Retinal Ganglion Cell Survival Research Articles

Targeting tRNA-Derived Non-Coding RNA Alleviates Diabetes-Induced Visual Impairment through Protecting Retinal Neurovascular Unit.

Diabetes is a major risk factor for compromised visual health, leading to retinal vasculopathy and neuropathy, both of which are hallmarks of neurovascular unit dysfunction. Despite the critical impact of diabetic retinopathy, the precise mechanism underlying neurovascular coupling and effective strategies to suppress neurovascular dysfunction remain unclear. In this study, the up-regulation of a tRNA-derived stress-induced RNA, 5'tiRNA-His-GTG, in response to diabetic stress is revealed. 5'tiRNA-His-GTG directly regulates Müller glia action and indirectly alters endothelial angiogenic effects and retinal ganglion cell (RGC) survival in vitro. Downregulation of 5'tiRNA-His-GTG alleviates diabetes-induced retinal neurovascular dysfunction, characterized by reduced retinal vascular dysfunction, decreased retinal neurodegeneration, and improved visually-guided behaviors in vivo. Mechanistically, 5'tiRNA-His-GTG acts as a key regulator of retinal neurovascular dysfunction, primarily by modulating arachidonic acid (AA) metabolism via the CYPs pathway. The 5'tiRNA-His-GTG-CYP2E1-19(S)-HETE signaling axis is identified as a key driver of retinal neurovascular dysfunction. Thus, targeting 5'tiRNA-His-GTG presents a promising therapeutic strategy for treating vasculopathy and neuropathy associated with diabetes mellitus. Modulating this novel signaling pathway can open up new avenues for intervention in diabetic retinopathy and its related complications.

To explore the protective effect and mechanism of KLF7 overexpression on retinal ganglion cells after optic nerve crush in mice.

To investigate the protective effect and mechanism of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) after optic nerve crush (ONC). Ninety 10-week-old C57BL/6J mice were randomly assigned to five groups. The blank control group (group A), IVT (intravitreal injection) of KLF7 (group B), IVT of phosphate-buffered saline after ONC (group C), IVT of KLF7 after ONC (group D), and IVT of green fluorescent protein after ONC (group E). Retinal electroretinography and immunofluorescence staining were performed to observe the function and survival rate of RGCs on days 3 and 7 after ONC. The Western Blot determined the expressions of JNK 1, ERK, P38, NF-κB, IL-1, IL-6, and TNF-α seven days after ONC. After ONC, KLF7 gradually increased from day 3 to day 14, with the peak noted a peak on day 7. On day 7 after ONC, the RGC survival rate in Group D was significantly higher than in groups C (P = 0.028) and E (P = 0.007). The negative PhNR wave amplitude significantly decreased in groups C (P = 0.03) and E (P = 0.04) compared to group D. Moreover, group D also showed significantly higher ERK levels among the groups (all P < 0.01); NF-κB, IL-1, IL-6, and TNF-α expression decreased significantly in group D compared with groups C and E (all P < 0.01). Overexpression of KLF7 improved the survival rate and function of RGCs in mice ONC models by activating the ERK pathway and inhibiting relevant inflammatory factors. KLF7 may be a promising protective factor against optic nerve damage.

Implantation of biomimetic polydopamine nanocomposite scaffold promotes optic nerve regeneration through modulating inhibitory microenvironment

Optic nerve regeneration remains challenging worldwide due to the limited intrinsic regenerative capacity of retinal ganglion cells (RGCs) and the inhibitory microenvironment. Oxidative stress, induced by excessive reactive oxygen species (ROS) following optic nerve injury, is associated with prolonged neuroinflammation, resulting in a secondary injury of RGCs and the impairment of axon regeneration. Herein, we developed a bionic nanocomposite scaffold (GA@PDA) with immunoregulatory ability for enhanced optic nerve regeneration. The ice-templating method was employed to fabricate biopolymer-based scaffolds with a directional porous structure, mimicking the optic nerve, which effectively guided the oriented growth of neuronal cells. The incorporation of bioinspired polydopamine nanoparticles (PDA NPs) further confers excellent ROS scavenging ability, thereby modulating the phenotype transformation of microglia/macrophages from pro-inflammatory M1 to anti-inflammatory M2. In a rat optic nerve crush model, the implantation of GA@PDA scaffold enhanced survival of RGCs and promoted axonal regeneration. Our study offers novel insights and holds promising potential for the advancement of engineered biomaterials in facilitating optic nerve regeneration.

Growth Hormone Neuroprotective Effects After an Optic Nerve Crush in the Male Rat.

Growth hormone (GH) has neuroprotective effects that have not been evaluated in the mammalian visual system. This study tested the hypothesis that GH administration can promote retinal neuroprotection in an optic nerve crush (ONC) model in male rats. The ON was compressed for 10 seconds, and bovine GH was injected concomitantly to injury for 14days (0.5µg/g every 12 hours). At 24 hours and 14 days after ONC, we evaluated the effects of GH upon several markers by quantitative PCR (qPCR), Western blot, and immunohistochemistry; the ON integrity was assessed using CTB Alexa 488 anterograde tracer, and retinal function was tested by full-field electroretinogram. GH partially prevented the ONC-induced death of retinal ganglion cells (RGCs), as well as the increase in gliosis marker GFAP at 14 days. Most of the ONC-induced changes in mRNA retinal levels of several neurotrophic, survival, synaptogenic, gliosis, and excitotoxicity markers were prevented by GH, both at 24 hours and 14 days, and treatment also stimulated the expression of antiapoptotic proteins Bcl-2 and Bcl-xL at 24 hours. Additionally, GH partially maintained the ON integrity and active anterograde transport, as well as retinal function by avoiding the reduced amplitude and slowing of the A- and B-waves and oscillatory potentials associated with the ONC at 14 days. GH has neuroprotective effects in the ONC model in male rats, it promoted RGC survival, gliosis reduction, and axonal transport increase, likely through the regulation of genes involved in neuroprotection, survival, and synaptogenesis. Furthermore, GH prevented functional impairment, indicating its potential as a therapeutic option for retinal neurodegenerative diseases.

NFATc4 Knockout Promotes Neuroprotection and Retinal Ganglion Cell Regeneration After Optic Nerve Injury.

Retinal ganglion cells (RGCs), neurons transmitting visual information via the optic nerve, fail to regenerate their axons after injury. The progressive loss of RGC function underlies the pathophysiology of glaucoma and other optic neuropathies, often leading to irreversible blindness. Therefore, there is an urgent need to identify the regulators of RGC survival and the regenerative program. In this study, we investigated the role of the family of transcription factors known as nuclear factor of activated T cells (NFAT), which are expressed in the retina; however, their role in RGC survival after injury is unknown. Using the optic nerve crush (ONC) model, widely employed to study optic neuropathies and central nervous system axon injury, we found that NFATc4 is specifically but transiently up-regulated in response to mechanical injury. In the injured retina, NFATc4 immunolocalized primarily to the ganglionic cell layer. Utilizing NFATc4-/- and NFATc3-/- mice, we demonstrated that NFATc4, but not NFATc3, knockout increased RGC survival, improved retina function, and delayed axonal degeneration. Microarray screening data, along with decreased immunostaining of cleaved caspase-3, revealed that NFATc4 knockout was protective against ONC-induced degeneration by suppressing pro-apoptotic signaling. Finally, we used lentiviral-mediated NFATc4 delivery to the retina of NFATc4-/- mice and reversed the pro-survival effect of NFATc4 knockout, conclusively linking the enhanced survival of injured RGCs to NFATc4-dependent mechanisms. In summary, this study is the first to demonstrate that NFATc4 knockout may confer transient RGC neuroprotection and decelerate axonal degeneration after injury, providing a potent therapeutic strategy for optic neuropathies.

RIP1 inhibition protects retinal ganglion cells in glaucoma models of ocular injury

AbstractReceptor-interacting protein 1 (RIP1, RIPK1) is a critical mediator of multiple signaling pathways that promote inflammatory responses and cell death. The kinase activity of RIP1 contributes to the pathogenesis of a number of inflammatory and neurodegenerative diseases. However, the role of RIP1 in retinopathies remains unclear. This study demonstrates that RIP1 inhibition protects retinal ganglion cells (RGCs) in preclinical glaucoma models. Genetic inactivation of RIP1 improves RGC survival and preserves retinal function in the preclinical glaucoma models of optic nerve crush (ONC) and ischemia–reperfusion injury (IRI). In addition, the involvement of necroptosis in ONC and IRI glaucoma models was examined by utilizing RIP1 kinase-dead (RIP1-KD), RIP3 knockout (RIP3-KO), and MLKL knockout (MLKL-KO) mice. The number of RGCs, retinal thickness, and visual acuity were rescued in RIP1-kinase-dead (RIP1-KD) mice in both models, while wild-type (WT) mice experienced significant retinal thinning, RGC loss, and vision impairment. RIP3-KO and MLKL-KO mice showed moderate protective effects in the IRI model and limited in the ONC model. Furthermore, we confirmed that a glaucoma causative mutation in optineurin, OPTN-E50K, sensitizes cells to RIP1-mediated inflammatory cell death. RIP1 inhibition reduces RGC death and axonal degeneration following IRI in mice expressing OPTN-WT and OPTN-E50K variant mice. We demonstrate that RIP1 inactivation suppressed microglial infiltration in the RGC layer following glaucomatous damage. Finally, this study highlights that human glaucomatous retinas exhibit elevated levels of TNF and RIP3 mRNA and microglia infiltration, thus demonstrating the role of neuroinflammation in glaucoma pathogenesis. Altogether, these data indicate that RIP1 plays an important role in modulating neuroinflammation and that inhibiting RIP1 activity may provide a neuroprotective therapy for glaucoma.

In Vitro and In Vivo Methods for Studying Retinal Ganglion Cell Survival and Optic Nerve Regeneration.

Glaucoma is marked by a progressive degeneration of the optic nerve and delayed loss of retinal ganglion cells (RGCs), the projection neurons of the eye. Because RGCs are not replaced and because surviving RGCs cannot regenerate their axons, the visual loss in glaucoma is largely irreversible. Here we describe methods to evaluate treatments that may be beneficial for treating glaucoma using in vitro cell culture models (immunopanning to isolate neonatal RGCs, dissociated mature retinal neurons, retinal explants) and in vivo models that test potential treatments or investigate underlying molecular mechanisms in an intact system. Potentially, the use of these models can help investigators continue to improve treatments to preserve RGCs and restore visual function in patients with glaucoma.

The broad-spectrum deubiquitinating enzyme inhibitor PR-619 protects retinal ganglion cell and augments parkin-mediated mitophagy in experimental glaucoma

Glaucoma is a leading cause of irreversible visual impairment worldwide, characterized by the progressive death of retinal ganglion cells (RGCs). Deubiquitinating enzyme (DUB) inhibitors have shown promise as pharmacological interventions for neurodegenerative disorders. Our study focuses on the pan-DUB inhibitor PR-619 and its potential neuroprotective effects on RGCs through modulation of parkin-mediated mitophagy in experimental glaucoma models. The results show that impaired mitophagy exists in RGCs of our experimental glaucomatous model. In vivo, PR-619 increased RGCs survival in glaucomatous rats. In vitro, it protected RGCs against excitotoxicity and reduced ubiquitin-specific protease (USP) 15 expression. Additionally, PR-619 upregulated parkin expression, increased LC3-II/LC3-I ratios, and elevated LAMP1 levels, indicating enhanced mitophagy in vivo and in vitro. Moreover, numbers of mitophagosomes were increased in optic nerves of PR-619-treated ocular hypertensive rats in vivo. Furthermore, parkin knockdown negated the salutary effects of PR-619 and attenuated expression of parkin-dependent mitophagy effectors in RGCs subjected to glutamate excitotoxicity in vitro. Collectively, these findings implicate augmented parkin-mediated mitophagy as the mechanistic substrate underscoring the neuroprotective capacity of PR-619 in experimental glaucoma. These revelations engender the prospect that pharmacological agents or biotherapeutics augmenting parkin-mediated mitophagy may proffer viable therapeutic modalities for glaucomatous neurodegeneration characterized by impaired mitophagy.

AIBP Protects Müller Glial Cells Against Oxidative Stress-Induced Mitochondrial Dysfunction and Reduces Retinal Neuroinflammation.

Glaucoma, an optic neuropathy with the loss of retinal ganglion cells (RGCs), is a leading cause of irreversible vision loss. Oxidative stress and mitochondrial dysfunction have a significant role in triggering glia-driven neuroinflammation and subsequent glaucomatous RGC degeneration in the context of glaucoma. It has previously been shown that apolipoprotein A-I binding protein (APOA1BP or AIBP) has an anti-inflammatory function. Moreover, Apoa1bp-/- mice are characterized by retinal neuroinflammation and RGC loss. In this study, we found that AIBP deficiency exacerbated the oxidative stress-induced disruption of mitochondrial dynamics and function in the retina, leading to a further decline in visual function. Mechanistically, AIBP deficiency-induced oxidative stress triggered a reduction in glycogen synthase kinase 3β and dynamin-related protein 1 phosphorylation, optic atrophy type 1 and mitofusin 1 and 2 expression, and oxidative phosphorylation, as well as the activation of mitogen-activated protein kinase (MAPK) in Müller glia dysfunction, leading to cell death and inflammatory responses. In vivo, the administration of recombinant AIBP (rAIBP) effectively protected the structural and functional integrity of retinal mitochondria under oxidative stress conditions and prevented vision loss. In vitro, incubation with rAIBP safeguarded the structural integrity and bioenergetic performance of mitochondria and concurrently suppressed MAPK activation, apoptotic cell death, and inflammatory response in Müller glia. These findings support the possibility that AIBP promotes RGC survival and restores visual function in glaucomatous mice by ameliorating glia-driven mitochondrial dysfunction and neuroinflammation.

IL-33 protects retinal structure and function via mTOR/S6 signaling pathway in optic nerve crush

This study demonstrated the functions and molecular mechanisms of the IL-33/ST2 axis in experimental optic neuropathy. C57BL/6J mice were used to establish an optic nerve crush (ONC) model. ONC mice were administered with IL-33 intraperitoneal injection, with PBS vehicle as control. Immunofluorescence, quantitative RT-PCR, and western blot techniques were utilized to assess the expression of the IL-33/ST2 axis. The electroretinography (ERG), optical coherence tomography (OCT), H&E, and luxol fast blue were used to assess the structural and functional changes. Western blot was employed to detect the activation of the mTOR/S6 pathway. The IL-33 expression level in the inner nuclear layer of the retina in ONC mice reached its peak on day 3, accompanied by a significant increase in IL-33 receptor ST2 expression. IL-33 treatment promoted the survival of retinal ganglion cells, restored the thickness of inner retina layer (IRL), alleviated the demyelination of the optic nerve, and recovered the decreased amplitude of b-wave in ONC mice. Furthermore, administration of IL-33 activated the mTOR/S6 signaling pathway in RGCs, which was significantly suppressed in the ONC condition. This study indicated that boosting the IL-33/ST2/mTOR/S6 pathway can protect against structural and functional damage to the retina and optic nerve induced by ONC. As a result, the IL-33/ST2 axis holds potential as a therapeutic option for treating various optic neuropathies.

Transcriptional responses in a mouse model of silicone wire embolization induced acute retinal artery ischemia and reperfusion.

Acute retinal ischemia and ischemia-reperfusion injury are the primary causes of retinal neural cell death and vision loss in retinal artery occlusion (RAO). The absence of an accurate mouse model for simulating the retinal ischemic process has hindered progress in developing neuroprotective agents for RAO. We developed a unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model using silicone wire embolization combined with carotid artery ligation. The survival of retinal ganglion cells and visual function were evaluated to determine the duration of ischemia. Immunofluorescence staining, optical coherence tomography, and haematoxylin and eosin staining were utilized to assess changes in major neural cell classes and retinal structure degeneration at two reperfusion durations. Transcriptomics was employed to investigate alterations in the pathological process of UPOAO following ischemia and reperfusion, highlighting transcriptomic differences between UPOAO and other retinal ischemia-reperfusion models. The UPOAO model successfully replicated the acute interruption of retinal blood supply observed in RAO. 60 min of Ischemia led to significant loss of major retinal neural cells and visual function impairment. Notable thinning of the inner retinal layer, especially the ganglion cell layer, was evident post-UPOAO. Temporal transcriptome analysis revealed various pathophysiological processes related to immune cell migration, oxidative stress, and immune inflammation during the non-reperfusion and reperfusion periods. A pronounced increase in microglia within the retina and peripheral leukocytes accessing the retina was observed during reperfusion periods. Comparison of differentially expressed genes (DEGs) between the UPOAO and high intraocular pressure models revealed specific enrichments in lipid and steroid metabolism-related genes in the UPOAO model. The UPOAO model emerges as a novel tool for screening pathogenic genes and promoting further therapeutic research in RAO.

Transcriptional responses in a mouse model of silicone wire embolization induced acute retinal artery ischemia and reperfusion

Acute retinal ischemia and ischemia-reperfusion injury are the primary causes of retinal neural cell death and vision loss in retinal artery occlusion (RAO). The absence of an accurate mouse model for simulating the retinal ischemic process has hindered progress in developing neuroprotective agents for RAO. We developed a unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model using silicone wire embolization combined with carotid artery ligation. The survival of retinal ganglion cells and visual function were evaluated to determine the duration of ischemia. Immunofluorescence staining, optical coherence tomography, and haematoxylin and eosin staining were utilized to assess changes in major neural cell classes and retinal structure degeneration at two reperfusion durations. Transcriptomics was employed to investigate alterations in the pathological process of UPOAO following ischemia and reperfusion, highlighting transcriptomic differences between UPOAO and other retinal ischemia-reperfusion models. The UPOAO model successfully replicated the acute interruption of retinal blood supply observed in RAO. 60 min of Ischemia led to significant loss of major retinal neural cells and visual function impairment. Notable thinning of the inner retinal layer, especially the ganglion cell layer, was evident post-UPOAO. Temporal transcriptome analysis revealed various pathophysiological processes related to immune cell migration, oxidative stress, and immune inflammation during the non-reperfusion and reperfusion periods. A pronounced increase in microglia within the retina and peripheral leukocytes accessing the retina was observed during reperfusion periods. Comparison of differentially expressed genes (DEGs) between the UPOAO and high intraocular pressure models revealed specific enrichments in lipid and steroid metabolism-related genes in the UPOAO model. The UPOAO model emerges as a novel tool for screening pathogenic genes and promoting further therapeutic research in RAO.

Pharmacological Advances in Glaucoma Management

Glaucoma is the leading cause of global irreversible blindness, with clinical diagnosis related to population-based low vision standards and subsequently to diminished vision-related quality of life. The main objective of the present study is to review the updates regarding pharmacological advances in glaucoma management. Pharmacological management to reduce intraocular pressure and inflammation and improve retinal ganglion cell survival has a significant impact on the overall management of glaucoma. The changes in the therapeutic pipelines include the targeting of both traditional and novel molecules. New medications in the offing either improve the already established targets or focus on newer avenues to confer better anti-glaucomatous effects with minimal side effects.

Bioengineering strategy to promote CNS nerve growth and regeneration via chronic glutamate signaling

Being part of the mature mammalian central nervous system, impairments of the retina and optic nerves caused by trauma or diseases often cannot be restored. Progressive degeneration of retinal ganglion cells (RGCs) in glaucoma and other optic neuropathies gradually leads to permanent vision loss, which currently has no cure. The purpose of this study is to develop a biocompatible scaffold to support RGC survival and guide axon growth, facilitating optic nerve repair and regeneration. We here report that electrical stimulation (ES) significantly promoted neurite outgrowth and elongation from primary RGCs, mediated through glutamate receptor signaling. To mimic prolonged glutamate stimulation and facilitate sustained nerve growth, we fabricated biocompatible poly-γ-benzyl-L-glutamate (PBG) scaffolds for controlled glutamate release. These PBG scaffolds supported RGC survival and robust long-distance nerve growth in both retinal explants and isolated RGC cultures. In contrast, control polycaprolactone (PCL) scaffolds with similar physical structures showed little benefits on RGC survival or nerve growth. Moreover, PBG scaffolds promoted the differentiation and neurite outgrowth from embryonic stem cell-derived RGC progenitors. The aligned PBG scaffold drove directed nerve elongation along the fiber alignment. Transplantation of PBG-coated biocompatible conduits induced robust optic nerve regeneration in adult mice following nerve transection. Together, the findings present the exciting possibility of driving optic nerve regeneration and RGC progenitor cell differentiation by imitating ES or glutamate signaling. PBG presents a permissive biomaterial in supporting robust and directed axon growth with promising clinical applications in the future. Statement of SignificanceWe here reported compelling findings that demonstrate the potent regenerative effects of a bioengineered scaffold incorporating poly-γ-benzyl-L-glutamate (PBG) on the optic nerve. Retinal ganglion cell (RGC) axons, which form the optic nerve, are incapable of regenerating in adulthood, posing a significant hurdle in restoring vision for patients with optic nerve diseases or injuries. Built upon the finding that electrical stimulation promotes RGC axonal growth through glutamate signaling, we developed PBG scaffolds to provide sustained glutamate stimulation and showed their exceptional effects on driving directed axonal elongation in cultured RGCs and neural progenitors, as well as supporting robust optic nerve regeneration after transection in vivo. The findings hold great promise for reversing vision loss in patients with optic nerve conditions.

The influence of polyacrylamide gel substrate elasticity on primary cultures of rat retinal ganglion cells

In vitro primary cell culture models of retinal ganglion cells (RGC) are widely used to study pathomechanisms of diseases such as glaucoma. The biomechanic interaction with the culture substrate is known to influence core cellular functions. RGC cultures, however, are usually grown on rigid plastic or glass substrates. We hypothesized that soft polyacrylamide gel substrates may alter survival and neurite outgrowth of primary cultured RGC.Primary retinal cultures from postnatal (day 1-6) Wistar rats were grown on glass coverslips or polyacrylamide (PA) gel substrate with different Young’s elastic moduli (0.75, 10 or 30 kPa). Substrates were coated with Poly-l-lysine and / or laminin. RGC were immunostained with anti-beta-III-tubulin. Total neurite length, growth cone morphology, RGC density, mitochondrial morphology and transport as well as pro-survival pathways (Erk1/2, Akt, CREB) were assessed.PA gel substrates of E = 10 kPa significantly increased the total neurite length by factor 1.5 compared to glass (p = 0.02). The growth cone area was significantly larger by factor 5.3 on 30 kPa gels (p = 0.01). The presence of a substrate coating was more important for neurite outgrowth and RGC survival on PA gels (poly-l-lysine > laminin) than on glass. Neither mitochondrial morphology and motility nor the activation of pro-survival pathways significantly differed between the four substrates.PA gel substrates significantly enhanced RGC neurite outgrowth. The signaling cascades mediating this effect remain to be determined.

Roles of Kdm6a and Kdm6b in regulation of mammalian neural regeneration.

Epigenetic regulation of neuronal transcriptomic landscape is emerging to be a key coordinator of mammalian neural regeneration. Here we investigated roles of two histone 3 lysine 27 (H3K27) demethylases Kdm6a/b in controlling neuroprotection and axon regeneration. Deleting either Kdm6a or Kdm6b led to enhanced sensory axon regeneration in PNS, whereas in the CNS only deleting Kdm6a in retinal ganglion cells (RGCs) significantly enhanced optic nerve regeneration. Moreover, both Kdm6a and Kdm6b functioned to regulate RGC survival but with different mechanisms. Mechanistically, Kdm6a regulates RGC regeneration via distinct pathway from that of Pten and co-deleting Kdm6a and Pten resulted in long distance optic nerve regeneration passing the optic chiasm. In addition, RNA-seq profiling revealed that Kdm6a deletion switched the RGC transcriptomics into a developmental-like state and suppressed several known repressors of neural regeneration. Klf4 was identified as a direct downstream target of Kdm6a-H3K27me3 signaling in both sensory neurons and RGCs to regulate axon regeneration. These findings not only revealed different roles of Kdm6a and Kdm6b in regulation of neural regeneration and their underlying mechanisms, but also identified Kdm6a-mediated histone demethylation signaling as a novel epigenetic target for supporting CNS neural regeneration.

Evaluation of Rho kinase inhibitor effects on neuroprotection and neuroinflammation in an ex-vivo retinal explant model

BackgroundGlaucoma is a leading cause of blindness, affecting retinal ganglion cells (RGCs) and their axons. By 2040, it is likely to affect 110 million people. Neuroinflammation, specifically through the release of proinflammatory cytokines by M1 microglial cells, plays a crucial role in glaucoma progression. Indeed, in post-mortem human studies, pre-clinical models, and ex-vivo models, RGC degeneration has been consistently shown to be linked to inflammation in response to cell death and tissue damage. Recently, Rho kinase inhibitors (ROCKis) have emerged as potential therapies for neuroinflammatory and neurodegenerative diseases. This study aimed to investigate the potential effects of three ROCKis (Y-27632, Y-33075, and H-1152) on retinal ganglion cell (RGC) loss and retinal neuroinflammation using an ex-vivo retinal explant model.MethodsRat retinal explants underwent optic nerve axotomy and were treated with Y-27632, Y-33075, or H-1152. The neuroprotective effects on RGCs were evaluated using immunofluorescence and Brn3a-specific markers. Reactive glia and microglial activation were studied by GFAP, CD68, and Iba1 staining. Flow cytometry was used to quantify day ex-vivo 4 (DEV 4) microglial proliferation and M1 activation by measuring the number of CD11b+, CD68+, and CD11b+/CD68+ cells after treatment with control solvent or Y-33075. The modulation of gene expression was measured by RNA-seq analysis on control and Y-33075-treated explants and glial and pro-inflammatory cytokine gene expression was validated by RT-qPCR.ResultsY-27632 and H-1152 did not significantly protect RGCs. By contrast, at DEV 4, 50 µM Y-33075 significantly increased RGC survival. Immunohistology showed a reduced number of Iba1+/CD68+ cells and limited astrogliosis with Y-33075 treatment. Flow cytometry confirmed lower CD11b+, CD68+, and CD11b+/CD68+ cell numbers in the Y-33075 group. RNA-seq showed Y-33075 inhibited the expression of M1 microglial markers (Tnfα, Il-1β, Nos2) and glial markers (Gfap, Itgam, Cd68) and to reduce apoptosis, ferroptosis, inflammasome formation, complement activation, TLR pathway activation, and P2rx7 and Gpr84 gene expression. Conversely, Y-33075 upregulated RGC-specific markers, neurofilament formation, and neurotransmitter regulator expression, consistent with its neuroprotective effects.ConclusionY-33075 demonstrates marked neuroprotective and anti-inflammatory effects, surpassing the other tested ROCKis (Y-27632 and H-1152) in preventing RGC death and reducing microglial inflammatory responses. These findings highlight its potential as a therapeutic option for glaucoma.

FK962 protects retinal ganglion cell under hypoxia/reoxygenation: Possible involvement of glial cell line-derived neurotrophic factor signaling pathway

Loss of retinal ganglion cells (RGCs) is the cause of visual impairment and blindness in glaucoma. Previously, our studies showed that FK962 (N-[1-acetylpiperidin-4-yl]-4-fluorobenzamide) promoted neurite elongation in rat RGCs and trigeminal ganglion (TG) cells. In TG cells, glial cell line-derived neurotrophic factor (GDNF) is known to be involved in the mechanism. The purpose of the present study is to investigate whether, 1) FK962 shows an RGC-protective effect under hypoxia/reoxygenation (H/R) and 2) GDNF is involved in the neuroprotective mechanism of FK962. Rat primary retinal cells were cultured under 24-h hypoxia/24-h reoxygenation conditions, with or without FK962, recombinant GDNF, GDNF antibody and RET receptor tyrosine kinase inhibitor, GSK3179106. Cells were co-immunostained with RBPMS and Neurofilament 200 as a RGC marker, and the number of survived RGCs was counted. Results showed H/R treatment decreased the number of survived RGCs. FK962 promoted RGC survival under H/R by a bell-shaped dose response, with the highest RGC-protective effect of 10−8 M. The protective effect was the same level with 10−12 M exogenous GDNF. Addition of GDNF antibody or GSK3179106 counteracted the neuroprotective effect of FK962. From these results, it is suggested that FK962 ameliorates RGC death under H/R, possibly via a GDNF signaling pathway.

Pharmacological Activation and Transgenic Overexpression of SIRT1 Attenuate Traumatic Optic Neuropathy Induced by Blunt Head Impact.

Resveratrol (RSV) is a nutraceutical compound known for its therapeutic potential in neurodegenerative and metabolic diseases. RSV promotes survival signals in retinal ganglion cells (RGCs) through activation of SIRT1, an NAD+-dependent deacetylase. RSV and SIRT1 reduce RGC loss induced by direct optic nerve injury, but effects in indirect models of traumatic optic neuropathy remain unknown and are examined in this study. An electromagnetic stereotaxic impactor device was used to impart five traumatic skull impacts with an inter-concussion interval of 48 hours to wild type (WT) and SIRT1 knock in (KI) C57BL/6J mice overexpressing the SIRT1 gene. A cohort of WT mice also received intranasal administration of RSV (16mg/kg) throughout the experimental period. Loss of righting reflex (RR), optokinetic response (OKR) scores, and immunolabeled RGC count are determined to assess optic neuropathy in this model of traumatic brain injury (TBI). TBI significantly decreases RGC survival and decreases OKR scores compared with control uninjured mice. Either RSV administration in WT mice, or SIRT1 overexpression in SIRT1 KI mice, significantly increases RGC survival and improves OKR scores. RR time increases after the first few impacts in all groups of mice subjected to TBI, demonstrating that RSV and SIRT1 overexpression are able to attenuate optic neuropathy following similar degrees of TBI. Intranasal RSV is effective in preserving visual function in WT mice following TBI. Constitutive overexpression of SIRT1 recapitulates the neuroprotective effect of RSV. Results support future exploration of RSV as a potential therapy for indirect traumatic optic neuropathy.

Age-related effects of optineurin deficiency in the mouse eye

Optineurin (OPTN) is a gene associated with familial normal tension glaucoma (NTG). While NTG involves intraocular pressure (IOP)-independent neurodegeneration of the visual pathway that progresses with age, how OPTN dysfunction leads to NTG remains unclear. Here, we generated an OPTN knockout mouse (Optn−/−) model to test the hypothesis that a loss-of-function mechanism induces structural and functional eye deterioration with aging. Eye anatomy, visual function, IOP, retinal histology, and retinal ganglion cell survival were compared to littermate wild-type (WT) control mice. Consistent with OPTN’s role in NTG, loss of OPTN did not increase IOP or alter gross eye anatomy in young (2–3 months) or aged (12 months) mice. When retinal layers were quantitated, young Optn−/− mice had thinner retina in the peripheral regions than young WT mice, primarily due to thinner ganglion cell-inner plexiform layers. Despite this, visual function in Optn−/− mice was not severely impaired, even with aging. We also assessed relative abundance of retinal cell subtypes, including amacrine cells, bipolar cells, cone photoreceptors, microglia, and astrocytes. While many of these cellular subtypes were unaffected by Optn deletion, more dopaminergic amacrine cells were observed in aged Optn−/− mice. Taken together, our findings showed that complete loss of Optn resulted in mild retinal changes and less visual function impairment, supporting the possibility that OPTN-associated glaucoma does not result from a loss-of-function disease mechanism. Further research using these Optn mice will elucidate detailed molecular pathways involved in NTG and identify clinical or environmental risk factors that can be targeted for glaucoma treatment.