T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

Grease-gun injury of the orbit: two cases report and literature review.

BACKGROUND: The grease-guns injury is an uncommon injury to the orbit. We present the twelfth and thirteenth cases of grease-gun injury to the orbit to be reported in the English language literature since 1964. Here we discus and review the presentation, investigation, and treatment of this unusual trauma. CASE PRESENTATION: Case 1 was a 29-year-old man who presented 1 day after a grease-gun injury of the left orbit with severe pain, marked periorbital swelling, and proptosis. Computed tomography (CT) revealed penetration of grease into his left orbit. Following surgical removal, proptosis decreased. The limitation of extraocular movement and loss of visual acuity to finger count was discovered after the initial surgery. Motility gradually returned. Visual acuity recovered after phacoemulsification, capsular tension ring and intraocular lens implantation for traumatic cataract and subluxation. Case 2 was a 6-year-old boy who was referred 2 months after a grease-gun injury for worsening swelling with sinus, necrosis and slight ptosis of the upper left eyelids. This is a case of orbital chronic inflammation from grease-gun injuries masquerading as orbital cellulitis. The imaging findings of CT and magnetic resonance imaging (MRI) are not typical. Surgical exploration and debridement was inevitable and actually relieved the symptoms. CONCLUSIONS: Grease-gun injuries can damage the orbit in different degrees. Careful history inquiry and taking is important to establish the diagnosis. Imaging examinations using CT or MRI are helpful to determine depth of trauma and foreign bodies in the orbit at diagnosis. We suggest that surgical exploration and debridement is a key step in the management.

Expression of glucose transporter-2 in murine retina: Evidence for glucose transport from horizontal cells to photoreceptor synapses.

The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.

Based on multiple machine learning to identify the ENO2 as diagnosis biomarkers of glaucoma.

PURPOSE: Glaucoma is a generic term of a highly different disease group of optic neuropathies, which the leading cause of irreversible vision in the world. There are few biomarkers available for clinical prediction and diagnosis, and the diagnosis of patients is mostly delayed. METHODS: Differential gene expression of transcriptome sequencing data (GSE9944 and GSE2378) for normal samples and glaucoma samples from the GEO database were analyzed. Furthermore, based on different algorithms (Logistic Regression (LR), Random Forest (RF), lasso regression (LASSO)) two diagnostic models are constructed and diagnostic markers are screened. GO and KEGG analyses revealed the possible mechanism of differential genes in the pathogenesis of glaucoma. ROC curve confirmed the effectiveness. RESULTS: LR-RF model included 3 key genes (NAMPT, ADH1C, ENO2), and the LASSO model outputted 5 genes (IFI16, RFTN1, NAMPT, ADH1C, and ENO2), both algorithms have excellent diagnostic efficiency. ROC curve confirmed that the three biomarkers ADH1C, ENO2, and NAMPT were effective in the diagnosis of glaucoma. Next, the expression analysis of the three diagnostic biomarkers in glaucoma and control samples confirmed that NAMPT and ADH1C were up-regulated in glaucoma samples, and ENO2 was down-regulated. Correlation analysis showed that ENO2 was significantly negatively correlated with ADH1C (cor = -0.865714202) and NAMPT (cor = -0.730541227). Finally, three compounds for the treatment of glaucoma were obtained in the TCMs database: acetylsalicylic acid, 7-o-methylisomucitol and scutellarin which were applied to molecular docking with the diagnostic biomarker ENO2. CONCLUSIONS: In conclusion, our research shows that ENO2, NAMPT, and ADH1C can be used as diagnostic markers for glaucoma, and ENO2 can be used as a therapeutic target.

Inhibition of the MAPK/c-Jun-EGR1 Pathway Decreases Photoreceptor Cell Death in the rd1 Mouse Model for Inherited Retinal Degeneration.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies that typically results in photoreceptor cell death and vision loss. Here, we explored the effect of early growth response-1 (EGR1) expression on photoreceptor cell death in Pde6brd1 (rd1) mice and its mechanism of action. To this end, single-cell RNA-seq (scRNA-seq) was used to identify differentially expressed genes in rd1 and congenic wild-type (WT) mice. Chromatin immunoprecipitation (ChIP), the dual-luciferase reporter gene assay, and western blotting were used to verify the relationship between EGR1 and poly (ADP-ribose) polymerase-1 (PARP1). Immunofluorescence staining was used to assess PARP1 expression after silencing or overexpression of EGR1. Photoreceptor cell death was assessed using the TUNEL assay following silencing/overexpression of EGR1 or administration of MAPK/c-Jun pathway inhibitors tanzisertib and PD98059. Our results showed differential expression of ERG1 in rd1 and WT mice via scRNA-seq analysis. The ChIP assay demonstrated EGR1 binding to the PARP1 promoter region. The dual-luciferase reporter gene assay and western blotting results revealed that EGR1 upregulated PARP1 expression. Additionally, the TUNEL assay showed that silencing EGR1 effectively reduced photoreceptor cell death. Similarly, the addition of tanzisertib and PD98059 reduced the expression of c-Jun and EGR1 and decreased photoreceptor cell death. Our study revealed that inhibition of the MAPK/c-Jun pathway reduced the expression of EGR1 and PARP1 and prevented photoreceptor cell death. These results highlight the importance of EGR1 for photoreceptor cell death and identify a new avenue for therapeutic interventions in RP.

Inherited FGFR2 mutation in a Chinese patient with Crouzon syndrome and luxation of bulbus oculi provoked by trauma: a case report.

BACKGROUND: Crouzon syndrome (CS), which results from fibroblast growth factor receptor 2 mutations, is associated with craniosynostosis, exophthalmos, and other symptoms. Herein, we report the genetic abnormalities detected in a Chinese family with autosomal dominant CS, combined with luxation of the eyeball. This luxation was a consequence of the trauma to the shallow orbits. CASE PRESENTATION: The proband was a 4-year-old boy. He accidentally fell, following which luxation of the bulbus oculi occurred immediately. Computed tomography and magnetic resonance imaging clearly revealed ocular proptosis. Upon physical examination, the proband, his father, and grandfather had ocular proptosis, shallow orbits, and mid-face hypoplasia. However, their hands and feet were clinically normal. Genomic DNA was extracted from the peripheral blood through a polymerase chain reaction performed for the target sequence. Genetic assessments revealed a heterozygous missense mutation (c.1012G > C, p.G338R) in exon 10 of the human FGFR2, cosegregated with the disease phenotype in this family. These findings confirmed the diagnosis of CS. DISCUSSION: CS is usually caused by FGFR2 mutations. While there are a few reports of luxation of the bulbus oculi in Chinese families with CS, the ocular proptosis, shallow orbits, combined with luxation of eyeball after trauma observed in this patient were particularly interesting. Our findings enhance the current knowledge of traumatic luxation concomitant with CS.

In Vivo Confocal Microscopic Evaluation of Corneal Endothelial Dysfunction Induced by Phacoemulcification in Rhesus Monkey Models.

OBJECTIVE: To observe the characteristic morphological changes of corneal endothelial dysfunction induced by phacoemulcification in rhesus monkey models under confocal microscope. METHODS: The corneal endothelial dysfunction models were established by phacoemulcification power on the central corneal of 7 to 9 mm diameter in the right eyes of 4 rhesus monkeys (the modeling group). The left eyes of 4 rhesus monkeys were set as blank control group. The structural changes in different corneal layers were evaluated by slit lamp microscope and in vivo confocal microscope before surgery and 1, 2, 3, and 4 weeks after surgery. SPSS 19.0 software was applied to analyze data. Paired-t test was used to compare the number of nerve plexus in Bowman's layer and corneal endothelial cell density. Analysis of variance (ANOVA) was used to analyze corneal thickness. RESULTS: After phacoemulcification, the changes of cornea occurred gradually in the endothelial layer, stroma, Bowman's membrane, and basal epithelial layer. In the early stage, the interspace of corneal endothelial cells enlarged and few activated stromal cells were detected in the stroma. The cell morphology of stroma altered. The thickness of stroma increased. Two weeks after surgery, the nerve plexus in Bowman's layer decreased and edema of stroma and endothelial layer increased. Three weeks after surgery, the interspace of basal epithelial cells increased with a few Langerhans' cells infiltration and edema of stroma and endothelial layer increased. Four weeks after the surgery, a large amount of Langerhans' cells presented in basal epithelial layer. Only a few nerve lexus could be seen in Bowman's layer. The stroma and endothelial cells had severe edema. A large number of activated stromal cells could be found in stromal layer. Two weeks after the surgery, the number of nerve plexus in Bowman's layer (t=6.9192, P=0.002) and corneal endothelial cell density (t=7.8936, P<0.0001) in the modeling group were significantly lower than that in control group. Compared with corneal thickness in control group, it was significantly larger in the modeling group at 1 (t=28.31, P<0.0001), 2 (t=63.56, P<0.0001), 3 (t=123.22, P<0.0001), and 4 weeks (t=180.80, P<0.0001) after the surgery. CONCLUSIONS: The changes in corneal endothelial dysfunction induced by phacoemulcification in rhesus monkey models can be clearly shown under in vivo confocal microscope. Gradual increase of endothelial cells interspace, activated stromal cells, increase of Langerhans' cells, and decrease of plexus in Bowman's layer are the main changes.

[Transplanted vascular endothelial cells to replace corneal endothelial cells by improved anterior chamber injection].

OBJECTIVE: To observe form and function changes of vascular endothelial cells (VEC) which were transplanted to the posterior surface of rhesus monkey cornea without Descemet's Membrane by anterior chamber injection, explore the feasibility of transplanting VEC to treat corneal endothelial injury, and find new method of corneal endothelial cell transplantation. METHODS: Cultured VEC to proliferate in vitro.Rhesus monkeys were randomly divided into two groups: Experimental group (6) and control group (6) according to a random number table. The experimental group:transplant the culured VEC to the posterior surface of rhesus monkey cornea without Descemet's membrane by anterior chamber injection. The control group:Tear out the Descemet's membrane by capsulotomy needle without VEC transplantation. A ultrasound apparatus was adopted to measure the postoperative thicknes of the cornea and Goldman intraocular pressure meter measuring intraocular pressure in the postoperative 7 days, 14 days, 30 days, 60 days, 90 days, organize the data and entered into the computer , applicated the software of SPSS 11.5 for data independent samples t-test and analysis of variance. The eyes were removed respectively in postoperative 30, 60, 90 days to do pathological HE dyeing and scanning electron microscopy (SEM) observation of VEC in the posterior surface of cornea graft. RESULTS: Corneal transpareney:In the experimental observation period (3 months), the experimental group had better transparency than the control group with normal anterior chamber depth and without bullous keratopathy. And the corneal neovascularization was exist in the cornea graft in experimental group in the third month. After 3 months, the corneal thickness of experimental group (500 ± 14) µm was significantly lower than the control group (618 ± 11) µm, Corneal thickness values between experimental and control groups were statistically significant differences in changes (all P < 0.05, t-values were -3.256, -4.419, -12.896 postoperative 1, 2, 3 months). Changes of intraocular pressure difference between the experimental group and the control was not statistically significant (all P > 0.05, t-values were -1.179, -2.166, -2.536 postoperative 1, 2, 3 months). The pathological:the cell layer was visible in the posterior surface of cornea graft. The control group:can't find the cell sample structure.SEM:Experimental group showed that VEC with irregular shape uniformly distributed on the inner surface of cornea and growing well, a small amount of white blood cells can be seen between VEC, and part of cellular debris exist in the trabecular meshwork. Control group showed a fiber material without VEC. CONCLUSIONS: Ultrasonic emulsification can established a repeatability and simple model of corneal endothelial injury in Rhesus monkeys.VEC can be transplanted to the corneal surface by Anterior chamber injection and the cells can grow on the surface and play a barrier role in maintaining the state of dehydration and transparency of the cornea to a certain extent. After transplantation, organizational structure and morphology of the anterior chamber angle does not produce pathological effects in the short term. Prompt that transplant the culured VEC to the posterior surface of rhesus monkey cornea without Descemet's Membrane by anterior chamber injection to substitute the function of the corneal endothelial cells may be a new idea for treatment of corneal endothelial damage.

Clinical and pathological characteristics of intraocular cysticercosis.

This study aimed to explore the clinical, radiological, and pathological characteristics of intraocular cysticercosis due to Taenia solium metacestode infection. Total 8 patients diagnosed with intraocular cysticercosis at the Red Cross Hospital of Yunnan Province, China were examined retrospectively. Patients with clear dioptic media had undergone fundus chromophotography. All patients underwent B ultrasonography of the ocular region (CT) successive scanning of the orbit and cerebral tissues. Parasites were extracted surgically and then examined pathologically. The fundus chromophotography showed a white and condensing scolex package in the vesicle. The B ultrasonic examination showed a vesicle-like echogenic mass in the vitreous chamber, in which the high-level echo spot was the cysticercus scolex. The pathological examinations showed that the vesicle wall exhibited hyaline degeneration, inflammatory cell infiltration, neuroglial fiber, and glial cell proliferation layers from the inside to the outside. The scolex is round and is composed of the outer tissue (the body wall) and the inner furrow tissue; these tissues migrated together. Primordially differentiated sucking discs were found in one case, but no hooklets were found. The inner scolex tissue was folded like a paper flower. The severity of intraocular disease is closely correlated with the pathophysiological processes of the cysticercus worm. Pathological examination of the intraocular lesions can help to evaluate the course of the disease as well as to provide a scientific basis for effective antiparasitic medication.

[Experimental study on the rhesus monkey corneal endothelial cells substituted by the allogeneic vascular endothelial cells cultivated in vitro].

OBJECTIVE: To Explore the feasibility of rhesus monkey vascular endothelial cell(RMVEC) transplantation to substitute the allogeneic corneal endothelial cells(CEC), through observe the morphologic and functional change of the vascular endothelial cell which was transplanted to the inner surface of cornea without Descemet's membrane. METHODS: It was an experimental study. The rhesus monkey vascular endothelial cell was cultivated to proliferation and marked by BrdU in vitro. The experimented monkeys are divided into 2 groups: experimental group (6 monkeys), control group (3 monkey). In experimental group: the cultured RMVEC, marked by BrdU, were transplanted onto the posterior surface of rhesus monkey cornea without Descemet's membrane though centrifugal sedimentation, then sew back the corneal graft. In control group: detach the corneal Descemet's Membrane of rhesus monkey but without cultured RMVEC transplantation. Corneal transparency of every monkey was frequently observed postoperation. On 30, 60, 90 postoperative days, corneal graft were respectively detached to observe the distribution, appearance and ultrastructures morphological structure of RMVEC on the inner surface of cornea, by pathological section, anti BrdU monoclonal antibody immunohistochemistry, scanning electronic microscope(SEM) and transmission election microscopy(TEM). RESULTS: Corneal transparency: In the experimental observation period (three months), the corneal graft of experimental group had better transparency than control group and without corneal neovascularization and bullous keratopathy. Pathological section: A layer of cells with BrdU staining positive was found on the posterior surface of cornea in experimental group, indicated the cells are RMVEC. And no cell-like structure was found in control group. SEM: Experimental group showed that RMVEC with irregular shape uniformly distributed on the inner surface of cornea and growing well, a small amount of white blood cells can be seen between RMVEC, and part of cellular debris exist in the trabecular meshwork. Control group showed a fiber material without RMVEC. TEM: The cultured RMVEC and which in posterior surface of cornea was irregular oblateness, a large number of desmosomes link were been seen between RMVEC. Abundant organelles and characteristic WPBs appear in cytoplasm, which suggest the characteristics and vitality of vascular endothelial cells in vivo, and no cell structure in the control group. CONCLUSIONS: Rhesus monkey endothelial cells can growth on the posterior surface of cornea without Descemet's membrane. The cultured cells, with similar ultrastructure to RMVEC in vivo, can play a role of barrier to keep the corneal dehydration and transparency to some extent.

Study of pathological processes of meibomian gland dysfunction by in vitro culture airlifting conditions.

Meibomian gland dysfunction (MGD) is a group of disorders linked by functional abnormalities of the meibomian glands. Current studies on MGD pathogenesis focus on meibomian gland cells, providing information on a single cell's response to experimental manipulation, and do not maintain the architecture of an intact meibomian gland acinus and the acinar epithelial cells' secretion state in vivo. In this study, rat meibomian gland explants were cultured by a Transwell chamber-assisted method under an air-liquid interface (airlift) in vitro for 96 h. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TUNEL assays, hematoxylin and eosin (H&E) staining, immunofluorescence, Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), transmission electron microscopy (TEM), and western blotting (WB). MTT, TUNEL, and H&E staining indicated better tissue viability and morphology than the submerged conditions used in previous studies. Levels of MGD biomarkers, including keratin 1 (KRT1) and 14 (KRT14) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), along with oxidative stress markers, including reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal, gradually increased over culture time. The MGD pathophysiological changes and biomarker expression of meibomian gland explants cultured under airlift conditions were similar to those reported by previous studies, indicating that abnormal acinar cell differentiation and glandular epithelial cell hyperkeratosis may contribute to obstructive MGD occurrence.

HDAC inhibition delays photoreceptor loss in Pde6b mutant mice of retinitis pigmentosa: insights from scRNA-seq and CUT&Tag.

Purpose: This research aimed to ascertain the neuroprotective effect of histone deacetylase (HDAC) inhibition on retinal photoreceptors in Pde6brd1 mice, a model of retinitis pigmentosa (RP). Methods: Single-cell RNA-sequencing (scRNA-seq) explored HDAC and poly (ADP-ribose) polymerase (PARP)-related gene expression in both Pde6b-mutant rd1 and wild-type (WT) mice. The CUT&Tag method was employed to examine the functions of HDAC in rd1 mice. Organotypic retinal explant cultures from WT and rd1 mice were exposed to the HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) postnatally, from day 5 to day 11. The terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay was applied to quantify the percentage of photoreceptor loss in the outer nuclear layer (ONL). HDAC activity was confirmed to be inhibited by SAHA through an HDAC activity assay. Moreover, the study evaluated PARP activity, a key driver of the initial response to DNA damage during photoreceptor degeneration, following HDAC inhibition. Results: The scRNA-seq revealed that diverse roles of HDAC and PARP isoforms in photoreceptor cell death. HDAC-related genes appeared to regulate cell death and primary immunodeficiency. Alterations in HDAC activity were consistent with the TUNEL-positive cells in the ONL at different time points. Notably, SAHA significantly postponed photoreceptor loss and decreased HDAC and PARP activity, thereby implicating both in the same degenerative pathway. Conclusions: This study highlights that the interaction between HDAC inhibition and PARP can delay photoreceptor cell death, proposing a promising therapeutic approach for RP.

Inherited Retinal Degeneration: Towards the Development of a Combination Therapy Targeting Histone Deacetylase, Poly (ADP-Ribose) Polymerase, and Calpain.

Inherited retinal degeneration (IRD) represents a diverse group of gene mutation-induced blinding diseases. In IRD, the loss of photoreceptors is often connected to excessive activation of histone-deacetylase (HDAC), poly-ADP-ribose-polymerase (PARP), and calpain-type proteases (calpain). Moreover, the inhibition of either HDACs, PARPs, or calpains has previously shown promise in preventing photoreceptor cell death, although the relationship between these enzyme groups remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type mice and rd1 mice as a model for IRD were treated with different combinations of inhibitors specific for HDAC, PARP, and calpain. The outcomes were assessed using in situ activity assays for HDAC, PARP, and calpain, immunostaining for activated calpain-2, and the TUNEL assay for cell death detection. We confirmed that inhibition of either HDAC, PARP, or calpain reduced rd1 mouse photoreceptor degeneration, with the HDAC inhibitor Vorinostat (SAHA) being most effective. Calpain activity was reduced by inhibition of both HDAC and PARP whereas PARP activity was only reduced by HDAC inhibition. Unexpectedly, combined treatment with either PARP and calpain inhibitors or HDAC and calpain inhibitors did not produce synergistic rescue of photoreceptors. Together, these results indicate that in rd1 photoreceptors, HDAC, PARP, and calpain are part of the same degenerative pathway and are activated in a sequence that begins with HDAC and ends with calpain.

Silencing of Rac1 expression via RNA interference inhibits retinal neovascularization in rats.

PURPOSE: To investigate the inhibitory effect of Ras-related C3 botulinum toxin substrate 1-small interfering RNA (Rac1-siRNA) on retinal neovascularization in a rat model. METHODS: Rac1-short hairpin RNA (shRNA) was synthesized, constructed, and transfected into HeLa cells. Reverse transcription polymerase chain reaction was then conducted to test for Rac1 gene expression. Retinal vein obstruction was performed in 25 Sprague-Dawley rats using the retinal photodynamic method. The vitrea bulbus of the eye in the shRNA rats group was transfected with the Rac1-shRNA vector, the other eye in the blank control group was transfected with the blank vector, and the interference control group was prepared by transfecting the Rac1-shRNA vector. Two weeks after transfection, the neonatal vessels were tested using fluorescein isothiocyanate-dextran retinal angiography. The number of endothelial cells beyond the internal limiting membrane was counted with hematoxylin and eosin staining. RESULTS: A large area of neovascularization and fluorescein isothiocyanate leakage was found in the positive control group. However, a small area of neovascularization and a little fluorescence leakage were found in the shRNA group, whereas the retinal vessels were normal in the negative control interference group. In the shRNA interference group, the mean number of endothelial cells beyond the internal limiting membrane was significantly higher than that in the positive control group or the interference negative control group (p<0.05). CONCLUSIONS: Silencing Rac1 expression with RNA interference inhibits retinal neovascularization in rats.

Organotypic Retinal Explant Cultures from Macaque Monkey.

Hereditary retinal degeneration (RD) is characterized by progressive photoreceptor cell death. Overactivation of the cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) pathway in photoreceptor cells causes photoreceptor cell death, especially in models harboring phosphodiesterase 6b (PDE6b) mutations. Previous studies on RD have used mainly murine models such as rd1 or rd10 mice. Given the genetic and physiological differences between mice and humans, it is important to understand to which extent the retinas of primates and rodents are comparable. Macaques share a high level of genetic similarity with humans. Therefore, wild-type macaques (aged 1-3 years) were selected for the in vitro culture of retinal explants that included the retina-retinal pigment epithelium (RPE)-choroid complex. These explants were treated with different concentrations of the PDE6 inhibitor zaprinast to induce the cGMP-PKG signaling pathway and simulate RD pathogenesis. cGMP accumulation and cell death in primate retinal explants were subsequently verified using immunofluorescence and the TUNEL assay. The primate retinal model established in this study may serve for relevant and effective studies into the mechanisms of cGMP-PKG-dependent RD, as well as for the development of future treatment approaches.

VR-10 polypeptide interacts with CD36 to induce cell apoptosis and autophagy in choroid-retinal endothelial cells: Identification of VR-10 as putative novel therapeutic agent for choroid neovascularization (CNV) treatment.

Choroid neovascularization (CNV) is important adverse pathological changes that contributes to the aggravation of hypoxic-ischemic eye diseases, and our preliminary work evidences that the thrombospondin-1 (TSP-1) synthetic polypeptide VR-10 may be the candidate therapeutic agent for the treatment of CNV, but its detailed effects and molecular mechanisms are not fully delineated. In this study, the CNV models in BN rats were established by using the laser photocoagulation method, which were further subjected to VR-10 peptide treatment. The RNA-seq and bioinformatics analysis suggested that VR-10 peptide significantly altered the expression patterns of genes in the rat ocular tissues, and the changed genes were especially enriched in the CD36-associated signal pathways. Next, by performing the Real-Time qPCR and Western Blot analysis, we expectedly found that VR-10 upregulated the anti-angiogenesis biomarker (PEDF) and downregulated pro-angiogenesis biomarkers (VEGF, HIF-1 and IL-17) in rat tissues. In addition, we evidenced that VR-10 downregulated CDK2, CDK4, CDK6, Cyclin D1 and Cyclin D2 to induce cell cycle arrest, upregulated cleaved Caspase-3, Bax and downregulated Bcl-2 to promote cell apoptosis, and increased LC3B-II/I ratio and facilitate p62 degradation to promote cell autophagy in RF/6A cells, which were all reversed by knocking down CD36. Moreover, VR-10 upregulated PEDF, and decreased the expression levels of VEGF, HIF-1 and IL-17 to block angiogenesis of RF/6A cells in a CD36-dependent manner. Taken together, VR-10 peptide interacts with its receptor CD36 to regulate the biological functions of RF/6A cells, and these data suggest that VR-10 peptide may be the putative therapeutic drug for the treatment of CNV in clinic.

Nestin is induced by hypoxia and is attenuated by hyperoxia in Müller glial cells in the adult rat retina.

This study investigated the reactive changes in Müller glial cells and astrocytes of the rat retinae, which had been subjected either to hypoxia or to hypoxia followed by hyperoxia treatments. Fifteen rats were used. Ten rats were exposed to 9% O(2) for 2 h. Of these, five rats were killed at 24 h later; the remaining five rats were immediately exposed to 80% O(2) for 2 h and then killed 24 h later. Double immunofluorescence was carried out between nestin and glutamine synthetase (GS) and between glial fibrilary acidic proteins (GFAP) and GS in normal and pathological retinae. Enhanced nestin expression was observed in reactive astrocytes following hypoxia treatment as revealed in whole mount sections. A novel finding was the induction of nestin expression in Müller glial cells. Remarkably, the nestin immunostaining was downregulated to levels comparable to those of the normal rats with immediate hyperoxia treatment. Induced nestin expression by hypoxia colabelled with GFAP in astrocytes, however, remained unaffected after hyperoxia treatment. The induced expression of nestin in Müller glial cells and astrocytes in hypoxia and differential downregulation after hyperoxia treatment suggest a structural plasticity of the cytoskeletal framework of these cells. The differential response after hyperoxia treatment may be related to the functional states of the cells.

Guinea pigs reared in a monochromatic environment exhibit changes in cone density and opsin expression.

This study aimed to determine if a monochromatic environment will affect the development of cones in a guinea pig model. Thirty 3-day-old guinea pigs were randomized into three groups and exposed to green, violet, and white light (control) for 8 weeks. The animals were sacrificed and the density of middle-wavelength cones (M cones) and short-wavelength sensitive (S cones) and expression of M-opsin and S-opsin were determined. The density of M cones was increased in the green light group as compared to the control group, and decreased in the violet light group as compared to the control group (both, p < 0.05). There was no significant difference in the density of the S cones among the groups (all, p > 0.05). The density of coexpressing cones in the middle retina was significantly increased in the green light group in comparison to the violet light group (p < 0.01). In addition, there was a significant increase in the level of M-opsin as determined by Western blotting and M-opsin mRNA expression as determined by PCR analysis in the green light group as compared to the control group and a significant decrease in violet light group as compared to the control group (all, p < 0.05). No significant difference in S-opsin level or S-opsin mRNA expression was noted among the groups. We concluded that monochromatic lighting affected the density of cones and expression of opsins in a guinea pig model, and this indicates that the retinal color visual system of the guinea pig possess developmental plasticity.

Ragweed pollen induces allergic conjunctivitis immune tolerance in mice via regulation of the NF-κB signal pathway.

AIM: To investigate the feasibility and mechanism of immune tolerance in allergic conjunctivitis. METHODS: The allergic conjunctivitis immune tolerance mice model was established by ragweed pollen (RW) and the related cytokines were detected. The mice were divided into 9 groups and the maslinic acid (MA) or PBS were given for different group after modeling. The expression levels of chemokine ligand 5 (CCL5) and P-65 in the conjunctival tissue were analyzed by immunohistochemistry, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. The percentage of interleukin-17 (IL-17) and CD4+CD25+ in the splenocyte supernatant was analyzed by flow cytometry. Furthermore, the serum and splenocyte supernatant concentration of total-IgE, interleukin-10 (IL-10), and IL-17 was analyzed by enzyme linked immune response (ELISA). RESULTS: After the model was established, symptoms of conjunctivitis were alleviated, the level of P-65, CCL5, IL-17, and total-IgE was raised, while the expression of IL-10, CD4+CD25+ was decreased. This result fully demonstrated that a typical IL-17/regulatory-T-cells (Treg cells) imbalance and NF-κB activation. When the NF-κB signal pathway was suppressed, it showed that there was a further relief of conjunctivitis in mice. At the same time, the expression of total-IgE, IL-17, and CCL5 was decreased and the expression of anti-inflammatory factor (IL-10, CD4+CD25+) was increased. CONCLUSION: In the state of immune tolerance, symptoms of conjunctivitis in mice are alleviated, the Th-17 cells of allergic conjunctivitis mice are inhibited, and Treg cells activity is enhanced.

RNA Biological Characteristics at the Peak of Cell Death in Different Hereditary Retinal Degeneration Mutants.

Purpose: The present work investigated changes in the gene expression, molecular mechanisms, and pathogenesis of inherited retinal degeneration (RD) in three different disease models, to identify predictive biomarkers for their varied phenotypes and to provide a better scientific basis for their diagnosis, treatment, and prevention. Methods: Differentially expressed genes (DEGs) between retinal tissue from RD mouse models obtained during the photoreceptor cell death peak period (Pde6b rd1 at post-natal (PN) day 13, Pde6b rd10 at PN23, Prph rd2 at PN29) and retinal tissue from C3H wild-type mice were identified using Illumina high-throughput RNA-sequencing. Co-expression gene modules were identified using a combination of GO and KEGG enrichment analyses and gene co-expression network analysis. CircRNA-miRNA-mRNA network interactions were studied by genome-wide circRNA screening. Results: Pde6b rd1 , Pde6b rd10 , and Prph rd2 mice had 1,926, 3,096, and 375 DEGs, respectively. Genes related to ion channels, stress, inflammatory processes, tumor necrosis factor (TNF) production, and microglial cell activation were up-regulated, while genes related to endoplasmic reticulum regulation, metabolism, and homeostasis were down-regulated. Differential expression of transcription factors and non-coding RNAs generally implicated in other human diseases was detected (e.g., glaucoma, diabetic retinopathy, and inherited retinal degeneration). CircRNA-miRNA-mRNA network analysis indicated that these factors may be involved in photoreceptor cell death. Moreover, excessive cGMP accumulation causes photoreceptor cell death, and cGMP-related genes were generally affected by different pathogenic gene mutations. Conclusion: We screened genes and pathways related to photoreceptor cell death. Additionally, up-stream regulatory factors, such as transcription factors and non-coding RNA and their interaction networks were analyzed. Furthermore, RNAs involved in RD were functionally annotated. Overall, this study lays a foundation for future studies on photoreceptor cell death mechanisms.