Castration results in a dampened local inflammatory response and reduced expression of proinflammatory TNF-α. (a) Upper panels are low-magnification (×20) images of inflammatory cell staining for Mac-3. Mac-3–positive cells (arrow, bottom panels) were increased in day 3 wounds of intact mice compared with their castrated littermates (magnification: ×100). (b) Quantification of Mac-3–positive cells per unit area mm² showed a significant increase in the intact animals at day 5 after wounding compared with castrated mice. (c) Expression of TNF-α was reduced at days 5 and 21 in the wounds of the castrated mice (C) compared with wounds of intact mice (I). Wound tissue was pooled from five mice, and the gel shown is representative of three experiments. Right panel illustrates a representative RNase protection assay (of three replicate experiments) showing no differences in tissue expression of IL-6, macrophage migration inhibitory factor (MIF), or TGF-β1 between intact and castrated mice. (d) TNF-α protein levels were increased in wounds of intact compared with castrated mice as illustrated by day 5 immunostaining (left panels, representative of six wounds stained per group). Quantification of immunostaining (graph) showed a significant increase at days 3, 5, and 21 in the wounds of the intact mice compared with the castrated mice. n = 6 per timepoint. Western blot analysis of wound tissue showed increased levels of TNF-α at days 5 and 21 in wounds of intact mice compared with those of castrated mice. Blot is representative of three experiments using five mice per group. *P < 0.05.