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Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium.

Rutar M, Natoli R, Chia RX, Valter K, Provis JM - J Neuroinflammation (2015)

Bottom Line: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10.In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE).This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Building 131, Garran Road, Canberra, ACT 2601, Australia. matt.rutar@anu.edu.au.

ABSTRACT

Background: Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration.

Methods: Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR.

Results: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

Conclusions: Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations.

No MeSH data available.


Related in: MedlinePlus

Changes the number of CD45+cells using fluorescence activated cell sorting (FACS), and their expression of chemokine receptors. A: Representative FACS plots, with gating strategies, for a 7-days postexposure sample stained for CD45. Gating methodology was applied equally for all samples. B: Histogram depicts changes in the population of retinal CD45+ cells following light damage (LD). Proportion of CD45+ cells roughly tripled following 24 hrs of LD (P <0.05), and continued to increase substantially during the post-exposure period after 3 days (P <0.05); a further increase at 7 days was not significant (P >0.05). The overall trend was significant by one-way ANOVA (P <0.05); n = 5. C-G: Representative images of PCR products via electrophoresis for Ccr1, Ccr2, Ccr5, Cxcr2, Cxcr3, in samples of CD45-sorted cells. Receptor expression was low in dim-reared control samples (C-F), and absent for Cxcr3 (G). Expression increased substantially following 24 hrs of LD for every receptor assessed (C-G), and was maintained through the post exposure period, with the exception of Cxcr3 (G).
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Fig7: Changes the number of CD45+cells using fluorescence activated cell sorting (FACS), and their expression of chemokine receptors. A: Representative FACS plots, with gating strategies, for a 7-days postexposure sample stained for CD45. Gating methodology was applied equally for all samples. B: Histogram depicts changes in the population of retinal CD45+ cells following light damage (LD). Proportion of CD45+ cells roughly tripled following 24 hrs of LD (P <0.05), and continued to increase substantially during the post-exposure period after 3 days (P <0.05); a further increase at 7 days was not significant (P >0.05). The overall trend was significant by one-way ANOVA (P <0.05); n = 5. C-G: Representative images of PCR products via electrophoresis for Ccr1, Ccr2, Ccr5, Cxcr2, Cxcr3, in samples of CD45-sorted cells. Receptor expression was low in dim-reared control samples (C-F), and absent for Cxcr3 (G). Expression increased substantially following 24 hrs of LD for every receptor assessed (C-G), and was maintained through the post exposure period, with the exception of Cxcr3 (G).

Mentions: Changes in the number of CD45+ monocytes/microglia following LD were identified using FACS; representative gating strategies and scatter blots are noted in Figure 7A. In dim-reared retinas, CD45+ cells comprised a relatively small population of the gated retinal isolates, at approximately 0.098% (Figure 7B). After 24 hrs of LD, the retinal CD45+ population rose substantially to 0.340% (P <0.05), then reaching 0.875% by 3 days (P < 0.05). A further increase to 1.24% was observed at 7 days, although this was not significantly different from the population size at 3 days (P >0.05).Figure 7


Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium.

Rutar M, Natoli R, Chia RX, Valter K, Provis JM - J Neuroinflammation (2015)

Changes the number of CD45+cells using fluorescence activated cell sorting (FACS), and their expression of chemokine receptors. A: Representative FACS plots, with gating strategies, for a 7-days postexposure sample stained for CD45. Gating methodology was applied equally for all samples. B: Histogram depicts changes in the population of retinal CD45+ cells following light damage (LD). Proportion of CD45+ cells roughly tripled following 24 hrs of LD (P <0.05), and continued to increase substantially during the post-exposure period after 3 days (P <0.05); a further increase at 7 days was not significant (P >0.05). The overall trend was significant by one-way ANOVA (P <0.05); n = 5. C-G: Representative images of PCR products via electrophoresis for Ccr1, Ccr2, Ccr5, Cxcr2, Cxcr3, in samples of CD45-sorted cells. Receptor expression was low in dim-reared control samples (C-F), and absent for Cxcr3 (G). Expression increased substantially following 24 hrs of LD for every receptor assessed (C-G), and was maintained through the post exposure period, with the exception of Cxcr3 (G).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig7: Changes the number of CD45+cells using fluorescence activated cell sorting (FACS), and their expression of chemokine receptors. A: Representative FACS plots, with gating strategies, for a 7-days postexposure sample stained for CD45. Gating methodology was applied equally for all samples. B: Histogram depicts changes in the population of retinal CD45+ cells following light damage (LD). Proportion of CD45+ cells roughly tripled following 24 hrs of LD (P <0.05), and continued to increase substantially during the post-exposure period after 3 days (P <0.05); a further increase at 7 days was not significant (P >0.05). The overall trend was significant by one-way ANOVA (P <0.05); n = 5. C-G: Representative images of PCR products via electrophoresis for Ccr1, Ccr2, Ccr5, Cxcr2, Cxcr3, in samples of CD45-sorted cells. Receptor expression was low in dim-reared control samples (C-F), and absent for Cxcr3 (G). Expression increased substantially following 24 hrs of LD for every receptor assessed (C-G), and was maintained through the post exposure period, with the exception of Cxcr3 (G).
Mentions: Changes in the number of CD45+ monocytes/microglia following LD were identified using FACS; representative gating strategies and scatter blots are noted in Figure 7A. In dim-reared retinas, CD45+ cells comprised a relatively small population of the gated retinal isolates, at approximately 0.098% (Figure 7B). After 24 hrs of LD, the retinal CD45+ population rose substantially to 0.340% (P <0.05), then reaching 0.875% by 3 days (P < 0.05). A further increase to 1.24% was observed at 7 days, although this was not significantly different from the population size at 3 days (P >0.05).Figure 7

Bottom Line: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10.In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE).This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Building 131, Garran Road, Canberra, ACT 2601, Australia. matt.rutar@anu.edu.au.

ABSTRACT

Background: Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration.

Methods: Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR.

Results: Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors.

Conclusions: Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations.

No MeSH data available.


Related in: MedlinePlus