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Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

Dot C, Parier V, Behar-Cohen F, Benezra D, Jonet L, Goldenberg B, Picard E, Camelo S, de Kozak Y, May F, Soubrane G, Jeanny JC - Mol. Vis. (2009)

Bottom Line: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups.Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression.The above results may provide some insight into possible therapeutic strategies in the future.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France.

ABSTRACT

Purpose: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation.

Methods: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls.

Results: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively.

Conclusions: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

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VEGF expression after photocoagulation in RPE-choroid complex. A: The amount of vascular endothelial growth factor (VEGF) transcripts was compared with 18S transcripts (y-axis) expressed in the same samples. Four eyes were assessed at each time point. Values in histogram are means±SEM B-C: Representative samples of PCR fragments (individual ratio VEGF:18S closest to the corresponding mean) were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light for 10–12-week-old mice (B) and 1-year-old mice (C). Prior to laser treatment, VEGF mRNA expression was lower in old control mice versus young control mice (p<0.001). At day 3 after PC, the highest level of VEGF mRNA was 2.7 times higher than the controls in old mice. In young mice the highest level of expression was reached at 16 h after photocoagulation and did not exceed 1.3 times the level observed in controls.
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f9: VEGF expression after photocoagulation in RPE-choroid complex. A: The amount of vascular endothelial growth factor (VEGF) transcripts was compared with 18S transcripts (y-axis) expressed in the same samples. Four eyes were assessed at each time point. Values in histogram are means±SEM B-C: Representative samples of PCR fragments (individual ratio VEGF:18S closest to the corresponding mean) were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light for 10–12-week-old mice (B) and 1-year-old mice (C). Prior to laser treatment, VEGF mRNA expression was lower in old control mice versus young control mice (p<0.001). At day 3 after PC, the highest level of VEGF mRNA was 2.7 times higher than the controls in old mice. In young mice the highest level of expression was reached at 16 h after photocoagulation and did not exceed 1.3 times the level observed in controls.

Mentions: After photocoagulation, kinetics of VEGF mRNA expression was also different in 10–12-week-old mice and 1-year-old mice. In 10–12-week-old mice (group III), VEGF mRNA reached its peak 16 h after photocoagulation, while in one-year-old mice (group IV) the peak was observed on day 3 and declined thereafter (Figure 9). In addition, the ratio of VEGF expression in RPE-choroid complex after photocoagulation in relation to RPE-choroid complex in control not photocoagulated was more important in one-year-old mice (group IV; 2.7 times) than in 10–12-week-old mice (group III; 1.3 times).


Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

Dot C, Parier V, Behar-Cohen F, Benezra D, Jonet L, Goldenberg B, Picard E, Camelo S, de Kozak Y, May F, Soubrane G, Jeanny JC - Mol. Vis. (2009)

VEGF expression after photocoagulation in RPE-choroid complex. A: The amount of vascular endothelial growth factor (VEGF) transcripts was compared with 18S transcripts (y-axis) expressed in the same samples. Four eyes were assessed at each time point. Values in histogram are means±SEM B-C: Representative samples of PCR fragments (individual ratio VEGF:18S closest to the corresponding mean) were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light for 10–12-week-old mice (B) and 1-year-old mice (C). Prior to laser treatment, VEGF mRNA expression was lower in old control mice versus young control mice (p<0.001). At day 3 after PC, the highest level of VEGF mRNA was 2.7 times higher than the controls in old mice. In young mice the highest level of expression was reached at 16 h after photocoagulation and did not exceed 1.3 times the level observed in controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664845&req=5

f9: VEGF expression after photocoagulation in RPE-choroid complex. A: The amount of vascular endothelial growth factor (VEGF) transcripts was compared with 18S transcripts (y-axis) expressed in the same samples. Four eyes were assessed at each time point. Values in histogram are means±SEM B-C: Representative samples of PCR fragments (individual ratio VEGF:18S closest to the corresponding mean) were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light for 10–12-week-old mice (B) and 1-year-old mice (C). Prior to laser treatment, VEGF mRNA expression was lower in old control mice versus young control mice (p<0.001). At day 3 after PC, the highest level of VEGF mRNA was 2.7 times higher than the controls in old mice. In young mice the highest level of expression was reached at 16 h after photocoagulation and did not exceed 1.3 times the level observed in controls.
Mentions: After photocoagulation, kinetics of VEGF mRNA expression was also different in 10–12-week-old mice and 1-year-old mice. In 10–12-week-old mice (group III), VEGF mRNA reached its peak 16 h after photocoagulation, while in one-year-old mice (group IV) the peak was observed on day 3 and declined thereafter (Figure 9). In addition, the ratio of VEGF expression in RPE-choroid complex after photocoagulation in relation to RPE-choroid complex in control not photocoagulated was more important in one-year-old mice (group IV; 2.7 times) than in 10–12-week-old mice (group III; 1.3 times).

Bottom Line: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups.Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression.The above results may provide some insight into possible therapeutic strategies in the future.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France.

ABSTRACT

Purpose: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation.

Methods: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls.

Results: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively.

Conclusions: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

Show MeSH
Related in: MedlinePlus