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Objectives: To investigate the expression changes of the hydrogen sulfide synthases cystathionine γ-lyase （CSE）, cystathionine β-synthase （CBS）, and 3-mercaptopyruvate sulfurtransferase （3-MST）, after optic nerve crush （ONC） in rat the retina., Methods: The rat model of ONC was established. Rats were divided into normal control, ONC, and sham control groups. Histopathologic changes in retina, the number of retinal ganglion cells （RGC） and retinal thickness of inner part （RTIP） were measured. The changes of CSE, CBS and 3-MST mRNA expression were detected with quantitative real-time PCR., Results: The retinal histostructure was normal in normal controls and with minor changes in sham controls, respectively. Compared with sham group, significant retina damages were found in the ONC group： a time-dependent reduction of RGC number and RTIP. Expressions of CSE, CBS and 3-MST mRNA in rat retina were detected in normal control. Compared with normal controls, the expressions of CSE, CBS and 3-MST mRNA did not show any significant changes in the sham controls. Compared with sham controls, CBS mRNA expressions showed a time-dependent increase at 3 d, 7 d and 14 d after crush in the ONC group; CSE mRNA expressions increased to the peak at 3 d and then slightly reduced at 14 d after crush; 3-MST mRNA expressions showed the trend of increase at 3 d and 7 d and then enhanced remarkably at 14 d after crush., Conclusions: Hydrogen sulfide synthases CSE, CBS and 3-MST expressions were up-regulated in rat retina following ONC, which may cause an increase in local endogenous hydrogen sulfide production in the retina and a compensatory protective effect., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Forensic Medicine.)

Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease that we identified as a nerve regeneration-associated molecule. The expression of DINE is upregulated in response to nerve injury in both the peripheral and central nervous systems, while its transcription is regulated by the activating transcription factor 3 (ATF3), a potent hub-transcription factor for nerve regeneration. Despite its unique hallmark of injury-induced upregulation, the physiological relevance of DINE in injured neurons has been unclear. In this study, we have demonstrated that the expression of DINE is upregulated in injured retinal ganglion cells (RGCs) in a coordinated manner with that of ATF3 after optic nerve injury, whereas DINE and ATF3 are not observed in any normal retinal cells. Recently, we have generated a mature DINE-deficient (KO Tg ) mouse, in which exogenous DINE is overexpressed specifically in embryonic motor neurons to avoid aberrant arborization of motor nerves and lethality after birth that occurs in the conventional DINE KO mouse. The DINE KO Tg mice did not show any difference in retinal structure and the projection to brain from that of wild-type (wild type) mice under normal conditions. However, injured RGCs of DINE KO Tg mice failed to regenerate even after the zymosan treatment, which is a well-known regeneration-promoting reagent. Furthermore, a DINE KO Tg mouse crossed with a Atf3:BAC Tg mouse, in which green fluorescent protein (GFP) is visualized specifically in injured RGCs and optic nerves, has verified that DINE deficiency leads to regeneration failure. These findings suggest that injury-induced DINE is a crucial endopeptidase for injured RGCs to promote axonal regeneration after optic nerve injury. Thus, a DINE-mediated proteolytic mechanism would provide us with a new therapeutic strategy for nerve regeneration.

After traumatic damage of the brain or spinal cord, many surviving neurons are disconnected, and recovery of function is limited by poor axon regeneration. Recent data have suggested that poly ADP-ribosylation plays a role in limiting axonal regrowth such that inhibition of poly (ADP-ribose) polymerase (PARP) may have therapeutic efficacy for neurological recovery after trauma. Here, we tested systemic administration of the PARP inhibitor, veliparib, and showed effective suppression of PARylation in the mouse CNS. After optic nerve crush injury or dorsal hemisection of the thoracic spinal cord in mice, treatment with veliparib at doses with pharmacodynamic action had no benefit for axonal regeneration or functional recovery. We considered whether PARP gene family specificity might play a role. In vitro mouse cerebral cortex axon regeneration experiments revealed that short hairpin RNA (shRNA)-mediated suppression of PARP1 promoted axonal regeneration, whereas suppression of other PARP isoforms either had no effect or decreased regeneration. Therefore, we examined recovery from neurological trauma in mice lacking PARP1. No increase of axonal regeneration was observed in Parp1 -/- mice after optic nerve crush injury or dorsal hemisection of the thoracic spinal cord, and there was no improvement in motor function recovery. Thus, comprehensive in vivo analysis reveals no indication that clinical PARP inhibitors will on their own provide benefit for recovery from CNS trauma., Competing Interests: S.M.S. is a co-founder of Axerion Therapeutics seeking to develop NgR1-based therapeutics.

Objective: The aim of this study was to study the impacts of Rho kinase inhibitor Fasudil on expressions of Rho/ROCK signaling pathway associated genes in rabbits with optic nerve injury (ONI), and to explore the therapeutic mechanisms towards ONI., Methods: The rabbit ONI model was established, then the rabbits were divided into model group (treated with saline), control group (treated with dexamethasone, Dex), and intervention group (treated with Fasudil, Fas). The eyeball and optic nerve were sampled at 3, 7, 14 and 21 days after injury. The morphological changes of retina and optic nerve were observed. The expressions of RhoA, Caspase-3, Rock 2 and Nogo-A gene were determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) methods., Results: At different time after injury, there were significant differences of RhoA, Caspase-3, Rock 2 and Nogo-A gene expression among three groups (P < 0.05)., Conclusions: After ONI, Fas can decrease the expression of Caspase-3 gene, and down-regulate the expressions of Nogo-A and Rock 2 gene. Therefore, it can treat ONI through affecting the Rho/ROCK signaling pathway.

Background: Axonal injury of the optic nerve (ON) is involved in various ocular diseases, such as glaucoma and traumatic optic neuropathy, which leads to apoptotic death of retinal ganglion cells (RGCs) and loss of vision. Caspases have been implicated in RGC pathogenesis. However, the role of caspase-7, a functionally unique caspase, in ON injury and RGC apoptosis has not been reported previously. The purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis., Results: C57BL/6 (wildtype, WT) and caspase-7 knockout (Casp7(-/-)) mice were used. We show that ON crush activated caspase-7 and calpain-1, an upstream activator of caspase-7, in mouse RGCs, as well as hydrolysis of kinectin and co-chaperone P23, specific substrates of caspase-7. ON crush caused a progressive loss of RGCs to 28 days after injury. Knockout of caspase-7 partially and significantly protected against the ON injury-induced RGC loss; RGC density at 28 days post ON crush in Casp7(-/-) mice was approximately twice of that in WT ON injured retinas. Consistent with changes in RGC counts, spectral-domain optical coherence tomography analysis revealed that ON crush significantly reduced the in vivo thickness of the ganglion cell complex layer (including ganglion cell layer, nerve fiber layer, and inner plexiform layer) in the retina. The ON crush-induced thinning of retinal layer was significantly ameliorated in Casp7(-/-) mice when compared to WT mice. Moreover, electroretinography analysis demonstrated a decline in the positive component of scotopic threshold response amplitude in ON crushed eyes of the WT mice, whereas this RGC functional response was significantly higher in Casp7(-/-) mice at 28 days post injury., Conclusion: Altogether, our findings indicate that caspase-7 plays a critical role in ON injury-induced RGC death, and inhibition of caspase-7 activity may be a novel therapeutic strategy for glaucoma and other neurodegenerative diseases of the retina.

Histones deacetylases (HDACs), besides their function as epigenetic regulators, deacetylate and critically regulate the activity of nonhistone targets. In particular, HDACs control partially the proapoptotic activity of p53 by balancing its acetylation state. HDAC inhibitors have revealed neuroprotective properties in different models, but the exact mechanisms of action remain poorly understood. We have generated a conditional knockout mouse model targeting retinal ganglion cells (RGCs) to investigate specifically the functional role of HDAC1 and HDAC2 in an acute model of optic nerve injury. Our results demonstrate that combined HDAC1 and HDAC2 ablation promotes survival of axotomized RGCs. Based on global gene expression analyses, we identified the p53-PUMA apoptosis-inducing axis to be strongly activated in axotomized mouse RGCs. Specific HDAC1/2 ablation inhibited this apoptotic pathway by impairing the crucial acetylation status of p53 and reducing PUMA expression, thereby contributing to the ensuing enhanced neuroprotection due to HDAC1/2 depletion. HDAC1/2 inhibition and the affected downstream signaling components emerge as specific targets for developing therapeutic strategies in neuroprotection., (© The Author(s) 2015.)

The dedicator of cytokinesis 3 (Dock3) is an atypical guanine nucleotide exchange factor that is predominantly expressed in the CNS. Dock3 exerts neuroprotective effects and stimulates optic nerve regeneration. The p38 mitogen-activated protein kinase acts downstream of apoptosis signal-regulating kinase 1 (ASK1) signaling and plays an important role in neural cell death. We assessed a therapeutic efficacy of Dock3 stimulation and p38 inhibition in retinal degeneration induced by optic nerve injury (ONI). In vivo retinal imaging using optical coherence tomography revealed that ONI-induced retinal degeneration was ameliorated in SB203580 (a p38 inhibitor)-treated WT mice and PBS-treated Dock3 overexpressing (Dock3 Tg) mice, and SB203580 further stimulated retinal protection in Dock3 Tg mice. In addition, SB203580 increased the number of regenerating axons after ONI in both WT and Dock3 Tg mice. ONI-induced phosphorylation of ASK1, p38 and the N-methyl-d-aspartate receptor 2B subunit were suppressed in the retina of Dock3 Tg mice. Inhibition of the ASK1 pathway in Dock3 Tg mice suggests that Dock3 may have an antioxidant-like property. These results indicate that overexpression of Dock3 and pharmacological interruption of p38 have synergistic effects for both neuroprotection and axon regeneration, thus combined application may be beneficial for the treatment of ONI., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Optic nerve damage initiates a series of early atrophic events in retinal ganglion cells (RGCs) that precede the BAX-dependent committed step of the intrinsic apoptotic program. Nuclear atrophy, including global histone deacetylation, heterochromatin formation, shrinkage and collapse of nuclear structure, and the silencing of normal gene expression, comprise an important obstacle to overcome in therapeutic approaches to preserve neuronal function. Several studies have implicated histone deacetylases (HDACs) in the early stages of neuronal cell death, including RGCs. Importantly, these neurons exhibit nuclear translocation of HDAC3 shortly after optic nerve damage. Additionally, HDAC3 activity has been reported to be selectively toxic to neurons., Results: RGC-specific conditional knockout of Hdac3 was achieved by transducing the RGCs of Hdac3fl/fl mice with an adeno-associated virus serotype 2 carrying CRE recombinase and GFP (AAV2-Cre/GFP). Controls included similar viral transduction of Rosa26fl/fl reporter mice. Optic nerve crush (ONC) was then performed on eyes. The ablation of Hdac3 in RGCs resulted in significant amelioration of characteristics of ONC-induced nuclear atrophy such as H4 deacetylation, heterochromatin formation, and the loss of nuclear structure. RGC death was also significantly reduced. Interestingly, loss of Hdac3 expression did not lead to protection against RGC-specific gene silencing after ONC, although this effect was achieved using the broad spectrum inhibitor, Trichostatin A., Conclusion: Although other HDACs may be responsible for gene expression changes in RGCs, our results indicate a critical role for HDAC3 in nuclear atrophy in RGC apoptosis following axonal injury. This study provides a framework for studying the roles of other prevalent retinal HDACs in neuronal death as a result of axonal injury.

Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.

Rho GTPases are molecular switches that modulate multiple intracellular signaling processes by means of various effector proteins. As a result, Rho GTPase activities are tightly spatiotemporally regulated in order to ensure homeostasis within the cell. Though the roles of Rho GTPases during neural development have been well documented, their participation during neurodegeneration has been far less characterized. Herein we discuss our current knowledge of the role and function of Rho GTPases and regulators during neurodegeneration, and highlight their potential as targets for therapeutic intervention in common neurodegenerative disorders., (© 2013 Elsevier Inc. All rights reserved.)

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1-3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is a potent hematopoietic cytokine. In the present study, we examined whether GM-CSF is neuroprotective in retinal ganglion cells (RGCs). First, we studied the expression of GM-CSF and the GM-CSF-alpha-receptor in rat and human retina and in RGC-5 cells. Then, RGC-5 cells were incubated with apoptosis-inducing agents (e.g., staurosporine, glutamate and NOR3). The cell death was assessed by Live-Death-Assays and apoptosis-related-proteins were examined by immunoblotting. In addition, the expression of phosphorylated ERK1/2-pathway-proteins after incubation with GM-CSF and after inhibiting MEK1/2 with U0126 was analyzed. To assess the in vivo-effect, first staurosporine or GM-CSF plus staurosporine was injected into the vitreous body of Sprague-Dawley rats. In a second axotomy model the optic nerve was cut and GM-CSF was injected into the vitreous body. In both models, the RGCs were labeled retrogradely with either Fluoro-Gold or 4-Di-10-Asp and counted. As a first result, we identified GM-CSF and the GM-CSF-alpha-receptor in rat and human retina as well as in RGC-5 cells. Then, in the RGC-5 cells GM-CSF counteracts induced cell death in a dose-and time-dependent manner. With respect to apoptosis, Western blot analysis revealed a decreased Bad-expression and an increased Bcl-2-expression after co-incubation with GM-CSF. Concerning signaling pathways, incubation with GM-CSF activates the ERK1/2 pathway, whereas inhibition of MEK1/2 with U0126 strongly decreased the phosphorylation downstream in the ERK1/2 pathway, and the antiapoptotic activity of GM-CSF in vitro. Like in vitro, GM-CSF counteracts the staurosporine-induced cell death in vivo and protects RGCs from axotomy-induced degeneration. Our data suggest that GM-CSF might be a novel therapeutic agent in neuropathic disease of the eye.

Adult spinal axons do not spontaneously regenerate after injury. However, if the peripheral branch of dorsal root ganglion neurons is lesioned before lesioning the central branch of the same neurons in the dorsal column, these central axons will regenerate and, if cultured, are not inhibited from extending neurites by myelin-associated inhibitors of regeneration such as myelin-associated glycoprotein (MAG). This effect can be mimicked by elevating cAMP and is transcription dependent. The ability of cAMP to overcome inhibition by MAG in culture involves the upregulation of the enzyme arginase I (Arg I) and subsequent increase in synthesis of polyamines such as putrescine. Now we show that a peripheral lesion also induces an increase in Arg I expression and synthesis of polyamines. We also show that the conditioning lesion effect in overcoming inhibition by MAG is initially dependent on ongoing polyamine synthesis but, with time after lesion, becomes independent of ongoing synthesis. However, if synthesis of polyamines is blocked in vivo the early phase of good growth after a conditioning lesion is completely blocked and the later phase of growth, when ongoing polyamine synthesis is not required during culture, is attenuated. We also show that putrescine must be converted to spermidine both in culture and in vivo to overcome inhibition by MAG and that spermidine can promote optic nerve regeneration in vivo. These results suggest that spermidine could be a useful tool in promoting CNS axon regeneration after injury.

A ROCK inhibitor Fasudil is widely administered to relieve vasospasm in patients after subarachnoid hemorrhage in Japan. We investigated the difference of Fasudil and Y-27632, a common ROCK inhibitor, on neurite regeneration in culture and axonal regeneration after injuring the optic nerve (OpN) in cats. The optimal dose of Y-27632, determined by counting the number and length of neurites in retinal explants, was found to be 100 microM: the only effect of Fasudil was to promote extension of glial processes. We next examined the effects of Fasudil (10 microM-100 microM) and Y-27632 (10 microM-300 microM) on axonal regeneration in the crushed OpN model in vivo. Immediately after crushing the left OpN, Fasudil or Y-27632 was injected into the vitreous and the crushed site. Injection of 10 microM and 100 microM Y-27632 induced extension of the optic axons beyond the crush site, with the latter dosage giving stronger regeneration. Very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection, and no axons elongated in the OpN with Fasudil injection.

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.

We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.

Fish CNS neurons can repair their axons following nerve injury, whereas mammalian CNS neurons cannot regenerate, and become apoptotic within 1-2 weeks after the nerve lesion. One explanation for these differences is that one, or several molecules are upregulated in fish CNS neurons during nerve regeneration, and this same molecule is downregulated in mammalian CNS neurons before the development of apoptosis caused by nerve injury. A molecule satisfying these criteria might successfully rescue and repair the mammalian CNS neurons. In this study, we looked for such a candidate molecule from goldfish retinas. Transglutaminase derived from goldfish retina (TG(R)) was characterized as a regenerating molecule after optic nerve injury. A full-length cDNA for TG(R) was isolated from the goldfish retinal cDNA library prepared from axotomized retinas. Levels of TG(R) mRNA and protein increased only in the retinal ganglion cells (RGCs) between 10 and 40 days after optic nerve transection. Recombinant TG(R) protein enhanced neurite outgrowth from adult fish RGCs in culture. Specific interference RNA and antibodies for TG(R) inhibited neurite outgrowth both in vitro and in vivo. In contrast, the level of TG(R) protein decreased in rat RGCs within 1-3 days after nerve injury. Furthermore, the addition of recombinant TG(R) to retinal cultures induced striking neurite outgrowth from adult rat RGCs. These molecular and cellular data strongly suggest that TG(R) promotes axonal elongation at the surface of injured RGCs after optic nerve injury.

We have shown that application of basic fibroblast growth factor (FGF-2) to axotomized optic nerve promotes the survival of frog retinal ganglion cells (RGCs). In the present study we used western blotting and immunocytochemistry to investigate the effects of this FGF-2 treatment upon the activation of the extracellular signal-regulated kinase (ERK) pathway, the amounts and distribution of Bcl-2 family proteins, and the activation of caspase-3. Axotomy alone temporarily increased ERK activation; FGF-2 treatment to the nerve prolonged this activation. This effect was blocked by U0126, a selective ERK kinase (MEK) inhibitor. Axotomy caused a decrease in Bcl-2 and a small increase in Bcl-x(L). FGF-2 treatment caused an ERK-dependent increase in Bcl-2 and an ERK-independent increase in Bcl-x(L). The pro-apoptotic Bax was increased by axotomy; FGF-2 treatment greatly decreased Bax levels, an effect that was inhibited by U0126. Axotomy induced the cleavage of caspase-3; FGF-2 treatment blocked this effect in an ERK-dependent manner. Finally, intraocular application of the MEK inhibitor caused a large reduction in the survival-promoting effect that FGF-2 application to the nerve stump had on RGCs. Our results suggest that FGF-2 acts, at least in part, via the ERK pathway to prevent apoptosis of axotomized RGCs not only by increasing amounts of anti-apoptotic proteins, but also by a striking reduction in the levels of apoptotic effectors themselves.

Retinal ganglion cells die by apoptosis after optic nerve crush. FK506 has been shown to be neuroprotective in this model but the mechanism(s) by which it exerts these actions remains unknown. We and others have shown that caspase 9 is cleaved in the retina in other injury models and we hypothesized that the neuroprotection observed with FK506 was mediated by interference with caspase 9 activation. The present study examined the cellular localization of caspase 9 cleavage after intraorbital optic nerve crush in rats, the time course of caspase 9 cleavage after optic nerve crush and the ability of orally administered FK506 to block caspase 9 cleavage after optic nerve crush. We show by immunohistochemistry that cleaved caspase 9 is present in retinal ganglion cells (identified by prior backlabelling) after optic nerve crush. Immunoblot analysis showed that caspase 9 cleavage is significantly elevated 5 and 8 days after optic nerve crush. We show that orally administered FK506 reaches the retina and is pharmacologically active in retinal tissue. Furthermore, the oral administration of FK506 5 mg kg(-1) day(-1) blocks the cleavage of caspase 9 at both time points. These data suggest that caspase 9 activation may play an important role in retinal ganglion cell death following optic nerve crush and that the neuroprotection seen with FK506 may be mediated by interfering with the activation of caspase 9.

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.

Purpose: To determine whether inducible nitric oxide synthase (NOS-2) is involved in glaucomatous optic neuropathy., Methods: Chronic elevation of rat intraocular pressure (IOP) leading to optic nerve damage was induced by episcleral injection of hypertonic saline, which caused sclerosis and blockade of aqueous humor outflow pathways. Expression of NOS-2 in the retina and optic nerve head (ONH) was evaluated by immunohistochemistry, gene array analysis, and quantitative PCR (Q-PCR). Immunohistochemistry was also used to assess the NOS-2 level in the ONH from primary open-angle glaucoma (POAG) and nonglaucomatous human eyes. Finally, an NOS-2 inhibitor, aminoguanidine, administered orally in the drinking water, was tested for its effect on optic nerve injury in rats with ocular hypertension., Results: Chronically elevated IOP in the rat produced optic nerve damage that correlated with pressure change (r(2) = 0.77), but did not increase NOS-2 immunoreactivity in the optic nerve, ONH, or ganglion cell layer. Retinal and ONH NOS-2 mRNA levels did not correlate with either IOP level or severity of optic nerve injury. Similarly, there was no difference in NOS-2 immunoreactivity in the optic nerve or ONH between POAG and nonglaucomatous eyes. Furthermore, aminoguanidine treatment did not affect the development of pressure-induced optic neuropathy in the rat., Conclusions: As demonstrated by several independent methods, glaucomatous optic neuropathy was not associated with a significant change in the expression of NOS-2 in the retina, ONH, or optic nerve.

Recently, receptor protein tyrosine phosphatase-sigma (RPTPsigma) has been shown to inhibit axon regeneration in injured peripheral nerves. Unlike the peripheral nervous system (PNS), central nervous system (CNS) neurons fail to regenerate their axons after injury or in disease. In order to assess the role of RPTPsigma in CNS regeneration, we used the retinocollicular system of adult mice lacking RPTPsigma to evaluate retinal ganglion cell (RGC) axon regrowth after optic nerve lesion. Quantitative analysis demonstrated a significant increase in the number of RGC axons that crossed the glial scar and extended distally in optic nerves from RPTPsigma (-/-) mice compared to wild-type littermate controls. Although we found that RPTPsigma is expressed by adult RGCs in wild-type mice, the retinas and optic nerves of adult RPTPsigma (-/-) mice showed no histological defects. Furthermore, the time-course of RGC death after nerve lesion was not different between knockout and wild-type animals. Thus, enhanced axon regrowth in the absence of RPTPsigma could not be attributed to developmental defects or increased neuronal survival. Finally, we show constitutively elevated activity of mitogen-activated protein kinase (MAPK) and Akt kinase in adult RPTPsigma (-/-) mice retinas, suggesting that these signaling pathways may contribute to promoting RGC axon regrowth following traumatic nerve injury. Our results support a model in which RPTPsigma inhibits axon regeneration in the adult injured CNS.

After injury to the central nervous system, a glial/collagen scar forms at the lesion site, which is thought to act as a physicochemical barrier to regenerating axons. We have shown that scar formation in the transected optic nerve (ON) is attenuated when robust growth of axons is stimulated. Matrix metalloproteases (MMP), modulated by tissue inhibitors of MMP (TIMP), degrade a wide variety of extracellular matrix components (ECM) and may be activated by growing axons to remodel the ECM to allow regeneration through the inhibitory environment of the glial or collagen scar. Here, we investigate whether MMP levels are modulated in a nonregenerating (scarring) versus a regenerating (nonscarring) model of ON injury in vivo. Western blotting and immunohistochemistry revealed that MMP-1, -2, and -9 levels were higher and TIMP-1 and TIMP-2 levels were lower in regenerating compared to nonregenerating ON and retinae. In situ zymography demonstrated significantly greater MMP-related gelatinase activity in the regenerating model, mainly colocalized to astrocytes in the proximal ON stump and around the lesion site. These results suggest that activation of MMP and coincident down-regulation of TIMP may act to attenuate the inhibitory scarring in the regenerating ON, thus transforming the ON into a noninhibitory pathway for axon regrowth.

We have evaluated the spatial and temporal expression patterns of three protein tyrosine phosphatases (PTPs), receptor PTPalpha (RPTPalpha), striatal enriched phosphatase (STEP), and leucocyte common antigen-related phosphatase (LAR), in the retina and optic nerve (ON) of adult rats in which the crushed ON was either regenerating after retinal ganglion cell (RGC) stimulation with intravitreal peripheral nerve (PN) grafting or lens injury (LI), or not regenerating (no treatment). In intact adult rats, all three PTPs were expressed by RGCs and ON glia. In both the regenerating and non-regenerating models, a postlesion rise in RPTPalpha, STEP, and LAR expression occurred in the RGC somata and in the ON. However, for RPTPalpha and LAR in the RGCs, and for RPTPalpha, STEP, and LAR in the ON, this postlesion increase was attenuated in the regenerating versus the non-regenerating models. ON PTP expression changes were localized to glia in the proximal and distal stumps, and to macrophages and extracellular matrix of the glial scar at the lesion site. Interestingly, neither RPTPalpha, STEP, nor LAR localized to intact or regenerating axons. One explanation of these findings is that RPTPalpha and LAR may modulate RGC survival, and that RPTPalpha, STEP, and LAR may modulate axon growth.

We have shown previously that intraocular elevation of cAMP using the cAMP analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) failed to promote axonal regeneration of axotomized adult retinal ganglion cells (RGCs) into peripheral nerve (PN) grafts but significantly potentiated ciliary neurotrophic factor (CNTF)-induced axonal regeneration. Using the PN graft model, we now examine the mechanisms underlying spontaneous and CNTF/CPT-cAMP-induced neuronal survival and axonal regrowth. We found that blockade of the cAMP pathway executor protein kinase A (PKA) using the cell-permeable inhibitor KT5720 did not affect spontaneous survival and axonal regeneration but essentially abolished the CNTF/CPT-cAMP-induced RGC survival and axonal regeneration. Blockade of CNTF signaling pathways such as phosphotidylinositol 3-kinase (PI3K)/akt by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) by 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059), or Janus kinase (JAK)/signal transducer and activators of transcription (STAT3) by tyrphostin AG490 also blocked the CNTF/CPT-cAMP-dependent survival and regeneration effects. PKA activity assay and Western blots showed that KT5720, LY294002, and PD98059 almost completely inhibited PKA, PI3K/akt, and MAPK/ERK signal transduction, respectively, whereas AG490 substantially decreased JAK/STAT3 signal transduction. Intraocular injection of CPT-cAMP resulted in a small PKA-dependent increase in CNTF receptor alpha mRNA expression in the retinas, an effect that may facilitate CNTF action on survival and axonal regeneration. Surprisingly, in the absence of CNTF/CPT-cAMP, LY294002, PD98059, and AG490, but not KT5720, significantly enhanced spontaneous RGC survival, suggesting differential roles of these pathways in RGC survival under different conditions. Our data suggest that CNTF/CPT-cAMP-induced RGC survival and axonal regeneration are a result of multiple pathway actions, with PKA as an essential component, but that these pathways can function in an antagonistic manner under different conditions.

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.

Phospholipase A(2) (PLA(2)) hydrolyzes phosphatidylcholine to lysophosphatidylcholine and arachidonic acid. The former can induce myelin breakdown and the latter, via eicosanoids, can stimulate inflammatory responses. Immunohistochemical analysis of secreted (sPLA(2)) and cytosolic (cPLA(2)) forms of the enzyme was assessed in the injured adult rat sciatic and optic nerves. sPLA(2) and cPLA(2) are expressed in the first 2 weeks in the injured sciatic nerve, which correlates with rapid Wallerian degeneration in peripheral nerves. In contrast, both forms of PLA(2) were not expressed in the optic nerve for the first 3 weeks after crush injury, which correlates with slow Wallerian degeneration in the central nervous system (CNS). In addition, PLA(2) is not expressed in the lesioned sciatic nerve of C57BL/Wld(s) mutant mice in which Wallerian degeneration is severely retarded. Blocking cPLA(2) in the transected sciatic nerve of C57BL/6 mice, which have a naturally occurring null mutation for the major from of sPLA(2), resulted in a marked slowing of myelin and axonal degradation and phagocytosis in the distal nerve segment. These results provide direct evidence of an important role for cPLA(2) in Wallerian degeneration.

This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity.

We have revealed previously that the survival rate of beta cells of cat retinal ganglion cells (RGCs) rapidly decreased to 29% on day 7 after optic nerve transection, whereas that of alpha cells slowly decreased to 64% on day 14 (Watanabe et al., 2001). The reason that beta cells die more rapidly than alpha cells was not clear. In the present study, we tested the possibility that the rapid death of beta cells is attributable to apoptosis, as shown for some axotomized RGCs in rats. The following results were obtained. First, the proportion of pyknotic cells in Nissl-stained cat retinas started to increase sharply starting on day 4 and reached a peak on day 6 after optic nerve transection. The time course of occurrence of pyknotic cells corresponded well with that of the rapid death of axotomized beta cells. Secondly, the proportion of pyknotic cells was the highest in the area centralis (AC), in which beta cells are densely distributed. The preferential death of axotomized RGCs in the AC was also confirmed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining in cross sections. Thirdly, after the intravitreal injection of caspase 3 inhibitor (z-DEVD-cmk) the survival of axotomized beta cells on day 7 was significantly enhanced, whereas no such survival-promoting effect was obtained in axotomized alpha cells. Taken together, these results suggest that the rapid death of axotomized beta cells is attributable mainly to apoptosis, which is mediated by caspase 3.

Extracellular matrix (ECM) remodeling after neuronal injury and reactive gliosis is carried out by activation of matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs). In glaucoma, there is a loss of retinal ganglion cells and extensive ECM remodeling (cupping) at the level of the optic nerve head, frequently associated with elevated intraocular pressure. To determine whether ECM remodeling in the glaucomatous optic nerve head occurs in response to loss of axons or to elevated intraocular pressure we compared the patterns of MMP and TIMP expression in the eyes of monkeys with laser-induced glaucoma or with optic nerve transection. MT1-MMP and MMP1 expression was markedly increased in reactive astrocytes in optic nerve heads with experimental glaucoma but not in the optic nerve head of transected eyes. In normal control eyes retinal ganglion cells expressed MMP2, TIMP1 and TIMP2 constitutively, and the proteins were detected in their axons. At the site of transection, MT1-MMP, MMP1, MMP2, TIMP1 and TIMP2 were expressed by reactive astrocytes. Inflammatory cells, fibroblasts and reactive astrocytes at the transected site expressed MMP3 and MMP9, which were undetectable in the retina and optic nerve head in any condition. Constitutive expression of MMP2, TIMP1 and TIMP2 in retinal ganglion cells suggests a role in maintenance of synaptic integrity and plasticity and maintenance of the periaxonal space. Increased MMP1 and MT1-MMP1 expression in the glaucomatous optic nerve head is specific to tissue remodeling due to elevated intraocular pressure and not secondary to loss of axons.

Purpose: To investigate changes in percentage of tyrosine kinase (trk)A-, trkB-, and trkC-immunopositive ((+)) retinal ganglion cells (RGCs) at various times after optic nerve (ON) axotomy; the proportion of RGCs regenerating axons into peripheral nerve (PN) grafts that are trkA(+), trkB(+), and trkC(+); whether intravitreal PN-ON implants affect trk immunoreactivity; and the levels of trk mRNAs in ON-injured retinas., Methods: The ON was transected intraorbitally. Proportions of trkA(+), trkB(+), and trkC(+) RGCs and levels of trk mRNAs were studied by using immunocytochemistry and Northern blot methods, respectively, in injured and RGC-regenerating retinas., Results: In normal retinas, only small numbers of trkB(+) and trkC(+), but not trkA(+), RGCs were seen. The optic fiber layer was intensively immunolabeled with trkB. After ON injury, the proportions of trkA(+), trkB(+), and trkC(+) RGCs rapidly increased and reached their peaks by 3 to 5 days. During the next 3 weeks, the proportion of trkA(+) or trkB(+) RGCs gradually decreased, but the proportion of trkC(+) RGCs remained high. Intravitreal implants of PN+ON segments transiently but significantly suppressed injury-induced increases in all these trk(+) RGC proportions for approximately 5 days. In contrast, 3 days after ON injury, quantitative retinal expression of trkA mRNA, and to a lesser extent trkC mRNA, was downregulated, whereas trkB mRNA expression remained unaffected. Higher proportions of trkA(+) and trkB(+) RGCs and higher levels of all trk mRNAs were seen in regenerating RGCs and retinas, respectively., Conclusions: This study provides a kinetic analysis of expression of trk in RGCs and retinas after ON injury and during regeneration.

The purpose of this study is to investigate the cytochrome oxidase (COX) activity in the retina and optic nerve following an optic nerve injury. The optic nerve crush of one eye was carried out in Balb/c mice. A semi-quantitative RT-PCR method was then adopted to evaluate the mRNA expression of cytochrome oxidase subunit 1 (COX1) in the retina after surgery. Up-regulation of COX1 mRNA in the retina was detected by RT-PCR at 24 hr following the optic nerve injury. Total retinal mitochondrial mass measured by fluorescent intensity of MitoTracker green was not altered following the injury. COX histochemistry performed on cryostat sections showed an elevated enzyme activity of COX in the retina and in the optic nerve. In the retina, elevation of the COX activity was observed in the retinal ganglion cell layer and the overlying nerve fibre layer. The increase of COX activity began from 24 hr after injury, peaked around day 3, and maintained up to 1 week after the operation. In the optic nerve, increase of COX activity was observed in regions distal to the crush line and distributed either randomly or in a cone shape. In conclusion, both the expression of COX1 mRNA in retina and the activity of COX in inner plexiform layer and retinal ganglion cell layer were elevated following optic nerve injury without affecting total retinal mitochondrial mass. These findings suggested that one of early responses in the retina and in the optic nerve after the optic nerve injury is to scale up the energy production., (Copyright 2002 Elsevier Science Ltd.)

The neuronal form of nitric oxide synthase (NOS-1) has been localized to several cell types in the retinas of experimental animals; however, localization in the human retina has not been definitive. By using in situ hybridization and immunohistochemistry, we have compared the cellular expression and localization of NOS-1 in the rat and human retinas. In both rat and human retinas, NOS-1 is expressed in the inner segments of photoreceptors, cells in the inner nuclear layer, particularly amacrine cells, and retinal ganglion cells. In human cones, NOS-1 is abundantly present in the outer segments. In the rat, optic nerve transection caused a loss of cells that were positive for NOS-1 in the ganglion cell layer. Although a retinal ganglion cell localization has not been reported consistently in the literature, our data clearly localize NOS-1 to the retinal ganglion cells of the rat and human retinas., (Copyright 2000 Wiley-Liss, Inc.)