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

BACKGROUND: Glaucoma 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. METHODS: Rat 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. RESULTS: Y-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-1ß, 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. CONCLUSION: Y-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.

Three-dimensional Microarchitecture of Lamina Cribrosa Pores in High and Normal-tension Glaucoma using Optical Coherence Tomography.

PRCIS: The lamina cribrosa pores of high-tension glaucoma subjects appear to take a more tortuous pathway than the LC pores of non-glaucomatous subjects. PURPOSE: To compare the lamina cribrosa pore microarchitecture in high-tension glaucoma (HTG), normal-tension glaucoma (NTG) and in non-glaucomatous (NG) subjects, by reconstructions of the lamina cribrosa made from tomographic images. PATIENTS AND METHODS: SD-OCT images of 52 eyes (18 NG, 18 HTG, 16 NTG) of 29 patients were analyzed. Pores were traced using segmentation software. Pore length, tortuosity and verticality were the three quantitative parameters compared between the three groups. Correlation analyses were performed to determine the effects of covariates on the three quantitative parameters. RESULTS: Pore tortuosity in HTG (1.419 +/- 0.093) was significantly higher (P=0.011) than in NG (1,347 +/- 0,034) but did not differ from that of NTG eyes (P=0.251). In addition, NTG had significantly shorter pores (P=0.005) than NG. No difference in pore tortuosity or verticality was found between NG and NTG (P=0.587 and P=0.120 respectively). Pore verticality and length in HTG eyes did not significantly differ from that of NG eyes (P=0.049 and P=0.033 respectively) and NTG eyes (P=0.827 and P=0.968 respectively). All of the quantitative parameters measured were not correlated with age, but were associated with glaucoma severity (VFI, MD, RNFL, GCC), except for pore verticality which was not correlated with RNFL. CONCLUSION: The LC pores of HTG subjects appear to be more tortuous than the pores of NG subjects and the pores of NTG patients are shorter than those of NG subjects. Changes in pore parameters appear to be associated with severity of the glaucomatous optic neuropathy.

The PRAMOS Study: PRostaglandin Analogues Monotherapy-Awareness Survey on Ocular Surface Involvement.

INTRODUCTION: Even though the local tolerance of prostaglandin (PG) analogues has improved drastically since the introduction of preservative-free (PF) eye drops, prescription patterns still vary widely among practitioners and between countries and could have an impact on the ocular surface of treated patients and, in consequence, their adherence. The aim of this study is to explore the prescribing patterns of PG analogues monotherapy in France and to evaluate their impact on ocular surface status. METHODS: This was a national multicenter cross-sectional observational study that was conducted by 18 glaucoma experts in France. Patients over 18 years of age and receiving monotherapy with topical PG analogues for the treatment of ocular hypertension and/or glaucoma, with no history of prior glaucoma surgery, were consecutively selected from the glaucoma outpatient clinics of participating physicians and underwent an ocular surface examination. RESULTS: A total of 344 eyes of 344 patients were enrolled between November 2022 and November 2023. Prescribed PG monotherapy was PF in 271 (78.7%) patients. Clinical history and ocular surface evaluation indicated that 79.4% of the study population (n = 273) presented with at least one symptom or clinical sign of dry eye and that three patients out of four had an unstable tear film. Subgroup analysis comparing preserved and PF PG analogues showed a higher prevalence of conjunctival hyperemia and corneal staining in the preserved group. Multivariate analysis identified conjunctival hyperemia as consistently associated with preservative use (odds ratio = 7.654; p = 0.003 for moderate conjunctival hyperemia). CONCLUSIONS: This study highlights the growing trend toward PF PG analogue prescriptions by specialists in France. However, ocular surface issues remain prevalent, impacting patient adherence and treatment efficacy. Comprehensive ocular surface examinations are crucial in glaucoma management to enhance long-term tolerance, compliance, and overall treatment success.