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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.

Aredo B, Zhang K, Chen X, Wang CX, Li T, Ufret-Vincenty RL - J Neuroinflammation (2015)

Bottom Line: Reverse-transcription quantitative PCR (RT-qPCR) was done for genes involved in oxidative stress, complement activation and inflammation.The number of yellow fundus spots correlated highly with subretinal Iba-1+ cells.In contrast, aging leads to a scavenging phenotype in the C57BL/6J subretinal microglia/macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. Bogale.Aredo@UTSouthwestern.edu.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig5: 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.

Mentions: Having seen that aging and the presence of the rd8 mutation cause phenotypic changes in the subretinal MG/MΦ of B6 mice, we wondered if these changes were accompanied by morphological signs of activation [36]. We confirmed that injection of lipopolysaccharides (LPS) intraperitoneally (i.p.) into WT mice led to a marked increase in the accumulation of subretinal MG/MΦ and that those cells demonstrated the typical morphologic changes associated with activation, including larger cell bodies, shorter extensions and a lower number of extensions (see Additional file 6: Figure S2). Thus, we decided to quantitatively analyze the morphological activation of subretinal MG/MΦ on the RPE flat mounts of rd8 mutant and B6-WT mice, by measuring the cell body area and the extension length using ImageJ software. We also counted the total number of extensions per cell and derived a single parameter, ‘microglial morphology activation value’ (MMAV): MMAV = (cell body area)/((largest extension length) × (# of extensions)). Subretinal MG/MΦ in B6 mice of both genotypes were divided into CD16+ and CD16- subgroups and separately analyzed using the MMAV. The CD16+ cells of old rd8 mutant mice showed a significant decrease in the number of extensions per cell, and the extension length (Figure 5A and B) when compared to both old WT and young rd8 mutant mice. There were not enough CD16+ cells in the central young B6-WT mice to include in the analyses. There were no differences in these measurements for CD16- cells among the groups (data not shown). Within each group of mice, we found that the CD16+ subpopulation of Iba-1+ cells had a significantly higher MMAV compared to the CD16- subpopulation of Iba-1+ cells. Furthermore, the magnitude and significance of the difference increased with age, and with the presence of the rd8 mutation (Figure 5C). When the difference in the activation morphology of CD16+ versus CD16- cells for each group of mice was quantified, by calculating the ratio (MMAV for CD16+)/(MMAV for CD16-), it was three times higher in old rd8 mutant mice compared to old WT mice (P <1 × 10−8, Figure 5D). Examples of the Iba-1 and CD16 staining of MG/MΦ cells in two C57BL/6J mice (Figure 5E,G,I,K) versus two C57BL/6N mice (Figure 5F,H,J,L) are shown.Figure 5


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.

Aredo B, Zhang K, Chen X, Wang CX, Li T, Ufret-Vincenty RL - J Neuroinflammation (2015)

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.
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Fig5: 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.
Mentions: Having seen that aging and the presence of the rd8 mutation cause phenotypic changes in the subretinal MG/MΦ of B6 mice, we wondered if these changes were accompanied by morphological signs of activation [36]. We confirmed that injection of lipopolysaccharides (LPS) intraperitoneally (i.p.) into WT mice led to a marked increase in the accumulation of subretinal MG/MΦ and that those cells demonstrated the typical morphologic changes associated with activation, including larger cell bodies, shorter extensions and a lower number of extensions (see Additional file 6: Figure S2). Thus, we decided to quantitatively analyze the morphological activation of subretinal MG/MΦ on the RPE flat mounts of rd8 mutant and B6-WT mice, by measuring the cell body area and the extension length using ImageJ software. We also counted the total number of extensions per cell and derived a single parameter, ‘microglial morphology activation value’ (MMAV): MMAV = (cell body area)/((largest extension length) × (# of extensions)). Subretinal MG/MΦ in B6 mice of both genotypes were divided into CD16+ and CD16- subgroups and separately analyzed using the MMAV. The CD16+ cells of old rd8 mutant mice showed a significant decrease in the number of extensions per cell, and the extension length (Figure 5A and B) when compared to both old WT and young rd8 mutant mice. There were not enough CD16+ cells in the central young B6-WT mice to include in the analyses. There were no differences in these measurements for CD16- cells among the groups (data not shown). Within each group of mice, we found that the CD16+ subpopulation of Iba-1+ cells had a significantly higher MMAV compared to the CD16- subpopulation of Iba-1+ cells. Furthermore, the magnitude and significance of the difference increased with age, and with the presence of the rd8 mutation (Figure 5C). When the difference in the activation morphology of CD16+ versus CD16- cells for each group of mice was quantified, by calculating the ratio (MMAV for CD16+)/(MMAV for CD16-), it was three times higher in old rd8 mutant mice compared to old WT mice (P <1 × 10−8, Figure 5D). Examples of the Iba-1 and CD16 staining of MG/MΦ cells in two C57BL/6J mice (Figure 5E,G,I,K) versus two C57BL/6N mice (Figure 5F,H,J,L) are shown.Figure 5

Bottom Line: Reverse-transcription quantitative PCR (RT-qPCR) was done for genes involved in oxidative stress, complement activation and inflammation.The number of yellow fundus spots correlated highly with subretinal Iba-1+ cells.In contrast, aging leads to a scavenging phenotype in the C57BL/6J subretinal microglia/macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9057, USA. Bogale.Aredo@UTSouthwestern.edu.

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.

No MeSH data available.


Related in: MedlinePlus