Materials and Reagents

Corning^TM Falcon^TM 50 mL conical centrifuge tubes (Fisher Scientific, catalog number: 14-432-22)
Covidien sterile gauze (Fisher Scientific, catalog number: 2187)
BD brand isopropyl alcohol swabs (Fisher Scientific, catalog number: 13-680-63)
Betadine solution swab stick (Fisher Scientific, catalog number: 19065534)
Covidien Telfa^TM non-adherent pads (Fisher Scientific, Covidien, 1961)
Tegaderm, 3 M, 1626W (VWR, catalog number: 56222-191)
Dental surgical ruler (DoWell Dental Products, catalog number: S1070)
C57/BL6 females (6–8 weeks old) (The Jackson Laboratory, catalog number: 000664)
B6.BKS(D)-Lepr^db/J females (6–8 weeks old) (The Jackson Laboratory, catalog number: 000697)
Buprenorphine SR (0.5 mg/mL) (Buprenex, Indivior Inc., catalog number: 12496-0757-1)
Isoflurane, USP (Dechra Veterinary Products, catalog number: 17033-094-25)
Puralube^® ophthalmic ointment (Dechra, NDC, catalog number: 17033-211-38)
Vetbond (3 M) (Saint Paul, MN, catalog number: 1469SB)
Depilatory cream (Nair Hair Remover Lotion, Church&Dwight, CVS, catalog number: 339823)
Ethanol 70% solution (Fisher Scientific, catalog number: 64-17-5)
Medequip Depot Silk Black Braided Sutr 6-0 Rx (Medequip Depot D707N, Fisher Scientific, catalog number: NCO835822)

Equipment

Tissue forceps 4¾ in. stainless 1 × 2 teeth (Mckesson, catalog number: 43-2-775)
Iris scissors 4½ in. stainless (McKesson, catalog number: 43-2-104)
Disposable biopsy punch 8 mm (Integra Miltex, catalog number: 33-37)
Needle holder 5 in. with serrated jaws (McKesson, catalog number: 43-2-842)
Keyes cutaneous punch 16 mm (Delasco, catalog number: KP-16)
Keyes cutaneous punch 10 mm (Delasco, catalog number: KP-10)
Silicone sheet 0.5 mm, no PSA (Sigma-Aldrich, Grace Bio-Labs CultureWell, GBL664581-5EA)
Liquid repellent drape 75 × 90 cm with adhesive hole 6 × 9 cm (Omnia S.p.A., catalog number: 12.T4362)
Inhalation anesthesia system (VetEquip, catalog number: 922130)
Aesculap Exacta mini trimmer (Aesculap)
Thermo-peep heating pad (K&H, Amazon)
Surgical skin marker (McKesson, 19-1451_BX)

Software

ImageJ (ImageJ, Wayne Rasband, imagej.net)
Prism 9 (GraphPad Holdings, LLC, graphpad.com)
R studio Desktop (RStudio PBC, rstudio.com, open-source software)
Excel (Microsoft Cooperation, Microsoft.com)

Procedure

Making splints (equipment shown in Figure 1A) (Table 1)

Time: Each splint takes approximately 30 s to make.

Remove the sticky coverings from both sides of the silicone sheets.
Place the silicone sheet on a soft base (e.g., folded cloth).
First, punch out the outer circle with the 16 mm cutaneous punch using circular movements and applying pressure on the silicone sheet.
Punch out the inner circle with the 10 mm cutaneous punch, also using circular movements and applying pressure on the silicone sheet.

Place splints in a 70% ethanol bath in a conical tube for disinfection (leave in the 70% ethanol solution until use).

Figure 1. Splints and Excisional Wounds. A. From left to right: biopsy punches used to make splints [10 mm (left) and 16 mm cutaneous punch (right)]; splints; all material necessary to make splints, with the silicone sheet on top. B. From left to right: shaved and depilated dorsum of mouse; excisional wounds on mouse dorsum; excisional wounds with splints; wound dressing (Telfa + Tegaderm).

Table 1. Making splints

Step	Procedure	Troubleshooting
1	Remove sticky coverings from both sides of the silicone sheets.
2	Place the silicone sheet on a soft base.	Folded cloth works best, as it has high resistance to punching forces.
3	First, punch out the outer circle with a 16 mm punch using circular movements and applying pressure on the silicone sheet.
4	Punch out the inner circle with a 10 mm punch, also using circular movements and applying pressure on the silicone sheet.
5	Place splints in a 70% ethanol bath in a conical tube for disinfection (leave in the 70% ethanol solution until use).	Replace ethanol bath regularly.

Excisional wounding (Table 2)

Time: 12–30 min per mouse (after sufficient practice), time varies depending on operator’s skills and mouse line. Diabetic mice (high BMI) tend to be easier to handle during surgery than thin wildtype mice.

Prepare surgical field with heating pad set at 40°C, surgical drape, and anesthesia system.
Anesthetize mouse with isoflurane at a flow rate of 5% in 100% oxygen (flow rate 1 L/min).
Inject buprenorphine SR 0.5 mg/kg subcutaneously 5 min prior to incision and apply ophthalmic ointment on both eyes.
Maintain anesthetized state with 1%–1.5% isoflurane; monitor mouse every 90 s for changes in breathing rate.
Place mouse in a prone position on the prepared surgical table.
Shave complete dorsum of mouse from the neck to the root of the tail and to the sides; shave until the beginning of all four extremities.
Apply depilatory cream on the shaved area for 30 s. Use wet gauze swabs to remove all remaining cream and fur.
Disinfect skin with three alternating betadine and alcohol wipes in a circular fashion.
Place sterile drape over the mouse, leaving the surgical area free.
Use the biopsy punch to outline the pattern for excision of skin on the dorsum of the mice (Figure 1B).
Create two wounds on the dorsum of the mice with the biopsy punch.
Push the biopsy punch firmly onto the skin and then twist fairly quickly in swift circulating motions to cut through the dermis.
Notes:
1. Fixate skin between fingers to create tension on the skin in order to excise biopsy.
2. Do not push too hard or you will wound the underlying muscle.
3. Wet the skin with an alcohol swab to ease friction force from the biopsy punch.
Excise skin (with the aid of scissors if necessary).
Take out the splint from ethanol bath and let it dry on sterile surgical area.
Apply minimal amounts of Vetbond to the splint and place it carefully around the wound.
Notes:
1. Leave an area of the splint without Vetbond for forceps to grab. Otherwise, the splint will be glued to the forceps.
2. Make sure to apply enough Vetbond on the silicone splints so that the splint sticks well to the skin, but care must be taken to not apply Vetbond onto the wound.
Use 6-0 sutures to apply eight interrupted sutures fixating the splint to the surrounding skin (Figure 1B).

Take a picture of the wound with a ruler for documentation and baseline for analysis of wound closure.

Note: Be sure to try to always take the images from the same angle, orientation, and distance from each wound to promote consistency.

Table 2. Excisional wounding

Step	Procedure	Troubleshooting
1	Prepare surgical field with heating pad set at 40 °C, surgical drape, and anesthesia system.	To limit anesthesia time for mice, accurate preparation is essential.
2	Anesthetize mouse with isoflurane at a flow rate of 5% in 100% oxygen (flow rate 1 L/min).	Monitor mouse carefully and take out of anesthesia box immediately when sufficient level of anesthesia has been reached. Exposure to high flow rates of isoflurane for too long can cause death.
3	Inject buprenorphine SR 0.5 mg/kg subcutaneously 5 min prior to incision and apply ophthalmic ointment on both eyes.	Do not start surgery immediately after injecting buprenorphine, as activation period needs to be accounted for.
4	Maintain anesthetized state with 1%–1.5% isoflurane; monitor mouse every 90 s for changes in breathing rate.	Maintaining flow rate as low as possible, but as high as necessary is imperative. An insufficient flow rate will cause peri-surgical awakening; however, an excessive flow rate will cause death during anesthesia.
5	Place mouse in a prone position on prepared surgical table.
6	Shave complete dorsum of mouse from the neck to the root of the tail and to the sides; shave until the beginning of all four extremities.	Make sure to shave a sufficient area, as Vetbond will not stick to unshaved skin.
7	Apply depilatory cream on the shaved area for 30 s. Use wet gauze swabs to remove all remaining cream and fur.	Do not leave depilatory cream on for longer than 30 s. Leaving it on too long will cause skin irritations that will likely result in wound dehiscence in a later stage.
8	Disinfect skin with three alternating betadine and alcohol wipes in a circular fashion.
9	Place sterile drape over the mouse, leaving the surgical area free.
10	Use biopsy punch to outline pattern for excision of skin on the dorsum of the mice (Figure 1B).
11	Create two wounds on the dorsum of the mice with biopsy punch.
12	Push the biopsy punch firmly onto the skin and then twist fairly quickly in swift circulating motions to cut through the dermis.	Fixate skin between fingers to create tension on the skin in order to excise biopsy. Do not push too hard or you will wound the underlying muscle. Wet the skin with an alcohol swab to ease friction forces from the biopsy punch.
13	Excise skin.	Scissors can be used to aid excision of skin if necessary.
14	Take out the splint from ethanol bath and let it dry on sterile surgical area.	Let splint fully dry before applying Vetbond.
15	Apply minimal amounts of Vetbond to the splint and place it carefully around the wound.	Leave an area of the split without Vetbond for forceps to grab. Otherwise, the splint will be glued to the forceps. Make sure to apply enough Vetbond on the silicone splints, so the splint sticks well to skin, but care must be taken to not apply Vetbond onto the wound.
16	Use 6-0 sutures to apply eight interrupted sutures fixating the splint to the surrounding skin (Figure 1B).
17	Take a picture of the wound with a ruler for documentation and baseline for analysis of wound closure.	Be sure to try and always take the images at the same angle, orientation, and distance from each wound to promote consistency.

Therapeutics and dressings (Table 3)

Time: Varies on applied treatment (approximately 2–15 min per mouse).

Injecting therapeutic (e.g., cells, pharmacologic) or no treatment (if using hydrogels, skip to step C2)
1. Inject therapeutic or vehicle control around the wound edge.
2. Wrap the wounds with an initial layer of Telfa.
3. Skip to step C3.
Hydrogel therapy (Figure 2A)
1. Apply piece of circular hydrogel onto the wound (with or without therapeutic imbued) (Figure 2B).
  Note: Hydrogel should be cut out so that it has the same measurements as the excisional wound.
2. Wrap the wounds with an initial layer of Tegaderm.
  Note: Initial layer keeps hydrogel protected, moist, and pressed against wound to maximize delivery.
3. Next, cover the wound with Telfa dressing.
4. Proceed to step C3.
Cut Tegaderm sheets in half and use the halves to wrap around the mice (Figure 1B).
Monitor mouse until it is fully awake.

Place in cage—single housing is necessary now.

Figure 2. Wound healing analysis. A. Pictures of wound healing over time in wildtype (WT) and diabetic mice (db/db). B. ImageJ settings for analysis of wound closure. Blue boxes demonstrate settings and wound area, green boxes demonstrate settings and measurement of 1 cm as scale bar.

Table 3. Therapeutics and dressings

Step	Procedure	Troubleshooting
1	Injecting therapeutic (e.g., cells, pharmacologic) or no treatment (if using hydrogels, skip to step 2)
1a	Inject therapeutic or vehicle control around the wound edge.
1b	Wrap the wounds with an initial layer of Telfa.
1c	Skip to step 3.
2	Hydrogel therapy (Figure 2A)
2a	Apply piece of circular hydrogel onto the wound (with or without therapeutic imbued) (Figure 2B).	Hydrogel should be cut out so that it has the same measurements as the excisional wound.
2b	Wrap the wounds with an initial layer of Tegaderm.	Initial layer keeps hydrogel protected, moist, and pressed against wound to maximize delivery.
2c	Next, cover the wound with Telfa dressing.
3	Cut Tegaderm sheets in half and use the halves to wrap around the mice (Figure 1B).
4	Monitor mouse until it is fully awake.
5	Place in cage—single housing is necessary now.

Monitoring and wound measurements (Table 4)

Time: Approximately 2–10 min per mouse (depending on status of splint; time increases if additional sutures have to be placed or treatment has to be applied).

Monitor mouse daily.
Every other day: measure wound using the following steps.
Anesthetize mouse as described in section B.
Carefully remove wound dressing (Tegaderm and Telfa).
Note: Be sure to remove the dressings carefully so as to not disturb the wound bed or tear off splints and sutures.
Take picture of wound for analysis as described in Step B17.
Check if splints are securely fixed to skin; otherwise, the wound will contract and cannot be used for analysis anymore.
If splint is not securely fixated to skin, reapply Vetbond and 6-0 interrupted suture(s).
Note: Examples of damaged splints and explanation on how to deal with them are shown in Figure 4A–F.

Re-apply dressings and/or therapeutics as described in section C.

Table 4. Monitoring and wound measurements

Step	Procedure	Troubleshooting
1	Monitor mouse daily.
2	Every other day: measure wound using the following steps.
3	Anesthetize mouse as described in section B.	Monitor mouse carefully and take out of anesthesia box immediately when sufficient level of anesthesia has been reached. Exposure to high flow rates of isoflurane for too long can cause death.
4	Carefully remove wound dressing (Tegaderm and Telfa).	Be sure to remove the dressings carefully so as to not disturb the wound bed or tear off splints and sutures.
5	Take a picture of wound for analysis as described in Step B17.
6	Check if splints are securely fixed to skin; otherwise, the wound will contract and cannot be used for analysis anymore.
7	If splint is not securely fixated to skin, reapply Vetbond and 6-0 interrupted suture(s).	Examples of damaged splints and explanation on how to deal with them are shown in Figure 4A–F.
8	Re-apply dressings and/or therapeutics as described in section C.

Explant and analysis (Table 5)

Time: Approximately 10 min should be allocated for each mouse to excise the wounded skin (steps E1–7). Time for consecutive analyses varies according to analysis technique.

Anesthetize mouse as described in section B.
Take off wound dressing.
Take a picture of wound for analysis as described in Step B17.
Mark area of excisional wound with a surgical skin marker.
Remove splints from underlying skin by carefully cutting sutures and peeling off.
Euthanize mouse using neck dislocation while still under anesthesia.
Note: Other approved methods of mouse euthanasia may also be used, depending on institutional rules.
Immediately after euthanizing, excise skin within marked area of the wound.

Excised tissue can now be further processed for desired analysis techniques.

Further data analysis techniques of the tissue can include:

Histological staining (H&E, Trichrome, etc.)
Immunofluorescent staining for proteins
Single cell RNA-sequencing

Mechanical testing

Table 5.Explant and analysis

Step	Procedure	Troubleshooting
1	Anesthetize mouse as described in section B.
2	Take off wound dressing.
3	Take a picture of wound for analysis as described in Step B17.
4	Mark area of excisional wound with a surgical skin marker.
5	Remove splints from underlying skin by carefully cutting sutures and peeling off.	Take care not to disturb wounded skin as this can lead to tissue damage.
6	Euthanize mouse using neck dislocation while still under anesthesia.
7	Immediately after euthanizing, excise skin within marked area of the wound.
8	Excised tissue can now be further processed for desired analysis techniques. Further data analysis techniques of the tissue can include: Histological staining (H&E, Trichrome, etc.) Immunofluorescent staining for proteins Single-cell RNA sequencing Mechanical testing

Data analysis

N = 5 per experimental group is needed to obtain statistically significant results.
Note: Exclusion criteria for the wounds are severe displacement of the splint, infection, or auto-mutilation of the wound (Figure 4).
Upload images into ImageJ in a TIFF format.
Note: Use images until full re-epithelization has occurred.
Create table in Microsoft Excel.
Take measurement of 1 cm by tracing along the ruler with the fixed drawing tool in ImageJ.
Trace the outer linings of the wound with the freestyle or polygon drawing tool in ImageJ.
Transfer the two values to the excel sheet.
Convert area into cm² with the following equation:
x cm²=(Wound Pixel Area)/(pixel length of 1 cm)²
Transfer data to GraphPad Prism 9.
1. Use an unpaired Student’s t-test for two experimental groups.
2. Use a 2-way ANOVA test for multiple (> two) groups.
3. Consider p < 0.05 as statistically significant.
Use a standard XY graph to create a wound healing curve [seen in figure 6H of the original publication (Henn et al., 2021) and Figure 3A of this protocol].
Multiple other comparisons can be performed using the same tests described in step 8:
1. Comparison of days until wound closure (Figure 3B).
2. Wound closure at specific post-operative days (POD) (Figure 3C and 3D).
  
  Figure 3. Statistical analysis of wound healing.
  A. Graph of wound size (closure) over time. Post-operative day (POD). x-axis represents the POD and y-axis the percentage of wound closure. B. Bar graph of days until wound closure. x-axis represents the experimental group and y-axis the days until wound closure. C. Bar graph of wound size at POD 8. x-axis represents the experimental group and y-axis the percentage of wound size to the initial wound size at POD 0. D. Bar graph of wound size at POD 10. x-axis represents the experimental group and y-axis the percentage of wound size to the initial wound size at POD 0.
  
  Figure 4. Troubleshooting images. A. Splint is partially broken. If the rest of the splint is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be included in experimental analyses. B. Sutures have come off. If splint is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be included. C. Splint is partially ripped. If the rest is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be used. D. Splint is entirely broken on one side; wound has to be excluded from further analysis. E. Splint has ripped through the ring. If splint is still adherent to the skin, apply more Vetbond and interrupted sutures; wound can still be used. (F) Splint is entirely broken on one side; wound has to be excluded from further analysis.

Acknowledgments

We thank Theresa Carlomagno for administrative support. This work was supported by the National Institute of Health RO1 grant (DK074095). Schematic figures were created with BioRender.com. The original research paper to this protocol is found here (DOI: 10.1126/sciadv.abi4528) (Henn et al., 2021).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were performed in accordance with both Stanford University and University of Arizona Institutional Animal Care and Use Committees (IACUC) and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The study was approved by the Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University (APLAC protocol number: 12080), as well at the University of Arizona under IACUC protocol 2021-0828.

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