TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles

Purpose: Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina.

Methods: Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs).

Results: Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced Ca2+ influx in retinal VSMCs was reversed by the TRPV2 inhibitor tranilast and the nonselective TRPP1/V2 antagonist amiloride. Inhibitors of TRPC1, M7, V1, and V4 had no effect. Hypoosmotic stretch-activated cation currents were similar in Na+ and Cs+ containing solutions suggesting no contribution by TRPP1 channels. Direct plasma membrane stretch triggered cation current activity that was blocked by tranilast and specific TRPV2 pore-blocking antibodies and mimicked by the TRPV2 activator, Δ9-tetrahydrocannabinol. Preincubation of retinal arterioles with TRPV2 blocking antibodies prevented the development of myogenic tone.

Conclusions: Our results suggest that retinal VSMCs express a range of mechanosensitive TRP channels, but only TRPV2 appears to contribute to myogenic signaling in this vascular bed.

Retinal nerve fiber layer analysis using GDx® in 49 patients with chronic phase DUSN

To describe retinal nerve fiber layer changes in late-stage diffuse unilateral subacute neuroretinitis eyes and compare these results with healthy eyes observed through nerve fiber analyzer (GDx®). Methods: This is a retrospective case-control study in which 49 eyes in late-stage diffuse unilateral subacute neuroretinitis were examined from May/97 to December/ 01. First, eyes with diffuse unilateral subacute neuroretinitis and healthy contralateral eyes (Control Group I) were statistically matched. Subsequently, eyes with diffuse unilateral subacute neuroretinitis were compared with eyes of healthy patients (Control Group II). Results: Eyes from Control Groups I and II had higher relative frequency of “within normal limits” status. Eyes from the diffuse unilateral subacute neuroretinitis (DUSN) Group had higher frequency of “outside normal limits” and “borderline” status. Control Groups I and II had absolute values different from the DUSN Group regarding all parameters (p<0.05), except for Symmetry in Control Groups I and II, Average thickness and Superior Integral in control group II. Conclusion: Patients with late-stage diffuse unilateral subacute neuroretinitis presented presumed decrease in nerve fiber layer thickness shown by GDx®. Retinal zones with larger vascular support and larger amount of nerve fibers presented higher decrease in the delay of the reflected light measured by the nerve fiber analyzer

RETINAL ANGIOMATOUS PROLIFERATION: A Quantitative Analysis of the Fundoscopic Features of the Fellow Eye

PURPOSE: To quantitatively analyze and compare the fundoscopic features between fellow eyes of retinal angiomatous proliferation and typical exudative age-related macular degeneration and to identify possible predictors of neovascularization. METHODS: Retrospective case-control study. Seventy-nine fellow eyes of unilateral retinal angiomatous proliferation (n = 40) and typical exudative age-related macular degeneration (n = 39) were included. Fundoscopic features of the fellow eyes were assessed using digital color fundus photographs taken at the time of diagnosis of neovascularization in the first affected eye. Grading was performed by two independent graders using RetmarkerAMD, a computer-assisted grading software based on the International Classification and Grading System for age-related macular degeneration. RESULTS: Baseline total number and area (square micrometers) of drusen in the central 1,000, 3,000, and 6,000 μm were considerably inferior in the fellow eyes of retinal angiomatous proliferation, with statistically significant differences (P < 0.05) observed in virtually every location (1,000, 3,000, and 6,000 μm). A soft drusen (≥125 μm) area >510,196 μm2 in the central 6,000 μm was associated with an increased risk of neovascularization (hazard ratio, 4.35; 95% confidence interval [1.56-12.15]; P = 0.005). CONCLUSION: Baseline fundoscopic features of the fellow eye differ significantly between retinal angiomatous proliferation and typical exudative age-related macular degeneration. A large area (>510,196 μm2) of soft drusen in the central 6,000 μm confers a significantly higher risk of neovascularization and should be considered as a phenotypic risk factor.

The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration

Retinitis pigmentosa (RP) is one of the most common retinal degenerative conditions affecting people worldwide, and is currently incurable. It is characterized by the progressive loss of photoreceptors, in which the death of rod cells leads to the secondary death of cone cells; the cause of eventual blindness. As rod cells die, retinal-oxygen metabolism becomes perturbed, leading to increased levels of reactive oxygen species (ROS) and thus oxidative stress; a key factor in the secondary death of cones. In this study, norgestrel, an FDA-approved synthetic analog of progesterone, was found to be a powerful neuroprotective antioxidant, preventing light-induced ROS in photoreceptor cells, and subsequent cell death. Norgestrel also prevented light-induced photoreceptor morphological changes that were associated with ROS production, and that are characteristic of RP. Further investigation showed that norgestrel acts via post-translational modulation of the major antioxidant transcription factor Nrf2; bringing about its phosphorylation, subsequent nuclear translocation, and increased levels of its effector protein superoxide dismutase 2 (SOD2). In summary, these results demonstrate significant protection of photoreceptor cells from oxidative stress, and underscore the potential of norgestrel as a therapeutic option for RP.

Best Practice Guidelines on molecular diagnostics in Duchenne/Becker muscular dystrophies

Meeting participants: Rosário dos Santos, Porto, Portugal

RETINAL ANGIOMATOUS PROLIFERATION: A Quantitative Analysis of the Fundoscopic Features of the Fellow Eye

PURPOSE: To quantitatively analyze and compare the fundoscopic features between fellow eyes of retinal angiomatous proliferation and typical exudative age-related macular degeneration and to identify possible predictors of neovascularization. METHODS: Retrospective case-control study. Seventy-nine fellow eyes of unilateral retinal angiomatous proliferation (n = 40) and typical exudative age-related macular degeneration (n = 39) were included. Fundoscopic features of the fellow eyes were assessed using digital color fundus photographs taken at the time of diagnosis of neovascularization in the first affected eye. Grading was performed by two independent graders using RetmarkerAMD, a computer-assisted grading software based on the International Classification and Grading System for age-related macular degeneration. RESULTS: Baseline total number and area (square micrometers) of drusen in the central 1,000, 3,000, and 6,000 μm were considerably inferior in the fellow eyes of retinal angiomatous proliferation, with statistically significant differences (P < 0.05) observed in virtually every location (1,000, 3,000, and 6,000 μm). A soft drusen (≥125 μm) area >510,196 μm2 in the central 6,000 μm was associated with an increased risk of neovascularization (hazard ratio, 4.35; 95% confidence interval [1.56-12.15]; P = 0.005). CONCLUSION: Baseline fundoscopic features of the fellow eye differ significantly between retinal angiomatous proliferation and typical exudative age-related macular degeneration. A large area (>510,196 μm2) of soft drusen in the central 6,000 μm confers a significantly higher risk of neovascularization and should be considered as a phenotypic risk factor.

Retinal nerve fiber layer analysis using GDx® in 49 patients with chronic phase DUSN

To describe retinal nerve fiber layer changes in late-stage diffuse unilateral subacute neuroretinitis eyes and compare these results with healthy eyes observed through nerve fiber analyzer (GDx®). Methods: This is a retrospective case-control study in which 49 eyes in late-stage diffuse unilateral subacute neuroretinitis were examined from May/97 to December/ 01. First, eyes with diffuse unilateral subacute neuroretinitis and healthy contralateral eyes (Control Group I) were statistically matched. Subsequently, eyes with diffuse unilateral subacute neuroretinitis were compared with eyes of healthy patients (Control Group II). Results: Eyes from Control Groups I and II had higher relative frequency of “within normal limits” status. Eyes from the diffuse unilateral subacute neuroretinitis (DUSN) Group had higher frequency of “outside normal limits” and “borderline” status. Control Groups I and II had absolute values different from the DUSN Group regarding all parameters (p<0.05), except for Symmetry in Control Groups I and II, Average thickness and Superior Integral in control group II. Conclusion: Patients with late-stage diffuse unilateral subacute neuroretinitis presented presumed decrease in nerve fiber layer thickness shown by GDx®. Retinal zones with larger vascular support and larger amount of nerve fibers presented higher decrease in the delay of the reflected light measured by the nerve fiber analyzer

Inherited Photoreceptor Degeneration Causes the Death of Melanopsin-Positive Retinal Ganglion Cells and Increases Their Coexpression of Brn3a

Purpose: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. Methods: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. Results: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). Conclusions: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.

Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function:implications for age-related macular degeneration (AMD)

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D. Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test. Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure. Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.

Retinal Macroglial Responses in Health and Disease

Due to their permanent and close proximity to neurons, glial cells perform essential tasks for the normal physiology of the retina. Astrocytes andM¨uller cells (retinal macroglia) provide physical support to neurons and supplement them with several metabolites and growth factors.Macroglia are involved in maintaining the homeostasis of extracellular ions and neurotransmitters, are essential for information processing in neural circuits, participate in retinal glucose metabolism and in removing metabolic waste products, regulate local blood flow, induce the blood-retinal barrier (BRB), play fundamental roles in local immune response, and protect neurons from oxidative damage. In response to polyetiological insults, glia cells react with a process called reactive gliosis, seeking to maintain retinal homeostasis. When malfunctioning, macroglial cells can become primary pathogenic elements. A reactive gliosis has been described in different retinal pathologies, including age-related macular degeneration (AMD), diabetes, glaucoma, retinal detachment, or retinitis pigmentosa. A better understanding of the dual, neuroprotective, or cytotoxic effect of macroglial involvement in retinal pathologies would help in treating the physiopathology of these diseases.The extensive participation of the macroglia in retinal diseases points to these cells as innovative targets for new drug therapies.

A Diagnostic Calculator for Detecting Glaucoma on the Basis of Retinal Nerve Fiber Layer, Optic Disc, and Retinal Ganglion Cell Analysis by Optical Coherence Tomography

Purpose: The purpose of this study was to develop and validate a multivariate predictive model to detect glaucoma by using a combination of retinal nerve fiber layer (RNFL), retinal ganglion cell-inner plexiform (GCIPL), and optic disc parameters measured using spectral-domain optical coherence tomography (OCT). Methods: Five hundred eyes from 500 participants and 187 eyes of another 187 participants were included in the study and validation groups, respectively. Patients with glaucoma were classified in five groups based on visual field damage. Sensitivity and specificity of all glaucoma OCT parameters were analyzed. Receiver operating characteristic curves (ROC) and areas under the ROC (AUC) were compared. Three predictive multivariate models (quantitative, qualitative, and combined) that used a combination of the best OCT parameters were constructed. A diagnostic calculator was created using the combined multivariate model. Results: The best AUC parameters were: inferior RNFL, average RNFL, vertical cup/disc ratio, minimal GCIPL, and inferior-temporal GCIPL. Comparisons among the parameters did not show that the GCIPL parameters were better than those of the RNFL in early and advanced glaucoma. The highest AUC was in the combined predictive model (0.937; 95% confidence interval, 0.911–0.957) and was significantly (P = 0.0001) higher than the other isolated parameters considered in early and advanced glaucoma. The validation group displayed similar results to those of the study group. Conclusions: Best GCIPL, RNFL, and optic disc parameters showed a similar ability to detect glaucoma. The combined predictive formula improved the glaucoma detection compared to the best isolated parameters evaluated. The diagnostic calculator obtained good classification from participants in both the study and validation groups.

Macroglial and retinal changes in hypercholesterolemic rabbits after normalization of cholesterol levels

This study evaluates hypercholesterolemic rabbits, examining the retinal changes in Müller cells and astrocytes as well as their variations after a period of normal blood-cholesterol values induced by a standard diet. New Zealand rabbits were divided into three groups: G0, fed a standard diet; G1A, fed a 0.5% cholesterol-enriched diet for 8 months; and G1B, fed as G1A followed by standard diet for 6 months. Eyes were processed for transmission electron microscopy and immunohistochemistry (GFAP). While G1B resembled G0 more than did G1A, they shared alterations with G1A: a) as in G1A, Müller cells were GFAP+, filled spaces left by axonal degeneration, formed glial scars and their nuclei were displaced to the nerve-fibre layer. The area occupied by the astrocytes associated with the nerve-fibre bundles (AANFB) and by perivascular astrocytes (PVA) in G1A and G1B was significantly lower than in controls. However, no significant differences in PVA were found between G1A and G1B. In G1B, type I PVA was absent and replaced by hypertrophic type II cells; b) Bruch's membrane (BM) was thinner in G1B than in G1A; c) the retinal pigment epithelium (RPE) cytoplasm contained fewer lipids in G1B than in G1A; d) in G1A and G1B choriocapillaris and retinal vessel showed alterations with respect to G0; e) cell death and axonal degeneration in the retina were similar in G1A and G1B. The substitution of a hyperlipemic diet by a standard one normalizes blood-lipid levels. However, the persistence of damage at retinal vessels and BM-RPE could trigger chronic ischemia.