Enhanced Neurogenic Biomarker Expression and Reinnervation in Human Acute Skin Wounds Treated by Electrical Stimulation Anil Sebastian, Susan W. Volk,

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Enhanced Neurogenic Biomarker Expression and Reinnervation in Human Acute Skin Wounds Treated by Electrical Stimulation Anil Sebastian, Susan W. Volk, Poonam Halai, James Colthurst, Ralf Paus, Ardeshir Bayat Journal of Investigative Dermatology Volume 137, Issue 3, Pages 737-747 (March 2017) DOI: 10.1016/j.jid.2016.09.038 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Increased reinnervation and neuropeptide synthesis in ES-treated wounds. (a) Representative Western blot of PGP9.5 expression. (b) Quantification of PGP9.5 expression in uninjured skin and cutaneous wounds after injury. (c) Quantification of immunoreactivity to PGP9.5 (%PGP9.5+ area) in uninjured skin (US), control, and ES-treated wounds. (d) IHC images of PGP9.5 expression. (e) Representative Western blot of SP expression. ES14 is ES day 14, C14 is control day 14. (f) Quantification of percentage of SP+ cells in uninjured skin and cutaneous wounds at 7 and 14 days after injury. (g) Quantification of IHC analysis of SP expression. (h) Representative IHC of SP expression in skin (indicated with arrows). Dotted lines indicate epidermal-dermal junction. Scale bar = 100 μm. Star indicates statistical significance of P < 0.05. C, control; D, day; DER, dermis; EP, epidermis; ES, electrical stimulation; IHC, immunohistochemistry; PGP9.5, protein gene product 9.5; SP, substance P; US, uninjured skin. Journal of Investigative Dermatology 2017 137, 737-747DOI: (10.1016/j.jid.2016.09.038) Copyright © 2016 The Authors Terms and Conditions

Figure 2 ES treatment increases TUBB3 expression in human cutaneous wounds, specifically in melanocytes. (a) Microarray analysis of uninjured skin (US) and ES-treated wounds on day 14 after injury (ES14) shows up-regulation of TUBB3. (b) Quantification of IHC analysis of TUBB3 expression. (c) Representative IHC of TUBB3 expression in uninjured skin, control (non–ES-treated), and ES-treated skin wounds at day 14. (d) Quantification of melanocytes indicated by percentage gp100+ cells in uninjured skin, control, and ES-treated skin wounds. (e) Quantification of TUBB3+ melanocytes. (f) IHC of TUBB3+ melanocytes in uninjured skin, control, and ES-treated skin wounds at day 14. Dotted line indicates the epidermal-dermal junction. Scale bar = 100 μm. Star indicates statistical significance of P < 0.05. ES, electrical stimulation; IHC, immunohistochemistry; US, uninjured skin; vol, volunteer. Journal of Investigative Dermatology 2017 137, 737-747DOI: (10.1016/j.jid.2016.09.038) Copyright © 2016 The Authors Terms and Conditions

Figure 3 NGF and FIG4 expression is up-regulated in human cutaneous wounds treated with ES. (a) Representative IHC and (b) quantification of NGF expression in uninjured skin (US), control (non–ES-treated), and ES-treated skin wounds at day 14. (c) Representative Western blot and (d) quantitation of NGF expression in uninjured skin, control, and ES-treated skin wounds at day 14; P < 0.05. (e) Representative IHC and (f) quantification of FIG4 expression in uninjured skin, control, and ES-treated skin wounds at day 14. (g) Representative WB and (h) quantitation of FIG4 expression in uninjured skin, control, and ES-treated skin wounds at day 14. Scale bar = 100 μm. Star indicates statistical significance of P < 0.05. C, control; ES, electrical stimulation; IHC, immunohistochemistry; NGF, nerve growth factor; US, uninjured skin. Journal of Investigative Dermatology 2017 137, 737-747DOI: (10.1016/j.jid.2016.09.038) Copyright © 2016 The Authors Terms and Conditions

Figure 4 ES treatment enhances human SHSY5Y neuroblastoma differentiation. (a) Representative Western blot and (b) quantitation for mediators of phosphatidylinositol (3,4,5)-triphosphate synthesis pathway in undifferentiated neurons (UDN) and neurons cultured under differentiating conditions with and without ES treatment. (c) Representative Western blot and (d) quantitation for mediators of phosphatidylinositol (3,4,5)-triphosphate synthesis pathway after FIG4 siRNA treatment. DN is differentiated neurons. Scale bar = 50 μm. Star indicates statistical significance of P < 0.05. BDNF, brain-derived neurotrophic factor; DN, differentiated neurons; ES, electrical stimulation; RA, retinoic acid; siRNA, small interfering RNA; UDN, undifferentiated neurons. Journal of Investigative Dermatology 2017 137, 737-747DOI: (10.1016/j.jid.2016.09.038) Copyright © 2016 The Authors Terms and Conditions

Figure 5 ES expands the pool of TUBB3+ Merkel cells and increases Merkel cell reinnervation during human cutaneous wound healing. (a) Representative IHC of TUBB3+ Merkel cells in uninjured skin (US), control (non–ES-treated), and ES-treated skin wounds at day 14. (b) Percentage of TUBB3+ Merkel (CK20+) cells in uninjured skin, control, and ES-treated skin wounds at days 3, 7, 10, and 14 after injury. (c) Representative IHC of PGP9.5+ Merkel cells in uninjured skin, control, and ES-treated skin wounds at day 14. (d) Percentage of PGP9.5+ Merkel (CK20+) cells in uninjured skin, control, and ES-treated skin wounds at days 3, 7, 10, and 14 after injury. The upper side of the dotted lines is epidermis and the bottom side is dermis. Scale bar = 100 μm. Star indicates statistical significance of P < 0.05. CK20, cytokeratin 20; ES, electrical stimulation; IHC, immunohistochemistry; PGP9.5, protein gene product 9.5; US, uninjured skin. Journal of Investigative Dermatology 2017 137, 737-747DOI: (10.1016/j.jid.2016.09.038) Copyright © 2016 The Authors Terms and Conditions