The standardized protocol can be used to assess endogenous cell migration and can be applied to several cell types as well as skeletal phenotypes. The advantage of this technique is that it allows in vivo cell migration to be tracked for individual cells rather than a population of cells in response to injury and/or cytokine treatment. Before beginning the procedure, confirm a lack of response to toe pinch in the anesthetized mouse and apply ointment to the animal's eyes.
Next, make a transverse, less than one centimeter incision from immediately medial to the right ear toward the left ear and make a second incision from the initial incision, approximately two to three millimeters past the right eye toward the nose. Use forceps to separate the skin from the periosteum and use scissors to gently cut through to connective tissue to separate the skin from the calvaria periosteum. Using a sterile cotton swab, apply ophthalmic ointment to the top of the skin flap and gently fold the flap over the left eye.
The intersection of the sagittal and coronal sutures should be clearly exposed. Flush the open surface with sterile PBS to clean the area of any blood and residual hair and place the tip of a bevel one to two needle widths toward the nose from the coronal suture and one to two needle widths to the right of the sagittal suture. Using a very small amount of downward pressure, carefully rotate the syringe clockwise one time and counterclockwise several times.
Then use a 27 gauge needle to widen the microfracture to approximately 40 micrometers. If bone fragments remain, flush the microfracture with PBS. If bone fragments still remain, use a 29 gauge needle to gently scoop the fragments out.
For CCL5 treatment, use a 100 nanogram per milliliter stock CCL5 solution and basement membrane matrix to obtain a 10 nanogram per milliliter working chemoattractant solution. To treat the injury, load a 220 microliter pipette with five microliters of the solution and apply two microliters of the chemokine to the suture. Then allow the matrix to solidify and gently cover the calvaria with the skin flap to ensure that the tissue does not become dehydrated during the one hour chemokine treatment.
For intravital imaging of periosteal stem cell migration in real time, before moving the mouse to the microscope, apply gentle pressure to the back of the mouse's head to check the visual plane of the calvaria. If the plane is not level, gently rotate the mouse restraint to the left or the right to adjust the position. Next, cover the entire calvaria in skin flap with sterile 2%methylcellulose in water and place the mouse on the XYZ axis motorized microscope stage.
Using the epifluorescent light, align the mouse so that it is facing the microscope and that the intersection of the coronal and sagittal sutures is the centered reference point of the stage. Then place a glass cover on the imaging area and double check the alignment. For time-lapse imaging of the migration into and out of the suture, select a low magnification water immersion lens to scan the calvaria.
Acquire a reference image of the sagittal and coronal suture intersection and using the SHG and fluorescent signals from the cells, locate each injury site and record the XYZ coordinates as well as the distances from the sagittal and coronal sutures. Select a location on either the coronal or sagittal sutures for long-term imaging and obtain a detailed ZStack image of this area from reference during imaging. Use the time-lapse software settings to record an image every one minute for at least one hour, comparing the current field of view with the initial field of view in the time between snapshots.
If the fields of view are different, use the ZStack image to determine in which direction the field of view needs to be adjusted. Recently, Mx1 tomato alpha-SMA GFP reporter mice were generated in which periosteal skeletal stem cells are marked by Mx1 positive alpha-SMA positive dual labeling. Little to no Mx1 positive alpha-SMA positive periosteal skeletal stem cell migration is observed over a one hour period of imaging, 24 hours after suture placement.
The CCL5 chemokine treatment of a calvaria defect 24 hours post injury induces directionally distinct migration away from the coronal suture and toward the injury site. In this representative analysis, within one hour of imaging, one of the Mx1 positive alpha-SMA positive periosteal skeletal stem cells migrated approximately 50 micrometers toward the injury. The most important aspect of this protocol to remember is to limit the CCL5 treatment to the injury site in order to stimulate cell-specific migration.
Once this procedure is complete, localized treatment can be continued for one week. A micro CT can be used to assess calvaria injury healing and mineral deposition.
