Open Access
Immunology and Microbiology  |   January 2025
C3aR1-Deletion Delays Retinal Degeneration in a White-Light Damage Mouse Model
Author Affiliations & Notes
  • Verena Behnke
    Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
  • Anne Wolf
    Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
    Center for Molecular Medicine Cologne (CMMC), Cologne, Germany
  • Mandy Hector
    Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
  • Thomas Langmann
    Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
    Center for Molecular Medicine Cologne (CMMC), Cologne, Germany
  • Correspondence: Thomas Langmann, Department of Ophthalmology, Laboratory for Experimental Immunology of the Eye, University of Cologne, Joseph-Stelzmann-Str. 9, Cologne D-50931, Germany; [email protected]
Investigative Ophthalmology & Visual Science January 2025, Vol.66, 15. doi:https://doi.org/10.1167/iovs.66.1.15
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Verena Behnke, Anne Wolf, Mandy Hector, Thomas Langmann; C3aR1-Deletion Delays Retinal Degeneration in a White-Light Damage Mouse Model. Invest. Ophthalmol. Vis. Sci. 2025;66(1):15. https://doi.org/10.1167/iovs.66.1.15.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: In the aging retina, persistent activation of microglia is known to play a key role in retinal degenerative diseases like age-related macular degeneration (AMD). Furthermore, dysregulation of the alternative complement pathway is generally accepted as the main driver for AMD disease progression and microglia are important producers of local complement and are equipped with complement receptors themselves. Here, we investigate the involvement of anaphylatoxin signaling, predominantly on Iba1+ cell activity, in light-induced retinal degeneration as a model for dry AMD, using anaphylatoxin receptor knockout (KO) mice.

Methods: Bright white light with an intensity of 10,000 lux was applied for 30 minutes to complement component 3a receptor 1 (C3ar1) or complement component 5a receptor 1 (C5ar1) KO and wildtype (WT) mice. Analyses of transcriptome changes and migration activity of Iba1+ cells as well as retinal thickness were performed 4 days after light exposure.

Results: Full body KO mice of either C3aR1 or C5aR1 were tested, but none led to mitigated migration of Iba1+ cells to the subretinal space or decreased expression of complement factors after light damage compared to WT mice. However, a partial rescue of retinal thickness was shown in C3aR1 KO mice, which was mirrored by significant less membrane attack complex (MAC) occurrence in the outer retina.

Conclusions: We conclude that deletion of the anaphylatoxin receptor C3aR1 cannot modulate mononuclear phagocytes but diminishes retinal degeneration through interference with the complement pathway and thus decreased MAC assembling. C3aR1-targeted therapy may be considered for patients with dry AMD.

A variety of complement proteins, receptors, and regulators are expressed in the retina.1 The cleavage of C3 and C5 by their respective convertases are the central steps in all complement pathways and generate toxic complement proteins C3a and C5a, respectively. Both so-called anaphylatoxins drive inflammation by engaging their corresponding chemotactic receptors C3aR1 and C5aR1 (also named CD88),24 which are primarily expressed on myeloid and lymphoid cells throughout the body.510 Anaphylatoxins are the most potent pro-inflammatory fragments generated during the complement pathway and function as chemoattractants for mononuclear phagocytes.11,12 
Hence, microglia are, alongside the RPE, important producers of local complement and are equipped with complement receptors themselves.13 Microglia are the resident immune cells of the central nervous system.14 In homeostasis, they build a regularly spaced network of ramified cells in the plexiform layers,15,16 whereas, in the aging retina, persistent activation of microglia is known to play a key role in retinal degenerative diseases like age-related macular degeneration (AMD).1719 
AMD is a major blinding disease in the elderly of the western world, with the number of affected people rising rapidly. In terminal stages of its two forms, dry and wet, AMD can lead to complete vision loss that severely decreases the patient's quality of life. Representing a multifactorial disease, main risk factors for AMD are advanced age, smoking, and variations in genes of the immune system, especially those regulating the complement system.20,21 Although numerous studies raise convincing evidence that the overly activated complement system in retinas can lead to AMD and a variety of complement regulators have been generated and tested in clinical studies,22 effective treatment options are lacking. Here, we investigate the involvement of anaphylatoxin signaling, predominantly on Iba1+ cell activity, during retinal degeneration using anaphylatoxin receptor knockout (KO) mice. 
Materials and Methods
Cell Culture
Murine BV-2 cells23 were cultured in T75 flasks at 37°C and 5% CO2 humidity. RPMI 1640 (Gibco, Waltham, MA, USA) culture medium contained 5% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 1% penicillin/streptomycin (Gibco, Waltham, MA, USA), 3 mM L-glutamin (Gibco, Waltham, MA, USA), and 50 µM β-Mercaptoethanol (Sigma-Aldrich, Darmstadt, Germany). Media was changed every 3 days and cells were split at 95% confluency. BV-2 cells were cultured in 6-well plates, with 3*105 cells per well and treated with 50 ng/mL lipopolysaccharide (LPS; InvivoGen, San Diego, CA, USA) for 6, 24, or 72 hours. 
Animals
All experimental protocols complied with the ARRIVE guidelines and were carried out in accordance to the German Animal Welfare Act, which is in line with the European Directive 2010/63/EU, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animal experiments used in this study were reviewed and approved by the governmental body responsible for animal welfare in the state of North Rhine-Westphalia, Germany (Landesamt für Natur, Umwelt, und Verbraucherschutz). 
BALB/cJRj mice were purchased from Janvier. C.129S4-C3ar1tm1Cge/J and C.129S4(B6)-C5ar1tm1Cge/J, further referred to as C3ar1 and C5ar1, were purchased from Jackson Laboratory (stock number 005712 and 006845, respectively), backcrossed two times with BALB/cJ and bred in-house. The animals were kept under SPF-conditions in an air-conditioned environment with a 12-hour light/dark cycle and water and food ad libitum. Eight to 10-week-old male and female BALB/cJ mice were used in the experiments.2426 For genotyping, DNA was isolated from ear punches via the HotSHOT Method.27 PCR was performed based on Jackson Laboratories’ protocols with the Taq-S PCR kit (Genaxxon). The primers used were as follows: C3ar1, forward common 5′-agccattctaggggcgtatt-3′, reverse wildtype (WT) 5′-tggggttatttcgtcttctgc-3′, reverse mutant 5′-tggatgtggaatgtgtgcgag-3′; C5ar1, forward WT 5′-ggtctctccccagcatcata-3′, forward mutant 5′-gccagaggccacttgtgtag-3′, and reverse common 5′-ggcaacgtagccaagaaaaa-3′. C3ar1 and C5ar1 PCR products were separated on a 2% / 1% (w/v) agarose gel regarding their size (WT = 250 bp and KO = 400 bp and WT = 386 bp and KO = 244 bp), respectively. 
Light Exposure
Littermates were dark-adapted for 16 hours prior to light exposure. The pupils were dilated with 2.5% phenylephrine and 1% tropicamide under dim red light and the mice were placed separately or in pairs in reflective, aluminum-foil-coated cages to prevent covering. Bright white light with an intensity of 10,000 lux for 30 minutes was applied. After light exposure, the animals were transferred to normal light cycle until further analysis. 
In Vivo Imaging Using Spectral-Domain Optical Coherence Tomography and BluePeak Autofluorescence
Retinal thickness using spectral domain optical coherence tomography (SD-OCT) and BluePeak autofluorescence (BAF) to investigate structural changes in the retina were performed on both eyes 4 days after light exposure with the Spectralis HRA + OCT device (Heidelberg, Germany). The mice were anesthetized with a mixture of ketamine (100 mg/kg body weight, Ketavet; Pfizer Animal Health) and xylazine (5 mg/kg body weight, 2% Rompun; Bayer HealthCare) diluted in 0.9% sodium chloride by intraperitoneal (IP) injection and their pupils were dilated with a topical drop of phenylephrine 2.5%–tropicamide 0.5% before image acquisition. Retinal thickness measurements were performed using the Heidelberg Eye Explorer Software using a circular ring scan (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (µm) of each quadruplet. 
RNA Isolation, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction
RNA from cultured BV-2 microglia cells was isolated with the RNA Isolation Kit following the manufacturer's instructions (Machery & Nagel, Düren, Germany). RNA from retinas was isolated with the Qiagen Micro isolation kit according to the manufacturer’s protocol. Purity and integrity of the RNA was assessed with a NANODrop 2000 machine (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was synthesized with the Thermo Fischer Reverse Transcriptase Kit according to the company's protocol (Thermo Fisher Scientific, Waltham, MA, USA). Subsequent quantitative real-time PCR (qRT-PCR) analysis was performed in duplicates with either the Takyon Probe Assay protocol (Eurogentec Deutschland GmbH, Cologne, Germany) or Takyon SYBR Green Assay protocol using the LightCycler 480 II (Roche, Basel, Switzerland). Primer sequences and Roche Universal Probe Library probe numbers for probe-based assay were as follows ATP synthase, H+-transporting, mitochondrial F1 complex, β polypeptide (Atp5b), forward primer 5′-ggcacaatgcaggaaagg-3′, reverse primer 5′-tcagcaggcacatagatagcc-3′, probe #77; Complement C1q A chain (C1qa), forward primer 5′-ggagcatccagtttgatcg-3′, reverse primer 5′-catccctgagaggtctccat-3′, probe #16; Complement component 3 (C3), forward primer 5′-accttacctcggcaagtttct-3′, reverse primer 5′-ttgtagagctgctggtcagg-3′, probe #76; complement component 6 (C6), forward primer 5′-AAGGAAGACACGTGCACCAA-3′, reverse primer 5′- TCCTTCACCGATTCTAGCCAC -3; complement component 7 (C7), forward primer 5′-GGTGTGCTTTATAGCAGCGTT-3′, reverse primer 5′-CTGAACGCCTTCGAGTCTGAG-3; complement component 8a (C8a), forward primer 5′-AAACGCCACCTGGTGTGTAA-3′, reverse primer 5′-AGGATGTTGTACCCCAAGGC-3; complement component 9 (C9), forward primer 5′-CAGCAGGCTATGGGATCAACA-3′, reverse primer 5′-CGGTCACAGAGTCCGTTGTA-3; complement factor b (Cfb) forward primer 5′-ctcgaacctgcagatccac-3′, reverse primer 5′-tcaaagtcctgcggtcgt-3′, probe #1; complement factor h (Cfh), forward primer 5′-gaaaaaccaaagtgccgaga-3′, reverse primer 5′-ggaggtgatgtctccattgtc-3′, probe #25; inducible nitric oxide synthase (iNos), forward primer 5′-ctttgccacggacgagac-3′, reverse primer 5′-tcattgtactctgagggctga-3′, probe #13; Translocator protein (Tspo), forward primer 5′-cccttgggtctctacactgg-3′, reverse primer 5′-aagcagaagatcggccaag-3′, probe #21. ATPase was used as reference gene and the ΔΔC method was applied using the LightCycler 480 software 1.2.1 for data evaluation. 
Immunofluorescence Staining
Eyes were enucleated and fixed in 4% paraformaldehyde (Roti Histofix; Roth, Karlsruhe, Germany) for 2 hours at room temperature. For retinal flat mounts, eyes were dissected and incubated with PERM/Block Buffer (5% NDS, 0.2% BSA, and 0.3% Triton X-100 in PBS) overnight at 4°C. For cryosections, eyes were transferred in 10%, 20%, and 30% sucrose for 1 hour each before embedding in optimal cutting temperature compound. For each eye, 10-µm sections were prepared with a Leica CM3050 S Cryostat (Leica Biosystems, Wetzlar, Germany). Frozen slides were thawed at room temperature, dehydrated in PBS, and unspecific antigens were blocked with BLOTTO (1% milk powder and 0.3% Triton X-100 in PBS) for 30 minutes at room temperature. Retinal flat mounts and sections were incubated with primary antibody overnight at 4°C. A 1:500 dilution of anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody (FUJIFILM Wako Chemicals Europe, Neuss, Germany) or C5b-9 (Abcam, Cambridge, United Kingdom) was used. Afterward, the tissue was incubated with anti-rabbit Alexa Fluor 488/ 647 (1:1000) secondary antibody for 1 hour at room temperature (Invitrogen, Carlsbad, CA, USA). Retinal flat mounts were mounted on microscopic slides and embedded with Vectashield H-1400 (Vector Laboratories, Burlingame, CA, USA). Sections were mounted in Fluoromount-G with Dapi (Thermo Fisher Scientific, Waltham, MA, USA). 
TUNEL Assay
Retinal cryosections were labeled with an in situ cell death detection kit RED (Roche) according to the manufacturer's instructions. Fluoromount-G with DAPI was used to counterstain the nuclei in mounted sections. 
Image Analysis
Images of the central retina were taken with a Zeiss Imager M.2 equipped with ApoTome.2 (Oberkochen, Germany). Autofluorescence was recorded at 594 nm in the red channel. The total number of Iba1+ cells was counted in four images of each retinal flat mount or five sections and averaged for one retinal n. TUNEL assay images were analyzed by counting the TUNEL+ cells and the number of photoreceptor nuclei in the outer nuclear layer (ONL). The percentage of TUNEL+ cells in the ONL was calculated as the ratio of TUNEL+ nuclei to the total number of photoreceptor nuclei in the ONL, multiplied by 100. Counting was performed with the particle analyzer plugin or the multi-point tool of ImageJ version 1.52a (Bethesda, MD, USA). 
Enzyme-Linked Immunosorbent Assay
The concentration of cytokines in total retinal lysates were measured by ELISA. Tissue samples were sonicated in 1× PBS supplemented with protease and phosphatase inhibitors (complete protease inhibitor cocktail; Roche). The CCL2 ELISA Kit was purchased from R&D Systems and performed according to the manufacturer’s protocol. Absorbance was measured with a TECAN infinite M1000. 
Statistical Analysis
All data were plotted and analyzed with GraphPad PRISM version 7.04. After the D’Agostino & Pearson normality tests, the data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Comparisons between all groups were made, however, if only two groups were compared in one experiment the Mann Whitney t-test was performed as indicated (no asterisk: not significant, *P < 0.05, **P < 0.01, ***P ≤ 0.001, and ****P < 0.0001). Error bars show mean ± SEM. 
Results
Anaphylatoxins C3a and C5a drive inflammation by engaging their corresponding chemotactic receptors C3aR1 and C5aR124 and attract mononuclear phagocytes as a consequence.11,12 Anaphylatoxin receptors were found significantly upregulated in LPS-treated BV-2 microglia cells and retinal lysates of BALB/c wildtype mice after light damage (Supplementary Fig. S1). Therefore, we used anaphylatoxin receptor KO mice to elucidate the involvement of anaphylatoxin signaling during retinal degeneration with regard to mononuclear phagocyte activity, with microglia being the resident immune cells of the retina.14 For this, 8 to 10-week-old WT or KO animals, determined using PCR (Supplementary Figs. S2A, S2B), of mouse lines C3ar1 and C5ar1 with BALB/cJ background, were dark adapted for 16 hours before light exposure to 10,000 lux for 30 minutes (Supplementary Fig. S2C). In the mouse model of light exposure, monocytes are known to infiltrate the retina from the blood stream and mix with residential microglia.28 Hence, we will refer to the Iba1+ population in the retina after light exposure as mononuclear phagocytes. All analysis assessed below were performed 4 days after light exposure. 
C3ar1 KO Mice Tend to a More Regulated Alternative Complement Pathway Gene Expression
To examine transcription changes of complement components and inflammation modulators, gene expression analysis via qRT-PCR of retinal tissue was performed (Fig. 1, Supplementary Fig. S4) Technical duplicates of one retinal expression measurement were averaged for one n. Four days after light exposure, the transcripts of the inflammation marker iNos and the early mononuclear phagocyte activation marker Tspo remained mostly unchanged (see Figs. 1A, 1B, 1G, 1H), with the exceptions of iNos in light-damaged C3ar1 and C5ar1 WT mice as well as Tspo in C5ar1 KO mice after light damage (see Figs. 1A, 1G, 1H). In the matter of the complement system, light damage significantly upregulated C3 and C1qa transcripts regardless of the genotype (see Figs. 1C, 1D, 1I, 1J). Cfb transcripts could not be detected in untreated samples; thus, data were normalized to light-exposed WT samples (see Figs. 1E, 1K). Cfb transcripts were unaltered between light-exposed WT and KO mice. Transcripts of regulatory Cfh displayed significant elevation due to light exposure (see Figs. 1F, 1L). C7 was the only terminal component found significantly upregulated in C3ar1 WT retinas (see Supplementary Figs. S4A–D), whereas In C5ar1 animals, C6, C7, and C8a transcripts were significantly enhanced in KO mice (see Supplementary Figs. S4E–H). These data indicate that light damage induces complement signaling accompanied by light inflammation in the retina, with tendencies of lower induction of central factor C3 and Cfb as well as higher levels of regulatory Cfh in C3aR1 KO mice compared to WT mice. 
Figure 1.
 
Expression analysis of retinal tissue. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. The qRT-PCR was performed and ΔΔCT analysis was used for quantification. ATP5B was used as reference gene. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test (n = WT, KO, light-damaged WT, and light-damaged KO; C3ar1: n = 11, 15, 13–14, 13; TSPO: 6, 11, 8, 13; CFB: 0, 0, 9, 8; C5aR: n = 12–13, 12, 13, 12–13, TSPO: 12, 9, 12, 12; and CFB: 0, 0, 7, 12).
Figure 1.
 
Expression analysis of retinal tissue. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. The qRT-PCR was performed and ΔΔCT analysis was used for quantification. ATP5B was used as reference gene. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test (n = WT, KO, light-damaged WT, and light-damaged KO; C3ar1: n = 11, 15, 13–14, 13; TSPO: 6, 11, 8, 13; CFB: 0, 0, 9, 8; C5aR: n = 12–13, 12, 13, 12–13, TSPO: 12, 9, 12, 12; and CFB: 0, 0, 7, 12).
Genetic Ablation of C3ar1 or C5ar1 Does Not Change Microgliosis
Systematic morphometric analysis of mononuclear phagocytes using Iba1-stained retinal flat mounts and cryosections revealed no change in microgliosis among light-challenged genotypes (Figs. 23). An evenly spaced network of ramified microglia was evident in the outer plexiform layer (OPL) of unchallenged mice (see Figs. 2A, 2G), which significantly increased after light damage (see Figs. 2C, 2I). Furthermore, light-challenged mononuclear phagocytes showed a more amoeboid shape with some rudiment protrusions. Untreated controls depicted very few activated microglia in the subretinal space (SR), whereas light damage significantly increased these numbers of migrated mononuclear phagocytes (see Figs. 2B, 2H). These data were further supported by CC-chemokine ligand 2 (CCL2) protein levels in the retina (see Figs. 2D, 2J). Concurrent to mononuclear phagocyte activation and migration, these cells became amoeboid and enhanced their phagocytic reactivity, shown by increased autofluorescence co-localized with subretinal mononuclear phagocytes (see Figs. 2B, 2H lower panels, see Supplementary Fig. S3). Nevertheless, there were no differences in mononuclear phagocyte numbers and autofluorescent area found in the SR of light-challenged retinas (see Figs. 2E, 2F, 2K, 2L). Iba1-stained cryosections confirmed the results from retinal flat mounts, that phagocytosed autofluorescent material co-localized with subretinal mononuclear phagocytes (see Figs. 3A, 3E; white arrows). Shredded photoreceptor disks and byproducts of the visual cycle are recycled in the RPE,29,30 which was visible as autofluorescent material in the RPE cell layer. Counting of mononuclear phagocytes in the ONL revealed migration after light damage in both WT and KO animals (see Figs. 3C, 3G). 
Figure 2.
 
Morphological and migration analysis of microglia in the retina. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, B, G, H) Mononuclear phagocytes were stained on retinal flat mounts against ionized calcium-binding adapter molecule 1 (Iba1). (C, E, F, I, K, L) Four images of the central retina were taken around the optic nerve of each eye and averaged for 1 n. Cell numbers were counted using the particle analyzer plugin of ImageJ. (C3ar1: n = 11 and C5ar1 n = 13). (D, J) ELISA was performed with full retinal lysates (n = 5, C5ar1 light damage KO: n = 4). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; CCL2, CC-chemokine ligand 2; OPL, outer plexiform layer; SR, subretinal space.
Figure 2.
 
Morphological and migration analysis of microglia in the retina. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, B, G, H) Mononuclear phagocytes were stained on retinal flat mounts against ionized calcium-binding adapter molecule 1 (Iba1). (C, E, F, I, K, L) Four images of the central retina were taken around the optic nerve of each eye and averaged for 1 n. Cell numbers were counted using the particle analyzer plugin of ImageJ. (C3ar1: n = 11 and C5ar1 n = 13). (D, J) ELISA was performed with full retinal lysates (n = 5, C5ar1 light damage KO: n = 4). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; CCL2, CC-chemokine ligand 2; OPL, outer plexiform layer; SR, subretinal space.
Figure 3.
 
Spatial analysis of microglia and complement membrane attack complex (MAC) in the eyes. The eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, E) Cryosections were stained with DAPI and against ionized calcium-binding adapter molecule 1 (Iba1). (B, F) Cryosections were stained with DAPI and against MAC (C5b-9). (C, D, G, H) Cell numbers were counted using the multi-point tool of ImageJ. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; SR, subretinal space.
Figure 3.
 
Spatial analysis of microglia and complement membrane attack complex (MAC) in the eyes. The eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, E) Cryosections were stained with DAPI and against ionized calcium-binding adapter molecule 1 (Iba1). (B, F) Cryosections were stained with DAPI and against MAC (C5b-9). (C, D, G, H) Cell numbers were counted using the multi-point tool of ImageJ. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; SR, subretinal space.
C3aR1 KO Leads to Decreased MAC Formation
Membrane attack complex (MAC; C5b-9) formation represents the terminal pathway of the complement cascade and either leads to pore formation on the cell surface and cell lysis31 or induces intracellular inflammatory signaling.32,33 Expression level analysis indicated lower terminal expression levels of terminal complement components in KO mice compared to WT mice (see Supplementary Figs. S4A–S4D). Cryosections stained for C5b-9 revealed increased MAC formation in the outer retina of light-challenged mice (see Figs. 3B, 3F; white arrows). Interestingly, significantly less MAC was detected in C3aR1 KO mice than WT mice (see Fig. 3D). 
C3aR1-Deficiency Delays Retinal Thinning
As DAPI-staining in cryosections already indicated light damage-induced thinning of the outer nuclear layer, we quantified the overall retinal thickness using SD-OCT scans (Fig. 4). Here, OCT scans confirmed thinning of the ONL with light damage, which changed the overall reflectance patterns of the retina (see Figs. 4A, 4F). As clearly indicated by color changes in retinal heatmaps (see Figs. 4B, 4G), analysis revealed a significant reduction of retinal thickness after light damage (see Figs. 4D, 4I). However, the retinal thickness of light-challenged C3aR1 KO animals significantly exceeded that of WT mice (see Fig. 4D). 
Figure 4.
 
Retinal thickness and OCT analysis. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, F) Representative scans and (B, G) corresponding heatmaps compiled by spectral-domain optical coherence tomography (SD-OCT) displayed retinal overviews. (C) BluePeak autofluorescence (BAF) images depict retinal background. (D, I) Retinal thickness was assessed in circular ring scans (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (µm) of each quadruplet. (C3aRr1: n = 36 and C5ar1: n = 32). (E, J) Percentage of autofluorescent area was measured with ImageJ with the same region of interest for all images (n = 18). Data were analyzed using 1-way ANOVA and Tukey's multiple comparison test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM.
Figure 4.
 
Retinal thickness and OCT analysis. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, F) Representative scans and (B, G) corresponding heatmaps compiled by spectral-domain optical coherence tomography (SD-OCT) displayed retinal overviews. (C) BluePeak autofluorescence (BAF) images depict retinal background. (D, I) Retinal thickness was assessed in circular ring scans (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (µm) of each quadruplet. (C3aRr1: n = 36 and C5ar1: n = 32). (E, J) Percentage of autofluorescent area was measured with ImageJ with the same region of interest for all images (n = 18). Data were analyzed using 1-way ANOVA and Tukey's multiple comparison test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM.
Genetic Anaphylatoxin Receptor Ablation Does Not Alter Autofluorescent Material Accumulation
Accumulation of autofluorescent material is a hallmark of AMD disease progression. Hyper-reflective foci appear as drusen in retinal fundus images, which are already detectable in patients with intermediate AMD.34 Complement components like Anaphylatoxins are prevalent components of drusen12,3539; therefore, BAF images were integrated in the study (see Figs. 4C, 4H). The autofluorescent area in fundus images was significantly increased after light damage, however, unaltered among genotypes (see Figs. 4E, 4J). 
Similar Cell Death in C3aR1 KO and WT Mice Retinas
OCT scans revealed that retinal thickness of light-challenged C3aR1 KO animals significantly exceeded that of WT mice. In addition, in situ retinal cell death was detected and quantified in cryosections of C3ar1 mice via TUNEL (Fig. 5). Following light damage, TUNEL+ cells were visible in the ONL (see Fig. 5A), without significant changes between WT and KO mice (see Fig. 5B). 
Figure 5.
 
Apoptosis of photoreceptors in the outer nuclear layer. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A) Apoptotic cells on cryosections were labeled with TUNEL assay and counterstained with DAPI. (B) Cell numbers were counted using the particle analyzer plugin of ImageJ in outer nuclear layer (ONL) specific ROIs, set individually for each image via DAPI staining. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Figure 5.
 
Apoptosis of photoreceptors in the outer nuclear layer. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A) Apoptotic cells on cryosections were labeled with TUNEL assay and counterstained with DAPI. (B) Cell numbers were counted using the particle analyzer plugin of ImageJ in outer nuclear layer (ONL) specific ROIs, set individually for each image via DAPI staining. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Discussion
Anaphylatoxins are potent inflammatory mediators, therefore are implicated in inflammatory diseases.40 C3a and C5a drive inflammation by engaging their corresponding chemotactic receptors C3aR1 and C5aR1, respectively.24 Thus, full body KO mice of either C3ar1 or C5ar1 were tested in an experimental mouse model mimicking some features of dry AMD. Here, we showed that none of the KOs led to mitigated mononuclear phagocyte migration to the subretinal space, albeit C3aR1 KO decelerated retinal thinning. 
Dysregulation of the alternative complement pathway is generally accepted as a main driver for AMD disease progression. Despite numerous experimental mouse models and clinical studies focused on complement inhibition in AMD, effective treatment options are still lacking. Although anaphylatoxin receptors C3aR1 and C5aR1 were found to be highly expressed on immune cells after light exposure, reduced microgliosis could only be detected in C5aR1-deficient mice, but not C3aR1-deficient mice, and neither of the KO mice were able to preserve retinal thickness after light exposure.41 In line with this, in another study, C3aR1 KO and C3aR1/C5aR1-double KO mice, but not C5aR1 KO mice, had progressive retinal cell loss and dysfunction after light damage.4 However, there is still a debate if one anaphylatoxin signaling axis is pro-inflammatory and the other one is protective or if both are mandatory for retinal function and structure. Our results observed no change in mononuclear phagocyte activity in the outer retina and only a partial rescue of the retinal thickness in C3aR1-deficient mice. Indeed, C3ar1, but not C5ar1, was found upregulated in BALB/c animals in our study (see Supplementary Fig. S1). All studies mentioned before used BALB/cJ mice, but Yu et al. furthermore compared their results with C57BL/6J mice and could not report differences, eliminating potential influences of the genetic background.4 However, major experimental settings as light regime and analysis time points as well as age and sex of mice differed among all studies. Noteworthy, while often termed a decoy receptor, pro-inflammatory functions have been proposed for the second C5a ligand C5aR2.42 Antibody blockade or targeted deletion of C5aR2 resulted in excessive C5a-mediated chemotaxis,43,44 suggesting the absence of C5aR1 in our model prohibits heterodimerization and therefore may alter C5aR2 function. Despite the fact that most pro-inflammatory effects of C5a binding occur through C5aR1,40 unchanged findings in C5aR1-deficient animals in this model could be possibly due to signaling through C5aR2. 
Here, we showed a slight thickening of the C3aR1 KO mice retinal structure compared to WT mice 4 days after light exposure. This was mirrored by significant less MAC staining in the outer retina of C3aR1-deficient mice as well as less pronounced transcripts of complement proteins. Nevertheless, in situ detection of retinal cell death on cryosections of C3ar1 mice via TUNEL did not indicate less apoptotic cell in KO mice compared to WT mice after 4 days. As retinal thinning is a gradual process, lower cell death levels comparing WT mice to KO mice could be expected at earlier time points. In fact, unpublished data of our group showed highest TUNEL+ cell counts 1 day after light exposure and only intermediate levels on day 4. Notably, also monocytes express C3a and C5a receptors, which could also influence retinal degeneration in this model. Furthermore, C3a stimulation of monocytes was shown to induce IL-1β secretion, subsequently to NLRP3 inflammasome activation,45 which would be a first line response in the tissue. In line with our data, C3aR1 was identified as a damaging neuroinflammatory factor in ocular hypertensive DBA/2J mice, influencing the microglial expression pattern and being associated with the risk of degeneration.46 Although drusen are a hallmark in the development of AMD and C3a trigger the formation of sub-RPE deposits in vitro,47 altered retinal structures in BAF fundus images could not be detected in our model. 
Studies targeting complement in inflammatory mouse models yield diverse results. In the laser model of wet AMD neutralization of anaphylatoxins, or their respective receptors, resulted in a reduced neovascular area.12 In another choroidal neovascularization (CNV) study, antibody blocking of the alternative complement pathway with the fusion protein CR2-fH, combining the iC3b/C3d-binding region of CR2 and the N-terminus of the regulatory CFH, normalized anaphylatoxin levels, and reduced lesion size.48 Contrarily, blockade of the anaphylatoxin receptors was not sufficient to alter the course of lesion repair significantly in the same study.48 Dual time-dependent effects were demonstrates for C5aR1/C5a signaling in a spinal cord injury model49 as well as for C3aR signaling during ischemic injury of the adult brain.50 In a mouse model of experimental autoimmune uveitis (EAU) less severe uveitis in C3aR1/C5aR1-double KO mice than control mice was evident, involving reduced T cell response.2 
Indeed, anaphylatoxin receptor signaling subsequently recruits immune cells and promotes the pro-inflammatory surrounding, which is considered the most important effect of complement dysregulation in AMD.51,52 Nevertheless, their signaling is also indispensable for tissue homeostasis and regeneration. Therefore, genetic compensatory mechanisms, as previously reported,53 due to full body KO used in this study, should be taken into account, when interpreting the study data. In vitro analyses of RPE cells suggest a combination of signaling between C3aR and C5aR1 in order to implement their precise immune regulatory functions.54 Future work will also be required to investigate the interplay among anaphylatoxin receptor signaling, complement activation, and MAC formation. 
Conclusions
Here, we tested full body KO mice of either C3ar1 or C5ar1 anaphylatoxin receptor in a mouse model mimicking some features of dry AMD. A partial rescue of retinal thickness was shown in C3aR1 KO mice, which was mirrored by a significantly less MAC occurrence in the outer retina. Furthermore, a tendency of a more regulated alternative complement pathway and inflammation was given. However, none of the tested conditions led to mitigated mononuclear phagocyte migration to the subretinal space. Whereas C3aR1-targeted therapy may be considered for patients with dry AMD, further work will be necessary to elucidate the optimal intervention point in the anaphylatoxin signaling axis. Furthermore, heterogeneity of disease progression may point to more patient-tailored therapy strategies, favoring from advanced technologies and imaging modalities. 
Acknowledgments
The authors thank Eva Scheiffert and Claudia Bismar for their expertise and technical assistance in animal experiments. 
Supported by the Helmut Ecker Foundation (03/17); the Deutsche Forschungsgemeinschaft (FOR2240, Project 6); and the Center for Molecular Medicine Cologne. 
Disclosure: V. Behnke, None; A. Wolf, None; M. Hector, None; T. Langmann, None 
References
Anderson DH, Radeke MJ, Gallo NB, et al. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited. Prog Retin Eye Res. 2010; 29: 95–112. [CrossRef] [PubMed]
Zhang L, Bell BA, Yu M, et al. Complement anaphylatoxin receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis. J Leukoc Biol. 2016; 99: 447–454. [CrossRef] [PubMed]
Engelke C, Wiese AV, Schmudde I, et al. Distinct roles of the anaphylatoxins C3a and C5a in dendritic cell–mediated allergic asthma. J Immunol. 2014; 193: 5387–5401. [CrossRef] [PubMed]
Yu M, Zou W, Peachey NS, McIntyre TM, Liu J. A novel role of complement in retinal degeneration. Invest Ophthalmol Vis Sci. 2012; 53: 7684–7692. [CrossRef] [PubMed]
Gasque P, Singhrao SK, Neal JW, et al. The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis. J Immunol. 1998; 160: 3543–3554. [CrossRef] [PubMed]
Gerard C, Bao L, Orozco O, Pearson M, Kunz D, Gerard NP. Structural diversity in the extracellular faces of peptidergic G-protein- coupled receptors: molecular cloning of the mouse C5a anaphylatoxin receptor. J Immunol. 1992; 149: 2600–2606. [CrossRef] [PubMed]
Quell KM, Karsten CM, Kordowski A, et al. Monitoring C3aR expression using a floxed tdTomato-C3aR reporter knock-in mouse. J Immunol. 2017; 199: 688–706. [CrossRef] [PubMed]
Laumonnier Y, Karsten CM, Köhl J. Novel insights into the expression pattern of anaphylatoxin receptors in mice and men. Mol Immunol. 2017; 89: 44–58. [CrossRef] [PubMed]
Soruri A, Kim S, Kiafard Z, Zwirner J. Characterization of C5aR expression on murine myeloid and lymphoid cells by the use of a novel monoclonal antibody. Immunol Lett. 2003; 88: 47–52. [CrossRef] [PubMed]
Ames RS, Li Y, Sarau HM, et al. Molecular cloning and characterization of the human anaphylatoxin C3a receptor. J Biol Chem. 1996; 271: 20231–20234. [CrossRef] [PubMed]
Brandstetter C, Holz FG, Krohne TU. Complement component C5a primes retinal pigment epithelial cells for inflammasome activation by lipofuscin-mediated photooxidative damage. J Biol Chem. 2015; 290: 31189–31198. [CrossRef] [PubMed]
Nozaki M, Raisler BJ, Sakurai E, et al. Drusen complement components C3a and C5a promote choroidal neovascularization. Proc Natl Acad Sci USA. 2006; 103: 2328–2333. [CrossRef] [PubMed]
Luo C, Chen M, Xu H. Complement gene expression and regulation in mouse retina and retinal pigment epithelium/choroid. Mol Vis. 2011; 17: 1588–1597. [PubMed]
Priller J, Flügel A, Wehner T, et al. Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment. Nat Med. 2001; 7: 1356–1361. [CrossRef] [PubMed]
Karlstetter M, Scholz R, Rutar M, Wong WT, Provis JM, Langmann T. Retinal microglia: just bystander or target for therapy? Prog Retin Eye Res. 2015; 45: 30–57. [CrossRef] [PubMed]
Karlstetter M, Langmann T. Microglia in the aging retina. In: Advances in Experimental Medicine and Biology. Vol. 801. New York, NY: Springer; 2014: 207–212.
Gupta N, Brown KE, Milam AH. Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration. Exp Eye Res. 2003; 76: 463–471. [CrossRef] [PubMed]
Levy O, Calippe B, Lavalette S, et al. Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration. EMBO Mol Med. 2015; 7: 211–226. [CrossRef] [PubMed]
Combadière C, Feumi C, Raoul W, et al. CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration. J Clin Invest. 2007; 117: 2920–2928. [CrossRef] [PubMed]
Fritsche LG, Igl W, Bailey JNC, et al. A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat Genet. 2016; 48: 134–143. [CrossRef] [PubMed]
Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005; 308: 385–389. [CrossRef] [PubMed]
Qin S, Dong N, Yang M, Wang J, Feng X, Wang Y. Complement inhibitors in age-related macular degeneration: a potential therapeutic option. J Immunol Res. 2021; 2021: 9945725. [PubMed]
Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F. Immortalization of murine microglial cells by a v-raf /v-myc carrying retrovirus. J Neuroimmunol. 1990; 27: 229–237. [CrossRef] [PubMed]
Scholz R, Caramoy A, Bhuckory MB, et al. Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration. J Neuroinflammation. 2015; 12: 201. [CrossRef] [PubMed]
Scholz R, Sobotka M, Caramoy A, Stempfl T, Moehle C, Langmann T. Minocycline counter-regulates pro-inflammatory microglia responses in the retina and protects from degeneration. J Neuroinflammation. 2015; 12: 209. [CrossRef] [PubMed]
Lückoff A, Caramoy A, Scholz R, Prinz M, Kalinke U, Langmann T. Interferon-beta signaling in retinal mononuclear phagocytes attenuates pathological neovascularization. EMBO Mol Med. 2016; 8: 670–678. [CrossRef] [PubMed]
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques. 2000; 29: 52–54. [CrossRef] [PubMed]
O'Koren EG, Yu C, Klingeborn M, et al. Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration. Immunity. 2019; 50: 723–737. [CrossRef] [PubMed]
Bok D. Retinal photoreceptor-pigment epithelium interactions. Invest Ophthalmol Vis Sci. 1985; 26: 1659–1694. [PubMed]
Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005; 85: 845–881. [CrossRef] [PubMed]
Lueck K, Wasmuth S, Williams J, et al. Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration. Eye. 2011; 25: 1074. [CrossRef] [PubMed]
Laudisi F, Spreafico R, Evrard M, et al. Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1β release. J Immunol. 2013; 191: 1006–1010. [CrossRef] [PubMed]
Triantafilou K, Hughes TR, Triantafilou M, Morgan PP. The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation. J Cell Sci. 2013; 126: 2903–2913. [PubMed]
Bird AC, Bressler NM, Bressler SB, et al. An international classification and grading system for age-related maculopathy and age-related macular degeneration. Surv Ophthalmol. 1995; 39: 367–374. [CrossRef] [PubMed]
Crabb JW, Miyagi M, Gu X, et al. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci USA. 2002; 99: 14682–14687. [CrossRef] [PubMed]
Gold B, Merriam JE, Zernant J, et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet. 2006; 38: 458–462. [CrossRef] [PubMed]
Mullins RF, Aptsiauri N, Hageman GS. Structure and composition of drusen associated with glomerulonephritis: Implications for the role of complement activation in drusen biogenesis. Eye. 2001;15: 390–395. [CrossRef] [PubMed]
Mullins RF, Dewald AD, Streb LM, Wang K, Kuehn MH, Stone EM. Elevated membrane attack complex in human choroid with high risk complement factor H genotypes. Exp Eye Res. 2011; 93: 565. [CrossRef] [PubMed]
Mullins RF, Schoo DP, Sohn EH, et al. The membrane attack complex in aging human choriocapillaris: relationship to macular degeneration and choroidal thinning. Am J Pathol. 2014; 184: 3142. [CrossRef] [PubMed]
Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Köhl J. The role of the anaphylatoxins in health and disease. Mol Immunol. 2009; 46: 2753–2766. [CrossRef] [PubMed]
Song D, Sulewski ME, Wang C, et al. Complement C5a receptor knockout has diminished light-induced microglia/macrophage retinal migration. Mol Vis. 2017; 23: 210–218. [PubMed]
Zhang T, Garstka MA, Li K. The controversial C5a receptor C5aR2: its role in health and disease. J Immunol Res. 2017; 2017: 8193932 [PubMed]
Bamberg CE, Mackay CR, Lee H, et al. The C5a receptor (C5aR) C5L2 is a modulator of C5aR-mediated signal transduction. J Biol Chem. 2010; 285: 7633–7644. [CrossRef] [PubMed]
Gerard NP, Lu B, Liu P, et al. An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2. J Biol Chem. 2005; 280: 39677–39680. [CrossRef] [PubMed]
Asgari E, Le Friec G, Yamamoto H, et al. C3a modulates IL-1β secretion in human monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome activation. Blood. 2013; 122: 3473–3481. [CrossRef] [PubMed]
Harder JM, Williams PA, Braine CE, et al. Complement peptide C3a receptor 1 promotes optic nerve degeneration in DBA/2J mice. J Neuroinflammation. 2020; 17: 336. [CrossRef] [PubMed]
Fernandez-Godino R, Pierce EA. C3a triggers formation of sub-retinal pigment epithelium deposits via the ubiquitin proteasome pathway. Sci Rep. 2018; 8: 9679. [CrossRef] [PubMed]
Parsons N, Annamalai B, Obert E, Schnabolk G, Tomlinson S, Rohrer B. Inhibition of the alternative complement pathway accelerates repair processes in the murine model of choroidal neovascularization. Mol Immunol. 2019; 108: 8–12. [CrossRef] [PubMed]
Brennan FH, Gordon R, Lao HW, et al. The complement receptor C5aR controls acute inflammation and astrogliosis following spinal cord injury. J Neurosci. 2015; 35: 6517–6531. [CrossRef] [PubMed]
Pekna M, Stokowska A, Pekny M. Targeting complement C3a receptor to improve outcome after ischemic brain injury. Neurochem Res. 2021; 46: 2626–2637. [CrossRef] [PubMed]
Behnke V, Wolf A, Langmann T. The role of lymphocytes and phagocytes in age-related macular degeneration (AMD). Cell Mol Life Sci. 2020; 77: 781–788. [CrossRef] [PubMed]
Ogura S, Baldeosingh R, Bhutto IA, et al. A role for mast cells in geographic atrophy. FASEB J. 2020; 34: 10117–10131. [CrossRef] [PubMed]
El-Brolosy MA, Stainier DYR. Genetic compensation: a phenomenon in search of mechanisms. PLoS Genet. 2017; 13: e1006780. [CrossRef] [PubMed]
Busch C, Annamalai B, Abdusalamova K, et al. Anaphylatoxins activate Ca2+, Akt/PI3-kinase, and FOXO1/FoxP3 in the retinal pigment epithelium. Front Immunol. 2017; 8: 703. [CrossRef] [PubMed]
Figure 1.
 
Expression analysis of retinal tissue. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. The qRT-PCR was performed and ΔΔCT analysis was used for quantification. ATP5B was used as reference gene. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test (n = WT, KO, light-damaged WT, and light-damaged KO; C3ar1: n = 11, 15, 13–14, 13; TSPO: 6, 11, 8, 13; CFB: 0, 0, 9, 8; C5aR: n = 12–13, 12, 13, 12–13, TSPO: 12, 9, 12, 12; and CFB: 0, 0, 7, 12).
Figure 1.
 
Expression analysis of retinal tissue. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. The qRT-PCR was performed and ΔΔCT analysis was used for quantification. ATP5B was used as reference gene. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test (n = WT, KO, light-damaged WT, and light-damaged KO; C3ar1: n = 11, 15, 13–14, 13; TSPO: 6, 11, 8, 13; CFB: 0, 0, 9, 8; C5aR: n = 12–13, 12, 13, 12–13, TSPO: 12, 9, 12, 12; and CFB: 0, 0, 7, 12).
Figure 2.
 
Morphological and migration analysis of microglia in the retina. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, B, G, H) Mononuclear phagocytes were stained on retinal flat mounts against ionized calcium-binding adapter molecule 1 (Iba1). (C, E, F, I, K, L) Four images of the central retina were taken around the optic nerve of each eye and averaged for 1 n. Cell numbers were counted using the particle analyzer plugin of ImageJ. (C3ar1: n = 11 and C5ar1 n = 13). (D, J) ELISA was performed with full retinal lysates (n = 5, C5ar1 light damage KO: n = 4). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; CCL2, CC-chemokine ligand 2; OPL, outer plexiform layer; SR, subretinal space.
Figure 2.
 
Morphological and migration analysis of microglia in the retina. Retinas were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, B, G, H) Mononuclear phagocytes were stained on retinal flat mounts against ionized calcium-binding adapter molecule 1 (Iba1). (C, E, F, I, K, L) Four images of the central retina were taken around the optic nerve of each eye and averaged for 1 n. Cell numbers were counted using the particle analyzer plugin of ImageJ. (C3ar1: n = 11 and C5ar1 n = 13). (D, J) ELISA was performed with full retinal lysates (n = 5, C5ar1 light damage KO: n = 4). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; CCL2, CC-chemokine ligand 2; OPL, outer plexiform layer; SR, subretinal space.
Figure 3.
 
Spatial analysis of microglia and complement membrane attack complex (MAC) in the eyes. The eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, E) Cryosections were stained with DAPI and against ionized calcium-binding adapter molecule 1 (Iba1). (B, F) Cryosections were stained with DAPI and against MAC (C5b-9). (C, D, G, H) Cell numbers were counted using the multi-point tool of ImageJ. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; SR, subretinal space.
Figure 3.
 
Spatial analysis of microglia and complement membrane attack complex (MAC) in the eyes. The eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, E) Cryosections were stained with DAPI and against ionized calcium-binding adapter molecule 1 (Iba1). (B, F) Cryosections were stained with DAPI and against MAC (C5b-9). (C, D, G, H) Cell numbers were counted using the multi-point tool of ImageJ. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. AF, autofluorescence; GCL, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; SR, subretinal space.
Figure 4.
 
Retinal thickness and OCT analysis. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, F) Representative scans and (B, G) corresponding heatmaps compiled by spectral-domain optical coherence tomography (SD-OCT) displayed retinal overviews. (C) BluePeak autofluorescence (BAF) images depict retinal background. (D, I) Retinal thickness was assessed in circular ring scans (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (µm) of each quadruplet. (C3aRr1: n = 36 and C5ar1: n = 32). (E, J) Percentage of autofluorescent area was measured with ImageJ with the same region of interest for all images (n = 18). Data were analyzed using 1-way ANOVA and Tukey's multiple comparison test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM.
Figure 4.
 
Retinal thickness and OCT analysis. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A, F) Representative scans and (B, G) corresponding heatmaps compiled by spectral-domain optical coherence tomography (SD-OCT) displayed retinal overviews. (C) BluePeak autofluorescence (BAF) images depict retinal background. (D, I) Retinal thickness was assessed in circular ring scans (circle diameters 3 and 6 mm), centered on the optic nerve head, which represents the average retinal thickness (µm) of each quadruplet. (C3aRr1: n = 36 and C5ar1: n = 32). (E, J) Percentage of autofluorescent area was measured with ImageJ with the same region of interest for all images (n = 18). Data were analyzed using 1-way ANOVA and Tukey's multiple comparison test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM.
Figure 5.
 
Apoptosis of photoreceptors in the outer nuclear layer. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A) Apoptotic cells on cryosections were labeled with TUNEL assay and counterstained with DAPI. (B) Cell numbers were counted using the particle analyzer plugin of ImageJ in outer nuclear layer (ONL) specific ROIs, set individually for each image via DAPI staining. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
Figure 5.
 
Apoptosis of photoreceptors in the outer nuclear layer. Eyes were analyzed 4 days after light damage with 10,000 lux for 30 minutes. (A) Apoptotic cells on cryosections were labeled with TUNEL assay and counterstained with DAPI. (B) Cell numbers were counted using the particle analyzer plugin of ImageJ in outer nuclear layer (ONL) specific ROIs, set individually for each image via DAPI staining. Five images of the central retina were taken of each eye and averaged for 1 n (n = 9). Data were analyzed using 1-way ANOVA followed by Tukey’s multiple comparison post-test. Graphs were plotted with GraphPad Prism version 7.04. Bars represent mean ± SEM. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×