Abstract Purpose Glaucoma is a group of neurodegenerative diseases characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Mitochondrial dynamics contribute to the maintenance of neuronal homeostasis, and abnormalities can lead to the development of glaucoma. SS31 is a mitochondria-targeting short peptide that has been found to protect mitochondrial function and thereby protect damaged retina-associated cells. The aim of this study is to investigate whether SS31 protects damaged retinal cells by activating the AMPK/DRP1 pathway. Methods The relationship between SS31 and oxidative stress was determined by live-cell imaging. The cells were inoculated in the well plate and treated with H2O2 (150 μM) for 24 h and pretreated with or without SS31 (100 nM), AICAR (17.5 μM), or dorsomorphin (7 μM) for 2 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were used to evaluate cell viability. Mitochondrial genetic stability was detected by droplet digital PCR. The expression of AMPK and DRP1 was detected by western blot. In addition, intraocular pressure and retinal thickness were evaluated in mice with acute ocular hypertension. Results SS31 inhibited H2O2-induced cell death of 661W through the AMPK signaling pathway and maintained mitochondrial genetic stability. SS31 regulates AMPK/Drp1 signaling in 661W cells after oxidative stress. SS31 reduced intraocular pressure and maintained normal retinal thickness in mice with acute ocular hypertension. Conclusions SS31 regulates mitochondrial dynamic network remodeling by activating AMPK/DRP1 signaling pathway and protects retinal-related cells.
Abstract Purpose To investigate the potential protective effects of fluvoxamine (FVX), a selective serotonin reuptake inhibitor with known anti-inflammatory properties, on corneal, limbal, and retinal tissues in a rat model of endotoxin-induced uveitis (EIU), clinical and histopathologic evaluations were performed. Methods Twenty-eight female Wistar Albino rats were divided into four groups: controls, EIU, EIU + FVX, and FVX (n = 7 per group). EIU was induced by a single intraperitoneal injection of lipopolysaccharide (LPS). FVX or normal saline (NS) was administered 30 min before LPS or NS injection. Clinical ocular inflammation was assessed using slit-lamp biomicroscopy 6 h postinjection. Following euthanasia, enucleated eyes were processed for histopathologic analysis of corneal, limbal, and retinal tissues. Results Total corneal and epithelial thickness were measured, while limbal and retinal lesions were semiquantitatively graded. The EIU group exhibited significantly increased clinical ocular inflammation scores compared to the control, EIU + FVX, and FVX groups (p < 0.0001 for all). At the microscopical analysis, the EIU group showed significant corneal epithelial thickening, cellular changes, edema, hemorrhage, and increased total corneal thickness compared to the control group (p < 0.001). In addition, histopathologic analysis revealed retinal hyperemia and edema in the EIU group but not in the EIU + FVX, FVX, or control groups. Histopathologic scores confirmed significant retinal changes in the EIU group compared to the others (p < 0.001). Conclusions FVX significantly reduced clinical signs of ocular inflammation and mitigated histopathologic damage in the cornea, limbus, and retina.
Abstract Purpose Both photoreceptors and the retinal pigment epithelium (RPE) are highly metabolically active cell types that require extensive molecular transport and adequate energy production to maintain their function. We tested the hypothesis that cargo adaptor protein Bicdl1 has a non-redundant role in retinal health. Methods Using an unbiased forward genetics approach, we identified an association between outer retinal thinning and a point mutation in Bicdl1. We generated Bicdl1 KI (knock-in) mice carrying the same point mutation identified in the mutagenesis pipeline and characterized the resulting retinal phenotype using optical coherence tomography (OCT), fundus photography, histology, immunofluorescence, electroretinography, electron microscopy (EM), and transcriptomics. Results Analysis of OCT images and histology sections demonstrated a mild decrease in outer retinal thickness and rod outer segment thickness. We found an increased number of fundus spots in retinal photographs and an increased number of Iba1+/Tmem119+ cells on RPE flat mounts of Bicdl1 KI mice. Electroretinography in aging mice showed a mild impairment in visual function, reflected by a reduction in a-wave amplitudes. EM revealed fewer RPE mitochondria in Bicdl1 KI mice. To elucidate the mechanistic basis of the observed retinal degeneration, transcriptomic studies were conducted, which suggested an association between low Bicdl1 expression and increased oxidative phosphorylation in photoreceptors. Conclusion Despite the expression of many cargo adaptor proteins and dynein activators in the retina, we identified a novel non-redundant role of Bicdl1 in retinal homeostasis. Pathway analysis of transcriptomic data suggested that Bicdl1 deficiency may affect energy metabolism.
Abstract Diabetic retinopathy (DR) is a leading cause of blindness in the working population of the Western world. Advances in our understanding of specific variables contributing to this condition is restricted by the lack of characterization available with in vitro model systems. In this study we establish a retinal porcine model and investigate the effect of hyperglycemia on the level of hypoxia inducible factor type 1 alpha in the porcine retina. Porcine retinae were excised from porcine eyes and cultured in 5 or 25 mM D-glucose in Dulbecco’s Minimal essential Media (DMEM), without a matrix (2D) or within a matrix (3D) consisting of a co-gel (0.5% agarose plus collagen 0.25mg/ml) in DMEM and maintained in a humidified atmosphere with 5% CO2 at 37 °C. This process took 30 min. Retinal integrity was monitored by visualization with a light microscope followed by hematoxylin and eosin staining and Gomori Trichrome. Matrix oxygen saturation (% O2) was recorded with a Microx TX3 oxygen microsensor at time zero and 24 h following incubation. Immunostaining for HIF-1α was performed using primary mouse anti-HIF-1α (1:20) followed by anti-mouse DyLight 488 goat secondary antibody (1:200) and visualized with an inverted fluorescent microscope. Retinal tissue and vessel integrity were morphologically preserved on 3D matrices versus 2D control evidenced by hematoxylin and eosin staining. A time dependent increase in collagen incorporation within the explant was seen with the Gomori Trichrome stain. Oxygen levels significantly increased within the explant during the 23h test period from 15.5% to 22.3% (p<0.001) in media alone and from 10% to 13% (p<0.05) with the matrix (n=6) with all values relative to atmospheric pressure. HIF-1α was detected by immune-histochemical analysis in explants at t=0 in 5 and 25mM glucose concentration, with staining of serial sections with DAPi. HIF-1α levels only remained elevated at 24 h in 25 mM D-glucose and was undetected in 5 mM D-glucose. This study presents a novel model of retinal explants demonstrating that retinal tissue can be cultured in non-hypoxic conditions and that the 3-dimensional matrix was able to sustain retinal structure. Retinal structure was, at least partially sustained by the incorporation of collagen into the retina. Differential expression of protein was identified in the different matrix environment; specifically, hyperglycemia sustained non-hypoxia induced HIF-1α expression in isolated retina.
Abstract Purpose Inflammatory processes in the aged retina may be exacerbated by systemic infection with viruses and bacteria, potentially aggravating age-related macular degeneration (AMD). Toll-like receptors (TLR) are key mediators in detecting pathogen-associated molecular patterns (PAMPs) and host-derived damage-associated molecular patterns (DAMPs). This study aimed to investigate TLR gene expression in cultured retinal pigment epithelial (RPE) cells, focusing particularly on TLR9. Methods TLR gene expression was assessed using semiquantitative qPCR, while protein expression was evaluated through western blotting, immunocytochemistry, and ELISA. Hypoxic conditions were simulated using CoCl2 or by incubation in a 0.1% O2 atmosphere. RPE cell proliferation and viability were examined using bromodeoxyuridine (BrdU) and MTT assays. Cell necrosis and apoptosis were analyzed with a cellular DNA fragmentation ELISA. Results Among the TLRs analyzed, TLR9 showed the most pronounced upregulation under hypoxic conditions. TLR9 protein expression was similarly induced by CoCl2 as well as in a 0.1% O2 atmosphere. Hypoxia increased the protein levels of γH2A.X, a marker for DNA damage and cellular senescence. The TLR9 agonist ODN 1826 induced the gene expression of downstream signaling molecules (MYD88, IRF7, RELA), inflammatory factors (IFNA, COX2), and MMP9 as well as the secretion of TGF-β1. These effects were reversed by TLR9 inhibitors (ODN 2088 and ODN INH-18). Transforming growth factor (TGF)-β1 significantly suppressed the TLR9 expression at both the mRNA and protein levels. This suppressive effect was reversed at the RNA level by inhibiting components of the TGF-β1 signaling pathway, including activin receptor-like kinase (ALK), Smad3, p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and phosphatidylinositol 3-kinase (PI3K) under hypoxic conditions. Conclusions TLR9 activation under hypoxic conditions initiates pro-inflammatory pathways in RPE cells. In the aging retina, viral and bacterial infections and/or DAMPs may further amplify these responses, worsening retinal degeneration. TGF-β1 may exert protective, anti-inflammatory effects by downregulating TLR9 expression.
