The role of Bax-inhibiting peptide in retinal ganglion cell apoptosis after optic nerve transection

Bax, a pro-apoptotic member of Bcl-2 family proteins, plays a central role in mitochondria-dependent apoptosis. Bax normally resides in the cytosol in a quiescent state. Bax-inhibiting peptide (BIP) is a membrane permeable peptide comprised of five amino acids designed from the Bax-binding domain of Ku70 [M. Sawada, P. Hayes, S. Matsuyama, Cytoprotective membrane-permeable peptides designed from the Bax-binding domain of Ku70, Nat. Cell Biol. 5 (2003) 352-357]. It inhibits Bax-mediated translocation of cytochrome c and suppresses mitochondria-dependent apoptosis. BIP was used in order to elucidate its role in preventing retinal ganglion cell (RGC) death from apoptosis after optic nerve transection (ONT) in adult Wistar rats. RGC survival was significantly higher in animals with intravitreal injection of BIP, when compared with control animals. These findings suggest that BIP prevented RGC apoptosis after ONT prompting the suggestion that Bax plays a central role in RGC apoptosis after ONT.     

Viability of retinal ganglion cells after optic nerve crush in adult rats

Summary The response of retinal ganglion cells to optic nerve crush was examined in the hooded rat. Intracranial nerve crush produces a transient shrinkage of the retinal ganglion cells during the first several weeks postoperatively but partial recovery of cell size then appears to occur. This transient response is considered to be a direct response to axotomy. Retrograde transport of horseradish peroxidase (HRP) is clearly demonstrated at 2 weeks postoperatively. Transport of newly synthesized protein progressively decreases over the first 2 postoperative months. The ganglion cell therefore retains viability for at least the first few weeks after axotomy. Loss of 60% of the neurons in the ganglion cell layer occurs between 3 and 7 months postoperatively. This late occurring retrograde response is considered to result at least in part from loss of sustaining trophic influences rather than as a direct result of the lesion.     

Qualitative and quantitative ultrastructural observations on retinal ganglion cell layer of rat after intraorbital optic nerve crush

Summary Rat retinal ganglion cell layer (GCL) was examined ultrastructurally 1–180 days after intraorbital crushing of one optic nerve. It was confirmed quantitatively that axotomized ganglion cells lost cisternal membranes of the rough endoplasmic reticulum (RER) and showed disintegration of Nissl bodies and ribosomal rosettes 3 days postoperatively. Between 60 and 180 days after neurotomy there was partial reversion of the RER towards normal. At postoperative intervals of 14–60 days, chromatin aggregation became conspicuous and some nuclei were prominently furrowed and contained electron-dense inclusions. Concurrently, profiles of dead ganglion cells were encountered. Mean mitochondrial area increased in axotomized neurons but mitochondrial density declined, while the Golgi apparatus, lamellar specializations of the RER and the size of nuclei did not change significantly. Cytoplasmic atrophy was profound, however. Small nerve cells of the GCL appeared morphologically distinct from ganglion cells and did not undergo appreciable alteration.A decline in neuronal density, approximating 35%, occurred between the third and seventh postoperative day and progressed slowly thereafter. Neuronal density was 32% of normal 180 days postoperatively. A temporary increase in glial density 3–28 days after operation was due to microglial hyperplasia. Müller cell and astrocytic processes hypertrophied, infiltrated nerve fibre bundles, and surrounded and intruded into neuronal somata. Bundles of unmyelinated small axons, invested by astrocytes and basal lamina, were present within the necrotic cavity of the lesioned nerve 28–90 days postoperatively and had cytologic features of regenerative axonal sprouts.We conclude that intraorbital optic nerve crush is followed by a noteworthy degree of regenerative axonal sprouting which occurs and persists against a background of slow but relentless decline in the retinal ganglion cell population. This slow decline follows a rapidly-sustained loss of approximately one-third of the axotomized retinal ganglion cells during the first postoperative week. Intraorbital, as opposed to intracranial, injury of the optic nerve appears, paradoxically, to induce both a greater degree of ganglion cell death and a greater amount of regenerative axonal sprouting. Cytologic changes in axotomized retinal ganglion cells resemble those described for other populations of mammalian intrinsic neurons subjected to like injury. However, they differ, especially with regard to patterns of nuclear, nucleolar and RER alteration, from changes observed in peripheral neurons of mammals and intrinsic neurons of submammalian vertebrates that successfully regenerate severed axons. The neuroglial response in the surround of retinal ganglion cells after optic nerve crush is characterized by hypertrophy of astrocytes and Müller cells and a transient, modest increase of microglia. The microglial reaction is clearly less pronounced than that which occurs in the surround of axotomized peripheral neurons of the rat. The data presented here provide qualitative and quantitative cytologic information against which any effects exerted on the axotomy response and optic nerve regeneration by growth-promoting agents may be assessed.     

A morphometric study of the retinal ganglion cell response to optic nerve severance in the frog Rana pipiens

Summary This study examines the cell body response to axotomy of retinal ganglion cells in the frogRana pipiens. Cell soma sizes were measured in carefully matched regions of Nissl-stained wholemounted retinae after either nerve crush, nerve cut with stump separation, nerve crush with intraocular nerve growth factor (NGF) or nerve cut with NGF applied to the proximal stump. The state of axonal regeneration was also assessed in each case by anterograde transport of HRP.Following nerve crush axons crossed the lesion by 7 days, reached the chiasma by 14 days and entered the tectum around 20–30 days. The normally evenly stained ganglion cells exhibited granular Nissl staining at 7 and 10 days but very little change in soma size. From 10 to 28 days the mean retinal ganglion cell area increased by 102% and maintained this size until at least 75 days. By 102 days soma size had nearly returned to normal. A population of displaced amacrine cells retained a normal appearance and soma size throughout regeneration.Following nerve cut and stump separation the retinal ganglion cells were slightly more reactive in appearance at 7 days after crush but otherwise the soma reaction developed in a similar manner. Axon tracing revealed no extension beyond the lesion site in these animals and therefore the state of axonal growth did not affect the early soma response.NGF applied at the time of the lesion had no detectable effect on the soma reaction.Although many retinal ganglion cells re-establish contact with visual centres after axotomy in the frog, a considerable proportion die. This contrasts with both the goldfish, where all cells regenerate successfully, and various mammals, where none do so and all retinal ganglion cells die. All retinal ganglion cells in the frog undergo reactive changes similar to those of goldfish and there is no sign of the cell shrinkage seen in mammals. Therefore the cell death in frog would appear to be different from that in mammalian retina but similar to that of mammalian peripheral nerve in which chromatolysis generally preceeds death.     

Long term survival of cat retinal ganglion cells after intracranial optic nerve transection

Summary After intracranial transection of the optic nerve in cats the retinal stump of the nerve was injected with HRP. Surviving retinal ganglion cells can be retrogradely labeled at least up to 15.5 months of postoperative survival.     

Factors affecting the survival of cat retinal ganglion cells after optic nerve injury

Summary After partial transection of one optic nerve in adult cats the majority of retinal ganglion cells degenerate and die 1 week after axotomy, whilst other cell classes degenerate slowly and survive for a long period after the lesion. We have investigated the effects of intravitreal and intraperitoneal injections of MK-801, a NMDA-glutamate receptor antagonist, on the early degeneration of retinal ganglion cells after partial optic nerve section. Control animals received saline intravitreal injections. Retinal flat mounts were retrogradely labelled with horseradish peroxidase and counterstained with Cresyl Violet. We evaluated the ganglion cell loss in the three experimental groups 1 week after lesion and compared them with normal uninjured controls and injured untreated retinae. In untreated retinae 49% of ganglion cells die 1 week after the lesion. Systemic MK-801 treatment prolonged survival of 41% of retinal ganglion cells that would die without treatment. Intravitreal MK-801 or saline prolonged survival of 71% of retinal ganglion cells that would die without treatment, but the results of saline administration had a larger range of variability. In untreated retinae many pyknotic cells were observed. The decreased in number after systemic MK-801 treatment and in some retinae treated with intravitreal injections of saline solution. There were no pyknotic cells after local, intravitreal MK-801 treatment. These results support the hypothesis that NMDA-receptor mediated neurotoxicity plays an important role in the early retinal ganglion cell death after retrobulbar axotomy. They also support the existence of an endogenous source of neurotrophins whose release is triggered by eyeball injury. We conclude that the early death of retinal ganglion cells after axotomy occurs by a mechanism that can be controlled by neurotrophins and antagonists to NMDA-glutamate receptors.To whom correspondence should be addressed.     

活化的巨噬细胞在体内对成年大鼠视神经夹伤后视网膜神经节细胞的保护作用

目的:评估活化的巨噬细胞对于视神经损伤后视网膜神经节细胞存活的影响。 方法:成年Wistar大鼠随机分为2组，A组左眼为正常对照组，右眼为单纯视神经夹伤组；B组按夹伤后眼内注射药物不同，每只大鼠右眼为酵母多糖组，左眼为PBS组。采用双上丘和外侧膝状体注射3%荧光金标记双眼视网膜神经节细胞7 d后，除正常对照组外均应用40 g力的视神经夹在大鼠眼球后2 mm处夹视神经9 s，观察视神经夹伤后不同时间，视网膜神经节细胞存活数。视网膜进行ED-1染色以鉴定是否有巨噬细胞被激活。 结果:正常对照组、视神经夹伤组及PBS组中未见ED-1阳性巨噬细胞，酵母多糖处理组中在视网膜表面可见ED-1阳性巨噬细胞。正常对照组及视神经夹伤对照组RGC逆行标记3、7、14、21 d细胞数不同。视神经夹伤组被标记的RGC分别相当于正常对照组的92.6%、82.9%、69.6%、57.6%。酵母多糖治疗组在夹伤后3、7、14、21 d标记的RGC数分别相当于正常对照组的99.6%、89.2%、72.6%、62.6%，均显著高于视神经夹伤组。 结论:玻璃体内注射酵母多糖可激活巨噬细胞，激活巨噬细胞可以促进视神经损伤后视网膜神经节细胞的存活。     

Retinal ganglion cell death during regeneration of the frog optic nerve is not accompanied by appreciable cell loss from the inner nuclear layer

Summary We estimated cell numbers in the ganglion cell and inner nuclear layers of adult frog (Hyla moorei) retinae, examining normal animals and those with regenerated optic nerves. Analysis of sections stained with cresyl violet showed that cell numbers in a nasotemporal strip, which included the area centralis and visual streak, were comparable between sides for both these cellular layers in normal animals. In line with our previous observations, after optic nerve regeneration cell numbers in the ganglion cell layer had fallen by 35–43% compared to the unoperated sides. By contrast cell numbers remained similar for the inner nuclear layers on the two sides. We conclude that retrograde transneuronal degeneration had not taken place in the inner nuclear layer in response to ganglion cell death.     

Regrowth of retinal ganglion cell axons into a peripheral nerve graft in the adult hamster is enhanced by a concurrent optic nerve crush

Summary Transplantation of a segment of peripheral nerve to the retina of the adult hamster resulted in regrowth of damaged ganglion cell axons into the graft, with the fastest regenerating axons extending at 2 mm/day after an initial delay of 4.5 days (Cho and So 1987b). In this study, the effect of making 2 lesions on the same axon (the conditioning lesion effect) on the regrowth of ganglion cell axons into the peripheral nerve graft was examined. When a conditioning lesion (first lesion) was made by crushing the optic nerve 7 or 14 days before the peripheral nerve grafting (the second lesion) to the retina, the distance of regrowth achieved by the fastest regenerating axons in the graft, measured at the 7th post-grafting day, was lower than in animals with a peripheral nerve grafted to a normal eye. This indicated that in contrast to the situation in peripheral nerve axons (Forman et al. 1980) and goldfish optic axons (Edwards et al. 1981), the conditioning lesion was unable to enhance the regrowth of mammalian retinal ganglion cell axons. However, when crushing of the optic nerve was followed immediately by peripheral nerve grafting, an enhancement in axonal regrowth could be observed. The initial delay time before the axons extended into the peripheral nerve graft was reduced by 1 day while the rate of elongation of the fastest regrowing axons in the graft apparently remained unchanged. Moreover, the shortening of the initial delay could still be observed even when the sequence of performing the 2 lesions was reversed. From these data, it was concluded that the classical conditioning lesion effect was not responsible for the enhancement observed. Rather it was suggested that changes in the intra-retinal environment brought about by crushing of the optic nerve might account for it.     

不同给药途径的聚乙二醇对大鼠视神经切断后视网膜神经节细胞存活的影响

目的:比较不同给药途径的聚乙二醇(PEG)对成年大鼠视神经切断后视网膜神经节细胞(节细胞)存活的影响。方法:72只成年SD大鼠左眼球后1.5 mm处横断视神经,眶侧断端留置浸有荧光金的明胶海绵以逆行标记存活的节细胞。术后立即尾静脉注射1 ml 30%PEG(尾静脉注射PEG组)或等体积生理盐水(尾静脉注射盐水对照组),或在视神经眶侧断端留置浸有50%PEG(局部PEG组)或生理盐水(局部盐水对照组)的明胶海绵。四组动物(n=18)分别存活2、7 d或14 d(每时间点,n=6)后处死,取术侧视网膜,平铺计数存活节细胞并计算出节细胞密度。结果:术后7 d尾静脉注射PEG组存活节细胞平均密度(1121.43 mm2±42.69/mm2)显著高于尾静脉注射盐水对照组(846.67/mm2±58.19/mm2,P<0.05),而2 d和14 d时间点两组节细胞密度间无显著性差异(P>0.05);局部PEG组节细胞密度在各时间点与局部盐水对照组相比均无显著性差异(P>0.05);在7 d点,尾静脉注射PEG组节细胞密度显著高于局部PEG组(774.43/mm2±50.49/mm2,P<0.05)。结论:PEG能在视神经切断后一定时间内延缓节细胞死亡,且这种神经保护作用有赖于PEG的给药途径。     

Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

The conditioning effect of optic nerve injury upon axonal regrowth from adult rat retinal ganglion cells explanted in vitro

An in vitro assay was used to determine the effects of conditioning nerve lesions on the regeneration of adult rat retinal ganglion cell (RGC) axons from retinal explants. Following the conditioning lesion (CL) of unilateral optic nerve transection, maximal regrowth was seen from RGC explanted from ipsilateral retinae 10 days post-CL. Explants from this group initiated axonal regrowth earlier and a greater percentage regrew axons when compared with explants from normal rats. Axonal regrowth from explants of retinae contralateral to CL was also seen earlier than normal. In further experiments, the effects of both exposure of the optic nerve sheath in the orbit and the incision of the dura without injury to optic nerve axons were studied. The conditioning effect of a dural incision was found to be the same as that of optic nerve transection, whilst exposure of the optic nerve sheath had no conditioning effect on RGC axonal regrowth in vitro.     

黄芪甲苷对成年大鼠视神经切断后视网膜节细胞存活的影响

目的:探讨黄芪甲苷(AST)在成年大鼠视神经切断后对视网膜节细胞(RGCs)存活的影响。方法:动物分为正常组、单纯切断视神经组、AST处理组和生理盐水对照组。用荧光金(FG)逆行示踪标记法及定量解剖学技术观察正常和经AST处理的SD大鼠于视神经切断后5、7、14 d的RGCs的密度。结果:正常组RGCs平均密度为(2 230±156)/mm~2。单纯视神经切断组RGCs平均密度与生理盐水对照组相比较,无显著性差异;AST处理组与单纯切断视神经组和生理盐水对照组相比较,在各个时间点上均存在显著性差异。结论:黄芪甲苷可提高成年大鼠视神经切断后视网膜节细胞短期存活。     

Parameters of optic nerve electrical stimulation affecting neuroprotection of axotomized retinal ganglion cells in adult rats

We previously showed the enhancement of survival of retinal ganglion cells (RGCs) by electrical stimulation (ES) of the optic nerve (ON) stump in adult rats. To elucidate the mechanisms underlying the survival enhancement, we determined whether the neuroprotective effect of ES is affected by the following parameters: stimulation time, frequency of current pulses and starting of ES. ES for 10 min immediately after ON transection was not effective in increasing the number of surviving RGCs 7 days after the transection, but that for 30 min was effective. ES at 20 Hz was the most effective, when applied just after axotomy. When the starting of ES to the ON was shifted either 3 h after or 4 h before the axotomy, the neuroprotective effect of ES was not observed. These results suggest that the electrical activation of RGCs and/or the transected ON interfere with early events after axotomy that leads to RGC death.     

Neither laminin nor prior optic nerve section are essential for the regeneration of adult mammalian retinal ganglion cell axons in vitro

Summary Retinal explants obtained from normal adult rats and from operated animals in which the optic nerve had been sectioned 10 days previously were cultured in either serum-containing or serum-free medium on poly-l-lysine and laminin substrata. Regenerating ganglion cell axons growing from these explants have been identified using monoclonal antibodies against Thy-1.1 cell surface glycoprotein and the 200-kDa subunit neurofilament protein. Irrespective of substratum or medium composition, axons regenerated from 28–49% of normal rat retinal explants. This percentage increased to 60–84% of explants from operated rats. There were no significant differences in percentages of explants from normal or operated rats showing neurite outgrowth when substrata of either poly-l-lysine or laminin were compared in serum-free medium. In serum-containing medium the results were less easily interpreted due to the presence of an outgrowth of non-neuronal (glia and mesenchymal) flat cells, which served as a preferred axonal substratum in many cases. Thus we show that adult rat retinal ganglion cell axons will regrowin vitro, and that a priming optic nerve section will increase this response. In neither case is the response laminin-dependent.     

Neuroprotective effect on retinal ganglion cells by transpupillary laser irradiation of the optic nerve head

This study demonstrates that subthreshold transpupillary thermotherapy (TTT) laser irradiation on optic nerve head protects retinal ganglion cells (RGCs) in an optic nerve crush (ONC) model. TTT was performed in right eyes with an 810-nm diode laser aimed at the center of the optic nerve head, using the following protocol: power 60 mW, duration 60 s, spot size 500 μm. Fluoro-Gold was injected into bilateral superior colliculi 5 days before sacrifice and fluorescent gold labeled RGCs were counted under fluorescence microscopy. In the ONC group, a progressive loss of RGCs was observed; however, in comparison with the ONC group, RGCs density was significantly higher (P = 0.001, independent samples t-test) at day 7 postoperative and only borderline significances were obtained at days 14 and 28 postoperative (P = 0.044 and P = 0.045, respectively, independent samples t-test) in ONC + TTT group, which implies the potential neuroprotective role of TTT. This protective effect seems to be heat shock proteins (HSPs) related, because intraperitoneal Quercetin (an inhibitor of HSPs, 4 mg/kg/day for 7 days) could completely abolish this protective effect at days 7, 14 and 28 postoperative (P = 0.012, P = 0.002, and P = 0.000, respectively, independent samples t-test). Minimal collateral damage of TTT on optic nerve head tissue, peripapillary RGCs and the myelin sheath of the optic nerve were observed under transmission electron microscopy. These findings suggested that subthreshold TTT might be a safe and practical approach to protect RGCs. The underlying mechanisms may involve TTT-induced HSPs in RGCs.     

Transplanted olfactory ensheathing cells incorporated into the optic nerve head ensheathe retinal ganglion cell axons: possible relevance to glaucoma

A mixture of olfactory ensheathing cells and fibroblasts cultured from the adult rat olfactory mucosa was transplanted through a scleral incision into the retina. A major stream of transplanted cells migrated through the stratum opticum and penetrated for up to about 0.5mm into the optic nerve head. This stream of transplanted cells consisted of a mixture of bipolar olfactory ensheathing cells with long processes which give rise to a non-myelinating ensheathment of single retinal ganglion cell axons, and olfactory nerve fibroblasts embedded in a dense fibronectin-positive extracellular matrix. A second stream of ovoid olfactory ensheathing cells with tufted processes and unaccompanied by fibroblasts or matrix migrate into the internal plexiform layer. The incorporation of olfactory ensheathing cells in the optic nerve head may suggest future possibilities for protection of the axons in this vulnerable region from mechanical damage, as in the raised intraocular pressure of glaucoma.