Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

Neuroprotective Gene Therapy by Overexpression of the Transcription Factor MAX in Rat Models of Glaucomatous Neurodegeneration.

Purpose: Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods: Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results: In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions: Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.

Gene Therapy Strategies for Glaucomatous Neurodegeneration.

Glaucoma leads to irreversible vision loss and current therapeutic strategies are often insufficient to prevent the progression of the disease and consequent blindness. Elevated intraocular pressure is an important risk factor, but not required for the progression of glaucomatous neurodegeneration. The demise of retinal ganglion cells represents the final common pathway of glaucomatous vision loss. Still, lifelong control of intraocular pressure is the only current treatment to prevent severe vision loss, although it frequently fails despite best practices. This scenario calls for the development of neuroprotective and pro-regenerative therapies targeting the retinal ganglion cells as well as the optic nerve. Several experimental studies have shown the potential of gene modulation as a tool for neuroprotection and regeneration. In this context, gene therapy represents an attractive approach as a persistent treatment for glaucoma. Viral vectors engineered to promote overexpression of a broad range of cellular factors have been shown to protect retinal ganglion cells and/or promote axonal regeneration in experimental models. Here, we review the mechanisms involved in glaucomatous neurodegeneration and regeneration in the central nervous system. Then, we point out the current limitations of gene therapy platforms and review a myriad of studies that use viral vectors to manipulate genes in retinal ganglion cells, as a strategy to promote neuroprotection and regeneration. Finally, we address the potential of combining neuroprotective and regenerative gene therapies as an approach to glaucomatous neurodegeneration.

Retinal changes in COVID-19 hospitalized cases.

The main objective of this study was to evaluate the retinas of severely or critically ill COVID-19 patients during their hospital stay, at varying time points after symptoms onset. This was a case series observed during May 2020 in two referral centers for COVID-19 treatment in Rio de Janeiro, Brazil. 47 eyes from 25 hospitalized patients with severe or critical confirmed illness were evaluated. A handheld retinal camera was used to acquire bilateral fundus images at several time points after symptoms onset. Electronic health records were retrospectively analyzed and clinical data collected. Severe and critical diseases were noticed in 52% (13/25) and 48% (12/25) of enrolled patients, respectively. Retinal changes were present in 12% (3/25) of patients: a 35 year-old male demonstrated bilateral nerve fiber layer infarcts and microhemorrhages in the papillomacular bundle, but required mechanical ventilation and developed severe anemia and systemic hypotension, acute kidney injury and neurologic symptoms during the course of the disease (critical illness); a 56 year-old male, who required full enoxaparin anticoagulation due to particularly elevated D-dimer (>5.0 mcg/mL), demonstrated unilateral and isolated flame-shaped hemorrhages; and a 49 year-old hypertensive male showed bilateral and discrete retinal dot and blot microhemorrhages. The other 22 patients evaluated did not demonstrate convincing retinal changes upon examination. There was no correlation between disease severity and admission serum levels of CRP, D-dimer and ferritin. This was the first study to show that vascular retinal changes may be present in not insignificant numbers of severe or critical COVID-19 inpatients. These retinal changes, only seen after morbid developments, were likely secondary to clinical intercurrences or comorbidities instead of a direct damage by SARS-CoV-2, and may be important and easily accessible outcome measures of therapeutic interventions and sentinels of neurologic and systemic diseases during COVID-19 pandemic.