Apoptotic Vesicles Modulate Endothelial Metabolism and Ameliorate Ischemic Retinopathy via PD1/PDL1 Axis.

Pathological angiogenesis with subsequent disturbed microvascular remodeling is a major cause of irreversible blindness in a number of ischemic retinal diseases. The current anti-vascular endothelial growth factor therapy can effectively inhibit angiogenesis, but it also brings significant side effects. The emergence of stem cell derived extracellular vesicles provides a new underlining strategy for ischemic retinopathy. Apoptotic vesicles (apoVs) are extracted from stem cells from human exfoliated deciduous teeth (SHED). SHED-apoVs are delivered into the eyeballs of oxygen-induced retinopathy (a most common model of angiogenic retinal dieseases) mice through intravitreal injection. The retinal neovascularization and nonperfusion area, vascular structure, and density changes are observed during the neovascularization phase (P17) and vascular remodeling phase (P21), and visual function is measured. The expression of extracellular acidification rate and lactic acid testing are used to detect endothelial cells (ECs) glycolytic activity. Furthermore, lentivirus and neutralizing antibody are used to block PD1-PDL1 axis, investigating the effects of SHED-apoVs on glycolysis and angiogenic activities. This work shows that SHED-apoVs are taken up by ECs and modulate the ECs glycolysis, leading to the decrease of abnormal neovessels and vascular remodeling. Furthermore, it is found that, at the molecular level, apoVs-carried PD1 interacts with PDL1 on hypoxic ECs to regulate the angiogenic activation. SHED-apoVs inhibit pathological angiogenesis and promote vascular remodeling in ischemic retinopathy partially by modulating ECs glycolysis through PD1/PDL1 axis. This study provides a new potential strategy for the clinical treatment of pathological retinal neovascularization.

Evaluation of retinal vascularization in retinopathy of prematurity regressed after intravitreal ranibizumab monotherapy or without treatment based on fluorescein angiography.

To investigate the fluorescein angiography (FA) findings and compare the extent of retinal vascularization in retinopathy of prematurity (ROP), recovered after intravitreal ranibizumab (IVR) monotherapy and those regressed spontaneously. Infants with a history of ROP who underwent FA between April 2018 and November 2021 were retrospectively included. The patients were divided into two groups based on whether they had received IVR (IVR group) or had ROP that regressed spontaneously without treatment (untreated group). The differences between the two groups in zone II ROP were also compared, to equalize the subgroups as much as possible in terms of disease severity. FA findings were recorded. The extent of vascularization was measured by the ratio of the distance from the center of the disk to the border of the vascularized zone (DB) and the distance from the center of the disk to the center of the fovea (DF). The width of the persistent avascular retina (PAR) was counted by disc diameters (DD). One hundred and ten eyes of 55 infants were included in the IVR group and 76 eyes of 38 babies in the untreated group. The ratio of abnormal shape of vessels was significantly higher in the IVR group than in the untreated group (50.9% vs. 35.5%; P = 0.038), while the linear choroidal filling pattern, tortuosity of vessels over the posterior pole, dye leakage, anomalous branching of vessels, circumferential vessels, arteriovenous shunt, abnormal capillary bed, and macular abnormalities were similarly. There was a smaller temporal DB/DF ratio (4.48 vs. 4.63; P = 0.003) and greater PAR (2.63 vs. 1.76; P < 0.001) in the IVR group compared to the untreated group. In zone II ROP, the progression of retinal vascularization was significantly larger in the IVR group than that in the untreated group (P = 0.003), while no statistical differences were observed in FA features, the DB/DF ratio, and PAR between the two subgroups. The residual vascular abnormalities and PAR may be common results of ROP regression. The DB/DF ratio of 4.0 temporally and 3.3 nasally could be used as the preliminary indicators for safe retinal vascularization in the completion of ROP regression.

The efficacy and safety of sodium nitroprusside in the treatment of schizophrenia: a meta-analysis.

Objective: Schizophrenia is a serious mental disease that brings not only serious burdens to patients and their families but also serious challenges to society. More research is needed to find better drugs to treat schizophrenia. This meta-analysis investigated the efficacy and safety of sodium nitroprusside in the treatment of schizophrenia. Methods: Randomized controlled trials comparing the efficacy and safety of sodium nitroprusside in the treatment of schizophrenia were searched via English and Chinese databases. The outcomes, including the Positive and Negative Syndrome Scale (PANSS) and Brief Psychiatric Rating Scale (BPRS), were recorded. RevMan 5.3 was used for the meta-analysis. Results: A total of six randomized controlled trials (174 patients) were included. The overall quality of the included studies was good. No statistically significant benefit of sodium nitroprusside over placebo was found when combined PANSS total and BPRS-18 (95% CI: -1.40, 0.02). Except for PANSS positive (95% CI: -1.86, -0.01), there was no significant difference in the scale score after sodium nitroprusside treatment compared with the control group in PANSS total (95% CI: -4.93, 0.23), PANSS general (95% CI: -2.53, 1.33), and PANSS negative (95% CI: -4.44, 0.89). The results of the sensitivity analysis excluding the study with clinical heterogeneity showed that sodium nitroprusside had no statistical benefit for the score of PANSS positive (95% CI: -2.19, 0.46). Moreover, there was also no significant difference in the BPRS-18 (95% CI: -3.23, -0.43). Conclusion: We conservatively believe that sodium nitroprusside does not alleviate the symptoms of schizophrenia compared with placebo. The subjects tolerated sodium nitroprusside well. Our findings provide a new idea for researchers to explore and solve the drug treatment of schizophrenia.

Microglial Galectin3 enhances endothelial metabolism and promotes pathological angiogenesis via Notch inhibition by competitively binding to Jag1.

Microglia were considered as immune cells in inflammation until their angiogenic role was widely understood. Although the pro-inflammatory role of microglia in retinal angiogenesis has been explored, little is known about its role in pro-angiogenesis and the microglia-endothelia interaction. Here, we report that galectin-3 (Gal3) released by activated microglia functions as a communicator between microglia and endothelia and competitively binds to Jag1, thus inhibiting the Notch signaling pathway and enhancing endothelial angiogenic metabolism to promote angiogenesis. These results suggest that Gal3 may be a novel and effective target in the treatment of retinal angiogenesis.

A novel lytic bacteriophage against colistin-resistant Escherichia coli isolated from different animals.

Escherichia coli is a common pathogen in human and veterinary clinical infection. With antibiotic resistance including colistin resistance increasing globally, few antibiotic treatments are available for use against multidrug-resistant strains of E. coli. Given such circumstances, bacteriophage (phage) therapy is once again being considered as a potential alternative or adjunct to antibiotic therapy. Here, we isolated 52 phages from 816 samples from pig, chicken and duck farms in 4 provinces in China and identified a novel Escherichia phage, vB_EcoStr-FJ63A, from pig feces. Morphological observation showed that phage vB_EcoStr-FJ63A had an icosahedral capsid and an inflexible tail. Whole-genome sequencing revealed a double-stranded DNA genome of 168,157 bp (including 271 coding sequences) with a GC content of 40.29%. Bioinformatic analysis classified phage vB_EcoStr-FJ63A as a Krischvirus, belonging to Straboviridae. The phage was relatively stable at pH 4-10 and below 60℃. It was lytic against a wide variety of colistin-resistant strains of E. coli from various animals, with one-step growth curves showing a latent period of 30 min and burst size of ∼11 PFU per infected cell. Maximum bactericidal activity was achieved within 2 h. No antibiotic resistance or virulence genes were detected in the phage genome. Further studies are warranted to develop phage vB_EcoStr-FJ63A as a potential biocontrol agent against colistin-resistant E. coli.

Efficacy of inferior oblique belly transposition combined with inferior oblique recession for asymmetric inferior oblique overaction.

OBJECTIVE: To evaluate the efficacy of inferior oblique belly transposition (IOBT) combined with inferior oblique (IO) recession in treating bilateral asymmetric inferior oblique overaction (IOOA). METHODS: A retrospective review. The data of 14 patients who underwent IOBT on the mild side of IOOA and IO recession on the severe side for bilateral asymmetric IOOA were analyzed retrospectively. The main surgical results including the correction of IOOA, hypertropia, horizontal deviation, V pattern, and fovea-disc angle (FDA) were observed. RESULTS: The IOBT corrected the preoperative grade (+1.86 ± 0.53) of the mild-side IOOA to a postoperative grade (+0.07 ± 0.27; p < 0.001), and the severe-side IOOA was corrected from a grade of +3.14 ± 0.53 to a postoperative grade of +0.14 ± 0.36 by the IO recession (p < 0.001). The vertical deviation at distance in the primary position was decreased from 8.43 ± 4.05 PD preoperatively to 1.21 ± 1.48 PD postoperatively (p < 0.001). The mean V pattern was 25.00 ± 11.62 PD preoperatively and 3.18 ± 2.18 PD postoperatively (p < 0.001). The mean preoperative FDA on the side where IOBT was performed was -10.47 ± 5.85 degrees, and the postoperative FDA was -7.82 ± 6.42 degrees (p = 0.023). The mean FDA on the side with IO recession was -11.05 ± 5.14 degrees before surgery and -6.09 ± 4.52 degrees after surgery (p = 0.001). The overall success rate was 71.4% (10 of 14). CONCLUSIONS: IOBT combined with IO recession is effective and safe in eliminating hypertropia, V pattern, and extorsion with bilateral asymmetric IOOA.

Certain Dietary Nutrients Reduce the Risk of Eye Affliction/Retinopathy in Individuals with Diabetes: National Health and Nutrition Examination Survey, 2003-2018.

As the global trend of diabetes intensifies, the burden of vision-threatening retinopathy, particularly diabetic retinopathy (DR), is increasing. There is an urgent need to seek strategies for early prevention and control of DR. This study attempted to comprehensively evaluate the relationship between dietary nutrient intake and the risk of DR to provide assistance for doctors in guiding the diet of diabetic patients. Data from eligible participants with diabetes from the US National Health and Nutrition Examination Survey (NHANES) from 2003-2018 were analyzed. Univariate logistic regression was used to assess the association between 58 dietary nutrient intakes and self-reported eye disease risk. Multivariate logistic regression model was used to further evaluate the relationship between the two groups after adjusting relevant confounding factors. A total of 4595 diabetic patients were included. People with self-reported eye affliction/retinopathy had lower dietary fiber, butanoic, octanoic, vitamin A, alpha-carotene, folate, magnesium, copper and caffeine intake compared to those without self-reported eye affliction/retinopathy. The pooled ORs (95% CIs) were 0.78 (0.62-0.98), 0.79 (0.63-0.99), 0.72 (0.58-0.91), 0.74 (0.59-0.93), 0.70 (0.55-0.88), 075 (0.60-0.95), 0.79 (0.64-0.99), 0.67 (0.54-0.84) and 0.80 (0.64-0.99). Dietary cholesterol and hexadecenoic intake were higher, with the pooled ORs (95% CIs) of 1.26 (1.01-1.58) and 1.27 (1.02-1.59), respectively. Our research found that among dietary nutrients, dietary fiber, butanoic, octanoic, vitamin A, alpha-carotene, folate, magnesium, copper and caffeine intake reduced the occurrence of DR. Cholesterol and hexadecenoic intake promoted the occurrence of DR. This suggests that certain dietary nutrients should be paid more attention in the prevention of DR.

Associations between psycho-behavioral risk factors and diabetic retinopathy: NHANES (2005-2018).

Introduction: Diabetes mellitus (DM) and diabetic retinopathy (DR) increase the global burden. Since their pathogenesis is complex, it is necessary to use the biopsychosocial model to discover the most effective strategies. The study is aimed to investigate the psycho-behavioral factors of DR and confirm the discrepancies from previous studies. Research design and methods: The study comprised seven cycles of cross-sectional data of the National Health and Nutrition Examination Survey (NHANES) from 2005-2006 to 2017-2018. Samples of DM were selected from this complex multi-stage probability sample and divided into the non-DR and DR groups, where 4,426 samples represented 18,990,825 individuals after weighting. This study comprehensively explored the biological, social, and psychological risk factors of DR, among which the biological factors included blood pressure, blood routine, HbA1c%, blood glucose, the duration of DM, family history, comorbidities, and treatment methods. Social aspects include gender, education, income, insurance, smoking, drinking, sleep habits, and recreational activities. The Patient Health Questionnaire-9 (PHQ-9) was used to assess the psychological state. Taylor series regression was used to examine the connection between factors and DR. Results: Men accounted for 55.5% of the DR group (P = 0.0174). Lymphocyte count, insulin treatment, heart failure, stroke, liver condition, and renal failure showed significant differences in DR (P < 0.05). The incidence of depression in DR was 40.5%. Mild to moderate depression [odds ratio was associated with DR [(OR) = 1.37, 95% confidence interval (CI): 1.06-1.79], but there was no statistical difference in severe depression (OR = 1.34, 95% CI: 0.83-2.17). Although ≤ 6 h of sleep was associated with DR (OR = 1.38, 95% CI: 1.01-1.88), we found no statistical differences in alcohol consumption, recreational activities, or sedentary time between the two groups in our current study (P > 0.05). Conclusions: The biological risk factors of DR are significant. It showed that stroke is associated with DR, and retinal exams have the potential value as a screening tool for the brain. Besides, psycho-behavioral risk factors of DR should also be paid attention. Our study highlights that mild and moderate depression and ≤6 h of sleep are distinguishably associated with DM complicated with DR. It indicates that psycho-behavioral risk factors confer a vital influence on diabetic health care and DR.

Distinguished Functions of Microglia in the Two Stages of Oxygen-Induced Retinopathy: A Novel Target in the Treatment of Ischemic Retinopathy.

Microglia is the resident immune cell in the retina, playing the role of immune surveillance in a traditional concept. With the heated focus on the mechanisms of microglia in pathological conditions, more and more functions of microglia have been discovered. Although the regulating role of microglia has been explored in ischemic retinopathy, little is known about its mechanisms in the different stages of the pathological process. Here, we removed microglia in the oxygen-induced retinopathy model by PLX5622 and revealed that the removal of activated microglia reduced pathological angiogenesis in the early stage after ischemic insult and alleviated the over-apoptosis of photoreceptors in the vessel remodeling phase. Our results indicated that microglia might play distinguished functions in the angiogenic and remodeling stages, and that the inhibition of microglia might be a promising target in the future treatment of ischemic retinopathy.

Rapid and Sensitive Detection of the Colistin Resistance Gene mcr-3 by Loop-Mediated Isothermal Amplification and Visual Inspection.

Objective: The distribution of colistin resistance in mcr-carrying bacteria poses a threat to global public health. In particular, the newly identified mcr-3 allele has spread globally, especially in China, second only to mcr-1. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid, sensitive, and visual detection of the presence of the mcr-3 gene. Materials and Methods: A total of 13 clinical bacterial strains and 11 negative strains were used in this study. We designed LAMP Primers, optimized reaction conditions, used three different methods to detect LAMP amplification products: (1) agarose gel electrophoresis, (2) LAMP-hydroxy naphthol blue (HNB) detection, (3) LAMP-SYBR Green I (LAMP-SGI) visual inspection, and evaluated its specificity and sensitivity. Results: The amplification reaction was completed in 1 hr at 62°C under isothermal conditions. The final optimized mixtures contained 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100, 1.2 µL HNB, and 0.5 µL SYBR Green I as additives to the initial reaction mixture. LAMP detection, including two visual methods, LAMP-HNB and LAMP-SGI, of mcr-3 possessed the same specificity and a 10-fold higher sensitivity compared with a conventional polymerase chain reaction assay using the same samples. Conclusion: We successfully established an mcr-3 LAMP detection with portability and rapidity of the reaction by the easily distinguishable color changes in the reaction tubes. This visual LAMP assay for mcr-3 detection was simple, time saving, and economical, especially suited to field laboratories.

Connective tissue growth factor promotes retinal pigment epithelium mesenchymal transition via the PI3K/AKT signaling pathway.

Proliferative vitreoretinopathy (PVR) is a disease leading to the formation of contractile preretinal membranes (PRMs) and is one of the leading causes of blindness. Connective tissue growth factor (CTGF) has been identified as a possible key determinant of progressive tissue fibrosis and excessive scarring. Therefore, the present study investigated the role and mechanism of action of CTGF in PVR. Immunohistochemical staining was performed to detect the expression of CTGF, fibronectin and collagen type III in PRMs from patients with PVR. The effects and mechanisms of recombinant human CTGF and its upstream regulator, TGF­ß1, on epithelial­mesenchymal transition (EMT) and the synthesis of extracellular matrix (ECM) by retinal pigment epithelium (RPE) cells were investigated using reverse transcription­quantitative PCR, western blotting and a [3H]proline incorporation assay. The data indicated that CTGF, fibronectin and collagen type III were highly expressed in PRMs. In vitro, CTGF significantly decreased the expression of the epithelial markers ZO­1 and E­cadherin and increased that of the mesenchymal markers fibronectin, N­cadherin and α­smooth muscle actin in a concentration­dependent manner. Furthermore, the expression of the ECM protein collagen type III was upregulated by CTGF. However, the trends in expression for the above­mentioned markers were reversed after knocking down CTGF. The incorporation of [3H]proline into RPE cells was also increased by CTGF. In addition, 8­Bromoadenosine cAMP inhibited CTGF­stimulated collagen synthesis and transient transfection of RPE cells with a CTGF antisense oligonucleotide inhibited TGF­ß1­induced collagen synthesis. The phosphorylation of PI3K and AKT in RPE cells was promoted by CTGF and TGF­ß1 and the latter promoted the expression of CTGF. The results of the present study indicated that CTGF may promote EMT and ECM synthesis in PVR via the PI3K/AKT signaling pathway and suggested that targeting CTGF signaling may have a therapeutic or preventative effect on PVR.

High Prevalence and Diversity Characteristics of blaNDM, mcr, and blaESBLs Harboring Multidrug-Resistant Escherichia coli From Chicken, Pig, and Cattle in China.

The objective of this study was to understand the diversity characteristics of ESBL-producing Escherichia coli (ESBL-EC) in chicken, pig, and cattle. A high prevalence of ESBL-EC (260/344) was observed in all food animals with prevalence rates of 78.6% (110/140) for chicken, 70.7% (58/82) for cattle, and 75.4% (92/122) for swine. However, the resistance rates presented significant differences in different animal origin ESBL-EC, where resistance to CTX, GEN, IMP, NEO, and OFL was the highest in chicken ESBL-EC, then in cattle, and the lowest in swine. Seriously, most ESBL-EC harbor multidrug resistance to antibiotics (MDR, ≥3 antibiotic categories), and the MDR rates of ESBL-EC were the highest in chicken (98.18%), followed by swine (93.48%), and the lowest in cow (58.62%), while the same trend also was observed in MDR of ≥5 antibiotic categories. This high prevalence and resistance can be partly interpreted by the high carriage rates of the ß-lactamases CTX-M (n = 89), OXA (n = 59), SHV (n = 7), and TEM (n = 259). A significant difference of ß-lactamase genes also presented in different animal species isolates, where the chicken origin ESBL-EC possessed higher carriage rates of almost all genes tested than cattle and swine. Notably, eight chicken origin ESBL-EC carried transferable plasmid-mediated blaNDM-1 or blaNDM-5, especially, of which four ESBL-EC also contained the colistin resistance gene mcr-1, as confirmed by genomic analysis. More interestingly, two deletion events with a 500-bp deletion in ΔISAba125 and a 180-bp deletion in dsbC were observed in three blaNDM-5 IncX3 plasmids, which, as far as we know, is the first discovery. This showed the instability and horizontal transfer of blaNDM genetic context, suggesting that blaNDM is evolving to "pack light" to facilitate rapid and stable horizontal transfer. Sequence types (STs) and PFGE showed diversity patterns. The most prevalent STs were ST48 (n = 5), ST189 (n = 5), ST206 (n = 4), ST6396 (n = 3), ST10 (n = 3), and ST155 (n = 3), where ST48 ESBL-EC originated from three food animal species. The STs of all blaNDM-positive ESBL-EC were attributed to three STs, namely, ST6396 (n = 2), ST206 (n = 2), and ST189 (n = 4), where ST189 was also the unique type for four mcr-1-carrying ESBL-EC. In conclusion, we suggest that the three animal species ESBL-EC show similar high prevalence, diversity in isolate lineages, and significant discrepancies in antibiotic resistance and resistance genes. This suggests that monitoring and anti-infection of different food animal origin ESBL-EC need different designs, which deserves more attention and further surveillance.

M2 macrophages promote vasculogenesis during retinal neovascularization by regulating bone marrow-derived cells via SDF-1/VEGF.

Macrophages promote vasculogenesis during retinal neovascularization (RNV) by increasing the recruitment and differentiation of bone marrow-derived cells (BMCs). Different subtypes of macrophages (M1 and M2 macrophages) are associated with RNV. However, the mechanism underlying the regulation of BMCs by different macrophage subtypes during RNV remains unclear. In the present study, we investigated the role and mechanism of action of different macrophage subtypes that regulate BMCs during the development of RNV. The retinal avascular area and neovascularization (NV) tuft area in M2 macrophage group in vivo were the largest compared to those in the control phosphate buffer saline (PBS), unpolarized-M0, and M1 macrophage groups. The number of recruited green fluorescent protein (GFP)-positive BMCs and the degree of differentiation of BMCs into CD31-positive endothelial cells (ECs) and alpha-smooth muscle actin (α-SMA)-positive smooth muscle cells (SMCs) were higher in the M2 macrophage group than in the other groups. M2-conditional medium (M2-CM) affected the in vitro migration and activation of bone marrow mesenchymal stem cells (BMSCs, a subset of BMCs) more than M1-CM. The expression of stromal cell-derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) in M2 macrophages and BMSCs cultured with M2-CM was also higher than that in M1 macrophages and BMSCs cultured with M1-CM. Migration of BMSCs was reduced after inhibiting the SDF-1 signaling pathway. Our results indicate that M2 macrophages may express significantly higher levels of SDF-1 and VEGF than M1 macrophages, thus regulating the recruitment and differentiation of BMCs and further aggravating vasculogenesis during RNV.

Exosomes from Microglia Attenuate Photoreceptor Injury and Neovascularization in an Animal Model of Retinopathy of Prematurity.

The role of microglia in the pathophysiology of ischemic retinal diseases has been studied extensively. Exosomes from microglial cells exert protective effects during several nervous system diseases, but their roles in hypoxia-induced retinopathy remain unclear. In our study, exosomes derived from microglial cells were injected into the vitreous body of mice with oxygen-induced retinopathy (OIR). Results showed that exosome-treated OIR mice exhibited smaller avascular areas and fewer neovascular tufts in addition to decreased vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGF-ß) expression. Moreover, photoreceptor apoptosis was suppressed by exosome injection. Mechanistically, exosomes from microglial cells were incorporated into photoreceptors in vitro and inhibited the inositol-requiring enzyme 1α (IRE1α)-X-box binding protein 1 (XBP1) cascade, which contributes to hypoxia-induced photoreceptor apoptosis. Furthermore, the exosomes also downregulated the mRNA and protein levels of VEGF and TGF-ß in hypoxia-exposed photoreceptors. A microRNA assay showed that microRNA-24-3p (miR-24-3p) levels were extremely high in exosomes from microglial cells, suggesting that this could be the key molecule that inhibits the hypoxia-induced expression of IRE1α in photoreceptors. These findings delineate a novel exosome-mediated mechanism of microglial cell-photoreceptor crosstalk that facilitates normal angiogenesis and visual function in OIR mice; thus, our results also suggest a potential therapeutic approach for retinopathy of prematurity.

Impact of minocycline on vascularization and visual function in an immature mouse model of ischemic retinopathy.

The role of microglia in the pathophysiology of ischemic retinal diseases has been extensively studied. Retinal microglial activation may be correlated with retinal neovascularization in oxygen-induced retinopathy (OIR), an animal model that has been widely used in retinopathy of prematurity (ROP) research. Minocycline is an antibiotic that decreases microglial activation following hyperoxic and hypoxic-ischemic phases in neonatal rodents. Here, we investigated the effects of minocycline on vascularization and visual function. In our results, we found that after the administration of minocycline, microglial reactivity was reduced in the retina, which was accompanied by an increase in the avascular area at P12, P14 and P17. Although microglial reactivity was reduced at P17, minocycline treatment did not attenuate retinal neovascularization. A changing trend in microglial number was observed, and the apoptosis and proliferation states on different days partly contributed to this change. Further study also revealed that although minocycline downregulated the levels of proinflammatory factors, visual function appeared to be significantly worsened. Collectively, we demonstrated that minocycline disturbed the physiological vascularization of the avascular area and exacerbated visual dysfunction, indicating that minocycline may not be an effective drug and may even be detrimental for the treatment of ischemic retinopathy in immature mammals.

Topical nutraceutical Optixcare EH ameliorates experimental ocular oxidative stress in rats.

PURPOSE: Based on the hypothesis that oral nutraceuticals do not adequately reach all ocular tissues in the anterior segment, we evaluated the ability of a 3% concentration of the ingredients in a topical nutraceutical antioxidant formulation called Optixcare Eye Health (Optixcare EH) to ameliorate oxidative stress in rat models of age-related ocular diseases. METHODS: Diabetes was induced by tail-vein injection of streptozotocin, and the development of cataracts was monitored by slit lamp. Young rats were exposed to ultraviolet (UV) light, and the reduction in lens glutathione (GSH) levels and increase in 4-hydroxynonenol (4-HNE) were measured. Oxidative stress in the neural retina was generated by exposure of dark-adapted rats to 1,000 lx of light, and oxidative stress markers were measured. Dry eye was induced in rats by twice daily (b.i.d.) subcutaneous scopolamine injections. Topical Optixcare EH was administered b.i.d. and compared in select experiments to the multifunctional antioxidant JHX-4, the topical aldose reductase inhibitor (ARI) Kinostat™, oral Ocu-GLO™, and the topical ocular comfort agents Optixcare Eye Lube, Optixcare Eye Lube + Hyaluron, and Idrop Vet Plus hyaluronic acid. RESULTS: In diabetic rats, topical ARI treatment prevented cataract formation while the nutraceuticals delayed their development with Optixcare EH>Ocu-GLO. In UV-exposed rats, the reduction of GSH and increase in 4-HNE in the lens were normalized in order JHX-4>Optixcare EH>Ocu-GLO. In the retina, oxidative stress markers were reduced better by oral JHX-4 compared with topical Optixcare EH. In the scopolamine-induced dry-eye rats, tear flow was maintained by Optixcare EH treatment, while none of the comfort agents examined altered tear flow. CONCLUSIONS: Topical administration of a 3% concentration of the ingredients in Optixcare EH reduces experimentally induced reactive oxygen species in rats exposed to several sources of ocular oxidative stress. In addition, Optixcare EH maintains tear volume in scopolamine-induced dry eye. This suggests that in the anterior segment, the ingredients in Optixcare EH may have clinical potential against ocular oxidative stress.

Novel transgenic mouse models develop retinal changes associated with early diabetic retinopathy similar to those observed in rats with diabetes mellitus.

Retinal capillary pericyte degeneration has been linked to aldose reductase (AR) activity in diabetic retinopathy (DR). Since the development of DR in mice and rats has been reported to differ and that this may be linked to differences in retinal sorbitol levels, we have established new murine models of early onset diabetes mellitus as tools for investigating the role of AR in DR. Transgenic diabetic mouse models were developed by crossbreeding diabetic C57BL/6-Ins2(Akita)/J (AK) with transgenic C57BL mice expressing green fluorescent protein (GFP), human aldose reductase (hAR) or both in vascular tissues containing smooth muscle actin-α (SMAA). Changes in retinal sorbitol levels were determined by HPLC while changes of growth factors and signaling were investigated by Western Blots. Retinal vascular changes were quantitatively analyzed on elastase-digestion flat mounts. Results show that sorbitol levels were higher in neural retinas of diabetic AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors VEGF, IGF-1, bFGF and TGFß, as well as signaling changes in P-Akt, P-SAPK/JNK, and P-44/42 MAPK. Increased loss of nuclei per capillary length and a significant increase in the percentage of acellular capillaries presented in 18 week old AK-SMAA-GFP-hAR mice. These changes are similar to those observed in streptozotocin-induced diabetic rats. Retinal changes in both mice and rats were prevented by inhibition of AR. These studies confirm that the increased expression of AR in mice results in the development of retinal changes associated with the early stages of DR that are similar to those observed in rats.

Novel diabetic mouse models as tools for investigating diabetic retinopathy.

OBJECTIVE: Mouse models possessing green fluorescent protein (GFP) and/or human aldose reductase (hAR) in vascular tissues have been established and crossed with naturally diabetic Akita mice to produce new diabetic mouse models. RESEARCH DESIGN AND METHODS: Colonies of transgenic C57BL mice expressing GFP (SMAA-GFP), hAR (SMAA-hAR) or both (SMAA-GFP-hAR) in vascular tissues expressing smooth muscle actin were established and crossbred with C57BL/6-Ins2(Akita)/J (AK) mice to produce naturally diabetic offspring AK-SMAA-GFP and AK-SMAA-GFP-hAR. Aldose reductase inhibitor AL1576 (ARI) was administered in chow. Retinal and lenticular sorbitol levels were determined by HPLC. Retinal functions were evaluated by electroretinography (ERGs). Growth factor and signaling changes were determined by Western Blots using commercially available antibodies. Retinal vasculatures were isolated from the neural retina by enzymatic digestion. Flat mounts were stained with PAS-hematoxylin and analyzed. RESULTS: Akita transgenics developed DM by 8 weeks of age with blood glucose levels higher in males than females. Sorbitol levels were higher in neural retinas of AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice also had higher VEGF levels and reduced ERG scotopic b-wave function, both of which were normalized by AL1576. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors bFGF, IGF-1, and TGFß, as well as signaling changes in P-Akt, P-SAPK/JNK and P-44/42 MAPK that were also reduced by ARI treatment. Quantitative analysis of flat mounts in 18 week AK-SMAA-GFP-hAR mice revealed increased loss of nuclei/capillary length and a significant increase in the percentage of acellular capillaries present which was not seen in AK-SMAA-GFP-hAR treated with ARI. CONCLUSIONS/SIGNIFICANCE: These new mouse models of early onset diabetes may be valuable tools for assessing both the role of hyperglycemia and AR in the development of retinal lesions associated with diabetic retinopathy.

Effects of bevacizumab on the neovascular membrane of proliferative diabetic retinopathy: reduction of endothelial cells and expressions of VEGF and HIF-1α.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) agents have recently been used intravitreally during the perioperative period for proliferative diabetic retinopathy (PDR). However, the mechanism of theraputic effects of the agents remains unclear. This study aimed to investigate the effects of intravitreal bevacizumab (IVB) on retinal vascular endothelial cells and expressions of VEGF and hypoxia inducible factor-1α (HIF-1α) in PDR. METHODS: Twenty-four patients with PDR were enrolled and randomized to two groups. Twelve eyes of 12 patients of each group received either an intravitreal injection of 1.25 mg bevacizumab or a sham injection 6 days before vitrectomy. Neovascular membranes (NVMs) were collected during pars plana vitrectomy. The numbers of vascular endothelial cells in the NVMs were counted after staining with hematoxylin and eosin and von Willebrand. The expressions of VEGF and HIF-1α in the NVMs were detected through immunohistochemistry. Ten epiretinal membrane specimens from patients with proliferative vitreoretinopathy (PVR) without IVB treatment were set as an additional control. RESULTS: The number of vascular endothelial cells in NVMs of the IVB pretreated group was significantly lower than that of the sham group (21.5±3.94 versus 41.33±7.44, p=0.003). The IVB pretreated group also showed significantly lower levels of VEGF and HIF-1α in NVMs than those of the sham group (P(HIF-1α)=0.02, P(VEGF)<0.001). A stepwise regression analysis showed that IVB was a significant negative predictor for the numbers of vascular endothelial cells (ß=-0.89, p<0.001) and the expressions of VEGF (ß=-0.85, p<0.001) and HIF-1α (ß=-0.64, p=0.001) in PDR patients. Epiretinal membranes of the PVR group showed negative staining of VEGF and HIF-1α. CONCLUSIONS: Pretreatment with IVB in patients with PDR significantly decreased vascular endothelial cells and expressions of VEGF and HIF-1α, which further supports preoperative use of IVB in such patients.