Vision-related quality of life in patients with retinal vein occlusion.

PURPOSE: This study aims to assess the vision-related quality of life in patients with retinal vein occlusion (RVO) among those referred to Labbafinejad Medical Center and Imam Hossein Hospital between 2019 and 2021. METHODS: This comparative study included 37 eligible patients diagnosed with various types of RVO, with an average age of 61 ± 9. To ensure data validity, we included 74 age- and sex-matched healthy individuals. Only cases with a definitive diagnosis of RVO, confirmed by two retina specialists (ND and RN), were included. We assessed the vision-related quality of life of our participants using the National Eye Institute Visual Functioning Questionnaire-25 (NEI-VFQ-25). All participants underwent interviews. RESULTS: In our study, we examined the vision-related quality of life in different subgroups of RVO patients. Overall, RVO patients had a significantly lower total VRQoL score compared to healthy individuals (P < 0.001), except in the subscale analysis of specific factors such as ocular pain, color vision, and driving, where no statistically significant difference was observed. A statistically significant difference was found in the comparison of subgroups, indicating lower VRQoL in central retinal vein occlusion (CRVO) patients (P = 0.010). Furthermore, a significant correlation was observed between lower VRQoL and decreased vision (P = 0.009) as well as longer disease duration (P = 0.011). CONCLUSION: Retinal vein occlusion can significantly reduce vision-related quality of life, particularly in more severe cases.

Design, construction and in vivo functional assessment of a hinge truncated sFLT01.

Gene therapy for the treatment of ocular neovascularization has reached clinical trial phases. The AAV2-sFLT01 construct was already evaluated in a phase 1 open-label trial administered intravitreally to patients with advanced neovascular age-related macular degeneration. SFLT01 protein functions by binding to VEGF and PlGF molecules and inhibiting their activities simultaneously. It consists of human VEGFR1/Flt-1 (hVEGFR1), a polyglycine linker, and the Fc region of human IgG1. The IgG1 upper hinge region of the sFLT01 molecule makes it vulnerable to radical attacks and prone to causing immune reactions. This study pursued two goals: (i) minimizing the immunogenicity and vulnerability of the molecule by designing a truncated molecule called htsFLT01 (hinge truncated sFLT01) that lacked the IgG1 upper hinge and lacked 2 amino acids from the core hinge region; and (ii) investigating the structural and functional properties of the aforesaid chimeric molecule at different levels (in silico, in vitro, and in vivo). Molecular dynamics simulations and molecular mechanics energies combined with Poisson-Boltzmann and surface area continuum solvation calculations revealed comparable free energy of binding and binding affinity for sFLT01 and htsFLT01 to their cognate ligands. Conditioned media from human retinal pigment epithelial (hRPE) cells that expressed htsFLT01 significantly reduced tube formation in HUVECs. The AAV2-htsFLT01 virus suppressed vascular development in the eyes of newborn mice. The htsFLT01 gene construct is a novel anti-angiogenic tool with promising improvements compared to existing treatments.

Prevalence of cataract and its contributing factors in Iranian elderly population: the Gilan eye study.

PURPOSE: To report the prevalence and the associated factors leading to cataract among the Iranian population living in Gilan Province, Iran. METHODS: This population-based cross-sectional study was performed from June to November 2014 on 2,975 residents aged ≥ 50 years old living in urban and rural regions of the Gilan Province in Iran. A representative sample of residents in the province was recruited into the study through door-to-door visiting, and baseline data were collected by questionnaire. All participants were referred to the medical center for comprehensive ophthalmic examination, laboratory tests, and blood pressure measurement. RESULTS: Among the population, 2,588 (86.99%) subjects were eligible to be included in this study, categorized either into the cataract or the non-cataract group. The mean age of participants was 62.59 ± 8.92 years, and 57.5% were female. Higher prevalence of cataract was found in individuals of older ages (odds ratio (OR) = 1.13; 95% confidence interval (CI) = 1.10 to 1.16; P < 0.001) and a history of previous ocular surgery (OR = 5.78; 95% CI = 2.28 to 14.63; P < 0.001). At the same time, a lower prevalence of cataract was seen in patients exposed to sunlight for more than 4 h per day (OR = 0.49; 95% CI = 0.32 to 0.73; P = 0.001). CONCLUSION: Cataract affects 50.50% of the study population, especially those over 80. The mildest form of cataract, grade zero, is the most common. Surgery for cataract has good outcomes. The risk of cataract is higher for those older or who have had eye surgeries. People not affected by cataract tend to be exposed to more sunlight.

Unraveling the genetic complexities of combined retinal dystrophy and hearing impairment.

Usher syndrome, the most prevalent cause of combined hereditary vision and hearing impairment, is clinically and genetically heterogeneous. Moreover, several conditions with phenotypes overlapping Usher syndrome have been described. This makes the molecular diagnosis of hereditary deaf-blindness challenging. Here, we performed exome sequencing and analysis on 7 Mexican and 52 Iranian probands with combined retinal degeneration and hearing impairment (without intellectual disability). Clinical assessment involved ophthalmological examination and hearing loss questionnaire. Usher syndrome, most frequently due to biallelic variants in MYO7A (USH1B in 16 probands), USH2A (17 probands), and ADGRV1 (USH2C in 7 probands), was diagnosed in 44 of 59 (75%) unrelated probands. Almost half of the identified variants were novel. Nine of 59 (15%) probands displayed other genetic entities with dual sensory impairment, including Alström syndrome (3 patients), cone-rod dystrophy and hearing loss 1 (2 probands), and Heimler syndrome (1 patient). Unexpected findings included one proband each with Scheie syndrome, coenzyme Q10 deficiency, and pseudoxanthoma elasticum. In four probands, including three Usher cases, dual sensory impairment was either modified/aggravated or caused by variants in distinct genes associated with retinal degeneration and/or hearing loss. The overall diagnostic yield of whole exome analysis in our deaf-blind cohort was 92%. Two (3%) probands were partially solved and only 3 (5%) remained without any molecular diagnosis. In many cases, the molecular diagnosis is important to guide genetic counseling, to support prognostic outcomes and decisions with currently available and evolving treatment modalities.

Neural differentiation of human retinal pigment epithelial cells on alginate/gelatin substrate.

Purpose: The development of biomaterials provides potent promise for the regeneration of neuroretinal cells in degenerative eye diseases and retinal tissue engineering. Biomimetic three-dimensional (3D) microenvironments and specific growth factors motivate the differentiation of human retinal pigment epithelial (hRPE) cells toward a retinal neural lineage. In this study, we evaluated alginate/gelatin (A/G) as a substrate for the culture of hRPE cells. Methods: hRPE cells were isolated from neonatal human cadaver globes and cultivated on A/G substrate under different culture conditions, including 30% human amniotic fluid (HAF), 10% fetal bovine serum (FBS), and serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12). The proliferation of cells in different culture conditions was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and a cell proliferation assay. Immunocytochemistry and real-time PCR were performed to evaluate the effect of the substrate on hRPE cell differentiation. Results: A significant increase in the cell proliferation rate was observed in hRPE cells cultivated on an A/G substrate. Continuous observations demonstrated that hRPE cells formed densely packed, suspended spheroids in DMEM/F12 culture conditions, with dominant transdifferentiation into amacrine cells. Small adherent clusters of hRPE cells in HAF- and FBS-treated cultures represented dedifferentiation toward retinal progenitor cells. These cultures generated amacrine, rod photoreceptors, and bipolar cells. Conclusions: These findings indicated that A/G substrate induced neural retinal cell propagation in cultures and would therefore be promising for RPE-based tissue engineering studies.

Network analysis and the impact of Aflibercept on specific mediators of angiogenesis in HUVEC cells.

Angiogenesis, inflammation and endothelial cells' migration and proliferation exert fundamental roles in different diseases. However, more studies are needed to identify key proteins and pathways involved in these processes. Aflibercept has received the approval of the US Food and Drug Administration (FDA) for the treatment of wet AMD and colorectal cancer. Moreover, the effect of Aflibercept on VEGFR2 downstream signalling pathways has not been investigated yet. Here, we integrated text mining data, protein-protein interaction networks and multi-experiment microarray data to specify candidate genes that are involved in VEGFA/VEGFR2 signalling pathways. Network analysis of candidate genes determined the importance of the nominated genes via different centrality parameters. Thereupon, several genes-with the highest centrality indexes-were recruited to investigate the impact of Aflibercept on their expression pattern in HUVEC cells. Real-time PCR was performed, and relative expression of the specific genes revealed that Aflibercept modulated angiogenic process by VEGF/PI3KA/AKT/mTOR axis, invasion by MMP14/MMP9 axis and inflammation-related angiogenesis by IL-6-STAT3 axis. Data showed Aflibercept simultaneously affected these processes and determined the nominated axes that had been affected by the drug. Furthermore, integrating the results of Aflibercept on expression of candidate genes with the current network analysis suggested that resistance against the Aflibercept effect is a plausible process in HUVEC cells.

Optogenetic control of neural differentiation in Opto-mGluR6 engineered retinal pigment epithelial cell line and mesenchymal stem cells.

In retinal degenerative disorders, when neural retinal cells are damaged, cell transplantation is one of the most promising therapeutic approaches. Optogenetic technology plays an essential role in the neural differentiation of stem cells via membrane depolarization. This study explored the efficacy of blue light stimulation in neuroretinal differentiation of Opto-mGluR6-engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs were selected for optogenetic study due to their capability to differentiate into retinal-specific neurons. BMSCs were isolated and phenotypically characterized by the expression of mesenchymal stem cell-specific markers, CD44 (99%) and CD105 (98.8%). mRPE culture identity was confirmed by expression of RPE-specific marker, RPE65, and epithelial cell marker, ZO-1. mRPE cells and BMSCs were transduced with AAV-MCS-IRES-EGFP-Opto-mGluR6 viral vector and stimulated for 5 days with blue light (470 nm). RNA and protein expression of Opto-mGluR6 were verified. Optogenetic stimulation-induced elevated intracellular Ca2+ levels in mRPE- and BMS-treated cells. Significant increase in cell growth rate and G1/S phase transition were detected in mRPE- and BMSCs-treated cultures. Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rxrg, Nr2f2, Ascl1, Hes5, and Sox8 were overexpressed in treated BMSCs and Barlh2, Rorb, and Sox8 were overexpressed in treated mRPE cells. Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal-specific neuron markers, in mRPE and BMS cell cultures were demonstrated. Differentiation of ganglion, amacrine, photoreceptor cells, and bipolar and Muller precursors were determined in BMSCs-treated culture and were compared with mRPE. mRPE cells represented more abundant terminal Muller glial differentiation compared with BMSCs. Our results also demonstrated that optical stimulation increased the intracellular Ca2+ level and proliferation and differentiation of Opto-mGluR6-engineered BMSCs. It seems that optogenetic stimulation of mRPE- and BMSCs-engineered cells would be a potential therapeutic approach for retinal degenerative disorders.

Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model.

Connective tissue growth factor (CTGF) is released by retinal pigment epithelial (RPE) cells and detectable in proliferative membranes (PrMs). This experimental study was performed to investigate the mRNA and protein levels of both CTGF and vascular endothelial growth factor A (VEGF-A) in a rabbit model of proliferative vitreoretinopathy (PVR). In addition, the effects of a single intravitreal injection of the safe dose of anti-CTGF or bevacizumab as monotherapy and in combination were evaluated. PVR was induced in the right eye of albino rabbits by intravitreal injection of cultured adult human RPE cells. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot analysis of CTGF and VEGF-A were performed on whole eye tissue in the PVR model versus controls at different time points. In the next step, the PVR models were assigned to five groups. The monotherapy groups received a single intravitreal injection of 0.1 ml of anti-CTGF 100 µg/ml (final concentration of 6.6 µg/ml in the vitreous) or 0.03 ml of 25 mg/ml bevacizumab. In the combined group, the abovementioned amounts of anti-CTGF and bevacizumab were injected intravitreally from separate sites in one session. No antibody injection was performed in the control group. Intravitreal injection of 0.1 ml of control IgG (1 mg/ml of isotype matched) antibody was performed in the placebo group. After 2 weeks, histologic evaluation including, trichrome staining for collagen, immunostaining by anti-alpha-smooth muscle actin for myofibroblasts, and anti-collagen type-1 antibody on paraffin embedded anterior-posterior sections was done. In addition, fundus photography was performed for clinically equivalent PVR staging. Twenty-four hours following PVR induction, CTGF mRNA and protein levels increased five- and- three-fold compared to controls, respectively (P < 0.001). VEGF-A mRNA and protein levels decreased significantly after 72 h of PVR induction compared to controls (P < 0.05). Means of PrM thickness and myofibroblast cell counts significantly decreased in the anti-CTGF group (P < 0.001 and P < 0.05, respectively). The mean area of collagen type-1 fibers of PrM in the mono- and combination therapy groups that received intravitreal anti-CTGF was significantly reduced (P < 0.001); in addition, mild PVR (stage-1 and 2) formation occurred in comparison with moderate to severe PVR (stage-4 and higher) in other groups. In conclusion, we found that intravitreal injection of CTGF neutralizing antibody resulted in a reduction in PrM thickness, collagen fibers and myofibroblast density in the PVR model. CTGF inhibition may represent a potential therapeutic target for PVR.

Peripheral blood CD163(+) monocytes and soluble CD163 in dry and neovascular age-related macular degeneration.

Macrophages are the main infiltrating immune cells in choroidal neovascularization (CNV), a hallmark of the human wet, or neovascular age-related macular degeneration (AMD). Due to their plasticity and ability to adapt to the local microenvironment in a tissue-dependent manner, macrophages display polar functional phenotypes characterized by their cell surface markers and their cytokine profiles. We found accumulation of hemoglobin-scavenging cluster of differentiation 163 (CD163)(+) macrophages in laser-induced CNV lesions and higher expression of CD163(+) monocytes in the peripheral blood on day 7 post injury in mice. In comparison, CD80(+) macrophages did not differ with laser-injury in young or aged mice and did not significantly change in the peripheral blood of CNV mice. We examined the percentages of CD163(+), CD206(+), and CD80(+) monocytes in the peripheral blood of patients with wet AMD, patients with dry AMD, and in age-matched individuals without AMD as controls. Percentages of peripheral blood CD163(+) monocytes in both dry AMD (P < .001) and wet AMD (P < .05) were higher than in age-matched non-AMD controls, while there was no difference between the groups in the percentages of peripheral CD206(+) and CD80(+) monocytes. Further, serum level of soluble CD163 (sCD163) was elevated only in patients with wet AMD (P < .05). An examination of 40 cytokine levels across the study groups revealed that anti-VEGF treated patients with wet AMD, who showed no exudative signs on the day of blood drawing had a cytokine profile that was similar to that of non-AMD individuals. These results indicate that CD163 could be further evaluated for its potential as a useful marker of disease activity in patients with neovascular AMD. Future studies will address the origin and potential mechanistic role of CD163(+) macrophages in wet AMD pathologies of angiogenesis and leakage of blood components.

New criteria for evaluation of electroretinogram in patients with retinitis pigmentosa.

BACKGROUND: Electroretinogram (ERG) plays an essential role in the diagnosis of retinal disease. Choosing appropriate methods could extract valuable information from ERG. In this study, a new criterion based on time-frequency domain analysis was proposed to investigate the retina in retinitis pigmentosa (RP) patients. MATERIALS AND METHODS: The total number of 16 eyes from eight RP patients and 20 eyes from age-matched healthy subjects were assessed. The signals included photopic and scotopic ERGs. Continuous wavelet transform was applied to ERGs. Dominant frequencies were extracted, and the contours related to these dominant frequencies were selected. As a new criterion, the areas related to dominant frequency contours were considered a feature to differentiate the RP and normal groups. To better evaluate the proposed criterion results, the time-domain analysis characteristics of ERG were also considered. RESULTS: The results showed an increase in implicit time and reduced amplitude in RP patients (P < 0.05). A significant decrease of dominant frequencies and increasing their occurrence time were seen in ERG of RP patients. Also, in RP patients, the third dominant frequency was disappeared from the three main frequencies observed in photopic ERGs of normal subjects. The area criterion showed a significant decrease in RP groups (P < 0.05). CONCLUSION: RP can cause changes in the time and time-frequency components of the ERG. The area index could represent a new view of the characteristics of the ERG in the time-frequency domain. This criterion can help the ophthalmologist to have a better evaluation of retinal disease.

Continuous wavelet transform analysis of ERG in patients with diabetic retinopathy.

PURPOSE: Diabetic retinopathy (DR) is one of the leading causes of blindness worldwide. Non-proliferative diabetic retinopathy (NPDR) is a stage of the disease that contains morphological and functional disruption of the retinal vasculature and dysfunction of retinal neurons. This study aimed to compare time and time-frequency-domain analysis in the evaluation of electroretinograms (ERGs) in subjects with NPDR. METHOD: The ERG responses were recorded in 16 eyes from 12 patients with NPDR and 24 eyes from 12 healthy subjects as the control group. The implicit time, amplitude, and time-frequency-domain parameters of photopic and scotopic ERGs were analyzed. RESULTS: The implicit times of b-waves in the dark-adapted 10.0 (P = 0.0513) and light-adapted 3.0 (P = 0.0414) were significantly increased in the NPDR group. The amplitudes of a- and b-wave showed a significantly decreased dark-adapted 10.0 (P = 0.0212; P = 0.0133) and light-adapted 3.0 (P = 0.0517; P = 0.0021) ERG of the NPDR group. The Cohen's d effect size had higher values in the amplitude of dark-adapted 10.0 b-wave (|d|= 1.8058) and amplitude of light-adapted 3.0 b-wave (|d|= 1.9662). The CWT results showed that the frequency ranges of the dominant components in dark-adapted 10.0 and light-adapted 3.0 ERG were decreased in the NPDR group compared to the healthy group (P < 0.05). The times associated with the NDPR group's dominant components were increased compared to normal eyes in both dark-adapted 10.0 and light-adapted 3.0 ERG (P < 0.05). All Cohen's d effect sizes of the implicit times and dominant frequency components were on a large scale (|d|> 1). CONCLUSION: These findings suggest that the time and time-frequency parameters of both photopic and scotopic ERGs can be good indicators for DR. However, time-frequency-domain analysis could present more information might be helpful in the assessment of the DR severity.

Interdisciplinary Approaches to COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has been a significant concern worldwide. The pandemic has demonstrated that public health issues are not merely a health concern but also affect society as a whole. In this chapter, we address the importance of bringing together the world's scientists to find appropriate solutions for controlling and managing the COVID-19 pandemic. Interdisciplinary cooperation, through modern scientific methods, could help to handle the consequences of the pandemic and to avoid the recurrence of future pandemics.

The urgent need for integrated science to fight COVID-19 pandemic and beyond.

The COVID-19 pandemic has become the leading societal concern. The pandemic has shown that the public health concern is not only a medical problem, but also affects society as a whole; so, it has also become the leading scientific concern. We discuss in this treatise the importance of bringing the world's scientists together to find effective solutions for controlling the pandemic. By applying novel research frameworks, interdisciplinary collaboration promises to manage the pandemic's consequences and prevent recurrences of similar pandemics.

A mutation in DOP1B identified as a probable cause for autosomal recessive Peters anomaly in a consanguineous family.

Purpose: Peters anomaly (PA) is a heterogeneous developmental disorder characterized by central corneal opacity and iridocorneal or corneolenticular adhesions. Although many causative genes have been identified, most screened patients do not have mutations in the known genes. We aimed to identify the genetic cause of Peters anomaly in a pedigree with three affected individuals. Methods: Slit-lamp biomicroscopy and ultrasound biomicroscopy were performed for definitive diagnosis. Exome sequencing was conducted on the DNA of all three patients. After identification of a candidate causative gene, expression of the gene was assessed with real-time PCR in various ocular tissues of three human embryos and three adults. Results: The patients were affected with isolated PA. The parents of the patients were related to one another. Inheritance of PA was autosomal recessive. After appropriate filtering of the exome data, a homozygous variation in DOP1B remained as the only candidate genetic cause of PA in the pedigree. The variant segregated with disease status in the pedigree and was absent among 800 control Iranians. The variant has been reported in various databases at frequencies of 0.006 or less only in the heterozygous state in some cohorts of African origin. The p.Val1660 amino acid affected by the mutation is completely conserved in mammals and birds during evolution. Expression of DOP1B was shown in all adult and embryonic lens, iris, cornea, sclera, and retina tissues that were tested. Conclusions: DOP1B that encodes DOP1 leucine zipper like protein B was identified as the putative PA-causing gene in pedigree PA-101. As DOP1B is positioned within the Down syndrome chromosomal region on chromosome 21, until now this gene has mostly been studied with respect to brain functions. However, members of the Dopey gene family have been shown to have roles in development in other organisms. Evidence of the expression of DOP1B in various PA-relevant eye tissues, which, to the best of our knowledge, is shown here for the first time, is to be noted. However, this finding does not necessarily implicate a specific role for DOP1B in eye development as the gene is expressed in many tissues. Ultimately, definitive assessment of the contribution of DOP1B to PA pathology awaits identification of mutations in the gene in unrelated patients with PA and functional studies.

Effects of intravitreal connective tissue growth factor neutralizing antibody on choroidal neovascular membrane-associated subretinal fibrosis.

Connective tissue growth factor (CTGF) plays an essential role in the regulation of extracellular matrix proteins and pro-fibrotic and angiogenic factors. This experimental research was conducted to evaluate if CTGF is elevated after induction of a choroidal neovascular membrane (CNVM) and whether intravitreal anti-CTGF without and with intravitreal bevacizumab (IVB) may have any effect on the CNVM associated sub-retinal fibrosis. In adherence to ARRIVE guidelines, CNVM was induced by laser spots in the right eye retinas of ninety-four pigmented rats. Quantitative real-time reverse transcription PCR (qRT-PCR) and western-blot analysis were performed on sclerochoroidal tissues of forty-four rats before and at different time intervals after laser application. The remaining fifty rats were randomly divided into five groups after laser application. Group A received intravitreal injection of 2  µl of the 50 µg/ml anti-CTGF. In group B, intravitreal injection of 2  µl of 25 mg/ml bevacizumab was performed. Group C received 1  µl intravitreal anti-CTGF and 1  µl IVB. Group D did not receive any intravitreal injection as the control group. In group E, intravitreal injection of 2  µl of nonspecific purified mouse IgG antibody was performed as the placebo group. After two weeks, double immunohistochemistry was performed by isolectin B4 and anti-collagen type1 on the sclerochoroidal flat-mounts. Masked measurement of the fluorescent images of the CNVM and CNVM associated sub-retinal fibrosis areas was performed using the image J software. Ctgf mRNA and CTGF protein levels increased to the maximum level in 24 h after laser application and remained higher than the control level up to the 14th day for the Ctgf mRNA and up to the 7th day for the CTGF protein level. Means of CNVM associated sub-retinal fibrosis areas in three treatment groups (A, B and C) were significantly less than the control (D) and placebo (E) groups (P < 0.001, <0.05, <0.001 respectively). For groups A and C, mean CNVM associated sub-retinal fibrosis areas were also significantly less than group B (P < 0.05 and < 0.01, respectively). In conclusion, this study showed significant reduction of the CNVM associated sub-retinal fibrosis via inhibition of the CTGF which mediates the final steps of fibrosis in various inflammatory and angiogenic pathways.

Repeated Injection of Methotrexate into Silicone Oil-Filled Eyes for Grade C Proliferative Vitreoretinopathy: A Pilot Study.

PURPOSE: To evaluate the effects of repeated intra-silicone oil (SO) injections of methotrexate (MTX) on the outcomes of surgery for rhegmatogenous retinal detachment (RRD) with grade C proliferative vitreoretinopathy (PVR-C). METHODS: In this prospective pilot case series, eyes with RRD and PVR-C underwent pars plana vitrectomy and intraocular injection of SO. At the conclusion of the procedure, 250 µg of MTX was injected into the SO-filled vitreous cavity. Intra-SO injection was repeated at weeks 3 and 6; the minimum follow-up period was 6 months. The main outcome measure was retinal reattachment rate. RESULTS: Eleven eyes of 11 patients (mean age, 52.73 ± 18.01 years) were included. The mean follow-up period was 9 ± 3 months (range, 6-15 months). Total retinal detachment with anterior and/or posterior PVR-C was present in all eyes before surgery. Mean preoperative best-corrected visual acuity (BCVA) was 2.62 ± 0.04 logMAR. All operated eyes exhibited retinal reattachment posterior to the equator during the follow-up period. Mean postoperative BCVA was significantly improved to 1.02 ± 0.51 logMAR (p = 0.003). No ocular or systemic side effects were observed. CONCLUSION: Repeated intra-SO injection of MTX as an adjunctive therapy for RRD complicated by PVR showed promising results and was not associated with adverse effects. Further studies are needed to confirm its possible beneficial effects on the final anatomic and functional outcomes in these cases.

Rhodopsin gene mutation analysis in Iranian patients with autosomal dominant retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is the most common hereditary retinal degeneration and an important cause of visual disability worldwide. Rhodopsin gene is one of the most important genes implicated in autosomal dominant RP (ADRP). In this study, we investigated rhodopsin gene mutations in Iranian patients with ADRP. METHODS: Twenty-one patients from 21 unrelated families with a total of 51 affected members were enrolled in this study. After complete history taking, ophthalmic examination and genetic counseling, peripheral blood samples were obtained. Following genomic DNA extraction, all five exons and intron-exon boundaries of RHO gene were sequenced using Sanger method. Interpretation of detected variants was carried out using appropriate databases and bioinformatic tools. Novel variants were screened in 150 unrelated healthy subjects. RESULTS: Results of direct sequencing revealed that five of 21 patients (23.8%) had mutation in the rhodopsin gene. Two of them had previously identified p.P347L mutation, and three had novel variants including p.L95P, p.R177K and p.N310K. None of these novel variants were detected in healthy controls. The p.L95P variant was associated with predominantly inferior retinal involvement. CONCLUSIONS: Our study showed that mutations of the rhodopsin gene are relatively frequent in Iranian patients with ADRP and could be considered in further researches in the future. The novel p.L95P variant may be associated with a specific pattern of retinal degeneration in this population.

Human organotypic retinal flat-mount culture (HORFC) as a model for retinitis pigmentosa11.

The splicing factor PRPF31 is the most commonly mutated general splicing factor in the retinitis pigmentosa. We used a rapid, convenient and cost effective transfection method with an efficient PRPF31 knockdown in HORFC in order to study the effect of PRPF31 downregulation on retinal gene expressions in an ex vivo model. Modified calcium phosphate method was used to transfect HORFC by PRPF31 siRNA. Different times and doses of siRNA for transfection were assayed and optimum condition was obtained. PRPF31 mRNA and protein downregulation were assessed by qRTPCR and Western blot. The tissue viability of HORFC was measured using the MTT. ImageJ analysis on stained retinal sections by immunohistochemistry was used for thickness measurement of outer nuclear photoreceptor layer. The PRPF31 gene downregulation effects on retinal specific gene expression were analyzed by qRTPCR. A total of 50 nM of PRPF31 siRNA transfection after 63 h in HORFC, showed the optimum reduction in the level of PRPF31 mRNA and protein as shown by qRTPCR and Western blot (over 90% and 50% respectively). The PRPF31 mRNA silencing with calcium phosphate had no effect on cell viability in the period of the experiment. Thickness measurement of outer nuclear photoreceptor layer with IHC showed the significant reduction after 63 h of study (P value = 0.02). siRNA induced PRPF31 knockdown, led to reduction of retinal specific mRNA gene expression involved in phototransduction (RHO, GNAT1, RP1), photoreceptor structure (ROM1, FSCN2, CA4, SEMA4) and transcription factor (CRX) (fold change >5), after 63 h.

AAV delivery of GRP78/BiP promotes adaptation of human RPE cell to ER stress.

Adeno associated virus (AAV)-mediated gene delivery of GRP78 (78 kDa glucose-regulated protein) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in Retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV-2 in primary human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell response to ER stress. The purpose of this work was studying potential stimulating effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress, as an in vitro model for RPE degeneration. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, hRPE primary cultures were transduced by recombinant virus rAAV/Grp78, and treated with ER stressor drug, tunicamycin. Expression changes of four UPR markers including GRP78, PERK, ATF6α, and GADD153/CHOP, were assessed by real-time PCR and western blotting. We found that GRP78 has a great contribution in modulation of UPR markers to favor adaptive response in ER-stressed hRPE cells. In fact, GRP78 overexpression affected adaptation and apoptotic phases of early UPR, through enhancement of two master regulators/ER stress sensors (PERK and ATF6α) and down-regulation of a key pro-apoptotic cascade activator (GADD153/CHOP). Together these findings demonstrate the promoting effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress. This protein with anti-apoptotic actions in the early UPR and important role in cell fate regulation, can be recruited as a useful candidate for future investigations of RPE degenerative diseases.

CHANGES IN CENTRAL CHOROIDAL THICKNESS AFTER TREATMENT OF DIABETIC MACULAR EDEMA WITH INTRAVITREAL BEVACIZUMAB CORRELATION WITH CENTRAL MACULAR THICKNESS AND BEST-CORRECTED VISUAL ACUITY.

PURPOSE: To assess the effect of intravitreal injection of bevacizumab on central choroidal thickness (CCT) and its relationship with central macular thickness (CMT) and best-corrected visual acuity (BCVA) changes in eyes with center-involving diabetic macular edema. METHODS: This prospective interventional case series included 20 eyes of 20 patients with center-involving diabetic macular edema naive to treatment. The BCVA assessment, complete eye examination, enhanced depth optical coherence tomography, and fluorescein angiography were performed at baseline followed by 3 monthly intravitreal injection of bevacizumab. The treated eyes underwent BCVA evaluation and enhanced depth optical coherence tomography at Months 1, 2, 3, and 6 after the first injection. Change of the CCT was the primary outcome measure. Secondary outcome measures included BCVA and CMT changes and their relationship with CCT changes. RESULTS: Mean age of patients was 63.1 ± 8.0 (range, 52-75) years. Mean baseline CCT was 265 ± 79 µm, which reduced to 251 ± 81 µm and 232 ± 82 µm at Months 3 and 6, respectively (P < 0.001). Corresponding values for CMT were 470 ± 107 µm, 392 ± 104 µm, and 324 ± 122 µm, respectively (P < 0.001). The BCVA improved from 20/60 at baseline to 20/50 at Month 3 and 20/40 at Month 6 (P = 0.007). Each 1 µm decrease in CCT was associated with 2.74 µm reduction in CMT and 0.1 Early Treatment Diabetic Retinopathy Study letter score improvement in BCVA (P < 0.001 and P = 0.001, respectively). CONCLUSION: After treatment of diabetic macular edema with intravitreal injection of bevacizumab, CCT decreased and this reduction significantly correlated with CMT reduction and vision improvement.