Anti-inflammatory and neuroprotective properties of the corticosteroid fludrocortisone in retinal degeneration.

The pathogenesis of outer retinal degenerations has been linked to the elevation of cytokines that orchestrate pro-inflammatory responses within the retinal milieu, and which are thought to play a role in diseases such as geographic atrophy (GA), an advanced form of AMD. Here we sought investigate the anti-inflammatory and mechanistic properties of fludrocortisone (FA), as well as triamcinolone acetonide (TA), on Müller cell-mediated cytokine expression in response to inflammatory challenge. In addition, we investigated the neuroprotective efficacy of FA and TA in a photo-oxidative damage (PD), a model of outer retinal degeneration. Expression of CCL2, IL-6, and IL-8 with respect to FA and TA were assessed in Müller cells in vitro, following simulation with IL-1ß or TNF-α. The dependency of this effect on mineralocorticoid and glucocorticoid signaling was also interrogated for both TA and TA via co-incubation with steroid receptor antagonists. For the PD model, C57BL/6 mice were intravitreally injected with FA or TA, and changes in retinal pathology were assessed via electroretinogram (ERG) and optical coherence tomography (OCT). FA and TA were found to dramatically reduce the expression of CCL2, IL-6, and IL-8 in Müller glia in vitro after inflammatory challenge with IL-1ß or TNF-α (P < 0.05). Though FA acts as both a mineralocorticoid and glucocorticoid receptor agonist, co-incubation with selective steroid antagonists revealed that the suppressive effect of FA on CCL2, IL-6, and IL-8 expression is mediated by glucocorticoid signaling (P < 0.05). In PD, intravitreal FA was found to ameliorate outer-retinal atrophy as measured by ERG and OCT (P < 0.05), while TA had no significant effect (P > 0.05). Our data indicate potent anti-inflammatory and mechanistic properties of corticosteroids, specifically FA, in suppressing inflammation and neurodegeneration degeneration associated with outer retinal atrophy. Taken together, our findings indicate that corticosteroids such as FA may have value as a potential therapeutic for outer retinal degenerations where such pro-inflammatory factors are implicated, including AMD.

Ablation of C3 modulates macrophage reactivity in the outer retina during photo-oxidative damage.

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.

Preservation of the Foveal Avascular Zone in Achromatopsia Despite the Absence of a Fully Formed Pit.

Purpose: To examine the foveal avascular zone (FAZ) in patients with congenital achromatopsia (ACHM). Methods: Forty-two patients with genetically confirmed ACHM were imaged either with Optovue's AngioVue system or Zeiss's Plex Elite 9000, and the presence or absence of a FAZ was determined. For images where a FAZ was present and could be confidently segmented, FAZ area, circularity index, and roundness were measured and compared with previously published normative values. Structural optical coherence tomography images were acquired to assess the degree of foveal hypoplasia (number and thickness of inner retinal layers present at the fovea). Results: A FAZ was present in 31 of 42 patients imaged (74%), although no determination could be made for 11 patients due to poor image quality (26%). The mean ± SD FAZ area for the ACHM retina was 0.281 ± 0.112 mm2, which was not significantly different from the previously published normative values (P = 0.94). However, their FAZs had decreased circularity (P < 0.0001) and decreased roundness (P < 0.0001) compared to the normative cohort. In the patients with ACHM examined here, the FAZ area decreased as the number and thickness of the retained inner retinal layers increased. Conclusions: Our data demonstrate that despite the presence of foveal hypoplasia, patients with ACHM can have a FAZ. This is distinct from other conditions associated with foveal hypoplasia, which generally show an absence of the FAZ. In ACHM, FAZ formation does not appear to be sufficient for complete pit formation, contrary to some models of foveal development.

The Role of Pyruvate in Protecting 661W Photoreceptor-Like Cells Against Light-Induced Cell Death.

PURPOSE: Light is a requirement for the function of photoreceptors in visual processing. However, prolonged light exposure can be toxic to photoreceptors, leading to increased reactive oxygen species (ROS), lipid peroxidation, and photoreceptor cell death. We used the 661W mouse cone photoreceptor-like cell line to study the effects of pyruvate in protecting these cells from light-induced toxicity. METHODS: 661W cells were exposed to 15,000 lux continuous bright light for 5 hours and incubated in Dulbecco's modified eagle medium (DMEM) with various concentrations of pyruvate. Following light damage, cells were assessed for changes in morphology, cell toxicity, viability, and ROS production. Mitochondrial respiration and anaerobic glycolysis were also assessed using a Seahorse Xfe96 extracellular flux analyzer. RESULTS: We found that cell death caused by light damage in 661W cells was dramatically reduced in the presence of pyruvate. Cells with pyruvate-supplemented media also showed attenuation of oxidative stress and maintained normal levels of ATP. We also found that alterations in the concentrations of pyruvate had no effect on mitochondrial respiration or glycolysis in light-damaged cells. CONCLUSIONS: Taken together, the results show that pyruvate is protective against light damage but does not alter the metabolic output of the cells, indicating an alternative role for pyruvate in reducing oxidative stress. Thus, sodium pyruvate is a possible candidate for the treatment against the oxidative stress component of retinal degenerations.

A model of progressive photo-oxidative degeneration and inflammation in the pigmented C57BL/6J mouse retina.

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.

Role of Chemokines in Shaping Macrophage Activity in AMD.

Age-related macular degeneration (AMD) is a multifactorial disorder that affects millions of individuals worldwide. While the advent of anti-VEGF therapy has allowed for effective treatment of neovascular 'wet' AMD, no treatments are available to mitigate the more prevalent 'dry' forms of the disease. A role for inflammatory processes in the progression of AMD has emerged over a period of many years, particularly the characterisation of leukocyte infiltrates in AMD-affected eyes, as well as in animal models. This review focuses on the burgeoning understanding of chemokines in the retina, and their potential role in shaping the recruitment and activation of macrophages in AMD. Understanding the mechanisms which promote macrophage activity in the degenerating retina may be key to controlling the potentially devastating consequences of inflammation in diseases such as AMD.

Spatiotemporal Cadence of Macrophage Polarisation in a Model of Light-Induced Retinal Degeneration.

BACKGROUND: The recruitment of macrophages accompanies almost every pathogenic state of the retina, and their excessive activation in the subretinal space is thought to contribute to the progression of diseases including age-related macular degeneration. Previously, we have shown that macrophages aggregate in the outer retina following damage elicited by photo-oxidative stress, and that inhibition of their recruitment reduces photoreceptor death. Here, we look for functional insight into macrophage activity in this model through the spatiotemporal interplay of macrophage polarisation over the course of degeneration. METHODS: Rats were exposed to 1000 lux light damage (LD) for 24 hrs, with some left to recover for 3 and 7 days post-exposure. Expression and localisation of M1- and M2- macrophage markers was investigated in light-damaged retinas using qPCR, ELISA, flow cytometry, and immunohistochemistry. RESULTS: Expression of M1- (Ccl3, Il-6, Il-12, Il-1ß, TNFα) and M2- (CD206, Arg1, Igf1, Lyve1, Clec7a) related markers followed discrete profiles following light damage; up-regulation of M1 genes peaked at the early phase of cell death, while M2 genes generally exhibited more prolonged increases during the chronic phase. Moreover, Il-1ß and CD206 labelled accumulations of microglia/macrophages which differed in their morphological, temporal, and spatial characteristics following light damage. CONCLUSIONS: The data illustrate a dynamic shift in macrophage polarisation following light damage through a broad swathe of M1 and M2 markers. Pro-inflammatory M1 activation appears to dominate the early phase of degeneration while M2 responses appear to more heavily mark the chronic post-exposure period. While M1/M2 polarisation represents two extremes amongst a spectrum of macrophage activity, knowledge of their predominance offers insight into functional consequences of macrophage activity over the course of damage, which may inform the spatiotemporal employment of therapeutics in retinal disease.

Identification of miRNAs in a Model of Retinal Degenerations.

PURPOSE: We investigated the expression profile of and identify all microRNAs (miRNAs) that potentially regulate inflammation in a light-induced model of focal retinal degeneration. METHODS: Sprague Dawley (SD) rats aged 90 to 140 postnatal days were exposed to 1000 lux white fluorescent light for 24 hours. At 24 hours, and 3 and 7 days after exposure, the animals were euthanized and retinas processed for RNA. Expression of 750 miRNAs at 24 hours of exposure was assessed using low density array analysis. Significantly modulated miRNAs and their target mRNAs were used to assess the potential biological effects. Expression of seven miRNAs, potentially modulating inflammation, was investigated across a protracted time course after light exposure using quantitative PCR. Photoreceptor cell death was analyzed using TUNEL. RESULTS: Intense light exposure for 24 hours led to differential expression of a number of miRNAs, 37 of which were significantly modulated by 2-fold or more. Of those, 19 may potentially regulate the inflammatory immune response observed in the model. MicroRNAs -125-3p, -155, -207, -347, -449a, -351, and -542-3p are all upregulated at 24 hours of exposure along with peak photoreceptor cell death. The MiRNAs -542-3p and -351 reached maximum expression at 7 days after exposure, while -125-3p, -155, -207, -347, and -449 reached a peak expression at 3 days. CONCLUSIONS: The results of the study show that miRNAs are modulated in response to light damage (LD). These miRNAs potentially regulate the inflammatory immune response, triggered as a result of the acute retinal damage, which is a key mediator of retinal degeneration in this model and age-related macular degeneration.

Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium.

BACKGROUND: Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration. METHODS: Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR. RESULTS: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors. CONCLUSIONS: Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations.

Retinal microglia: just bystander or target for therapy?

Resident microglial cells can be regarded as the immunological watchdogs of the brain and the retina. They are active sensors of their neuronal microenvironment and rapidly respond to various insults with a morphological and functional transformation into reactive phagocytes. There is strong evidence from animal models and in situ analyses of human tissue that microglial reactivity is a common hallmark of various retinal degenerative and inflammatory diseases. These include rare hereditary retinopathies such as retinitis pigmentosa and X-linked juvenile retinoschisis but also comprise more common multifactorial retinal diseases such as age-related macular degeneration, diabetic retinopathy, glaucoma, and uveitis as well as neurological disorders with ocular manifestation. In this review, we describe how microglial function is kept in balance under normal conditions by cross-talk with other retinal cells and summarize how microglia respond to different forms of retinal injury. In addition, we present the concept that microglia play a key role in local regulation of complement in the retina and specify aspects of microglial aging relevant for chronic inflammatory processes in the retina. We conclude that this resident immune cell of the retina cannot be simply regarded as bystander of disease but may instead be a potential therapeutic target to be modulated in the treatment of degenerative and inflammatory diseases of the retina.

Synthesis and propagation of complement C3 by microglia/monocytes in the aging retina.

INTRODUCTION: Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging. METHODS: SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes. RESULTS: C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines. CONCLUSIONS: Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.

Adaptation of the central retina for high acuity vision: cones, the fovea and the avascular zone.

Presence of a fovea centralis is directly linked to molecular specification of an avascular area in central retina, before the fovea (or 'pit') begins to form. Modelling suggests that mechanical forces, generated within the eye, initiate formation of a pit within the avascular area, and its later remodelling in the postnatal period. Within the avascular area the retina is dominated by 'midget' circuitry, in which signals are transferred from a single cone to a single bipolar cell, then a single ganglion cell. Thus in inner, central retina there are relatively few lateral connections between neurons. This renders the region adaptable to tangential forces, that translocate of ganglion cells laterally/centrifugally, to form the fovea. Optical coherence tomography enables live imaging of the retina, and shows that there is greater variation in the morphology of foveae in humans than previously thought. This variation is associated with differences in size of the avascular area and appears to be genetically based, but can be modified by environmental factors, including prematurity. Even when the fovea is absent (foveal hypoplasia), cones in central retina adopt an elongated and narrow morphology, enabling them to pack more densely to increase the sampling rate, and to act as more effective waveguides. Given these findings, what then is the adaptive advantage of a fovea? We suggest that the advantages of having a pit in central retina are relatively few, and minor, but together work to enhance acuity.

Evaluation of normal human foveal development using optical coherence tomography and histologic examination.

OBJECTIVE: To assess outer retinal layer maturation during late gestation and early postnatal life using optical coherence tomography and histologic examination. METHODS: Thirty-nine participants 30 weeks' postmenstrual age or older were imaged using a handheld optical coherence tomography system, for a total of 102 imaging sessions. Foveal images from 16 participants (21 imaging sessions) were normal and evaluated for inner retinal excavation and the presence of outer retinal reflective bands. Reflectivity profiles of central, parafoveal, and parafoveal retina were extracted and were compared with age-matched histologic sections. RESULTS: The foveal pit morphologic structure in infants was generally distinguishable from that in adults. Reflectivity profiles showed a single hyperreflective band at the fovea in all the infants younger than 42 weeks' postmenstrual age. Multiple bands were distinguishable in the outer retina at the peri fovea by 32 weeks' postmenstrual age and at the fovea by 3 months' postterm. By 17 months' postnatal, the characteristic appearance of 4 hyperreflective bands was evident across the foveal region. These features are consistent with previous results from histologic examinations. A "temporal divot" was present in some infants, and the foveal pit morphologic structure and the extent of inner retinal excavation were variable. CONCLUSIONS: Handheld optical coherence tomography is a viable technique for evaluating neonatal retinas. In premature infants who do not develop retinopathy of prematurity, the foveal region seems to follow a developmental time course similar to that associated with in utero maturation. CLINICAL RELEVANCE: As pediatric optical coherence tomography becomes more common, a better understanding of normal foveal and macular development is needed. Longitudinal imaging offers the opportunity to track postnatal foveal development among preterm infants in whom poor visual outcomes are anticipated or to follow up treatment outcomes in this population.

Small interfering RNA-mediated suppression of Ccl2 in Müller cells attenuates microglial recruitment and photoreceptor death following retinal degeneration.

BACKGROUND: The recruitment and activation of inflammatory cells is thought to exacerbate photoreceptor death in retinal degenerative conditions such as age-related macular degeneration (AMD). We investigated the role of Müller cell-derived chemokine (C-C motif) ligand (Ccl)2 expression on monocyte/microglia infiltration and photoreceptor death in light-mediated retinal degeneration, using targeted small interfering (si)RNA. METHODS: Adult Sprague-Dawley rats were injected intravitreally with 1 µg of either Ccl2 siRNA or scrambled siRNA, and were then exposed to 1000 lux of light for a period of 24 hours. The mice were given an overdose of barbiturate, and the retinas harvested and evaluated for the effects of bright-light exposure. Ccl2 expression was assessed by quantitative PCR, immunohistochemistry, and in situ hybridization. Monocytes/microglia were counted on retinal cryostat sections immunolabeled with the markers ED1 and ionized calcium binding adaptor (IBA)1, and photoreceptor apoptosis was assessed using terminal dUTP nick end labeling. RESULTS: Intravitreal injection of Ccl2 siRNA significantly reduced the expression of Ccl2 following light damage to 29% compared with controls. In retinas injected with Ccl2 siRNA, in situ hybridization and immunohistochemistry on retinal cryostat sections showed a substantial decrease in Ccl2 within Müller cells. Cell counts showed significantly fewer ED1-positive and IBA1-positive cells in the retinal vasculature and outer nuclear layer of Ccl2 siRNA-injected retinas, compared with controls. Moreover, there was significantly less photoreceptor apoptosis in Ccl2 siRNA-injected retinas compared with controls. CONCLUSIONS: Our data indicate that Ccl2 expression by Müller cells promotes the infiltration of monocytes/microglia, thereby contributing to the neuroinflammatory response and photoreceptor death following retinal injury. Modulation of exaggerated chemokine responses using siRNA may have value in reducing inflammation-mediated cell death in retinal degenerative disease such as AMD.

Analysis of complement expression in light-induced retinal degeneration: synthesis and deposition of C3 by microglia/macrophages is associated with focal photoreceptor degeneration.

PURPOSE: To investigate the expression and localization of complement system mRNA and protein in a light-induced model of progressive retinal degeneration. METHODS: Sprague-Dawley (SD) rats were exposed to 1000 lux of bright continuous light (BCL) for up to 24 hours. At time points during (1-24 hours) and after (3 and 7 days) exposure, the animals were euthanatized and the retinas processed. Differential expression of complement genes at 24 hours of exposure was assessed using microarray analysis. Expression of complement genes was validated by quantitative PCR, and expression of selected genes was investigated during and after BCL exposure. Photoreceptor apoptosis was assessed using TUNEL and C3 was further investigated by spatiotemporal analysis using in situ hybridization and immunohistochemistry. RESULTS: Exposure to 24 hours of BCL induced differential expression of a suite of complement system genes, including classic and lectin components, regulators, and receptors. C1qr1, MCP, Daf1, and C1qTNF6 all modulated in concert with photoreceptor death and AP-1 expression, which reached a peak at 24 hours exposure. C1s and C4a reached peak expression at 3 days after exposure, while expression of C3, C3ar1, and C5r1 were maximum at 7 days after exposure. C3 mRNA was detected in ED1- and IBA1-positive microglia/macrophages, in the retinal vessels and optic nerve head and in the subretinal space, particularly at the margins of the emerging lesion. CONCLUSIONS: The data indicate that BCL induces the prolonged expression of a range of complement genes and show that microglia/macrophages synthesize C3 and deposit it in the ONL after BCL injury. These findings have relevance to the role of complement in progressive retinal degeneration, including atrophic AMD.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

Early focal expression of the chemokine Ccl2 by Müller cells during exposure to damage-inducing bright continuous light.

PURPOSE: To investigate the time course and localization of Ccl2 expression and recruitment of inflammatory cells associated with light-induced photoreceptor degeneration. METHODS: Sprague-Dawley (SD) rats were exposed to 1000 lux light for up to 24 hours, after which some animals were allowed to recover in dim light (5 lux) for 3 or 7 days. During and after exposure to light, the animals were euthanatized and the retinas processed. Ccl2 expression was assessed by qPCR, immunohistochemistry, and in situ hybridization at each time point. Counts were made of perivascular monocytes/microglia immunolabeled with ED1, and photoreceptor apoptosis was assessed with TUNEL. RESULTS: Upregulation of Ccl2 expression was evident in the retina by 12 hours of exposure and correlated with increased photoreceptor death. Ccl2 expression reached its maximum at 24 hours, coinciding with peak cell death. Immunohistochemistry and in situ hybridization showed that Ccl2 is expressed by Müller cells from 12 hours of exposure, most intensely in the superior retina, in the region of the incipient light-induced lesion. After the Müller cell-driven expression of Ccl2, there was a substantial recruitment of monocytes to the local retina and choroidal vasculature. This coincided spatially with the expression of Ccl2 in the superior retina. Peak monocyte infiltration followed maximum Ccl2 expression by up to 3 days. Furthermore, Ccl2 immunoreactivity was observed in many infiltrating monocytes after a 24-hour exposure. CONCLUSIONS: The data indicate that photoreceptor death promotes region-specific expression of Ccl2 by Müller cells, which facilitates targeting of monocytes to sites of injury. The data suggest that recruitment of monocytes to developing lesions is secondary to signaling events in the retina.

Differential effects of TGF-ß and FGF-2 on in vitro proliferation and migration of primate retinal endothelial and Müller cells.

PURPOSE: During retinal development, the pattern of blood vessel formation depends upon the combined effects of proliferation and migration of endothelial cells, astrocytes and Müller cells. In this study, we investigated the potential for transforming growth factor-ß (TGF-ß) and fibroblast growth factor (FGF-2) to influence this process by regulating proliferation and migration of retinal endothelial and macroglial cells. METHODS: We assessed the effects of exogenous TGF-ß and FGF-2 on the proliferation and migration of cultured endothelial (RF/6A) and Müller cell (MIO-M1) lines. Cell proliferation was measured using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay over 72 hr. Cell migration was measured using a scratch-wound assay over 72 hr. RESULTS: Transforming growth factor-ß inhibited the proliferation of endothelial and Müller cells and inhibited the migration of Müller cells, but not endothelial cells, compared to untreated controls. Conversely, FGF-2 increased endothelial cell proliferation but inhibited endothelial cell migration. Fibroblast growth factor-2 increased migration of Müller cells but had little effect on proliferation except at higher concentrations (20 ng/ml). CONCLUSION: Taken together, these observations indicate that TGF-ß and FGF could work in concert to inhibit endothelial cell proliferation and migration, respectively; this may have implications for establishing and maintaining the avascular zone of primate fovea.

Brief exposure to damaging light causes focal recruitment of macrophages, and long-term destabilization of photoreceptors in the albino rat retina.

PURPOSE: To characterize the long-term spatiotemporal features of light-mediated retinal degeneration. METHODS: Sprague-Dawley rats were exposed to 1000 lux for 24 h, then kept in dim light (5 lux), for up to 56 days. Animals were killed at 0, 3, 7, 28, and 56 days post-exposure, and retinas were prepared for immunohistochemistry. Outer nuclear layer (ONL) thickness and TUNEL labeling were used to quantify photoreceptor death. Antibodies to opsins, glial fibrillary acidic protein (GFAP), fibroblast growth factor-2 (FGF-2), and ED1 were used to assess the retina. RESULTS: At 0 days post-exposure, we detected photoreceptor death 2 mm superior to the optic disc (the "hotspot"), and ED1-positive macrophages in the retinal vasculature and underlying choroid. By 3 days, the ONL was thinner and there was gliosis in the outer retina, where ED1 positive macrophages were also present. Few ED1 positive cells remained at 28 days. At 56 days, there were TUNEL-positive nuclei in the penumbra, and increased FGF-2, and GFAP expression by Müller cells (MCs). In inferior retina, outer segment length was initially reduced, but recovered to near-normal by 28 days. CONCLUSIONS: Short exposure to damaging light destabilizes the retina adjacent to a hotspot of degeneration, so that the damaged region expands in size over time. Recruitment of macrophages is associated with the early phase of damage, but not with the longer term photoreceptor loss in the penumbra. Features of the focal and progressive retinal damage in this model are reminiscent of the progression of age-related macular degeneration (AMD).

The cellular expression of antiangiogenic factors in fetal primate macula.

PURPOSE: To characterize the cellular expression patterns of antiangiogenic factors differentially regulated in the fetal human macula. METHODS: RNA was extracted from macular, nasal, and surround biopsies of three human fetal retinas at midgestation. Relative levels of expression of pigment epithelium-derived factor (PEDF), brain natriuretic peptide (BNP), collagen type IValpha2 (COL4A2), and natriuretic peptide receptors A and C (NPRA and NPRC) were determined with quantitative PCR. Cellular expression of PEDF and BNP was investigated by in situ hybridization on retinal sections from monkeys aged between fetal day 55 and 11 years. BNP, COL4A2, and NPRA proteins were localized by immunohistochemistry. Labeling was imaged and quantified by confocal microscopy and optical densitometry. RESULTS: Quantitative PCR confirmed higher levels of PEDF and BNP and lower levels of COL4A2 in the macula at midgestation. PEDF mRNA was detected in ganglion cells (GCs) and the pigment epithelium (RPE). BNP mRNA was detected in GCs and macroglia, although BNP immunoreactivity (IR) was predominantly perivascular. COL4A2-IR was detected in large blood vessels and NPRA-IR on the retinal vascular endothelium, GC axons in fetal retinas, and cone axons at all ages. Optical densitometry showed a graded expression of PEDF and BNP at all ages, with highest levels of expression in GCs in the developing fovea. CONCLUSIONS: Because the retinal vessels initially form in the GC layer, it is likely that PEDF has a key role in defining and maintaining the foveal avascular area. The precise role of BNP is unclear, but it may include both antiangiogenic and natriuretic functions.