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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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Evolution of the CNV reaction with aging. A: This reaction reaches its peak during the first two weeks after photocoagulation (PC), diminishes progressively after, and is still present on month 4. Choroidal neovascularization is rare and difficult to quantify in 4-week-old mice (group I). It is more prominent in 10–12-week-old mice and 1-year-old mice. It diminishes more markedly in young adult mice after two months. On month 4, the difference is statistically significant between young adult and old mice. A p<0.0001 was considered significant. B: The mean CNV area during the first two weeks after PC is significantly larger in old mice in comparison to young adult mice according to t-test.
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f6: Evolution of the CNV reaction with aging. A: This reaction reaches its peak during the first two weeks after photocoagulation (PC), diminishes progressively after, and is still present on month 4. Choroidal neovascularization is rare and difficult to quantify in 4-week-old mice (group I). It is more prominent in 10–12-week-old mice and 1-year-old mice. It diminishes more markedly in young adult mice after two months. On month 4, the difference is statistically significant between young adult and old mice. A p<0.0001 was considered significant. B: The mean CNV area during the first two weeks after PC is significantly larger in old mice in comparison to young adult mice according to t-test.

Mentions: On serial sections, vW-positive cells in the choroid around the lesion (CNV formation) were detected in the four groups of mice already three days after photocoagulation. These vW-positive cells were more evident from day 7 onward, reaching a peak on the second week after photocoagulation. In 4-week-old mice, a few mice present new vessels after photocoagulation, but the great majority of them do not show any CNV. In mice 6 weeks of age or older, every time we did laser photocoagulation we obtained CNV. The extent of the CNV was more prominent in the eyes of the one-year-old mice (group IV): 3,851 µm2±101.6 versus 2,930 µm2±205.3 in young adult group (group III), 734.9 µm2±79.7 in group II and 171 µm2±44.7 in group I. These differences were highly statistically significant between the four groups: p=0.004 between groups III and IV; p<0.0001 between groups II and III; p<0.0001 between groups I and II (Figure 6B). In this model, CNV reaction was present for several months after photocoagulation and decreased progressively. It was still present at four months for the oldest groups III and IV (2,999 µm2±115 for group IV versus 1,282 µm2±90 for group III; p<0.0001), and at eight months (2,571 µm2±116 in group IV versus 1,226 µm2±59 in group III; p<0.0001, data not shown; Figure 6A).


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)

Evolution of the CNV reaction with aging. A: This reaction reaches its peak during the first two weeks after photocoagulation (PC), diminishes progressively after, and is still present on month 4. Choroidal neovascularization is rare and difficult to quantify in 4-week-old mice (group I). It is more prominent in 10–12-week-old mice and 1-year-old mice. It diminishes more markedly in young adult mice after two months. On month 4, the difference is statistically significant between young adult and old mice. A p<0.0001 was considered significant. B: The mean CNV area during the first two weeks after PC is significantly larger in old mice in comparison to young adult mice according to t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Evolution of the CNV reaction with aging. A: This reaction reaches its peak during the first two weeks after photocoagulation (PC), diminishes progressively after, and is still present on month 4. Choroidal neovascularization is rare and difficult to quantify in 4-week-old mice (group I). It is more prominent in 10–12-week-old mice and 1-year-old mice. It diminishes more markedly in young adult mice after two months. On month 4, the difference is statistically significant between young adult and old mice. A p<0.0001 was considered significant. B: The mean CNV area during the first two weeks after PC is significantly larger in old mice in comparison to young adult mice according to t-test.
Mentions: On serial sections, vW-positive cells in the choroid around the lesion (CNV formation) were detected in the four groups of mice already three days after photocoagulation. These vW-positive cells were more evident from day 7 onward, reaching a peak on the second week after photocoagulation. In 4-week-old mice, a few mice present new vessels after photocoagulation, but the great majority of them do not show any CNV. In mice 6 weeks of age or older, every time we did laser photocoagulation we obtained CNV. The extent of the CNV was more prominent in the eyes of the one-year-old mice (group IV): 3,851 µm2±101.6 versus 2,930 µm2±205.3 in young adult group (group III), 734.9 µm2±79.7 in group II and 171 µm2±44.7 in group I. These differences were highly statistically significant between the four groups: p=0.004 between groups III and IV; p<0.0001 between groups II and III; p<0.0001 between groups I and II (Figure 6B). In this model, CNV reaction was present for several months after photocoagulation and decreased progressively. It was still present at four months for the oldest groups III and IV (2,999 µm2±115 for group IV versus 1,282 µm2±90 for group III; p<0.0001), and at eight months (2,571 µm2±116 in group IV versus 1,226 µm2±59 in group III; p<0.0001, data not shown; Figure 6A).

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