TNF-α通过刺激RF/6A细胞表达SDF-1促进血管生成

目的 研究肿瘤坏死因子α(TNF-α)对体外培养的猴脉络膜/视网膜内皮细胞(RF/6A)表达基质细胞衍生因子-1(SDF-1)及细胞增殖、迁移和管腔形成的影响,探讨TNF-α参与视网膜血管形成的初步机制.方法 取体外培养的生长良好的RF/6A细胞用于实验,将细胞随机分为对照组、不同浓度TNF-α组(1、10、100 ng/ml)、TNF-α+AMD3100组(SDF-1拮抗剂AMD3100预处理1h,培养液中最终浓度为1μg/ml,然后加入TNF-α).培养24h、48h后采用ELISA法检测细胞上清液中SDF-1含量,MTT法检测细胞增殖,细胞划痕法检测细胞迁移,基质胶(Matrigel)法检测管腔形成.结果 ①TNF-α(1 ng/ml)作用24和48 h,RF/6A的SDF-1表达量与对照组无明显差异;TNF-α(10、100ng/ml)作用后两个时间点SDF-1表达量均明显高于对照组(P＜0.01);TNF-α (10 ng/nl)+AMD3100组SDF-1表达量较TNF-α (10 ng/ml)组明显减少(P＜0.05).②TNF-α不同浓度作用24 h的细胞相对增殖率均高于对照组(P＜0.05),且随着TNF-α浓度的升高而增加;除1 ng/ml浓度组外,TNF-α各组作用48h的相对增殖率均较对照组升高(P＜0.05);TNF-α(10 ng/ml)+AMD3100组的细胞相对增殖率在24h和48h时均明显小于TNF-α(10 ng/ml)组(P＜0.05).③TNF-α (10 ng/ml)组RF/6A细胞24h的迁移距离明显大于对照组(P＜0.05),TNF-α (10ng/ml)+AMD3100组迁移距离较TNF-α(10ng/nl)组减小(P＜0.05);培养48h后,各组之间的差异与24h的结果类似(P＜0.05).④TNF-α (10 ng/ml)组管腔形成数明显多于对照组(P ＜0.01),TNF-α (10ng/ml)+ AMD3100组明显少于TNF-α(10 ng/ml)组(P＜0.01).结论 TNF-α能够促进RF/6A细胞表达SDF-1,诱导细胞增殖、迁移和管腔形成,拮抗SDF-1的作用可明显抑制TNF-α诱导的血管生成.提示TNF-α促进RF/6A细胞的血管生成过程可能是通过刺激细胞产生SDF-1实现的.     

川芎嗪促进大鼠视网膜神经节细胞轴突再生的实验研究

目的通过在视网膜组织三维立体培养系统中加入不同浓度的川芎嗪溶液,观察川芎嗪对视网膜神经节细胞轴突再生的作用。方法建立体外培养的大鼠视网膜组织三维立体培养系统,将1～3d新生远交群SD（Sprague Dawley）大鼠视网膜切成0．5mm×0．5mm大小的视网膜组织,加入不同浓度（0．125、0．25、0．5、1．0g／L）川芎嗪溶液后,在相差显微镜下动态观察视网膜神经节细胞轴突的生长情况,于加药后第3、6和9天记录再生轴突的数目及长度。结果与对照组相比,各浓度川芎嗪对视网膜神经节细胞轴突生长均有促进作用,以0．5g／L浓度效果最明显,差异有统计学意义（P〈0．05）。免疫组织化学染色显示视网膜神经节细胞的轴突具有明显再生现象。结论一定剂量范围的川芎嗪可促进视网膜神经节细胞轴突再生和伸长。（中国跟耳鼻喉科杂志,2009,9：86—88）     

BDNF/GDNF对体外三维培养的成熟视网膜神经节细胞轴突再生的促进作用

目的建立成熟视网膜神经节细胞(RGCs)的体外三维培养模型,并应用此方法研究BDNF/GDNF对体外三维培养的成熟RGCs轴突再生的促进作用。方法取10周龄大鼠,每只鼠眼视网膜切成0·5mm×0·5mm大小16片。将视网膜片神经纤维层向下浸入12孔培养板的胶原溶液层中,胶原溶液凝固后,培养孔内加入无血清MEM培养液。实验组另外分别添加BDNF/GDNF100ng/ml。相差显微镜观察生长情况,记数每片视网膜外生神经突起数。应用羊抗鼠FITC-Thy-1·1单克隆抗体鉴定培养的神经节细胞突起。结果RGCs突起在凝胶中呈螺旋迂曲生长,无神经因子可存活2周以上,加入神经因子可存活3~4周。培养6d后计数每片视网膜外生神经突起数,BDNF对照组和实验组分别为24·4±11·7和51·2±15·6。GDNF对照组和实验组分别为19·9±11·6和33·6±18·5。免疫组织化学荧光显色神经节细胞突起呈阳性反应。结论应用凝胶做支持物可以成功地对成熟RGCs进行三维培养,BDNF/GDNF可以明显地促进神经节细胞的神经突起再生。     

B族维生素对体外培养大鼠视网膜神经细胞及节细胞的神经营养作用

目的评价维生素B1、B6、B12及其衍生物甲钴胺对视网膜神经细胞及神经节细胞(RGCs)的生存和轴突再生伸长的影响。方法采用新生大鼠视网膜神经细胞体外原代培养技术，与不同浓度的B族维生素共同培养，MTT比色法检测细胞活力，进行HE染色和抗Thy1免疫细胞化学染色，测量视网膜神经细胞和RGCs轴突长度，比较各种维生素对细胞轴突伸长的影响。结果维生素B1、B6、B12和甲钴胺的最有效作用浓度分别为100、100、1．0、1．0μmol／L；高密度培养该营养作用更明显。用该浓度作用于细胞，维生素B6、B12和甲钴胺可促进视网膜神经细胞和RGCs轴突伸长。结论B族维生素在体外短期内能够显著提高视网膜神经细胞和RGCs的活力，促进上述细胞轴突再生伸长。     

SDF-1和VEGF在糖尿病大鼠视网膜病变中的表达及AMD3100干预作用

目的:探讨不同时期糖尿病大鼠视网膜中基质细胞衍生因子-1(stromal cell derived factor-1,SDF-1)和血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA的表达,以及不同剂量SDF-1拮抗剂3100对SDF-1表达的抑制作用。方法:将实验大鼠分为正常组、拮抗剂组、糖尿病组。腹腔注射链脲佐菌素建立大鼠糖尿病模型。拮抗剂组、糖尿病组分别于成模后1wk玻璃体腔注射AMD3100和PBS。随后各组球后多次球后注射相应药物。实验方法采用RT-PCR和Western-Blot。RT-PCR实验共饲养三组大鼠60只,各组均于1,3,5mo处死部分大鼠,各组提取标本,RT-PCR检测HE染色观察视网膜血管增生情况。Western-Blot实验将18只大鼠分为正常对照组、4组不同剂量AMD3100的拮抗剂组及糖尿病组共6组,饲养时间均为3mo。结果:RT-PCR检测1,3,5mo各同龄组中三组间大鼠视网膜SDF-1和VEGF mRNA的表达均有显著统计学差异(P<0.01),且均以糖尿病组表达最高,拮抗剂组的表达低于糖尿病组。随着糖尿病病程的延长,SDF-1和VEGF mRNA的表达进一步增加,视网膜HE染色切片上突破内界膜的内皮细胞核数明显增加。Western-Blot检测表明,在一定范围内随着拮抗剂AMD3100浓度的增大,SDF-1和VEGF的蛋白表达量逐渐减少,差异具有显著统计学差异(P<0.01);当继续增加浓度(玻璃体腔注射>10μg/μL),SDF-1和VEGF的蛋白表达量无明显改变,差异无统计学意义(4和5组相比,P>0.05)。结论:随着糖尿病视网膜病变病程的进展,糖尿病大鼠视网膜组织里VEGF和SDF-1 mRNA的表达量增加。SDF-1受体拮抗剂AMD3100可降低SDF-1和VEGF的蛋白表达。这种拮抗作用在一定的浓度范围内,其抑制作用有剂量依从性,剂量越大,抑制作用越强。     

睫状神经营养因子对视神经损伤修复作用研究进展

视神经损伤后,视网膜神经节细胞进行性损害,轴突变性坏死,再生困难.睫状神经营养因子是一种非靶源性神经营养因子,在视网膜神经节细胞的生长发育中起重要作用,同时对损伤的视网膜神经节细胞有促进存活及轴突再生的作用.     

新生大鼠视网膜神经元及节细胞体外短期培养方法

目的　建立新生大鼠视网膜神经元原代培养体系 ,观察视网膜神经元和神经节细胞 (retinalganglioncells,RGC)在体外短期培养的生长特点。方法　将新生大鼠视网膜细胞悬液接种于预先用鼠尾胶原包被的 96孔细胞培养板中 ,按 4 0 0× 10 3 ·cm-2 (高密度 )及 2 0 0× 10 3 ·cm-2 (低密度 ) 2种密度接种 ,抑制非神经元生长 ,MTT比色法评价细胞活力 ;上述细胞悬液按高密度接种于 2 4孔细胞培养板中 ,进行HE染色和Thy1单克隆抗体的免疫化学染色 ,测量视网膜神经元和RGC轴突的长度。结果　视网膜神经细胞有聚集成簇生长的倾向 ,细胞活力在高密度培养时明显高于低密度培养 ;第 3天细胞活力最高 ;视网膜神经元在体外形态多样 ,最初 2d轴突生长速度最快 ,超过 12 .0 μm·d-1;大多数RGC从第 1天就再生出 2个或 2个以上的突起 ,第 1天的平均轴突长度为 12 .5 μm·d-1,从第 2天起保持在8 0 μm·d-1。第 4天的平均轴突长度为 2 9.4 5 μm。结论　以鼠尾胶原为支持物 ,经过酶消化法得到的新生大鼠视网膜神经元 ,包括RGC ,能够在短期内表现出较高的细胞活力并再生出较长的轴突 ;高密度培养 ( 4 0 0× 10 3 ·cm-2 )更有利于视网膜神经元在体外生长存活。     

巨噬细胞激活促进视神经损伤后修复的研究

目的研究巨噬细胞激活状态与视神经损伤后视网膜节细胞(retinal ganglion cell,RGC)存活及神经轴突再生的关系。设计实验性研究。研究对象费希尔大鼠23只。方法在费希尔大鼠上建立视神经损伤及自体外周神经嫁接模型,在玻璃体腔分别注射巨噬细胞激活剂酵母多糖(zymosan,ZYM)或联合巨噬细胞抑制因子(macrophage inhibitory factor,MIF)。大鼠存活3周后灌流固定取视网膜,灌流之前3天进行荧光金标记。对取出的视网膜进行免疫荧光染色,平铺视网膜后在荧光显微镜下分别计数存活RGC、轴突再生性RGC和巨噬细胞的数量。实验分为生理盐水对照组、ZYM组、MIF组及ZYM联合MIF组。主要指标巨噬细胞、存活RGC及轴突再生性RGC的数量。结果对照组巨噬细胞、存活及轴突再生性RGC数量分别为91±6.1/mm~2、185±9.0/mm~2及35±2.9/mm~2,与对照组相比,ZYM组可使眼内巨噬细胞(883±93.9/mm~2)的数量增至近十倍,同时可明显促进RGC存活(299±13.1/mm~2)及神经轴突再生(99±13.5/mm~2);MIF组本身对RGC的存活及轴突再生情况无明显影响。当巨噬细胞被激活后,联合应用MIF虽未能减少眼内巨噬细胞的数量(828±72.9/mm~2),但可有效降低这些巨噬细胞对RGC轴突再生(67±2.2/mm~2)的促进作用。结论巨噬细胞被酵母多糖激活后具有促进RGC的存活及神经轴突的再生,而巨噬细胞抑制因子对巨噬细胞的抑制作用可使其促神经再生的作用明显降低。     

基质细胞衍生因子-1α在增生性糖尿病视网膜病变继发新生血管性青光眼中的作用

目的 观察基质细胞衍生因子-1α(SDF-1α)在增生性糖尿病视网膜病变(PDR)继发新生血管性青光眼(NVG)中的作用,并探讨其作用机制.方法 采集PDR患者25例31只眼的玻璃体标本.其中,继发NVG者13只眼作为实验组,无NVG者18只眼作为对照组.配制含10、100、1000 ng/ml SDF-1α和10 ng/m1血管内皮生长因子(VEGF)培养液,并以此分组;测量各浓度组与体外对照组人脐静脉内皮细胞(HUVEC)管腔样结构及毛细血管样结构全长.配制含10、100、1000 ng/ml SDF-1α和100 ng/ml血管内皮生长因子(VEGF)培养液,并以此分组;采用5'-溴2'-脱氧尿嘧啶(BrdU)掺入法行细胞增生检测,分析各浓度组与体外对照组吸光度[A,旧称光密度(OD)]值.采用酶联免疫吸附试验(ELISA)检测玻璃体标本实验组和玻璃体标本对照组患者玻璃体标本中VEGF和SDF-1α含量.结果 体外血管生成检测显示,10、100、1000 ng/ml SDF-1α和10 ng/ml VEGF组HUVEC管腔样和毛细血管样结构长度均较体外对照组长,差异均有统计学意义(P＜0.05).细胞增生检测显示,10、100、1000 ng/ml SDF-1α和100 ng/ml VEGF 组A值均较体外对照组增高,差异有统计学意义(P＜0.05).ELISA法检测显示,玻璃体标本实验组患者玻璃体标本中SDF-1α和VEGF含量均明显高于玻璃体标本对照组,差异有统计学意义(P＜0.01).结论 SDF-1α参与了PDR继发NVG的形成过程,可能与SDF-1α促进血管内皮细胞增生而促进新生血管形成有关.     

基质细胞衍生因子-1在Wistar大鼠视网膜上的生理性表达

目的研究正常成体大鼠视网膜基质细胞衍生因子（SDF-1）的生理性表达情况。方法取正常Wistar成年大鼠视网膜进行抗SDF-1和抗PCK免疫组织化学染色,镜下观察结合半定量图像分析;实时定量RT-PCR测定视网膜神经上皮层SDF-1mRNA含量,与内参基因βactin比较。结果正常大鼠视网膜神经上皮可见SDF-1表达,其阳性结果多位于视网膜内层,视神经无明显染色。PCK在视网膜神经上皮上无明显表达。实时定量RT-PCR证实在健康成体大鼠视网膜神经上皮存在一定量的SDF-1mRNA。结论正常成年大鼠视网膜神经上皮层存在SDF-1的生理性表达,其生理作用及调控机制有待进一步研究。     

Lens epithelium supports axonal regeneration of retinal ganglion cells in a coculture model in vitro

The purpose of this study was to determine whether the lens epithelium influences the survival or axonal growth of regenerating retinal ganglion cells. The optic nerves of adult albino rats were injured in order to induce axonal regeneration, and axon growth was then studied in retinal explants in the presence of cocultivated lens capsules carrying living epithelial cells. In the first series of experiments, cocultivation of retinal explants with lens epithelium in immediate proximity resulted in penetration of fibers into the lens epithelium, indicating that it supported axonal growth. In the second series of experiments, co-explants were placed 0.5-1.0mm from each other. The numbers of outgrowing retinal axons were determined both with respect to the retinal eccentricity and the topological relationship with the lenticular co-explant. The Wilcoxon matched-pairs signed-rank test was used to determine if the numbers of axons differed significantly between four regions of the explants. Significantly more axons grew out from the retinal edge facing the lenticular explant than from its opposite side, indicating that the lens epithelium supports axon growth. The numbers of surviving retinal ganglion cells in culture were determined after retrograde prelabelling with a neuroanatomical tracer. The number of fluorescent ganglion cells within the retinal explants did not significantly differ between the groups (Mann-Whitney test). These findings indicate that the lens epithelium influences both the amount of axonal regeneration and the direction of growth without affecting the survival rate of retinal ganglion cells in vitro.     

Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture

PURPOSE: To examine and quantify neuroprotective and neurite-promoting activity on retinal ganglion cells (RGCs) after injury of the lens. METHODS: In adult albino rats, penetrating lens injury was performed by intraocular injection. To test for injury-induced neuroprotective effects in vivo, fluorescence-prelabeled RGCs were axotomized by subsequent crush of the optic nerve (ON) with concomitant lens injury to cause cataract. The numbers of surviving RGCs were determined in retinal wholemounts and compared between the different experimental and control groups. To examine axonal regeneration in vivo, the ON was cut and replaced with an autologous piece of sciatic nerve (SN). Retinal ganglion cells with axons that had regenerated within the SN under lens injury or control conditions were retrogradely labeled with a fluorescent dye and counted on retinal wholemounts. Neurite regeneration was also studied in adult retinal explants obtained either after lens injury or without injury. The numbers of axons were determined after 1 and 2 days in culture. Putative neurotrophins (NTs) were studied within immunohistochemistry and Western blot analysis. RESULTS: Cataractogenic lens injury performed at the same time as ON crush resulted in highly significant rescue of 746 +/- 126 RGCs/mm(2) (mean +/- SD; approximately 39% of total RGCs) 14 days after injury compared with controls without injury or with injection of buffer into the vitreous body (30 +/- 18 RGCs/mm(2)). When lens injury was performed with a delay of 3 days after ON crush, 49% of RGCs survived, whereas delay of 5 days still rescued 45% of all RGCs. In the grafting paradigm virtually all surviving RGCs after lens injury appeared to have regenerated an axon within the SN graft (763 +/- 114 RGCs/mm(2) versus 79 +/- 17 RGCs/mm(2) in controls). This rate of regeneration corresponds to approximately 40% of all RGCs. In the regeneration paradigm in vitro preceding lens injury and ON crush 5 days previous resulted in a maximum of regeneration of 273 +/- 39 fibers/explant after 1 day and 574 +/- 38 fibers/explant after 2 days in vitro. In comparison, in control retinal pieces without lens injury 28 +/- 13 fibers/explant grew out at 1 day, and 97 +/- 37 fibers/explant grew out at 2 days in culture. Immunohistochemical and Western blot analysis of potential NTs in the injured lens revealed no expression of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), NT-4, nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). CONCLUSIONS: The findings indicate that the lens contains high neuroprotective and neuritogenic activity, which is not caused by NT. Compared with the data available in the literature, this neuroprotection is quantitatively among the highest ever reported within the adult rat visual system.     

Axonal regeneration of retinal ganglion cells depending on the distance of axotomy in adult hamsters

PURPOSE: To examine the relationship between the distance of axotomy and axonal regeneration of injured retinal ganglion cells (RGCs) systematically and the effect of a predegenerated (pretransected or precrushed) peripheral nerve (PN) graft on axonal regeneration of RGCs axotomized at a definite distance (0.5 mm from the optic disc) in comparison with a normal PN graft. METHODS: The optic nerve (ON) was transected intraorbitally at 0.5, 1, 1.5, 2, or 3 mm or intracranially at 6 to 8 mm from the optic disc, and a PN graft was transplanted onto the ocular ON stump in adult hamsters. Four weeks after grafting, the number of RGCs regenerating their injured axons into the PN graft was investigated in all animals. RESULTS: The number of regenerating RGCs decreased significantly when the distance of axotomy increased from 0.5 to 7 mm. A precrushed PN graft was shown to enhance more injured RGCs to regenerate axons than a normal or pretransected PN graft. CONCLUSIONS: The distance of axotomy on the ON of adult hamsters is critical in determining the number of regenerating RGCs. Thus, experimental strategies to repair the damaged ON by PN transplantation is to attach a precrushed PN graft as close to the optic disc as possible to obtain optimal axonal regeneration of the axotomized RGCs.     

CNTF, not other trophic factors, promotes axonal regeneration of axotomized retinal ganglion cells in adult hamsters

PURPOSE: To investigate the in vivo effects of trophic factors on the axonal regeneration of axotomized retinal ganglion cells in adult hamsters. METHODS: The left optic nerve was transected intracranially or intraorbitally, and a peripheral nerve graft was apposed or sutured to the axotomized optic nerve to enhance regeneration. Trophic factors were applied intravitreally every 5 days. Animals were allowed to survive for 3 or 4 weeks. Regenerating retinal ganglion cells (RGCs) were labeled by applying the dye Fluoro-Gold to the distal end of the peripheral nerve graft 3 days before the animals were killed. RESULTS: Intravitreal application of ciliary neurotrophic factor substantially enhanced the regeneration of damaged axons into a sciatic nerve graft in both experimental conditions (intracranial and intraorbital optic nerve transections) but did not increase the survival of distally axotomized RGCs. Basic fibroblast growth factor and neurotrophins such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 failed to enhance axonal regeneration of distally axotomized RGCs. CONCLUSIONS: Neurons of the adult central nervous system can regenerate in response to trophic supply after injury, and ciliary neurotrophic factor is at least one of the trophic factors that can promote axonal regeneration of axotomized RGCs.     

Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitally at 2 mm (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.     

Optic Nerve Engraftment of Neural Stem Cells

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm² vs. 199 ± 58 RGCs/mm²; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.     

Axonal regeneration induced by repetitive electrical stimulation of crushed optic nerve in adult rats

Purpose  To investigate whether electrical stimulation promoted axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush in adult rats. Methods  Transcorneal electrical stimulation (TES), which stimulates the retina with current from a corneal contact lens electrode, was used to stimulate the eye. TES was applied for 1 h immediately after ON crush. Axonal regeneration was determined by anterograde labeling of RGC axons. To examine whether the axonal regeneration was mediated by insulin-like growth factor 1 (IGF-1) receptors, an IGF-1 receptor antagonist, JB3, was injected intraperitoneally before each TES application. Immunostaining for IGF-1 was performed to examine the effects of TES. To test the survival-promoting effects of TES applied daily, the mean density of retrogradely labeled RGCs was determined on day 12 after ON crush. Results  Compared with sham stimulation, the mean number of regenerating axons significantly increased at 250 μm distal from the lesion and increased IGF-1 immunoreactivity was observed in retinas treated daily with TES. Preinjection of an IGF-1 receptor antagonist significantly blocked axonal regeneration by TES applied daily. TES applied daily also markedly enhanced the survival of RGCs 12 days after ON crush. Conclusion  TES applied daily promotes both axonal regeneration and survival of RGCs after ON crush.     

Regeneration of retinal ganglion cell axons in organ culture is increased in rats with hereditary buphthalmos

This study used organ cultures to examine whether retinal ganglion cells (RGCs) retain their ability to regenerate axons in buphthalmos. A rat mutant with hereditary buphthalmos was used to (1) determine whether the extent of RGC loss corresponds to the severity and duration of elevated intraocular pressure (IOP), (2) examine whether RGCs exposed to an elevated IOP are able to regenerate their axons in a retina culture model, and (3) analyze the proteome of the regenerating retina in order to identify putative regeneration-associated proteins. Retrograde labeling of RGCs revealed a decrease in their numbers in the retinas of buphthalmic eyes that increased with age. Quantification of axons growing out of retinal explants taken at different stages of the disease demonstrated that buphthalmic RGCs possess a remarkable potential to regrow axons. As expected, immunohistochemistry and immunoblotting revealed that elevated IOP was associated with upregulation of certain known proteins, such as growth-associated protein 43, glial fibrillary acidic protein, and endothelin-1. In addition, two-dimensional polyacrylamide gel electrophoresis and mass spectrometry revealed several spots corresponding to proteins that were specifically regulated when buphthalmic RGCs were permitted to regrow their axons. Out of the proteins identified, heat-shock protein (HSP)-60 was constantly expressed during axonal growth at all stages of the disease. Antibodies against HSP-60 reduced axonal growth, indicating the involvement of this protein in regenerative axonal growth. These data are the first to show that diseased retinal neurons can grow their axons, and that HSP-60 supports neuritogenesis. This model may help to elucidate the fundamental mechanisms of optic neuropathy at stages preceding death caused by chronic injury, and aid in the development of neuroprotective strategies.