Clinical and Immunological Impacts of Latent Toxoplasmosis on COVID-19 Patients.

Background Parasites are well-known immune-modulators. They inhibit some aspects of the immune system to ensure persistence inside the host for a long time; meanwhile, they stimulate other immune aspects to assure the survival of the host. Wide variations in the severity of coronavirus disease 2019 (COVID-19) among developed and developing countries were reported during the COVID-19 pandemic. Parasitic infections, including Toxoplasma gondii (T. gondii), were claimed to contribute to such variations. Methods To explore a possible relationship between latent toxoplasmosis and COVID-19 severity, our study included 44 blood samples from moderate/severe COVID-19 patients, who were admitted to Mansoura University Hospitals, Egypt, during the pandemic. Patients' sera were screened for Toxoplasma IgG antibodies using ELISA (Roche Diagnostics, Indianapolis, USA), and the gene expression of important immune markers (iNOS, arginase-1, IFN-γ, TNF-α, IL-6, IL-10, and TGF-ß) was checked using real-time quantitative PCR. Clinical and laboratory data were obtained from the patients' medical records. Results Toxoplasma IgG antibodies were detected in 33 (75%) of patients. None of the studied clinical or laboratory parameters showed any significant changes in relation to toxoplasmosis seroprevalence. Further classification of the patients according to COVID-19 severity and Toxoplasma seroprevalence did not reveal any changes related to toxoplasmosis as well. Conclusion Our study indicates that latent toxoplasmosis has no effect on the severity of COVID-19.

Bone Morphogenetic Protein-4 Impairs Retinal Endothelial Cell Barrier, a Potential Role in Diabetic Retinopathy.

Bone Morphogenetic Protein 4 (BMP4) is a secreted growth factor of the Transforming Growth Factor beta (TGFß) superfamily. The goal of this study was to test whether BMP4 contributes to the pathogenesis of diabetic retinopathy (DR). Immunofluorescence of BMP4 and the vascular marker isolectin-B4 was conducted on retinal sections of diabetic and non-diabetic human and experimental mice. We used Akita mice as a model for type-1 diabetes. Proteins were extracted from the retina of postmortem human eyes and 6-month diabetic Akita mice and age-matched control. BMP4 levels were measured by Western blot (WB). Human retinal endothelial cells (HRECs) were used as an in vitro model. HRECs were treated with BMP4 (50 ng/mL) for 48 h. The levels of phospho-smad 1/5/9 and phospho-p38 were measured by WB. BMP4-treated and control HRECs were also immunostained with anti-Zo-1. We also used electric cell-substrate impedance sensing (ECIS) to calculate the transcellular electrical resistance (TER) under BMP4 treatment in the presence and absence of noggin (200 ng/mL), LDN193189 (200 nM), LDN212854 (200 nM) or inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2; SU5416, 10 µM), p38 (SB202190, 10 µM), ERK (U0126, 10 µM) and ER stress (Phenylbutyric acid or PBA, 30 µmol/L). The impact of BMP4 on matrix metalloproteinases (MMP2 and MMP9) was also evaluated using specific ELISA kits. Immunofluorescence of human and mouse eyes showed increased BMP4 immunoreactivity, mainly localized in the retinal vessels of diabetic humans and mice compared to the control. Western blots of retinal proteins showed a significant increase in BMP4 expression in diabetic humans and mice compared to the control groups (p < 0.05). HRECs treated with BMP4 showed a marked increase in phospho-smad 1/5/9 (p = 0.039) and phospho-p38 (p = 0.013). Immunofluorescence of Zo-1 showed that BMP4-treated cells exhibited significant barrier disruption. ECIS also showed a marked decrease in TER of HRECs by BMP4 treatment compared to vehicle-treated HRECs (p < 0.001). Noggin, LDN193189, LDN212854, and inhibitors of p38 and VEGFR2 significantly mitigated the effects of BMP4 on the TER of HRECs. Our finding provides important insights regarding the role of BMP4 as a potential player in retinal endothelial cell dysfunction in diabetic retinopathy and could be a novel target to preserve the blood-retinal barrier during diabetes.

Alteration of Aquaporins 1 and 4 immunohistochemical and gene expression in the cerebellum of diabetic albino rat.

Aquaporins (AQPs) are a family of transmembrane channel proteins. AQP1 and AQP4 are expressed in cerebellum amongst others. This study was designed to assess the effect of diabetes on AQP1 and AQP4 expression in cerebellum of rats. Diabetes was induced by a single intraperitoneal injection of Streptozotocin 45 mg/kg in 24 adult male Sprague Dawley rats. Six rats from control and diabetic groups were sacrificed at one, four, and eight weeks post diabetic confirmation. After eight weeks, measurement of malondialdehyde (MDA), reduced glutathione (GSH) concentrations, and cerebellar mRNA expression for AQP1 and AQP4 genes were performed. Immunohistochemical evaluation of AQP1, AQP4, and glial fibrillary acidic protein (GFAP) for cerebellar sections was performed for all groups. Diabetes caused degenerative changes in Purkinje cells with a significant increase in the cerebellar level of MDA and AQP1 immunoreactivity and a significant decrease in GSH level and AQP4 expression levels. However, the alteration in the AQP1 mRNA level was not statistically significant. GFAP immunoreactivity was increased in 8 W diabetic rats following its decrease in 1 W diabetic rats. Diabetes caused some alteration in the AQPs 1 and 4 expression in the cerebellum of diabetic rats which may contribute to diabetes-induced cerebellar complications.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

The hepatoprotective effects of n3-polyunsaturated fatty acids against non-alcoholic fatty liver disease in diabetic rats through the FOXO1/PPARα/GABARAPL1 signalling pathway.

AIM: We compared the efficacy of n3-polyunsaturated fatty acids (n3-PUFAs) and metformin in halting the progression of non-alcoholic fatty liver disease (NAFLD) developed in the milieu of insulin deficiency. MAIN METHODS: NAFLD was induced by a chronic high-fat diet (HFD) in male Sprague Dawley rats, rendered diabetic by a low dose streptozotocin (STZ). Diabetic rats were treated with n3-PUFAs (300 mg/kg/d) or metformin (150 mg/kg/d) for 8 weeks. Improvements in the NAFLD score and hepatic insulin resistance (IR) were addressed and correlated to changes in the hepatic expression of Forkhead box protein O1 (FOXO-1), microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) genes. Hepatic peroxisome proliferator-activated receptor alpha (PPAR-α), and B-cell lymphoma 2 (Bcl-2) protein expression was also assessed. KEY FINDINGS: Driven by insulin deficiency and HFD, the FOXO-1 gene along with its downstream targets, MAP1LC3B and GABARAPL1, were highly expressed in the liver tissue of the HFD/STZ group. Meanwhile, hepatic expression of PPAR-α and Bcl-2 was markedly decreased. These abnormalities coincided with a marked increase in the hepatic IR and NAFLD activity. Comparable to metformin, n3-PUFAs were able to rearrange hepatic PPAR-α and FOXO-1 expression in HFD/STZ rats, resulting in improved diabetic/steatotic liver phenotype. SIGNIFICANCE: Along with the enhancement of PPAR-α expression, inhibition of FoxO1/GABARAPL1/MAP1LC3B transcription is suggested as a core mechanism for the protective effects of n3-PUFAs on hepatic IR and NAFLD. Under conditions of insulin deficiency, n3-PUFAs retain their potential as a safe and promising approach for the control of NAFLD.

Bone Morphogenetic Proteins and Diabetic Retinopathy.

Bone morphogenetic proteins (BMPs) play an important role in bone formation and repair. Recent studies underscored their essential role in the normal development of several organs and vascular homeostasis in health and diseases. Elevated levels of BMPs have been linked to the development of cardiovascular complications of diabetes mellitus. However, their particular role in the pathogenesis of microvascular dysfunction associated with diabetic retinopathy (DR) is still under-investigated. Accumulated evidence from our and others' studies suggests the involvement of BMP signaling in retinal inflammation, hyperpermeability and pathological neovascularization in DR and age-related macular degeneration (AMD). Therefore, targeting BMP signaling in diabetes is proposed as a potential therapeutic strategy to halt the development of microvascular dysfunction in retinal diseases, particularly in DR. The goal of this review article is to discuss the biological functions of BMPs, their underlying mechanisms and their potential role in the pathogenesis of DR in particular.

Role of Arginase 2 in Murine Retinopathy Associated with Western Diet-Induced Obesity.

Western diet-induced obesity is linked to the development of metabolic dysfunctions, including type 2 diabetes and complications that include retinopathy, a leading cause of blindness. Aberrant activation of the inflammasome cascade leads to the progression of obesity-induced pathologies. Our lab showed the critical role of arginase 2 (A2), the mitochondrial isoform of this ureahydrolase, in obesity-induced metabolic dysfunction and inflammation. A2 deletion also has been shown to be protective against retinal inflammation in models of ischemic retinopathy and multiple sclerosis. We investigated the effect of A2 deletion on western diet-induced retinopathy. Wild-type mice fed a high-fat, high-sucrose western diet for 16 weeks exhibited elevated retinal expression of A2, markers of the inflammasome pathway, oxidative stress, and activation of microglia/macrophages. Western diet feeding induced exaggerated retinal light responses without affecting visual acuity or retinal morphology. These effects were reduced or absent in mice with global A2 deletion. Exposure of retinal endothelial cells to palmitate and high glucose, a mimic of the obese state, increased expression of A2 and inflammatory mediators and induced cell death. These effects, except for A2, were prevented by pretreatment with an arginase inhibitor. Collectively, our study demonstrated a substantial role of A2 in early manifestations of diabetic retinopathy.

Bone Morphogenetic Protein-2 Induces Non-Canonical Inflammatory and Oxidative Pathways in Human Retinal Endothelial Cells.

The mechanisms of diabetic retinopathy (DR), are not yet fully understood. We previously demonstrated an upregulation of retinal bone morphogenetic protein-2 (BMP2) in experimental diabetes and in retinas of diabetic human subjects. The purpose of current study was to investigate the role of non-canonical inflammatory pathway in BMP2-induced retinal endothelial cell (REC) barrier dysfunction. For this purpose, we used RT-PCR and western blotting to evaluate the levels of BMP2 signaling components (BMP2, BMP4, BMP receptors), VEGF, phosphorylated p38 MAPK and NFκB, and oxidative stress markers in cultured human retinal endothelial cells (HRECs) subjected to BMP2 (50ng/ml) for up to 24 h. Also, effect of high glucose (HG, 30mM D-glucose) on the expression of BMP2 and its downstream genes was examined in HRECs. H2-DCF is a fluorogenic dye that measures the levels of cellular reactive oxygen species (ROS) was used to measure the pro-oxidative effect of BMP2. Moreover, we evaluated the effect of inhibiting p38 and VEGF signaling on BMP2-induced HRECs barrier dysfunction by measuring the trans-endothelial cell electrical resistance (TER) using electric cell-substrate impedance sensing (ECIS). We also tested the effect of HG on the integrity of HRECs barrier in the presence or absence of inhibitors of BMP2 signaling. Our data reveals that BMP2 and high glucose upregulates BMP components of the BMP signaling pathway (SMAD effectors, BMP receptors, and TGFß ligand itself) and induces phosphorylation of p38 MAPK and NFκB with nuclear translocation of NFκB. Inhibition of p38 or NFκB attenuated BMP2-induced VEGF expression and barrier dysfunction in HRECs. Also, inhibition of VEGFR2 attenuated BMP2-induced barrier dysfunction. Moreover, BMP2 induces generation of ROS and endothelial nitric oxide synthase (eNOS) expression and activity in HRECs. Finally, HG upregulated BMP2 and its downstream genes (SMAD, BMP4, ALKs, and TGF-ß) in HRECs and BMP2 inhibitors attenuated HG-induced HRECs barrier dysfunction. Our results suggest that in addition to the regular canonical SMAD signaling BMP2 induces non-canonical inflammatory pathway in HRECs via activation of p38/NFκB pathway that causes the upregulation of VEGF and the disruption of HRECs. Inhibition of BMP2 signaling is a potential therapeutic intervention to preserve endothelial cell barrier function in DR.

Bioactive lipids and pathological retinal angiogenesis.

Angiogenesis, disruption of the retinal barrier, leukocyte-adhesion and oedema are cardinal signs of proliferative retinopathies that are associated with vision loss. Therefore, identifying factors that regulate these vascular dysfunctions is critical to target pathological angiogenesis. Given the conflicting role of bioactive lipids reported in the current literature, the goal of this review is to provide the reader a clear road map of what has been accomplished so far in the field with specific focus on the role of polyunsaturated fatty acids (PUFAs)-derived metabolites in proliferative retinopathies. This necessarily entails a description of the different retina cells, blood retina barriers and the role of (PUFAs)-derived metabolites in diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration as the most common types of proliferative retinopathies.

A Controlled Impact of Optic Nerve as a New Model of Traumatic Optic Neuropathy in Mouse.

Purpose: Traumatic optic neuropathy (TON) is the most feared visual consequence of head and ocular trauma in both military and civilian communities, for which standard treatment does not exist. Animal models are critical for the development of novel TON therapies as well as the understanding of TON pathophysiology. However, the models currently used for TON have some limitations regarding consistency and mirroring the exact pathological progression of TON in closed ocular trauma. In this study, we modified the model of controlled cortical impact and adapted it for studying TON. Methods: We defined new standardized procedures to induce TON in mice, wherein the optic nerve is reproducibly exposed to a graded controlled impact of known velocity to produce a graded deficit in retinal ganglion cell (RGC) electrophysiological functions. Results: The key results of validating this newly modified model, "controlled orbital impact (COI)," included (1) the injury parameters (velocity as well as contusion depth and time), which were quantifiable and manageable to generate a wide range of TON severities; (2) a reproducible endpoint of diminished positive scotopic threshold response (pSTR) has been achieved without the interference of surgical variability and destruction of surrounding tissues; (3) the contralateral eyes showed no significant difference to the eyes of naïve mice, allowing them to be used as an internal control to minimize interindividual variability among mice; and (4) the occurrence of injury-associated mortality and/or ocular comorbidity was rare. Conclusions: Taken together, this model overcomes some limitations of prior TON mouse models and provides an innovative platform to identify therapeutic targets for neuroprotection and/or neurorestoration following traumatic ocular injury.

PDL-1 Blockade Prevents T Cell Exhaustion, Inhibits Autophagy, and Promotes Clearance of Leishmania donovani.

Leishmania donovani is a causative pathogen of potentially fatal visceral leishmaniasis (VL). Therapeutic agents are available; however, their use is limited because of high cost, serious side effects, and development of antimicrobial resistance. Protective immunity against VL depends on CD4+ Th1 cell-mediated immunity. Studies have shown that progression of VL is due to exhaustion of T cells; however, the mechanism involved is not clearly understood. Here, we examined the role of PD1/PDL-1 in the pathogenesis of VL by using a murine model of VL. Our data indicate that L. donovani is able to elicit initial expansion of gamma interferon-producing CD4+ Th1 and CD8+ T cells at day 7 postinfection (p.i.); however, the frequency of those cells and inflammatory response decreased at day 21 p.i., despite persistence of parasites. Persistent infection-induced expansion of interleukin-10+ FOXP3+ Treg and CD4+ and CD8+ T cells expressing PD1. Blocking of PDL-1 signaling in vivo resulted in restoration of protective type 1 responses by both CD4+ and CD8+ T cells, which resulted in a significant decrease in the parasite burden. Mechanistically, PDL-1 blocking inhibited autophagy, a cellular degradation process hijacked by Leishmania to acquire host cell nutrients for their survival. Inhibition of autophagy was marked by decreased lipidation of microtubule-associated protein 1 light chain 3, a marker of autophagosome formation, and P62 accumulation. Together, our findings show for the first time that anti-PDL-1 antibody is an effective therapeutic approach for restoration of effector arms of protective immunity against VL and subsequent parasite clearance.

Epigenetic modifications in hyperhomocysteinemia: potential role in diabetic retinopathy and age-related macular degeneration.

To study Hyperhomocysteinemia (HHcy)-induced epigenetic modifications as potential mechanisms of blood retinal barrier (BRB) dysfunction, retinas isolated from three- week-old mice with elevated level of Homocysteine (Hcy) due to lack of the enzyme cystathionine ß-synthase (cbs-/- , cbs+/- and cbs+/+ ), human retinal endothelial cells (HRECs), and human retinal pigmented epithelial cells (ARPE-19) treated with or without Hcy were evaluated for (1) histone deacetylases (HDAC), (2) DNA methylation (DNMT), and (3) miRNA analysis. Differentially expressed miRNAs in mice with HHcy were further compared with miRNA analysis of diabetic mice retinas (STZ) and miRNAs within the exosomes released from Hcy-treated RPEs. Differentially expressed miRNAs were further evaluated for predicted target genes and associated pathways using Ingenuity Pathway Analysis. HHcy significantly increased HDAC and DNMT activity in HRECs, ARPE-19, and cbs mice retinas, whereas inhibition of HDAC and DNMT decreased Hcy-induced BRB dysfunction. MiRNA profiling detected 127 miRNAs in cbs+/- and 39 miRNAs in cbs-/- mice retinas, which were significantly differentially expressed compared to cbs+/+ . MiRNA pathway analysis showed their involvement in HDAC and DNMT activation, endoplasmic reticulum (ER), and oxidative stresses, inflammation, hypoxia, and angiogenesis pathways. Hcy-induced epigenetic modifications may be involved in retinopathies associated with HHcy, such as age-related macular degeneration and diabetic retinopathy.

Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy.

AIMS/HYPOTHESIS: Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. METHODS: We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo-/-); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2-/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. RESULTS: Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. CONCLUSIONS/INTERPRETATION: ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress.

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-ß-synthase is deficient (cbs -/-), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

Adenosine Deaminase-2-Induced Hyperpermeability in Human Retinal Vascular Endothelial Cells Is Suppressed by MicroRNA-146b-3p.

Purpose: We recently demonstrated that adenosine deaminase-2 (ADA2) contributes to diabetic retinopathy (DR) via up-regulating the production of inflammatory cytokines in macrophages. Also, microRNA (miR)-146b-3p has the ability to inhibit ADA2. The goal of this study was to investigate the potential role of ADA2 and therapeutic benefit of miR-146b-3p in retinal inflammation and endothelial barrier dysfunction during diabetes. Methods: Adenosine deaminase-2 activity was determined by colorimetric method in diabetic human vitreous. Human monocyte cell line U937 was differentiated into macrophages and then treated with amadori glycated albumin (AGA), and conditioned medium (CM) was used to assess the changes in ADA2 activity and TNF-α and IL-6 levels by ELISA. Also, macrophages were transfected with miR-146b-3p before treatment with AGA. Permeability of human retinal endothelial cells (hRECs) was assessed by electric cell-substrate impedance sensing (ECIS) after treatment with macrophage CM. Zonula occludens (ZO)-1 was examined by immuno-fluorescence in hRECs. Leukocyte adhesion was assessed in hRECs by measuring myeloperoxidase (MPO) activity and intercellular adhesion molecule-1 (ICAM-1) expression. Results: Adenosine deaminase-2 activity was significantly increased in diabetic human vitreous. ADA2 activity and TNF-α and IL-6 levels were significantly increased in human macrophages by AGA treatment. Amadori glycated albumin-treated macrophage CM significantly increased hREC permeability, disrupted ZO-1 pattern, and increased leukocyte adhesion to hRECs through up-regulating ICAM-1. All these changes were reversed by miR-146b-3p. Conclusions: Adenosine deaminase-2 is implicated in breakdown of the blood-retinal barrier (BRB) in DR through macrophages-derived cytokines. Therefore, inhibition of ADA2 by miR-146b-3p might be a useful tool to preserve BRB function in DR.