Injured Optic Nerve Research Articles

Augmenting fibronectin levels in injured adult CNS promotes axon regeneration in vivo

In an attempt to repair injured central nervous system (CNS) nerves/tracts, immune cells are recruited into the injury site, but endogenous response in adult mammals is insufficient for promoting regeneration of severed axons. Here, we found that a portion of retinal ganglion cell (RGC) CNS projection neurons that survive after optic nerve crush (ONC) injury are enriched for and upregulate fibronectin (Fn)-interacting integrins Itga5 and ItgaV, and that Fn promotes long-term survival and long-distance axon regeneration of a portion of axotomized adult RGCs in culture. We then show that, Fn is developmentally downregulated in the axonal tracts of optic nerve and spinal cord, but injury-activated macrophages/microglia upregulate Fn while axon regeneration-promoting zymosan augments their recruitment (and thereby increases Fn levels) in the injured optic nerve. Finally, we found that Fn's RGD motif, established to interact with Itga5 and ItgaV, promotes long-term survival and long-distance axon regeneration of adult RGCs after ONC in vivo, with some axons reaching the optic chiasm when co-treated with Rpl7a gene therapy. Thus, experimentally augmenting Fn levels in the injured CNS is a promising approach for therapeutic neuroprotection and axon regeneration of at least a portion of neurons.

Differential response of injured and healthy retinas to syngeneic and allogeneic transplantation of a clonal cell line of immortalized olfactory ensheathing glia: a double-edged sword.

JOURNAL/nrgr/04.03/01300535-202508000-00029/figure1/v/2024-09-30T120553Z/r/image-tiff Olfactory ensheathing glia promote axonal regeneration in the mammalian central nervous system, including retinal ganglion cell axonal growth through the injured optic nerve. Still, it is unknown whether olfactory ensheathing glia also have neuroprotective properties. Olfactory ensheathing glia express brain-derived neurotrophic factor, one of the best neuroprotectants for axotomized retinal ganglion cells. Therefore, we aimed to investigate the neuroprotective capacity of olfactory ensheating glia after optic nerve crush. Olfactory ensheathing glia cells from an established rat immortalized clonal cell line, TEG3, were intravitreally injected in intact and axotomized retinas in syngeneic and allogeneic mode with or without microglial inhibition or immunosuppressive treatments. Anatomical and gene expression analyses were performed. Olfactory bulb-derived primary olfactory ensheathing glia and TEG3 express major histocompatibility complex class II molecules. Allogeneically and syngenically transplanted TEG3 cells survived in the vitreous for up to 21 days, forming an epimembrane. In axotomized retinas, only the allogeneic TEG3 transplant rescued retinal ganglion cells at 7 days but not at 21 days. In these retinas, microglial anatomical activation was higher than after optic nerve crush alone. In intact retinas, both transplants activated microglial cells and caused retinal ganglion cell death at 21 days, a loss that was higher after allotransplantation, triggered by pyroptosis and partially rescued by microglial inhibition or immunosuppression. However, neuroprotection of axotomized retinal ganglion cells did not improve with these treatments. The different neuroprotective properties, different toxic effects, and different responses to microglial inhibitory treatments of olfactory ensheathing glia in the retina depending on the type of transplant highlight the importance of thorough preclinical studies to explore these variables.

Inflammatory Mediators of Axon Regeneration in the Central and Peripheral Nervous Systems.

Although most pathways in the mature central nervous system cannot regenerate when injured, research beginning in the late 20th century has led to discoveries that may help reverse this situation. Here, we highlight research in recent years from our laboratory identifying oncomodulin (Ocm), stromal cell-derived factor (SDF)-1, and chemokine CCL5 as growth factors expressed by cells of the innate immune system that promote axon regeneration in the injured optic nerve and elsewhere in the central and peripheral nervous systems. We also review the role of ArmC10, a newly discovered Ocm receptor, in mediating many of these effects, and the synergy between inflammation-derived growth factors and complementary strategies to promote regeneration, including deleting genes encoding cell-intrinsic suppressors of axon growth, manipulating transcription factors that suppress or promote the expression of growth-related genes, and manipulating cell-extrinsic suppressors of axon growth. In some cases, combinatorial strategies have led to unprecedented levels of nerve regeneration. The identification of some similar mechanisms in human neurons offers hope that key discoveries made in animal models may eventually lead to treatments to improve outcomes after neurological damage in patients.

Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration

Injured axons in the central nervous system (CNS) usually fail to regenerate, causing permanent disabilities. However, the knockdown of Pten knockout or treatment of neurons with hyper-IL-6 (hIL-6) transforms neurons into a regenerative state, allowing them to regenerate axons in the injured optic nerve and spinal cord. Transneuronal delivery of hIL-6 to the injured brain stem neurons enables functional recovery after severe spinal cord injury. Here we demonstrate that the beneficial hIL-6 and Pten knockout effects on axon growth are limited by the induction of tubulin detyrosination in axonal growth cones. Hence, cotreatment with parthenolide, a compound blocking microtubule detyrosination, synergistically accelerates neurite growth of cultured murine CNS neurons and primary RGCs isolated from adult human eyes. Systemic application of the prodrug dimethylamino-parthenolide (DMAPT) facilitates axon regeneration in the injured optic nerve and spinal cord. Moreover, combinatorial treatment further improves hIL-6-induced axon regeneration and locomotor recovery after severe SCI. Thus, DMAPT facilitates functional CNS regeneration and reduces the limiting effects of pro-regenerative treatments, making it a promising drug candidate for treating CNS injuries.

Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration.

Injured axons in the central nervous system (CNS) usually fail to regenerate, causing permanent disabilities. However, the knockdown of Pten knockout or treatment of neurons with hyper-IL-6 (hIL-6) transforms neurons into a regenerative state, allowing them to regenerate axons in the injured optic nerve and spinal cord. Transneuronal delivery of hIL-6 to the injured brain stem neurons enables functional recovery after severe spinal cord injury. Here we demonstrate that the beneficial hIL-6 and Pten knockout effects on axon growth are limited by the induction of tubulin detyrosination in axonal growth cones. Hence, cotreatment with parthenolide, a compound blocking microtubule detyrosination, synergistically accelerates neurite growth of cultured murine CNS neurons and primary RGCs isolated from adult human eyes. Systemic application of the prodrug dimethylamino-parthenolide (DMAPT) facilitates axon regeneration in the injured optic nerve and spinal cord. Moreover, combinatorial treatment further improves hIL-6-induced axon regeneration and locomotor recovery after severe SCI. Thus, DMAPT facilitates functional CNS regeneration and reduces the limiting effects of pro-regenerative treatments, making it a promising drug candidate for treating CNS injuries.

The oncomodulin receptor ArmC10 enables axon regeneration in mice after nerve injury and neurite outgrowth in human iPSC-derived sensory neurons.

Oncomodulin (Ocm) is a myeloid cell-derived growth factor that enables axon regeneration in mice and rats after optic nerve injury or peripheral nerve injury, yet the mechanisms underlying its activity are unknown. Using proximity biotinylation, coimmunoprecipitation, surface plasmon resonance, and ectopic expression, we have identified armadillo-repeat protein C10 (ArmC10) as a high-affinity receptor for Ocm. ArmC10 deletion suppressed inflammation-induced axon regeneration in the injured optic nerves of mice. ArmC10 deletion also suppressed the ability of lesioned sensory neurons to regenerate peripheral axons rapidly after a second injury and to regenerate their central axons after spinal cord injury in mice (the conditioning lesion effect). Conversely, Ocm acted through ArmC10 to accelerate optic nerve and peripheral nerve regeneration and to enable spinal cord axon regeneration in these mouse nerve injury models. We showed that ArmC10 is highly expressed in human-induced pluripotent stem cell-derived sensory neurons and that exposure to Ocm altered gene expression and enhanced neurite outgrowth. ArmC10 was also expressed in human monocytes, and Ocm increased the expression of immune modulatory genes in these cells. These findings suggest that Ocm acting through its receptor ArmC10 may be a useful therapeutic target for nerve repair and immune modulation.

Post-injury born oligodendrocytes incorporate into the glial scar and contribute to the inhibition of axon regeneration.

Failure of central nervous system (CNS) projection neurons to spontaneously regenerate long-distance axons underlies irreversibility of white matter pathologies. A barrier to axonal regenerative research is that the axons regenerating in response to experimental treatments stall growth before reaching post-synaptic targets. Here, we test the hypothesis that the interaction of regenerating axons with live oligodendrocytes, which were absent during developmental axon growth, contributes to stalling axonal growth. To test this hypothesis, first, we used single cell RNA-seq (scRNA-seq) and immunohistology to investigate whether post-injury born oligodendrocytes incorporate into the glial scar after optic nerve injury. Then, we administered demyelination-inducing cuprizone and stimulated axon regeneration by Pten knockdown (KD) after optic nerve crush. We found that post-injury born oligodendrocyte lineage cells incorporate into the glial scar, where they are susceptible to the demyelination diet, which reduced their presence in the glial scar. We further found that the demyelination diet enhanced Pten KD-stimulated axon regeneration, and localized cuprizone injection promoted axon regeneration. We also present a website for comparing the gene expression of scRNA-seq-profiled normal and injured optic nerve oligodendrocyte lineage cells.

Research progress on optic nerve injury and regeneration

<p indent="0mm">Humans have a good visual system. Retinal ganglion cells (RGCs), which connect the eyeball to the brain, fail to regenerate after injury, eventually leading to blindness. Difficulty in regeneration can be attributed in part to inhibitory molecules in glial scar and myelin and insufficient regenerative capacity intrinsic to RGCs. In addition, RGCs undergo apoptosis after optic nerve injury, which makes the regeneration of visual system more difficult. Here, we review the causes of regeneration failure and the progress achieved in repair of the visual system. Some of the encouraging findings may be applied clinically in the future to achieve meaningful regeneration of injured optic nerves.

Identification of a Novel Axon Regeneration Role for Noncanonical Wnt Signaling in the Adult Retina after Injury.

Canonical and noncanonical Wnt signaling pathways are essential for development and maintenance of the CNS. Whereas the roles of canonical Wnt pathways in neuronal survival and axonal regeneration in adult CNS have been described, the functions of noncanonical Wnt pathways are not well understood. Furthermore, the role of noncanonical Wnt ligands in the adult retina has not been investigated. Noncanonical Wnt signaling shares receptors with canonical Wnt ligands but functions through calcium and c-Jun N-terminal kinase (JNK) signaling pathways. Noncanonical ligands, such as the prototypic ligand Wnt5a, have varying effects in the developing CNS, including inhibiting or promoting axonal growth. To identify a role for noncanonical Wnt signaling in the developed retina after injury, we characterized the effect of Wnt5a on neurite outgrowth in cultured retinal ganglion cell (RGC) neurons and on axonal regeneration in the injured optic nerve in the mouse. Endogenous Wnt5a was upregulated after injury and exogenous Wnt5a significantly enhanced neurite growth of primary RGCs and led to extensive axonal regeneration after optic nerve crush (ONC) injury. Wnt5a also significantly increased RGC survival. Furthermore, Wnt5a induced phosphorylation of CamKII and JNK and induced expression of their downstream pathway components. Therefore, these results demonstrate for the first time that Wnt5a promotes axonal growth and protects RGCs in the adult retina.

Retinal ganglion cell expression of cytokine enhances occupancy of NG2 cell-derived astrocytes at the nerve injury site: Implication for axon regeneration

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

Neuroinflammation, Microglia and Implications for Retinal Ganglion Cell Survival and Axon Regeneration in Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) refers to a pathological condition caused by a direct or indirect insult to the optic nerves, which often leads to a partial or permanent vision deficit due to the massive loss of retinal ganglion cells (RGCs) and their axonal fibers. Retinal microglia are immune-competent cells residing in the retina. In rodent models of optic nerve crush (ONC) injury, resident retinal microglia gradually become activated, form end-to-end alignments in the vicinity of degenerating RGC axons, and actively internalized them. Some activated microglia adopt an amoeboid morphology that engulf dying RGCs after ONC. In the injured optic nerve, the activated microglia contribute to the myelin debris clearance at the lesion site. However, phagocytic capacity of resident retinal microglia is extremely poor and therefore the clearance of cellular and myelin debris is largely ineffective. The presence of growth-inhibitory myelin debris and glial scar formed by reactive astrocytes inhibit the regeneration of RGC axons, which accounts for the poor visual function recovery in patients with TON. In this Review, we summarize the current understanding of resident retinal microglia in RGC survival and axon regeneration after ONC. Resident retinal microglia play a key role in facilitating Wallerian degeneration and the subsequent axon regeneration after ONC. However, they are also responsible for producing pro-inflammatory cytokines, chemokines, and reactive oxygen species that possess neurotoxic effects on RGCs. Intraocular inflammation triggers a massive influx of blood-borne myeloid cells which produce oncomodulin to promote RGC survival and axon regeneration. However, intraocular inflammation induces chronic neuroinflammation which exacerbates secondary tissue damages and limits visual function recovery after ONC. Activated retinal microglia is required for the proliferation of oligodendrocyte precursor cells (OPCs); however, sustained activation of retinal microglia suppress the differentiation of OPCs into mature oligodendrocytes for remyelination after injury. Collectively, controlled activation of retinal microglia and infiltrating myeloid cells facilitate axon regeneration and nerve repair. Recent advance in single-cell RNA-sequencing and identification of microglia-specific markers could improve our understanding on microglial biology and to facilitate the development of novel therapeutic strategies aiming to switch resident retinal microglia’s phenotype to foster neuroprotection.

Optic Nerve Engraftment of Neural Stem Cells.

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.

The role of PGS/PCL scaffolds in promoting differentiation of human embryonic stem cells into retinal ganglion cells

The stem cell-based retinal ganglion cells (RGCs) replacement therapy offers a potential to restore vision in progressive optic neuropathies including glaucoma by replacing degenerated RGCs and by simulating axonal regeneration. Injured optic nerve axons do not regenerate owing to the limited intrinsic capacity of the neurons and the inhibitory environment at the injury site. Polymeric tissue scaffolds are able to modulate the physical environment while providing structural support for transplanted cells, however, their application specific to the RGC generation has been far from conclusive. The successful generation of clinically safe and functional RGCs that can appropriately integrate into the hosts’ retinas still remain largely unresolved.Our study reports on a process that enables generation of RGCs from human embryonic stem cells (hESCs) that is simple, straightforward and repeatable and, investigates the influence of the aligned poly(glycerol sebacate) (PGS)/poly(ε-caprolactone) (PCL) scaffold on this differentiation process. Our findings demonstrate that PGS/PCL scaffold promotes differentiation of hESCs into RGC-like cells possibly by the simulation of cell active environmental signalling and, facilitates the growth of RGCs neurites along their lengths. Statement of significanceGlaucoma can lead to the degeneration of retinal ganglion cells (RGCs), with consequential vision loss. RGCs are incapable of self-renewal, replacement of diseased RGCs with healthy cells has been a goal to restore vision in glaucoma patients. In this regard, stem cell RGC replacement therapy has been shown to improve vision in animal models of glaucoma, which could be facilitated by using tissue-engineered polymeric scaffolds.In this study, we generated homogenous stem cell-derived RGCs via a straightforward differentiation protocol and evaluated the effects of PGS/PCL scaffold on RGCs differentiation and growth of RGCs neurites. Our study contributes to the knowledge on how biomaterial scaffolds are able to support the regeneration of RGC neurites (i.e., axons or dendrites) as a part of a possible future clinical therapy for the treatment of glaucoma.

Effect of taxifolin on cisplatin-associated oxidative optic nerve damage in rats

Aim To investigate the effect of taxifolin on cisplatin-induced oxidative and proinflammatory optic nerve damage in rats. Methods A total of 18 albino Wistar male rats were assigned into 3 groups, as follows; Group 1: Control group, Group 2: Only cisplatin administered group for 14 days (Cisplatin group), and Group 3: Taxifolin + cisplatin administered group for 14 days (CIS + TAX group). Serum malondialdehyde (MDA), total Glutathione (tGSH), Nuclear Factor-Kappa B (NF-ƘB), Total Oxidative Status (TOS) and Total Antioxidant Status (TAS) levels were collected from the left eyes of rats. Rats’ right eyes were enucleated for histopathological evaluations of optic nerves. Results NF-ƘB, MDA and TOS levels were statistically significantly higher (p < 0.001) in cisplatin group when compared to other 2 groups, the tGSH and TAS levels of which were statistically significantly lower (p < 0.001). Regarding these parameters, in cisplatin group NF-ƘB, MDA and TOS levels were statistically significantly increased with cisplatin administration and giving taxifolin concomitantly with cisplatin prevented this elevation. On the other hand, tGSH and TAS levels were statistically significantly decreased with cisplatin administration and routine simultaneous application of taxifolin with cisplatin prevented this decrease. In histopathological findings, haemorrhage was observed in the perineum of the injured optic nerves in the cisplatin treated group. And also edoema and degeneration in nerve fascicles in damaged optic nerves were seen in the cisplatin group. In the taxifolin treated group histopathological examinations were close to normal appearance, except mild edoema in nerve fascicles. Conclusion Cisplatin causes oxidative stress on the rat optic nerves, and these changes lead to significant histopathological damage. Taxifolin, which we used to prevent oxidative damage to the optic nerves caused by cisplatin, has been emphasized as a powerful antioxidant agent in many previous scientific investigations. Concomitant administration of taxifolin may prevent these adverse effects of cisplatin, as well as histopathological damage. Further studies are needed to fully determine the effects of cisplatin and taxifolin on the eye.

Robust Myelination of Regenerated Axons Induced by Combined Manipulations of GPR17 and Microglia

Myelination facilitates rapid axonal conduction, enabling efficient communication across different parts of the nervous system. Here we examined mechanisms controlling myelination after injury and during axon regeneration in the central nervous system (CNS). Previously, we discovered multiple molecular pathways and strategies that could promote robust axon regrowth after optic nerve injury. However, regenerated axons remain unmyelinated, and the underlying mechanisms are elusive. In this study, we found that, in injured optic nerves, oligodendrocyte precursor cells (OPCs) undergo transient proliferation but fail to differentiate into mature myelination-competent oligodendrocytes, reminiscent of what is observed in human progressive multiple sclerosis. Mechanistically, we showed that OPC-intrinsic GPR17 signaling and sustained activation of microglia inhibit different stages of OPC differentiation. Importantly, co-manipulation of GPR17 and microglia led to extensive myelination of regenerated axons. The regulatory mechanisms of stage-dependent OPC differentiation uncovered here suggest a translatable strategy for efficient de novo myelination after CNS injury.

Nrn1 Overexpression Attenuates Retinal Ganglion Cell Apoptosis, Promotes Axonal Regeneration, and Improves Visual Function Following Optic Nerve Crush in Rats.

Neuritin (Nrn1) is a small highly conserved extracellular membrane protein involved in the process of neural cell survival and differentiation, axonal and dendritic growth, and synapse formation and maturation. Previous studies have demonstrated that intravitreal injection of recombinant Nrn1 as a gene therapy could alleviate retinal ganglion cell (RGC) apoptosis and promote optic nerve axon regeneration after optic nerve crush (ONC). However, the mechanism underlying the repairing effect of Nrn1 against optic never injury remains elusive. In this study, a rAAV2-mediated Nrn1 overexpression vector (AAV2-Nrn1) was applied to treat ONC through intravitreal injection for the purpose of further exploring the effect and mechanism of Nrn1 in repairing the injured optic nerve. The results showed that AAV2-Nrn1 was mainly transfected into RGCs without affecting astrocytes. Nrn1 overexpression effectively reduced RGC apoptosis and promoted optic nerve regeneration and visual function restoration as demonstrated by retinal imaging, histopathological analysis, and physiological function detection in vivo following ONC. Immunoblot assay revealed that functional molecules of Nrn1 activated the Akt1 and Stat3 pathways and inhibited the mitochondrial apoptotic pathway. The results of the present study may provide experimental evidence for further application of Nrn1 to the clinical treatment of optic nerve injury.

Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

GFAP expression in the optic nerve and increased H2S generation in the integration centers of the rainbow trout (Oncorhynchus mykiss) brain after unilateral eye injury.

Hydrogen sulfide (H2S) is considered as a protective factor against cardiovascular disorders. However, there are few reports on the effects of H2S in the central nervous system during stress or injury. Previous studies on goldfish have shown that astrocytic response occurs in the damaged and contralateral optic nerves. Glial fibrillary acidic protein (GFAP) concentration in the optic nerves of rainbow trout has not been measured previously. This study further characterized the astrocytic response in the optic nerve and the brain of a rainbow trout (Oncorhynchus mykiss) after unilateral eye injury and estimated the amount of H2S-producing enzyme cystathionine β-synthase (CBS) in the brain of the rainbow trout. Within 1 week after unilateral eye injury, a protein band corresponding to a molecular weight of 50 kDa was identified in the ipsi- and contralateral optic nerves of the rainbow trout. The concentration of GFAP in the injured optic nerve increased compared to the protein concentration on the contralateral side. The results of a quantitative analysis of GFAP+ cell distribution in the contralateral optic nerve showed the largest number of GFAP+ cells and fibers in the optic nerve head. In the damaged optic nerve, patterns of GFAP+ cell migration and large GFAP+ bipolar activated astrocytes were detected at 1 week after unilateral eye injury. The study of H2S-producing system after unilateral eye injury in the rainbow trout was conducted using enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry of polyclonal antibodies against CBS in the integrative centers of the brain: telencephalon, optic tectum, and cerebellum. Enzyme-linked immunosorbent assay results showed a 1.7-fold increase in CBS expression in the rainbow trout brain at 1 week after unilateral eye injury compared with that in intact animals. In the ventricular and subventricular regions of the rainbow trout telencephalon, CBS+ radial glia and neuroepithelial cells were identified. After unilateral eye injury, the number of CBS+ neuroepithelial cells in the pallial and subpallial periventricular regions of the telencephalon increased. In the optic tectum, unilateral eye injury led to an increase in CBS expression in radial glial cells; simultaneously, the number of CBS+ neuroepithelial cells decreased in intact animals. In the cerebellum of the rainbow trout, neuroglial interrelationships were revealed, where H2S was released, apparently, from astrocyte-like cells. The organization of H2S-producing cell complexes suggests that, the amount of glutamate produced in the rainbow trout cerebellum and its reuptake was controlled by astrocyte-like cells, reducing its excitotoxicity. In the dorsal matrix zone and granular eminences of the rainbow trout cerebellum, CBS was expressed in neuroepithelial cells. After unilateral eye injury, the level of CBS activity increased in all parts of the cerebellum. An increase in the number of H2S-producing cells was a response to oxidative stress after unilateral eye injury, and the overproduction of H2S in the cerebellum occurred to neutralize reactive oxygen species, providing the cells of the rainbow trout cerebellum with a protective effect. A structural reorganization in the dorsal matrix zone, associated with the appearance of an additional CBS+ apical zone, and a decrease in the enzyme activity in the dorsal matrix zone, was revealed in the zones of constitutive neurogenesis. All experiments were approved by the Commission on Biomedical Ethics, A.V. Zhirmunsky National Scientific Center of Marine Biology (NSCMB), Far Eastern Branch, Russian Academy of Science (FEB RAS) (approval No. 1) on July 31, 2019.

Axon transport as a target for promoting RGC survival and optic nerve regeneration

AbstractAdult CNS neurons have a weak intrinsic capacity for regeneration after injury, but the underlying cellular mechanisms preventing regeneration are not completely understood. Increasing intrinsic growth ability is a critical part of enabling injured optic nerve axons to regenerate. The work presented here is part of a large body of research into understanding the role of membrane protein trafficking and transport in the control of regenerative ability. We have found that as CNS neurons mature, there is a developmental decline in the axonal transport of integrins (cell surface adhesion and guidance molecules) and their endosomal transporters, Rab11 positive recycling endosomes. This is controlled by a molecular switch called ARF6. Switching off ARF6 in mature CNS axons leads to an increase in the anterograde axonal transport of integrins and Rab11 endosomes, leading to a restoration of the regenerative capacity that is lost as axons mature. We have been targeting these mechanisms to stimulate axon regeneration in the optic nerve, and neuroprotection of retinal ganglion neurons during raised IOP. Here we present novel data from two new lines of investigation into facilitating CNS axonal transport, neuroprotection and regeneration, focusing on signaling pathways and organelle motility. Firstly, by investigating signaling upstream of ARF6, we have identified an isoform of PI3 Kinase which we have used to stimulate axon transport, neuroprotection and regeneration; both after laser axotomy in vitro and after optic nerve crush. Secondly, we present exciting new data from studies into a trafficking molecule which resides in the endoplasmic reticulum (ER) and demonstrate that this can be targeted to enable robust optic nerve regeneration and neuroprotection in the retina. These findings confirm integrins and Rab11 as critical facilitators of optic nerve regeneration whilst establishing an important new role for the axonal ER in this process.

Change in phospholipid species of retinal layer in traumatic optic neuropathy model.

Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury (TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine(PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI.