1. Exploring the “Hair Growth–Wound Healing Connection”: Anagen Phase Promotes Wound Re-Epithelialization 
David M. Ansell, Jennifer E. Kloepper, Helen A. Thomason, Ralf Paus, Matthew J. Hardman 
Journal of Investigative Dermatology 
Volume 131, Issue 2, Pages (January 2011)
DOI: /jid
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Wounds made in the anagen stage of hair cycle are smaller, indicating accelerated healing. (a) Macroscopic images 3 days post-wounding show wounds of reduced size and hair regrowth in anagen skin. (b) Image analysis was used to measure day 3 wounds in female C57/Bl6 mice. Mice wounded during the anagen phase of the hair cycle display accelerated healing as quantified by decreased wound area, (c) and increased percentage of re-epithelialization (re-ep). The number on re-epithelialization bars denotes percentage of mice exhibiting fully re-epithelialized wounds. (d) Hematoxylin and eosin-stained day 3 wound 5μm wax sections from C57/Bl6 mice, arrows denote wound margins (composite images). (e) Macroscopic images of excisional wound timecourse. (f) Mice excisionally wounded in anagen display reduced planimetric wound area, (g) and increased histologically measured re-epithelialization. Bar=5mm (a), 400μm (d), 1cm (e). A, anagen; P, postnatal day; T, telogen. Mean data±SEM. *P<0.05, **P<0.01. Pairwise Mann–Whitney U-test. n=6–14.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Accelerated healing in anagen hair-cycle stage is accompanied by attenuated proliferation. (a) Representative histology of incisional wound timecourse. (b) Image analysis reveals increased re-epithelialization (re-ep) in anagen compared with telogen wounds 48 and 72 hours post wounding. BrdU immunohistochemistry (d), box denotes regions used for immunohistomorphometric measurements (outer root sheath of hair follicle (HF) and basal keratinocytes of interfollicular epidermis (IFE)). Telogen wounds displayed decreased initial HF proliferation followed by a substantially increased proliferative response (c). Bar=100μm (a), 200μm (d), and 50μm (d enlarged areas). A, anagen; T, telogen. Mean±SEM. **P<0.01. n=5.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Altered keratinocyte differentiation in anagen skin wounds. Representative immunohistochemical localization of (a) keratin 14, (b) keratin 1, (c) keratin 6, and (d) loricrin in normal skin and wounds from anagen and telogen cycle stages. (d) Delayed loricirin induction was observed in telogen wounds. Box indicates comparable region used for immunohistomorphometric quantification. Bar=400μm (a, b, d, c normal skin), 800 μm (c wound), 50μm (inset a, b, d).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Anagen wounds display reduced inflammatory cell infiltration. (a) Representative immunohistochemistry (neutrophil) indicating healing phenotype, dotted line indicates area where high-power images for scoring (i.e., box) were taken. Minimum four fields per wound, two wounds per mouse. (b, c) Quantification of inflammatory cell numbers in granulation tissue of day 3 wounds from anagen (A; postnatal day 35) and telogen (T; postnatal day 45) cycle stage. Increased neutrophil (b) and macrophage (c) numbers were observed in telogen wounds. Arginase-1 (alternatively, activated macrophages) (d), migration inhibitory factor (MIF) (e), and CD44 (g) were also significantly increased in telogen wounds. (f) CD74 (MIF receptor) was equally expressed in anagen and telogen wounds. Bar=400μm (a), 50μm (b–g). Mean±SEM. *P<0.05, **P<0.01, t-test, n=6.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Anagen skin wounds show increased angiogenesis and collagen deposition, but equivalent smooth muscle actin (SMA) expression. (a–f) Quantification of mean vessel length and diameter (PECAM-1 immunohistochemistry) in anagen and telogen mouse normal skin (a–c) and wound margins (d–f). Mean vessel length/diameter was assessed in 200 vessels in anagen (P35) and telogen (P45). (g) Picrosirius red-stained sections show increased collagen (red) deposition in anagen wounds with no difference in normal skin. (h) Representative immunohistochemistry for SMA reveals no difference in wound myofibroblast numbers. A, anagen; NS, normal skin; T, telogen. Bar=50μm (c, f), 400μm (g), 50μm (h). n=4, mean±SEM, *P<0.05, **P<0.01, Mann–Whitney U-test.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Comparative analysis of previously published microarray data sets reveals that telogen hair cycle correlates with delayed healing at the level of gene expression. (a) Schematic outline of analysis strategy. Briefly, comparison of independently generated microarray data sets from normal versus delayed healing wounds and anagen versus telogen skin revealed 180 common probe sets. Probe sets/genes were subsequently clustered and analyzed for biologically relevant overrepresentation. (b) The number of genes within each identified cluster. The majority were upregulated (blue; cluster 1) or downregulated (red; cluster 2) in both telogen and delayed healing. Only 5% of genes fell in cluster 3 (yellow) or cluster 4 (green). (c) Selected gene ontology groups specifically overrepresented in cluster 1 or cluster 2 with associated P-value. (d) Quantitative PCR validation confirms microarray changes in hair-cycle-associated wound repair. (e, f) Immunohistochemical profiling of array-identified proteins Cd34 and Cx43. (e) Cd34 localizes to HF bulge and suprabasal wound edge keratinocytes, and shows increased dermal endothelial staining in anagen skin. (f) Cx43 localizes to outer root sheath HF keratinocytes and basal IFE keratinocytes. Neoepidermis in anagen wounds displays reduced Cx43 expression. Bar=50μm (e), 25μm (f, NS and WE), 50μm (f, HF). Fold change±SEM. n=4. A, anagen; E, endothelial; HF, hair follicle; NS, normal skin; T, telogen; WE, wound edge. Dotted line denotes basement membrane; arrows denote immunohistochemical staining.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions