Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury.Valproate is a histone deacetylase inhibitor and multitarget drug,which has been demonstrated to protect retinal neurons.In this study,we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling.We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope.Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein,phosphorylated eukaryotic translation initiation factor 2α,and caspase-12 in the endoplasmic reticulum of retinal ganglion cells.These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress.These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.

Protective Effect of Paeoniflorin against Optic Nerve Crush

In order to evaluate the efficacy of traditional paeonia extract paeoniflorin against optic nerve crush, 16 Brown Norway rats were divided into two groups at random, with 8 rats in each group. In paeoniaflorin-treated group, 2 mg paeoniaflorin （total volum： 1 mL） was injected into rat＇s peritoneum one time a day for a period of 8 days. Rats in untreated group were given a single dose of vehicle. The optic nerve was crushed by a special forceps for 30 s in the left eye and a sham procedure was performed in the right eye on the 2nd day after the first injection. The retrograde fluorogold labeling of ganglion cells was conducted 5 days after optic nerve crush. The whole retina was flat-mounted thereafter. The ganglion cells that survived the crush were counted under fluorescent microscope by using an automatic counting software. As compared with the contralateral eye, the survival rate of ganglion cells in the left eye increased from 40.22% to 64.53% with a significant difference found between them （t=2.55, P=0.023）. The results suggested that the paeonia extract paeoniflorin possessed a protective effect against optic nerve crush.

Erythropoietin upregulates growth associated protein-43 expression and promotes retinal ganglion cell axonal regeneration in vivo after optic nerve crush

In this study, we established a rat model of optic nerve crush to explore the effects of erythropoietin on retinal ganglion cell axonal regeneration. At 15 days after injury in erythropoietin treated rats, retinal ganglion cell densities in regions corresponding to the 1/6, 3/6 and 5/6 ratios of the retinal radius were significantly increased. In addition, the number of growth associated protein-43 positive axons was significantly increased at different distances （50, 250 and 500 pm） from the crush site after erythropoietin treatment. Erythropoietin significantly increased growth associated protein-43 protein levels in the retina after crush injury, as determined by westem blot and immunofluorescence analysis. These results demonstrate that erythropoietin protects injured retinal ganglion cells and promotes axonal regeneration.

Protective effects of Achyranthes bidentata polypeptides on retinal ganglion cells post-optic nerve crush in rats

Achyranthes bidentata polypeptides（ABPP） have been reported to inhibit apoptosis of retinal ganglion cells（RGCs）.The present study investigated the protective effects of ABPP on RGCs in a rat model of optic nerve injury.With prolonged injury time,RGC densities were gradually decreased.ABPP（5 μg） significantly increased RGC densities and upregulated growth associated protein 43 expression in rats with optic nerve injury.Results demonstrate that ABPP can protect RGCs and promote axonal growth after optic nerve crush.

Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

Several studies have found that transplantation of neural progenitor cells(NPCs)promotes the survival of injured neurons.However,a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application.Small extracellular vesicles(sEVs)contain bioactive molecules for neuronal protection and regeneration.Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases.In this study,we intravitreally transplanted sEVs derived from human induced pluripotent stem cells(hiPSCs)and hiPSCs-differentiated NPCs(hiPSC-NPC)in a mouse model of optic nerve crush.Our results show that these intravitreally injected sEVs were ingested by retinal cells,especially those localized in the ganglion cell layer.Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration,and regulated the retinal microenvironment by inhibiting excessive activation of microglia.Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells,which had protective effects on RGCs after optic nerve injury.These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics

Axonal regeneration in the central nervous system is an energy-intensive process.In contrast to mammals,adult zebrafish can functionally recover from neuronal injury.This raises the question of how zebrafish can cope with this high energy demand.We previously showed that in adult zebrafish,subjected to an optic nerve crush,an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair.We postulate a‘dendrites for regeneration’paradigm that might be linked to intraneuronal mitochondrial reshuffling,as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously.Here,we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments(dendrites,somas,and axons)during the regenerative process.Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction,whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth.Upon dendritic regrowth in the retina,mitochondrial density inside the retinal dendrites returned to baseline levels.Moreover,a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage.Taken together,these findings suggest that during optic nerve injury-induced regeneration,mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury

Ras homolog enriched in brain(Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1(mTORC1).Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR.S6K1 and4E-BP1 are important downstream effectors of mTORC1.In this study,we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1in the protection of retinal ganglion cells.We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration.We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute(14 days) and chronic(21 and 42 days) stages of injury.We also found that either co-expression of the dominant-negative S6K1mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells.This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration.However,only S6K1 activation,but not 4E-BP1 knockdown,induced axon regeneration when applied alone.Furthermore,S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury,whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days postinjury.Ove rexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury.Likewise,co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury.These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rhe b/mTOR.Together,our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity.Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

Altered Energy Metabolism During Early Optic Nerve Crush Injury:Implications of Warburg-Like Aerobic Glycolysis in Facilitating Retinal Ganglion Cell Survival

Neurons,especially axons,are metabolically demanding and energetically vulnerable during injury.However,the exact energy budget alterations that occur early after axon injury and the effects of these changes on neuronal survival remain unknown.Using a classic mouse model of optic nerve-crush injury,we found that traumatized optic nerves and retinas harbor the potential to mobilize two primary energetic machineries,glycolysis and oxidative phosphorylation,to satisfy the robustly increased adenosine triphosphate(ATP) demand.Further exploration of metabolic activation showed that mitochondrial oxidative phosphorylation was amplified over other pathways,which may lead to decreased retinal ganglion cell(RGC) survival despite its supplement to ATP production.Gene set enrichment analysis of a microarray(GSE32309) identified significant activation of oxidative phosphorylation in injured retinas from wild-type mice compared to those from mice with deletion of phosphatase and tensin homolog(PTEN),while PTEN-/-mice had more robust RGC survival.Therefore,we speculated that the oxidation-favoring metabolic pattern after optic nervecrush injury could be adverse for RGC survival.After redirecting metabolic flux toward glycolysis(magnifying the Warburg effect) using the drug meclizine,we successfully increased RGC survival.Thus,we provide novel insights into a potential bioenergetics-based strategy for neuroprotection.

Promotion of axon regeneration and inhibition of astrocyte activation by alpha A-crystallin on crushed optic nerve

AIM：To explore the effects of αA-crystallin in astrocyte gliosis after optic nerve crush（ONC） and the mechanism of α-crystallin in neuroprotection and axon regeneration.METHODS：ONC was established on the SpragueDawley rat model and αA-crystallin（10 -4 g/L,4 μL） was intravitreously injected into the rat model.Flash-visual evoked potential（F-VEP） was examined 14 d after ONC,and the glial fibrillary acidic protein（GFAP） levels in the retina and crush site were analyzed 1,3,5,7 and 14 d after ONC by immunohistochemistry（IHC） and Western blot respectively.The levels of beta Tubulin（TUJ1）,growth-associated membrane phosphoprotein-43（GAP-43）,chondroitin sulfate proteoglycans（CSPGs） and neurocan were also determined by IHC 14 d after ONC.RESULTS：GFAP level in the retina and the optic nerve significantly increased 1d after ONC,and reached the peak level 7d post-ONC.Injection of αA-crystallin significantly decreased GFAP level in both the retina and the crush site 3d after ONC,and induced astrocytes architecture remodeling at the crush site.Quantification of retinal ganglion cell（RGC） axons indicated αAcrystallin markedly promoted axon regeneration in ONC rats and enhanced the regenerated axons penetrated into the glial scar.CSPGs and neurocan expression also decreased 14 d after αA-crystallin injection.The amplitude（N1-P1） and latency（P1） of F-VEP were also restored.CONCLUSION：Our results suggest α-crystallin promotes the axon regeneration of RGCs and suppresses the activation of astrocytes.

Pan-retinal ganglion cell markers in mice, rats, and rhesus macaques

Univocal identification of retinal ganglion cells(RGCs) is an essential prerequisite for studying their degeneration and neuroprotection. Before the advent of phenotypic markers, RGCs were normally identified using retrograde tracing of retinorecipient areas. This is an invasive technique, and its use is precluded in higher mammals such as monkeys. In the past decade, several RGC markers have been described. Here, we reviewed and analyzed the specificity of nine markers used to identify all or most RGCs, i.e., pan-RGC markers, in rats, mice, and macaques. The best markers in the three species in terms of specificity, proportion of RGCs labeled, and indicators of viability were BRN3A, expressed by vision-forming RGCs, and RBPMS, expressed by vision-and non-vision-forming RGCs. NEUN, often used to identify RGCs, was expressed by non-RGCs in the ganglion cell layer, and therefore was not RGC-specific. γ-SYN, TUJ1, and NF-L labeled the RGC axons, which impaired the detection of their somas in the central retina but would be good for studying RGC morphology. In rats, TUJ1 and NF-L were also expressed by non-RGCs. BM88, ERRβ,and PGP9.5 are rarely used as markers, but they identified most RGCs in the rats and macaques and ERRβ in mice. However, PGP9.5 was also expressed by non-RGCs in rats and macaques and BM88 and ERRβ were not suitable markers of viability.

Srgap2 suppression ameliorates retinal ganglion cell degeneration in mice

Slit-Robo GTPase-activating protein 2(SRGAP2) plays important roles in axon guidance, neuronal migration, synapse formation, and nerve regeneration. However, the role of SRGAP2 in neuroretinal degenerative disease remains unclear. In this study, we found that SRGAP2 protein was first expressed in the retina of normal mice at the embryonic stage and was mainly located in the mature retinal ganglion cell layer and the inner nuclear layer. SRGAP2 protein in the retina and optic nerve increased after optic nerve crush. Then, we established a heterozygous knockout(Srgap2+/–) mouse model of optic nerve crush and found that Srgap2 suppression increased retinal ganglion cell survival, lowered intraocular pressure, inhibited glial cell activation, and partially restored retinal function. In vitro experiments showed that Srgap2 suppression activated the mammalian target of rapamycin signaling pathway. RNA sequencing results showed that the expression of small heat shock protein genes(Cryaa, Cryba4, and Crygs) related to optic nerve injury were upregulated in the retina of Srgap2+/– mice. These results suggest that Srgap2 suppression reduced the robust activation of glial cells, activated the mammalian target of rapamycin signaling pathway related to nerve protein, increased the expression of small heat shock protein genes, inhibited the degeneration of retinal ganglion cells, and partially restored optic nerve function.

Mechanisms implicated in the contralateral effect in the central nervous system after unilateral injury:focus on the visual system

The retina,as part of the central nervous system is an ideal model to study the response of neurons to injury and disease and to test new treatments.During the last decade is becoming clear that unilateral lesions in bilateral areas of the central nervous system trigger an inflammatory response in the contralateral uninjured site.This effect has been better studied in the visual system where,as a rule,one retina is used as experimental and the other as control.Contralateral retinas in unilateral models of retinal injury show neuronal degeneration and glial activation.The mechanisms by which this adverse response in the central nervous system occurs are discussed in this review,focusing primarily on the visual system.

OTX2 stimulates adult retinal ganglion cell regeneration

Retinal ganglion cell(RGC) axons provide the only link between the light sensitive and photon transducing neural retina and visual centers of the brain.RGC axon degeneration occurs in a number of blinding diseases and the ability to stimulate axon regeneration from surviving ganglion cells could provide the anatomic substrate for restoration of vision.OTX2 is a homeoprotein transcription factor expressed in the retina and previous studies showed that,in response to stress,exogenous OTX2 increases the in vitro and in vivo survival of RGCs.Here we examined and quantified the effects of OTX2 on adult RGC axon regeneration in vitro and in vivo.The results show that exogenous OTX2 stimulates the regrowth of axons from RGCs in cultures of dissociated adult retinal cells and from explants of adult retinal tissue and that RGCs respond directly to OTX2 as regrowth is observed in cultures of purified adult rat RGCs.Importantly,after nerve crush in vivo,we observed a positive effect of OTX2 on the number of regenerating axons up to the optic chiasm within 14 days post crush and a very modest level of acuity absent in control mice.The effect of OTX2 on RGC survival and regeneration is of potential interest for degenerative diseases affecting this cell type.All animal procedures were approved by the local "Comié d'éιthique en expérimentation animale n°59" and authorization n° 00702.01 delivered March 28,2014 by the French "Ministére de l'enseignement supérieur et de la recherche".