RGC具有一定的保护作用。      

超声微泡造影剂联合美金胺增强视神经损伤大鼠视网膜神经节细胞保护作用

目的 探讨超声微泡造影剂联合美金胺对视神经损伤大鼠视网膜神经节细胞( RGC)的保护作用.方法 将Sprague-Dawley(SD)雄性成年大鼠40只随机分为正常对照组(A组),假手术组(B组),空白对照组(C组),玻璃体腔单独注射美金胺组(D组),玻璃体腔注射美金胺加超声微泡组(E组)5个组,每组8只大鼠,再将各组随机分为视神经损伤后1、2周2个亚组,各亚组4只大鼠.A组不做任何处理;B组只暴露视神经,不进行钳夹,玻璃体腔注射生理盐水,立即用超声波辐照大鼠眼球;C～E组建立视神经钳夹伤模型后,处理方式分别为C组玻璃体腔注射生理盐水,D组玻璃体腔注射美金胺,E组玻璃体腔注射超声微泡造影剂及美金胺,立即用超声波辐照大鼠眼球.视神经损伤1、2周时,各组行逆行荧光金标记RGC并计数;闪光视觉诱发电位(F-VEP)检测,记录P100波潜伏期及振幅;荧光电子显微镜下观察视网膜细胞形态学改变.结果 逆行荧光金标记RGC结果显示,各处理组视网膜定向铺片上均可见金黄色着染的RGC.A、B组RGC数间差异无统计学意义(q=0.018,0.011;P=0.986,0.873);C～E组RGC数均较A组减少,差异具有统计学意义(F=85.944,P=0.012);D组RGC数多于C组,差异具有统计学意义(q=1.721,1.924;P=0.043,0.037);E组RGC数明显高于C、D组,差异具有统计学意义(q=1.128,1.482,P=0.027,0.008;q=1.453,1.855,P=0.031,0.010).F-VEP检测发现,A、B组P100波潜伏期及振幅间差异无统计学意义(q=0.008,0.019,P=0.981,0.946;q=0.072,0.052,P=0.737,0.851) ;C～E组P100波潜伏期较A组延长,振幅较A组降低,差异具有统计学意义(F=134.312,106.312;P=0.017,0.009).荧光电子显微镜下观察发现,A、B组大鼠视网膜各层结构完整,排列整齐,RGC排列紧密整齐,细胞核均匀深染,胞核大小一致.C～E组大鼠的视网膜不同程度水肿变厚,RGC有不同程度的排列紊乱,空泡化及细胞数目减少.结论 超声微泡造影剂联合美金胺能抑制视神经损伤后大鼠RGC的丢失,促进其视功能的恢复,对视神经损伤大鼠的RGC具有保护作用.     

Evaluation of visual fields by ophthalmologists and by OCTOSMART program

The introduction of Goldmann perimetry standardized measuring conditions as much as possible. In spite of this, it had been possible for the perimetrist to influence the results of perimeter measurements. The introduction of computer-controlled perimetry, however, has largely eliminated the influence of the investigator on perimetry results. Nevertheless, the interpretation of a perimetric result in the everyday clinical situation is still extensively subjectively coloured and is liable to vary, depending on the doctor carrying it out. The OCTOPUS Program G1 was introduced a few years ago and used above all for glaucoma. This program greatly simplified visual field assessment thanks to its visual field indices. The indices make it possible to compare visual field results with those of a normal population. The present introduction of the OCTOSMART program represents a further step forward. This program analyses measured visual fields with the aid of standardized, statistical criteria based on a large, normal value study. This analysis standardizes and thereby simplifies the interpretation of visual field results. This study compares the outcome of the OCTOSMART program with visual field interpretations by eye doctors.     

Inhibition by indomethacin of the increased facility of outflow induced by adrenaline

Topical adrenaline lowers intraocular pressure (IOP) in the rabbit largely due to an increase in facility of outflow of aqueous humour. This paper studies the inhibition by indomethacin or piroxicam of the adrenaline-induced rise in facility of outflow. Topical indomethacin is shown to reduce the acute IOP changes induced by adrenaline in conscious rabbits; both the early rise and the prolonged fall in pressure were inhibited. In anaesthetized rabbits, indomethacin pretreatment prevented the large rise in facility of outflow which normally follows topical adrenaline. Indomethacin did not block the mydriasis induced by adrenaline, nor did it significantly alter aqueous humour protein levels. Piroxicam, a cyclo-oxygenase inhibitor which, unlike indomethacin, does not block Ca2+ movements in some tissues, also blocked the adrenaline-induced rise in facility of outflow, suggesting that this increased facility depends on cyclo-oxygenase and not on Ca2+ movements. Verapamil, a drug which blocks Ca2+ channels, was shown to inhibit the brief ocular hypertensive effect of adrenaline in the conscious rabbit, but to leave the hypotensive phase unchanged. It is concluded that the hypotensive mechanism of adrenaline may depend on synthesis of a prostaglandin, since inhibition of the adrenaline-induced rise in facility is achieved by inhibitors of cyclooxygenase. Despite previous reports that a prostaglandin may be responsible for the brief hypertensive phase, the present evidence suggests that Ca2+ movements may be involved, perhaps in activation of the extraocular muscles.     

Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.     

Synthesis of chondroitin sulfate by fibrotic vitreous induced by monocytes and lymphocytes

Vitreous fibrosis was induced in rabbit eyes by intravitreal injection of monocytes and lymphocytes. The fibrotic vitreous and normal vitreous removed from experimental animals were then incubated with [3H]-glucosamine at 37 degrees C for 24 hr. The newly synthesized 3H-labeled glycosaminoglycans (GAGs) were isolated by 4 M GuHCl extraction followed by pronase digestion. The 3H-labeled GAGs were then characterized by gel-filtration column chromatography and by specific enzymatic degradation, i.e. hyaluronidase, chondroitinase AC and/or chondroitinase ABC. The disaccahrides derived from chondroitinase ABC degradation were identified by thin-layer chromatography. Our results indicated that 91% of the total glycosaminoglycan synthesized by normal vitreous was hyaluronic acid. In contrast, in the fibrotic vitreous, the synthesis of hyaluronic acid was decreased to 30% whereas the synthesis of chondroitin sulfate increased to 47% of the total newly synthesized glycosaminoglycans. Control vitreous which was injected with freeze-thawed monocytes and lymphocytes synthesized 70% hyaluronic acid and 12% chondroitin sulfate although no fibrosis was observed.