Figure 5

Morphological analysis of subretinal microglia/macrophages in B6-mice. There is an increased microglia/macrophages (MG/MΦ) activation morphology in the rd8/rd8 mice, which is accentuated in old age. (A) The average number of extensions per MG/MΦ cell is decreased in old rd8/rd8 mice compared to both old wild-type (WT) mice and young rd8/rd8 mice. (B) The average length of the MG/MΦ cell extensions (measured using imageJ, http://imageJ.nih.gov/ij/index.html, and expressed as standard arbitrary units) of old rd8/rd8 mice is decreased relative to both old WT mice and young rd8/rd8 mice. (C) Quantification of MG/MΦ activation using the new parameter, microglial morphology activation value (MMAV) is shown. MMAV combines several morphological changes known to be associated with MG/MΦ activation into a single value, and is defined as the area of the MG/MΦ cell body divided by the product of the number of extensions and the average extension length. MMAV is increased in FcγIII/II Receptor (CD16/CD32, abbreviated as CD16) positive cells, particularly in old rd8 mutant mice. (D). The ratio of MMAV for CD16+ to MMAV for CD16- cells is markedly increased in both young and old rd8 mutant mice compared to old WT mice. Two similar experiments were combined (see methods; n = 3 to 5 eyes per group, and 3 to 5 photographic fields per eye, containing 3 to 5 cells with intact cell body per field, which were randomly selected by a masked investigator). Examples of the ionized calcium binding adaptor (Iba)-1 (E,F,I,J) and CD16 (G,H,K,L) staining of MG/MΦ in two C57BL/6J (E,G,I,K) versus two C57BL/6N mice (F,H,J,L) are shown. Mice 2 to 8 months of age were classified as ‘young’, while mice 14 to 20 months of age were classified as ‘old’. *P <0.05, **P <0.01, ***P <0.001, #P = 0.051.