Immunology and Infection
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Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.
Chapters
Summary March 25th, 2020
Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.
Transcript
These models can be implemented to assess various aspects of the wound healing response, including cellular and cytokine kinetics and wound closure. The PVA sponge model allows the recovery of millions of wound leukocytes for phenotypic and functional analyzes, while the tail skin excision model allows the easy visualization of slow-healing wounds. The PVA sponge and excisional tail wound models can be used in conjunction with comorbid conditions, such as diabetes or pneumonia, to understand their impact on wound healing.
Demonstrating the procedures with Meredith Crane will be Bill Henry, a research assistant from my lab. Before beginning the experiment, use scissors to cut sheets of PVA sponge into eight-by-eight-by-four-millimeter pieces. Rehydrate the sponge pieces in sterile PBS in a beaker for 10 minutes before autoclave sterilizing the sponges in the PBS.
When the sponges have cooled, store the sponges submerged in PBS at four degrees Celsius. On the day of the procedure, place six sponges per experimental animal into a sterile culture dish in a sterile laminar flow hood, and confirm a lack of response to toe pinch in an anesthetized, eight-to 12-week-old, male, C57-Black/6 mouse. Have an assistant use clippers to remove the hair along the dorsum and sterile gauze to apply povidone-iodine solution to the shaved area two times, followed by a single 70%ethanol application.
The assistant should then transfer the mouse onto sterile surgical drapes placed over a heated pad. Wearing sterile gloves, use forceps to pull the dorsal skin away from the underlying tissue, and use sterile surgical scissors to make a two-centimeter incision along the dorsal midline approximately two centimeters anterior to the base of the tail. Holding the incision open with sterile forceps, use sterile, curved, blunt-tipped surgical scissors to form a subcutaneous pocket along the dorsum in one of the positions as indicated in the figure.
Once the scissors have been inserted, open and close the tips two times to form a pocket big enough to hold an inserted sponge. When the pocket has been created, use sterile surgical scissors to gently squeeze one PVA sponge in the culture dish to remove excess PBS. Next, lift the sponge by one corner, and, leading with the corner held by the scissors, place the sponge into the subcutaneous pocket.
When all six sponges have been placed as demonstrated, use sterile forceps to pinch the incised dorsal skin together, and close the incision with two stainless steel wound clips. For PVA sponge fluid isolation, one to 14 days after implantation, remove the surgical staples, and open the incision with toothed forceps. Use scissors to extend the incision along the dorsal midline, and use forceps to extract one sponge from its subcutaneous pocket.
Place the sponge into the barrel of a five-milliliter syringe nested in a 16-milliliter culture tube on ice, and use scissors to disassociate any connective tissue that remains adherent to the surface of the sponge, as necessary. When all of the sponges have been extracted and transferred as demonstrated, centrifuge the culture tube to collect the wound fluid. To isolate the cells from the PVA sponges, after collecting the sponges as just demonstrated, place them into a 15-milliliter conical tube containing five milliliters of HBSS collection medium.
When all of the sponges have been collected, decant the sponge suspension into an 80-milliliter blender bag, and hang the bag from the hatch of the paddle blender so that the paddles will strike the sponges and medium. Set the paddle blender to run on high for 60 seconds, and press start. When the blender has stopped, squeeze the sponges in the bag to completely release the medium, and use a pipette to transfer the medium from the blender bag back into the 15-milliliter tube.
Adjust the settings of the paddle blender to run for 30 seconds on high, and add five milliliters of medium to the blender bag. Repeat the stomaching process two more times before centrifuging the tube of collected medium. A red pellet of cells and red blood cells should be visible at the bottom of the conical tube.
Mix 900 microliters of distilled water with the cells for three to five seconds before neutralizing the lysis with 100 microliters of 10x PBS and four milliliters of 1x PBS. Collect the cells by centrifugation, and resuspend the white cell pellet in the appropriate medium for downstream analysis. To create a tail skin wound, after confirming a lack of response to toe pinch in the anesthetized, eight-to 12-week-old, male, C57-Black/6 mouse, use sterile gauze to apply povidone-iodine solution two times to the surgical site, followed by one application of 70%ethanol.
Using a permanent marker and a premade template, trace a 10-by-three-millimeter section on the dorsal surface of the tail, 10 millimeters from the tail base, and place the mouse on sterile surgical drapes. Wearing sterile surgical gloves, use a sterile scalpel blade to make a full-thickness incision along the right, bottom, and left edges of the wound area. Using sterile forceps, peel the excised skin away from the tail, and use sterile surgical scissors to cut away the top edge of the wound area.
Use sterile gauze to apply pressure to the wound to stop bleeding, and apply a spray barrier film to the wound bed. Then, photograph the wounds from a fixed distance at regular time intervals, and analyze the photographs by planimetric analysis to determine the wound area measurements. PVA sponge implantation surgery generates a systemic inflammatory response, as demonstrated by the induction of IL-6 in the plasma one day after wounding.
The number of cells that can be recovered from PVA sponge wounds increases over time, with neutrophils, monocytes, and macrophages comprising the primary cellular infiltrating populations within the sponges. Neutrophils are identified as Ly6G-positive, Siglec-F-negative cells. Siglec-F-positive cells are primarily eosinophils.
Gating on the Ly6G-negative, Siglec-F-negative cells allows the identification of F4/80-positive monocytes and macrophages. F4/80-positive cells can be further differentiated by their Ly6C expression to distinguish Ly6C high inflammatory monocytes and Ly6C low monocyte-derived macrophages. The excisional tail skin model provides an alternative to the dorsal skin punch biopsy method to study wound closure in firm skin lacking dense fur.
Wound closure can be quantified by measuring the area of the wound bed over time. The tail skin wound can also be observed in cross section by histological analysis via H and E and Masson's trichrome staining. In these images, the lateral margins of the excised skin are indicated by the arrowheads on the dorsal surface of the tail.
To understand steady-state wound healing, therapeutic compounds or interventions can also be tested through their injection directly into the PVA sponge or through delivery to the tail wound bed.
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