Retinal Histopathology Research Articles

Early life stress induces visual dysfunction and retinal structural alterations in adult mice.

Early life stress (ELS) is defined as a period of severe and/or chronic trauma, as well as environmental/social deprivation or neglect in the prenatal/early postnatal stage. Presently, the impact of ELS on the retina in the adult stage is unknown. The long-term consequences of ELS at retinal level were analyzed in an animal model of maternal separation with early weaning (MSEW), which mimics early life maternal neglect. For this purpose, mice were separated from the dams for 2h at postnatal days (PNDs) 4-6, for 3h at PNDs 7-9, for 4h at PNDs 10-12, for 6h at PNDs 13-16, and weaned at PND17. At the end of each separation period, mothers were subjected to movement restriction for 10min. Control pups were left undisturbed from PND0, and weaned at PND21. Electroretinograms, visual evoked potentials, vision-guided behavioral tests, retinal anterograde transport, and retinal histopathology were examined at PNDs 60-80. MSEW induced long-lasting functional and histological effects at retinal level, including decreased retinal ganglion cell function and alterations in vision-guided behaviors, likely associated to decreased synaptophysin content, retina-superior colliculus communication deficit, increased microglial phagocytic activity, and retinal ganglion cell loss through a corticoid-dependent mechanism. A treatment with mifepristone, injected every 3 days between PNDs 4 and16, prevented functional and structural alterations induced by MSEW. These results suggest that retinal alterations might be included among the childhood adversity-induced threats to life quality, and that an early intervention with mifepristone avoided ELS-induced retinal disturbances.

Diosgenin, a Natural Steroidal Sapogenin, Alleviates the Progression of Diabetic Retinopathy in Diabetic Mice.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and a major cause of blindness in working-age adults. Diosgenin (DG), a natural steroidal sapogenin extracted from fenugreek seeds and wild yam roots, has hypolipidemic, hypoglycemic, anticancer, and anti-inflammatory properties. Given its pharmacological effects, we speculated that DG may be a promising treatment for DR. Therefore, this study was aimed at evaluating the effectiveness of DG in preventing or slowing DR progression in a mouse model (+Leprdb/+Leprdb strain) of type 2 diabetes (T2D). DG (5.0 mg/kg body weight) or phosphate-buffered saline (PBS) was administered to 8-week-old T2D mice via oral gavage daily for 24 weeks. Paraffin-embedded eye tissues from the mice were collected and stained with hematoxylin and eosin to evaluate retinal histopathology. Apoptosis-related proteins BCL2-associated X (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved caspase-3 were evaluated by western blotting of mouse retinas. Body weight was slightly reduced in the DG-treated group; however, glucose levels were not markedly different between the DG- and PBS-treated groups. Total retinal thickness, thickness of the photoreceptor and outer nuclear layers, and loss of ganglion cells significantly improved in the retina of the DG-treated T2D mice compared with those in the PBS-treated T2D mice. Cleaved caspase-3 level significantly decreased in the retina of the DG-treated T2D mice. Conclusion: DG alleviates DR pathology and exerts a protective effect on the T2D mouse retina. The inhibitory effects of DG on DR may involve mechanisms of the anti-apoptotic pathway.

Exploration of the mechanisms underlying the beneficial effect of Luo Tong formula on retinal function in diabetic rats via the “gut microbiota–inflammation–retina” axis

BackgroundDiabetic retinopathy (DR) is a common microvascular complication of diabetes. Luo Tong formula (LTF), a classical traditional Chinese medicine (TCM) prescription, consists of four plants that have been widely and effectively used to treat DR. Previous work in our laboratory has confirmed that LTF can effectively ameliorate DR. However, the potential mechanism underlying the therapeutic effect of LTF on DR has not been fully elucidated. To explore the potential mechanism of action through which LTF prevents and alleviates DR from an inflammation and gut microbiota perspective.Materials and methodsMetabolite profiling of LTF was performed using liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS). Type 1 diabetes was induced in male Sprague Dawley (SD) rats via tail vein injection of 45 mg/kg streptozotocin. Next, 100 SD rats were randomly divided into four groups, normal control; diabetic control; diabetic + insulin + calcium dobesilate; and diabetic + insulin + LTF. After 12 weeks of treatment, glucose metabolism, fundus oculi, blood-retinal barrier permeability, retinal thickness, microvascular damage, as well as cell junction expression in retinas were measured and the changes observed in different groups were compared. Finally, the alteration in gut microbiota and inflammatory cytokine expression in serum and tissues were monitored, and their correlation was analyzed.ResultsA total of 1024 valid peaks were obtained for LTF using GC–MS. The HbA1c and fasting blood glucose (FBG) levels in the LTF group were slightly decreased. LTF exerted protective effects on fundus oculi and the retina structure to different degrees. LTF attenuated systemic and local retinal inflammation by significantly decreasing the levels of seven pro-inflammatory cytokines, including ICAM-1, IL-6, IL-8, MCP-1, VCAM-1, VEGF, and IL-1β. LTF restored the intestinal microbiota of diabetic rats to levels that were similar to those of normal rats. Further analysis revealed that Enterobacteriales, Prevotellaceae, Enterobacteriaceae, Bacteroides, and Klebsiella were significantly and positively correlated with the inflammatory factors in DR after LTF treatment.ConclusionsOur results revealed the mechanisms underlying the preventive effects of LTF on DR development and progression. LTF inhibited pathological changes in retinal histopathology, cell composition, and cell junction proteins while effectively ameliorating systemic and local retinal inflammation via regulating pivotal gut microbiota.

Traumatic Brain Injury Exacerbates Retinal Alzheimer’s Disease Histopathology in the TgF344 Rat Model

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative condition of the central nervous system that affects both the brain and the retina, and can lead to visual impairment. However, the factors that modulate AD pathophysiology leading to the wide range of clinical presentations are not well understood. Notably, traumatic brain injury (TBI), which can arise from sport concussions, military combat and other causes, is associated with a 2.3‐fold higher risk of developing AD and AD‐related dementias. Yet the pathophysiological link between TBI and AD remains poorly understood. In this study we set out to evaluate the effects of TBI, AD, and their combination, on retinal histopathology, by taking advantage of a transgenic rat model (Tg‐F344‐AD) shown to recapitulate the main features of human AD pathology, and combining it with a controlled cortical impact paradigm of repetitive mild TBI (rmTBI).MethodsMale Tg‐F344‐AD rats and F344 wild‐type controls were aged to 12 months and randomly selected to undergo a two‐time unilateral controlled cortical impact (2xCCI; one week apart) that mimics rmTBI, or a sham procedure. The animals were sacrificed at four months post‐CCI to evaluate long‐term effects of the procedure, and the eyes were collected and analyzed using immunofluorescence and standard histological staining techniques to evaluate Alzheimer’s histopathology.ResultsHistopathological analyses at four months post‐impact confirm the presence of AD markers in Tg‐F344‐AD retinas. Moreover, we found that AD increases Aβ deposition and neuroinflammation in the retina, and that TBI enhances the severity of this phenotype by increasing the formation of dense core amyloid plaques.ConclusionsWe have developed and characterized a model to study the pathophysiology of AD in relation to rmTBI in the retina, which could lead to better treatments and to the development of retinal biomarkers for improved AD diagnosis.

VEGF promotes diabetic retinopathy by upregulating the PKC/ET/NF-κB/ICAM-1 signaling pathway.

Diabetic retinopathy (DR) is a common microvascular complication in patients with diabetes mellitus. DR is caused by chronic hyperglycemia and is characterized by progressive loss of vision because of damage to the retinal microvasculature. In this study, we investigated the regulatory role and clinical significance of the vascular endothelial growth factor (VEGF)/protein kinase C (PKC)/endothelin (ET)/nuclear factor-κB (NF-κB)/intercellular adhesion molecule 1 (ICAM-1) signaling pathway in DR using a rat model. Intraperitoneal injections of the VEGF agonist, streptozotocin (STZ) were used to generate the DR model rats. DR rats treated with the VEGF inhibitor (DR+VEGF inhibitor) were used to study the specific effects of VEGF on DR pathology and the underlying mechanisms. DR and DR+VEGF agonist rats were injected with the PKCβ2 inhibitor, GF109203X to determine the therapeutic potential of blocking the VEGF/PKC/ET/NF-κB/ICAM-1 signaling pathway. The body weights and blood glucose levels of the rats in all groups were evaluated at 16 weeks. DR-related retinal histopathology was analyzed by hematoxylin and eosin staining. ELISA assay was used to estimate the PKC activity in the retinal tissues. Western blotting and RT-qPCR assays were used to analyze the expression levels of PKC-β2, VEGF, ETs, NF-κB, and ICAM-1 in the retinal tissues. Immunohistochemistry was used to analyze VEGF and ICAM-1 expression in the rat retinal tissues. Our results showed that VEGF, ICAM-1, PKCβ2, ET, and NF-κB expression levels as well as PKC activity were significantly increased in the retinal tissues of the DR and DR+VEGF agonist rat groups compared to the control and DR+VEGF inhibitor rat groups. DR and DR+VEGF agonist rats showed significantly lower body weight and significantly higher retinal histopathology scores and blood glucose levels compared to the control and DR+VEGF inhibitor group rats. However, treatment of DR and DR+VEGF agonist rats with GF109203X partially alleviated DR pathology by inhibiting the VEGF/ PKC/ET/NF-κB/ICAM-1 signaling pathway. In summary, our data demonstrated that inhibition of the VEGF/ PKC/ET/NF-κB/ICAM-1 signaling pathway significantly alleviated DR-related pathology in the rat model. Therefore, VEGF/PKC/ET/NF-κB/ICAM-1 signaling axis is a promising therapeutic target for DR.

Beneficial Actions of Essential Fatty Acids in Streptozotocin-Induced Type 1 Diabetes Mellitus.

The essential fatty acids (EFA), n3 alpha-linolenic acid (ALA), and n6 linoleic acid (LA) are of benefit in diabetes mellitus, but their mechanisms of action are unknown. We, therefore, examined the effects of EFAs on the metabolism, gut microbiota, and inflammatory and retinal histopathology indices in streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) animals, and we assessed the levels of vitreal lipoxin A4 (LXA4)—derived from LA—in subjects with diabetic retinopathy (DR). STZ-induced T1DM rats received LA or ALA 100 μg/day intraperitoneally on alternate days for 21 days, and their blood glucose; lipid profile; plasma, hepatic, and retinal fatty acid profiles (by gas chromatography); retinal histology; activities of hepatic and retinal desaturases; and inflammatory markers (by qRT-PCR) were evaluated. Gut microbiota composition was assayed by 16S rDNA sequencing technology of the fecal samples, and their short-chain fatty acids and bile acids were assayed by gas chromatography, liquid chromatography coupled with tandem mass spectrometry, respectively. The human vitreal fatty acid profiles of subjects with proliferative DR and LXA4 levels were measured. LA and ALA significantly improved the plasma glucose and lipid levels; increased the abundance of Ruminococcaceae (the ALA-treated group), Alloprevotella, Prevotellaceae_Ga6A1_group, Ruminococcaceae_UCG_010, and Ruminococcus_1 (the LA-treated group) bacteria; enhanced acetate and butyrate levels; and augmented fecal and hepatic concentrations of cholic acid, chenodeoxycholic acid, and tauro ursodeoxycholic acid in ALA- and LA-treated animals. Significant STZ-induced decreases in plasma LA, gamma-linolenic acid, arachidonic acid, and ALA levels reverted to near normal, following LA and ALA treatments. Significant changes in the expression of desaturases; COX-2, 5-LOX, and 12-LOX enzymes; and cytokines in T1DM were reverted to near normal by EFAs. DR subjects also had low retinal LXA4 levels. The results of the present study show that ALA and LA are of significant benefit in reversing metabolism, gut microbiota, and inflammatory and retinal index changes seen in T1DM, suggesting that EFAs are of benefit in diabetes mellitus.

Yiqi Tongluo Fang could preventive and delayed development and formation of diabetic retinopathy through antioxidant and anti-inflammatory effects

BackgroundYiqi Tongluo Fang (YQTLF) is an effective prescription for the treatment of diabetic retinopathy (DR), but its mechanism of action remains unclear. MethodThe content of YQTLF was determined using liquid and gas chromatography-mass spectrometry (LC-MS and GC-MS, respectively). Twenty-five Sprague Dawley (SD) rats were randomly selected as the normal control group. One hundred SD streptozotocin-induced diabetes (type 1) rats were randomly divided into diabetic control, diabetic+insulin+ calcium dobesilate (CaD), and diabetic+insulin+ YQTLF groups, with 25 rats in each group. Bodyweight level was measured every 2 weeks. After 12 weeks of gavage, the glucose levels, lipids, oxidative stress, inflammation, retinal histopathology, and the blood-retinal barrier were assessed in each group. The p38 MAPK pathway was changed to explore its internal mechanism. The measurement data were expressed as mean ± standard deviation, and different statistical methods were used according to a normal distribution, square error, or not. ResultsA total of 1024 valid peaks were identified in YQTLF using GC-MS. YQTLF significantly lowered the fasting blood glucose levels in diabetic rats. YQTLF early inhibited changes in retinal histology, capillaries, cells, and tight junction proteins (such as ZO-1, occludin, claudin-5, and VE-cadherin) before the formation and development of DR. These findings correlated with the alleviation of glucolipid metabolism, inflammation, and oxidative stress. The lncRNA MALAT1 and the PRC 2/p38 MAPK-related pathway, such as the expression of EZH2, SUZ12, EED, p38 MAPK, MMP-9, and VEGFR, were also correlated. ConclusionWe have demonstrated the molecular and cellular mechanisms underlying the preventive and delayed development and formation of DR. YQTLF prevents changes in dyslipidemia, retinal histology, capillaries, cells, and tight junction proteins. These protective effects appear to be linked to its antioxidant and anti-inflammatory effects, which prevent the activation of intracellular signaling pathways, such as the lncRNA MALAT1 and PRC 2/p38 MAPK-related pathway.

Captopril alleviates oxidative damage in diabetic retinopathy

AimsTo primarily explore the mechanism of captopril in oxidative stress and investigate the link between captopril alleviated oxidative damage and diabetic retinopathy (DR). Main methodsHuman retinal microvascular endothelial cells (HRMECs) were used for in vitro experiments and cultured in a 5.5 mM or 30 mM glucose medium. Sprague-Dawley rats were used for in vivo experiments, and parts of the rats were established for diabetic groups by injected streptozotocin (n = 10, each group). Both experiments had a captopril-treated group. The levels of total cholesterol (TC), reactive oxygen species (ROS), nitric oxide (NO), and human 3-nitrotyrosine (3-NT) were detected in assay kits and ELISA. Western blotting was used to detect the expression of steroid regulatory element binding protein 2 (SREBP2), inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS). Hematoxylin-eosin staining and Evans blue were used to describe retinal histopathology. Key findingsThe levels of TC, ROS, NO, and 3-NT were increased in the higher glucose groups compared with the normal controls during in vivo and in vitro experiments. Western blotting showed a higher level of SREBP2, iNOS, and VEGF and a lower eNOS level in the higher glucose groups. These results were reversed by captopril. Captopril relieved diabetic retinal vascular leakage. SignificanceOur study suggested that captopril alleviates oxidative damage in DR due to creating lower peroxynitrite by decreasing ROS and NO, which may provide a visible direction for DR research.

Traumatic Brain Injury Exacerbates Alzheimer's Disease Pathology in the Retina of Rats

Alzheimer's disease (AD) is a neurodegenerative condition that affects 5.7 million people in the U.S. alone, representing the leading form of dementia. Additionally, traumatic brain injury (TBI), which can arise from sport concussions, military combat and other causes, is associated with a 2.3-fold higher risk of developing AD and AD-related dementias. Moreover, AD can also lead to visual impairment, and recent studies have found that AD histopathology manifests in the retina, which is an extension of the central nervous system. However, the pathophysiological link between TBI and AD remains poorly understood. In this study we set out to evaluate the effects of TBI, AD, and their combination, on retinal histopathology. Several animal models have been developed to investigate the mechanisms of AD but have been limited by imperfect recapitulation of human pathology. Here we take advantage of a transgenic rat model (Tg-F344) that more faithfully mimics human AD pathology, and subject it to a TBI paradigm. Wild-type and transgenic rats were randomly selected to undergo a unilateral controlled cortical impact (CCI). At 15 months of age, the rats were sacrificed and the eyes subsequently removed, fixed, and processed. Retinas were sectioned and analyzed using immunofluorescent and standard histological staining techniques. Our results confirm the presence of AD markers in transgenic retinas, and an increased severity of AD pathology due to TBI. This has meaningful implications in understanding the pathophysiology of AD in relation to TBI within the retina, which could lead to better treatments and to the development of retinal biomarkers for improved AD diagnosis.

Ocular Versus Oral Propranolol for Prevention and/or Treatment of Oxygen-Induced Retinopathy in a Rat Model.

Purpose: Propranolol, a nonselective B1/B2 adrenoceptor antagonist, promotes the regression of infantile hemangiomas likely through suppression of vascular endothelial growth factor (VEGF), which prompted its use for the prevention of retinopathy of prematurity. We tested the hypothesis that topical ocular propranolol is safe and effective for reducing the severity of oxygen-induced retinopathy (OIR) in the neonatal rat intermittent hypoxia (IH) model. Methods: At birth (P0), rat pups were randomly assigned to room air or neonatal intermittent hypoxia (IH) consisting of 50% O2 with brief episodes of hypoxia (12% O2) from P0 to P14, during which they received a single daily dose of oral propranolol (1 mg/kg/day in 50 μL in sterile normal saline) or topical ocular propranolol (0.2% in 10 μL in normal saline) from P5 to P14. Placebo-controlled littermates received 50 μL oral or 10 μL topical ocular sterile normal saline. Retinal vascular and astrocyte integrity; retinal histopathology and morphometry; and angiogenesis biomarkers were determined. Results: Topical ocular propranolol improved retinal vascular damage and preserved the astrocytic template, but did not completely prevent OIR. The beneficial effects of propranolol were associated with reduced ocular VEGF and increased endogenous soluble inhibitor, sVEGFR-1, when administered topically. Conclusions: Propranolol failed to completely prevent severe OIR, however, it prevented astrocyte degeneration resulting from neonatal IH-induced damage. We conclude that the mechanisms of propranolol's beneficial effects in neonatal IH may involve in part, astrocyte preservation.

Retinal safety evaluation of photoacoustic microscopy

Photoacoustic microscopy (PAM) has significant potential as a promising diagnostic method for eye diseases and can provide anatomic and functional information of the retinal and choroidal vasculature. However, there are no FDA-approved PAM systems for ophthalmic imaging. In this study, a comprehensive safety evaluation was performed to evaluate the safety of PAM retinal imaging and whether PAM causes damage to retinal structure or function in rabbit eyes. 12 Dutch-Belted pigmented rabbits received photoacoustic imaging to 57% of the retinal surface area with a laser energy of 5% of the ANSI safety limit for five consecutive days and followed before imaging and 3 days, 1, 2, 3, and 4 weeks post imaging. Retinal morphologic analyses using slit lamp examination, fundus photography, red free, FA, FAF, ICGA, and OCT showed no retinal hemorrhage, edema, detachment, vascular abnormalities, or pigmentary abnormalities in the retina or choroid after PAM imaging. Full-field ERG analysis showed no significant difference in scotopic or photopic a- and b-wave amplitudes or implicit times between the control and experimental eyes over time (n = 6, P values > 0.05). Retinal ultrastructural evaluation using TEM showed normal structure of organelles and nuclei, and no significant loss of cells after PAM. TUNEL assay showed no evidence of cells apoptosis in retina. Retinal histopathology indicated that the architecture and thickness of the retinal layers was well preserved in all experimental eyes. A positive control at 500% of the ANSI limit demonstrated significant damage. The comprehensive retinal safety evaluation demonstrated no damage to retinal structure or function for 4 weeks after PAM imaging in rabbits.

Histological and Behavioral Toxicity of Tributyltin in the Tropical Guppy Poecilia vivipara.

The tropical estuarine guppy Poecilia vivipara was used to address fish early life stage toxicity caused by the antifouling contaminant tributyltin. Six-day-old P. vivipara were exposed for 7 d to control water and waterborne tributyltin at 15.8, 83.8, 716, and 818 ng tin (Sn) L-1 . After exposure, swimming, feeding, growth, and eye histological endpoints were evaluated. Histopathological analysis of the retinal pigment epithelium (RPE) indicated alterations in pigment positioning at all tributyltin concentrations. A dose-dependent increase in photoreceptor layer disorganization and iris melanin hyperpigmentation was verified, and high frequencies of RPE invaginations and iris epithelial cell atrophy were observed even at the lowest exposure concentration of 15.8 ng Sn L-1 . At the highest exposure level (818 ng Sn L-1 ) fish also presented reductions in swimming speed, swimming resistance, daily capture of Artemia nauplii, and growth in weight of 85, 60, 33, and 56% relative to controls, respectively. This association between retinal histopathology and reduced swimming and foraging behavior can reduce recruitment to the adult population. Environ Toxicol Chem 2020;39:1953-1963. © 2020 SETAC.

Neutrophil Activities in Human Ocular Toxoplasmosis: An In Vitro Study With Human Cells.

Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.

Functional Regulation of an Oxidative Stress Mediator, Rac1, in Diabetic Retinopathy.

Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires activation of a small molecular weight G protein Rac1. Rac1 activation is regulated by guanine exchange factors and guanine nucleotide-dissociation inhibitors, and post-translational modifications assist in its association with exchange factors and dissociation inhibitors. The goal of this study is to investigate the mechanisms of Rac1 activation in the development of diabetic retinopathy. The levels of the dissociation inhibitor, prenylating enzyme (farnesyltransferase, FNTA), and exchange factor Vav2 were quantified in human retinal endothelial cells, incubated in normal or high glucose for 96h. The roles of prenylation and Vav2 in Rac1-Nox2-ROS mitochondrial damage were confirmed in FNTA-siRNA-transfected cells and using the Vav2 inhibitor EHop, respectively. Retinal histopathology and functional changes associated with diabetic retinopathy were analyzed in diabetic mice receiving EHop for 6months. Key parameters of Rac1 activation were confirmed in the retinal microvasculature from human donors with diabetic retinopathy. In HRECs, glucose increased FNTA and Vav2 and decreased the dissociation inhibitor. FNTA-siRNA and EHop inhibited glucose-induced activation of Rac1-Nox2-ROS signaling. In diabetic mice, EHop ameliorated the development of retinopathy and functional/structural abnormalities and attenuated Rac1-Nox2-mitochondrial damage. Similar alterations in Rac1 regulators were observed in retinal microvasculature from human donors with diabetic retinopathy. In diabetes, Rac1 prenylation and its interactions with Vav2 contribute to Nox2-ROS-mitochondrial damage, and the pharmacological inhibitors to attenuate Rac1 interactions with its regulators could have the potential to halt/inhibit the development of diabetic retinopathy. Graphical Abstract Activation of prenylating enzyme farnesyltransferase (FNTA) in diabetes, prenylates Rac1. The binding of Rac1 with guanine nucleotide-dissociation inhibitor (GDI) is decreased, but its association with the guanine exchange factor, Vav2, is increased, resulting in Rac1 activation. Active Rac1 helps in the assembly of Nox2 holoenzyme, and Nox2 activation increases cytosolic ROS production, damaging the mitochondria. Damaged mitochondria accelerate capillary cell apoptosis, and ultimately, results in the development of diabetic retinopathy.

Ginsenoside Rb1 attenuates diabetic retinopathy in streptozotocin-induced diabetic rats1.

Purpose To investigated the effects of ginsenoside Rb1 on diabetic retinopathy in streptozotocin-induced diabetic rats. Methods Diabetes was induced by a single intraperitoneal injection of streptozotocin (80 mg/kg) in male Wistar rats. Ginsenoside Rb1 (20, 40 mg/kg) was injected (i.p.) once a day for 4 weeks. Then, using fundus photography, the diameter and vascular permeability of retinal vessels were investigated. Retinal histopathology was undertaken. Contents of malondialdehyde (MDA) and glutathione (GSH) in retinas were assayed. Levels of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione cysteine ligase catalytic subunit (GCLC), and glutathione cysteine ligase modulatory subunit (GCLM) were measured. Results Treatment with ginsenoside Rb1 attenuated the diabetes-induced increase in the diameter of retinal blood vessels. Ginsenoside Rb1 reduced extravasation of Evans Blue dye from retinal blood vessels. Ginsenoside Rb1 partially inhibited the increase in MDA content and decrease in GSH level in rat retinas. Nrf2 levels in the nuclei of retinal cells and expression of GCLC and GCLM were increased significantly in rats treated with ginsenoside Rb1. Conclusion These findings suggest that ginsenoside Rb1 can attenuate diabetic retinopathy by regulating the antioxidative function in rat retinas.

Toxicity and in vivo release profile of sirolimus from implants into the vitreous of rabbits' eyes.

To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes. PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1-8weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24h, 1, 4, 6, and 8weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka-Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized. After 24 and 72h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8ng/mL) released from the device occurred within 4weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants. Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8weeks.

Antisense oligonucleotides targeting mutant Ataxin-7 restore visual function in a mouse model of spinocerebellar ataxia type 7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.

The Neuroprotective and antioxidative effects of submicron and blended Lycium barbarum in experimental retinal degeneration in rats.

The object is to determine the neuroprotective and antioxidative effects of submicron and blended Lycium barbarum (LB) on retinal degeneration as evaluated by ERG, retinal histopathology and assays of antioxidant (total GSH) and peroxidant (MDA) in the retina. A rat model of light-induced retinal degeneration was used to assess the protective effect of different forms of Lycium barbarum (LB) on retinal degeneration. Rats were divided into four experimental groups, normal control, light-induced untreated, submicron LB and blended LB treated. The rats of submicron and blended groups were treated with 250 mg/kg LB orally once daily for 54 days, followed by induction of retinal degeneration. Retinal function was assessed by electroretinography (ERG). Enzyme-linked immunosorbent assay of the retina lysates was measured for the levels of antioxidants, reduced glutathione and glutathione disulfide, and peroxidants, malondialdehyde, in the retina. The ERG results showed a protective effect in LB treated groups with a greater effect observed in submicron LB treated group than the blended LB treated group. There were higher levels of GSH plus GSSG and lower MDA in submicron LB treated group than other groups. In conclusion, LB provided protective and antioxidative effects on the rat retina with light-induced retinal degeneration. Submicron LB protected degenerative retina better than blended LB. LB is effective against oxidative stress in the degenerative retina.

Viral Retinopathy in Experimental Models of Zika Infection.

PurposeEmerging evidence has shown that both congenital and adult Zika virus (ZIKV) infection can cause eye diseases. The goals of the current study were to explore mechanisms and pathophysiology of ZIKV-induced eye defects.MethodsWild-type or A129 interferon type I receptor–deficient mice were infected by either FSS13025 or Mex1-7 strain of ZIKV. Retinal histopathology was measured at different time points after infection. The presence of viral RNA and protein in the retina was determined by in situ hybridization and immunofluorescence staining, respectively. Growth curves of ZIKV in permissive retinal cells were assessed in cultured retinal pigment epithelial (RPE) and Müller glial cells.ResultsZIKV-infected mice developed a spectrum of ocular pathologies that affected multiple layers of the retina. A primary target of ZIKV in the eye was Müller glial cells, which displayed decreased neurotrophic function and increased expression of proinflammatory cytokines after infection. ZIKV also infected RPE; and both the RPE and Müller cells expressed viral entry receptors TYRO3 and AXL. Retinitis, focal retinal degeneration, and ganglion cell loss were observed after the clearance of viral particles.ConclusionsOur data suggest that ZIKV can infect infant eyes with immature blood–retinal barrier and cause structural damages to the retina. The ocular findings in microcephalic infants may not be solely caused by ZIKV-induced impairment of neurodevelopment.

Histological Evaluation of Diabetic Neurodegeneration in the Retina of Zucker Diabetic Fatty (ZDF) Rats

In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes.