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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

Photographs of central and peripheral retina in C57BL/6N (rd8/rd8) and C57BL/6 J (wild-type) mice. A change in the distribution of fundus spots on B6-mice due to both age and the presence of the rd8 mutation is seen. Yellow spots are shown in central (A, C, E, and G) and peripheral (B, D, F, and H) fundus photographs of representative young (A-D) and old (E-H) B6-mice. Mice 2 to 8 months of age were classified as ‘young’, while mice 14 to 20 months of age were classified as ‘old’. Note that the number of central fundus spots is increased in old age for both rd8 mutant (G versus C) and wild-type (WT) (E versus A) mice. Furthermore, rd8/rd8 mice show a marked increase in central spots compared to WT, both in the young (C versus A) and old (G versus E) age groups.
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Fig1: Photographs of central and peripheral retina in C57BL/6N (rd8/rd8) and C57BL/6 J (wild-type) mice. A change in the distribution of fundus spots on B6-mice due to both age and the presence of the rd8 mutation is seen. Yellow spots are shown in central (A, C, E, and G) and peripheral (B, D, F, and H) fundus photographs of representative young (A-D) and old (E-H) B6-mice. Mice 2 to 8 months of age were classified as ‘young’, while mice 14 to 20 months of age were classified as ‘old’. Note that the number of central fundus spots is increased in old age for both rd8 mutant (G versus C) and wild-type (WT) (E versus A) mice. Furthermore, rd8/rd8 mice show a marked increase in central spots compared to WT, both in the young (C versus A) and old (G versus E) age groups.

Mentions: Fundus examination of C57BL/6 mice revealed yellow spots in mice of all ages (Figure 1). In young B6 mice (2- to 8 mo) of both genotypes, the vast majority of the spots were located in the far retinal periphery, close to the ora serrata (Figure 1B and D). In this age group, a small number of yellow spots were usually seen in the posterior retina of C57BL6/N mice. However, these spots were seen only rarely in the posterior retina of young C57BL/6J mice (Figure 1A and C). As mice aged, the distribution of yellow spots shifted. In old mice (14-to 20 mo), the geographic distribution of these spots shifted towards the mid-peripheral and central retina (Figure 1E,G, and black bars in Figure 2C) in both genotypes. However, this change was most accelerated in the C57BL/6N rd8 mutant mice. The number of yellow spots in the central retina was significantly higher in rd8 mutant compared to WT mice (Figure 1A versus C, E versus G, and black bars in Figure 2C) for both age groups.Figure 1


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)

Photographs of central and peripheral retina in C57BL/6N (rd8/rd8) and C57BL/6 J (wild-type) mice. A change in the distribution of fundus spots on B6-mice due to both age and the presence of the rd8 mutation is seen. Yellow spots are shown in central (A, C, E, and G) and peripheral (B, D, F, and H) fundus photographs of representative young (A-D) and old (E-H) B6-mice. Mice 2 to 8 months of age were classified as ‘young’, while mice 14 to 20 months of age were classified as ‘old’. Note that the number of central fundus spots is increased in old age for both rd8 mutant (G versus C) and wild-type (WT) (E versus A) mice. Furthermore, rd8/rd8 mice show a marked increase in central spots compared to WT, both in the young (C versus A) and old (G versus E) age groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4305240&req=5

Fig1: Photographs of central and peripheral retina in C57BL/6N (rd8/rd8) and C57BL/6 J (wild-type) mice. A change in the distribution of fundus spots on B6-mice due to both age and the presence of the rd8 mutation is seen. Yellow spots are shown in central (A, C, E, and G) and peripheral (B, D, F, and H) fundus photographs of representative young (A-D) and old (E-H) B6-mice. Mice 2 to 8 months of age were classified as ‘young’, while mice 14 to 20 months of age were classified as ‘old’. Note that the number of central fundus spots is increased in old age for both rd8 mutant (G versus C) and wild-type (WT) (E versus A) mice. Furthermore, rd8/rd8 mice show a marked increase in central spots compared to WT, both in the young (C versus A) and old (G versus E) age groups.
Mentions: Fundus examination of C57BL/6 mice revealed yellow spots in mice of all ages (Figure 1). In young B6 mice (2- to 8 mo) of both genotypes, the vast majority of the spots were located in the far retinal periphery, close to the ora serrata (Figure 1B and D). In this age group, a small number of yellow spots were usually seen in the posterior retina of C57BL6/N mice. However, these spots were seen only rarely in the posterior retina of young C57BL/6J mice (Figure 1A and C). As mice aged, the distribution of yellow spots shifted. In old mice (14-to 20 mo), the geographic distribution of these spots shifted towards the mid-peripheral and central retina (Figure 1E,G, and black bars in Figure 2C) in both genotypes. However, this change was most accelerated in the C57BL/6N rd8 mutant mice. The number of yellow spots in the central retina was significantly higher in rd8 mutant compared to WT mice (Figure 1A versus C, E versus G, and black bars in Figure 2C) for both age groups.Figure 1

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