肌苷毫微粒对成年大鼠视网膜节细胞的保护作用

目的 研究载有肌苷的毫微粒对视神经切断后视网膜节细胞(RGC)存活的影响。方法 制备肌苷毫微粒，体外测定理化性质。将等体积的肌苷毫微粒、空载毫微粒或生理盐水溶液分别注入成年大鼠左眼内，对照组未经任何治疗。1d后于眶内切断所有动物左侧视神经，术后7d取左视网膜，计数荧光金逆行标记的存活RGC。结果 肌苷毫微粒形态规整，具有缓释特点。同对照相比，肌苷毫微粒能显著提高存活RGC的密度，而空载体和生理盐水无此作用；空载毫微粒与生理盐水、对照之间以及空载毫微粒和肌苷毫微粒两组间RGC密度均无显著差异。结论 注入眼球的肌苷毫微粒至少在7d内能有效缓释肌苷，进而对轴突损伤RGC发挥显著的神经保护作用。     

Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system

PURPOSE: To investigate the effect of transcorneal electrical stimulation (TES) on the survival of axotomized RGCs and the mechanism underlying the TES-induced neuroprotection in vivo. METHODS: Adult male Wistar rats received TES after optic nerve (ON) transection. Seven days after the ON transection, the density of the surviving RGCs was determined, to evaluate the neuroprotective effect of TES. The levels of the mRNA and protein of insulin-like growth factor (IGF)-1 in the retina after TES were determined by RT-PCR and Northern and Western blot analyses. The localization of IGF-1 protein in the retina was examined by immunohistochemistry. RESULTS: TES after ON transection increased the survival of axotomized RGCs in vivo, and the degree of rescue depended on the strength of the electric charge. RT-PCR and Northern and Western blot analyses revealed a gradual upregulation of intrinsic IGF-1 in the retina after TES. Immunohistochemical analysis showed that IGF-1 immunoreactivity was localized initially in the endfeet of Muller cells and then diffused into the inner retina. CONCLUSIONS: TES can rescue the axotomized RGCs by increasing the level of IGF-1 production by Muller cells. These findings provide a new therapeutic approach to prevent or delay the degeneration of retinal neurons without the administration of exogenous neurotrophic factors.     

Survival of retinal ganglion cells after transection of the optic nerve in adult cats: a quantitative study within two weeks

We have previously reported that a small number of retinal ganglion cells (RGCs) of adult cats survive 2 months after transection of the optic nerve (ON) and that alpha cells have the greatest ability to survive among different types of RGCs (Watanabe et al., 1995). Here we report the time course of RGC survival within 15 days after ON transection using retrograde labeling with DiI injected into the bilateral lateral geniculate nuclei of cats. The density of DiI-labeled RGCs in the central retina as well as in the periphery did not change until day 3 after ON transection, then decreased rapidly, to 43% of the original density on day 7, and falling to 19% by day 14. We then intracellularly injected Lucifer yellow into the DiI-labeled RGCs to examine the difference in the time course between surviving alpha and beta cells. Similar to the density change in total surviving RGCs, the proportion of surviving beta cells did not change until day 3, then decreased rapidly to 65% of the original density on day 4, falling to 12% by day 14. By contrast, 64% of alpha cells survived for 14 days after axotomy. Analysis of regression lines for survival time courses indicated that death of beta cells was characterized with a rapid period phase from day 3 to day 7 after axotomy whereas that of alpha cells lacked it. Axon-like sprouting from surviving beta cells was first recognized in the nerve fiber layer on day 3, and were later more conspicuous.     

CNTF and BDNF have similar effects on retinal ganglion cell survival but differential effects on nitric oxide synthase expression soon after optic nerve injury

PURPOSE: To investigate the effect of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and nitric oxide synthase (NOS) expression in the retina during the early phase of optic nerve (ON) injury, and to examine whether intraperitoneal application of the NOS scavenger nitro-l-arginine (l-NA) could protect the injured RGCs. METHODS: RGCs were retrogradely labeled with granular blue 3 days before the ON was intraorbitally transected. RGC survival was examined 1 week after ON transection and intraocular injection of CNTF and/or BDNF, or 1 to 2 weeks after daily intraperitoneal injection of the NOS inhibitor l-NA. NOS expression was examined by NADPH-diaphorase histochemistry and neuronal NOS (nNOS) immunohistochemistry, and nNOS-positive cells were identified by various staining approaches. RESULTS: Both CNTF and BDNF significantly increased RGC survival 1 week after ON injury. In the ganglion cell layer (GCL), CNTF did not increase the number of NADPH-diaphorase positive ((+)) cells but appeared to reduce the intensity of NADPH-diaphorase staining, whereas BDNF increased the number of NADPH-diaphorase(+) cells and also appeared to enhance the intensity of NADPH-diaphorase staining. In the GCL, amacrine cells but not RGCs were nNOS(+). Some macrophages were also nNOS(+). In contrast, no amacrine cells were nNOS(+) in the inner nuclear layer. Daily intraperitoneal injection of l-NA at appropriate concentrations promoted RGC survival for 1 or 2 weeks after ON injury. CONCLUSIONS: Both CNTF and BDNF protected RGCs after ON injury. CNTF and BDNF acted differently on NOS expression in the GCL. Intraperitoneal injections of l-NA at appropriate dosages enhance RGC survival.     

Neuroprotective effect of nipradilol on axotomized rat retinal ganglion cells

PURPOSE: To determine whether nipradilol, a new anti-glaucoma drug, can protect retinal ganglion cells (RGCs) from secondary cell death caused by transection of the optic nerve (ON). METHODS: The ON was transected 0.7 mm from its exit from the eye in Sprague Dawley rats. Nipradilol (1 x 10(-8) - 10(-3) M), timolol, prazosin, or sodium nitroprusside (SNP) (1 x 10(-6) - 10(-4) M) was injected intravitreally fifteen-minutes before the ON transection. Control eyes received the same amount of phosphate buffered (PB). The RGCs were labeled retrogradely by placing gelfoam soaked in fluoro-gold (FG) on the stump of ON. RGCs density was determined by counting the FG-labeled RGCs in flat-mounted retinas 3 to 14 days post-transection. To determine whether the neuroprotective action of nipradilol was due to its NO-donor property, carboxy-PTIO, a NO-scavenger, or KT5832, a protein kinase G inhibitor, was injected with the nipradilol. RESULTS: After ON transection, the number of surviving RGCs after intravitreal injection of 1 x 10(-4) M nipradilol was significantly higher than that following PB injection. This protective activity was dose-dependent. Neither timolol nor prazosin had a neuroprotective effect but SNP protected RGCs in a dose-dependent manner. Carboxy-PTIO and KT5832 decreased the neuroprotective effect of nipradilol. CONCLUSIONS: These results indicate that nipradilol has a possibility of neuroprotective effect on axotomized RGCs, and the effect depended mainly on its NO-donor property.     

A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transection

PURPOSE: To use a rat model of optic nerve injury to differentiate primary and secondary retinal ganglion cell (RGC) injury. METHODS: Under general anesthesia, a modified diamond knife was used to transect the superior one third of the orbital optic nerve in albino Wistar rats. The number of surviving RGC was quantified by counting both the number of cells retrogradely filled with fluorescent gold dye injected into the superior colliculus 1 week before nerve injury and the number of axons in optic nerve cross sections. RGCs were counted in 56 rats, with 24 regions examined in each retinal wholemount. Rats were studied at 4 days, 8 days, 4 weeks, and 9 weeks after transection. The interocular difference in RGCs was also compared in five control rats that underwent no surgery and in five rats who underwent a unilateral sham operation. It was confirmed histologically that only the upper optic nerve had been directly injured. RESULTS: At 4 and 8 days after injury, superior RGCs showed a mean difference from their fellow eyes of -30.3% and -62.8%, respectively (P = 0.02 and 0.001, t-test, n = 8 rats/group), whereas sham-operation eyes had no significant loss (mean difference between eyes = 1.7%, P = 0.74, t-test). At 8 days, inferior RGCs were unchanged from control, fellow eyes (mean interocular difference = -4.8%, P = 0.16, t-test). Nine weeks after transection, inferior RGC had 34.5% fewer RGCs than their fellow eyes, compared with 41.2% fewer RGCs in the superior zones of the injured eyes compared with fellow eyes. Detailed, serial section studies of the topography of RGC axons in the optic nerve showed an orderly arrangement of fibers that were segregated in relation to the position of their cell bodies in the retina. CONCLUSIONS: A model of partial optic nerve transection in rats showed rapid loss of directly injured RGCs in the superior retina and delayed, but significant secondary loss of RGCs in the inferior retina, whose axons were not severed. The findings confirm similar results in monkey eyes and provide a rodent model in which pharmacologic interventions against secondary degeneration can be tested.     

Effects of different neurotrophic factors on the survival of retinal ganglion cells after a complete intraorbital nerve crush injury: A quantitative in vivo study

We examined in adult Sprague Dawley rats the loss of retinal ganglion cells (RGCs) induced by complete intraorbital optic nerve crush (IONC) as well as the effects of several neurotrophic factors to prevent IONC-induced RGC loss. Completeness of the IONC lesion was assessed by investigating the orthograde and retrograde transport of neuronal tracers applied to the origin and termination of the retinotectal pathway. RGC survival after IONC alone or combined with intraocular injection of the neurotrophic factors NT-4, BDNF or CNTF was quantified at survival intervals ranging from 5 to 12 days post-lesion (dpl) by identifying RGCs that had been pre-labelled with fluorogold (FG). RGC loss first appeared at 7 dpl and by 12 dpl only 32% of the RGC population remained in the retina. Intraocular administration of NT-4, BDNF or CNTF resulted in almost a complete protection against IONC-induced RGC loss by 7 dpl, and the protection remained significant by 12 dpl only for NT-4 and BDNF. We have analyzed these results taking into account our previous studies on the loss of RGCs induced by intraorbital optic nerve transection (IONT) and concluded that RGC loss induced by IONC is slower and less severe than that following IONT. Moreover, as for IONT-induced RGC loss, IONC-induced RGC loss may also be prevented with administration of NT-4, BDNF or CNTF, though for NT-4 and CNTF their neuroprotective effects differ depending on the injury type. Overall this data underscore the importance of the type of ON injury on the pattern of RGC degeneration as well as in their response to neuroprotective treatments.     

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.     

Neuroprotective effect of sulfhydryl reduction in a rat optic nerve crush model

PURPOSE: The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy. METHODS: RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia. RESULTS: The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone. CONCLUSIONS: The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.     

Different responses of macrophages in retinal ganglion cell survival after acute ocular hypertension in rats with different autoimmune backgrounds

Recently macrophages were shown to play a protective role in retinal ganglion cells (RGCs) after optic nerve (ON) injury. In the present study, we investigated how macrophages responded after acute intraocular pressure (IOP) elevation in experimental autoimmune encephalomyelitis (EAE)-resistant Fischer 344 (F344) and Sprague Dawley (SD) rats and EAE-vulnerable Lewis rats. Acute IOP elevation was performed at 110mmHg for 2h to mimic acute glaucoma. Phagocytic cells in the eye were removed by intravitreal application of clodronate liposomes whereas macrophage activation was achieved by intravitreal injection of zymosan, a yeast wall preparation. Fluorescence dye, FluoroGold, was applied behind the eyeballs to retrogradely label surviving RGCs 40h before animal sacrifice. Macrophages in the retina were identified by ED1 immunostaining. Loss of 25% RGCs in F344 but over 90% in Lewis rats was seen 2 weeks after acute IOP elevation. Significant increase in the number of macrophages in the retina was seen to accompany the great RGC loss in Lewis rats; removal of these macrophages reduced the extent of RGC loss, suggesting the involvement of macrophages in RGC death in Lewis strain. Low numbers of macrophages were seen in F344 retinas after acute IOP elevation, and removal of macrophages did not show clear effect on RGC viability. Whereas macrophage activation by zymosan protected RGCs after ON axotomy in F344 rats, the same macrophage activation became detrimental to RGCs after acute IOP elevation. The extent of RGC loss 3 weeks after acute IOP elevation or after macrophage activation by zymosan in EAE-resistant SD rats was similar to that in F344 rats. We thus demonstrate that macrophages in rats with different autoimmune backgrounds react differently to acute IOP elevation and differentially modulate RGC loss, a phenomenon contrary to the protective action in RGCs after ON axotomy. These data suggest that autoimmune background plays a role in modulating vulnerability of RGCs to acute IOP elevation.     

Superoxide signaling and cell death in retinal ganglion cell axotomy: effects of metallocorroles

Injury to retinal ganglion cell (RGC) axons within the optic nerve causes apoptosis of the soma. We previously demonstrated that in vivo axotomy causes elevation of superoxide anion within the RGC soma, and that this occurs 1-2 days before annexin-V positivity, a marker of apoptosis. Pegylated superoxide dismutase delivery to the RGC prevents the superoxide elevation and rescues the soma. Together, these results imply that superoxide is an upstream signal for apoptosis after axonal injury in RGCs. We then studied metallocorroles, potent superoxide dismutase mimetics, which we had shown to be neuroprotective in vitro and superoxide scavengers in vivo for RGCs. RGCs were retrograde labeled with the fluorescent dye 4Di-10Asp, and then axotomized by intraorbital optic nerve transection. Iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole (Fe(tpfc)(SO(3)H)(2)) (Fe-corrole) was injected intravitreally. Longitudinal imaging of RGCs was performed and the number of surviving RGCs enumerated. There was significantly greater survival of labeled RGCs with Fe-corrole, but the degree of neuroprotection was relatively less than that predicted by their ability to scavenge superoxide-This implies an unexpected complexity in signaling of apoptosis by reactive oxygen species.     

Optimal parameters of transcorneal electrical stimulation (TES) to be neuroprotective of axotomized RGCs in adult rats

We previously showed that transcorneal electrical stimulation (TES) promoted the survival of axotomized retinal ganglion cells (RGCs) of rats. However the relationship between the parameters of TES and the neuroprotective effect of TES on axotomized RGCs was unclear. In the present study, we determined whether the neuroprotective effect of TES is affected by the parameters of TES. Adult male Wistar rats received TES just after transection of the left optic nerve (ON). The pulse duration, current intensity, frequency, waveform, and numbers of sessions of the TES were changed systematically. The alterations of the retina were examined histologically seven days or fourteen days after the ON transection. The optimal neuroprotective parameters were pulse duration of 1 and 2 ms/phase (P < 0.001, each), current intensity of 100 and 200 μA (P < 0.05, each), and stimulation frequency of 1, 5, and 20 Hz (P < 0.001, respectively). More than 30 min of TES was necessary to have a neuroprotective effect (P < 0.001). Symmetric pulses without an inter-pulse interval were most effective (P < 0.001). Repeated TES was more neuroprotective than a single TES at 14 days after ON transection (P < 0.001). Our results indicate that there is a range of optimal neuroprotective parameters of TES for axotomized RGCs of rats. These values will provide a guideline for the use of TES in patients with different retinal and optic nerve diseases.     

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.     

Optic nerve transection in monkeys may result in secondary degeneration of retinal ganglion cells

PURPOSE: Interest in neuroprotection for optic neuropathies is, in part, based on the assumption that retinal ganglion cells (RGCs) die, not only as a result of direct (primary) injury, but also indirectly as a result of negative effects from neighboring dying RGCs (secondary degeneration). This experiment was designed to test whether secondary RGC degeneration occurs after orbital optic nerve injury in monkeys. METHODS: The superior one third of the orbital optic nerve on one side was transected in eight cynomolgus monkeys (Macaca fascicularis). Twelve weeks after the partial transection, the number of RGC bodies in the superior and inferior halves of the retina of the experimental and control eyes and the number and diameter of axons in the optic nerve were compared by detailed histomorphometry. Vitreous was obtained for amino acid analysis. A sham operation was performed in three additional monkeys. RESULTS: Transection caused loss of 55% +/- 13% of RGC bodies in the superior retina of experimental compared with fellow control eyes (mean +/- SD, t-test, P < 0.00,001, n = 7). Inferior RGCs, not directly injured by transection, decreased by 22% +/- 10% (P = 0.002). The loss of superior optic nerve axons was 83% +/- 12% (mean +/- SD, t-test, P = 0.0008, n = 5) whereas, the inferior loss was 34% +/- 20% (P = 0.02, n = 5). Intravitreal levels of glutamate and other amino acids in eyes with transected nerves were not different from levels in control eyes 12 weeks after injury. Fundus examination, fluorescein angiography, and histologic evaluation confirmed that there was no vascular compromise to retinal tissues by the transection procedure. CONCLUSIONS: This experiment suggests that primary RGC death due to optic nerve injury is associated with secondary death of surrounding RGCs that are not directly injured.     

RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection

PURPOSE: To establish a method for morphometric analysis of retrogradely labeled retinal ganglion cells (RGCs) of the mouse retina, to be used for the study of molecular aspects of RGC survival and neuroprotection in this model; to evaluate the effect of overexpression of Cu-Zn-superoxide dismutase (CuZnSOD) on RGC survival after severe crush injury to the optic nerve, and to assess the effect of the alpha2-adrenoreceptor agonist brimonidine, recently shown to be neuroprotective, on RGC survival. METHODS: A severe crush injury was inflicted unilaterally in the orbital portion of the optic nerves of wild-type and transgenic (Tg-SOD) mice expressing three to four times more human CuZnSOD than the wild type. In each mouse all RGCs were labeled 72 hours before crush injury by stereotactic injection of the neurotracer dye FluoroGold (Fluorochrome, Denver, CO) into the superior colliculus. Survival of RGCs was then assessed morphometrically, with and without systemic injection of brimonidine. RESULTS: Two weeks after crush injury, the number of surviving RGCs was significantly lower in the Tg-SOD mice (596.6 +/- 71.9 cells/mm(2)) than in the wild-type control mice (863. 5 +/- 68 cells/mm(2)). There was no difference between the numbers of surviving RGCs in the uninjured retinas of the two strains (3708 +/- 231.3 cells/mm(2) and 3904 +/- 120 cells/mm(2), respectively). Systemic injections of brimonidine significantly reduced cell death in the Tg-SOD mice, but not in the wild type. CONCLUSIONS: Overexpression of CuZnSOD accelerates RGC death after optic nerve injury in mice. Activation of the alpha2-adrenoreceptor pathway by brimonidine enhances survival of RGCs in an in vivo transgenic model of excessive oxidative stress.     

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。     

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG⁺ or DTMR⁺RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR⁺RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR⁺RGCs and FG⁺RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.     

Survival and axonal regeneration of retinal ganglion cells in adult cats

Axotomized retinal ganglion cells (RGCs) in adult cats offer a good experimental model to understand mechanisms of RGC deteriorations in ophthalmic diseases such as glaucoma and optic neuritis. Alpha ganglion cells in the cat retina have higher ability to survive axotomy and regenerate their axons than beta and non-alpha or beta (NAB) ganglion cells. By contrast, beta cells suffer from rapid cell death by apoptosis between 3 and 7 days after axotomy. We introduced several methods to rescue the axotomized cat RGCs from apoptosis and regenerate their axons; transplantation of the peripheral nerve (PN), intraocular injections of neurotrophic factors, or an antiapoptotic drug. Apoptosis of beta cells can be prevented with intravitreal injections of BDNF+CNTF+forskolin or a caspase inhibitor. The injection of BDNF+CNTF+forskolin also increases the numbers of regenerated beta and NAB cells, but only slightly enhances axonal regeneration of alpha cells. Electrical stimulation to the cut end of optic nerve is effective for the survival of axotomized RGCs in cats as well as in rats. To recover function of impaired vision in cats, further studies should be directed to achieve the following goals: (1) substantial number of regenerating RGCs, (2) reconstruction of the retino-geniculo-cortical pathway, and (3) reconstruction of retinotopy in the target visual centers.     

Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat

The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5mul intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500mum concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500mum concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.