This protocol uses histological analysis to obtain robust morphometric measurements of mouse excisional wounds. Using this technique, we can analyze the entire wound using a subset of serial sections, allowing us to more accurately detect defects that might otherwise be missed. Demonstrating the procedure will be Lindsey Rhea, a research associate from my laboratory.
After confirming a lack of response to pedal reflex in an anesthetized adult mouse, apply ointment to the animal's eyes and use an electric razor clipper in a caudal-rostral motion to remove the fur on the back of the mouse at the shoulder level. Use a razor blade held at a 20 degree angle from the back in a rostral-caudal motion to remove any remaining hair from the exposed skin, then clean the skin with one povidone iodine and one 70%isopropyl alcohol prep pad wipe. For wound induction, drape a clean paper-based towel onto a flat surface covered with dental wax and position the mouse onto the towel in the prone position.
Pinch the skin of the mouse between the shoulder blades along the dorsal midline and pull the sandwiched skin fold away from the body. Switching the mouse to a side-lying position, place a biopsy punch of the desired wound size as close to the body as possible and allow the skin to relax. Press down on the punch using a rocking motion to puncture all of the layers of the skin and remove the punch biopsies from the wound using sterile scissors and tweezers to free the punch from the surrounding skin as necessary.
Then allow the mouse to recover with monitoring and analgesia. For morphometric analysis of the wound bed, open a digital stained wound image. To set the scale and measurement preferences, under Analyze, select Set Scale and enter the distance in pixels, the known distance, and the unit of length.
Select Global and OK to keep the scale the same for each open image and select Analyze and Set Measurements to confirm that the area box is checked. To measure the wound length, select freehand, and use the tool to trace along the dermal-epidermal junction, starting from the last hair follicle of the uninjured tissue on one side of the wound to the first hair follicle of the uninjured tissue on the other side. If the epidermis does not cover the entire wound, follow the dermal-epidermal junction on one side of the wound where the migrating tongue ends and continue following the superior aspect of the granulation tissue or the junction between the granulation tissue and the scab until the migrating tongue and the first hair follicle of the uninjured tissue on the other side of the wound are reached.
Then click Analyze and Measure. The length of the measurement will appear in the same units as set in the scale. If the epidermis does not cover the entire wound, measure the distance between each epidermal leading edge following the superior aspect of the granulation tissue or the junction between the granulation tissue and the scab to the first hair follicle.
To measure the wound area, use the freehand tool to trace along the superior aspect of the epidermis or the superior aspect of the granulation tissue starting from the last hair follicle of the uninjured tissue on one side of the wound to the first hair follicle of the uninjured tissue on the other side. Continue to trace vertically along the hair follicle into the granulation tissue. Once the opposite hair follicle and adipose tissue or muscle are reached, follow the inferior border of the granulation tissue to the opposite side of the wound and join the starting point along the hair follicle to close the area.
If the wound is not fully epithelialized, trace along the superior aspect of the epidermis until the leading edge and return to the starting point following the dermal-epidermal junction. As illustrated, when taking 2D measurements throughout the entire wound, we were able to calculate parameters not possible with 2D analysis on the middle of the wound only, which is a commonly used method in the field. In this experiment, 2D measurements of wound area were used and averaged over the whole wound or a middle subset of the wound and revealed no significant difference between experimental groups or the methods of analysis.
The calculated wound volume, however, was significantly different between the experimental groups, demonstrating the importance of an in-depth histological analysis of wound healing parameters. Generating consistently sized wounds for histological analysis and performing rigorous serial sectioning are critical for an accurate morphometric analysis. Unstained sections can be used for immunofluorescence or Masson's trichrome staining to measure other aspects of wound healing while flash frozen wounds can be used alongside morphometry for quantitative protein analysis.
