Our research focuses on a standardizing macroscopic wound measurement using a controlled imaging set up by ensuring consistency and light position and distance or protocols aims to minimize variability and improve the currency of wound close measurement in preclinical models. We address the lack of the standardization in microscopic wound measurements. Current methodologies often lack control conditions leading to inconsistent data.
Our protocol offers our applicable setup that ensures uniform lighting, positioning and distance reducing probability and enhancing reproducibility. Our protocol eliminates common sources of measurement bias by standardizing imaging conditions. Unlike manual methods, it ensures consistency across experiments, enabling accurate comparisons and reducing variability in area and perimeter measurements of wounds.
Our result enable more precise and reproducible wound measurement data by providing a standardized methodology. This is the basis for evaluating therapeutic interventions and preclinical wound research. To begin, fix a slotted aluminum profile on the inner area of the side edge of the roof.
Place two rectangular aluminum plates with polyethylene centers into the grooves of the roof profiles to enable sliding through both profiles. Mount a 20 centimeter RGB LED tube light at a 45 degree angle on the underside edge of the first panel using double-sided tape. Then, mount a rectangular foam board panel with a central circular cutout of 7.82 centimeters on each roof panel to allow the installation of the camera lens.
For floor construction, first cut squares with side lengths of 2.54 centimeters from each corner of the 40 by 40 centimeter white foam board. This step ensures that the foam board fits properly within the floor area profiles. After trimming the corners, place the foam board on top of the floor area profiles.
Attach hook and loop fastener strips to aluminum profiles A and B to easily manipulate internal photo booth elements. A fixed adhesive fasteners one centimeter from the edge of two foam cardboard squares on one side and position them on the front and rear walls. Using silicone, glue two panels to sides C and D to form the left and right side walls.
Place a 3D printed mask 11.5 centimeters from the edge of a precut 40 by 28 centimeters white foam board rectangle to maintain the mice under inhalation anesthesia. Then, glue it to the center of the reference base. Fix the 3D printed ruler block one centimeter from the left side of the mouse base to accommodate the measurement reference element for digital image processing.
Then, attach a 15 centimeter stainless steel graduated ruler to the ruler block and install the complete reference base inside the cabin for image scaling. After anesthetizing the mouse, use a sterile five millimeter biopsy punch to apply pressure with circular movements to create a dermo epidermal incision at the shoulder blade level. Remove the flap with serrated forceps and cut with iris tip scissors.
Using jeweler forceps, place a one centimeter diameter circular dermal film over the wound to prevent contamination and contraction. To capture images of the wounds, position the reference base at the center of the photo booth floor. Then, position the anesthetized mouse in a prone position on the mouse platform.
Install the front wall of the photo booth and capture macroscopic images. To open the created backup image in imagej, navigate to File, Open and Search. Then, click Open.
Now maximize the image, select the straight line tool, then using the plus key zoom in on the ruler next to the mouse. Draw a 10 millimeter straight line on the ruler image. Go to analyze and set scale.
Enter 10 as the known distance. Set the unit of length to millimeters and click Okay. Next, use the rectangle tool to select the area around the wound with a width and a height of 150 and record the X and Y values.
Then, right click on the rectangle and select duplicate. Name the duplicated image and press enter. In the new image, press plus twice to zoom in.
To separate the image into color channels, navigate to Image, Type and RGB Stack. In the red channel image, right click on the image, select duplicate and press Okay in the duplicate window. Then, click on the red channel image, select image, adjust, and threshold to segment it.
Choose Default and click Apply to apply the thresholding. Rectify the region of interest or ROI to ensure complete wound coverage and avoid segmentation distortions. To define the perimeter of the area of interest, select Edit, then go to selection and choose Create Selection.
After that, select Edit, then Selection and click on Add to Manager to add it to the ROI manager. To validate the segmentation, open the ROI manager window and select the ROI to review. Click More, Translate, enter the recorded X and Y values and press Okay.
Maximize the original image then select the ROI in the ROI manager. Use the arrow keys to adjust the ROI until it aligns with the wound. Once the segmentation is confirmed, obtain the wound measurements by selecting Analyze and Measure.
The wound closure percentage initially increased to 138.04%on day two due to inflammatory processes before decreasing and progressing toward repair and consolidation phases with consistent closure observed after day seven. The wound edge retraction distance increased initially during the inflammatory phase, but started to decrease after day three as shown by Gilman's Equation indicating gradual wound contraction.
