Evaluation of neuroprotective and immunomodulatory properties of mesenchymal stem cells in an ex vivo retinal explant model.

BACKGROUND: Glaucoma is a blinding degenerative neuropathy in which the death of retinal ganglion cells (RGCs) causes progressive loss of visual field and eventually vision. Neuroinflammation appears to be a key event in the progression and spread of this disease. Thus, microglial immunomodulation represents a promising therapeutic approach in which mesenchymal stem cells (MSCs) might play a crucial role. Their neuroprotective and regenerative potentials have already raised hope in animal models. Yet no definitive treatment has been developed, and some safety concerns have been reported in human trials. In the present study, we investigated the neuroprotective and immunomodulatory properties as well as the safety of MSCs in an ex vivo neuroretina explant model. METHODS: Labeled rat bone marrow MSCs were placed in coculture with rat retinal explants after optic nerve axotomy. We analyzed the neuroprotective effect of MSCs on RGC survival by immunofluorescence using RBPMS, Brn3a, and NeuN markers. Gliosis and retinal microglial activation were measured by using GFAP, CD68, and ITGAM mRNA quantification and GFAP, CD68, and Iba1 immunofluorescence stainings. We also analyzed the mRNA expression of both 'M1' or classically activated state inflammatory cytokines (TNFα, IL1ß, and IL6), and 'M2' or alternatively activated state microglial markers (Arginase 1, IL10, CD163, and TNFAIP6). RESULTS: The number of RGCs was significantly higher in retinal explants cultured with MSCs compared to the control group at Day 7 following the optic nerve axotomy. Retinal explants cultured with MSCs showed a decrease in mRNA markers of gliosis and microglial activations, and immunostainings revealed that GFAP, Iba1, and CD68 were limited to the inner layers of the retina compared to controls in which microglial activation was observed throughout the retina. In addition, MSCs inhibited the M1 phenotype of the microglia. However, edema of the explants was observed in presence of MSCs, with an increase in fibronectin labeling at the surface of the explant corresponding to an epiretinal membrane-like phenotype. CONCLUSION: Using an ex vivo neuroretina model, we demonstrated a neuroprotective and immunomodulatory effect of MSCs on RGCs. Unfortunately, the presence of MSCs also led to explant edema and epiretinal membrane formation, as described in human trials. Using the MSC secretome might offer the beneficial effects of MSCs without their potential adverse effects, through paracrine signaling.

PreserFlo MicroShunt® exposure: a case series.

BACKGROUND: PreserFlo® MicroShunt (PM) (also known as InnFocus® MicroShunt) is a subconjunctival stent implanted ab externo via a minimally invasive surgical procedure. The current indication is progressive, mild to moderate, open angle glaucoma uncontrolled on topical medications. According to the literature, adverse events are rare, mild and transient. CASE PRESENTATION: Two cases of stand-alone PreserFlo MicroShunt® implantation in patients with uncontrolled open-angle glaucoma are reported. Exposure occurred 7 days and 3 months respectively after implantation. These cases shared common features including preexisting blepharitis and the lack of a Tenon's flap. In both cases, removal of the device was required after several attempts at repair. CONCLUSIONS: PreserFlo MicroShunt® exposure is a potentially vision-threatening complication because of the risk of endophthalmitis. Potential risk factors include the absence of a Tenon's flap and pre-existing ocular surface inflammation. Ocular surface inflammation should be detected and treated prior to PM implantation. If a deficiency in Tenon's capsule is noted intraoperatively, close monitoring should be performed because of the higher risk of PM exposure.

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.

An Overview of Current Glaucomatous Trabecular Meshwork Models.

PURPOSE: To provide an overview of the existing alternative models for studying trabecular meshwork (TM). METHODS: Literature review. RESULTS: The TM is a complex tissue that regulates aqueous humor outflow from the eye. Dysfunction of the TM is a major contributor to the pathogenesis of open-angle glaucoma, a leading cause of irreversible blindness worldwide. The TM is a porous structure composed of trabecular meshwork cells (TMC) within a multi-layered extracellular matrix (ECM). Although dysregulation of the outflow throughout the TM represents the first step in the disease process, the underlying mechanisms of TM degeneration associate cell loss and accumulation of ECM, but remain incompletely understood, and drugs targeting the TM are limited. Therefore, experimental models of glaucomatous trabeculopathy are necessary for preclinical screening, to advance research on this disease's pathophysiology, and to develop new therapeutic strategies targeting the TM. Traditional animal models have been used extensively, albeit with inherent limitations, including ethical concerns and limited translatability to humans. Consequently, there has been an increasing focus on developing alternative in vitro models to study the TM. Recent advancements in three-dimensional cell culture and tissue engineering are still in their early stages and do not yet fully reflect the complexity of the outflow pathway. However, they have shown promise in reducing reliance on animal experimentation in certain aspects of glaucoma research. CONCLUSION: This review provides an overview of the existing alternative models for studying TM and their potential for advancing research on the pathophysiology of open-angle glaucoma and developing new therapeutic strategies.

EYERUBBICS: The Eye Rubbing Cycle Study.

Purpose: Describe the cognitive and behavioral symptomatology of patients with chronic ocular rubbing in keratoconus (KC) and Ocular Surface Disease (OSD) using a self-questionnaire. Methods: A prospective study was conducted in a tertiary ophthalmology center between May and July 2021. We consecutively included all patients presenting with one of the following conditions: KC and OSD. A questionnaire including the evaluation of Goodman and CAGE-modified criteria for eye rubbing was given to patients in consultation to evaluate their ocular symptoms and medical background. Results: We included 153 patients in the study. Of these, 125 (81.7%) patients reported eye rubbing. The average Goodman score was 5.8 ± 3.1 and was ≥ 5 in 63.2% of cases. The CAGE score was ≥ 2 in 74.4% of patients. Addiction (p = 0.045) and psychiatric family history (p = 0.03) were more frequent in patients with higher scores. Ocular symptoms and eye rubbing were significantly more frequent and intense in patients with higher scores; Conclusion: Eye rubbing presents addictive-like cognitive and behavioral characteristics in patients with KC or OSD. The eye rubbing cycle could play an essential role in the onset and progression of keratoconus and could be a factor in the maintenance of dry eye.

The Dual Effect of Rho-Kinase Inhibition on Trabecular Meshwork Cells Cytoskeleton and Extracellular Matrix in an In Vitro Model of Glaucoma.

The trabecular meshwork (TM) is the main site of drainage of the aqueous humor, and its dysfunction leads to intraocular pressure elevation, which is one of the main risk factors of glaucoma. We aimed to compare the effects on cytoskeleton organization and extracellular matrix (ECM) of latanoprost (LT) and a Rho-kinase inhibitor (ROCKi) on a transforming growth factor beta2 (TGF-ß2)-induced glaucoma-like model developed from primary culture of human TM cells (pHTMC). The TGF-ß2 stimulated pHTMC were grown and incubated with LT or a ROCKi (Y-27632) for 24 h. The expression of alpha-smooth muscle actin (αSMA) and fibronectin (FN), and phosphorylation of the myosin light chain (MLC-P) and Cofilin (Cofilin-P) were evaluated using immunofluorescence and Western blot. The architectural modifications were studied in a MatrigelTM 3D culture. TGF-ß2 increased the expression of αSMA and FN in pHTMC and modified the cytoskeleton with cross-linked actin network formation. LT did not alter the expression of αSMA but decreased FN deposition. The ROCKi decreased TGF-ß2-induced αSMA and FN expression, as well as MLC-P and Cofilin-P, and stimulated the cells to recover a basal cytoskeletal arrangement. In the preliminary 3D study, pHTMC organized in a mesh conformation showed the widening of the TM under the effect of Y-27632. By simultaneously modifying the organization of the cytoskeleton and the ECM, with fibronectin deposition and overexpression, TGF-ß2 reproduced the trabecular degeneration described in glaucoma. The ROCKi was able to reverse the TGF-ß2-induced cytoskeletal and ECM rearrangements. LT loosened the extracellular matrix but had no action on the stress fibers.

Is the Xen® Gel Stent really minimally invasive?

PURPOSE: To describe a case of severe Streptococcus pneumoniae endophthalmitis in a patient with a cystic, avascular filtering bleb who had been implanted with a Xen® Gel Stent 21 months previously. OBSERVATIONS: A 64-year-old woman with open-angle glaucoma developed severe endophthalmitis 21 months after Xen® Gel Stent implantation. On presentation, visual acuity was limited to light perception. Examination revealed a 100% hypopyon, blebitis and an exposed stent, along with orbital cellulitis. Immediate explantation of the exposed Xen® was performed, and intravitreal antibiotics were administered. S. pneumoniae was isolated from an anterior chamber paracentesis. Based on the antibiogram, the patient was treated with topical fortified piperacillin, gentamicin and vancomycin along with appropriate systemic antibiotics (intravenous imipenem and oral levofloxacin). After 3 days of antibiotics, she received a daily intravenous bolus of methylprednisolone at a dose of 1 mg/kg/day for three days. Despite these measures, the patient's condition declined, with purulent melting of the globe requiring evisceration. CONCLUSIONSAND IMPORTANCE: As for other filtering surgeries, blebitis and severe endophthalmitis can occur after Xen® Gel Stent implantation. Patients with thin conjunctiva and/or cystic blebs over the stent should be followed particularly closely.

Assessment of corneal epithelial thickness mapping in epithelial basement membrane dystrophy.

PURPOSE: To investigate the corneal epithelial thickness topography with optical coherence tomography (OCT) and its relationship with vision quality in epithelial basement membrane dystrophy (EBMD). METHODS: 45 eyes of EBMD patients, 26 eyes of dry eye (DED) patients and 22 eyes of normal subjects were enrolled. All participants were subjected to 9-mm corneal epithelial mapping with OCT and vision quality was assessed with the optical quality analysis system using the objective scatter index (OSI). Central, superior, inferior, minimum, maximum, and standard deviation of epithelium thickness (Irregularity), were analysed and correlations with the OSI were calculated. RESULTS: The mean (±SD) central, inferior and maximum epithelial thicknesses of the EBMD patients (respectively, 56.4 (±8.1) µm, 58.9 (±6.4) µm, and 67.1 (±8.3) µm) were thicker compared to DED patients (P<0.05) and normal subjects (P<0.05). We found greater irregularity of epithelial thickness in EBMD (5.1±2.5 µm) compared to DED patients (2.6±1.0 µm) (P = 4.4.10-6) and normal subjects (2.1±0.7 µm) (P = 7.6.10-7). The mean OSI was worse in EBMD patients than in DED patients (P = 0.01) and compared to normal subjects (P = 0.02). The OSI correlated with the epithelial thickness irregularity (Spearman coefficient = 0.54; P = 2.65.10-5). CONCLUSIONS: The OCT pachymetry map demonstrated that EBMD patients had thicker corneal epithelium in the central and inferior region. These changes were correlated with objective measurements of vision quality. This OCT characterisation of the EMBD provides a better understanding of the epithelial behaviour in this dystrophy and its role in vision quality.

Efficacy and safety at 6 months of the XEN implant for the management of open angle glaucoma.

The purpose of this study is to evaluate the efficacy and complications of the XEN implant as a solo procedure or in association with cataract surgery in patients with open angle glaucoma (OAG). All patients who received a XEN implant between June 2017 and June 2018 were included in the study. The primary and secondary outcomes were: the reduction of the intraocular pressure (IOP) at 6 months postoperatively, the decrease of the glaucoma medications 6 months after surgery, the clinical success rate (eyes (%) achieving ≥20% IOP reduction on the same or fewer medications without secondary surgical intervention), the frequency and type of postoperative interventions as well as the complication rate. We included one hundred and seven eyes from 97 patients with primary OAG (79%), or secondary OAG (21%). Seventy-seven patients (72%) received a standalone XEN implantation and 30 (28%) underwent XEN implantation combined with phacoemusification. The IOP decreased from 20.4 mm Hg ± 6.4 preoperatively to 15.4 mm Hg ± 5.3 six months after the surgery, which represented a reduction of 24.5% (P = 1.4.10-7). It was associated with a lowering of glaucoma medications from 2.8 ± 1.0 preoperatively to 0.6 ± 1.0 six months postoperatively (P = 3.6.10-34). The clinical success rate was 67.2% six months after the surgery. The most frequent complications were: IOP spikes >30 mmHg (16.8%), improper position or angled drain (14.0%) and transient minimal hyphema (<1 week) (11.2%). During the follow-up, the needling was required in 34.6% of cases and a total of 10 eyes (9.4%) required a new glaucoma surgery. To conclude XEN implantation appears to be an effective short- and mid-term surgical technique to control IOP in OAG with a low risk of complication. However postoperative maneuvers were frequently required to maintain efficiency.