Caspase-9: involvement in secondary death of axotomized rat retinal ganglion cells in vivo

Recently we have shown that adult rat retinal ganglion cells (RGCs) die by apoptosis following optic nerve (ON) transection, activating caspase-3. In the present study, we report that caspase-9, known to be an important activator of caspase-3, becomes activated in the axotomized adult rat retina as revealed by immunoblot analysis and protease activity assays. Reduction of caspase-9 activity by repeated intraocular injection of specific inhibitors significantly prevented RGC death. Caspase-9 activity was effectively blocked by inhibitor treatment and by application of IGF-I and BDNF, neurotrophic factors which have been shown to be highly neuroprotective in this model. Taken together, our data suggest that caspase-9 plays a critical role in apoptosis induction in axotomized RGCs in vivo and is regulated under treatment with growth and survival factors. Thus, providing more insight into the mechanisms underlying neuronal death and survival following trauma might serve as a basis to improve future therapeutic strategies preventing or at least reducing the severe consequences of neuronal injury.     

Protection of axotomized retinal ganglion cells by adenovirally delivered BDNF in vivo

Following intraorbital transection of the optic nerve (ON) in rats, more than 80% of the retinal ganglion cell (RGC) population die by apoptosis within 14 days. Repeated intraocular injection of brain-derived neurotrophic factor (BDNF) has been efficient in enhancing RGC survival following ON axotomy. The present study was designed to define a potential survival-promoting effect of adenovirally administered BDNF on axotomized RGCs. A single injection of an adenoviral vector expressing the human BDNF gene from a CMV promoter/enhancer (Ad-BDNF) enhanced RGC survival 14 days after axotomy by 40.3%. Moreover, a combinatory treatment regimen consisting of intraocular Ad-BDNF administration and systemic application of the free radical scavenger, N-tert-butyl-(2-sulphophenyl)-nitrone (S-PBN), enhanced RGC survival by 63.0%. Our data demonstrate that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced RGC death. Further, as shown for S-PBN, therapeutic regimens that combine local virus-mediated gene delivery with systemic administration of protective compounds, may offer promising strategies for future treatment also in human neurodegenerative conditions.     

Electrical stimulation enhances the survival of axotomized retinal ganglion cells in vivo

In view of recent reports on the survival promotion of damaged spiral ganglion cells and motoneurons by electrical stimulation, we hypothesized that an electrical stimulation of the cut optic nerve (ON) may promote the survival of axotomized retinal ganglion cells (RGCs) in vivo. To test this hypothesis, we examined 1 week after ON transection the RGC densities in the retinas with or without electrical stimulation. The densities of surviving RGCs in the retinas with the electrical stimulation increased as compared with those without the electrical stimulation. We concluded that electrical stimulation of the ON enhances the survival of axotomized RGCs in vivo, probably due to electrical activation of their soma.     

电刺激对大鼠横断视神经视网膜节细胞的影响

目的在建立SD大鼠视神经横断和慢性电刺激模型的基础上,探讨电刺激对视网膜节细胞（RGCs）的保护作用。方法将大鼠随机分成电刺激治疗组和正常对照组,分离暴露右眼视神经并于球后1．5mm处进行横断。电刺激治疗组为分离横断视神经后给予电刺激,正常对照组为横断视神经后给予假性电刺激,在处死前2d断端处放人蘸有5％的荧光金（FG）的明胶海绵,以逆行标记RGCs。在3d、7d、14d不同的时间点处死动物并取材,进行HE染色并用荧光显微镜观察计数视网膜内存活的节细胞。结果在7d、14d电刺激治疗组存活的视网膜神经节细胞数目明显高于正常对照组,差异有显著意义（P〈0．05）。结论电刺激对视神经损伤有一定的保护作用。     

Regulation of caspase activation in axotomized retinal ganglion cells

Transection of the optic nerve initiates massive death of retinal ganglion cells (RGCs). Interestingly, despite the severity of the injury, RGC loss was not observed until several days after axotomy. The mechanisms responsible for this initial lack of RGC death remained unknown. In the current study, immunohistochemical analysis revealed that caspases-3 and -9 activation in the RGCs were not detected until day 3 post-axotomy, coinciding with the onset of axotomy-induced RGC loss. Interestingly, elevated Akt phosphorylation was observed in axotomized retinas during the absence of caspase activation. Inhibiting the increase in Akt phosphorylation by intravitreal injection of wortmannin and LY294002, inhibitors of PI3K, resulted in premature nuclear fragmentation, caspases-3 and -9 activation in the ganglion cell layer. Our findings thus indicate that the PI3K/Akt pathway may serve as an endogenous regulator of caspase activation in axotomized RGCs, thereby, contributing to the late onset of RGC death following axotomy.     

RNA content of normal and axotomized retinal ganglion cells of rat and goldfish

The responses of rat and goldfish retinal ganglion cells to axotomy were examined by a quantitative cytochemical method for RNA and by morphometric measurement 1-60 (rat) and 3-90 (goldfish) days after interruption of one optic nerve or tract intracranially. Unoperated control animals were studied also. The RNA content of axotomized neurons of rat fell 7-60 days postoperatively. Additionally, atrophy of the axotomized somas occurred. Over time, neuronal atrophy approximately paralleled the loss of RNA, and mean cell area and RNA content were reduced by about 25% 60 days after axotomy. Incorporation of 3H-uridine by axotomized neurons declined also. Axotomized retinal ganglion cells of goldfish behaved differently from those of the rat and showed increases in RNA content, most conspicuously 14-60 days postoperatively. Enlargement of axotomized fish neurons occurred but was less proportionately than concomitant increases in RNA content. The nonaxotomized ganglion cells of goldfish displayed statistically significant increases in size and RNA content 14-49 days after unilateral optic nerve or tract lesions. In contrast, alterations in rat retinal ganglion cells contralateral to interruption of one optic nerve were of limited and questionable significance. The contrasting reactions to axotomy by the retinal ganglion cells of these two vertebrates, one of which regenerates optic axons and one of which does not, may support the proposition that the somal response to axon injury has an important bearing upon the success or failure of CNS regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-dependent rescue of axotomized rat retinal ganglion cells by adenovirus-mediated expression of glial cell-line derived neurotrophic factor in vivo

Adult rat retinal ganglion cells undergo degeneration after optic nerve transection. Repeated intraocular injection of glial cell-line derived neurotrophic factor (GDNF) has been shown to be efficient in enhancing retinal ganglion cell survival following optic nerve axotomy. In the present study we evaluated the potential survival-promoting effect of adenovirally administered GDNF on axotomized retinal ganglion cells. A single intravitreal injection [7 x 107 plaque-forming units (pfu) or 7 x 108 pfu] of an adenoviral vector expressing the rat GDNF gene from a cytomegalovirus promoter enhanced retinal ganglion cell survival 14 days after axotomy by 67 and 125%, respectively, when compared to control animals. Intraocular administration of the vector rescued 12.6 and 23%, respectively, of the retinal ganglion cells which would otherwise have died after axotomy. An increase in retinal GDNF protein and specific virally transduced GDNF mRNA expression was detected following intraocular vector application. Our data support previous findings showing that adenoviral delivery of neurotrophic factors to the vitreous body is a feasible approach for the prevention of axotomy-induced retinal ganglion cell death in vivo and may constitute a relevant strategy for future treatment in traumatic brain injury and ensuing neurodegeneration.     

Morphometrical analysis of dendritic arborization in axotomized retinal ganglion cells

It has been reported that section of the optic nerve in mammals causes death in >90% of the retinal ganglion cells (RGCs). The cells which survive the section experience an irreparable loss of many of their dendritic segments and a rapid retraction of the dendritic tree. However, some growth cones and abnormal processes have been also reported. Our aim was to make a quantitative study of the morphological changes found in rabbit RGCs after optic nerve section. The morphometrical analysis of the RGCs which survived the axotomy showed an increase in the diameter of the soma and a significant increase in the area of the dendritic field; also, the length of the dendritic segments was significantly longer in axotomized RGCs than in control cells. Terminal dendritic segments (T) and preterminal segments (PT) were both measured in control and axotomized cells; the length ratio of T : PT segments was significantly greater in the axotomized cells than in the controls. We conclude that RGCs which survived the axotomy experienced a significant growth of their terminal dendritic branches.     

Artemin augments survival and axon regeneration in axotomized retinal ganglion cells

Artemin, a recently discovered member of the glial cell line‐derived neurotrophic factor (GDNF) family, has neurotrophic effects on damaged neurons, including sympathetic neurons, dopamine neurons, and spiral ganglion neurons both in vivo and in vitro. However, its effects on retinal cells and its intracellular signaling remain relatively unexplored. During development, expression of GFRα3, a specific receptor for artemin, is strong in the immature retina and gradually decreases during maturation, suggesting a possible role in the formation of retinal connections. Optic nerve damage in mature rats causes levels of GFRα3 mRNA to increase tenfold in the retina within 3 days. GFRα3 mRNA levels continue to rise within the first week and then decline. Artemin, a specific ligand for GFRα3, has a neuroprotective effect on axotomized retinal ganglion cells (RGCs) in vivo and in vitro via activation of the extracellular signal‐related kinase? and phosphoinositide 3‐kinase?Akt signaling pathways. Artemin also has a substantial effect on axon regeneration in RGCs both in vivo and in vitro, whereas other GDNF family members do not. Therefore, artemin/GFRα3, but not other GDNF family members, may be of value for optic nerve regeneration in mature mammals. © 2014 Wiley Periodicals, Inc.     

Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult rats in vivo

After transection of the optic nerve (ON) in adult rats, retinal ganglion cells (RGC) progressively degenerate until, after two months, a residual population of only about 5% of these cells survives. In this study, we investigated the effect of regeneration-associated factors from sciatic nerve (ScN), BDNF, and CNTF on the survival of adult rat RGC after intraorbital ON transection. Neurotrophic factors were injected into the vitreous body. Rats were allowed to survive 3, 5, or 7 weeks, and the remaining viable RGC were then labelled by retrograde staining with the carbocyanine dye, 4Di-10Asp, which was applied onto the proximal nerve stump in vivo. The animals were sacrificed 3 days later and RGC counted in retinal whole mounts. Due to progressive degeneration following nerve transection the number of surviving RGC decreased to about 10% of the initially labelled population after 3 weeks, to about 8% after 5 weeks, and to about 5% after 7 weeks. Survival of axotomized cells could be prolonged using either of the neurotrophic factors: after 3 weeks a 2–3-fold increase in the number of viable RGC could be obtained compared to uninjected controls and to those which received injection of buffer. The prolonged survival effect vanished after 5 and 7 weeks, and no additive effect could be seen when combining brain-derived neurotrophic factor (BDNF) and ciliary neuronotrophic factor (CNTF) treatment. Morphometric analysis of labelled cells revealed that all neurotrophic factors supported predominantly large RGC with somal areas > 250 μm². In retinae from rats that survived the ON transection for several months, a characteristic population of axotomy-resistant RGC remained alive. Their few, very large, and often curled dendrites showed signs of placticity in the depleted inner nuclear layer of the adult rat retina. We conclude that the intraocular injection of CNTF, BDNF, and ScN-derived medium, which retard the process of lesion-induced RGC degeneration, may be successfully used as a subsidiary strategy in transplantation protocols. This would result in larger populations of RGC which can be recruited to regenerate their axons and provide a basis for functional recovery.     

Simvastatin promotes heat shock protein 27 expression and Akt activation in the rat retina and protects axotomized retinal ganglion cells in vivo

Heat shock proteins (Hsps) are stress proteins that mediate protein stabilization in various tissues and protect cells from environmental stress. Novel evidence suggests that overexpression of the small heat shock protein 27 (Hsp27) in neurons protects against neurotoxic stimuli and may act as an inhibitor of neurodegeneration. Overexpression of Hsps has been achieved by different means including pharmacological induction. Here, we show that intravitreal injection of the 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor simvastatin induces Hsp27 expression in axotomized retinal ganglion cells (RGCs) and enhances RGC survival 7 and 14 days after optic nerve (ON) axotomy by 90% and 19%, respectively. The flavonoid quercetin inhibited Hsp27 induction and abrogated simvastatin-mediated neuroprotection. Simvastatin increased Akt phosphorylation in vivo, indicating that the PI3K/Akt pathway contributes to central nervous system (CNS) protective effects achieved. We propose the use of statins as a feasible approach to reduce lesion-induced CNS neuronal degeneration in vivo.     

Adenovirus-mediated expression of ciliary neurotrophic factor (CNTF) rescues axotomized rat retinal ganglion cells but does not support axonal regeneration in vivo

Rat optic nerve (ON) transection leads to mainly apoptotic cell death of about 85% of the retinal ganglion cell (RGC) population within 14 days after lesion. In the present study, we tested the effect of adenovirally delivered CNTF (Ad-CNTF) on survival and regeneration of axotomized adult RGCs in vivo. Single intravitreal Ad-CNTF injection led to stable CNTF mRNA and protein expression for at least 18 days and significantly enhanced RGC survival by 155% when compared to control animals 14 days after ON transection. ON stump application of Ad-CNTF also resulted in an increased number of surviving RGCs. Ad-CNTF injection led to better preservation of intraretinal RGC axons but did not support regeneration of axotomized RGCs into a peripheral nerve graft. Thus, adenovirus-mediated neurotrophic factor supply is a suitable approach for reducing axotomy-induced RGC death in vivo and may constitute a relevant strategy for clinical treatment of traumatic brain injury.     

Long-term effect of inhibition of ced 3-like caspases on the survival of axotomized retinal ganglion cells in vivo.

There is growing evidence that caspase inhibition exerts neuroprotective effects in various models of neuronal injury in vivo. However, whether caspase inhibition provides long-term neuroprotection is not known yet. In the present study, we therefore investigated the effects of prolonged caspase inhibition on the survival of adult rat retinal ganglion cells (RGCs) following optic nerve (ON) transection. Four weeks following ON transection the number of surviving RGCs in untreated animals declined to 11% of controls. Treatment for the initial 2 weeks with z-DEVD-cmk, an irreversible inhibitor of ced 3-like caspases, increased the number of surviving RGCs 4 weeks postlesion to 24%. Z-DEVD-cmk treatment over the entire experimental period of 4 weeks had no additional effect. Thus, we still found a neuroprotective effect of caspase inhibition on axotomized RGCs after extended survival time. However, in comparison to our recent observations 2 weeks after optic nerve transection, in which z-DEVD-cmk rescued 46% of RGCs (P. Kermer, N. Kl?cker, M. Labes, and M. B?hr, 1998, J. Neurosci. 18(12), 4656-4662) the positive effect clearly decreased. In conclusion, our results indicate that the therapeutical approach presented here results in a significant delay of secondary death rather than providing a permanent and complete rescue of axotomized RGCs.     

Polarization and orientation of retinal ganglion cells in vivo

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.     

In vivo imaging of murine retinal ganglion cells

Current methods for in vivo retinal ganglion cells (RGCs) imaging involve either retrograde or intravitreal injection of chemical or biological tracers, which are invasive and may require repeated injection for serial long-term assessment. We have developed a confocal scanning laser ophthalmoscope technique (blue-light CSLO or bCSLO) to image retinal ganglion cells (RGCs) in mice expressing cyan fluorescent protein under the control of a Thy-1 promoter. Fluorescent spots corresponding to CFP-expressing retinal ganglion cells were discernable with the bCSLO. 96.1+/-2.6% of CFP expressing cells also were retrograde labeled with DiI indicating the bCSLO imaged fluorescent spots are RGCs. The imaging of Thy-1 promoter-driven CFP expression in these mice could serve as a sensitive indicator to reflect the integrity of RGCs, and provides a non-invasive method for longitudinal study of the mechanism of RGC degeneration and the effect of neuroprotective agents.     

Environmental light enhances survival and axonal regeneration of axotomized retinal ganglion cells in adult cats

A segment of peripheral nerve was transplanted to the cut stump of the optic nerve to facilitate axonal regeneration of retinal ganglion cells (RGCs) in adult cats. The cats were reared under different light environment: 12 h light-12 h darkness, additional flash light under conventional light cycle, or 24 h darkness. After 60 days, the density and morphology of RGCs with regenerated axons were examined with retrograde labeling by fluoro-ruby and intracellular injections of Lucifer Yellow. In the retina of cats reared in darkness, densities of RGCs with regenerated axons were 11-42% of those in the retina of cats reared under conventional light and dark cycle. More than half of the labeled RGCs were degenerative in the retina of cats reared in darkness, while most RGCs were normal under conventional environment or flash light. We conclude that environmental light is essential for the survival and axonal regeneration of axotomized RGCs.     

Degeneration of axotomized retinal ganglion cells as a model for neuronal apoptosis in the central nervous system - molecular death and survival pathways

Programmed cell death (PCD) or apoptosis is a phenomenon important for proper development and morphological as well as functional fine tuning of the nervous system. In the past two decades it became evident that the same apoptotic machinery, which has crucial functions in during development, can be reactivated under pathological circumstances in the adult nervous system and contribute to neuronal cell loss due to various neurological disorders like ischemic stroke, neurodegenerative diseases or brain traumata. In this review, we present the optic nerve transection paradigm as a valuable model for investigation of apoptotic neuronal cell death in the central nervous system (CNS). We review and summarize the most important discoveries regarding molecular pathways and mechanisms of neuronal apoptosis during the past few years, and outline contributions that have been made investigating the death of retinal ganglion cells (RGCs) following transection of the optic nerve.     

Effects of low level laser treatment on the survival of axotomized retinal ganglion cells in adult Hamsters

Injury to axons close to the neuronal bodies in the mammalian central nervous system causes a large proportion of parenting neurons to degenerate. It is known that optic nerve transection close to the eye in rodents leads to a loss of about half of retinal ganglion cells in 1 week and about 90% in 2 weeks. Using low level laser treatment in the present study, we demonstrated that treatment with helium-neon(660 nm) laser with 15 m W power could delay retinal ganglion cell death after optic nerve axotomy in adult hamsters. The effect was most apparent in the first week with a short period of treatment time(5 minutes) in which 65–66% of retinal ganglion cells survived the optic nerve axotomy whereas 45–47% of retinal ganglion cells did so in optic nerve axotomy controls. We also found that single dose and early commencement of laser irradiation were important in protecting retinal ganglion cells following optic nerve axotomy. These findings thus convincingly show that appropriate laser treatment may be neuroprotective to retinal ganglion cells.     

Protection of axotomized ganglion cells by salicylic acid

Neuronal survival is influenced by the redox environment, and it has been shown that antioxidants protect developing neurons from the effects of axotomy. Here, we show that the intraocular injection of salicylic acid (SA) reduces the number of dying axotomized ganglion cells in the chick embryo. The antioxidant properties of SA are probably responsible for its protective effects, whose U-shaped dose-dependency matches that of several other antioxidants. We conclude that SA protects axotomized neurons by maintaining the redox status near an optimal set-point.     

Inwardly rectifying potassium channels in rat retinal ganglion cells

Inwardly rectifying potassium channels (Kir channels) are important for neuronal signalling and membrane excitability. In the present work we characterized, for the first time, Kir channels in rat retinal ganglion cells (RGCs), the output neurons in the retina, using immunocytochemical and patch-clamp techniques. Various subunits of Kir channels (Kir1.1, 2.1, 2.3, 3.1, 3.2 and 3.3) were expressed in RGCs, but with distinct subcellular localization. Kir1.1 was mainly expressed in axons of RGCs. Kir2.1 and Kir2.3 were both present in somata of RGCs. Whereas staining for Kir3.1 was profoundly present in an endoplasmic reticulum-like structure and Kir3.2 was strongly expressed in the cytoplasm and the cytomembrane of somata, dendrites and axons of RGCs, faint, sparse labelling for Kir3.3 was seen in the cytomembrane. Immunoreactivity for Kir4.1 and Kir4.2 was not detectable in RGCs. Whole-cell currents mediated by Kir channels were recorded in isolated RGCs and they differed from hyperpolarization-activated currents (I(h)) by showing full activation in < 10 ms, no inactivation, and being significantly suppressed by 300 microM Ba2+. Unlike in retinal horizontal cells and bipolar cells, these currents were mainly mediated by G-protein-coupled Kir3 (GIRK) channels, as demonstrated by the fact that GDP(beta)S and GTP(gamma)S included in the pipette solution markedly decreased and increased the currents, respectively. Furthermore, the GIRK channels were probably coupled to GABA(B) receptors, because baclofen considerably increased the Kir currents and the increased currents were suppressed by Ba2+. These characteristics of the Kir currents provide more versatility for signalling of RGCs.