title: Differences in the distribution, phenotype and gene expression of subretinal microglia/macrophages in C57BL/6N (Crb1 rd8/rd8) versus C57BL6/J (Crb1 wt/wt) mice.
abstract: BACKGROUND
  Microglia/macrophages (MG/MΦ) are found in the subretinal space in both mice and humans. Our goal was to study the spatial and temporal distribution, the phenotype, and gene expression of subretinal MG/MΦ in mice with normal retinas and compare them to mice with known retinal pathology.
  
  
  METHODS
  We studied C57BL/6 mice with (C57BL/6N), or without (C57BL/6J) the rd8 mutation in the Crb1 gene (which, in the presence of yet unidentified permissive/modifying genes, leads to a retinal degeneration), and documented their fundus appearance and the change with aging. Immunostaining of retinal pigment epithelium (RPE) flat mounts was done for 1) Ionized calcium binding adaptor (Iba)-1, 2) FcγIII/II Receptor (CD16/CD32, abbreviated as CD16), and 3) Macrophage mannose receptor (MMR). Reverse-transcription quantitative PCR (RT-qPCR) was done for genes involved in oxidative stress, complement activation and inflammation.
  
  
  RESULTS
  The number of yellow fundus spots correlated highly with subretinal Iba-1+ cells. The total number of subretinal MG/MΦ increased with age in the rd8 mutant mice, but not in the wild-type (WT) mice. There was a centripetal shift in the distribution of the subretinal MG/MΦ with age. Old rd8 mutant mice had a greater number of CD16+ MG/MΦ. CD16+ cells had morphological signs of activation, and this was most prominent in old rd8 mutant mice (P < 1 × 10(-8) versus old WT mice). Subretinal MG/MΦ in rd8 mutant mice also expressed iNOS and MHC-II, and had ultrastructural signs of activation. Finally, rd8 mutant mouse RPE/ MG/MΦ RNA isolates showed an upregulation of Ccl2, CFB, C3, NF-kβ, CD200R and TNF-alpha. The retinas of rd8 mutant mice showed upregulation of HO-1, C1q, C4, and Nrf-2.
  
  
  CONCLUSIONS
  When compared to C57BL/6J mice, C57BL/6N mice demonstrate increased accumulation of subretinal MG/MΦ, displaying phenotypical, morphological, and gene-expression characteristics consistent with a pro-inflammatory shift. These changes become more prominent with aging and are likely due to the combination of the rd8 mutation and yet unidentified permissive/modulatory genes in the C57BL/6N mice. In contrast, aging leads to a scavenging phenotype in the C57BL/6J subretinal microglia/macrophages.
authors.affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. Bogale.Aredo@UTSouthwestern.edu.
authors.forename: Bogale
authors.initials: B
authors.lastname: Aredo
authors.affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. Zhangkaiyan0898@hotmail.com.|Current address: Department of Ophthalmology, Hainan Provincial People's Hospital, Haikou, Hainan, 570203, PR China. Zhangkaiyan0898@hotmail.com.
authors.forename: Kaiyan
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. cxfn817@163.com.|Current address: Department of Ophthalmology, Wuhan General Hospital of Guangzhou Military Command, Wuhan, 430070, PR China. cxfn817@163.com.
authors.forename: Xiao
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. cynthiax.wang@utsouthwestern.edu.
authors.forename: Cynthia Xin-Zhao
authors.initials: CX
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. Tao.Li@UTSouthwestern.edu.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. Rafael.Ufret-Vincenty@UTSouthwestern.edu.
authors.forename: Rafael L
authors.initials: RL
authors.lastname: Ufret-Vincenty
mesh_terms: D000367:Age Factors; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002452:Cell Count; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000235:genetics*; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D017628:Microglia / Q000378:metabolism* / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009434:Neural Pathways / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000473:pathology*; D012162:Retinal Degeneration / Q000235:genetics / Q000473:pathology*; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology
chemical_list: C491049:Aif1 protein, mouse; D002135:Calcium-Binding Proteins; C453730:Crb1 protein, mouse; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D017452:Receptors, IgG