June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Characterization of YFP+ cells in the retina of CD11c-eYFP transgenic mice: the effects of breeding out the Crb1rd8 mutation
Author Affiliations & Notes
  • Samantha Dando
    Department of Anatomy and Developmental Biology, School of Biomedical Science, Monash University, Clayton, VIC, Australia
  • Xiangting Chen
    Department of Anatomy and Developmental Biology, School of Biomedical Science, Monash University, Clayton, VIC, Australia
  • Marc Ruitenberg
    School of Biomedical Sciences, University of Queensland, St Lucia, QLD, Australia
  • Paul G McMenamin
    Department of Anatomy and Developmental Biology, School of Biomedical Science, Monash University, Clayton, VIC, Australia
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2078. doi:
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      Samantha Dando, Xiangting Chen, Marc Ruitenberg, Paul G McMenamin; Characterization of YFP+ cells in the retina of CD11c-eYFP transgenic mice: the effects of breeding out the Crb1rd8 mutation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2078.

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

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Abstract

Purpose: The normal CNS parenchyma is traditionally thought to be devoid of professional antigen presenting cells (APCs); however, a population of putative dendritic cells (DCs) expressing yellow fluorescent protein (YFP) are present in the neural retina of C57Bl/6N CD11c-eYFP mice. We have previously shown that these mice carry the Crb1rd8 mutation, which results in retinal dystrophic lesions. We hypothesized that the accumulation of YFP+ putative DCs within the retina may be the result of the pathology associated with the Crb1rd8 mutation.

Methods: C57Bl/6N CD11c-eYFP mice were outcrossed with C57Bl/6J mice to create Crb1rd8/wt offspring; heterozygous offspring were then intercrossed to generate CD11c-eYFP Crb1wt/wt mice. At 6 weeks of age, CD11c-eYFP Crb1wt/wt mice (n=50) underwent clinical fundus examination in brightfield and fluorescent modes. At 8 weeks of age, eyes were collected from perfusion-fixed mice (n=5) and retinal wholemounts were processed for confocal microscopy. Single cell suspensions were prepared from pooled retinas (n=10 mice), stained with an antibody panel for the identification of APC surface markers and subsequently characterized by flow cytometry.

Results: In vivo examination of the eyes of CD11c-eYFP Crb1wt/wt mice revealed normal appearance of the fundus in brightfield mode, with no retinal lesions observed. However, fluorescent fundus imaging demonstrated the continued presence of YFP+ cells in the retina. Quantitative analysis of retinal wholemounts by confocal microscopy revealed a density of 94±3.3 (mean ± SEM) YFP+ cells/mm2. These YFP+ cells, which were predominantly distributed within the plexiform and nerve fiber-ganglion cell layers of the retina, co-expressed the microglial markers CD11b, F4/80 and Iba-1, whereas few YFP+ cells expressed the APC markers CD11c, CD80, CD86, CD103, CD8, DEC205, 33D1 and MHC class II.

Conclusions: The detection of YFP+ cells in the retina of CD11c-eYFP Crb1wt/wt mice indicates that the presence of these cells is not wholly a consequence of the Crb1rd8 mutation. Retinal YFP+ cells displayed an immunophenotype that is consistent with microglia, and not DCs, suggesting that the YFP+ cells in the retina of CD11c-eYFP Crb1wt/wt mice represent a subset of microglia.

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