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 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.     

Chemotactic effect of ciliary neurotrophic factor on macrophages in retinal ganglion cell survival and axonal regeneration

PURPOSE: To examine whether ciliary neurotrophic factor (CNTF) has a chemotactic effect on macrophages and whether macrophages are involved in CNTF-induced retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury. METHODS: Adult Fischer 344 rats received an autologous peripheral nerve graft onto transected ON for injured axons to grow. CNTF was applied intravitreally. When needed, clodronate liposomes were applied intravitreally or intravenously to deplete macrophages in the eye. A chemotaxis microchamber system was used to examine whether CNTF has a chemotactic effect on macrophages in vitro, whereas immunohistochemistry was used to identify the location of macrophages/microglia in the retina. The effects of CNTF on RGC neurite outgrowth and macrophage/microglia proliferation were tested in retinal explants. RESULTS: Intravitreal CNTF significantly enhanced RGC survival and axonal regeneration as well as the number of macrophages in the eye. Removal of macrophages significantly reduced CNTF-induced RGC survival and axon regeneration. A chemotaxis assay showed a clear chemotactic effect of CNTF on blood-derived but not peritoneal macrophages. Immunohistochemistry revealed that local microglia was located in a region from the nerve fiber layer (NFL) to the inner nuclear layer, whereas blood-derived macrophages were in the NFL. In vitro experiments revealed that CNTF did not enhance neurite outgrowth or macrophage/microglia proliferation in retinal explants. CONCLUSIONS: CNTF is a chemoattractant but not a proliferation enhancer for blood-derived macrophages, and blood-borne macrophages recruited into the eye by CNTF participate in RGC protection. This finding thus adds an important category to the existing understanding of the biological actions of CNTF.     

Survival of axotomized retinal ganglion cells in peripheral nerve-grafted ferrets.

PURPOSE: Peripheral nerve (PN) grafting to the optic nerve stump stimulates not only axonal regeneration of the axotomized retinal ganglion cells (RGCs) into the grafted PN but also their survival. The purpose of the present study was to determine the number, distribution, and soma diameter of only surviving RGCs without regenerated axons and surviving RGCs with regenerated axons in PN-grafted mammals. METHODS: A segment of PN was grafted to the optic nerve stump of adult ferrets. Two months after the PN grafting, surviving RGCs with regenerated axons were retrogradely labeled with granular blue (GB) and stained with RGC-specific antibody C38. Surviving RGCs without regenerated axons were identified as C38-positive cells without GB labeling. RESULTS: Twenty-one percent of RGCs survived axotomy after PN grafting in the area centralis (AC), whereas 47% survived in the peripheral retina. Twenty-six percent of surviving RGCs in the AC exhibited axonal regeneration, which was higher than that in the peripheral retina. Soma diameter histograms revealed that RGCs with regenerated axons showing both GB and C38 positivity were in the large soma diameter ranges. In contrast, the soma diameter distribution of surviving RGCs that did not have regenerated axons showed a peak in the smaller soma diameter ranges. CONCLUSIONS: The present data suggest that PN grafting promotes survival of axotomized RGCs more effectively in the peripheral retina than in the AC. Among surviving RGCs, the larger cells exhibited axonal regeneration into the grafted PN, whereas the axons of smaller cells did not to regenerate in either the AC or the peripheral retina.     

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.     

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.     

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.     

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.     

Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells

PurposeThe goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice.MethodsJNK2^−/− and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2^−/− and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections.ResultsIntravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 ^−/− mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2^−/− mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes.ConclusionsJNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2^−/− mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.     

Neuron stress and loss following rodent anterior ischemic optic neuropathy in double-reporter transgenic mice

PURPOSE: Nonarteritic anterior ischemic optic neuropathy (NAION) is an optic nerve infarct involving axons of retinal ganglion cell (RGC) neurons. The rodent NAION model (rAION) can use transgenic mouse strains to reveal unique characteristics about the effects of sudden optic nerve ischemia on RGCs and their axons. The impact of rAION on RGC stress patterns, RGC loss, and their axons after axonal infarct were evaluated. METHODS: A double-transgenic mouse strain was used, containing a construct with cyan fluorescent protein (CFP) under Thy-1 promoter control, and a construct with beta-galactosidase (lacZ) linked to the stress gene c-fos promoter. Thy-1 in the retina is expressed predominantly in RGCs, enabling stereologic analysis of CFP(+) RGC numbers and loss post-rAION-using confocal microscopy. RGC loss was correlated with axonal counts using transmission electron microscopy (TEM). LacZ immunohistochemistry was used to evaluate retinal cell stress after rAION. RESULTS: The 45,000 CFP(+) cells in the RGC layer of control animals compared with previous RGC quantitative estimates. rAION produced RGC stress, defined as lacZ expression, in patterns corresponding with later RGC loss. rAION-associated RGC loss correlated with regional nerve fiber layer loss. Axonal loss correlates with stereologically determined RGC loss estimates in transgenic mice retinas. CONCLUSIONS: Post-ON infarct RGC stress patterns correlate with regional RGC loss. Cellular lacZ levels in most RGCs are low, suggesting rAION-affected RGCs express c-fos only transiently. CFP(+) cell loss correlates closely with quantitative axonal loss, suggesting that the Thy-1 (CFP) transgenic mouse strain is appropriate for RGC stereologic analyses.     

The neurobiology of cell death in glaucoma

Glaucoma is an optic neuropathy in which the optic nerve axons are damaged, resulting in death of retinal ganglion cells (RGCs). The primary region of damage is thought to be the optic nerve head (ONH), with the lateral geniculate nucleus (LGN) and optic radiations to the visual cortex being secondarily affected. Neurotrophin deprivation resulting from optic nerve injury is thought to cause RGCs to die by apoptosis by inhibition of cell survival pathways. However, disruption of retrograde axonal transport is not the only mechanism associated with optic nerve damage and RGC death, and thus, an additional mechanism of injury is likely to be involved in glaucomatous optic neuropathy.     

Axotomized mouse retinal ganglion cells containing melanopsin show enhanced survival, but not enhanced axon regrowth into a peripheral nerve graft

Melanopsin is found in only approximately 2% of mouse retinal ganglion cells (RGCs), making these RGCs uniquely and directly photosensitive. Given that the majority of RGCs die after axotomy and that grafting of a peripheral nerve to the eye provides a permissive environment for axon regrowth, the present study examined the survival and axonal regrowth of melanopsin-containing RGCs in mice. One month after optic nerve transection and grafting, RGCs with regrown axons were labeled from the grafts and retinae were processed to visualize melanopsin and TUJ1. Melanopsin-positive and negative RGCs were counted and compared to axotomized RGCs from ungrafted eyes and uninjured RGCs. Melanopsin-positive RGCs showed a 3-fold increase in survival rate compared to non-melanopsin RGCs. Despite this enhanced survival, melanopsin-containing RGCs did not show increased axon regrowth into nerve grafts.     

Heparan sulfate regulates intraretinal axon pathfinding by retinal ganglion cells

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.     

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.     

Autophagy in axonal degeneration in glaucomatous optic neuropathy

The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.     

Gene therapy and transplantation in CNS repair: the visual system

Normal visual function in humans is compromised by a range of inherited and acquired degenerative conditions, many of which affect photoreceptors and/or retinal pigment epithelium. As a consequence the majority of experimental gene- and cell-based therapies are aimed at rescuing or replacing these cells. We provide a brief overview of these studies, but the major focus of this review is on the inner retina, in particular how gene therapy and transplantation can improve the viability and regenerative capacity of retinal ganglion cells (RGCs). Such studies are relevant to the development of new treatments for ocular conditions that cause RGC loss or dysfunction, for example glaucoma, diabetes, ischaemia, and various inflammatory and neurodegenerative diseases. However, RGCs and associated central visual pathways also serve as an excellent experimental model of the adult central nervous system (CNS) in which it is possible to study the molecular and cellular mechanisms associated with neuroprotection and axonal regeneration after neurotrauma. In this review we present the current state of knowledge pertaining to RGC responses to injury, neurotrophic and gene therapy strategies aimed at promoting RGC survival, and how best to promote the regeneration of RGC axons after optic nerve or optic tract injury. We also describe transplantation methods being used in attempts to replace lost RGCs or encourage the regrowth of RGC axons back into visual centres in the brain via peripheral nerve bridges. Cooperative approaches including novel combinations of transplantation, gene therapy and pharmacotherapy are discussed. Finally, we consider a number of caveats and future directions, such as problems associated with compensatory sprouting and the reformation of visuotopic maps, the need to develop efficient, regulatable viral vectors, and the need to develop different but sequential strategies that target the cell body and/or the growth cone at appropriate times during the repair process.     

移植含嗅鞘细胞和体外溃变的周围神经对成年大鼠视网膜神经节细胞轴突再生作用的比较

目的比较含嗅鞘细胞（OEC）或（和）体外溃变的周围神经移植对成年大鼠视网膜神经节细胞（RGC）轴突再生的影响。方法将24只成年雄性Sprague-Dawley大鼠随机分为4组，每组各6只大鼠：A组（周围神经对照组）：将取出的一段自体坐骨神经与眶内切断的左侧视神经近侧断端吻合；B组（OEC注入周围神经组）：自取出的坐骨神经两端注入10 μl OEC悬液后移植于视神经断端。C组（周围神经体外溃变组）：将取出的坐骨神经在体外单独培养5 d后植于视神经断端；D组（OEC-周围神经共培养组）：将取出的坐骨神经与OEC共培养5d后植于视神经断端。移植术后4周处死动物，计数各组以5%荧光金逆行标记的再生RGC数量。结果B、C、D三组RGC均数1481±268、1235±266和1464±285显著高于A组799±109（P值分别为0.0002、0.0010和0.0003）；B、C、D三组间差异无统计学意义（P值分别为0.3644、0.9167和0.4344）。结论OEC具有促进RGC轴突在新鲜周围神经移植物中再生的作用，但这种作用与体外溃变的周围神经相比无明显差异，二者亦无协同作用。（中华眼底病杂志，2007，23：130-132）