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.     

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.     

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.     

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.     

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.     

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.     

Anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush are PI3K/AKT-dependent

The purpose of present study is to dissect the role of PI3K/AKT signaling in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on rat retinal ganglion cells (RGCs) after optic nerve (ON) crush. The ONs of seventy-two adult male Wistar rats were crushed by a standardized method. Control eyes received a sham operation. G-CSF or phosphate-buffered saline (PBS) was immediately administrated after the ON event for 5 days. Twelve rats were used to investigate the signaling pathways using western blot analysis. In other sixty rats, each eye also received intravitreal injections of PI3K/AKT inhibitor (LY294002) or PBS immediately after the experiments. Rats were euthanized at 1 or 2 weeks after the experiment. RGC density was counted by retrograde labeling with Fluorogold. Western blot analysis of p-AKT, TUNEL assays, and immunohistochemistry of the retinas were conducted. Two weeks after ON injury, RGC densities in the central and mid-peripheral retinas of ON-crushed, G-CSF treated rats were significantly higher than those of corresponding ON-crushed, G-CSF-treated and LY294002-injected rats (survival rates of 60% vs. 39% and 43% vs. 33%, respectively; p < 0.01). Decreased TUNEL staining and the up-regulations of p-AKT signaling in retinas of ON-crushed, G-CSF-treated rats were blocked by intravitreal injections of LY294002. The double staining showed that p-AKT expression co-localized with RGCs in the ON crushed, G-CSF treated retinas. In conclusion, the anti-apoptotic effects of G-CSF on RGCs are PI3K/AKT signaling dependent in the retinas to rescue RGCs after ON crush injury.     

钙通道抑制介导轴突稳定对视神经损伤修复作用的研究进展

视神经损伤是常见的神经性疾病,其基本的病理特征为轴突变性和视网膜神经节细胞(RGCs)凋亡,从而导致视觉功能障碍等症状的出现。轴突变性是神经发育、轴突重塑和损伤反应的重要过程,包括轴突选择性退化、轴突横断诱导的Wallerian变性和凋亡诱导的轴突变性(轴突凋亡)。轴突变性是许多创伤性神经系统疾病的初始步骤之一,损伤的轴突一般无法再生,从而进一步导致神经元胞体凋亡。神经元凋亡导致胞体和轴突两者的退化,在发育过程中广泛发生并且应对神经元的各种损伤。近年来有研究证实,钙是轴突变性的主要调控因子。在视神经挤压伤(ONC)发生后,通过钙通道抑制剂阻止钙离子涌入轴突,可以减弱急性轴突变性(AAD)的程度,提高RGCs的存活率以及促进轴突的再生。