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

[摘要] Purpose: To analyze the influence of ageon retinochoroidal wound healing processes and on glial growth factorand cytokine mRNA expression profiles observed after argon laserphotocoagulation. Methods: A cellular and morphometricstudy was performed that used 44 C57Bl/6J mice: 4-week-old mice (groupI, n=8), 6-week-old mice (group II, n=8), 10–12-week-old mice (groupIII, n=14), and 1-year-old mice (group IV, n=14). All mice in thesegroups underwent a standard argon laser photocoagulation (50 µm, 400mW, 0.05 s). Two separated lesions were created in each retina using aslit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months afterphotocoagulation, mice from each of the four groups were sacrificed bycarbon dioxide inhalation. Groups III and IV were also studied at 6, 7,and 8 months after photocoagulation. At each time point the enucleatedeyes were either mounted in Tissue Tek (OCT), snap frozen and processedfor immunohistochemistry or either flat mounted (left eyes of groupsIII and IV). To determine, by RT–PCR, the time course of glialfibrillary acidic protein (GFAP), vascular endothelial growthfactor (VEGF), and monocyte chemotactic protein-1 (MCP-1)gene expression, we delivered ten laser burns (50 µm, 400 mW, 0.05 s)to each retina in 10–12-week-old mice (group III’, n=10) and 1-year-oldmice (group IV’, n=10). Animals from Groups III’ and IV’ had the sameage than those from Groups III and IV, but they received ten laserimpacts in each eye and served for the molecular analysis. Mice fromGroups III and IV received only two laser impacts per eye and servedfor the cellular and morphologic study. Retinal and choroidal tissuesfrom these treated mice were collected at 16 h, and 1, 2, 3, and 7 daysafter photocoagulation. Two mice of each group did not receivephotocoagulation and were used as controls. Results: In the cellular and morphologicstudy, the resultant retinal pigment epithelium interruption expansewas significantly different between the four groups. It was moreconcise and smaller in the oldest group IV (112.1 µm±11.4 versus 219.1µm±12.2 in group III) p<0.0001 between groups III and IV. Bycontrast, while choroidal neovascularization (CNV) was mild and notreadily identifiable in group I, at all time points studied, CNV wasmore prominent in the (1-year-old mice) Group IV than in the othergroups. For instance, up to 14 days after photocoagulation, CNVreaction was statistically larger in group IV than in group III((p=0.0049 between groups III and IV on slide sections and p<0.0001between the same groups on flat mounts). Moreover, four months afterphotocoagulation, the CNV area (on slide sections) was 1,282 µm2±90for group III and 2,999 µm2±115 for group IV (p<0.0001between groups III and IV). Accordingly, GFAP, VEGF,and MCP-1 mRNA expression profiles, determined by RT–PCR at 16h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging.In 1-year-old mice (group IV), GFAP mRNA expression was alreadysignificantly higher than in the younger (10–12 week) group III beforephotocoagulation. After laser burns, GFAP mRNA expressionpeaked at 16–24 h and on day 7, decreasing thereafter. VEGFmRNA expression was markedly increased after photocoagulation in oldmice eyes, reaching 2.7 times its basal level at day 3, while it wasonly slightly increased in young mice (1.3 times its level in untreatedyoung mice 3 days postphotocoagulation). At all time points afterphotocoagulation, MCP-1 mRNA expression was elevated in oldmice, reaching high levels of expression at 16 h and day 3respectively. Conclusions: Our results were based onthe study of four different age groups and included not only data frommorphological observations but also from a molecular analysis of thevarious alterations of cytokine signaling and expression. One-year-oldmice demonstrated more extensive CNV formation and a slower pace ofregression after laser photocoagulation than younger mice. These wereaccompanied by differences in growth factors and cytokine expressionprofiles indicate that aging is a factor that aggravates CNV. The aboveresults may provide some insight into possible therapeutic strategiesin the future.

[学科分类] 生物化学/生物物理