Jiawen Fan

High-salt loading exacerbates increased retinal content of aquaporins AQP1 and AQP4 in rats with diabetic retinopathy

In the neural retina, glial cells control formation of ionic gradients by mediating transmembrane water fluxes through aquaporin (AQP) water channels. Retinal content and immunolocalization of two water channels, AQP1 and AQP4, in the diabetic rat retinas during high-salt loading were examined in this study. Diabetes was induced by an intraperitoneal injection of streptozotocin. Diabetic and control animals were observed after varying lengths of exposure to normal- and high-salt conditions. Ultrathin sections of retinal tissue, stained with uranyl acetate and lead citrate, were photographed using a transmission electron microscope (TEM). Retinal wholemounts were immunostained with AQP1 and AQP4 antibody to detect the immunolocalization changes by confocal microscopy. AQP1 and AQP4 content were evaluated by Western blot analysis. In the retinas of high-salt loading diabetic animals, obviously increased intracellular edema was observed by TEM in ganglion cells and mitochondrial swelling was observed in glial cells. Immunolocalization of AQP1 increased from the posterior to peripheral retina. Western blot results indicated that a high-salt diet may cause increased retinal content of AQP4 and may exacerbate increased retinal content of AQP1 caused by diabetic retinopathy. High-salt loading may increase neural retinal edema in rats with diabetic retinopathy, and altered glial cell mediated water transport via AQP channels in the retina may play an important role in the neural retinal edema formation and resolution.

Ras Homolog Enriched in the Brain is Linked to Retinal Ganglion Cell Apoptosis after Light Injury in Rats

Ras homolog enriched in the brain (Rheb) is a small GTPase of the Ras family. It has been confirmed that Rheb activation not only regulates cell growth and migration but also induces neuron apoptosis after toxic stimuli. However, the function of Rheb in the retina is still not fully understood. To find out whether Rheb was involved in retinal neuron death, the expression profile of Rheb in light-damaged retinal ganglion cells (RGCs) of adult rats was investigated. Western blotting showed the expression of Rheb was significantly upregulated in the injured retina. Rheb was mainly detected in apoptotic RGCs by using double immunofluorescent staining. Active caspase-3 was upregulated and co-labeled with Rheb. Meanwhile, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that Rheb-positive RGCs underwent apoptosis after light exposure, which suggested that Rheb might be relevant to RGC apoptosis following phototoxicity. Furthermore, Western blotting and immunofluorescence showed that the expression profiles of CyclinD1 and cyclin-dependent kinase 4 (CDK4) were parallel with that of Rheb in a time–space dependent manner. Based on this study, it is speculated that Rheb might play an important role in physiological and pathological process in light-induced retina damage, which might provide a potential therapeutic avenue of retinal degeneration.

High salt loading alters the expression and localization of glial aquaporins in rat retina.

In the neural retina, glial cells control the ionic concentrations in part by mediation of transmembrane water fluxes through aquaporin (AQP) water channels. The expression and immunolocalization of two water channels, AQP1 and AQP4, in the rat retina during experimental high salt loading were investigated in this study. Six-week-old Wistar rats were allowed free access to rat chow with 8% NaCl concentration. Of these rats, 6 were killed after 2, 6, 10 and 20 weeks. Twelve-week-old and 26-week-old Wistar rats with a normal diet (0.5% NaCl concentration) were used as controls. Retinal tissues were collected. Ultrathin sections stained with uranyl acetate and lead citrate were photographed using a transmission electron microscope (TEM). Retinal whole mounts and cryosections were immunostained with AQP1 and AQP4 antibodies to detect the immunolocalization changes by confocal microscopy. The AQP1 and AQP4 contents were evaluated by western blot analysis. In control tissues, no intracellular edema and mitochondria swelling were observed by TEM. The immunoreactive AQP4 was expressed by glial cells (Müller cells and astrocytes) predominantly in the inner retina, and AQP1 was expressed in the outer retina. In the retinas of high salt loading animals, obvious intracellular edema was observed by TEM in retinal ganglion cell (RGC) and mitochondria swelling was observed in glial cells. Strong expression of AQP1 was found in glial cells located in the innermost retinal layers, mainly in astrocytes. The superficial retinal vessels were surrounded by AQP4 in control retinas, but by both AQP4 and AQP1 in retina of high salt loading animals. A similar alteration in the localization of AQP1 has been described in the rat retina after transient ischemia and diabetes. Western blot results supported the conclusion that the AQP1 expression increased during high salt diet. Our findings indicate that high salt loading may induce neural retina edema, and that altered glial cell-mediated water transport via AQP channels in the retina may be one of the reasons for intracellular edema in the neural retina.

Aquaporin Changes during Diabetic Retinopathy in Rats Are Accelerated by Systemic Hypertension and Are Linked to the Renin-Angiotensin System

PURPOSE We explored the relationship between the renin-angiotensin system (RAS) and aquaporins (AQP1 and AQP4 in Müller glia and astrocytes) in diabetic retinopathy (DR) with and without systemic hypertension. METHODS Diabetes was induced in spontaneously hypertensive rats (SHR) and normotensive control Wistar Kyoto (WKY) rats by intraperitoneal injections of streptozotocin. The diabetic and control non-diabetic rats were assigned randomly to receive no anti-hypertension treatment, or to be treated with the angiotensin II receptor blocker (ARB), valsartan (40 mg/kg/d) or the beta-blocker, metoprolol (50 mg/kg/day). Eight weeks later, retinas were evaluated by immunohistochemistry and Western blot to detect changes in the expression of AQP1, AQP4, and glial fibrillary acidic protein (GFAP). RESULTS Hypertension increased expression of glial GFAP and AQP4 (P < 0.01), but not AQP1 (P > 0.05) in diabetic rats. Valsartan and metoprolol decreased GFAP, AQP1, and AQP4 expression in diabetic SHR rats (P < 0.01). Valsartan decreased GFAP and AQP1 expression in diabetic WKY rats (P < 0.01), while metoprolol did not. CONCLUSIONS Activation of Müller glia and astrocytes was involved in the mechanism by which systemic hypertension affects DR. AQPs and macroglia were linked to changes in the RAS in DR. Changes in aquaporin expression in DR were increased by hypertension. This provides additional support for the early use of an ARB in the treatment of DR, especially in cases with retinal edema.

Gene expression changes under cyclic mechanical stretching in rat retinal glial (Müller) cells

Objective The retina is subjected to tractional forces in various conditions. As the predominant glial element in the retina, Müller cells are active players in all forms of retinal injury and disease. In this study, we aim to identify patterns of gene expression changes induced by cyclic mechanical stretching in Müller cells. Methods Rat Müller cells were seeded onto flexible bottom culture plates and subjected to a cyclic stretching regimen of 15% equibiaxial stretching for 1 and 24 h. RNA was extracted and amplified, labeled, and hybridized to rat genome microarrays. The expression profiles were analyzed using GeneSpring software, and gene ontology analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to select, annotate, and visualize genes by function and pathway. The selected genes of interest were further validated by Quantitative Real-time PCR (qPCR). Results Microarray data analysis showed that at 1 and 24 h, the expression of 532 and 991 genes in the Müller cells significantly (t-test, p<0.05) differed between the mechanically stretched and unstretched groups. Of these genes, 56 genes at 1 h and 62 genes at 24 h showed more than a twofold change in expression. Several genes related to response to stimulus (e.g., Egr2, IL6), cell proliferation (e.g., Areg, Atf3), tissue remodeling (e.g., PVR, Loxl2), and vasculogenesis (e.g., Epha2, Nrn1) were selected and validated by qPCR. KEGG pathway analysis showed significant changes in MAPK signaling at both time points. Conclusions Cyclic mechanical strain induces extensive changes in the gene expression in Müller cells through multiple molecular pathways. These results indicate the complex mechanoresponsive nature of Müller cells, and they provide novel insights into possible molecular mechanisms that would account for many retinal diseases in which the retina is often subjected to mechanical forces, such as pathological myopia and proliferative vitreoretinopathy.

Mechanical stretching induces matrix metalloproteinase-2 expression in rat retinal glial (Müller) cells.

Pathological myopia, as one of the leading causes of blindness, is characterized by excessive and progressive elongation of the eyeball with concomitant degenerative changes in the posterior segment of the eye. During the progressive distension of the posterior pole, the retina, choroid, and sclera are subjected to constant mechanical force, as a result of which, tissue remodeling occurs. Active remodeling of the sclera in myopia has been studied intensively. By comparison, retinal remodeling under mechanical stretching has attracted little attention, and further research is therefore required. In this study, we showed that constant mechanical stretching of rat retinal Müller cells for 24 h led to a significant increase in the intracellular matrix metalloproteinase-2 mRNA and protein levels. The extracellular secretory matrix metalloproteinase-2 protein levels and activity were also enhanced. These results suggest a possible novel molecular mechanism that would account for retinal remodeling in many ocular diseases in which the retina is often overstretched, such as pathological myopia and proliferative vitreoretinopathy.

Naloxone Inhibition of Lipopolysaccharide-Induced Activation of Retinal Microglia is Partly Mediated via the p38 Mitogen Activated Protein Kinase Signalling Pathway

OBJECTIVES: To investigate the effects and underlying mechanism of action of naloxone on lipopolysaccharide (LPS)-induced activation of retinal microglia in vitro. METHODS: Rat retinal microglia primary cultures were divided into four treatment groups: untreated; 1 μg/ml LPS for 12 h; 0.5, 1.0 or 2.0 μM naloxone for 30 min before LPS; 2.5 or 5.0 μM SB203580 for 12 h before LPS and naloxone. Levels of tumour necrosis factor (TNF)-α and interleukin (IL)-1β were determined by enzyme-linked immuno sorbent assay. Western blot analysis and double immunofluorescence were used to examine activation of the mitogen activated protein kinase (MAPK) signalling pathway. RESULTS: LPS induced an increase in TNF-α and IL-1β in culture supernatants, which was dose-dependently inhibited by naloxone. Naloxone also dose-dependently inhibited LPS-induced increases in phosphorylated p38 MAPK. Pretreatment of cells with SB203580 attenuated the inhibitory effect of naloxone on TNF-α and IL-1β production. CONCLUSIONS: Naloxone suppressed LPS-induced activation of cultured retinal microglia and this suppression appeared to occur partly through the p38 MAPK signalling pathway. Naloxone may have therapeutic potential in neurodegenerative diseases characterized by the activation of microglia.

Upregulation of SOX9 in Glial (Müller) cells in retinal light damage of rats.

Recent evidence suggests that SOX9 [sex-determining region Y (SRY) box 9], a transcription factor, plays a pivotal role in acquired diseases, revealing its importance in roles beyond development. However, whether SOX9, which is one of the key regulators of retinal Müller cell development, also participates in the pathological process of retinal degenerative diseases remains unknown. In the present study, we hypothesized that SOX9 was upregulated in Müller cells in retinal degeneration. Retinal light damage (LD) was used as a model for retinal degeneration. On day 3, 7, 14, 21, and 28 after LD in adult Sprague Dawley (SD) rats, the spatial distribution of SOX9 in the retina was observed by immunohistochemistry; the expression levels of SOX9 were measured by real-time PCR and Western blot analysis. Moreover, type 1 collagen (COL1) and cone-rod homeobox (CRX) protein levels, which are two downstream targets of SOX9, were also assessed by Western blot analysis. Colabeling for SOX9 and glutamine synthetase (GS), a specific Müller cell maker, indicated that SOX9 was expressed in the Müller cell nucleus in both control and LD groups. Significantly enhanced SOX9 expression was observed as early as day 3 after LD, and it persisted for at least 28 days. COL1 and CRX protein levels also increased after LD. Our study demonstrates the involvement of SOX9 in acquired retinal degeneration triggered by LD, which might provide novel insights into possible molecular mechanisms that would account for the involvement of Müller cells in retinal degenerative diseases.

Wogonin induces retinal neuron-like differentiation of bone marrow stem cells by inhibiting Notch-1 signaling

Age-related macular degeneration and retinitis pigmentosa are major causes of irreversible vision loss in the elderly and, despite sustained efforts, current treatments are largely ineffective. Wogonin is a bioactive plant flavonoid possessing a range of beneficial properties, including neuroprotective effects. We investigated the ability of wogonin to promote retinal neuron-like differentiation of bone marrow stem cells (BMSCs) and assessed the involvement of Notch-1 signaling in this process. Cultured mouse BMSCs were left untreated or exposed to neurotrophic factors in the presence or absence of wogonin, and western blotting, RT-PCR and immunofluorescence were used to identify changes in molecular markers of stemness and neuroretinal differentiation. Proteins in the Notch-1 signaling pathway, a main negative regulator of neurogenesis, were also examined by western blotting. We found that expression of stem cell markers was reduced, while markers of mature retinal neurons, bipolar cells and photoreceptors were increased in wogonin-treated BMSCs. Wogonin also dose-dependently decreased expression of Notch-1 signaling proteins. Moreover, blockade of Notch-1 both mimicked and enhanced the effect of wogonin to facilitate BMSC differentiation into retinal neuron-like cells. Wogonin thus appears to promote retinal neuron-like differentiation of BMSCs by antagonizing the inhibitory actions of Notch-1 signaling on neurogenesis and may be useful in the treatment of retinal degenerative diseases.