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Retinal GFAP expression in WT and CCL2 2/ 2 CX3CR1 GFP/GFP mice. Cryosections of mouse eyes were stained for GFAP (red) and DAPI (blue) and observed by confocal microscopy. (A) 3month WT mice. (B) 3-month CCL2 2/2 CX3CR1 GFP/GFP mice. (C) 18month WT mice, (D) 18-month CCL2 2/2 CX3CR1 GFP/GFP mice. GL, ganglion layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar = 20 mm. doi:10.1371/journal.pone.0061381.g006
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Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2-/-CX3CR1GFP/GFP mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2-/-CX3CR1GFP/GFP mice younger than 6 months. Patches...
Contexts in source publication
Context 1
... investigation of retinal sections showed that GFAP was expressed in the ganglion layer in 3-month old WT ( Figure 6A) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 6B), although expression in the latter slightly extended to the inner nuclear layer (INL, Figure 6B). In aged (18-month) WT ( Figure 5C) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 5D) the expression was significantly increased and extended to the outer nuclear layers. ...
Context 2
... investigation of retinal sections showed that GFAP was expressed in the ganglion layer in 3-month old WT ( Figure 6A) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 6B), although expression in the latter slightly extended to the inner nuclear layer (INL, Figure 6B). In aged (18-month) WT ( Figure 5C) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 5D) the expression was significantly increased and extended to the outer nuclear layers. ...
Context 3
... investigation of retinal sections showed that GFAP was expressed in the ganglion layer in 3-month old WT ( Figure 6A) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 6B), although expression in the latter slightly extended to the inner nuclear layer (INL, Figure 6B). In aged (18-month) WT ( Figure 5C) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 5D) the expression was significantly increased and extended to the outer nuclear layers. ...
Context 4
... aged (18-month) WT ( Figure 5C) and CCL2 2/2 CX3CR1 GFP/GFP mice ( Figure 5D) the expression was significantly increased and extended to the outer nuclear layers. The increment was more significant in aged CCL2 2/ 2 CX3CR1 GFP/GFP mice ( Figure 6D) compared to WT mice ( Figure 6C). ...
Citations
... Interestingly, CD3 + T cells (Fig. 2B) were not detected in RPE flatmounts from mice between 16 and 24 months old. Isolectin-B4 labels active microglia (Chen et al., 2010(Chen et al., , 2013a and a proportion of Iba-1 + cells are Isolectin-B4 + (Fig. 2C), suggesting the heterogeneous activation of subretinal phagocytes. ...
The retina is an immune privileged tissue, which is protected from external and internal insults by its blood-retinal barriers and immune suppressive microenvironment. Apart from the avoidance and tolerance strategies, the retina is also protected by its own defense system, i.e., microglia and the complement system. The immune privilege and defense mechanisms work together to maintain retinal homeostasis. During aging, the retina is at an increased risk of developing various degenerative diseases such as age-related macular degeneration, diabetic retinopathy, and glaucomatous retinopathy. Previously, we have shown that aging induces a para-inflammatory response in the retina. In this review, we explore the impact of aging on retinal immune regulation and the connection between homeostatic control of retinal immune privilege and para-inflammation under aging conditions and present a view that may explain why aging puts the retina at risk of developing degenerative diseases.
... Phagocytic activity of macrophages was assessed using the pHrodo S.aureus bioparticles conjugate phagocytosis kit and the Dextran-FITC phagocytosis system (Thermo- Fisher Scientific, UK) according to the manufacture's instruction and previous publication [3]. Macrophages were seeded onto a black-walled 96-well plate for 2 h at a density of 5×10 4 /well followed by incubation with 2- Deoxy-D-glucose (2-DG) or oligomycin. ...
... Culture medium alone with bioparticles was served as negative control. The net phagocytosis was calculated by subtracting the average fluorescence intensity of the no-cell negative controls from the experimental wells [3]. 1×10 5 macrophages were incubated with Dextran-FITC (1 mg/ml) at 37 °C for 60 mins, and counter-stained with F4/80 antibody and analysed via FACSCanto II. ...
Background
Peritoneal macrophages are widely used in immunological studies. The cells can be collected under non-elicited (resident) or elicited (e.g., with Brewer thioglycollate broth injection) conditions, and their phenotype and functions differ. Recent studies have shown that macrophage phenotype and function are related to their metabolic states, and metabolic reprogramming has been an emerging concept for controlling macrophage function. In this study, we examined the metabolic state of resident and elicited macrophages and investigated how their metabolic state may affect cell function, including phagocytosis. FindingsFlow cytometry showed that elicited macrophages expressed higher levels of MHC-II, LFA-1 and CD64 but lower levels of F4/80 compared to naïve resident peritoneal macrophages, suggesting a more mature and active phenotype. Elicited macrophages had significantly higher levels of phagocytic activity compared to that of resident macrophages. Metabolic studies showed that the Extracellular Acidification Rates (ECAR) and Oxygen Consumption Rates (OCR) were both significantly higher in elicited macrophages than those in resident macrophages. The treatment of macrophages with 2-Deoxy-D-glucose suppressed glycolysis and reduced phagocytosis, whereas treatment with oligomycin enhanced glycolysis and increased phagocytosis in elicited macrophages. Conclusion
Naïve resident peritoneal macrophages are less metabolically active compared to elicited macrophages. Elicited macrophages had higher levels of glycolysis and oxidative phosphorylation, which may be related to their increased phagocytic capacity and higher levels of maturation and activation. Further understanding of the molecular links between metabolic pathways and cell function would be crucial to develop strategies to control macrophage function through metabolic reprogramming.
... As a result, the aging RPE matures into a heterogeneous monolayer of different-sized cells with multiple nuclei in which some of the very large cells may be moribund, but overall a functioning RPE monolayer is preserved. Evidence for this precarious balance can be seen in both experimental models with age (Chen et al., 2013; Kim et al., 2014) and in human retinal degenerative disease including AMD (Mullins et al., 2014; Ozaki et al., 2014). We propose therefore that increase in cell size and multinucleation may be a strategy for the RPE cell monolayer to repair damage during aging (Fig. 6). ...
Background:
The aim of this study was to investigate the plasma levels of complement C3a, C4a, and C5a in different types of neovascular age-related macular degeneration (nAMD) and whether the levels were related to patients' responsiveness to anti-VEGF therapy.
Results:
Ninety-six nAMD patients (including 61 with choroidal neovascularisation (CNV), 17 with retinal angiomatous proliferation (RAP), 14 with polypoidal choroidal vasculopathy (PCV) and 4 unclassified patients) and 43 controls were recruited to this case-control study. Subretinal fibrosis was observed in 45 nAMD patients and was absent in 51 nAMD patients. In addition, the responsiveness to anti-VEGF (Lucentis) therapy was also evaluated in nAMD patients. Forty-four patients were complete responders, 48 were partially responders, and only 4 patients did not respond to the therapy. The plasma levels of C3a, C4a and C5a were significantly higher in nAMD patients compared to controls. Further analysis of nAMD subgroups showed that the levels of C3a, C4a and C5a were significantly increased in patients with CNV but not RAP and PCV. Significantly increased levels of C3a, C4a and C5a were also observed in nAMD patients with subretinal fibrosis but not in those without subretinal fibrosis. Higher levels of C3a were observed in nAMD patients who responded partially to anti-VEGF therapy.
Conclusions:
Our results suggest increased systemic complement activation in nAMD patients with CNV but not RAP and PCV. Our results also suggest that higher levels of systemic complement activation may increase the risk of subretinal fibrosis in nAMD patients.
... As a result, the aging RPE matures into a heterogeneous monolayer of different-sized cells with multiple nuclei in which some of the very large cells may be moribund, but overall a functioning RPE monolayer is preserved. Evidence for this precarious balance can be seen in both experimental models with age (Chen et al., 2013;Kim et al., 2014) and in human retinal degenerative disease including AMD (Mullins et al., 2014;Ozaki et al., 2014). ...
Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.
... Mice were sacrificed by CO 2 inhalation and eyes dissected and fixed in 2% paraformaldehyde for 2 h. Eyes were then processed for immunohistochemistry as previously described [26,27]. Briefly, 14 mm-thick retinal cryosections were incubated overnight (4uC) with primary antibodies (Table 1) diluted in 0.5% TritonX-100 with 10% normal donkey serum in PBS. ...
Retinal neurodegeneration is a key component of diabetic retinopathy (DR), although the detailed neuronal damage remains ill-defined. Recent evidence suggests that in addition to amacrine and ganglion cell, diabetes may also impact on other retinal neurons. In this study, we examined retinal degenerative changes in Ins2Akita diabetic mice. In scotopic electroretinograms (ERG), b-wave and oscillatory potentials were severely impaired in 9-month old Ins2Akita mice. Despite no obvious pathology in fundoscopic examination, optical coherence tomography (OCT) revealed a progressive thinning of the retina from 3 months onwards. Cone but not rod photoreceptor loss was observed in 3-month-old diabetic mice. Severe impairment of synaptic connectivity at the outer plexiform layer (OPL) was detected in 9-month old Ins2Akita mice. Specifically, photoreceptor presynaptic ribbons were reduced by 25% and postsynaptic boutons by 70%, although the density of horizontal, rod- and cone-bipolar cells remained similar to non-diabetic controls. Significant reductions in GABAergic and glycinergic amacrine cells and Brn3a+ retinal ganglion cells were also observed in 9-month old Ins2Akita mice. In conclusion, the Ins2Akita mouse develops cone photoreceptor degeneration and the impairment of synaptic connectivity at the OPL, predominately resulting from the loss of postsynaptic terminal boutons. Our findings suggest that the Ins2Akita mouse is a good model to study diabetic retinal neuropathy.
... In addition to the Ccl2 − / − /Cx3cr1 − / − double knockout, the C57BL/6N mouse line has the Crb1 (crumbs-like 1) mutation in homozygous form (Mattapallil et al., 2012). The DKO rd8 mouse has earlier onset and higher penetrance than Ccl2 and Cx3cr1 single knockout strains ( Chu et al., 2012;Chen et al., 2013). Although it is not a perfect model for AMD ( Chu et al., 2012;Mattapallil et al., 2012), these mice develop certain features resembling human AMD lesions ( Chu et al., 2012). ...
Age-related macular degeneration (AMD) is a neurodegenerative disease causing irreversible central blindness in the elderly. Apoptosis and inflammation play important roles in AMD pathogenesis. Pigment epithelium-derived factor (PEDF) is a potent neurotrophic and anti-inflammatory glycoprotein that protects the retinal neurons and photoreceptors against cell death caused by pathological insults. We studied the effects of PEDF on focal retinal lesions in Ccl2-/-/Cx3cr1-/- on C57BL/6N [Crb1rd8] (DKO rd8) mice, a model for progressive, focal retinal degeneration. First, we found a significant decrease in PEDF transcript expression in DKO rd8 mouse retina and retinal pigment epithelium (RPE) than wild type (WT, C57BL/6N). Next, cultured DKO rd8 RPE cells secreted lower levels of PEDF protein in the media than WT. Then the right eyes of DKO rd8 mice were injected intravitreously with recombinant human PEDF protein (1 µg), followed by a subconjunctival injection of PEDF (3 µg) 4 weeks later. The untreated left eyes served as controls. The effect of PEDF was assessed by fundoscopy, ocular histopathology and A2E levels, as well as apoptotic and inflammatory molecules. The PEDF-treated eyes showed slower progression or attenuation of the focal retinal lesions, fewer and/or smaller photoreceptor and RPE degeneration, and significantly lower A2E, relative to the untreated eyes. Additionally, lower expression of apoptotic and inflammatory molecules were detected in the PEDF-treated than untreated eyes. Our results establish that PEDF potently stabilizes photoreceptor degeneration via suppression of both anti-apoptotic and anti-inflammatory pathways. The multiple beneficial effects of PEDF represent a novel approach for potential AMD treatment.
... Because this model recapitulates many of the key morphological and immunological characteristics of AMD, DKO rd8 can be used to study AMD pathogenesis as well as to test new therapeutic compounds. Furthermore, Ccl2 -/-/Cx3cr1 -/-mice that were generated from C57BL/6J (without Crb1 rd8 ) can also develop localized retinal atrophy, similar to human geographic atrophy AMD [22]. Some authors have since argued that DKO rd8 may not be a model for AMD, suggesting the Crb1 rd8 mutation, instead of mutations in Ccl2 and Cx3cr1, is the source of the AMD-like pathology found in the DKO rd8 model232425. ...
... C57BL/6N genetic background is important to allow for full expression of the rd8 phenotype [20]. Our data, in conjunction with other reports, suggest that the C57BL/6N genetic background is also important in the phenotypes seen in DKO rd8 [22,23,25]. ...
Although the mouse has no macula leutea, its neuroretina and retinal pigment epithelium (RPE) can develop lesions mimicking certain features of age-related macular degeneration (AMD). Differences between the Ccl2 and Cx3cr1 double deficient mouse on Crb1rd8 (rd8) background (DKO
rd8
) and the Crb1rd8 mouse in photoreceptor and RPE pathology, as well as ocularA2E contents and immune responses, show that DKO
rd8
recapitulates some human AMD-like features in addition to rd8 retinal dystrophy/degeneration. Different therapeutic interventions have been demonstrated to be effective on the AMD-like features of DKO
rd8
mice. The use of the DKO
rd8
model and C57BL/6N (wild type, WT) mice as group controls (4 groups) to test treatments such as high omega-3 polyunsaturated fatty acid (n-3) diet has, for example, shown the beneficial effect of n-3 on AMD-like lesions by anti-inflammatory action of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The use of self-control in the DKO
rd8
mouse by treating one eye and using the contralateral eye as the control for the same mouse allows for appropriate interventional experiments and evaluates various novel therapeutic agents. Three examples will be briefly presented and discussed: (1) tumor necrosis factor-inducible gene 6 recombinant protein (TSG-6) arrests the AMD-like lesions via modulation of ocular immunological gene expression, e.g., Il-17a; (2) adeno-associated virus encoding sIL-17R (AAV2.sIL17R) stabilizes the AMD-like lesions; and (3) pigment epithelium-derived factor (PEDF) ameliorates the AMD-lesions by its anti-inflammatory, anti-apoptotic and neuroprotective roles. Therefore, the DKO
rd8
mouse model can be useful and appropriate for therapeutic compound screening in the management of human AMD.
... Because this model recapitulates many of the key morphological and immunological characteristics of AMD, DKO rd8 can be used to study AMD pathogenesis as well as to test new therapeutic compounds. Furthermore, Ccl2 -/-/Cx3cr1 -/-mice that were generated from C57BL/6J (without Crb1 rd8 ) can also develop localized retinal atrophy, similar to human geographic atrophy AMD [22]. Some authors have since argued that DKO rd8 may not be a model for AMD, suggesting the Crb1 rd8 mutation, instead of mutations in Ccl2 and Cx3cr1, is the source of the AMD-like pathology found in the DKO rd8 model232425. ...
... C57BL/6N genetic background is important to allow for full expression of the rd8 phenotype [20]. Our data, in conjunction with other reports, suggest that the C57BL/6N genetic background is also important in the phenotypes seen in DKO rd8 [22,23,25]. ...
There are no disease-modifying treatments available for geographic atrophy (GA), the advanced form of dry age-related macular degeneration. Current murine models fail to fully recapitulate the features of GA and thus hinder drug discovery. Here we describe a novel mouse model of retinal degeneration with hallmark features of GA. We used an 810 nm laser to create a retinal lesion with central sparing (RLCS), simulating parafoveal atrophy observed in patients with progressive GA. Laser-induced RLCS resulted in progressive GA-like pathology with the development of a confluent atrophic lesion. We demonstrate significant changes to the retinal structure and thickness in the central unaffected retina over a 24-week post-laser period, confirmed by longitudinal optical coherence tomography scans. We further show characteristic features of progressive GA, including a gradual reduction in the thickness of the central, unaffected retina and of total retinal thickness. Histological changes observed in the RLCS correspond to GA pathology, which includes the collapse of the outer nuclear layer, increased numbers of GFAP + , CD11b + and FcγRI + cells, and damage to cone and rod photoreceptors. We demonstrate a laser-induced mouse model of parafoveal GA progression, starting at 2 weeks post-laser and reaching confluence at 24 weeks post-laser. This 24-week time-frame in which GA pathology develops, provides an extended window of opportunity for proof-of-concept evaluation of drugs targeting GA. This time period is an added advantage compared to several existing models of geographic atrophy.
Chronic oxidative stress and immune dysregulation are key mechanisms involved in the pathogenesis of most retinal degenerative diseases, including age-related macular degeneration. The Ccl2−/−/Cx3cr1−/−/Crb1rd8/rd8 mouse model develops a progressive degeneration phenotype, with photoreceptor atrophy, drusen-like lesions or pigment alterations at an early age; however, the role of oxidative stress and immune function in the pathogenesis of the model is poorly understood. We performed a comprehensive characterization of the Ccl2−/−/Cx3cr1−/−/Crb1rd8/rd8 mouse to evaluate how these pathways influence pathogenesis. We generated a Ccl2−/−/Cx3cr1−/− double-knockout (DKO) mouse on a C57BL/6N background (with the rd8 mutation of the Crb1 gene), assessed its retina status and function during 9 months in both in vivo and post-mortem analysis, and performed a comprehensive transcriptomic analysis. DKOrd8 mice presented focal retinal lesions with increased infiltration of microglia and involvement of Müller cells. Lesions progressed to thinning of the photoreceptor nuclear layer, causing a loss in retinal function. Transcriptomics analysis revealed major differential expression of genes involved in oxidative stress and neuronal function, in particular genes related to the mitochondrial electron transport chain and antioxidant cellular response. Our results suggest that alterations in chemokine signaling combined with the rd8 mutation in Ccl2−/−/Cx3cr1−/−/Crb1rd8/rd8 mice involve early changes in several pathways associated with age-related macular degeneration, highlighting the relevance of these processes in the pathological retinal degeneration in the DKOrd8 model.
