Title: The Complement Regulatory Protein CD46 Deficient Mouse Spontaneously Develops Dry-Type Age-Related Macular Degeneration–Like Phenotype
Abstract: In the mouse, membrane cofactor protein (CD46), a key regulator of the alternative pathway of the complement system, is only expressed in the eye and on the inner acrosomal membrane of spermatozoa. We noted that although Cd46−/− mice have normal systemic alternative pathway activating ability, lack of CD46 leads to dysregulated complement activation in the eye, as evidenced by increased deposition of C5b-9 in the retinal pigment epithelium (RPE) and choroid. A knockout of CD46 induced the following cardinal features of human dry age-related macular degeneration (AMD) in 12-month-old male and female mice: accumulation of autofluorescent material in and hypertrophy of the RPE, dense deposits in and thickening of Bruch's membrane, loss of photoreceptors, cells in subretinal space, and a reduction of choroidal vessels. Collectively, our results demonstrate spontaneous age-related degenerative changes in the retina, RPE, and choroid of Cd46−/− mice that are consistent with human dry AMD. These findings provide the exciting possibility of using Cd46−/− mice as a convenient and reliable animal model for dry AMD. Having such a relatively straight-forward model for dry AMD should provide valuable insights into pathogenesis and a test model system for novel drug targets. More important, tissue-specific expression of CD46 gives the Cd46−/− mouse model of dry AMD a unique advantage over other mouse models using knockout strains. In the mouse, membrane cofactor protein (CD46), a key regulator of the alternative pathway of the complement system, is only expressed in the eye and on the inner acrosomal membrane of spermatozoa. We noted that although Cd46−/− mice have normal systemic alternative pathway activating ability, lack of CD46 leads to dysregulated complement activation in the eye, as evidenced by increased deposition of C5b-9 in the retinal pigment epithelium (RPE) and choroid. A knockout of CD46 induced the following cardinal features of human dry age-related macular degeneration (AMD) in 12-month-old male and female mice: accumulation of autofluorescent material in and hypertrophy of the RPE, dense deposits in and thickening of Bruch's membrane, loss of photoreceptors, cells in subretinal space, and a reduction of choroidal vessels. Collectively, our results demonstrate spontaneous age-related degenerative changes in the retina, RPE, and choroid of Cd46−/− mice that are consistent with human dry AMD. These findings provide the exciting possibility of using Cd46−/− mice as a convenient and reliable animal model for dry AMD. Having such a relatively straight-forward model for dry AMD should provide valuable insights into pathogenesis and a test model system for novel drug targets. More important, tissue-specific expression of CD46 gives the Cd46−/− mouse model of dry AMD a unique advantage over other mouse models using knockout strains. Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in individuals >50 years of age in the United States and around the world.1Friedman D.S. O'Colmain B.J. Muñoz B. Tomany S.C. McCarty C. de Jong P.T. Nemesure B. Mitchell P. Kempen J. Prevalence of age-related macular degeneration in the United States.Arch Ophthalmol. 2004; 122: 564-572Crossref PubMed Scopus (77) Google Scholar, 2Coleman H.R. Chan C.C. Ferris III, F.L. Chew E.Y. Age-related macular degeneration.Lancet. 2008; 372: 1835-1845Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Age-related macular degeneration: emerging pathogenetic and therapeutic concepts.Ann Med. 2006; 38: 450-471Crossref PubMed Scopus (488) Google Scholar, 4Christoforidis J.B. Tecce N. Dell'Omo R. Mastropasqua R. Verolino M. Costagliola C. Age related macular degeneration and visual disability.Curr Drug Targets. 2011; 12: 221-233Crossref PubMed Scopus (30) Google Scholar, 5Rein D.B. Wittenborn J.S. Zhang X. Honeycutt A.A. Lesesne S.B. Saaddine J. Vision Health Cost-Effectiveness Study GroupForecasting age-related macular degeneration through the year 2050: the potential impact of new treatments.Arch Ophthalmol. 2009; 127: 533-540Crossref PubMed Scopus (264) Google Scholar This disease causes a progressive destruction of the macula, leading to the loss of central vision. AMD is a chronic degenerative process with multiple risk factors.2Coleman H.R. Chan C.C. Ferris III, F.L. Chew E.Y. Age-related macular degeneration.Lancet. 2008; 372: 1835-1845Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Age-related macular degeneration: emerging pathogenetic and therapeutic concepts.Ann Med. 2006; 38: 450-471Crossref PubMed Scopus (488) Google Scholar, 6Husain D. Ambati B. Adamis A.P. Miller J.W. Mechanisms of age-related macular degeneration.Ophthalmol Clin North Am. 2002; 15: 87-91Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 7Smith W. Assink J. Klein R. Mitchell P. Klaver C.C. Klein B.E. Hofman A. Jensen S. Wang J.J. de Jong P.T. Risk factors for age-related macular degeneration: pooled findings from three continents.Ophthalmology. 2001; 108: 697-704Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar, 8Evans J.R. Risk factors for age-related macular degeneration.Prog Retin Eye Res. 2001; 20: 227-253Crossref PubMed Scopus (285) Google Scholar, 9Khandhadia S. Cherry J. Lotery A.J. Age-related macular degeneration.Adv Exp Med Biol. 2012; 724: 15-36Crossref PubMed Scopus (67) Google Scholar, 10Yates J.R. Moore A.T. Genetic susceptibility to age related macular degeneration.J Med Genet. 2000; 37: 83-87Crossref PubMed Scopus (82) Google Scholar, 11Zarbin M.A. Rosenfeld P.J. Pathway-based therapies for age-related macular degeneration: an integrated survey of emerging treatment alternatives.Retina. 2010; 30: 1350-1367Crossref PubMed Scopus (126) Google Scholar, 12Querques G. Rosenfeld P.J. Cavallero E. Borrelli E. Corvi F. Querques L. Bandello F.M. Zarbin M.A. Treatment of dry age-related macular degeneration.Ophthalmic Res. 2014; 52: 107-115Crossref PubMed Scopus (34) Google Scholar, 13Bora N.S. Jha P. Lyzogubov V.V. Bora P.S. Emerging role of complement in ocular diseases.Curr Immunol Rev. 2011; 7: 360-367Crossref Scopus (4) Google Scholar, 14Bora N.S. Jha P. Bora P.S. The role of complement in ocular pathology.Semin Immunopathol. 2008; 30: 85-95Crossref PubMed Scopus (63) Google Scholar, 15Anderson D.H. Radeke M.J. Gallo N.B. Chapin E.A. Johnson P.T. Curletti C.R. Hancox L.S. Hu J. Ebright J.N. Malek G. Hauser M.A. Rickman C.B. Bok D. Hageman G.S. Johnson L.V. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.Prog Retin Eye Res. 2010; 29: 95-112Crossref PubMed Scopus (585) Google Scholar, 16Johnson L.V. Leitner W.P. Staples M.K. Anderson D.H. Complement activation and inflammatory processes in drusen formation and age related macular degeneration.Exp Eye Res. 2001; 73: 887-896Crossref PubMed Scopus (512) Google Scholar, 17Seddon J.M. Yu Y. Miller E.C. Reynolds R. Tan P.L. Gowrisankar S. Goldstein J.I. Triebwasser M. Anderson H.E. Zerbib J. Kavanagh D. Souied E. Katsanis N. Daly M.J. Atkinson J.P. Raychaudhuri S. Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration.Nat Genet. 2013; 45: 1366-1370Crossref PubMed Scopus (253) Google Scholar Nearly two million individuals in the United States alone are currently afflicted with AMD. This number is expected to grow in part because of increasing life expectancy.5Rein D.B. Wittenborn J.S. Zhang X. Honeycutt A.A. Lesesne S.B. Saaddine J. Vision Health Cost-Effectiveness Study GroupForecasting age-related macular degeneration through the year 2050: the potential impact of new treatments.Arch Ophthalmol. 2009; 127: 533-540Crossref PubMed Scopus (264) Google Scholar Clinically, AMD is usually classified into two forms—nonexudative (dry type) and exudative (wet type). Although the dry form of AMD is more prevalent (approximately 85% of the cases) and a precursor to wet AMD, the fundamental processes underlying dry AMD are particularly not well understood.2Coleman H.R. Chan C.C. Ferris III, F.L. Chew E.Y. Age-related macular degeneration.Lancet. 2008; 372: 1835-1845Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Age-related macular degeneration: emerging pathogenetic and therapeutic concepts.Ann Med. 2006; 38: 450-471Crossref PubMed Scopus (488) Google Scholar, 4Christoforidis J.B. Tecce N. Dell'Omo R. Mastropasqua R. Verolino M. Costagliola C. Age related macular degeneration and visual disability.Curr Drug Targets. 2011; 12: 221-233Crossref PubMed Scopus (30) Google Scholar, 6Husain D. Ambati B. Adamis A.P. Miller J.W. Mechanisms of age-related macular degeneration.Ophthalmol Clin North Am. 2002; 15: 87-91Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 7Smith W. Assink J. Klein R. Mitchell P. Klaver C.C. Klein B.E. Hofman A. Jensen S. Wang J.J. de Jong P.T. Risk factors for age-related macular degeneration: pooled findings from three continents.Ophthalmology. 2001; 108: 697-704Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar, 8Evans J.R. Risk factors for age-related macular degeneration.Prog Retin Eye Res. 2001; 20: 227-253Crossref PubMed Scopus (285) Google Scholar, 9Khandhadia S. Cherry J. Lotery A.J. Age-related macular degeneration.Adv Exp Med Biol. 2012; 724: 15-36Crossref PubMed Scopus (67) Google Scholar, 10Yates J.R. Moore A.T. Genetic susceptibility to age related macular degeneration.J Med Genet. 2000; 37: 83-87Crossref PubMed Scopus (82) Google Scholar Several agents to treat dry AMD are currently in the developmental stage. However, no effective treatment option is currently available.11Zarbin M.A. Rosenfeld P.J. Pathway-based therapies for age-related macular degeneration: an integrated survey of emerging treatment alternatives.Retina. 2010; 30: 1350-1367Crossref PubMed Scopus (126) Google Scholar, 12Querques G. Rosenfeld P.J. Cavallero E. Borrelli E. Corvi F. Querques L. Bandello F.M. Zarbin M.A. Treatment of dry age-related macular degeneration.Ophthalmic Res. 2014; 52: 107-115Crossref PubMed Scopus (34) Google Scholar Novel therapeutic targets for dry AMD need to be discovered. Studies during the past decade have shown that the alternative pathway (AP) of the complement system is a critical player in AMD pathogenesis. A simple interpretation of the accumulating data from animal and especially human studies is that in AMD there is overactivation of the AP, leading to adverse consequences.1Friedman D.S. O'Colmain B.J. Muñoz B. Tomany S.C. McCarty C. de Jong P.T. Nemesure B. Mitchell P. Kempen J. Prevalence of age-related macular degeneration in the United States.Arch Ophthalmol. 2004; 122: 564-572Crossref PubMed Scopus (77) Google Scholar, 2Coleman H.R. Chan C.C. Ferris III, F.L. Chew E.Y. Age-related macular degeneration.Lancet. 2008; 372: 1835-1845Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Gehrs K.M. Anderson D.H. Johnson L.V. Hageman G.S. Age-related macular degeneration: emerging pathogenetic and therapeutic concepts.Ann Med. 2006; 38: 450-471Crossref PubMed Scopus (488) Google Scholar, 4Christoforidis J.B. Tecce N. Dell'Omo R. Mastropasqua R. Verolino M. Costagliola C. Age related macular degeneration and visual disability.Curr Drug Targets. 2011; 12: 221-233Crossref PubMed Scopus (30) Google Scholar, 6Husain D. Ambati B. Adamis A.P. Miller J.W. Mechanisms of age-related macular degeneration.Ophthalmol Clin North Am. 2002; 15: 87-91Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 13Bora N.S. Jha P. Lyzogubov V.V. Bora P.S. Emerging role of complement in ocular diseases.Curr Immunol Rev. 2011; 7: 360-367Crossref Scopus (4) Google Scholar, 14Bora N.S. Jha P. Bora P.S. The role of complement in ocular pathology.Semin Immunopathol. 2008; 30: 85-95Crossref PubMed Scopus (63) Google Scholar, 15Anderson D.H. Radeke M.J. Gallo N.B. Chapin E.A. Johnson P.T. Curletti C.R. Hancox L.S. Hu J. Ebright J.N. Malek G. Hauser M.A. Rickman C.B. Bok D. Hageman G.S. Johnson L.V. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.Prog Retin Eye Res. 2010; 29: 95-112Crossref PubMed Scopus (585) Google Scholar, 16Johnson L.V. Leitner W.P. Staples M.K. Anderson D.H. Complement activation and inflammatory processes in drusen formation and age related macular degeneration.Exp Eye Res. 2001; 73: 887-896Crossref PubMed Scopus (512) Google Scholar, 17Seddon J.M. Yu Y. Miller E.C. Reynolds R. Tan P.L. Gowrisankar S. Goldstein J.I. Triebwasser M. Anderson H.E. Zerbib J. Kavanagh D. Souied E. Katsanis N. Daly M.J. Atkinson J.P. Raychaudhuri S. Rare variants in CFI, C3 and C9 are associated with high risk of advanced age-related macular degeneration.Nat Genet. 2013; 45: 1366-1370Crossref PubMed Scopus (253) Google Scholar, 18Bora P.S. Sohn J.H. Cruz J.M. Jha P. Nishihori H. Wang Y. Kaliappan S. Kaplan H.J. Bora N.S. Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization.J Immunol. 2005; 174: 491-497Crossref PubMed Scopus (221) Google Scholar, 19Bora N.S. Kaliappan S. Jha P. Cu Q. Sohn J.H. Dhaulakhandi D.B. Kaplan H.J. Bora P.S. Complement activation via alternative pathway is critical in the development of laser-induced choroidal neovascularization: role of factor B and factor H.J Immunol. 2006; 177: 1872-1878Crossref PubMed Scopus (118) Google Scholar, 20Bora N.S. Kaliappan S. Jha P. Xu Q. Sivasankar B. Harris C.L. Morgan B.P. Bora P.S. CD59, a complement regulatory protein, controls choroidal neovascularization in a mouse model of wet-type age-related macular degeneration.J Immunol. 2007; 178: 1783-1790Crossref PubMed Scopus (86) Google Scholar, 21Kaliappan S. Jha P. Lyzogubov V.V. Tytarenko R.G. Bora N.S. Bora P.S. Alcohol and nicotine consumption exacerbates choroidal neovascularization by modulating the regulation of complement system.FEBS Lett. 2008; 582: 3451-3458Crossref PubMed Scopus (20) Google Scholar, 22Bora N.S. Jha P. Lyzogubov V.V. Kaliappan S. Liu J. Tytarenko R.G. Fraser D.A. Morgan B.P. Bora P.S. Recombinant membrane-targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice.J Biol Chem. 2010; 285: 33826-33833Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 23Lyzogubov V.V. Tytarenko R.G. Jha P. Liu J. Bora N.S. Bora P.S. 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Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46).J Biol Chem. 2000; 275: 37692-37701Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Recently, we reported that CD46 is expressed in the neurosensory retina, retinal pigment epithelium (RPE), and choroid of the mouse eye.28Lyzogubov V. Wu X. Jha P. Tytarenko R. Triebwasser M. Kolar G. Bertram P. Bora P.S. Atkinson J.P. Bora N.S. Complement regulatory protein CD46 protects against choroidal neovascularization in mice.Am J Pathol. 2014; 184: 2537-2548Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Our goal was to investigate if the Cd46−/− mouse spontaneously develops a phenotype that closely resembles the dry form of human AMD. Herein, we describe that Cd46−/− male and female mice have normal systemic AP activating ability, but the lack of ocular CD46 leads to dysregulated complement activation in the retina and choroid. Furthermore, aged Cd46−/− mice spontaneously develop cardinal features of human dry AMD. We have previously described the generation of a mouse with homozygous deficiency of Cd46 (Cd46−/−) on the C57BL/6 background.28Lyzogubov V. Wu X. Jha P. Tytarenko R. Triebwasser M. Kolar G. Bertram P. Bora P.S. Atkinson J.P. Bora N.S. Complement regulatory protein CD46 protects against choroidal neovascularization in mice.Am J Pathol. 2014; 184: 2537-2548Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar C3−/−, Cfb−/−, and Cfp−/− mice were generated, as previously reported.33Stover C.M. Luckett J.C. Echtenacher B. Dupont A. Figgitt S.E. Brown J. Männel D.N. Schwaeble W.J. Properdin plays a protective role in polymicrobial septic peritonitis.J Immunol. 2008; 180: 3313-3318Crossref PubMed Scopus (68) Google Scholar, 34Circolo A. Garnier G. Fukuda W. Wang X. Hidvegi T. Szalai A.J. Briles D.E. Volanakis J.E. Wetsel R.A. Colten H.R. Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.Immunopharmacology. 1999; 42: 135-149Crossref PubMed Scopus (146) Google Scholar, 35Matsumoto M. Fukuda W. Circolo A. Goellner J. Strauss-Schoenberger J. Wang X. Fujita S. Hidvegi T. Chaplin D.D. Colten H.R. Abrogation of the alternative complement pathway by targeted deletion of murine factor B.Proc Natl Acad Sci U S A. 1997; 94: 8720-8725Crossref PubMed Scopus (176) Google Scholar Mice were bred and housed in a pathogen-free, temperature-controlled environment. Genotyping was performed by PCR analysis using tail-derived DNA. The following primers were used in PCR analysis: CD46, 5′-ATGCCTGTGAACTACCACGGCCATTTGAAG-3′ (forward) and 5′-AACTTTAATATAGCTCCAGTGCCAGTTGCA-3′ (reverse); Neo, 5′-AACAGACAATCGGCTGCTCTGATG-3′ (forward) and 5′-GCTCTTCGTCCAGATCATCCTGATCG-3′ (reverse); C3, 5′-GATCCCCAGAGCTAATG-3′ (V787) and 5′-AGGGACCAGCCCAGGTTCAG-3′ (V789); Neo, 5′-TCGTCCTGCAGTTCATTCAG-3′ (V788); FB, 5′-CCGAAGCATTCCTATCCTCC-3′ (forward 1), 5′-GTAGTCTTGTCTGCTTTCTCC-3′ (reverse 1), and 5′-CGAATGGGTGACCGCTTCC-3′ (Neo). For properdin (P), the following neoprimers were initially used in PCR analysis: Neo F371, 5′-AACAGACAATCGGCTGCTCTGATG-3′; Neo R779, 5′-GCTCTTCGTCCAGATCATCCTGATCG-3′. Western blotting using rabbit anti-mouse properdin36Bertram P. Akk A.M. Zhou H.F. Mitchell L.M. Pham C.T. Hourcade D.E. Anti-mouse properdin TSR 5/6 monoclonal antibodies block complement alternative pathway-dependent pathogenesis.Monoclon Antib Immunodiagn Immunother. 2015; 34: 1-6Crossref PubMed Scopus (12) Google Scholar was used to establish the absence of properdin in the Cfp−/− mouse. Protocols for animal breeding, housing, and handling were approved by the Division of Comparative Medicine at Washington University (St. Louis, MO) and Institutional Animal Care and Use Committee, University of Arkansas for Medical Sciences (Little Rock, AR). All mice were kept under a 12-hour dark and 12-hour light cycle with ad libitum access to food and water. Homozygous Cd46 knockout mice were backcrossed into C57BL/6 for at least eight generations before use. Male and female C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and served as a wild-type control. Two-month-old (adult) and 12-month-old (aged) mice were used. The eyes from the following eight groups of mice were analyzed: group 1, wild type (WT) male, 2-month-old; group 2, WT female, 2-month-old; group 3, Cd46−/− male, 2-month-old; group 4, Cd46−/− female, 2-month-old; group 5, WT male, 12-month-old; group 6, WT female, 12-month-old; group 7, Cd46−/− male, 12-month-old; and group 8, Cd46−/− female, 12-month-old. Eyes for histological investigation were processed as described.24Lyzogubov V.V. Tytarenko R.G. Liu J. Bora N.S. Bora P.S. Polyethylene glycol (PEG)-induced mouse model of choroidal neovascularization.J Biol Chem. 2011; 286: 16229-16237Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 26Lyzogubov V.V. Bora N.S. Tytarenko R.G. Bora P.S. Polyethylene glycol induced mouse model of retinal degeneration.Exp Eye Res. 2014; 127: 143-152Crossref PubMed Scopus (14) Google Scholar, 28Lyzogubov V. Wu X. Jha P. Tytarenko R. Triebwasser M. Kolar G. Bertram P. Bora P.S. Atkinson J.P. Bora N.S. Complement regulatory protein CD46 protects against choroidal neovascularization in mice.Am J Pathol. 2014; 184: 2537-2548Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Animals were sacrificed between 4 and 7 hours after the light was turned on using carbon dioxide inhalation, and the eyes were harvested immediately. One eye from each animal was fixed in 4% buffered paraformaldehyde (pH 7.4 in 0.05 mol/L phosphate-buffered saline) and embedded in paraffin. Three composite blocks were formed to contain two eyes from each group (total six eyes from each group) and serial sections (5 μm thick) were cut. Paraffin sections were stained with hematoxylin and eosin or were subsequently used in immunofluorescence (IF) analysis as described below. One eye from each animal was fixed with 2.5% glutaraldehyde (Polysciences Inc., Warrington, PA) for 1 hour. The anterior part of the eye (including the ciliary body and lens) was removed, whereas the posterior part of the eye was fixed in 2.5% glutaraldehyde for additional 3 hours. Samples were washed in 7.5% sucrose overnight. The posterior part of the eye was sliced in two parts by a sagittal cut close to the optic nerve and post fixed in 1% osmium tetroxide (Polysciences Inc.) in 0.05 mol/L phosphate-buffered saline for 1 hour at room temperature. Samples were then treated with saturated solution of uranyl acetate (Polysciences Inc.) prepared in 50% ethanol for 1 hour at room temperature. Ethanol was used to dehydrate the tissue. Electron microscopy grade acetone (Polysciences Inc.) was used as an intermediate medium between ethanol and resin. The Embed 812 epon resin kit (Electron Microscopy Sciences, Hatfield, PA) was used for embedding. All samples were oriented with sagittal slice facing the cutting surface in silicon molds before polymerization (24 hours at 45°C and 24 hours at 57°C). Semithin (1 μm thick) sections from each plastic block (two blocks per eye) were cut and stained using Toluidine Blue and Basic Fuchsin (Electron Microscopy Sciences) for 1 minute at 60°C, dried, and embedded in Canada balsam (Alfa Aesar, Heysham, England). These stains differentiate basophilic structures (pink) and nuclei (blue). Semithin (1 μm thick) sections were used for light microscopy, whereas ultrathin (60 to 80 nm thick) sections were used for electron microscopy. Serial sections of composite blocks were used in IF studies for membrane attack complex (MAC; C5b-9) and detection of autofluorescence. Using sections containing two eyes from each of the eight groups generated identical conditions for each step and all samples were processed at the same time. Sections were deparaffinized, hydrated, and treated with antigen unmasking solution (Vector Laboratories, Burlingame, CA). For detection of MAC, sections were treated with rabbit polyclonal anti-MAC (C9 neoepitope) antibody (Ab; primary Ab) provided by Prof. B. P. Morgan (University of Wales College of Medicine, Cardiff, UK), followed by AF488-conjugated donkey anti-rabbit IgG (H + L) (Molecular Probes, Eugene, OR). To identify RPE cells on the same sections, we used DyLight 649 conjugated mouse monoclonal anti-RPE65 (IgG1 κ) from Novus Biological (Littleton, CO). Negative control sections were stained with an isotype-matched control Ab at identical concentrations to those of the primary Ab. To reduce autofluorescence, we treated paraffin sections with 1% Sudan Black after immunostaining as described.23Lyzogubov V.V. Tytarenko R.G. Jha P. Liu J. Bora N.S. Bora P.S. Role of ocular complement factor H in a murine model of choroidal neovascularization.Am J Pathol. 2010; 177: 1870-1880Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar To investigate autofluorescence in RPE, sections (not treated with Sudan Black) were mounted in ProLong antifade reagent with DAPI (Invitrogen, Grand Island, NY). Images were captured using the laser confocal microscope LSM510. Beam Splitters were set up as follows: 405-nm laser (10%) window, 20 to 480 nm; 488-nm laser (10%) window, 505 to 530 nm; and 561-nm laser (15%) window, 575 to 615 nm. Eight-bit images were obtained using the microscope in sequential mode with line average of eight and format of 1024 × 1024 pixels for autofluorescence investigation and 512 × 512 pixels for C5b-9 IF. We captured a single 1-μm optical slice of each section stained for C5b-9 using the Plan-Apochromat 40×/1.4 or 63×/1.4 oil differential interference contrast objectives. We obtained a 3-μm optical slice for investigation of autofluorescence using Plan-Apochromat 63×/1.4 oil differential interference contrast objective. All images were captured with the same settings. Differential interference contrast images were captured to facilitate localization of histological structures of the eye. Intensity of C5b-9 positive staining on RPE and choroid was observed by two independent investigators in a masked manner. Integrated (total) intensity of the signal was used to evaluate C5b-9 positive fluorescence. C5b-9 positive fluorescence (green channel) was measured using ImageJ software version 1.50b (NIH, Bethesda, MD) in RPE-choroid. Autofluorescence (red channel) of paraffin sections was measured using ImageJ in RPE65 positive structures. To investigate choroidal vasculature, WT male and Cd46−/− male mice (at 2 and 12 months of age) were first anesthetized with ketamine/xylazine cocktail. Animals were then perfused (through the heart) with 0.75 mL of phosphate-buffered saline containing 50 mg/mL of fluorescein isothiocyanate (FITC)-labeled dextran (2 × 106 molecular weight; Sigma, St. Louis, MO) before they were sacrificed.18Bora P.S. Sohn J.H. Cruz J.M. Jha P. Nishihori H. Wang Y. Kaliappan S. Kaplan H.J. Bora N.S. Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization.J Immunol. 2005; 174: 491-497Crossref PubMed Scopus (221) Google Scholar, 19Bora N.S. Kaliappan S. Jha P. Cu Q. Sohn J.H. Dhaulakhandi D.B. Kaplan H.J. Bora P.S. Complement activation via alternative pathway is critical in the development of laser-induced choroidal neovascularization: role of factor B and factor H.J Immunol. 2006; 177: 1872-1878Crossref PubMed Scopus (118) Google Scholar, 20Bora N.S. Kaliappan S. Jha P. Xu Q. Sivasankar B. Harris C.L. Morgan B.P. Bora P.S. CD59, a complement regulatory protein, controls choroidal neovascularization in a mouse model of wet-type age-related macular degeneration.J Immunol. 2007; 178: 1783-1790Crossref PubMed Scopus (86) Google Scholar, 28Lyzogubov V. Wu X. Jha P. Tytarenko R. Triebwasser M. Kolar G. Bertram P. Bora P.S. Atkinson J.P. Bora N.S. Complement regulatory protein CD46 protects against choroidal neovascularization in mice.Am J Pathol. 2014; 184: 2537-2548Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Harvested eyes were dissected, and the posterior part of the eye was gently scratched with a bine brush to destroy RPE cells and remove pigment. Choroid-sclera complexes were flat mounted in ProLong antifade reagent with DAPI (Invitrogen). Z-stack images of flat mounts were captured (in a consistent location), and a three-dimensional model of choroidal vasculature was built using ZEN software version 2009