Müller Cells Research Articles

Poster Session II: Intravitreal gene therapy in primate reaches extrafoveal cones.

Intravitreal delivery of gene therapy vectors to the retina carries lower risk of adverse events versus subretinal injections, but efficiently targeting cones is a challenge. We used a new adeno-associated vector (AAV) to deliver genes to primate cone photoreceptors. The vector carries a cassette directing expression of an engineered 493 nm opsin to long- and middle-wavelength (L/M) cones, and was injected into the vitreous of the left eye of an adult macaque. An identical AAV carrying a fusion of the engineered opsin to green fluorescent protein (GFP) was injected into the right eye. Electroretinograms were performed on the left eye before and after injection to measure isolated 493 nm light responses; 5 weeks post-injection, response increased modestly. A central strip of the right eye was prepared for histology with cryosections; we found ~30% of cones in the fovea had been transduced, with a preference toward L/M cones (see https://iovs.arvojournals.org/article.aspx?articleid=2782955). Upon close examination of GFP in the peripheral retina, we were surprised to find extensive expression in cones across the retina. Here, we report patches of expression from the perifovea to the retinal margin which reaches ~10% of cones. Expression patches appeared stochastically, or in regions containing blood vessels or disrupted Muller cells. This demonstrates that extrafoveal expression is attainable using intravitreal injection of gene therapy vectors in an adult primate.

ASCL1 induces neurogenesis in human Müller glia

In mammals, loss of retinal cells due to disease or trauma is an irreversible process that can lead to blindness. Interestingly, regeneration of retinal neurons is a well established process in some non-mammalian vertebrates and is driven by the Müller glia (MG), which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury or disease. Progress has been made to restore this mechanism in mammals to promote retinal regeneration: MG can be stimulated to generate new neurons invivo in the adult mouse retina after the over-expression of the pro-neural transcription factor Ascl1. In this study, we applied the same strategy to reprogram human MG derived from fetal retina and retinal organoids into neurons. Combining single cell RNA sequencing, single cell ATAC sequencing, immunofluorescence, and electrophysiology we demonstrate that human MG can be reprogrammed into neurogenic cells invitro.

Idiopathic Macular Hole: Algorithm for Nonsurgical Closure Based on Literature Review.

Our purpose is to review the closure time and optical coherence tomography (OCT) biomarkers that result in the non-surgical repair of idiopathic full-thickness macular holes (IFTMH). Our methodology consisted of a comprehensive literature review of the nonsurgical resolution of IFTMH followed by the calculation of the estimated closure time using the structural equation model. Forty-nine studies were found eligible yielding 181 eyes with IFTMH: 81.1% being small holes (250 µm) with a median diameter of 166 µm. Final vision (mean 20/41) was related to initial vision (mean 20/65) and mean age (67 years). The hole diameter was correlated with initial vision and closure time (mean 3.9 months). Closure time was related to hole diameter and initial vision in the following algorithm: Closure time (month)= 0.057 + 0.008 diameter (µm) + 0.021 age (year) + 2.153 initial vision (logMAR). Biomarkers by OCT for self-closure included in decreasing frequency: pointed edge, de-turgescence of cystic macular edema (CME) with reversal of bascule bridge, and vitreomacular traction (VMT) release. The crucial function of Muller cell bridging in sealing the hole attests to its exceptional capacity for regeneration. After the hole has begun to close; however in less than 5%, a delayed restoration of the ellipsoid layer or a persistent outer foveal defect may prevent visual recovery and reopening of the hole is possible. In conclusion, eyes with small-size IFTMH and good baseline vision can have the additional option of close OCT monitoring for biomarkers of self-sealing biomarkers. When rehabilitative activity seems to be lacking, surgery is therefore mandatory.

Neuroglia Cell-Originated Vitreous Cyst in a Retinitis Pigmentosa Patient.

A 47-year-old female presented with a complaint of a dark shadow floating in front of her right eye. She was diagnosed with retinitis pigmentosa in both eyes 15 years ago in Southwest Hospital. On examination, her presenting visual acuity was 20/400 in both eyes. Visual acuity did not improve even with pinhole and best refractive correction. On slitlamp examination, anterior segment of both eyes was normal. Intraocular pressure of both eyes was within reference limit. On posterior segment examination, fundus evaluation of both eyes revealed typical bone-spicule pigment, and a free-floating cyst of about 6 mm in diameter was seen in the vitreous cavity of the right eye (Fig. 1A). The patient expressed a strong desire for surgery to relieve the dark shadow in her right eye.FIGURE 1: A, Funduscopy showed a free-floating cyst in the vitreous cavity and bone-spicule pigment in the retina. The cyst was gray-white and about 6 mm in diameter. B, Hematoxylin-eosin staining and immunohistochemical staining (GFAP, syn, and S100) of the capsule wall. GFAP indicates glial fibrillary acidic protein; S100, calcium binding protein; syn, synuclein.The cyst was removed by 25-G pars plana vitrectomy (Supplementary Digital Content Video, https://links.lww.com/APJO/A195). Immunohistochemical staining of the capsule wall showed glial fibrillary acidic protein (GFAP), S100 (calcium binding protein), and synuclein (syn) positive (Fig. 1B). GFAP and S100 are markers of neuroglia cells, which are expressed in astrocytes and Muller glia cells of the retina. Syn can also be expressed in activated neuroglia cells. While our staining also showed melanocyte markers (HMB45, MelanA), epithelial cell marker (cytokeratin), macrophage marker (CD68), and lymphocyte marker (CD45) were all negative. These suggested the vitreous cyst originated from the neuroglia cell. Vitreous cysts are rare clinical findings, which can be congenital or acquired. Congenital ones have been reported to be associated with the residual hyaloid vascular system. Acquired ones are usually associated with ocular trauma, uveitis, intraocular infection, retinitis pigmentosa, and retinal detachment.1 Most vitreous cysts can be observed without any treatment. Laser disruption or vitrectomy may be performed when the patient has obvious symptoms and a strong desire for therapy. In our case, the cyst occurred in retinitis pigmentosa and originated from the neuroglia cell. The patient no longer complained about the floater after the operation.

The effect of low-frequency ultrasound on the functional and morphological state of the retina of rabbits

Currently, three groups of scientists are developing the introduction and use of lowfrequency ultrasound in retinal and vitreous surgery. There is not enough data and work to study the effects of this type of energy on the functional activity and structural state of the retina. In this regard, we conducted experimental and diagnostic studies using modern and objective methods. The main purpose of this article is analyzing the parameters of the electroretinogram of the rabbit retina and compare it with histological data after removal of the vitreous body using low-frequency ultrasound and mechanical action. Experiments were conducted on Chinchilla rabbits (n=40). In the experimental group (n=20), vitreous removal was performed using low-frequency ultrasound, in the control group (n=20) using a guillotine mechanism. On the 1st, 7th, 14th and 14th days, the parameters of the a- and b-waves of the electroretinogram were recorded. The structural state of the retina was also evaluated on histological sections. On the 1st day after surgery in both study groups there was a decrease in all parameters of the electroretinogram, the evaluation of histological data showed the presence of edema in most layers of the retina. On days 7 and 14, the parameters of the a- and b-waves were restored, but did not return to normal in both groups, a decrease in edema in all layers of the retina was observed on histological preparations. On day 30, the a- and b-wave indicators returned to normal, which indicates the restoration of the functional properties of photoreceptor cells, bipolar and Muller cells in both groups, the retinal layers were clearly visualized on histological sections without signs of edema. Analysis of electroretinogram parameters and histological data showed that the use of low-frequency ultrasound for vitreous removal can be considered safe and promising for further development.

Fullerol rescues the light-induced retinal damage by modulating Müller glia cell fate

Excessive light exposure can damage photoreceptors and lead to blindness. Oxidative stress serves a key role in photo-induced retinal damage. Free radical scavengers have been proven to protect against photo-damaged retinal degeneration. Fullerol, a potent antioxidant, has the potential to protect against ultraviolet-B (UVB)-induced cornea injury by activating the endogenous stem cells. However, its effects on cell fate determination of Müller glia (MG) between gliosis and de-differentiation remain unclear. Therefore, we established a MG lineage-tracing mouse model of light-induced retinal damage to examine the therapeutic effects of fullerol. Fullerol exhibited superior protection against light-induced retinal injury compared to glutathione (GSH) and reduced oxidative stress levels, inhibited gliosis by suppressing the TGF-β pathway, and enhanced the de-differentiation of MG cells. RNA sequencing revealed that transcription candidate pathways, including Nrf2 and Wnt10a pathways, were involved in fullerol-induced neuroprotection. Fullerol-mediated transcriptional changes were validated by qPCR, Western blotting, and immunostaining using mouse retinas and human-derived Müller cell lines MIO-M1 cells, confirming that fullerol possibly modulated the Nrf2, Wnt10a, and TGF-β pathways in MG, which suppressed gliosis and promoted the de-differentiation of MG in light-induced retinal degeneration, indicating its potential in treating retinal diseases.

Dynamics of morphological changes in the cellular layers of retina in the long-term experimental opioid exposure

Relevance of research. There is enough information in the foreign and domestic specialized literature on the issue of morphological reorganization of cells and tissues during experimental opioid exposure and other types of toxic effects on the body today. At present, the issue of structural reorganization in the layers of the retina and disruption of its blood supply processes during subchronic and chronic periods of experimental opioid exposure remains unclear. That is why we decided to supplement, expand and deepen information about these processes by conducting our own research in this direction. The purpose of the study is to clarify the dynamics of pathomorphological changes in the layers of the retina and the links of its hemomicrocirculatory channel in the long term of experimental opioid exposure.Material and methods. The research material was sexually mature, outbred, white male rats, 56 animals in number, weighing 160-270 g, aged 4.5-7.5 months. The initial dose of nalbuphine during the first 2 weeks was 0.212 mg/kg, the next 2 weeks (III-IV weeks) – 0.225 mg/kg, the next (V-VI weeks) – 0.252 mg/kg, the next (VI-VIII weeks ) – 0.260 mg/kg, the next (VIII – X weeks) – 0.283 mg/kg, the next (X – XII weeks) – 0.3 mg/kg, and during (XII – XIV weeks) – 0.45 mg/ kg. Before taking the material of the experimental site, the animal was removed from the experiment with the help of diethyl ether. Rat eyeballs were used as a material for ultrastructural research, considering the preservation of the topographic ratio of the eye membranes. Ultrastructural preparations were prepared according to the generally accepted method. Research results. At the end of the eighth week and the tenth week, persistent hyperemia of the choroidal vessels, degenerative and necrotic changes in the cells of the pigment layer, degeneration of the outer segments of photoreceptors, shortening and fragmentation of mitochondrial cristae of the inner segments of photoreceptors, necrotic changes in the nuclei of individual photoreceptors, degeneration of axons of the outer and inner reticular layers, as well as degenerative and necrotic changes of bipolar and amacrine cells, hypertrophy of processes of Müller cells, degeneration of ganglion cells, hyperemia and perivascular edema of vessels of the outer and inner reticular layers were revealed. After twelve and fourteen weeks, the initial stages of development with subsequent progression of sclerotic changes of Bruchʹs membrane were first detected. The phenomena of degenerative and necrotic changes in the cells of the pigment layer, destruction of the membrane of the outer segments of photoreceptors, degenerative changes in the axons of the outer and inner reticular layers and the layer of nerve fibers, degenerative and necrotic changes in bipolar and amacrine cells, hypertrophic changes in processes of Muller cells, hyperemia and stasis gradually increased, perivascular swellings in the vessels of the inner reticular layer, ganglionic layer and layer of nerve fibers, reactive changes in Muller cells and protoplasmic astrocytes. Conclusion. In the long-term course of experimental opioid exposure, irreversible pathomorphological changes occurred and subsequently progressed, which were caused by an increase in dyscirculatory manifestations in the superficial and deep vascular networks of the retina, causing the deepening of edema phenomena with further compression and hypoxia in the thickness of the retinal layers.

CHANGES IN THE STRUCTURAL ORGANIZATION OF CELLULAR COMPONENTS IN THE RETINAL LAYERS AND LINKS OF ITS HEMOMICOCIRCULATORY BED DURING THE CHRONIC PERIOD OF EXPERIMENTAL OPIOID EXPOSURE AND THE CORRECTION OF THESE CHANGES AT THE ULTRASTRUCTURAL LEVEL

Over the past twenty years, the experimental direction of research has been the most relevant in morphology to study the dynamics of the course of nosologies. In terms of the dynamics of the occurrence of pathomorphological manifestations at the cellular, tissue, organ, and organism levels, it off ers a larger variety of pathology investigations. The study of the retina in the norm and the processes of modifying its structural reorganisation under the eff ect of various nosologies are accorded high importance in the scientifi c literature written by both domestic and international professionals. Despite these eff orts, the problem of opioid retinopathy on prolonged experimental opioid exposure remains essential. It is still unknown, in particular, if the correction of the chronic period of experimental opioid exposure may stabilise pathomorphological symptoms in the retinal layers and linkages of its hemomycrocirculatory bed. The purpose of the research is to fi nd out the features of structural reorganization in the retinal layers and links of its hemomicrocirculatory bed in the chronic period of experimental opioid exposure and the correction of these changes with pentoxifylline.Material and methods. The research used 34 male, sexually mature, non-breeding rats that ranged in weight from 160 to 270 g and were 4.5 to 7.5 months old as its subject matter. The correction dose of pentoxifylline was 2.857 mg/kg. At the end of the 10th week, the material was taken for ultrastructural examination of the retina. Before the sampling of the experimental site material, the animal was withdrawn from the experiment using diethyl ether. The eyeballs of rats were used as a material for ultrastructural examination. Ultrastructural preparations were prepared according to the generally accepted method. As a result of our research of the retina at the end of the tenth week in rats, which were injected with an opioid for six weeks, and subsequently discontinued for four weeks, the phenomena of degeneration of the pigment epithelium, destruction of the membrane discs of the outer segments of the photoreceptors, degenerative changes in the inner segments of the photoreceptors, degenerative and necrotic changes of individual photoreceptors developed. Degeneration and necrosis processes were detected in bipolar and amacrine neurons. Rats were injected with the opioid for six weeks, with its cancellation and subsequent four-week correction with pentoxifylline. After ten weeks of the experiment, degenerative changes in the apical microvilli of the pigment epithelium, destruction of the membrane discs of the outer segments of the photoreceptors, degeneration of bipolar and amacrine neurons, moderately pronounced discirculatory changes, and reactive processes of glial elements were detected. In rats given an opioid for six weeks, followed by four weeks of an opioid with pentoxifylline administration, fl ushing, stasis, and perivascular oedema in the choroidal arteries were established ten weeks later. Degeneration and necrosis of the pigment epithelium, degeneration and destruction of the membrane discs of the outer segments of photoreceptors with separation of individual outer segments, degeneration of the inner segments of photoreceptors, degeneration and necrotic changes of photoreceptors, degeneration and necrotic changes of bipolar and amacryne neurons, Muller cells, degeneration of ganglion neurons, hyperemia, stasis and perivasal oedema in the vessels of the ganglion layer, the layer of nerve fi bers, in the inner mesh layer.

Oral administration of S-nitroso-L-glutathione (GSNO) provides anti-inflammatory and cytoprotective effects during ocular bacterial infections.

Bacterial endophthalmitis is a severe complication of eye surgeries that can lead to vision loss. Current treatment involves intravitreal antibiotic injections that control bacterial growth but not inflammation. To identify newer therapeutic targets to promote inflammation resolution in endophthalmitis, we recently employed an untargeted metabolomics approach. This led to the discovery that the levels of S-nitroso-L-glutathione (GSNO) were significantly reduced in an experimental murine Staphylococcus aureus (SA) endophthalmitis model. In this study, we tested the hypothesis whether GSNO supplementation via different routes (oral, intravitreal) provides protection during bacterial endophthalmitis. Our results show that prophylactic administration of GSNO via intravitreal injections ameliorated SA endophthalmitis. Therapeutically, oral administration of GSNO was found to be most effective in reducing intraocular inflammation and bacterial burden. Moreover, oral GSNO treatment synergized with intravitreal antibiotic injections in reducing the severity of endophthalmitis. Furthermore, in vitro experiments using cultured human retinal Muller glia and retinal pigment epithelial (RPE) cells showed that GSNO treatment reduced SA-induced inflammatory mediators and cell death. Notably, both in-vivo and ex-vivo data showed that GSNO strengthened the outer blood-retinal barrier during endophthalmitis. Collectively, our study demonstrates GSNO as a potential therapeutic agent for the treatment of intraocular infections due to its dual anti-inflammatory and cytoprotective properties.

The Value of Electroretinography in Identifying Candidate Genes for Inherited Retinal Dystrophies: A Diagnostic Guide.

Inherited retinal dystrophies (IRDs) are a group of heterogeneous diseases caused by genetic mutations that specifically affect the function of the rod, cone, or bipolar cells in the retina. Electroretinography (ERG) is a diagnostic tool that measures the electrical activity of the retina in response to light stimuli, and it can help to determine the function of these cells. A normal ERG response consists of two waves, the a-wave and the b-wave, which reflect the activity of the photoreceptor cells and the bipolar and Muller cells, respectively. Despite the growing availability of next-generation sequencing (NGS) technology, identifying the precise genetic mutation causing an IRD can be challenging and costly. However, certain types of IRDs present with unique ERG features that can help guide genetic testing. By combining these ERG findings with other clinical information, such as on family history and retinal imaging, physicians can effectively narrow down the list of candidate genes to be sequenced, thereby reducing the cost of genetic testing. This review article focuses on certain types of IRDs with unique ERG features. We will discuss the pathophysiology and clinical presentation of, and ERG findings on, these disorders, emphasizing the unique role ERG plays in their diagnosis and genetic testing.

A comparative analysis of gene and protein expression in chronic and acute models of photoreceptor degeneration in adult zebrafish.

Background: Adult zebrafish are capable of photoreceptor (PR) regeneration following acute phototoxic lesion (AL). We developed a chronic low light (CLL) exposure model that more accurately reflects chronic PR degeneration observed in many human retinal diseases. Methods: Here, we characterize the morphological and transcriptomic changes associated with acute and chronic models of PR degeneration at 8 time-points over a 28-day window using immunohistochemistry and 3'mRNA-seq. Results: We first observed a differential sensitivity of rod and cone PRs to CLL. Next, we found no evidence for Müller glia (MG) gliosis or regenerative cell-cycle re-entry in the CLL model, which is in contrast to the robust gliosis and proliferative response from resident MG in the AL model. Differential responses of microglia between the models was also observed. Transcriptomic comparisons between the models revealed gene-specific networks of PR regeneration and degeneration, including genes that are activated under conditions of chronic PR stress. Finally, we showed that CLL is at least partially reversible, allowing for rod and cone outer segment outgrowth and replacement of rod cell nuclei via an apparent upregulation of the existing rod neurogenesis mechanism. Discussion: Collectively, these data provide a direct comparison of the morphological and transcriptomic PR degeneration and regeneration models in zebrafish.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

Serous Maculopathy Due to Aspecific Choroidopathy (SMACH).

Serous Maculopathy Due to Aspecific Choroidopathy (SMACH).

Assessment of Macular Function by Multifocal Electroretinography Following Idiopathic Epiretinal Membrane Surgery with Internal Limiting Membrane Peeling

Purpose To evaluate macular function before and after vitrectomy for idiopathic epiretinal membrane (ERM) with internal limiting membrane (ILM) peeling by multifocal electroretinography (mfERG) and their correlations with visual acuity (VA) and central subfoveal thickness (CST) by optical coherence tomography (OCT). Settings and Design A prospective interventional case series of 15 eyes with idiopathic ERM of 14 patients who underwent pars plana vitrectomy (PPV) for ERM removal with ILM peeling. Methods Multifocal electroretinography (mfERG) and OCT were performed preoperatively and one month after surgery. The response amplitude densities and implicit times of mfERG were analyzed. Correlation analyses among visual acuity, CST and mfERG values in the five concentric rings were performed. Results The first order responses of mfERG recorded preoperatively showed diffuse reduction in P1 amplitudes in all 5 rings with no statistically significant differences in mfERG responses after ERM surgery. Correlation coefficients showed a significant correlation between postoperative VA and P1 amplitude of ring 2 (P = 0.005). There was no significant correlation between postoperative CST and P1 amplitude for all rings Conclusions The mfERG responses in eyes with idiopathic ERM show macular function abnormalities and correlate with VA after surgery. The residual retinal distortion along with the damage to inner retinal layers and Muller cell dysfunction associated with ILM peeling affect the mfERG responses after surgery. The incorporation of mfERG into the pre- and post-operative assessment of patients with iERM, allow for identification of functional impairment and predict visual outcomes.

Biochemical reactions remote-sensitized by excitons propagating across macroscopic distances along biological fibers: Isomerization of 11-cis-retinal

Presently we report a second example of a biochemical reaction sensitized remotely by excitons (electronic excited states) efficiently transferred along intermediate filaments (IFs) over macroscopic distances. IFs are excellent conductors of energy in the form of excitons, with the efficiency of ca. 0.53 reported in vitro. Excitons were generated by visible light and propagated along Müller cell (MC) intermediate filaments, about 100 μm long. These experiments used a capillary matrix filled by MC IFs extracted from porcine retina. Excitons induced efficient isomerization of 11-cis- to all-trans-retinal, with the reaction quantum yield φcis/trans = 0.347 obtained for 11-cis-retinal concentration of 1.0 × 10−3 M (0.284 g/L) using 546 nm light at 6.87 mW/cm2 power density. Exciton quantum yield φexc at 546 nm was also measured in function of radiation power density, with nonlinearity indicative of biphotonic processes. This is the first case of biphotonic processes occurring at such low light intensities with steady-state illumination. The present results support quantum mechanism of high-contrast vision of vertebrate eyes.

Different responses of the MIO‑M1 Mueller cell line to angiotensin II under hyperglycemic or hypoxic conditions.

Members of the renin-angiotensin aldosterone system (RAAS) are expressed by various retinal tissues including Mueller glial cells. As the RAAS is hypothesized to play an important role in the pathogenesis of diseases that threaten vision, such as diabetic macular edema or retinal vein occlusion, the possible changes induced by exposure of the human cell line MIO-M1, an established model of Mueller cells, to angiotensin II or aldosterone for 6 h under hypoxic and/or hyperglycemic conditions were investigated. The mRNA expression levels of the members of the RAAS were assessed by reverse transcription-quantitative PCR, and the secretion of cytokines was assessed by ELISA. Under hyperglycemic conditions, the mRNA expression levels of the angiotensin-converting enzyme 2 (ACE2), angiotensin II receptors, AT1 and AT2, and the receptor of angiotensin (1-7) MAS1 were significantly higher after exposure to angiotensin II, and the expression of ACE2, AT2, and IL-6 (a marker of inflammation) was significantly increased after treatment with aldosterone; the expression of the other targets investigated remained unchanged. Significantly more IL-6 was secreted by MIO-M1 cells exposed to hyperglycemia and angiotensin. When cells were cultured in a hypoxic environment, additional treatment with aldosterone significantly increased the mRNA expression levels of ACE, but significantly more ACE2 mRNA was expressed in the presence of angiotensin II. Under hypoxic plus hyperglycemic conditions, significantly less ACE but more AT2 was expressed after treatment with angiotensin II, which also led to strongly elevated expression of IL-6. The mRNA expression levels of the angiogenic growth factor VEGF-A and secretion of the encoded protein were notably increased under hypoxic and hypoxic plus hyperglycemic conditions, irrespective of additional treatment with angiotensin II or aldosterone. These findings suggest that angiotensin II induces a pro-inflammatory response in MIO-M1 cells under hyperglycemic conditions despite activation of the counteracting ACE2/MAS1 signaling cascade. However, hypoxia results in an increased expression of angiogenic VEGF-A by these cells, which is not altered by angiotensin II or aldosterone.

Single Cell RNA Sequencing Analysis of Mouse Retina Identifies a Subpopulation of Muller Glia Involved in Retinal Recovery From Injury in the FCD-LIRD Model.

Although retinal light injury models have been useful in understanding aspects of retinal degeneration and retinal oxidative stress, information on retinal recovery from oxidative/photoinflammatory retinal injury is scarce. The fundus camera-delivered light-induced retinal degeneration model is a simple and reproducible retinal light injury model developed to recapitulate not only the retinal degeneration aspect, but also the retinal recovery from injury. In this study, we used the fundus camera-delivered light-induced retinal degeneration model to perform cell type-specific analyses of the acute and subacute retinal responses to light injury. C57BL/6J eyes were collected before or after light injury (4 hours, 48 hours, and day 5). Retina samples were processed into single-cell suspensions. Droplet-based encapsulation of single cells was performed to generate libraries for sequencing. Gene expression analysis generated 23 clusters encompassing all known major retinal cell populations. Using unbiased analyses, we identified genes and pathways that were significantly altered in each cell type after light injury, including some cellular processes suggestive of activation of pathways for retinal recovery (e.g., synaptogenesis signaling, ephrin receptor signaling, and Reelin signaling in neurons). More importantly, our data show that a subpopulation of Muller glia cells may play an important role in the cellular recovery process. This work identifies acute and subacute cell type-specific responses to retinal photo-oxidative injury. A subpopulation of Muller glia seems to initiate the cellular recovery process. A better understanding of these responses may be helpful in identifying therapeutic approaches to minimize retinal damage and maximize recovery after exposure to injury.

Phenomenology of spontaneous closure in degenerative and mixed type lamellar macular hole.

To the describe OCT imaging characteristics of a cohort of patients showing spontaneously closing degenerative or mixed type lamellar macular holes (LMH) and to compare them to the ones of a sex and age matched group showing stable lesions. Patients diagnosed with degenerative and mixed type LMHs showing OCT-documented spontaneous anatomical closure were retrospectively selected from 3 specialized retina centres. An equal number of age and sex matching subjects were randomly selected among patients with anatomically stable lesions. Eleven (11) spontaneously closing (SC group) and 11 stable (ST group) degenerative LMH with a mean follow up of 4 years were recruited. Hyperreflective inner border (HIB) and linear hyperreflectivity in the outer plexiform layer (LHOP) at baseline were significantly more prevalent in SC group in processed images (respectively p = 0.007 and p = 0.003). A borderline significance in lamellar hole associated epiretinal proliferation (LHEP) at last follow up was detected (p = 0.085). As for mixed type LMH, 10 patients for SC group and 10 for ST group were recruited. LHOP at baseline in processed images was significantly more prevalent in SC group (p = 0.005). Spontaneously closing LMHs show higher prevalence of HIB and LHOP at the beginning of the closing process, a difference which is enhanced by image processing. These signs might be a signal of microglial and Muller cells coordinated activation.

Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

Association of OCT biomarkers and visual impairment in patients with diabetic macular oedema with vitreomacular adhesion.

To analyse the distribution of spectral domain optical coherence tomography (SD-OCT) biomarkers in different types of vitreomacular adhesion (VMA) associated visual impairment in diabetic macular oedema. A total of 317 eyes of 202 patients were enrolled. Cases were divided into two groups focal VMA and broad VMA and subjects with no VMA were enrolled as controls. A grading platform was used for evaluating the morphology of diabetic macular oedema (DME), using good-quality SD-OCT images. Grading was done for VMA and the biomarkers. Best corrected visual acuity (BCVA), central retinal thickness (CRT) and central subfield thickness (CSFT) was also recorded. The CRT (p = <0.001) and CSFT (p = <0.001) values were statistically significant between the groups. Except for Inner Nuclear Layer Cysts (p = <0.001), absence of Bridging Tissue that is composed of muller cell fibers and bipolar cells (p<0.001), and Hyper Reflective Dots (HRD) in cyst (p = 0.006) there were no significant differences in the distribution of OCT biomarkers among the 3 groups (focal VMA, broad VMA and no VMA). Only Disorganization of Retinal Inner Layers (DRIL) (p = 0.044) showed significant association with vision impairment in all the 3 groups. The distribution of OCT biomarkers was similar across all eyes of cases and controls. However, they were more likely to be associated with visual impairment in the presence of VMA than no VMA.