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In a previous study we showed that Leydig cell-specific gene-inactivated Grem1-GFP-TG mice developed severe diaphragmatic dysfunction and sutural remodeling. Here, we investigated the time-course and mechanisms of this severe diaphragmatic injury. Grem1-GFP-TG and wild-type (WT) mice were immersed in water for 10, 20, 40 and 80 d and analyzed at days 4, 8, 13 and 25. In the diaphragm muscle, the number of fibroblasts increased significantly at day 4, peaked at day 13, and decreased at day 25. The collagen content also increased at day 4, but remained higher than in WT at days 13 and 25. Grem1 expression was initially unchanged, then increased gradually until day 25, at which point the Grem1-GFP-TG diaphragm demonstrated a higher Grem1 expression level than WT. The density of F4/80-positive cells in the diaphragm muscle of Grem1-GFP-TG mice was higher than in WT at all time points. IL-6 mRNA levels were higher in
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