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Purpose Dysregulation of the complement cascade contributes to a variety of retinal dystrophies, including age-related macular degeneration (AMD). The central component of complement, C3, is expressed in abundance by macrophages in the outer retina, and its ablation suppresses photoreceptor death in experimental photo-oxidative damage. Whether this also influences macrophage reactivity in this model system, however, is unknown. We investigate the effect of C3 ablation on macrophage activity and phagocytosis by outer retinal macrophages during photo-oxidative damage. Methods Age-matched C3 knockout (KO) mice and wild-type (WT) C57/Bl6 mice were subjected to photo-oxidative damage. Measurements of the outer nuclear layer (ONL) thickness and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess pathology and photoreceptor apoptosis, respectively. Macrophage abundance and phagocytosis were assessed with immunolabeling for pan-macrophage and phagocytic markers, in conjunction with TUNEL staining in cohorts of C3 KO and WT mice. Results The C3 KO mice exhibited protection against photoreceptor cell death following photo-oxidative damage, which was associated with a reduction in immunoreactivity for the stress-related factor GFAP. In conjunction, there was a reduction in IBA1-positive macrophages in the outer retina compared to the WT mice and a decrease in the number of CD68-positive cells in the outer nuclear layer and the subretinal space. In addition, the engulfment of TUNEL-positive and -negative photoreceptors by macrophages was significantly lower in the C3 KO mice cohort following photo-oxidative damage compared to the WT cohort. Conclusions The results show that the absence of C3 mitigates the phagocytosis of photoreceptors by macrophages in the outer retina, and the net impact of C3 depletion is neuroprotective in the context of photo-oxidative damage. These data improve our understanding of the impact of C3 inhibition in subretinal inflammation and inform the development of treatments for targeting complement activation in diseases such as AMD.

Purpose: Given the role of corneal sensory nerves during epithelial wound repair, we sought to examine the relationship between immune cells and polymodal nociceptors following corneal injury. Methods: Young C57BL/6J mice received a 2 mm corneal epithelial injury. One week later, corneal wholemounts were immunostained using β-tubulin-488, TRPV1 (transient receptor potential ion channel subfamily V member-1, a nonselective cation channel) and immune cell (MHC-II, CD45 and CD68) antibodies. The sum length of TRPV1+ and TRPV1- nerve fibers, and their spatial association with immune cells, was quantified in intact and injured corneas. Results: TRPV1+ nerves account for ∼40% of the nerve fiber length in the intact corneal epithelium and ∼80% in the stroma. In the superficial epithelial layers, TRPV1+ nerve terminal length was similar in injured and intact corneas. In intact corneas, the density (sum length) of basal epithelial TRPV1+ and TRPV1- nerve fibers was similar, however, in injured corneas, TRPV1+ nerve density was higher compared to TRPV1- nerves. The degree of physical association between TRPV1+ nerves and intraepithelial CD45+ MHC-II+ CD11c+ cells was similar in intact and injured corneas. Stromal leukocytes co-expressed TRPV1, which was partially localized to CD68+ lysosomes, and this expression pattern was lower in injured corneas. Conclusions: TRPV1+ nerves accounted for a higher proportion of corneal nerves after injury, which may provide insights into the pathophysiology of neuropathic pain following corneal trauma. The close interactions of TRPV1+ nerves with intraepithelial immune cells and expression of TRPV1 by stromal macrophages provide evidence of neuroimmune interactions in the cornea.

Purpose: The purpose of this study was to assess the morphological and phenotypic responses of corneal epithelial dendritic cells (DCs) to acute topical hyperosmolar stress, given a pathogenic role for tear hyperosmolarity in dry eye disease (DED). Methods: C57BL/6J mice were anesthetized and received 350 mOsm/L (physiological; n = 5 mice), 450 mOsm/L (n = 6), or 600 mOsm/L (n = 6) saline on a randomly assigned eye. Corneas were harvested 2 hours later. Immunofluorescent staining was performed using CD45, CD86, and CD68 antibodies to investigate DC morphology (density, viability, field area, circularity, and dendritic complexity) and immunological phenotype. Flow cytometry was used to confirm CD86 and CD68 expression in CD11c+ DCs, using C57BL/6J mice that received topical applications of 350 mOsm/L, 450 mOsm/L, or 600 mOsm/L (n = 5 per group) bilaterally for 2 hours. Results: Following exposure to 450 mOsm/L topical saline for 2 hours, DCs in the central and peripheral cornea were larger (field area: Pcentral = 0.005, Pperipheral = 0.037; circularity: Pcentral = 0.026, and Pperipheral = 0.013) and had higher expression of CD86 compared with 350 mOsm/L controls (immunofluorescence: P < 0.0001; flow cytometry: P = 0.0058). After application of 600 mOsm/L saline, DC morphology was unchanged, although the percentage of fragmented DCs, and phenotypic expression of CD86 (immunofluorescence: P < 0.0001; and flow cytometry: P = 0.003) and CD68 (immunofluorescence: P = 0.024) were higher compared to 350 mOsm/L controls. Conclusions: Short-term exposure to mild hyperosmolar saline (450 mOsm/L) induced morphological and phenotypic maturation in corneal epithelial DCs. More severe hyperosmolar insult (600 mOsm/L) for 2 hours appeared toxic to these cells. These data suggest that hyperosmolar conditions activate corneal DCs, which may have implications for understanding DC activation in DED.

Additional file 1: Figure S1. Experiment 2: CD45+ dendritic cells (DCs) after 6-hours of topical treatment (i.e., 3 × doses, 2 hours apart) in CD11c-eYFP mice. (A) CD45+ CD11c+ DCs in peripheral cornea after decorin treatment. (B) CD45+ CD11c+ DCs in peripheral cornea after saline treatment. Scale bar (50 μm) applies to all images. Figure S2. Experiment 1: Initial corneal abrasion area at baseline (Time 0h). Figure S3. Experiment 1: Corneal thickness and epithelial cell proliferation after 1-week of topical treatment, dosed 3 times/day. (A) Representative SD-OCT images of the anterior segment from naïve mice. Blue curved line represents the area of central cornea and blue dashed line represents peripheral cornea. Scale bar is 200 μm. (B) A higher magnification image of the boxed area in A. Orange and green double arrows indicate epithelial and stromal thickness respectively. (C) Corneal epithelial and stromal thickness. There were no significant inter-group differences. (D) Representative en face confocal image of Ki67 staining in the central cornea after 1-week treatment of decorin. Scale bar is 50 μm. (E-F) Density of proliferative epithelial cells in the central and peripheral cornea. Summary data are shown as mean ± SD. Each data point represents one cornea. Red symbols represent the contralateral eye of the decorin-treated eye. Legend: DCN, decorin; Gel, fluid gel. Figure S4. Experiment 2: Corneal re-epithelialisation and stromal thickness after 6-hours of topical treatment (i.e., 3 × doses, 2 hours apart). (A) Representative en face OCT image at baseline (0h after abrasion). (B) Representative en face OCT image after 6-hours of treatment. Red dashed lines in panels A and B indicate the margin of the injured epithelium. Scale bar in B is 0.5 mm. (C) Percentage of re-epithelialised corneal area after 6-hours of treatment. (D) Corneal stromal thicknesses after 6-hours of treatment. Red symbols in panels C and D represent the contralateral eye of the decorin-treated eye. Summary data are shown as mean ± SD. Each data point represents one cornea. P-values for each of the inter-group comparisons are provided in Table 2. Legend: DCN, decorin; Gel, fluid gel. Figure S5. Experiment 4: Effect of topical decorin applied before injury (DCN) on corneal immune cells and nerve regeneration. (A-B) Density of DCs in the central and peripheral corneal epithelium after topical application of prophylactic decorin on intact corneas. (C-D) Sum length of the SNTs and SBNP in the central cornea, at 1 week after prophylactic application of decorin. (E-F) Density of DCs in the peripheral epithelium and macrophages in the central stroma, at 1 week after prophylactic application of topical decorin. Summary data are shown as mean ± SD. * indicates a statistically significant difference between saline-treated and decorin-treated eyes. Each data point represents one cornea. Legend: DC, dendritic cell; DCN, decorin; SBNP, sub-basal nerve plexus; SNT, superficial nerve terminal; WT, wild-type. Figure S6 (A) Comparison of initial abrasion area between Experiment 1 and 3. (B) Relationship between the initial abrasion area and the re-epithelialised area at 6h.

Light-induced degeneration in rodent retinas is an established model for of retinal degeneration, including the roles of oxidative stress and neuroinflammatory activity. In these models, photoreceptor death is elicited via photo-oxidative stress, and is exacerbated by recruitment of subretinal macrophages and activation of immune pathways including complement propagation. Existing light damage models have relied heavily on albino rodents, and mostly using acute light stimuli. These albino models have proven valuable in uncovering the pathogenic mechanisms of such pathways in the context of retinal disease. However, their inherent albinism hinders comparability to normal retinal physiology, and also makes gene technology analysis time-consuming due to the predominance of the pigmented mouse strains in these applications. In this study, we characterise a new light damage model utilising C57BL/6J mice over a 7 day period of chronic light exposure. We use high-efficiency LED technology to deliver a sustained intensity of 100 k lux with negligible modulation of ambient temperature. We show that in the C57BL/6J mouse, chronic light exposure elicits the cardinal features of light damage including photoreceptor degeneration, atrophy of the choriocapillaris, decreased retinal function and increases in oxidative stress markers 4-HNE and 8-OHG, which emerge progressively over the 7 day period of exposure. These changes are accompanied by robust recruitment of IBA1+ and F4/80 + microglia/macrophages to the ONL and subretinal space, followed the strong up-regulation of monocyte-chemoattractants Ccl2, Ccl3, and Ccl12, as well as increases in expression of complement component C3. These findings are in agreement with prior damage models conducted in albino rodents such as Balb/c mice, and support the use of this new model in further investigating the causative features of oxidative stress and inflammation in retinal disease.

International audience; Purpose: Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between obesity and retinal inflammation has yet to be assessed. We used the leptin-deficient (ob/ob) mouse to further our understanding of the contribution of obesity to retinal oxidative stress and inflammation.Methods: Retinas from ob/ob mice were compared to age-matched wild-type controls for retinal function (electroretinography) and gene expression analysis of retinal stress (), oxidative stress ( and ), and complement activation (, , , and ). Oxidative stress was further quantified using a reactive oxygen species and reactive nitrogen species (ROS and RNS) assay. Retinal microglia and macrophage migration to the outer retina and complement activation were determined using immunohistochemistry for IBA1 and C3, respectively. Retinas and sera were used for metabolomic analysis using QTRAP mass spectrometry.Results: Retinal function was reduced in ob/ob mice, which correlated to changes in markers of retinal stress, oxidative stress, and inflammation. An increase in C3-expressing microglia and macrophages was detected in the outer retinas of the ob/ob mice, while gene expression studies showed increases in the complement activators ( and ) and a decrease in a complement regulator (). The expression of several metabolites were altered in the ob/ob mice compared to the controls, with changes in polyunsaturated fatty acids (PUFAs) and branched-chain amino acids (BCAAs) detected.Conclusions: The results of this study indicate that oxidative stress, inflammation, complement activation, and lipid metabolites in the retinal environment are linked with obesity in ob/ob animals. Understanding the interplay between these components in the retina in obesity will help inform risk factor analysis for acquired retinal degenerations, including AMD.

Muller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Muller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1β was analysed in retinas. In vitro, primary Muller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Muller cells only, and Muller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Muller cells. Following PD, 670 nm light-treated Muller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1β and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Muller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Muller cell gene expression. Our data indicates that 670 nm light reduces Muller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations.

Introduction Light is the primary stimulus for vision, but may also cause damage to the retina. Pre-exposing the retina to sub-lethal amount of light (or preconditioning) improves chances for retinal cells to survive acute damaging light stress. Objectives This study aims at exploring the changes in retinal metabolome after mild light stress and identifying mechanisms that may be involved in preconditioning. Methods Retinas from 12 rats exposed to mild light stress (1000 lux × for 12 h) and 12 controls were collected one and seven days after light stress (LS). One retina was used for targeted metabolomics analysis using the Biocrates p180 kit while the fellow retina was used for histological and immunohistochemistry analysis. Results Immunohistochemistry confirmed that in this experiment, a mild LS with retinal immune response and minimal photoreceptor loss occurred. Compared to controls, LS induced an increased concentration in phosphatidylcholines. The concentration in some amino acids and biogenic amines, particularly those related to the nitric oxide pathway (like asymmetric dimethylarginine (ADMA), arginine and citrulline) also increased 1 day after LS. 7 days after LS, the concentration in two sphingomyelins and phenylethylamine was found to be higher. We further found that in controls, retina metabolome was different between males and females: male retinas had an increased concentration in tyrosine, acetyl-ornithine, phosphatidylcholines and (acyl)-carnitines. Conclusions Besides retinal sexual metabolic dimorphism, this study shows that preconditioning is mostly associated with re-organisation of lipid metabolism and changes in amino acid composition, likely reflecting the involvement of arginine-dependent NO signalling. Electronic supplementary material The online version of this article (doi:10.1007/s11306-016-1156-9) contains supplementary material, which is available to authorized users.

Purpose Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1β, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1β, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.