Molecular pathways in experimental glaucoma models

Glaucoma is a complex and progressive disease that primarily affects the optic nerve axons, leading to irreversible vision loss. Although the exact molecular mechanisms underlying glaucoma pathogenesis are not fully understood, it is believed that except increased intraocular pressure, a combination of genetic and environmental factors play a role in the development of the disease. Animal models have been widely used in the study of glaucoma, allowing researchers to better understand the underlying mechanisms of the disease and test potential treatments. Several molecular pathways have been implicated in the pathogenesis of glaucoma, including oxidative stress, inflammation, and excitotoxic-induced neurodegeneration. This review summarizes the most important knowledge about molecular mechanisms involved in the glaucoma development. Although much research has been done to better understand the molecular mechanisms underlying this disease, there is still much to be learned to develop effective treatments and prevent vision loss in those affected by glaucoma

Longitudinal observation of the retinal nerve fibre layer in glaucoma patients treated with brimonidine combined with timolol or timolol alone

INTRODUCTION. The aim of the study was to evaluate the retinal nerve fibre layer (RNFL) thickness loss in primary open-angle glaucoma (POAG) patients treated topically with anti-glaucoma drops containing brimonidine and timolol combination or solely timolol. MATERIALS AND METHODS. Retrospective case series study of patients with POAG diagnosis followed up for a five-year period. Inclusion criteria were fulfilled by a group of 98 patients consisting of 53 combination and 45 monotherapy treatments. Intraocular pressure (IOP) at the level of 21 mm Hg or below for each measurement was observed in 52 patients, while incidences of pressure above 21 mm Hg were measured in 46 patients. POAG diagnosis was based on standard optical coherence tomography, IOP, and visual field examinations. RESULTS. Mean annual loss of RNFL thickness in the overall study group (if IOP levels are not taken into consideration) treated with timolol monotherapy was 1.8 ± 1.5 μm, while in group treated with brimonidine + timolol combination therapy it was 1.7 ± 1.5 μm (p > 0.05). In selected groups of patients with incidents of pressure rises, the mean annual loss of retinal nerve fibre layer thickness was 1.8 ± 1.6 and 1.9 ± 1.4 μm, respectively, for the monotherapy and combination therapy groups (p > 0.05). In the group of patients with no reported IOL rises, mean annual loss of RNFL thickness was 1.8 ± 0.9 and 1.1 ± 0.4 μm, respectively, for the monotherapy and combination therapy groups (p < 0.01). No significant differences were observed for the visual field mean deviation. CONCLUSIONS. POAG patients with low values of IOP might achieve slower progression of RNFL thinning on brimonidine combined with timolol therapy.

The Role of Endogenous Neuroprotective Mechanisms in the Prevention of Retinal Ganglion Cells Degeneration

Retinal neurons are not able to undergo spontaneous regeneration in response to damage. A variety of stressors, i.e., UV radiation, high temperature, ischemia, allergens, and others, induce reactive oxygen species production, resulting in consecutive alteration of stress-response gene expression and finally can lead to cell apoptosis. Neurons have developed their own endogenous cellular protective systems. Some of them are preventing cell death and others are allowing functional recovery after injury. The high efficiency of these mechanisms is crucial for cell survival. In this review we focus on the contribution of the most recently studied endogenous neuroprotective factors involved in retinal ganglion cell (RGC) survival, among which, neurotrophic factors and their signaling pathways, processes regulating the redox status, and different pathways regulating cell death are the most important. Additionally, we summarize currently ongoing clinical trials for therapies for RGC degeneration and optic neuropathies, including glaucoma. Knowledge of the endogenous cellular protective mechanisms may help in the development of effective therapies and potential novel therapeutic targets in order to achieve progress in the treatment of retinal and optic nerve diseases

Predegenerated Schwann cells-a novel prospect for cell therapy for glaucoma : neuroprotection, neuroregeneration and neuroplasticity

Glaucoma is an optic neuropathy that leads to irreversible blindness. Because the current therapies are not sufficient to protect against glaucoma-induced visual impairment, new treatment approaches are necessary to prevent disease progression. Cell transplantation techniques are currently considered to be among the most promising opportunities for nervous system damage treatment. The beneficial effects of undifferentiated cells have been investigated in experimental models of glaucoma, however experiments were accompanied by various barriers, which would make putative treatment difficult or even impossible to apply in a clinical setting. The novel therapy proposed in our study creates conditions to eliminate some of the identified barriers described for precursor cells transplantation and allows us to observe direct neuroprotective and pro-regenerative effects in ongoing optic neuropathy without additional modifications to the transplanted cells. We demonstrated that the proposed novel Schwann cell therapy might be promising, effective and easy to apply, and is safer than the alternative cell therapies for the treatment of glaucoma.Peer reviewe

FluoroGold-Labeled Organotypic Retinal Explant Culture for Neurotoxicity Screening Studies

Preclinical toxicity screening of the new retinal compounds is an absolute requirement in the pathway of further drug development. Since retinal neuron cultivation and in vivo studies are relatively expensive and time consuming, we aimed to create a fast and reproducible ex vivo system for retinal toxicity screening. For this purpose, we used rat retinal explant culture that was retrogradely labeled with the FluoroGold before the isolation. Explants were exposed to a toxic concentration of gentamicin and ciliary neurotrophic factor (CNTF), a known neuroprotective agent. The measured outcomes showed the cell density in retinal ganglion cell layer (GCL) and the activity of lactate dehydrogenase (LDH) in the culture medium. Gentamicin-induced oxidative stress resulted in retinal cell damage and rapid LDH release to the culture medium (p<0.05). Additional CNTF supplementation minimized the cell damage, and the increase of LDH release was insignificant when compared to LDH levels before gentamicin insult (p>0.05). As well as this, the LDH activity was directly correlated with the cell count in GCL (R=−0.84, p<0.00001), making a sensitive marker of retinal neuron damage. The FLOREC protocol could be considered as a fast, reproducible, and sensitive method to detect neurotoxicity in the screening studies of the retinal drugs

Electrical synapses interconnecting axons revealed in the optic nerve head – a novel model of gap junctions’ involvement in optic nerve function

Abstract Purpose To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. Methods Twenty-five Wistar rats were killed by CO2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 ?m meclofenamic acid solution (gap junctions? blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. Results Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with ?3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p Peer reviewe

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38 MAPK

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR’s role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1 alpha in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1 alpha dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1 alpha dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.Peer reviewe

The effectiveness and safety of one-stage iStent-based micro-invasive glaucoma surgery—A retrospective study

PurposeMicro-invasive glaucoma surgery involves a group of treatment methods associated with a low rate of side effects and good effectiveness outcomes. One of the most frequently performed procedures belonging to this group is iStent microstent implantation. The aim of this study was to perform a retrospective evaluation of the safety and efficacy of a combined procedure involving cataract phacoemulsification and single iStent microstent implantation, performed simultaneously.Materials and methodsThe complete medical records of 62 patients (91 eyes) were analyzed retrospectively, including the best corrected visual acuity, intraocular pressure, the mean defect of visual fields, and the number of active substances used in eye drops. The follow-up times were 1, 3, 6, 9, and 12 months after the surgical procedure.ResultsA significant improvement in the best corrected visual acuity and a reduction of the intraocular pressure were achieved after the surgery. On average, after 12 months, the best corrected visual acuity improved from 0.70 (0.25) to 0.91 (0.18; p = 0.001), the intraocular pressure reduced from 17.76 (3.95) to 14.91 (3.04; p = 0.0001), and the number of active substances used in eye drops reduced from 2.07 (1.08) to 0.70 (0.06; p = 0001). In addition, we found that patients who initially showed higher intraocular pressure values did not benefit from surgery in the aspect of the number of active substances used in their eye drops. Intraoperative and postoperative adverse events were transient and ultimately did not affect the outcomes.ConclusionSimultaneous cataract phacoemulsification with single iStent implantation in patients with open-angle glaucoma is a safe and effective method for reducing intraocular pressure and the number of topical medications that must be used. Having initially higher intraocular pressure values may limit the beneficial effects of iStent implantation by subordinating patients from topical treatment; thus, single iStent implantation may not be the most favorable choice in uncontrolled glaucoma cases