Figure 1.
Visualization of eGFP macrophages in wound tissue.
eGFP expressing macrophages were injected in full-thickness excisional skin wounds and tissue was isolated (A) 3 days, (B) 4 days, (C) 6 days and (D) 8 days after injection, and visualized for eGFP expression by fluorescence microscopy.
Figure 2.
Phenotypic characterization of polarized macrophages. (A) Gene expression analysis for M2 markers in M0 control, M2a and M2c bone marrow derived macrophages (BMDM) after 24 h of IL-4 (for M2a) or IL-10 (for M2c) polarization. (B-D) Gene expression analysis for inflammatory markers in M0 control, M2a and M2c polarized cells after 24 h of polarization and an additional 24 h of LPS stimulation. Cells were analyzed in triplicates and the mean expression of 2–3 independent experiments is indicated. M0 control levels were arbitrarily set to 1 in each experiment and for each gene and statistical significance was evaluated analyzing M2a and M2c expression levels compared to M0, using a one-sample t-test analysis against the hypothetical value 1 (M0). *p≤0.05.
Figure 3.
Wound healing in M2 macrophage injected wounds generated in C57BL/6 mice.
(A) Macroscopic appearance of representative saline, M0, M2a, and M2c injected wounds. Day 0 pictures were taken immediately after wounding. (B) Quantification of wound closure rate. At the indicated days, wound areas were determined using image analysis and expressed as percentage of wound area immediately post-injury as described in methods (n = 6 mice/group). Statistical significance was evaluated with a two-way ANOVA with Bonferroni post-test.
Figure 4.
Wound healing in M2 macrophage injected wounds generated in diabetic db/db mice.
(A) Macroscopic appearance of representative saline, M0, M2a, and M2c injected wounds. Day 0 pictures were taken immediately after wounding. (B) Quantification of wound closure rate. At the indicated days, wound areas were determined using image analysis and expressed as percentage of wound area immediately post-injury as described in methods (n = 6 mice/group). Statistical significance was evaluated with a two-way ANOVA with Bonferroni post-test. * p<0.05 M0 vs. M2a and M0 vs. M2c. ** p<0.01 saline vs. M2a and saline vs. M2c.
Figure 5.
Gene expression analysis of db/db wounds isolated at day 15 post-wounding.
Wounds were isolated from db/db mice 15 days after wounding and analyzed by real-time PCR for expression levels of (A) macrophage M2 markers Arginase-1, YM-1, Fizz-1 and HMOX-1; (B) inflammatory markers TNF, IL-6, IL-12, IL-1β and IL-10; (C) chemokines CXCL1 and CXCL2 and (D) matrix metalloproteinases MMP2 and MMP9. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test. *p<0.05. n = 6 mice (wounds)/group.
Figure 6.
Histological analysis of db/db wounds isolated at day 15 post-wounding.
Wounds from saline or macrophage injected db/db mice were isolated 15 days after wounding and processed for generation of paraffin sections. (A-D) H&E stained sections indicating migrating epithelial tongues (dotted line) in M2a and M2c wounds while complete re-epithelialization is present in saline and M0 wounds. (E-H) MSB trichrome staining indicating increased presence of fibrin (marked by arrowheads) and erythrocytes (marked by stars) in M2 macrophage injected wounds. Magnification 40x with 100x insets. Graphs are quantifications of (A) re-epithelialization and (B) fibrin/erythrocyte staining respectively. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test. n = 6 mice (wounds)/group.
Figure 7.
Immunohistochemical analysis of db/db wounds isolated at day 15 post-wounding.
Immunohistochemical stainings were performed in cryosections. (A-D) NIMP staining for wound neutrophils indicated higher numbers of neutrophils in M2a and M2c treated wounds compared to saline and M0 groups. Mice analyzed per group: salines = 3; M0 = 5; M2a = 6; M2c = 5. (E-H) F4/80 staining for macrophages indicated not significant differences between the groups. Mice analyzed per group: salines = 4; M0 = 4; M2a = 6; M2c = 6. (I-L) CD31 staining indicated similar staining for endothelial cells in the different groups. Mice analyzed per group: salines = 4; M0 = 5; M2a = 5; M2c = 4. Magnification 40x with 100x insets. Graphs are quantifications of (A) NIMP, (B) F4/80 and (C) angiogenesis staining respectively. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test.