依托咪酯促进大鼠视神经损伤后再生的研究

目的探讨依托咪酯对成年大鼠视神经损伤后再生的影响。方法选取25只成年SD大鼠,按随机数字表法随机分为依托咪酯治疗组（腹腔注射依托咪酯脂肪乳注射液,15只）、治疗对照组f腹腔注射脂肪乳,5只）和空白对照组（5只）;治疗组又分为低（2mg／kg）、中（4mg／kg）和高（6mg／kg）剂量3个亚组,每亚组5只。采用自体坐骨神经移植模型和荧光金逆行标记再生视网膜神经节细胞（RGCs）。自体坐骨神经移植术后4周,采用视网膜平铺技术计数再生RGCs。结果空白对照组每张视网膜中再生RGCs数量平均为（1032±147）个,治疗对照组为（1114±179）个,两者之间无明显差异fP〉0．05）。低、中和高剂量依托咪酯治疗组每张视网膜中再生RGCs数量分别为（2054±349）个、（2853±498）个和（4118±615）个,与空白对照组和治疗对照组相比均显著增高（P〈0．01）,而且不同剂量之间均差异显著（P〈0．01）。结论依托咪酯能显著促进大鼠视神经损伤后轴突再生,且具有剂量依赖性。     

管内段视神经损伤后视网膜的形态学改变及GAP-43的表达

背景：视神经损伤后,视网膜发生一系列复杂的病理变化,从而造成功能丧失。 目的：观察管内段视神经损伤后视网膜的病理变化,比较视神经管减压术和激素对管内段视神经损伤的治疗作用。 设计、时间及地点：不同实验因素作用下视网膜病理形态学对比观察。实验于2008-09/11在潍坊医学院解剖学教研室完成。 材料：健康成年家兔36只,应用金属圆柱体自由落体冲击法建立兔右眼管内段视神经损伤模型。 方法：随机抽签法将实验兔分为单纯损伤组（n=24）,治疗组（n=12）。利用免疫组织化学、图像分析等技术,观察神经损伤后1、3、5、7、14 d及视神经管减压术、地塞米松、视神经管减压术＋地塞米松治疗14 d后,视网膜的形态学改变及GAP-43的表达。 主要观察指标：视神经损伤后RGCs记数及视网膜GAP-43表达阳性细胞记数的变化。 结果：纳入实验兔36只均进入结果分析。视神经损伤1 d后,RGCs平均记数轻度下降;损伤3、5、7 d时,RGCs数分别为对照组的84.48% 、72.23% 、57.46%;14 d时,节细胞仅有15.43％存活。损伤后3 d,视网膜切片中可见GAP-43表达阳性的细胞;伤后5 d阳性细胞增多;伤后7 d阳性细胞数和积分光密度值达最高峰;伤后14 d阳性细胞数下降。经手术减压、激素治疗、激素＋手术治疗后,RGCs存活率分别为36.01%、32.78%、56.98%。 结论：管内段视神经损伤后RGCs出现渐进性退变,数量逐渐减少;视神经管减压术和激素对管内段视神经损伤有一定的治疗作用,其疗效明显优于只采用一种方法。     

神经干细胞移植对大鼠视神经损伤后节细胞的保护作用

目的探讨神经干细胞（NSCs）移植对视神经受损SD大鼠视网膜节细胞（RGCs）的保护作用。方法将48只健康成年SD大鼠随机分为N组（NSCs移植组）和C组（对照组），均使用精确校准方法在右眼造成部分视神经损伤，左眼作为正常对照。从胚胎SD大鼠海马分离NSCs。利用细胞培养和体内移植技术。将培养后的NSCs注入N组大鼠右眼玻璃体内，C组大鼠右眼玻璃体内注入同等体积的PBS。处死前3d在双上丘注射3％快蓝逆行标记双眼RGCs。将大鼠分别于注射NSCs或PBS后7d、14d、21d、28d处死，各分为4组。每组6只。分离视网膜置于荧光显微镜下，摄影输入计算机图像分析仪计数RGC。计算RGC标识率。另取6只健康成年SD大鼠作NSCs移植，分别于移植后7d、28d处死，每时间段3只。通过视网膜免疫荧光切片观察NSCs在视网膜的存活、整合情况。结果N组大鼠各时间段RGCs标识率与C组同时间段比较均增高，有显著性差异（P〈0．01）；N组与C组RGCs标识率均随时间延长而降低。且前后段时间比较有显著性差异，但N组降低速度明显慢于C组。NSCs移植7d后部分移植细胞迁移进入视网膜的内网层和节细胞层。28d后可见移植细胞广泛整合至宿主视网膜内。结论NSCs移植入视神经损伤大鼠视网膜后可提高视网膜神经节细胞的存活率．对受损的节细胞具有一定的保护作用。     

神经干细胞移植后在视神经损伤大鼠视网膜内表达BDNF的研究

目的探讨神经干细胞(NSC)移植入视神经损伤(ONI)的SD大鼠玻璃体下腔后在视网膜内表达脑源性神经营养因子(BDNF)的情况,为进一步探索NSC移植治疗视神经损伤提供实验依据。方法体外培养NSC,其上清液采用酶联免疫吸附实验(ELISA)定量分析BDNF含量。取34只SD大鼠,其中4只作为正常对照,另外30只制作成右眼ONI模型并随机等分为N组和P组。ONI术后随即向N组大鼠右眼玻璃体下腔注入定量NSC,P组注入等量PBS,并采用半定量RT-PCR方法检测正常大鼠视网膜及N组、P组大鼠术后第3天、1周、2周、3周、4周时视网膜BDNFmRNA的表达水平,行统计学分析。结果正常视网膜内可见BDNF表达;N组与P组大鼠视网膜内表达BDNF的量除第1周差异无统计学意义外,余时间段N组均高于P组。结论NSC能促使视神经损伤大鼠视网膜高表达BDNF,NSC移植治疗视神经损伤值得进一步深入研究。     

MAPK/ERK信号通路在大鼠视神经损伤后小胶质细胞活化中的作用及机制研究

目的 探讨丝裂原激活蛋白激酶(MAPK)/细胞外信号调节蛋白激酶(ERK)信号通路在大鼠视神经损伤后小胶质细胞活化中的作用及机制。方法 将100只成年雄性大鼠随机分为实验组50只和对照组50只,实验组采用荧光金双侧上丘逆行标记视网膜神经节细胞并在培养7d后制作视神经损伤模型,免疫组织化学方法检测2组视网膜铺片小胶质细胞计数情况,应用蛋白免疫印记法检测2组MAPK通路的ERK蛋白表达水平及磷酸化蛋白水平,并应用免疫组化法检测2组视网膜白介素10(IL-10)和肿瘤坏死因子(TNF-α)等表达水平; 向实验组视网膜铺片添加ERK1/2通路阻断剂PD98059,再次检测其IL-10和TNF-α等表达水平及小胶质细胞计数情况。结果 与对照组比较,实验组ERK蛋白表达水平及磷酸化蛋白水平降低,视网膜铺片小胶质细胞计数降低,IL-10阳性率和TNF-α阳性率升高(P<0.05)。与阻断前比较,实验组阻断后6 h的IL-10和TNF-α等表达水平升高,小胶质细胞计数降低(P<0.05)。结论 MAPK/ERK信号通路在大鼠视神经损伤后小胶质细胞活化中具有保护作用,其机制可能通过抑制炎症因子IL-10和TNF-α等表达而减少其视神经损伤并促进小胶质细胞活化。     

移植时间对大鼠视神经损伤后神经干细胞迁移的影响

目的探讨移植时间对大鼠视神经损伤后神经干细胞(NSCs)在视网膜内迁移的影响。方法 18只SD大鼠行右侧视神经损伤后按随机数字表法随机平均分为3组,并分别于损伤后当天及伤后7d和14d采用微量注射法行右眼视网膜下腔移植2μl(105个/μl)转染绿色荧光蛋白基因的NSCs(GFP-NSCs)。4周后处死大鼠,取右眼球做冰冻切片,对迁移到视网膜内的移植细胞进行计数。各组切片分别用胸腺细胞表面糖蛋白、胶质纤维酸性蛋白、β微管蛋白、视紫红质抗体进行免疫标记,观察各组移植细胞在视网膜上的分化情况。结果损伤当天及伤后7和14d移植组视网膜上的GFP阳性细胞数分别为(163441.14±16876.81)个、(145736.57±27449.07)个和(117291.33±15849.19)个,两两相较,均差异显著(P<0.01)。三组的移植细胞均可在宿主视网膜内存活、迁移,且可分化为神经胶质细胞、神经元和视网膜神经节样细胞。结论大鼠视神经损伤后随着移植时间的推迟,迁移到宿主视网膜内的移植细胞量下降。     

兔视神经损伤后谷氨酸对视网膜的兴奋毒性损伤作用研究

目的探讨家兔视神经损伤后谷氨酸浓度变化对视网膜的兴奋毒性及其作用机制。方法 40只健康雄性日本大耳白家兔随机分成2组,A组35只,B组5只。A组家兔左眼为实验制备视神经损伤模型,右眼为对照眼。B组家兔双眼同时制备视神经损伤模型。分别于第1、3、7、14及28d随机处死A组7只家兔及B组1只家兔,检测家兔实验眼和对照眼视网膜谷氨酸浓度,并观察B组家兔视网膜病理形态学改变。结果家兔视神经损伤后视网膜内谷氨酸浓度逐渐升高,3d达到高峰,14d时家兔实验眼视网膜谷氨酸浓度仍高于对照眼(p<0.05),28d时其视网膜谷氨酸浓度无统计学差异(p>0.05)。家兔视网膜病理形态学观察:视神经损伤后1d,神经节细胞排列紊乱;3～7d,视网膜节细胞大量空泡化;7d～14d,视网膜厚度变薄,神经节细胞减少;14d后视网膜各层细胞改变逐步趋于稳定。结论家兔视神经损伤后,视网膜谷氨酸浓度升高是视网膜神经节细胞继发损伤的原因之一。     

骨髓间充质干细胞对癫痫模型认知及海马区GABAA受体表达的影响

目的探究骨髓间充质干细胞(BMSCs)移植对大鼠癫痫模型认知功能及海马CA1区域γ氨基丁酸A受体(GABAARs)的表达的影响。方法成年雄性SD大鼠30只随机分为正常组(n=10),模型组(n=10)和实验组(n=10)。采用匹鲁卡品诱导癫痫模型,2 h后移植进行BMSCs移植,28 d后水迷宫检测认知变化,免疫组化和实时荧光定量PCR(RT-PCR)检测大鼠海马CA1区GABAARs的表达变化。结果 RT-PCR和免疫组化结果显示,模型组相比正常组GABAARα1表达水平(mRNA以及免疫组化染色强度)下调(P0.05)而GABAARα4上调(P0.05),移植组GABAARs相关改变比模型组轻微(P0.05),且认知功能改善明显(P0.05)。结论 BMSCs移植可调节大鼠癫痫模型中海马区域GABAARs表达水平并减轻癫痫相关认知障碍。     

猫视神经慢性受压后视网膜神经节细胞的动态观察

目的 观察猫视神经慢性受压后视网膜神经节细胞(RGCs)的动态变化.方法 30只成年家猫按随机数字表法等分为正常对照组、假手术组、压迫1周组、压迫2周组、压迫4周组和压迫8周组,每组5只.利用球囊植入法建立慢性视神经损伤模型.所有动物在术前2周用Dil逆行标记RGCs.取各组动物视网膜进行光镜、电镜观察,并在荧光显微镜下对RGCs进行计数.结果 正常成年猫视网膜HE染色可见三层细胞核,各层间界限较为清楚,从玻璃体腔向巩膜依次为神经节细胞层、双极细胞层和感光细胞层;视神经慢性受压4周时视网膜变化仍不明显:受压8周时可见RGCs核明显稀疏,大而染色浅的细胞核基本消失,视网膜总厚度变薄,胶质细胞增生.电镜下正常猫RGCs核呈卵圆形、核大、核质均匀,有时可见核仁,胞浆占细胞比例小,但细胞器丰富;受压2周时视网膜无明显改变;受压4周和8周时可见RGCs胞浆成分疏松、内质网扩张、线粒体肿胀、质膜下出现空泡、核膜皱缩内陷、胞浆空化、染色质边聚.正常组DiI逆行标记的RGCs的密度在406～527个/mm2之间,平均为(465±38)个/mm2,中央区密度高于周边区;视神经受压后前4周RGCs数量无明显改变;受压8周时RGCs数量有显著下降,平均密度为(293±32)个/mm2.下降约37%.结论 视神经慢性受压后RCJCs将出现延迟的变性.为视神经受压后RGCs的保护提供了时机.     

酒精戒断对清醒大鼠海马DG区部分兴奋性氨基酸的影响

目的探讨在清醒状态下大鼠长期饮酒停饮后出现戒断症状时海马DG(dentate gyrus)区部分兴奋性氨基酸含量变化。方法成年Wistar大鼠(300g~350g)40只随机分成对照组(n=10)和酒精组(n=30)。酒精组用50%酒精按10ml/kg/d灌胃1周(n=10)、2周(n=10)、4周(n=10),对照组给予同等剂量的生理盐水灌胃4周。用微透析法和高效液相色谱法分别测定末次饮酒6h、30h、48h、72h海马DG区谷氨酸及天冬氨酸含量,同时观察大鼠撤除酒精后不同时间戒断症状。结果2周酒精组及4周酒精组在末次饮酒30h、48h时,清醒大鼠海马DG区天冬氨酸及谷氨酸水平较末次饮酒6h显著上升(P<0.05),末次饮酒72h与末次饮酒6h无明显变化;兴奋性氨基酸含量升高时间与戒断症状明显出现时间基本一致;1周酒精组、对照组在上述各时段天冬氨酸及谷氨酸水平无显著变化。结论(1)酒精戒断症状与饮酒时间长短呈正相关;(2)酒精戒断综合征与海马DG区细胞外液兴奋性氨基酸神经递质含量增加有关。     

Math5 (Atoh7) gene dosage limits retinal ganglion cell genesis

The basic helix-loop-helix factor Math5 (Atoh7) is critical for the determination of retinal ganglion cell (RGC) fate in mice. Recently, genome-wide association studies have identified the ATOH7 locus as a major determinant of variation in the human optic disc area, which is directly correlated with the RGC number. These studies suggest that the level of Math5 expression may determine the ultimate number of RGCs. To test this hypothesis, we systematically compared optic nerve area and RGC axon number in C57BL/6J congenic Math5+/- and +/+ mice at young adult and neonatal ages by transmission electron microscopy. Optic disc area and RGC abundance were not significantly different in adults, but heterozygotes had thinner optic nerves and 25-30% fewer RGCs at birth than wild-type littermates (P<0.05). Our results suggest that Math5 dosage is important for the genesis, but not the ultimate number, of RGCs. Our findings highlight the importance of ganglion cell culling as a compensatory mechanism for retinal homeostasis, and support a quantitative role for Math5 in RGC specification.     

Dose-dependent and combined effects of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine on the survival of retinal ganglion cells in adult hamsters

This study investigated the effects of daily intraperitoneal injections of N-methyl-D-aspartate receptor antagonist MK-801 and nitric oxide synthase inhibitor nitro-L-arginine (L-NA) on the survival of retinal ganglion cells (RGCs) at 1 and 2 weeks after unilateral optic nerve transection in adult hamsters. The left optic nerves of all animals were transected intraorbitally 1 mm from the optic disc and RGCs were retrogradely labeled with Fluorogold before they received different daily dosages of single MK-801 or L-NA as well as daily combinational treatments of these two chemicals. All experimental and control animals survived for 1 or 2 weeks after optic nerve transection. Our results revealed that the mean numbers of surviving RGCs increased and then decreased when the dosage of MK-801 (1.0, 3.0 and 4.5 mg/kg) and L-NA (1.5, 3.0, 4.5 and 6.0 mg/kg) increased at both 1 and 2 weeks survival time points. Daily combinational use of 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA lead to a highest RGC number that was even higher than the sum of the RGC numbers in 1.0 mg/kg MK-801 and 1.5 mg/kg L-NA subgroups at 2 weeks. These findings indicated that both MK-801 and L-NA can protect axotomized RGCs in a dose-dependent manner and combinational treatment of these chemicals possesses a potentiative and protective effect.     

Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3⁺ microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENT Despite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.     

Topography of ganglion cells and photoreceptors in the sheep retina

Retinal topographies of some cell types and distribution of the tapetum lucidum in the sheep's eye were investigated in this study. The tapetum was observed macroscopically in the fundus. The topographical distributions of retinal ganglion cells (RGCs), cones, and rods were simultaneously analyzed in retinal whole mounts stained with cresyl violet. Short‐wavelength‐sensitive (S) cones were immunocytochemically identified in retinal whole mounts. The tapetum was located dorsal to the optic disc, with the nasal part elongated horizontally and the temporal part expanded dorsally. RGCs were distributed densely in the area centralis, horizontal visual streak, and anakatabatic area. The highest density in the area centralis was approximately 18,000 RGCs/mm². Cones showed high density in the horizontal area crossing the optic disc and dorsotemporal area, whereas rods showed high density in the horizontal area, which was greater in height than the horizontal area of high cone density. S cones showed high density in the dorsotemporal retina. The rod/cone ratios were high horizontally in the dorsal retina to the optic disc, with a mean value of 11:1. The cone/RGC and rod/RGC ratios were lower in the horizontal and dorsotemporal retina, and the rod/cone/RGC ratio was lowest in the area centralis (9:1:1). The retinal topographies and distribution of the tapetum were specialized in the horizontal and dorsotemporal fundus. This suggests that sheep have better visual acuity in horizontal and anteroinferior visual fields and that this specialization is related to the visual ecology of sheep. J. Comp. Neurol. 518:2305–2315, 2010. © 2010 Wiley‐Liss, Inc.     

Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.     

EphA5 and ephrin-A2 expression during optic nerve regeneration: a 'two-edged sword'

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.     

Virally delivered,constitutively active NFκB improves survival of injured retinal ganglion cells

As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno‐associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2‐p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P‐value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P‐value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration.     

Effect of bilateral tectum lesions on retinal ganglion cell morphology in rats.

We have examined morphological changes of retinal ganglion cells (RGCs) during postnatal development in albino rats. Somatic diameter, dendritic field diameter, and branching frequencies of RGCs of normal rats were compared with those of animals that had received bilateral lesions of the tectum immediately after birth. Bilateral lesions of the tectum at P1 (first postnatal day) induced a dramatic increase in RGC death during the time of naturally occurring cell death in the first postnatal week. RGC densities in adult experimental animals were found to be reduced to about 55% of normal. RGCs of normal and operated animals were retrogradely stained with crystals of the fluorescent dye DiI, which was applied to the optic disc of flat mounted and fixed retinae. In normal rats, the somatic and the dendritic field diameters of the RGCs increased and the branching frequency of type I and III RGCs decreased from P1 to P14. By P14, neither the somatic diameter nor the dendritic field size had yet reached adult values and the branching frequencies were still higher than those of adult rat RGCs. In animals with bilateral lesions of the tectum, all cell types showed an increase in somatic sizes, and in type I and II RGCs an expansion of dendritic territories could be observed. The branching frequencies, however, were significantly lower than those of normal rats of the same age. The dendritic morphology in type III RGCs in operated animals was not significantly different from controls. These findings demonstrate a potential plasticity of type I and II RGCs, which respond to a loss of neighbouring cells by expansion of their dendritic field during postnatal development.     

Precrushed sciatic nerve grafts enhance the survival and axonal regrowth of retinal ganglion cells in adult rats.

We have examined the influence of normal and precrushed ("conditioned") sciatic nerve grafts on the survival and axonal growth of retinal ganglion cells (RGCs) in adult rats. Normal sciatic nerves (group A) or sciatic nerves which had been crushed 1 week before transplantation (group B, conditioned grafts) were used as grafts. The nerves were removed and sutured to the proximal stump of intraorbitally axotomized optic nerves. Neuronal survival and axon growth were determined by counting the numbers of surviving, DiI-prelabled RGCs, cresyl violet-stained RGCs and the numbers of axons which had grown into the grafts 3 and 6 months after transplantation. Counting of axons was performed by combined use of light and electron microscopy. We observed that the use of conditioned grafts (group B) significantly enhanced RGC survival and axonal regrowth as compared to normal grafts 3 months after transplantation. Six months after grafting, RGC survival (as determined in DiI-stained retinae) and axonal growth were not significantly different in both groups. These results suggest that the functional status of a peripheral nerve used for grafting in the CNS influences neuronal viability and axonal reelongation especially during the first 3 months after grafting. Very long-term RGC survival, however, may be determined by functional reconnection of regenerating RGC axons rather than by the graft itself.