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. 2017 May;25(3):366-376.
doi: 10.1111/wrr.12531. Epub 2017 Apr 27.

Wound Healing in Mac-1 Deficient Mice

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Free PMC article

Wound Healing in Mac-1 Deficient Mice

Lin Chen et al. Wound Repair Regen. .
Free PMC article

Abstract

Mac-1 (CD11b/CD18) is a macrophage receptor that plays several critical roles in macrophage recruitment and activation. Because macrophages are essential for proper wound healing, the impact of Mac-1 deficiency on wound healing is of significant interest. Prior studies have shown that Mac-1-/- mice exhibit deficits in healing, including delayed wound closure in scalp and ear wounds. This study examined whether Mac-1 deficiency influences wound healing in small excisional and incisional skin wounds. Three millimeter diameter full thickness excisional wounds and incisional wounds were prepared on the dorsal skin of Mac-1 deficient (Mac-1-/- ) and wild type (WT) mice, and wound healing outcomes were examined. Mac-1 deficient mice exhibited a normal rate of wound closure, generally normal levels of total collagen, and nearly normal synthesis and distribution of collagens I and III. In incisional wounds, wound breaking strength was similar for Mac-1-/- and WT mice. Wounds of Mac-1 deficient mice displayed normal total macrophage content, although macrophage phenotype markers were skewed as compared to WT. Interestingly, amounts of TGF-β1 and its downstream signaling molecules, SMAD2 and SMAD3, were significantly decreased in the wounds of Mac-1 deficient mice compared to WT. The results suggest that Mac-1 deficiency has little impact on the healing of small excisional and incisional wounds. Moreover, the findings demonstrate that the effect of single genetic deficiencies on wound healing may markedly differ among wound models. These conclusions have implications for the interpretation of the many prior studies that utilize a single model system to examine wound healing outcomes in genetically deficient mice.

Conflict of interest statement

Conflict of interest: None.

Figures

Figure 1
Figure 1
Wound closure in Mac-1 deficient and WT mice. A) CD11b expression on neutrophils in day 1 wounds of WT and Mac-1−/− mice. B) CD11b expression on macrophages in day 1 wounds of WT and Mac-1−/− mice. Bar=20μm C) Representative photomicrographs of wounds from days 0 to 8 post-wounding. Six 3mm full thickness excisional wounds were made on the dorsal skin of WT C57BL/6 and Mac-1 −/− mice. Bar=3mm. D) Representative photomicrographs of HE stained histologic sections of day 1, 3 and 7 wounds. Bar=200μm. Arrows indicate wound edges. E) Wound closure expressed as percent of closure as calculated from photomicrographs of wounds. N=10 in each group. F) Wound re-epithelialization at days 1 and 3 using histological analysis. N=6 in each group except that n=5 in WT group at day 3. G). Volume of granulation tissue at days 5 and 7. N=6 in each group except that n=5 in Mac-1 −/− group at day 5. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 2
Figure 2
Macrophage content, CCL2 levels, and expression of macrophage phenotype markers in wounds of Mac-1 deficient and WT mice. A) Time course of macrophage density in wounds. B Protein levels of CCL-2 determined by multiplex ELISA, expressed as pg per mg of homogenized tissue. C) mRNA expression of the anti-inflammatory macrophage marker YM1 during the course of wound healing. D) mRNA expression of the pro-inflammatory macrophage marker, iNOS during the course of wound healing. NS: normal or unwounded skin, N=6 in each group.
Figure 3
Figure 3
Neutrophil content and levels of CXCL-1, IL-1β, IL-6, and TNF-α in wounds of Mac-1 deficient and WT mice. A) Time course of the number of neutrophils in wounds. B, C, D, and E) Protein levels of CXCL-1, IL-1β, IL-6, and TNF-α determined by multiplex ELISA, expressed as pg per mg of homogenized tissue. NS: normal or unwounded skin. N=6 in each group.
Figure 4
Figure 4
Biochemical and mRNA analysis of wound collagen content in Mac1 deficient and WT mice. A) Levels of hydroxyproline in wounds over time. Concentrations of hydroxyproline were determined using a hydroxyproline assay kit. B, C) mRNA expression of collagen I and collagen III over the time course of the healing. NS: normal or unwounded skin. N=6 in each group.
Figure 5
Figure 5
Histologic assessment of collagen content in wounds of Mac-1 deficient and WT mice. A) Representative photomicrographs of Masson’s Trichrome stained sections of the wound bed at days 7, 14, 21, and 28. Bar=200μm. B) Quantification of Masson’s Trichrome collagen staining in wounds. The density of collagen in wound bed was quantified using Image J. C) Representative photomicrographs of picrosirius red staining sections of normal skin (NS), days 7 and 28 wounds. Bar=100μm. D) Summary of quantification of picrosirius red staining. The relative density of collagen I and collagen III in wound bed was quantified using Image J. NS: normal or unwounded skin. N=5 in each group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 6
Figure 6
Myofibroblast content and wound breaking strength in wounds of Mac-1 deficient and WT mice. A) Myofibroblast content in day 7 wound bed, assessed as the % of wound area. With α-SMA+ staining. B) Wound breaking strength, in lbs, over the time course of healing. NS: normal or unwounded skin. N=6 in each group.
Figure 7
Figure 7
Amount of activated TGF- β1 protein and mRNA expression of SMAD2&3 in wounds of Mac-1 deficient and WT mice. A) Protein concentrations of activated TGF-β1 in day 7 wounds as determined by ELISA. B) mRNA expression of SMAD2 and SMAD3 in day 7 wounds as determined by quantitative RT-PCR. N=6 in each group.

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