Retinal Photoreceptor Cells Research Articles

Photoreceptor phosphodiesterase (PDE6): activation and inactivation mechanisms during visual transduction in rods and cones.

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to-and relieves inhibition of-PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9-1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells.

Defining Phenotype, Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in Crb1.

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

Mechanisms of vitamin A metabolism and deficiency in the mammalian and fly visual system

Vitamin A deficiency can cause human pathologies that range from blindness to embryonic malformations. This diversity is due to the lack of two major vitamin A metabolites with very different functions: the chromophore 11-cis-retinal (vitamin A aldehyde) is a critical component of the visual pigment that mediates phototransduction, while the signaling molecule all-trans-retinoic acid regulates the development of various tissues and is required for the function of the immune system.Since animals cannot synthesize vitamin A de novo, they must obtain it either as preformed vitamin A from animal products or as carotenoid precursors from plant sources. Due to its essential role in the visual system, acute vitamin A deprivation impairs photoreceptor function and causes night blindness (poor vision under dim light conditions), while chronic deprivation results in retinal dystrophies and photoreceptor cell death. Chronic vitamin A deficiency is the leading cause of preventable childhood blindness according to the World Health Organization. Due to the requirement of vitamin A for retinoic acid signaling in development and in the immune system, vitamin A deficiency also causes increased mortality in children and pregnant women in developing countries.Drosophila melanogaster is an excellent model to study the effects of vitamin A deprivation on the eye because vitamin A is not essential for Drosophila development and chronic deficiency does not cause lethality. Moreover, genetic screens in Drosophila have identified evolutionarily conserved factors that mediate the production of vitamin A and its cellular uptake. Here, we review our current knowledge about the role of vitamin A in the visual system of mammals and Drosophila melanogaster. We compare the molecular mechanisms that mediate the uptake of dietary vitamin A precursors and the metabolism of vitamin A, as well as the consequences of vitamin A deficiency for the structure and function of the eye.

Efficacy and safety of Bu Jing Yi Shi tables for high myopia: A protocol of systematic review and meta-analysis.

Background:High myopia is a kind of ametropia with diopter more than −6.00D or axial length ≥26 mm. With the change of the modern environment, the incidence rate is increasing year by year. At present, the pathogenesis of high myopia is not clear. Some current studies indicate that it may be related to the environment and genetics. A Chinese patent medicine named Bu Jing Yi Shi Tablets (BJYST) has many functions including anti-oxidation, expansion of blood vessels, anti-inflammatory, immune regulation, inhibition of retinal photoreceptor cell apoptosis, and promotion of retinal repair. A large number of existing studies have shown that this prescription can relieve the clinical manifestations of high myopia and its complications, but its true efficacy and safety are still unclear. To certify this point, a protocol for a systematic review and meta-analysis of BJYST for high myopia will be performed.Methods and analysis:Articles that have been identified by electronically searching of 9 English and 5 Chinese databases from their inception to December 4, 2020 will be incorporated into the study. This study only adopts Chinese and English. Two researchers will take charge of completing the selection of research, the extraction of data as well as the assessment of research quality independently. The primary outcomes will be an average change in refractive error measured in diopters and an average change in axial length measured in millimeters. Data analysis will be performed via the RevMan 5.3 software, and Grading of Recommendations Assessment, Development, and Evaluation (GRADE) will help to assess the evidence level.Results:The results of this study will be published in a peer-reviewed journal.Conclusion:This study will conclude whether BJYST is safe and effective in treating high myopia on the basis of evidence-based medicine.Registration:The Open Science Framework (OSF) registration number is osf.io/dpk5b.

Long-term corticosteroid-induced chronic glaucoma model produced by intracameral injection of dexamethasone-loaded PLGA microspheres

Purpose To evaluate a new chronic glaucoma model produced by intracameral injection of dexamethasone-loaded poly lactic-co-glycolic acid microspheres (Dex-PLGA-Ms) over six months. Methods Healthy rats received two injections (at baseline and Week 4) of Dex-PLGA-Ms into the anterior chamber of the right eye. Clinical signs and intraocular pressure (IOP) were weekly recorded. The structure of the retina and optic nerve was in vivo evaluated using optical coherence tomography (OCT) every two weeks and functionally using dark- and light-adapted electroretinography at 0–12–24 weeks. Histological studies were also performed. Results IOP progressively increased up to hypertension (23.22 ± 3.63 mmHg) in both eyes but did so later in left eyes. OCT quantified a decrease in full-thickness retina posterior pole (R), retinal-nerve-fiber layer (RNFL), and ganglion-cell layer (GCL) thickness up to 24 weeks. Right eyes showed higher neuroretinal thickness loss up to week 8. RNFL experienced the highest percentage thickness loss at the inferior-superior axis, while in GCL the inner sectors of the horizontal axis (Nasal-Temporal) suffered the greatest decrease in thickness. Retinal ganglion cell, photoreceptor, and intermediate cell functionality decreased over time. Increased deposition of collagen IV was also found in zonular fibers and the ciliary body. Conclusions This work shows the usefulness of drug delivery systems, not to treat pathology but to induce it. Only two injections of Dex-PLGA-Ms in the anterior chamber of rat eyes were enough to progressively create ocular hypertension and subsequent functional and structural neuroretinal degeneration, at least over 6 months.

Comparative cytotoxic and antiproliferative profile of methotrexate and fluorouracil on different ocular cells

To analyse the cytotoxic and antiproliferative effect of methotrexate (MTX) and fluorouracil (5-FU) invitro on fibroblasts, retinal pigment epithelial (RPE) and photoreceptor cells as an adjunct for reducing the incidence of proliferative vitreoretinopathy (PVR) after rhegmatogenous retinal detachment surgery. Methotrexate and 5-FU were dissolved separately in balanced salt solution (BSS) with concentrations ranging from 0-8000µg/ml and 0-4000µg/ml, respectively. All solutions were analysed in terms of pH and osmolarity and applied for 1h to fibroblasts (BJ), RPE (ARPE-19) and photoreceptor (661W) cell lines adherently cultivated in 96-well cell culture plates (10000cells/well). 24h after incubation, the proliferative (BrdU), metabolic (CellTiter-Glo) and apoptotic (Caspase 3/7) activity of the cells were examined invitro. 5-FU had an antiproliferative effect on BJ and ARPE-19 cells starting from low concentrations (2µg/ml). However, the viability of 661W cells decreased and apoptosis was induced with increasing 5-FU concentration. In contrast, MTX up to a concentration of 266µg/ml did neither result in a significant loss of viability nor in increased caspase 3/7 activity of BJ, ARPE-19 and 661W cells and inhibited the proliferation of ARPE-19 already at low concentrations starting from 8µg/ml. Methotrexate dissolved in BSS is biocompatible up to a concentration of 266µg/ml and may act as an intraoperative rinse solution to inhibit RPE proliferation in PVR-diseased eyes. Contrary, the use of 5-FU within the posterior segment of the eye is limited by its cell-damaging effect on photoreceptor cells.

Light Adaptation of Retinal Rods, Adaptation Memory, and Afterimages

The GPCR signal cascade in retinal photoreceptor cells – rods and cones – supports powerful signal amplification and allows rods to respond reliably to single light quanta. Rods remain functional at light fluxes down to 105 quanta/sec, which is supported by the highly effective light adaptation system. Light adaptation is based on negative feedback circuits, mostly via changes in intracellular Ca2+ ion concentrations. Three calcium regulation loops have been reliably identified – acceleration of quenching of light-activated rhodopsin, acceleration of cGMP synthesis by guanylate cyclase, and increased affinity of cGMP-controlled ion cannels for nucleotide. However, one or two further highly effective mechanisms of adaptation are known to operate, one of which regulates the lifetime of activated phosphodiesterase; for others, the target of regulation is unknown. The mediators of this regulation are also unknown. Our studies of these mechanisms revealed a novel phenomenon, which has been missed in previous studies. We found that recovery of the dark current of rods after cessation of non-saturating adapting light can take 20–30 sec. Furthermore, after formal return of the membrane current to the dark level, cell sensitivity to test stimuli remains decreased for a further 1–2 min. We termed this phenomenon “adaptation memory.” Adaptation memory is reminiscent of the phenomenology of afterimages. The gradual return of the membrane current to the dark level may correspond to fading of the positive afterimage. Prolonged decreases in photoreceptor sensitivity to additional stimulation can create a negative afterimage. As far as we know, this is the first experimental physiological demonstration of the ability to generate afterimages at the level of single photoreceptors.

Spatially Aware Dense-LinkNet Based Regression Improves Fluorescent Cell Detection in Adaptive Optics Ophthalmic Images.

Retinal pigment epithelial (RPE) cells play an important role in nourishing retinal neurosensory photoreceptor cells, and numerous blinding diseases are associated with RPE defects. Their fluorescence signature can now be visualized in the living human eye using adaptive optics (AO) imaging combined with indocyanine green (ICG), which motivates us to develop an automated RPE detection method to improve the quantitative evaluation of RPE status in patients. This paper proposes a spatially-aware, Dense-LinkNet-based regression approach to improve the detection of in vivo fluorescent cell patterns, achieving precision, recall, and F1-Score of 93.6 ± 4.3%, 81.4 ± 9.5%, and 86.7 ± 5.7%, respectively. These results demonstrate the utility of incorporating spatial inputs into a deep learning-based regression framework for cell detection.

Long noncoding RNA U90926 is crucial for herpes simplex virus type 1 proliferation in murine retinal photoreceptor cells

Long non-coding RNAs (lncRNAs) play vital roles in the pathogenesis of infectious diseases, but the role of lncRNAs in herpes simplex virus 1 (HSV-1) infection remains unknown. Using RNA sequencing analysis, we explored lncRNAs that were highly expressed in murine retinal photoreceptor cell-derived 661W cells infected with HSV-1. U90926 RNA (522 nucleotides) was the most upregulated lncRNA detected post HSV-1 infection. The level of U90926 RNA was continuously increased post HSV-1 infection, reaching a 100-fold increase at 24 h. Cellular fractionation showed that U90926 RNA was located in the nucleus post HSV-1 infection. Downregulation of U90926 expression by RNA interference markedly suppressed HSV-1 DNA replication (80% reduction at 12 h post infection) and HSV-1 proliferation (93% reduction at 12 h post infection) in 661W cells. The survival rates of U90926-knockdown cells were significantly increased compared to those of control cells (81% and 21%, respectively; p < 0.0001). Thus, lncRNA U90926 is crucial for HSV-1 proliferation in retinal photoreceptor cells and consequently leads to host cell death by promoting HSV-1 proliferation.

Effect of autologous growth factors on apoptosis and thickness of the outer nuclear layer in an experimental retinal degeneration model

Retinal pigment epithelium and photoreceptor cells are a microenvironment where 90 different peptides are synthesized for transduction, visual cycle, intracellular electron transport chain, and removal of metabolic wastes. Depending on the inheritance pattern, either mutant proteins accumulate inside the cells or the energy cycle is disrupted. Disruption of homeostasis causes the cells to switch to the dormant phase; if the improper conditions last longer, then apoptosis eventually develops resulting in a loss of visual function. In neural tissues, growth factors such as neural growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, and insulin-like growth factor are regulatory peptides for intracellular energy cycle and intracellular digestion. In this study, it has been shown histopathologically that autologous growth factors can prevent apoptosis and prevent loss of outer retinal thickness in the retinal degeneration model created with sodium iodate.

Astaxanthin Protects Retinal Photoreceptor Cells against High Glucose-Induced Oxidative Stress by Induction of Antioxidant Enzymes via the PI3K/Akt/Nrf2 Pathway.

Diabetic retinopathy (DR) is a major microvascular complication that can lead to severe visual impairment in patients with diabetes. The elevated oxidative stress and increased reactive oxygen species (ROS) production induced by hyperglycemia have been reported to play an important role in the complex pathogenesis of DR. Astaxanthin (AST), a natural carotenoid derivative, has been recently recognized as a strong free radical scavenger and might, therefore, be beneficial in different diseases, including DR. In this study, we evaluated the potential role of AST as an antioxidative and antiapoptotic agent in protecting retinal cells and also investigated the involvement of the PI3K/Akt/Nrf2 pathway in AST-mediated effects. We treated high glucose-cultured mouse photoreceptor cells (661W) with different concentrations of AST and analyzed ROS production and cell apoptosis in the different regimens. Moreover, we also analyzed the expression of PI3K, Akt, Nrf2, and Phase II enzymes after AST treatment. Our results showed that AST dose-dependently reduced ROS production and attenuated 661W cell apoptosis in a high glucose environment. Importantly, its protective effect was abolished by treatment with PI3K or Nrf2 inhibitors, indicating the involvement of the PI3K/Akt/Nrf2 pathway. These results suggest AST as a nutritional supplement that could benefit patients with DR.

Label-free microfluidic enrichment of photoreceptor cells

Inherited retinal degenerative disorders such as retinitis pigmentosa and Usher syndrome are characterized by progressive death of photoreceptor cells. To restore vision to patients blinded by these diseases, a stem cell-based photoreceptor cell replacement strategy will likely be required. Although retinal stem cell differentiation protocols suitable for generating photoreceptor cells exist, they often yield a rather heterogenous mixture of cell types. To enrich the donor cell population for one or a few cell types, scientists have traditionally relied upon the use of antibody-based selection approaches. However, these strategies are quite labor intensive and require animal derived reagents and equipment that are not well suited to current good manufacturing practices (cGMP). The purpose of this study was to develop and evaluate a microfluidic cell sorting device capable of exploiting the physical and mechanical differences between retinal cell types to enrich specific donor cell populations such as Retinal Pigment Epithelial (RPE) cells and photoreceptor cells. Using this device, we were able to separate a mixture of RPE and iPSC-derived photoreceptor precursor cell lines into two substantially enriched fractions. The enrichment factor of the RPE fraction was 2 and that of the photoreceptor precursor cell fraction was 2.7. Similarly, when human retina, obtained from 3 independent donors, was dissociated and passed through the sorting device, the heterogeneous mixture could be reliably sorted into RPE and photoreceptor cell rich fractions. In summary, microfluidic cell sorting is a promising approach for antibody free enrichment of retinal cell populations.

Light or tyrosine phosphorylation recruits retinal rod outer segment proteins to lipid rafts

Lipid rafts are localized liquid-ordered regions of the plasma membrane that contain high levels of cholesterol and glycosphingolipids, and are resistant to extraction with nonionic detergents. Retinal photoreceptor cells contain detergent-resistant membrane microdomains (DRM), which were isolated here from bovine rod outer segments (ROS) under dark and light conditions. Rhodopsin (R) was present in both DRM and detergent soluble fractions (DSF), and detergent-insoluble ROS rafts were enriched in caveolin 1 (Cav-1) and c-Src. In the dark, arrestin and its 44-kDa truncated form (p44) were present mainly in DSF; however, p44 was translocated to DRM under illumination. Similarly, transducin (T) was mainly present in DSF in the dark, but it was recruited toward the DRM fraction following photolysis. DRM were also prepared in the absence or presence of Mg-ATP, guanosine 5′-3-O-(thio)triphosphate (GTPγS), or both. Although GTPγS released T into DSF in the light, GTPγS-activated T was retained in DRM when Mg2+ and ATP were added. Moreover, T was always tyrosine-phosphorylated under light conditions, which suggested that T phosphorylation prevents its GTPγS-induced release from DRM. In addition, treatment with the tyrosine kinase inhibitor genistein prevented the segregation of T to the rafts. In contrast, no localization difference was seen in the presence of Mg-ATP for Cav-1, c-Src, R and both forms of arrestin. Interestingly, immunoprecipitation assays followed by Western blot analyses under light conditions showed the formation of multimeric complexes containing R, T, c-Src, p44 and Cav-1 in DRM, where T and c-Src were tyrosine-phosphorylated.

Caspase-3 mRNA expression in rats with apoptosis of retinal photoreceptor cells**

BACKGROUNDApoptosis of retinal photoreceptor cells is a commonly pathological characteristic of various eye diseases, while caspase-3 is an important regulating gene and plays a key role in apoptosis. OBJECTIVETo measure the expression of caspase-3 mRNA in rats with apoptosis of retinal photoreceptor cells by using real-time fluorescent quantitative polymerase chain reaction and compare with those of the normal rats. DESIGNRandomized controlled animal study. SETTINGZhongshan Ophthalmological Center of Sun Yat-sen University. MATERIALSA total of 36 female SD rats of 50 days old and clean grade and weighing (150±10) g were provided by Experimental Animal Center of Northern Area of Sun Yat-sen University. All rats were randomly divided into normal control group (n = 6) and N-methyl-N-nitrosourea (MNU) group (n = 30), and they were observed at 12 hours, 1, 2, 3 and 5 days after model establishment, with 6 rats at each time point. METHODSThe experiment was carried out at Zhongshan Ophthalmological Center, Key Laboratory of Ophthalmology by State Ministry of Education from March to December 2004. Rats in the normal control group were intraperitoneally injected with saline and rats in the MNU group were intraperitoneally injected with 40 mg/kg MNU. And then, retinal photoreceptor injured models were established. At 12 hours, 1, 2, 3 and 5 days after model establishment, the rats were sacrificed for enucleating right eyeballs, isolating retina immediately and extracting total RNA. The expression of caspase-3 mRNA in retina was measured with real-time fluorescent quantitative polymerase chain reaction. MAIN OUTCOME MEASURESExpression of caspase-3 mRNA in retina of rats in the two groups. RESULTSA total of 36 SD rats were involved in the final analysis. The expressions of caspase-3 mRNA in the rat retina of both groups at the five time points (12 hours, 1, 2, 3 and 5 days) after model establishment were 1.52×105, 18.35×105, 25.14×105, 29.25×105, 13.72×105 and 12.24×105, respectively. The expression of caspase-3 mRNA in the MNU group increased after 12 hours of intraperitoneal injection, and rose to the top on the 2nd day, which was 19 times as many as that of the normal control group. Then, it decreased gradually and was still 8 times as many as that of the normal control group on the 5 day. CONCLUSIONThe expression of caspase-3 mRNA is related to apoptosis of retinal photoreceptor cells, while caspase-3 plays an important role in occurrence and development of apoptosis of retinal photoreceptor cells.

Non-photoreceptor Expression of Tulp1 May Contribute to Extensive Retinal Degeneration in Tulp1-/- Mice.

Mutations in tubby like protein 1 gene (TULP1) are causative of early-onset recessive inherited retinal degenerations (IRDs); similarly, the Tulp1-/- mouse is also characterized by a rapid IRD. Tulp1 mRNA and protein expression was analyzed in wild type mouse retinas and expression data sets (NCBI) during early postnatal development. Comparative histology was undertaken in Tulp1-/-, rhodopsin-/- (Rho-/-) and retinal degeneration slow-/- (Rds-/-) mouse retinas. Bioinformatic analysis of predicted TULP1 interactors and IRD genes was performed. Peak expression of Tulp1 in healthy mouse retinas was detected at p8; of note, TULP1 was detected in both the outer and inner retina. Bioinformatic analysis indicated Tulp1 expression in retinal progenitor, photoreceptor and non-photoreceptor cells. While common features of photoreceptor degeneration were detected in Tulp1-/-, Rho-/-, and Rds-/- retinas, other alterations in bipolar, amacrine and ganglion cells were specific to Tulp1-/- mice. Additionally, predicted TULP1 interactors differed in various retinal cell types and new functions for TULP1 were suggested. A pilot bioinformatic analysis indicated that in a similar fashion to Tulp1, many other IRD genes were expressed in both inner and outer retinal cells at p4–p7. Our data indicate that expression of Tulp1 extends to multiple retinal cell types; lack of TULP1 may lead to primary degeneration not only of photoreceptor but also non-photoreceptor cells. Predicted interactors suggest widespread retinal functions for TULP1. Early and widespread expression of TULP1 and some other IRD genes in both the inner and outer retina highlights potential hurdles in the development of treatments for these IRDs.

Autophagy and post-ischemic conditioning in retinal ischemia

ABSTRACT Retinal ischemia is a major cause of vision loss and a common underlying mechanism associated with diseases, such as diabetic retinopathy and central retinal artery occlusion. We have previously demonstrated the robust neuroprotection in retina induced by post-conditioning (post-C), a brief period of ischemia, 24 h, following a prolonged and damaging initial ischemia. The mechanisms underlying post-C-mediated retinal protection are largely uncharacterized. We hypothesized that macroautophagy/autophagy is a mediator of post-C-induced neuroprotection. This study employed an in vitro model of oxygen glucose deprivation (OGD) in the retinal R28 neuronal cell line, and an in vivo rat model of retinal ischemic injury. In vivo, there were significant increases in autophagy proteins, MAP1LC3-II/LC3-II, and decreases in SQSTM1/p62 (sequestosome 1) in ischemia/post-C vs. ischemia/sham post-C. Blockade of Atg5 and Atg7 in vivo decreased LC3-II, increased SQSTM1, attenuated the functional protective effect of post-C, and increased histological damage and TUNEL compared to non-silencing siRNA. TUNEL after ischemia in vivo was found in retinal ganglion, amacrine, and photoreceptor cells. Blockade of Atg5 attenuated the post-C neuroprotection by a brief period of OGD in vitro. Moreover, in vitro, post-C attenuated cell death, loss of cellular proliferation, and defective autophagic flux from prolonged OGD. Stimulating autophagy using Tat-Beclin 1 rescued retinal neurons from cell death after OGD. As a whole, our results suggest that autophagy is required for the neuroprotective effect of retinal ischemic post-conditioning and augmentation of autophagy offers promise in the treatment of retinal ischemic injury. Abbreviations: BECN1: Beclin 1, autophagy related; DAPI: 4’,6-diamidino-2-phenylindole; DR: diabetic retinopathy; EdU: 5-ethynyl-2’-deoxyuridine; ERG: Electroretinogram; FITC: Fluorescein isothiocyanate; GCL: Ganglion cell layer; GFAP: Glial fibrillary acidic protein; INL: Inner nuclear layer; IPL: Inner plexiform layer; MAP1LC3/LC3: Microtubule-associated protein 1 light chain 3; OGD: Oxygen-glucose deprivation; ONL: Outer nuclear layer; OP: Oscillatory potential; PFA: Paraformaldehyde; PL: Photoreceptor layer; post-C: post-conditioning; RFP: Red fluorescent protein; RGC: Retinal ganglion cell; RPE: Retinal pigment epithelium; RT-PCR: Real-time polymerase chain reaction; SEM: Standard error of the mean; siRNA: Small interfering RNA; SQSTM1: Sequestosome 1; STR: Scotopic threshold response; Tat: Trans-activator of transcription; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling

The Photoreceptor Cells in Retina and Pineal Gland of Amphibian Hoplobatrachus rugulosus

Hoplobatrachus rugulosus, commonly referred to as tiger‐skinned frog, is to be chosen the animal model in this study. It has been found that both chicken α‐internexin and zebrafish inaa (an ortholog of mammalian α‐internexin) were developmentally distributed not only in photoreceptors of retina but also in the photoreceptor‐like cells of the pineal gland from our previous studies. However, from an evolutionary point of view, there is still lack of evidence to illustrate the temporal and spatial distribution of frog α‐internexin protein in the developing pineal gland. According to other studies about Xenopus laevis, the animal model that is usually used for amphibian developmental research, the mRNA sequence which encodes Xenopus α‐internexin had been identified as “xefiltin”. Although H. rugulosus is not the same species with X. laevis, both of them are classified in the same order. The aims of this study is to characterize the expression pattern of α‐internexin‐like proteins, internexin neuronal intermediate filament protein alpha S homeolog (ina.S) and low molecular weight neuronal intermediate filament S homeolog (nif.S) which might be expressed in the developing retina and the pineal gland of the tiger‐skinned frog. Thus, the specific antibodies were generated to detect the frog α‐internexin‐like proteins and to confirm the distribution of these proteins in the developing frog retina as well as the pineal gland by western blot and immunohistochemistry. Furthermore, the markers applied to detect the photoreceptor cell in the retina and the pineal gland are recoverin and XAP1, both of them are reported to be specific to photoreceptors. Based on the distribution patterns in frog retina, immunopositive staining of anti‐nif.S could be observed in the outer segment of photoreceptors, but anti‐ina.S could only be found in retinal neurons. In the pineal gland, both photoreceptor markers, recoverin and XAP1, could be detected. The α‐internexin‐like protein, ina.S and nif.S, could be detected only in a few photoreceptor‐like cells at the early stage of the frog pineal gland. In summary, the distribution patterns of photoreceptor cells in retina and in pineal gland of developing tiger‐skinned frogs has been characterized in this study. It can be concluded that XAP1 could be a good marker to identify the photoreceptor cells in the developing pineal gland of frogs.Support or Funding InformationGrant SponsorMinistry of Science and Technology, Taiwan; Grant Number: MOST 108‐2312‐B‐002‐030‐MY2.

In silico analysis of a novel causative mutation in Cadherin23 gene identified in an Omani family with hearing loss

BackgroundHereditary hearing loss is a heterogeneous group of complex disorders with an overall incidence of one in every 500 newborns presented as syndromic and non-syndromic forms. Cadherin-related 23 (CDH23) is one of the listed deafness causative genes. It is found to be expressed in the stereocilia of hair cells and in the retina photoreceptor cells. Defective CDH23 have been associated mostly with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic (USH1D) or non-syndromic SNHL (DFNB12) deafness. The purpose of this study was to identify causative mutations in an Omani family diagnosed with severe-profound sensorineural hearing loss by whole exome sequencing technique and analyzing the detected variant in silico for pathogenicity using several in silico mutation prediction software. ResultsA novel homozygous missense variant, c.A7436C (p. D2479A), in exon 53 of CDH23 was detected in the family while the control samples were all negative for the detected variant. In silico mutation prediction analysis showed the novel substituted D2479A to be deleterious and protein destabilizing mutation at a conserved site on CDH23 protein. ConclusionIn silico mutation prediction analysis might be used as a useful molecular diagnostic tool benefiting both genetic counseling and mutation verification. The aspartic acid 2479 alanine missense substitution might be the main disease-causing mutation that damages CDH23 function and could be used as a genetic hearing loss marker for this particular Omani family.

Effects and mechanisms of water-soluble Semen cassiae polysaccharide on retinitis pigmentosa in rats

Abstract This study aimed to investigate the effects and mechanisms of water-soluble Semen cassiae polysaccharide (SCP) on retinitis pigmentosa (RP) in rats. One hundred rats were randomly divided into control, model, and 50, 100 and 200 mg/kg SCP groups. The 50, 100 and 200 mg/kg SCP groups were treated with 50, 100 and 200 mg/kg SCP for 5 days, respectively. On the sixth day, the model, and 50, 100 and 200 mg/kg SCP groups were treated with N-nitroso-N-methylurea (MNU). After 12 h and 5 days, the electroretinogram, retinal thickness, apoptosis of retinal photoreceptor cells, and expressions of apoptosis related protein in retina tissue were determined. Results showed that, after 12 h and on 5 days from MNU treatment, compared with model group, the a-wave and b-wave amplitude and retinal thickness in SCP groups were significantly increased, the apoptosis rate of retinal photoreceptor cells was significantly decreased, the Bcl-2 protein expression level in retinal tissue was significantly increased, and the Bax and Caspase-3 protein level were significantly decreased (all P < 0.05). In conclusion, SCP has obvious protective effects on RP in rats. The mechanism may be related to it regulation of Bcl-2, Bax and Caspase-3 protein expression in retina tissue.

Multipotency of mouse trophoblast stem cells

BackgroundIn a number of disease processes, the body is unable to repair injured tissue, promoting the need to develop strategies for tissue repair and regeneration, including the use of cellular therapeutics. Trophoblast stem cells (TSCs) are considered putative stem cells as they differentiate into other subtypes of trophoblast cells. To identify cells for future therapeutic strategies, we investigated whether TSCs have properties of stem/progenitor cells including self-renewal and the capacity to differentiate into parenchymal cells of fetal organs, in vitro and in vivo.MethodsTSCs were isolated using anti-CD117 micro-beads, from embryonic day 18.5 placentas. In vitro, CD117+ TSCs were cultured, at a limiting dilution in growth medium for the development of multicellular clones and in specialized medium for differentiation into lung epithelial cells, cardiomyocytes, and retinal photoreceptor cells. CD117+ TSCs were also injected in utero into lung, heart, and the sub-retinal space of embryonic day 13.5 fetuses, and the organs were harvested for histological assessment after a natural delivery.ResultsWe first identified CD117+ cells within the labyrinth zone and chorionic basal plate of murine placentas in late pregnancy, embryonic day 18.5. CD117+ TSCs formed multicellular clones that remained positive for CD117 in vitro, consistent with self-renewal properties. The clonal cells demonstrated multipotency, capable of differentiating into lung epithelial cells (endoderm), cardiomyocytes (mesoderm), and retinal photoreceptor cells (ectoderm). Finally, injection of CD117+ TSCs in utero into lungs, hearts, and the sub-retinal spaces of fetuses resulted in their engraftment on day 1 after birth, and the CD117+ TSCs differentiated into lung alveolar epithelial cells, heart cardiomyocytes, and retina photoreceptor cells, corresponding with the organs in which they were injected.ConclusionsOur findings demonstrate that CD117+ TSCs have the properties of stem cells including clonogenicity, self-renewal, and multipotency. In utero administration of CD117+ TSCs engraft and differentiate into resident cells of the lung, heart, and retina during mouse development.