Using methanol to preserve retinas for immunostaining.

BACKGROUND: Experimental studies on retinal vasculature and retinal ganglion cells (RGCs) investigating the developmental and pathological conditions of the retina mainly rely on whole-mount retinal immunostaining. Methanol, an auxiliary fixed medium for retinal whole-mount preparations, has been used in some studies; however, its application in short- and long-term storage of retinas for further study has not been well described. We aimed to evaluate methanol use as a preservation treatment for further immunostaining of the retina. METHODS: We generated oxygen-induced retinopathy (OIR) and optic nerve crush (ONC) mouse models and used their retinas for analysis. We pipetted cold methanol (-20°C) on the surface of the retina to help fix the tissues while promoting permeability, after which the retinas were stored in cold methanol (-20°C) for 1, 6, or 12 months before being evaluated using various optical techniques. Thereafter, retinal whole-mount immunostaining was performed to analyse retinal neovascularisation and retinal hypoxia in OIR model, and retinal ganglion cell survival rate in ONC model. RESULTS: Quantitative analysis revealed no significant differences in the fixed retinas after long-term storage in terms of retinal vasculature or retinal hypoxia in the OIR model. Similarly, no significant difference was found in RGC survival rate after long-term storage in methanol. These results suggest that methanol can be used as a storage medium when preserving retinal whole-mount samples. CONCLUSIONS: Cold (-20°C) methanol can serve as an effective medium for long-term storage of fixed retinas, which is useful for further research.

Vitreous metabolomics profiling of proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: Proliferative diabetic retinopathy (PDR) with retinal neovascularisation (NV) is a leading cause of vision loss. This study identified a set of metabolites that were altered in the vitreous humour of PDR patients compared with non-diabetic control participants. We corroborated changes in vitreous metabolites identified in prior studies and identified novel dysregulated metabolites that may lead to treatment strategies for PDR. METHODS: We analysed metabolites in vitreous samples from 43 PDR patients and 21 non-diabetic epiretinal membrane control patients from Japan (age 27-80 years) via ultra-high-performance liquid chromatography-mass spectrometry. We then investigated the association of a novel metabolite (creatine) with retinal NV in mouse oxygen-induced retinopathy (OIR). Creatine or vehicle was administered from postnatal day (P)12 to P16 (during induced NV) via oral gavage. P17 retinas were quantified for NV and vaso-obliteration. RESULTS: We identified 158 metabolites in vitreous samples that were altered in PDR patients vs control participants. We corroborated increases in pyruvate, lactate, proline and allantoin in PDR, which were identified in prior studies. We also found changes in metabolites not previously identified, including creatine. In human vitreous humour, creatine levels were decreased in PDR patients compared with epiretinal membrane control participants (false-discovery rate <0.001). We validated that lower creatine levels were associated with vascular proliferation in mouse retina in the OIR model (p = 0.027) using retinal metabolomics. Oral creatine supplementation reduced NV compared with vehicle (P12 to P16) in OIR (p = 0.0024). CONCLUSIONS/INTERPRETATION: These results suggest that metabolites from vitreous humour may reflect changes in metabolism that can be used to find pathways influencing retinopathy. Creatine supplementation could be useful to suppress NV in PDR. Graphical abstract.

Pharmacological restoration of visual function in a zebrafish model of von-Hippel Lindau disease.

Von Hippel-Lindau (VHL) syndrome is a rare, autosomal dominant disorder, characterised by hypervascularised tumour formation in multiple organ systems. Vision loss associated with retinal capillary hemangioblastomas remains one of the earliest complications of VHL disease. The mortality of Vhl-/- mice in utero restricted modelling of VHL disease in this mammalian model. Zebrafish harbouring a recessive germline mutation in the vhl gene represent a viable, alternative vertebrate model to investigate associated ocular loss-of-function phenotypes. Previous studies reported neovascularisation of the brain, eye and trunk together with oedema in the vhl-/- zebrafish eye. In this study, we demonstrate vhl-/- zebrafish almost entirely lack visual function. Furthermore, hyaloid vasculature networks in the vhl-/- eye are improperly formed and this phenotype is concomitant with development of an ectopic intraretinal vasculature. Sunitinib malate, a multi tyrosine kinase inhibitor, market authorised for cancer, reversed the ocular behavioural and morphological phenotypes observed in vhl-/- zebrafish. We conclude that the zebrafish vhl gene contributes to an endogenous molecular barrier that prevents development of intraretinal vasculature, and that pharmacological intervention with sunitinib can improve visual function and hyaloid vessel patterning while reducing abnormally formed ectopic intraretinal vessels in vhl-/- zebrafish.

Systemic Rho-kinase inhibition using fasudil in mice with oxygen-induced retinopathy.

PURPOSE: To investigate the influence of the selective Rho-kinase (ROCK) inhibitor, fasudil, on the mRNA level of proinflammatory factors and the retinal vascular development in mice with oxygen-induced retinopathy (OIR). METHODS: C57BL/6J mice underwent standard protocol for OIR induction from postnatal days 7 to 12. Subsequently, they received a daily intraperitoneal injection of fasudil or sodium chloride from P12 to P16. Analyses were performed using vascular staining on retinal flat mounts, RNA expression by qPCR, and immunohistochemistry on paraffin sections. RESULTS: On retinal flat mounts, the proportion of avascular area and tuft formation did not differ between the fasudil and NaCl group. Immunohistochemical staining revealed a less intense staining with inflammatory markers after fasudil. Nevertheless, there were no differences on RNA level between the two groups. CONCLUSIONS: In conclusion, our findings support that daily systemic application of fasudil does not decrease retinal neovascularization in rodents with oxygen-induced retinopathy. The results of our study together with the controversial results on the effects of different ROCK inhibitors from the literature makes it apparent that effects of ROCK inhibition are more complex, and further studies are necessary to analyze its potential therapeutic effects.

Folic acid modified gold nanoparticle for targeted delivery of Sorafenib tosylate towards the treatment of diabetic retinopathy.

Preparing a controlled release dosage form for specific locations within the body is one of the most challenging tasks in the present times. Moreover, green synthesis of metal nanoparticles is attaining greater significance because of their high biocompatibility and negligible toxicity. Gold nanoparticles, among them, are considered to be one of the best modes of drug delivery system for the treatment of various diseases. The excellent properties of gold nanoparticles make them a potential carrier for carrying and releasing drugs at the specified location in a controlled manner. In this research, gold nanoparticle (AuNPs) has been prepared by green synthesis for the treatment of diabetic retinopathy. These AuNPs has been further modified with FA-b-PEG copolymer for the targeted delivery of the drug Sorafenib tosylate. The selected drug works on VEGF receptors in the region of retinal neovascularisation. The optimized dosage form prepared from gold chloride and ginger extract has been characterized. Histopathological studies as well as fundus photography has been used for the in-vivo characterisation of the prepared dosage form and it has been found that it can be quite an effective treatment for retinal neovascularisation in patients of diabetic retinopathy.

Takayasu's arteritis: Ischaemic retinal arterial occlusion as a possible initial presentation. / Arteritis de Takayasu: obstrucción arterial retiniana isquémica como posible presentación inicial.

Ocular manifestations are observed in 25% of patients with Takayasu's arteritis. Its signs and symptoms can be very variable. The case is presented of a 41-year-old woman with progressive vision loss in her right eye secondary to ischaemic retinal arterial occlusion. After a systematic study, a protein purified derivative (PPD) skin test compatible with tuberculosis was found to be the only alteration. After ruling out other causes, and based on the initial suspicion of tuberculous retinal vasculitis, treatment was started with antimicrobial agents and systemic corticosteroids, without any therapeutic response. Eighteen months later, the patient developed acute kidney failure, secondary to right renal artery stenosis. The CT-angiography revealed a thickening of the aortic arch and its branches, and Takayasús arteritis was finally diagnosed. Therefore, emphasis is made on the importance of the ophthalmologist in the diagnosis of Takayasús arteritis, in which its ophthalmological manifestations can be an early sign of the disease.

Peripheral ischaemic retinopathy and neovascularisation in a patient with subacute streptococcus mitis-induced bacterial endocarditis.

Objective: To describe a patient with peripheral retinal ischaemia and neovascularisation who was diagnosed with streptococcus mitis-induced bacterial endocarditis. Methods: Retrospective analysis of case report. A 57-year-old man presented with a history of a rapidly progressive, bilateral, painless visual loss. He also suffered from pain in the neck and lower back and a weight loss of 10 kg. He underwent a full ophthalmologic work-up, laboratory investigations, and imaging of the spine. Results: BCVA was reduced to 20/40 in the right eye and 20/32 in the left eye. Fundoscopy showed rare intra-retinal haemorrhages including few Roth spots and cotton wool lesions. Fluorescein angiography demonstrated large areas of peripheral retinal ischaemia and neovascularisation. Imaging of the spine showed spondylodiscitis on several levels. Further imaging and blood cultures confirmed bacterial endocarditis of the mitral valve. Streptococcus mitis was subsequently identified as the causative organism. Conclusion: Peripheral retinal ischaemia and neovascularisation were previously unrecognised as a feature of infectious endocarditis. Therefore, their presence, apart from the classic Roth spots, should prompt the consideration of infectious endocarditis in the etiologic work-up.

Choroidal melanoma treated with stereotactic fractionated radiotherapy and prophylactic intravitreal bevacizumab: The Dunedin Hospital experience.

INTRODUCTION: Choroidal melanoma (CM) is the most common primary ocular malignancy in adults. This study reviewed the Dunedin Hospital (DH) experience in the treatment of CM with stereotactic fractionated radiotherapy (SRT) and the outcome of prophylactic use of intravitreal injection bevacizumab (PIB) in preventing radiation retinopathy (RR). METHODS: A retrospective study was conducted of patients at DH who underwent SRT for CM with and without PIB from 1 January 2001 to 31 January 2012. In DH, some patients who had SRT following the introduction of intravitreal bevacizumab in December 2006 were also treated with PIB with the expectation that this might reduce the risk of developing RR, although the evidence of its effectiveness in this respect is not clear. The primary outcome measure was local progression as monitored with regular ultrasound. Secondary outcome measures were metastatic progression incidence, enucleation incidence, no functional vision incidence, overall survival, disease-specific mortality, incidence of RR, and radiotherapy to clinical diagnosis of RR time. RESULTS: Twenty-seven patients who were followed up at DH were reviewed after a mean follow-up of 5.1 years (range 0.4-12.6). Fourteen patients received PIB. The local progression, metastatic progression and enucleation rate were 4%, 8% and 11%, respectively. The no functional vision (hand movements or less) rate was 62%. Overall survival was 63%, but only three (11%) deaths were due to metastatic choroidal melanoma. Incidence of RR was 57% and 54% for those that received PIB and those who did not, respectively. PIB did not reduce the rate of RR (P = 1.00). CONCLUSION: This study reaffirmed that SRT achieves very good local control and eye retention rates. PIB did not appear to reduce the radiation retinopathy rate in this study, and more studies are required especially Phase II and III trials to determine PIB efficacy in preventing RR.