P3 amputation is a preclinical model for investigating mammalian epimorphic regeneration and a powerful tool for identifying critical cell populations and mechanisms that control endogenous regeneration. The P3 model allows for the identification of early and late blastemal cell populations, the study of revascularization during regeneration, and the investigation of intramembranous ossification without complex bone stabilization device requirements. Demonstrating the procedure with me will be Osama Qureshi, Alyssa Falck, and Katherine Zimmel, technician, and Regina Brunaeur, a Research Assistant Professor all from our laboratory.
After confirming a lack of response to toe pinch in an eight to 12-week-old CD-1 mouse, Apply ointment to the animal's eyes and place the mouse under a 10X dissection microscope. Use surgical povidone iodine and 70%ethanol to sterilize the digits of each hind limb and use microscissors to trim hair away from the surgical site. Apply one microliter of topical bupivacaine locally to anesthetize the surgical site, and gently splay one hind paw to expose the medial digit surface.
Then holding the scalpel at an angle parallel to the fat pad, use a sterile number 10 scalpel to amputate the distal tip of the terminal phalanx of digits two and four. Apply one additional microliter of bupivacaine locally to each amputated digit and return the mouse to a clean cage to allow the wounds to heal without wound dressing and with monitoring until full recumbency. To assure a successful P3 amputation plane, hold the scalpel parallel to the fat pad during the amputation and perform micro-CT scanning to confirm the correct amputation length.
At the appropriate experimental endpoint, use a scalpel to sever the digit midway through the middle phalanx bone at approximately the second ventral fat pad indent. Transfer the tissue to a 20-milliliter scintillation vial containing 10 milliliters of fresh buffered zinc formalin fixative for 24 to 48 hours. And use a microtome to acquire four to five micrometer thick sections of each digit, placing the slices into a 38 to 41 degrees Celsius water bath supplemented with a histological adhesive solution as they are obtained.
Then capture the slices on adhesive slides and place the slides on a 37-degree Celsius slide warmer to dry. When the slides have been deparaffinized immerse the samples in a staining jar of an appropriate antigen retrieval solution, and heat the slides to 95 degrees Celsius for 25 minutes making sure that boiling does not occur. At the end of the incubation, allow the jar to come to room temperature, and lay the slides flat in a one-inch deep covered plastic slide box with moistened tissue paper at the base.
Add 100 microliters of prewarmed proteinase K to each sample. Cover each slide with a small strip of parafilm to ensure the sections do not become dry. After 12 minutes at 37 degrees Celsius, carefully remove the paraffin film, and gently drain the slides on tissue paper to remove excess blocking solution.
Next, add 100 to 200 microliters of the primary antibody solution of interest to each slide. Return the sites to the humidifying chamber covered with paraffin film for an overnight incubation at four degrees Celsius. The next morning after washing the slides in TRIS-buffered saline plus TWEEN or TBST label the slides with an appropriate secondary antibody solution for a 45-minute incubation at room temperature protected from light.
At the end of the incubation, wash the slides in TBST and allow them to dry. Then use 100 microliters of anti-fade mounting medium to carefully place coverslips onto the dried slides and store the slides flat at room temperature overnight protected from light to allow the mounting medium to dry. For sequential in vivo micro-computed tomography or micro-CT outfit the micro-CT imager with tubing to enable oxygen flow into and out of the micro-CT animal chamber, making sure that the outflowing oxygen is equipped with an activated charcoal filter to trap the isoflurane.
Set the Voxelsize to 10.5 micrometers at 55 peak kilovoltage and 145 microamps with 1, 000 projections per 180 degrees and continuous rotation with an Integration Time of 200 milliseconds, resulting in a maximum of 213 slices per scan. When the scanner is ready, gently place the anesthetized mouse in the micro-CT animal chamber with the hind limb digits arranged in flat, close association ventral side up with the left paw on the left side and the right on the right side of the scanning platform. Then apply surgical tape to gently secure the digits in place.
Apply ophthalmic ointment to the animal's eyes before initiating the scan. After scanning, return animal to a clean cage with monitoring until full recumbency, and allow up to several hours for micro-CT image reconstruction. For analysis of the reconstructed 3D bone image, using the software provided with the micro-CT, convert the image files to a series of DICOM files.
When all of the files have been converted, download the DICOM series in a single folder using a batch file downloader in a web browser. Load the BoneJ plug-in into the ImageJ plug-in folder. Drag-and-drop the DICOM file series folder into the ImageJ bar to open the files.
A 16-bit image stack displaying all of the gray values will be opened. To display bone only, click Image Adjust and Threshold. Slide the upper threshold bar to the far right and adjust the lower threshold bar until only the bone is highlighted red.
The lower threshold value will be approximately 10, 000 to 13, 000 at a maximal signal intensity of 32, 767. Click Apply and in the new dialog box, click Black background. Next, click OK.An eight-bit image displaying black and white values will be generated.
With the white corresponding to bone. Draw a rectangle around the P3 bone to select it and duplicate the stack. To generate a 3D image, click Plugins and 3D Viewer and use the freehand selection tool to circle the bone to be deleted.
Then right-click and select Fill selection to crop any unnecessary bone from the image. To quantify the bone volume from the 3D rendering, open the BoneJ plug-in and select Volume Fraction. A results window will appear and the bone volume displayed in millimeters cubed.
To quantify the bone length from the 3D rendering, use the ImageJ multipoint tool. To capture an image of the 3D rendering, click View and take a snapshot. Then save the image as a TIF or JPEG file.
In these images sections of an adult mouse regenerating P3 digit were immunostained with antibodies to visualize intramembranous bone regeneration and blastema formation at day six to seven, nine and 10 post-amputation. Here, representative micro-CT renderings of digits scanned prior to amputation and at various time points over the course of regeneration with landmarks used to identify length measurements are shown. The mouse digit model is a powerful system for analyzing a pro-regenerative wound environment that triggers blastema formation when amputated at a distal level.
Conversely, proximal amputation of the digits serves as a model system for investigating regenerative failure as well a site for testing strategies for enhancing regeneration.
