A neuroprotective herbal mixture inhibits caspase-3-independent apoptosis in retinal ganglion cells.

It was previously demonstrated that Menta-FX, a mixture of Panax quinquefolius L. (PQE), Ginkgo biloba (GBE), and Hypericum perforatum extracts (HPE), enhances retinal ganglion cell survival after axotomy. However, the mechanisms of neuroprotection remain unknown. The aim of this study is to elucidate the neuroprotective mechanisms of Menta-FX. Since PQE, GBE and HPE have all been observed to display anti-oxidative property, the involvement of anti-oxidation in Menta-FX's neuroprotective effect was investigated. Menta-FX lowered nitric oxide (NO) content in axotomized retinas without affecting nitric oxide synthase activity, suggesting that Menta-FX possibly exhibited a NO scavenging property. In addition, the effect of Menta-FX on the frequency of axotomy-induced nuclear fragmentation and caspase-3 activation was investigated. Menta-FX treatment significantly reduced nuclear fragmentation in axotomized retinas. Surprisingly, Menta-FX had no effect on caspase-3 activation, but selectively lowered caspase-3-independent nuclear fragmentation in axotomized retinal ganglion cells. In addition, inhibition of PI3K activity by intravitreal injection of wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor, completely abolished the neuroprotective effect of Menta-FX, indicating that Menta-FX's neuroprotective effect was PI3K-dependent. Data here suggest that Menta-FX displayed a PI3K-dependent, selective inhibition on a caspase-3-independent apoptotic pathway in axotomized RGCs, thus, highlighting the potential use of herbal remedies as neuroprotective agents for other neurodegenerative diseases.

Expression of telomerase reverse transcriptase in adult goldfish retina.

Telomerase, a specialized reverse transcriptase that maintains telomere during cell division, is commonly associated with cell proliferation. Increasing evidence suggests that telomerase may bear functions other than telomere elongation. We investigated whether telomerase is expressed in the continuously growing goldfish retina. Telomeric repeat amplification protocol (TRAP) assay reveals telomerase activity in goldfish retina. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot show that telomerase catalytic subunit (TERT) is expressed at both mRNA and protein levels. Localization of TERT by immunohistochemistry indicates prominent expression of TERT in the outer nuclear layer, the inner nuclear layer, and, in a small population of cells, in the ganglion cell layer. Coexpression of TERT with proliferative cell nuclear antigen (PCNA) immunoreactivity is found in rod progenitor cells. These results suggest the role of telomerase in vertebrate central nervous system (CNS) other than telomere maintenance, such as regulation of cell cycle progression and maintenance of retinal cell phenotypes.

Endothelial nitric oxide synthase is expressed in amacrine cells of developing human retinas.

PURPOSE: To examine the expression and cellular distribution pattern of endothelial nitric oxide synthase (eNOS) in the developing human retina and to compare its expression with that in rats. METHODS: Expression of eNOS was examined by immunohistochemistry in retinas of humans ranging from 8.5 to 28 weeks of gestation (WG) and of rats. RESULTS: In the developing human retina, eNOS expression was first detected in the proximal margin of the neuroblastic layer in the incipient fovea-surrounding area at 12 WG. At 17 to 28 WG, eNOS-immunoreactive cells were located in the innermost part of the inner nuclear layer and in the ganglion cell layer, expanding to both temporal and nasal retinas and the processes projecting into the inner plexiform layer. These eNOS-positive cells coexpressed syntaxin and glutamate decarboxylase, and are probably GABAergic amacrine cells. The onset of eNOS expression in developing amacrine cells, however, preceded the invasion of retinal vasculature, long before vascular function involving these cells can be expected, suggesting that eNOS has a role not only in vasoregulation but also in retinal development. From 20 WG on, eNOS was also detected in the photoreceptors adjacent to the fovea. eNOS expression in amacrine cells and photoreceptors was observed in the central-to-peripheral and temporal-to-nasal gradients. However, in the developing rat retina, eNOS was expressed exclusively in the vascular endothelial cells. CONCLUSIONS: The results support that eNOS plays a role, not only in the regulation of vascular function but also in the process of retinal development in humans.

CNTF and BDNF have similar effects on retinal ganglion cell survival but differential effects on nitric oxide synthase expression soon after optic nerve injury.

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.

Different optic nerve injury sites result in different responses of retinal ganglion cells to brain-derived neurotrophic factor but not neurotrophin-4/5.

In this study, we investigated whether brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) can achieve prolonged protection on retinal ganglion cells (RGCs) and whether site of axon injury modulates RGC response to neurotrophins. Two optic nerve (ON) injury paradigms, proximal and distal transections, were used. Autologous sciatic nerves were grafted onto ON stump in some animals to provide a suitable environment for axons to regrow. Multiple intravitreal injections of saline, BDNF, or NT-4/5 were performed. Immunohistochemistry was used to determine the proportion of RGCs that were expressing trkB. Twenty days after proximal injury, both BDNF and NT-4/5 promoted RGC survival; this protection diminished 30 days after injury. One month after distal injury, BDNF, but not NT-4/5, promoted RGC survival (by 2-fold). No difference in the proportion of trkB expressing RGCs among the viable ones was seen between the two injury models or after BDNF treatment. Interestingly, the mean size of RGC somata was larger after proximal injury than distal injury. This study demonstrates that (1) RGCs respond differently to neurotrophins under different injury conditions, (2) BDNF but not NT-4/5 significantly enhances survival of distally but not proximally injured RGCs over a prolonged period.

c-Jun expression in surviving and regenerating retinal ganglion cells: effects of intravitreal neurotrophic supply.

PURPOSE: To investigate c-jun expression in surviving and axon-regenerating retinal ganglion cells (RGCs) and the effect of intravitreal neurotrophic supply on c-jun expression. METHODS: All animals underwent optic nerve transection (ONT) 0.5 mm behind the eyeball. Some animals underwent a replacement of the optic nerve with an autologous sciatic nerve graft (SNG) to allow axonal regrowth. To provide a neurotrophic supply, a peripheral nerve (PN) segment or brain-derived neurotrophic factor (BDNF)/ciliary neurotrophic factor (CNTF) was applied intravitreally. The time course of c-jun expression was first examined in both surviving and regenerating RGCs. Then, c-jun expression was examined in surviving and regenerating RGCs 3 weeks after intravitreal BDNF/CNTF treatment. Animals with vehicle eye injection were used as the control. Fluorescent dye was used for retrograde labeling of surviving (applied behind the eyeball) and regenerating (applied at the distal end of the SNG) RGCs. All retinas were immunohistochemically stained for c-jun. RESULTS: c-Jun was not detected in normal RGCs, but weak expression was seen in surviving RGCs after ON injury. The proportion of c-jun-positive (+) RGCs among surviving cell population was 52.6% to 86.5% 2 to 6 weeks after ONT. Among regenerating RGCs, more than 80% expressed c-jun in all treatment groups, a proportion that was significantly higher after CNTF treatment (90.7%). In addition, c-jun expression was much stronger in intensity and the c-jun(+) nuclei were much larger in regenerating than in surviving RGCs. CONCLUSIONS: c-Jun expression in RGCs was upregulated after injury. Most regenerating RGCs were c-jun(+), and the intensity of c-jun expression was higher in regenerating than in surviving RGCs. CNTF also upregulated c-jun expression in RGCs.

Expression of trkA, trkB, and trkC in injured and regenerating retinal ganglion cells of adult rats.

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.

Synergistic effect of Nogo-neutralizing antibody IN-1 and ciliary neurotrophic factor on axonal regeneration in adult rodent visual systems.

The presence of Nogo axon regeneration inhibitory molecules in the central nervous system (CNS) and the counteracting effect of IN-1 antibodies have been widely reported. In this study, we examined the effect of IN-1-producing hybridoma cells on axon regeneration in adult rodent retinal ganglion cells (RGCs) after various types of optic nerve (ON) injury, evaluating therein whether ciliary neurotrophic factor (CNTF) potentiated the effect of IN-1. We found that application of IN-1 alone failed to enhance regeneration of intracranially or intraorbitally transected RGC axons in a peripheral nerve (PN) graft. IN-1 hybridoma cells also failed to significantly promote intraorbitally crushed ON axons to reenter the distal part of the ON. However, a combined application of IN-1 and CNTF had a synergistic effect in both intracranial PN and intraorbital ON crush paradigms. This study suggests that the action of IN-1 antibodies in promoting axon regeneration in the CNS could be more effective when coupled with other appropriate factors.

Enhanced survival and regeneration of axotomized retinal ganglion cells by a mixture of herbal extracts.

The aim of this study is to investigate the effects of Panax quinquefolius L. extract (PQE), Ginkgo biloba extract (GBE), and Hypericum perforatum extract (HPE), in combination or alone, on the survival and regeneration of axotomized retinal ganglion cells (RGCs) in an optic nerve transection model in adult hamsters. Unilateral transection of the optic nerve was performed to evaluate the effects of herbal extracts on the survival of axotomized RGCs. Effects of the herbal extracts on axonal regeneration of axotomized RGCs, on the other hand, were studied by attaching a peripheral nerve graft onto the transected ocular stump to induce regeneration. Operated animals received daily oral administration of vehicle or herbal extracts (PQE, GBE, and HPE), alone or in combination, for 7 and 21 days, respectively, in the survival and regeneration experiments. Surviving and regenerating RGCs were retrogradely labeled with Fluoro-Gold. The eyes were then enucleated and the retinas were flat-mounted for the counting of the labeled RGCs. Treatment with PQE, GBE and HPE alone failed to offer neuroprotection to injured RGCs. However, treatment with Menta-FX, a mixture of PQE, GBE, and HPE, significantly augmented RGC survival 7 days postaxotomy. Treatment with Menta-FX also induced a significant (87%) increase in the number of regenerating RGCs 21 days after optic nerve transection. This study demonstrates that herbs can act as a potential neuroprotective agent for damaged RGCs. It also suggests that the therapeutic value of herbal remedies can be maximized by the use of mixtures of appropriate herbs.

Development of the regenerative capacity of postnatal axotomized rat spinal motoneurons.

The present study examined whether a peripheral nerve (PN) graft can rescue developing motoneurons from degeneration and determined when immature motoneurons begin to express a regenerative capacity. Transplantation of a PN graft was unable to rescue motoneurons from degeneration if spinal root avulsion was performed in animals younger than P14. However, this procedure did enhance motoneuron survival when root avulsion was performed at P14 or later. Immature (P1 or P7) motoneurons were unable to regenerate their axons into the transplanted PN graft following root avulsion, whereas in older animals (P14-P28) motoneurons were able to regenerate axons into the PN graft. The percentage of regenerated motoneurons increased from P21 to P28 and was similar to that of adult animals. Therefore, the regenerative capacity of rat spinal motoneurons first begins at about P14, which seems to be critical.

Axotomy induces cytochrome c release in retinal ganglion cells.

Activation of caspase-3 and -9 has been implicated in the death of axotomized retinal ganglion cells (RGCs). The upstream pathways involved in the activation of these caspases, however, remain unknown. The aim of the current study is to examine the role of cytochrome c release in axotomized RGC death using immuno-histochemistry. We found that while only a low level of cytochrome c immunoreactivity was evident in normal retina, cytochrome c immunoreactivity increased markedly at 1 day post-axotomy, peaked at 3 days post-axotomy, and decreased thereafter. In addition, cytochrome c immunoreactivity localized almost exclusively to RGCs, suggesting that the cytochrome c release observed was injury-related. Our data indicate that cytochrome c release potentially contributes to the death of axotomized RGCs.

Retinal stem cells and regeneration of vision system.

The vertebrate retina is a well-characterized model for studying neurogenesis. Retinal neurons and glia are generated in a conserved order from a pool of mutlipotent progenitor cells. During retinal development, retinal stem/progenitor cells (RPC) change their competency over time under the influence of intrinsic (such as transcriptional factors) and extrinsic factors (such as growth factors). In this review, we summarize the roles of these factors, together with the understanding of the signaling pathways that regulate eye development. The information about the interactions between intrinsic and extrinsic factors for retinal cell fate specification is useful to regenerate specific retinal neurons from RPCs. Recent studies have identified RPCs in the retina, which may have important implications in health and disease. Despite the recent advances in stem cell biology, our understanding of many aspects of RPCs in the eye remains limited. PRCs are present in the developing eye of all vertebrates and remain active in lower vertebrates throughout life. In mammals, however, PRCs are quiescent and exhibit very little activity and thus have low capacity for retinal regeneration. A number of different cellular sources of RPCs have been identified in the vertebrate retina. These include PRCs at the retinal margin, pigmented cells in the ciliary body, iris, and retinal pigment epithelium, and Müller cells within the retina. Because PRCs can be isolated and expanded from immature and mature eyes, it is possible now to study these cells in culture and after transplantation in the degenerated retinal tissue. We also examine current knowledge of intrinsic RPCs, and human embryonic stems and induced pluripotent stem cells as potential sources for cell transplant therapy to regenerate the diseased retina.

Neuroprotective signaling mechanisms of telomerase are regulated by brain-derived neurotrophic factor in rat spinal cord motor neurons.

Telomerase can promote neuron survival and can be regulated by growth factors such as brain-derived neurotrophic factor (BDNF). Increases of BDNF expression and telomerase activity after brain injury suggest that telomerase may be involved in BDNF-mediated neuroprotection. We investigated BDNF regulation of telomerase in rat spinal cord motor neurons (SMNs). Our results indicate that BDNF increases telomerase expression and activity levels in SMNs and activates mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2 and phosphatidylinositol-3-OH kinase/protein kinase B signals, and their downstream transcription factors nuclear factor-κB, c-Myc, and Sp1. Administration of the tyrosine kinase receptor B inhibitor K-252a, the mitogen-activated protein kinase 1 inhibitor PD98059, and the phosphatidylinositol-3-OH kinase inhibitor LY294002 abolished BDNF-induced upregulation of these transcription factors and telomerase expression. The nuclear factor-κB inhibitor Bay11-7082 also attenuated c-Myc and Sp1 expression and increased telomerase promoter activity. Spinal cord motor neurons with higher telomerase levels induced by BDNF became more resistant to apoptosis; survival of SMNs that overexpressed the catalytic protein component of telomerase with reverse transcriptase activity was also enhanced against apoptosis. The neuronal survival-promoting effect of telomerase was mediated through the regulation of Bcl-2, Bax, p53, and maintenance of mitochondrial membrane potential. Taken together, these data suggest that the neuroprotective effect of BDNF via telomerase is mediated by inhibition of apoptotic pathways.

Regulation of retinal progenitor cell differentiation by bone morphogenetic protein 4 is mediated by the smad/id cascade.

PURPOSE. Bone morphogenetic proteins (BMPs) are secreted signaling molecules that are implicated in the control of multiple events during mouse eye development. However, little is known about the mechanisms by which BMP signaling regulates these retinal developmental processes. METHODS. Real-time PCR, Western blot, and immunohistochemistry were used to investigate the expression of components of BMP signaling in the mouse retina. Retinal progenitor cells (RPCs) were used to study the effects of BMP4 on retinal cell differentiation and regulation of Id protein expression. RESULTS. Results showed that BMP2, -4, and -7; BMP receptor (BMPRIb) mRNAs; and proteins and downstream signaling molecule Smad1/5/8 proteins were all highly expressed in the mouse retina during the embryonic (E13.5-E18.5) and early postnatal (P)1 stage and that the expression was downregulated in the adult. On stimulation with BMP4, cultured mouse RPCs differentiated into neuronal lineage whereas astrocyte cell differentiation was inhibited. BMP4 mainly stimulated production of retinal ganglion cells (RGCs). Results also revealed that BMPs and BMPRIb were co-localized with inhibitors of differentiation (Id) (mainly Id1 and -3) in RGCs in the adult mouse retina. Exposure of RPCs to BMP4 upregulated Id1-3 expression levels, mediated through the phosphorylation of Smad1/5/8 proteins. CONCLUSIONS. These results suggest that Id genes are one of the potential targets of BMP signaling in the differentiation of RPCs.

Bone morphogenetic proteins mediate cellular response and, together with Noggin, regulate astrocyte differentiation after spinal cord injury.

Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of pSmad1/5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced pSmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury.

Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts.

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitally at 2 mm (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.

Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons.

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.

Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats.

We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases.

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.

CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway.

We examined the neuroprotective effect of ciliary neurotrophic factor (CNTF) on retinal ganglion cells (RGCs) in a rat glaucoma model with increased intraocular pressure (IOP) and studied the CNTF-mediated activation of Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. Elevated IOP was induced by laser photocoagulation of the episcleral and limbal veins. The survival of RGCs was studied using Fluoro-Gold labelled in ocular hypertensive eyes with or without CNTF intravitreal injection. Immunochemical staining and immunoblot analysis for CNTF and phosphorylated STAT3 (pSTAT3) were performed. There was a significant and progressive loss of RGCs in the retinas following the induction of elevated IOP. A single intravitreal injection of 2 microg in 2 microL CNTF significantly protected RGCs up to 4 weeks. pSTAT3 was only transiently expressed in ocular hypertensive eyes. However, in eyes treated with CNTF, pSTAT3 was observed up to 2 weeks after the induction of elevated IOP. In ocular hypertensive eyes, CNTF-positive cells were found in the inner nuclear layer (INL), and there was a transient increase in the pSTAT3 cells in the ganglion cell layer and INL. Immunoblots showed that STAT3 was transiently phosphorylated after IOP increase, but with an injection of CNTF, pSTAT3 protein was observed up to 2 weeks after hypertensive glaucoma induction. Laser-induced chronic ocular hypertension in rats resulted in the death of RGCs and a transient activation of STAT3 in the retina. Intravitreal injection of CNTF showed a significant protection of RGCs, and the JAK-STAT signalling could be one of the important pathways that underlie the mechanism of CNTF neuroprotection in this rat glaucoma model.