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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

Central fundus spots increase with age in both B6 groups, most prominently in rd8/rd8 mice. (A) A retinal pigment epithelium (RPE) flat mount of an old rd8 mouse stained for ionized calcium binding adaptor (Iba)-1 is shown to demonstrate the area counted as the ‘central flat mount’ (large white square). The ‘central flat mount’ is made up of four higher magnification photos (magnification 10X/1.6X optivar) taken around the disc (smaller squares). (B) One of those higher magnification photos is shown. (C) The number of yellow spots (black bars) in the central fundus (within a circle with a 5 disc diameter (DD) radius and centered on the disc) is very similar to the number of Iba-1+ cells (gray bars) on the corresponding central flat mounts. There is an increase in central fundus spots in rd8/rd8 mice compared to wild-type (WT) mice in each age group. Furthermore, there is an increase in the number of central yellow spots with age in both genotypes (young WT (n = 11), young rd8 (n = 3); old WT (n = 11), old rd8 (n = 10)). (D) Linear regression showing a significant correlation between the number of yellow spots in central fundus (5 DD radius) and the number of Iba-1 positive cells in the corresponding flat mount area (n = 19 eyes). 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 <0.001.
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Fig2: Central fundus spots increase with age in both B6 groups, most prominently in rd8/rd8 mice. (A) A retinal pigment epithelium (RPE) flat mount of an old rd8 mouse stained for ionized calcium binding adaptor (Iba)-1 is shown to demonstrate the area counted as the ‘central flat mount’ (large white square). The ‘central flat mount’ is made up of four higher magnification photos (magnification 10X/1.6X optivar) taken around the disc (smaller squares). (B) One of those higher magnification photos is shown. (C) The number of yellow spots (black bars) in the central fundus (within a circle with a 5 disc diameter (DD) radius and centered on the disc) is very similar to the number of Iba-1+ cells (gray bars) on the corresponding central flat mounts. There is an increase in central fundus spots in rd8/rd8 mice compared to wild-type (WT) mice in each age group. Furthermore, there is an increase in the number of central yellow spots with age in both genotypes (young WT (n = 11), young rd8 (n = 3); old WT (n = 11), old rd8 (n = 10)). (D) Linear regression showing a significant correlation between the number of yellow spots in central fundus (5 DD radius) and the number of Iba-1 positive cells in the corresponding flat mount area (n = 19 eyes). 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 <0.001.

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)

Central fundus spots increase with age in both B6 groups, most prominently in rd8/rd8 mice. (A) A retinal pigment epithelium (RPE) flat mount of an old rd8 mouse stained for ionized calcium binding adaptor (Iba)-1 is shown to demonstrate the area counted as the ‘central flat mount’ (large white square). The ‘central flat mount’ is made up of four higher magnification photos (magnification 10X/1.6X optivar) taken around the disc (smaller squares). (B) One of those higher magnification photos is shown. (C) The number of yellow spots (black bars) in the central fundus (within a circle with a 5 disc diameter (DD) radius and centered on the disc) is very similar to the number of Iba-1+ cells (gray bars) on the corresponding central flat mounts. There is an increase in central fundus spots in rd8/rd8 mice compared to wild-type (WT) mice in each age group. Furthermore, there is an increase in the number of central yellow spots with age in both genotypes (young WT (n = 11), young rd8 (n = 3); old WT (n = 11), old rd8 (n = 10)). (D) Linear regression showing a significant correlation between the number of yellow spots in central fundus (5 DD radius) and the number of Iba-1 positive cells in the corresponding flat mount area (n = 19 eyes). 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 <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Central fundus spots increase with age in both B6 groups, most prominently in rd8/rd8 mice. (A) A retinal pigment epithelium (RPE) flat mount of an old rd8 mouse stained for ionized calcium binding adaptor (Iba)-1 is shown to demonstrate the area counted as the ‘central flat mount’ (large white square). The ‘central flat mount’ is made up of four higher magnification photos (magnification 10X/1.6X optivar) taken around the disc (smaller squares). (B) One of those higher magnification photos is shown. (C) The number of yellow spots (black bars) in the central fundus (within a circle with a 5 disc diameter (DD) radius and centered on the disc) is very similar to the number of Iba-1+ cells (gray bars) on the corresponding central flat mounts. There is an increase in central fundus spots in rd8/rd8 mice compared to wild-type (WT) mice in each age group. Furthermore, there is an increase in the number of central yellow spots with age in both genotypes (young WT (n = 11), young rd8 (n = 3); old WT (n = 11), old rd8 (n = 10)). (D) Linear regression showing a significant correlation between the number of yellow spots in central fundus (5 DD radius) and the number of Iba-1 positive cells in the corresponding flat mount area (n = 19 eyes). 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 <0.001.
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