4-aminopyridine promotes accelerated skin wound healing

The discovery of ways to enhance skin wound healing is of great importance due to the 31 frequency of these lesions. We discovered that 4-aminopyridine (4-AP), a potassium channel 32 blocker, greatly enhances skin wound healing. Benefits include faster wound closure, restoration 33 of normal-appearing skin architecture and epidermal thickness, increased vascularization and 34 increases in K14 + keratinocytes. Hair follicle number was increased, both histologically and by 35 analysis of K15 and K17 expression. Levels of vimentin (which marks fibroblasts) and α - 36 smooth muscle actin ( α -SMA, which marks collagen-producing myofibroblasts) increased, as did 37 α -SMA + cell numbers. 4-AP also increased numbers of axons and S-100 + Schwann cells, and 38 increased expression of p75-NTR and SOX10. Treatment also increased levels of nerve growth 39 factor, transforming growth factor-β , Substance P and PGP9.5, important modulators of wound 40 healing. 43


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Dermal Schwann cell isolation, culture conditions and characterization: The separated dermis 3 9 6 was minced into small pieces and placed in a petri dish containing collagenase (Gibco, # 17018-3 9 7 029) in DMEM basal medium at 37 o C for 2.5 hours. The dermis was then dissociated cells were 3 9 8 collected and centrifuged at 1500 rpm for 5 minutes. The pellet was resuspended in complete containing DMEM complete medium and incubated at 37 o C in 5% CO 2 incubator for 24 hours. incubator overnight 47 . The next day, adherent cells were refed with complete fibroblast medium, 4 1 0 and grown at 37 o C in 5% CO 2 incubator until cells reached ~95% confluency. hours 20,24,30,52 . The cell viability following 4-AP treatment was assayed by MTT assay according (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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After scratching, the cells were washed with PBS and the respective cell media was added with 4 2 6 or without 1 mM of 4-AP 24,30,52 . The plate was incubated in the IncuCyte™ automated imaging 4 2 7 system, and wound healing and cell migration was monitored by time-lapse photography Software Module (Essen BioScience) and the percent of wound healing was calculated from the 4 3 1 area measured after scratching relative to the basal area as expressed in pixels.  The cells were grown in their respective complete medium in the presence or absence of 4-AP 4 3 7 for 72 hours. Cells then were fixed with 4% paraformaldehyde followed by 0.1% tritonX-100 4 3 8 and stained with primary antibodies used against, cytokeratin-14, keratin-10, keratin-17, S100, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Next, wound scratches were created and cells were grown with or without 4-AP in the medium.

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Cells were imaged every hour until 24 hours and analyzed for percent of closure as described healing mouse skin sections. The following primary antibodies were used: mouse Cytokeratin14 4 5 6 antibody (# ab7800; IF-1:100), rat CD31 antibody (# 553370; IF-1:100), mouse S100 antibody incubated overnight with 5% BSA in 0.1% PBS-T for IF stain and/or 5% skimmed milk in 0.1% used as nuclear counterstain. The immunofluorescence stained sections were imaged using 4 7 2 ZEISS Axio Observer 7-Axiocam 506 mono -Apotome.2 microscope. The image analysis and 4 7 3 quantification was carried out using ZEN 2.6 pro (Zeiss) imaging software or ImageJ-1.53e  Protein isolation and western blot. For protein isolation the harvested skin tissue was flash 4 7 6 frozen immediately. The frozen skin tissue was ground to a fine power using a liquid nitrogen  incubated with HRP-conjugated secondary antibodies (dilution,1:3000) for 1 hour.

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Immunoreactivity was then detected using chemiluminescent substrate (Thermo Scientific™ 4 8 9 SuperSignal™ West Pico PLUS, # 34577). The intensities of the bands were quantified using 4 9 0 Gel-imaging software (Bio-Rad Laboratories Inc., Image Lab 6.1). The quantified band 4 9 1 intensities were normalized using GAPDH and expressed either as normalized intensity or as 4 9 2 ratios with respect to saline treated mice. analyses. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.  Representative images of the wound healing in control (saline treated) and 4-AP treated mice at  staining intensity in the 4-AP treated group was significantly higher than that of control.
The copyright holder for this preprint this version posted November 3, 2021. ; https://doi.org/10.1101/2021.10.31.465317 doi: bioRxiv preprint replicates/group, and *P = 0.01 to 0.05, **P = 0.01 to 0.001, ***P < 0.0002, and ****P < 0.0001 closure was calculated as the ratio of the remaining wound gap at the given time point. Mean ± 6 1 8 SEM, N = 5 wound scratch replicates/group, and *P = 0.01 to 0.05, **P = 0.01 to 0.001, ***P < 6 1 9 0.0002, and ****P < 0.0001 one-way ANOVA Sidak's multiple comparisons test. technical support. We thank Dr. Pierre A Coulombe for providing the K17 antibody. We would 6 2 4 also like to acknowledge staff technical assistance from the Penn State College of Medicine  Funding: This work was supported by grants from the NIH (K08 AR060164-01A) and DOD    that covers "Method and materials for treating nerve injury and/or wound healing" and Peripheral Therapeutics, a company that has licenced a previous patent on the use of 4-AP for 6 4 0 traumatic peripheral nerve injury and nerve continuity diagnosis. All other authors declare that 6 4 1 they have no competing financial interests. 6 4 2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 3, 2021. ; https://doi.org/10.1101/2021.10.31.465317 doi: bioRxiv preprint Data and supplementary materials availability: All data associated with this study are 6 4 3 described and shown in the paper or the Supplementary Materials.

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References: (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 3, 2021. Immunofluorescence staining of control and healed wound sections for pan-neuronal marker PGP-9.5 (red) and nuclear stain DAPI (blue). Scale bars = 20 μm. (D) Quantification of PGP-9.5 protein expressing cells showed significantly increased PGP-9.5 intensity in the 4-AP treated group compared to the saline treated group at day 14. PGP 9.5 in 4-AP-treated mice was not significantly different from seen in uninjured (control) tissue. Mean ± SEM; N = 6 animal wound tissues/group (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 3, 2021. of wound closure was calculated as the ratio of the remaining wound gap at the given time point compared to time 0. Mean ± SEM, N = 5 wound scratch replicates/group, and *P = 0.01 to 0.05, **P = 0.01 to 0.001, ***P < 0.0002, and ****P < 0.0001 one-way ANOVA Sidak's multiple comparisons test. (C) Co-immunostaining of dermal Schwann cells exposed to 4-AP or no treatment for 72 hours and immunostained against SC markers including the Schwann cell marker S100 (green), a de-differentiation marker p75-NTR (red) and myelin basic protein (MBP-yellow). DAPI (blue) was used as nuclear counterstaining. Scale bars, 100 µm. (D -F) A representative western blot and normalized integrated densities for SOX10, p75-NTR, NGF and GAPDH.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 3, 2021. Images were recorded every one hour. Scale bar = 100 µm.
Supplementary Movie 9. An example of time-lapse phase contrast images depicting the migration of co-cultured keratinocytes and fibroblasts without treatment (control) during wound scratch closure. Images were recorded every one hour. Scale bar = 100 µm.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.