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• W3036691716 abstract "Myopia is a leading cause of visual impairment worldwide. This sight-compromising condition is associated with scleral thinning, extracellular matrix remodeling, and inappropriate optical axial length elongation. Although macrophages are present in the sclera, their involvement in this condition is unknown. By using a form-deprivation myopia (FDM) mouse model, we found that both the scleral macrophage density and their matrix metalloproteinase-2 (MMP-2) expression levels increased in myopic eyes. Partial scleral macrophage depletion by clodronate shifted the refraction toward hyperopia in both the form-deprived and the untreated fellow eyes compared with their respective counterparts in the vehicle-injected control mice. However, this procedure did not alter susceptibility to FDM. FDM development was 59% less in the macrophage-specific Mmp2 deletion (LysMCreMmp-2fl/fl) mice than in their Cre-negative littermates (Mmp2fl/fl mice). Moreover, the expression of scleral C-C motif chemokine ligand-2 (CCL2), which is a potent monocyte chemoattractant recruiting monocytes to tissue sites, was increased during myopia progression. However, the increase in the density of scleral macrophages and myopia development were suppressed in fibroblast-specific Ccl2 deletion mice. These declines suggested that the increase in scleral macrophage density in myopic eyes stems from the up-regulation of scleral Ccl2 expression in fibroblasts, which, in turn, promotes monocytes recruitment. In summary, scleral monocyte–derived macrophages contribute to myopia development through enhancing MMP-2 expression in mice. Myopia is a leading cause of visual impairment worldwide. This sight-compromising condition is associated with scleral thinning, extracellular matrix remodeling, and inappropriate optical axial length elongation. Although macrophages are present in the sclera, their involvement in this condition is unknown. By using a form-deprivation myopia (FDM) mouse model, we found that both the scleral macrophage density and their matrix metalloproteinase-2 (MMP-2) expression levels increased in myopic eyes. Partial scleral macrophage depletion by clodronate shifted the refraction toward hyperopia in both the form-deprived and the untreated fellow eyes compared with their respective counterparts in the vehicle-injected control mice. However, this procedure did not alter susceptibility to FDM. FDM development was 59% less in the macrophage-specific Mmp2 deletion (LysMCreMmp-2fl/fl) mice than in their Cre-negative littermates (Mmp2fl/fl mice). Moreover, the expression of scleral C-C motif chemokine ligand-2 (CCL2), which is a potent monocyte chemoattractant recruiting monocytes to tissue sites, was increased during myopia progression. However, the increase in the density of scleral macrophages and myopia development were suppressed in fibroblast-specific Ccl2 deletion mice. These declines suggested that the increase in scleral macrophage density in myopic eyes stems from the up-regulation of scleral Ccl2 expression in fibroblasts, which, in turn, promotes monocytes recruitment. In summary, scleral monocyte–derived macrophages contribute to myopia development through enhancing MMP-2 expression in mice. Worldwide, myopia is the most prevalent refractive error.1Holden B.A. Fricke T.R. Wilson D.A. Jong M. Naidoo K.S. Sankaridurg P. Wong T.Y. Naduvilath T.J. Resnikoff S. Global prevalence of myopia and high myopia and temporal trends from 2000 through 2050.Ophthalmology. 2016; 123: 1036-1042Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar,2Dolgin E. The myopia boom.Nature. 2015; 519: 276-278Crossref PubMed Scopus (321) Google Scholar In less severe cases, distance vision is blurred, whereas more severe cases can lead to a variety of sight-threatening impairments, including retinal detachment, cataract, subretinal neovascularization, glaucoma, and even blindness.3Ikuno Y. Overview of the complications of high myopia.Retina. 2017; 37: 2347-2351Crossref PubMed Scopus (107) Google Scholar Although orthokeratology and low concentrations of atropine may slow myopia progression in children, each of these treatments can have severe adverse effects.4Leo S.W. Scientific Bureau of World Society of Paediatric Ophthalmology and Strabismus (WSPOS)Current approaches to myopia control.Curr Opin Ophthalmol. 2017; 28: 267-275Crossref PubMed Scopus (38) Google Scholar These limitations are prompting efforts to identify novel targets whose modulation can potentially improve therapeutic management of this condition in a clinical setting. In human myopia and mammalian myopia models, excessive axial length (AL) elongation and increases in vitreous chamber depth (VCD) accompany declines in collagen biosynthesis and increases in proteolysis of the collagenous structural framework and extracellular matrix (ECM) remodeling in the sclera, particularly at the posterior region of the eye.5Rada J.A. Shelton S. Norton T.T. The sclera and myopia.Exp Eye Res. 2006; 82: 185-200Crossref PubMed Scopus (382) Google Scholar Scleral matrix metalloproteinase-2 (MMP-2) up-regulation is one of the proteolytic changes contributing to ECM remodeling.6Zhao F. Zhou Q. Reinach P.S. Yang J. Ma L. Wang X. Wen Y. Srinivasalu N. Qu J. Zhou X. Cause and effect relationship between changes in scleral matrix metallopeptidase-2 expression and myopia development in mice.Am J Pathol. 2018; 188: 1754-1767Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 7Guggenheim J.A. McBrien N.A. Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew.Invest Ophthalmol Vis Sci. 1996; 37: 1380-1395PubMed Google Scholar, 8Jones B.E. Thompson E.W. Hodos W. Waldbillig R.J. Chader G.J. Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation.Exp Eye Res. 1996; 63: 369-381Crossref PubMed Scopus (38) Google Scholar, 9Rada J.A. Perry C.A. Slover M.L. Achen V.R. Gelatinase A and TIMP-2 expression in the fibrous sclera of myopic and recovering chick eyes.Invest Ophthalmol Vis Sci. 1999; 40: 3091-3099PubMed Google Scholar, 10Siegwart J.T. Norton T.T. The time course of changes in mRNA levels in tree shrew sclera during induced myopia and recovery.Invest Ophthalmol Vis Sci. 2002; 43: 2067-2075PubMed Google Scholar, 11Qian L. Zhao H. Li X. Yin J. Tang W. Chen P. Wang Q. Zhang J. Pirenzepine inhibits myopia in guinea pig model by regulating the balance of MMP-2 and TIMP-2 expression and increased tyrosine hydroxylase levels.Cell Biochem Biophys. 2015; 71: 1373-1378Crossref PubMed Scopus (20) Google Scholar It is a member of the zinc-dependent endopeptidase family, which degrades type I collagen and most of the ECM components under both physiological and pathologic conditions. Fibroblasts, which are the predominant scleral cell type, contribute to MMP-2 up-regulation and type I collagen degradation in a form-deprivation myopia (FDM) mouse model.6Zhao F. Zhou Q. Reinach P.S. Yang J. Ma L. Wang X. Wen Y. Srinivasalu N. Qu J. Zhou X. Cause and effect relationship between changes in scleral matrix metallopeptidase-2 expression and myopia development in mice.Am J Pathol. 2018; 188: 1754-1767Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Despite such increases, myopia was suppressed by only 27% in the fibroblast-specific Mmp2 deletion mice.6Zhao F. Zhou Q. Reinach P.S. Yang J. Ma L. Wang X. Wen Y. Srinivasalu N. Qu J. Zhou X. Cause and effect relationship between changes in scleral matrix metallopeptidase-2 expression and myopia development in mice.Am J Pathol. 2018; 188: 1754-1767Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar One possible explanation for this relatively small inhibition is that there are other cell types besides fibroblasts contributing to scleral MMP-2 up-regulation. This supposition was in accord with reports describing the presence of a large number of macrophages in the scleras of humans12Schlereth S.L. Kremers S. Schrödl F. Cursiefen C. Heindl L.M. Characterization of antigen-presenting macrophages and dendritic cells in the healthy human sclera.Invest Ophthalmol Vis Sci. 2016; 57: 4878-4885Crossref PubMed Scopus (17) Google Scholar,13Schlereth S.L. Neuser B. Caramoy A. Grajewski R.S. Koch K.R. Schrödl F. Cursiefen C. Heindl L.M. Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-positive macrophages around blood vessels in the normal human sclera.Invest Ophthalmol Vis Sci. 2014; 55: 865-872Crossref PubMed Scopus (28) Google Scholar and rodents,14Xu H. Chen M. Reid D.M. Forrester J.V. LYVE-1-positive macrophages are present in normal murine eyes.Invest Ophthalmol Vis Sci. 2007; 48: 2162-2171Crossref PubMed Scopus (80) Google Scholar especially at the posterior region. In other tissues, these immune cells play important roles in ECM remodeling and tissue homeostasis under both physiological and pathologic conditions.15Wynn T.A. Barron L. Macrophages: master regulators of inflammation and fibrosis.Semin Liver Dis. 2010; 30: 245-257Crossref PubMed Scopus (811) Google Scholar, 16Krzyszczyk P. Schloss R. Palmer A. Berthiaume F. The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes.Front Physiol. 2018; 9: 419Crossref PubMed Scopus (280) Google Scholar, 17Mahdavian Delavary B. van der Veer W.M. van Egmond M. Niessen F.B. Beelen R.H. Macrophages in skin injury and repair.Immunobiology. 2011; 216: 753-762Crossref PubMed Scopus (424) Google Scholar Therefore, macrophages may contribute to myopia progression because MMP-2 up-regulation is associated with increases in their infiltration in some other tissues.18Aoki T. Kataoka H. Morimoto M. Nozaki K. Hashimoto N. Macrophage-derived matrix metalloproteinase-2 and -9 promote the progression of cerebral aneurysms in rats.Stroke. 2007; 38: 162-169Crossref PubMed Scopus (208) Google Scholar,19Oviedo-Orta E. Bermudez-Fajardo A. Karanam S. Benbow U. Newby A.C. Comparison of MMP-2 and MMP-9 secretion from T helper 0, 1 and 2 lymphocytes alone and in coculture with macrophages.Immunology. 2008; 124: 42-50Crossref PubMed Scopus (52) Google Scholar Herein, we show that scleral macrophages are another MMP-2 source because increases in their density parallel increases in MMP-2 expression during myopia progression in a well-established FDM mouse model. These increases are driven by the up-regulation of C-C motif chemokine ligand-2 (Ccl2) expression in fibroblast. This chemokine, in turn, induces infiltration of circulating monocytes into the sclera, and they subsequently differentiate into MMP-2–secreting macrophages. Taken together, scleral monocyte–derived macrophages contribute to scleral MMP-2 up-regulation during myopia progression. All animals were bred in the animal breeding unit at Wenzhou Medical University (Wenzhou, China) and raised in standard mouse cages at 22°C ± 2°C with a 12-hour light/12-hour dark cycle. The lights were turned on at 8:30 am every day. During the light phase, the luminance was approximately 100 to 200 lux. All animal experiments were approved by the Animal Care and Ethics Committee at Wenzhou Medical University and conducted according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research. LysMCre mice20Clausen B.E. Burkhardt C. Reith W. Renkawitz R. Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice.Transgenic Res. 1999; 8: 265-277Crossref PubMed Scopus (1399) Google Scholar [C57BL/6J background, B6.129P2-Lyz2tm1(cre)Ifo/J; stock number 004781; Jackson Laboratory, Bar Harbor, ME] were crossed with Rosa26-stop-flox-tdTomato mice21Madisen L. Zwingman T.A. Sunkin S.M. Oh S.W. Zariwala H.A. Gu H. Ng L.L. Palmiter R.D. Hawrylycz M.J. Jones A.R. Lein E.S. Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci. 2010; 13: 133-140Crossref PubMed Scopus (3167) Google Scholar [developed on a C57BL/6J background, B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J; stock number 007909; Jackson Laboratory] to generate LysMCreRosa26fl/fl mice. In these mice, the STOP cassette is deleted in LysM-expressing cells (including monocytes and macrophages), resulting in red tdTomato fluorescence in these cells. Mmp2fl/fl mice were developed on a C57BL/6J background at the Model Animal Research Center of Nanjing University (Nanjing, China).6Zhao F. Zhou Q. Reinach P.S. Yang J. Ma L. Wang X. Wen Y. Srinivasalu N. Qu J. Zhou X. Cause and effect relationship between changes in scleral matrix metallopeptidase-2 expression and myopia development in mice.Am J Pathol. 2018; 188: 1754-1767Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Mmp2fl/fl mice were crossed with LysMCre mice to generate macrophage-specific Mmp2 deletion mice (LysMCreMmp2fl/fl) and their Cre-negative littermates (designated Mmp2fl/fl). Ccl2fl/fl mice22Takahashi M. Galligan C. Tessarollo L. Yoshimura T. Monocyte chemoattractant protein-1 (MCP-1), not MCP-3, is the primary chemokine required for monocyte recruitment in mouse peritonitis induced with thioglycollate or zymosan A.J Immunol. 2009; 183: 3463-3471Crossref PubMed Scopus (63) Google Scholar [C57BL/6NJ background, B6N.129S1(FVB)Ccl2tm1.2Tyos/J; stock number 023347; Jackson Laboratory] were crossed with S100a4Cre mice23Tsutsumi R. Xie C. Wei X. Zhang M. Zhang X. Flick L.M. Schwarz E.M. O'Keefe R.J. PGE2 signaling through the EP4 receptor on fibroblasts upregulates RANKL and stimulates osteolysis.J Bone Miner Res. 2009; 24: 1753-1762Crossref PubMed Scopus (48) Google Scholar [BALB/cByJ background, BALB/c-Tg(S100a4-cre)1Egn/YunkJ; stock number 012641; Jackson Laboratory] to generate fibroblast-specific Ccl2 deletion mice (S100a4CreCcl2fl/fl) and their Cre-negative littermates (designated Ccl2fl/fl). PCR analysis of tail-extracted genomic DNA using specific primers (Table 1) confirmed all mouse genotype identities. Monocular FDM was induced by covering the right eye of each mouse with a handmade white translucent occluder.24Schaeffel F. Burkhardt E. Howland H.C. Williams R.W. Measurement of refractive state and deprivation myopia in two strains of mice.Optom Vis Sci. 2004; 81: 99-110Crossref PubMed Scopus (137) Google Scholar,25Zhou X. Shen M. Xie J. Wang J. Jiang L. Pan M. Qu J. Lu F. The development of the refractive status and ocular growth in C57BL/6 mice.Invest Ophthalmol Vis Sci. 2008; 49: 5208-5214Crossref PubMed Scopus (57) Google Scholar It was carefully attached with polystyrene glue to the fur around the eye so as to prevent exposure to any direct unfiltered light. To prevent the mice from removing the occluder, a collar made of thin plastic was fitted around the neck. The form-deprivation (FD)–treated right eyes were designated as FD-T, and the untreated left fellow eyes were designated as FD-F. Ocular refraction without cycloplegia was measured using an eccentric infrared photorefractor in a dark room. Each measurement was repeated three times to obtain a final mean refraction.24Schaeffel F. Burkhardt E. Howland H.C. Williams R.W. Measurement of refractive state and deprivation myopia in two strains of mice.Optom Vis Sci. 2004; 81: 99-110Crossref PubMed Scopus (137) Google Scholar The ocular biometric parameters, AL (refers to the distance from the corneal apex to the nerve fiber layer of the retina), VCD (refers to the distance from the back of the lens to the nerve fiber layer of the retina), lens thickness (refers to the distance from its anterior to posterior surface), and anterior chamber depth (refers to the distance between the posterior surface of the cornea and the anterior surface of the lens), were measured using a custom-made optical coherence tomography instrument.24Schaeffel F. Burkhardt E. Howland H.C. Williams R.W. Measurement of refractive state and deprivation myopia in two strains of mice.Optom Vis Sci. 2004; 81: 99-110Crossref PubMed Scopus (137) Google Scholar Supplemental Figure S1 provides a diagrammatic representation of the experimental design. Four-week–old wild-type male C57BL/6 mice with anisometropia (the difference in refraction between right and left eyes) of <3 diopters (D), and free of injuries and infections, were used in this study. The mice were randomly divided into FD and age-matched control groups. In the FD group, the right eye (designated FD-T) of each mouse was form deprived for 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 7, or 14 days, and the untreated left eye constituted the fellow eye control group (designated FD-F). Age-matched mice with no FD induction served as normal controls (designated normal). After 14 days of FD induction, scleral tissues of the mice were prepared for co-immunofluorescence labeling of MMP-2 and F4/80, a macrophage-specific antigen.26Austyn J.M. Gordon S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1242) Google Scholar MMP-2 is synthesized and secreted in a latent form and converted into an active form in the extracellular environment.27Brinckerhoff C.E. Matrisian L.M. Matrix metalloproteinases: a tail of a frog that became a prince.Nat Rev Mol Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (930) Google Scholar However, there are no specific antibodies available to distinguish between these two forms. To assess if macrophages contribute to scleral MMP-2 up-regulation, frozen scleral sections were used to evaluate macrophage-specific (F4/80+) MMP-2 expression in FD-treated mice. Quantitative real-time RT-PCR (RT-qPCR) determined the time-dependent (1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 7, and 14 days) differential expression levels of scleral MMP-2 and macrophage-related protein encoding genes. To determine if these scleral gene expression levels follow a circadian rhythm, their expression levels were measured at 3:00 am, 9:00 am, 3:00 pm, and 9:00 pm across a single day in 4-week–old C57BL/6J mice. At these time points, the scleral tissues were isolated and prepared for RT-qPCR analysis. Scleral monocyte and macrophage densities were also evaluated on the basis of immunofluorescence staining of the endogenous LysM gene promoter, which is a specific marker of these cell types. Red tdTomato fluorescence evaluated LysM promoter-driven Cre recombinase expression during FDM progression in LysMCreRosa26fl/fl mice. In addition, F4/80 colabeling was used to validate the correspondence between tdTomato expression and scleral macrophage presence. Four-week–old LysMCreRosa26fl/fl mice were randomly divided into the FD and normal groups. In the FD group, the right eye of each mouse was form deprived (FD-T eyes) for 2, 7, or 14 days, whereas the left fellow eyes (FD-F eyes) were untreated. Age-matched untreated LysMCreRosa26fl/fl mice served as the normal controls (normal). To assess the effect of macrophage-specific Mmp2 deletion on normal refractive development, the ocular measurements were performed from postnatal day (P) 28 (week 4) to P70 (week 10) in both Mmp2fl/fl (n = 25) and LysMCreMmp-2fl/fl (n = 22) mice. To assess the effect of macrophage-specific Mmp2 deletion on FDM development, 4 weeks of FD were performed in Mmp2fl/fl (n = 14) and LysMCreMmp-2fl/fl (n = 24) mice. The FD treatment was initiated at 4 weeks of age. Another 10 untreated animals from each genotype served as the age-matched controls. Ocular measurements were recorded before and after 4 weeks of FD. The scleral tissues obtained from these mice were prepared for RT-qPCR analysis of Mmp2 expression and immunofluorescence labeling of F4/80 and MMP-2. Clodronate (CLOD) encapsulated within the liposomes (Liposoma BV, Amsterdam, the Netherlands) has been widely used in many immunologic studies to selectively deplete macrophages with high efficiency.28van Rooijen N. van Kesteren-Hendrikx E. Clodronate liposomes: perspectives in research and therapeutics.J Liposome Res. 2002; 12: 81-94Crossref PubMed Scopus (118) Google Scholar,29Feng B. Jiao P. Nie Y. Kim T. Jun D. van Rooijen N. Yang Z. Xu H. Clodronate liposomes improve metabolic profile and reduce visceral adipose macrophage content in diet-induced obese mice.PLoS One. 2011; 6: e24358Crossref PubMed Scopus (91) Google Scholar To determine the effect of scleral macrophage depletion on FDM development, i.p. injection (described below) of liposomes containing CLOD (CLOD; n = 13) or the control liposomes containing phosphate-buffered saline (PBS; LIP; n = 15) were administered every 2 days for 2 weeks in 4-week–old male wild-type mice. FDM was induced for 2 weeks beginning after the first 24 hours of CLOD or LIP injection. Ocular measurements were recorded before and after 2 weeks of FDM induction. Peripheral blood was drawn and prepared for flow cytometry, whereas the sclera was collected for F4/80 immunofluorescence labeling. CLOD treatment is highly efficacious in depleting liver macrophage content.30Moreno S.G. Depleting macrophages in vivo with clodronate-liposomes.Methods Mol Biol. 2018; 1784: 259-262Crossref PubMed Scopus (16) Google Scholar Accordingly, the liver was used as a positive control to validate that CLOD-encapsulated liposome treatment has such an effect in this tissue. The assessment entailed performing F4/80 immunofluorescence staining to estimate declines in macrophage density in frozen sections of liver. To assess the effect of fibroblast-specific Ccl2 deletion on normal refractive development, the ocular measurements were performed from P31 (week 4.5) to P73 (week 10.5) in both Ccl2fl/fl mice (n = 22) and the scleral S100a4CreCcl2fl/fl mice (n = 26). To assess the effect of fibroblast-specific Ccl2 deletion on FDM development, 2 weeks of FD were performed in Ccl2fl/fl (n = 16) and S100a4CreCcl2fl/fl (n = 12) mice. FD treatment was initiated at 4.5 weeks of age. Untreated animals from Ccl2fl/fl (n = 8) and S100a4CreCcl2fl/fl (n = 11) served as the age-matched controls. Ocular measurements were recorded before and after 2 weeks of FD. The tissues obtained from these mice were prepared for RT-qPCR analysis of Ccl2 expression and immunofluorescence labeling of F4/80. In this case, the ocular measurements and the FDM induction in S100a4CreCcl2fl/fl mice were initiated at 4.5 weeks rather than 4 weeks of age. This delay was chosen because at 4 weeks of age, it was difficult to accurately measure their refraction. It is possible that this difficulty was encountered because S100a4CreCcl2fl/fl mice had a lower body weight than the Ccl2fl/fl mice (Supplemental Figure S2). For experiments using the transgenic mice, both male and female mice were randomly distributed into treatment groups within each genotype. Macrophage depletion was achieved by i.p. injection of CLOD. CLOD (50 mg/kg body weight) or an equal volume of the control LIP was injected31Bu L. Gao M. Qu S. Liu D. Intraperitoneal injection of clodronate liposomes eliminates visceral adipose macrophages and blocks high-fat diet-induced weight gain and development of insulin resistance.AAPS J. 2013; 15: 1001-1011Crossref PubMed Scopus (54) Google Scholar 24 hours before FD, and then once every 2 days. To estimate the CLOD depletion efficiency, flow cytometry was used to monitor its effects on CD45+CD11b+CD115+ monocyte densities in the peripheral blood, as previously described.32Sunderkotter C. Nikolic T. Dillon M.J. Van Rooijen N. Stehling M. Drevets D.A. Leenen P.J. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (810) Google Scholar Briefly, anticoagulated blood samples (100 μL) from the two groups were incubated for 20 minutes at room temperature in the dark with rat anti-mouse CD45 peridinin-chlorophyll proteins-cyanine 5.5 (1:100; clone 30-F11; eBioscience, San Diego, CA), rat anti-mouse CD11b phosphatidylethanolamine (1:50; clone M1/70; BD Bioscience, San Jose, CA), and rat anti-mouse CD115-allophycocyanin (1:50; clone AFS98; eBioscience). Then, 1 mL of Erythrolyse Red Cell Lysing Buffer (BD Bioscience) was added, and the samples were incubated in the dark for 10 minutes at room temperature. After incubation, samples were centrifuged at 400 × g for 5 minutes, and the supernatants were discarded. Cell pellets were resuspended with 200 μL cold PBS to achieve a concentration of 1 × 105 to 1 × 107 cells/mL. Cells were detected by a BD FACS ARIA II cell sorter (BD Bioscience) and analyzed using BD FACSDiva software version 6.1.3 (BD Bioscience). After different treatments, mice were sacrificed, and their eyes were enucleated. Frozen scleral sections were prepared, as previously described.6Zhao F. Zhou Q. Reinach P.S. Yang J. Ma L. Wang X. Wen Y. Srinivasalu N. Qu J. Zhou X. Cause and effect relationship between changes in scleral matrix metallopeptidase-2 expression and myopia development in mice.Am J Pathol. 2018; 188: 1754-1767Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, the cornea, conjunctiva, lens, and vitreous were removed and discarded. The remaining eyecup, including the retina, choroid, and sclera, was then fixed in freshly prepared 4% paraformaldehyde for 20 minutes at room temperature and dehydrated in an ascending series of 10% (2 hours), 20% (2 hours), and 30% (overnight) sucrose solutions at 4°C. The eyecups were then embedded in Neg 50 Frozen Section Medium (Thermo Fisher Scientific, Waltham, MA) and frozen in liquid nitrogen followed by storage at −80°C before sectioning. The eyecups were cut into sections (12 μm thick) using a freezing microtome (Leica CM 1860 UV; Leica Microsystems, Solms, Germany). After rinsing the slides three times in PBS for 5 minutes each, a 0.1 mol/L PBS blocking solution containing 6% normal donkey serum, 1% bovine serum albumin, and 0.3% Triton X-100 was applied for 2 hours at room temperature to block non-specific binding. Primary antibodies against F4/80 (1:200; ab6640; CI:A3-1; Abcam, Cambridge, UK) and MMP-2 (1:400; SC-10736; H-76; Santa Cruz Biotechnology, Dallas, TX) were diluted in the dilution buffer (containing 3% normal donkey serum, 0.5% bovine serum albumin, and 0.3% Triton X-100 in 0.1 mol/L PBS), which covered the sections for overnight incubation at 4°C. After washing the sections three times with PBS for 5 minutes each, they were incubated with secondary antibodies for 2 hours at room temperature with either donkey anti-rat IgG (H+L) conjugated to Alexa Fluor 488 (1:400; A-21208; Invitrogen, Carlsbad, CA) or donkey anti-rabbit IgG (H+L) conjugated to Alexa Fluor 555 (1:400; A-31572; Invitrogen). The secondary antibodies were diluted in the same solution as the primary antibody. Finally, cell nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). The frozen sections were visualized, and the images were captured with a Zeiss LSM 710 confocal microscope (ZEISS, Gottingen, Germany) or AXIO IMAGER Z1 microscope (ZEISS) under a ×20 magnification objective. Negative controls were established by omitting the primary F4/80 and/or MMP-2 antibodies and processing the frozen scleral sections in the same manner as the other scleral sections. Supplemental Figure S3 shows the negative control images, in which there was an absence of F4/80 macrophage and/or MMP-2 staining. Supplemental Figure S4 shows the posterior region (referring to the anatomic location; ie, closest to the posterior pole of the eye), which was subjected to cell counting of ×20 imaging fields in stained scleral frozen sections. F4/80+ or tdTomato+ signal that overlapped with DAPI staining was assumed to be indicative of a macrophage in each image. The region of interest subjected to the image analysis encompassed the circumscribed area between the optic nerve and the scleral periphery. This delineation is relevant because we took into account the possibility that the scleral macrophage density in the posterior region may be different from that in the peripheral region. The region of interest for cell counting was outlined in each immunofluorescence image. The measurement of the region of interest area and the cell counting of the scleral macrophages were performed by utilizing the ImageJ software version 1.48 (NIH, Bethesda, MD; https://imagej.nih.gov/ij). Three discontinuous sections from each eyeball were assessed in a blinded manner (Y.W., Y.L., and L.M.), and the average cell density was taken as the sample value. Data analyses were performed on groups each containing four to seven mice (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, 8, and 9, and Supplemental Figures S5 and S7). The Mmp2 mRNA expression levels in the sclera, retina, cornea, and lens of 10-week–old LysMCreMmp-2fl/fl and Mmp2fl/fl mice were determined by RT-qPCR analysis. Such analysis also determined the time-dependent differential expression levels of Mmp2 and macrophage-related protein-encoding genes after 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 7, and 14 days of FD induction in wild-type mice. Furthermore, this analysis determined Ccl2 mRNA expression levels in sclera, retina, cornea, and lens of 10.5-week–old S100a4CreCcl2fl/fl and Ccl2fl/fl mice. The sclera, cornea, and lens were dissected following eye enucleation. Subsequently, they were homogenized separately using a ball mill, and total RNA was extracted using the RNeasy Fibrous Tissue MiniKit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Total RNA from the retina was extracted using TRIZOL reagent (Invitrogen), according to the manufacturer's protocol. After treating with RQ1 RNase-Free DNase (Promega, Madison, WI), the RNAs were subjected to reverse transcription with random primers and M-MLV Reverse Transcriptase (Promega) to synthesize the respective cDNAs.33Zhou X. Ji F. An J. Zhao F. Shi F. Huang F. Li Y. Jiao S. Yan D. Chen X. Chen J. Qu J. Exper" @default.
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• W3036691716 title "Up-Regulation of Matrix Metalloproteinase-2 by Scleral Monocyte–Derived Macrophages Contributes to Myopia Development" @default.
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• W3036691716 doi "https://doi.org/10.1016/j.ajpath.2020.06.002" @default.
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