Generating A Cortical Stab Injury in a Mouse Model to Induce Reactive Astrogliosis

Secure an anesthetized mouse in a stereotaxic frame.

Shave its head and disinfect the site. Apply eye ointment to prevent corneal drying.

Make an incision to expose the skull.

Remove the overlying membranes, and clean the surface with saline.

Mark a region on the skull, targeting the cortex.

Drill along the marked region. Apply saline to maintain tissue hydration.

Remove the loosened bone piece, exposing the underlying cortex.

Place saline-soaked gel foam to prevent tissue drying and absorb blood.

Position a sterile blade attached to a probe holder over the cortex. Then, remove the gel foam.

Lower the blade to penetrate the cortex and move it horizontally in repeated strokes to create a mechanical injury.

Remove the blade and use gel foam to absorb blood. Suture the surgical incision.

At the injury site, astrocytes undergo reactive gliosis, where they activate and proliferate, releasing protective molecules that reduce inflammation and support tissue repair.

In this step, place a fully sedated mouse in the stereotaxic frame. Secure its nose in the nose cone, which is connected to isoflurane. Next, tighten the ear bars and ensure that the head is stable.

Shave the head from the eye level to the ear level. Then sterilize the skin with three alternating scrubs of isopropyl alcohol and Betadine iodine solution. Afterward, apply ointment to both eyes to prevent them from drying.

Monitor the depth of the animal's anesthesia by pinching its toe or tail. The mouse is in the appropriate surgical plane when there is no response and the respiration is slow and even. Make a parasagittal skin incision from just behind the eyes to almost the point between the ears in one single firm cut. Move the skin aside and clip the right side with a hemostat.

Under the microscope, clear the overlying membrane on the skull using the dull side of the scalpel and cotton-tipped applicators. Then clean the skull with a cotton-tipped applicator dipped in 0.9% saline solution and allow it to dry completely. Using a small ruler and a permanent marker, mark the anterior border of the craniotomy at 1 millimeter caudal to the coronal suture and then mark the left edge of the craniotomy at 1 millimeter lateral to the sagittal suture.

Next, mark the right and caudal borders of the craniotomy at 4 millimeters from the sagittal and coronal sutures, respectively. Using a 0.5 drill bit, carefully drill on the permanent marker outline and be sure not to break through the skull completely. Press gently on the isolated piece of parietal bone with forceps. The thinned bone is ready for removal when it is sufficiently weak around the perimeter.

Now, using a 10-milliliter syringe fitted with a 23-gauge needle, apply a small amount of 0.9% saline to the isolated bone and drilled area. Attach the manipulator arm to the stereotaxic apparatus. Then attach a new scalpel blade to the probe holder with the sharp side facing rostrally.

Keeping the manipulator arm out of the way, carefully lift the bone piece with a pair of angled forceps by inserting the tip into the side of the bone and pulling it off in one movement. Then place a small piece of the soaked absorbable gel foam on the uncovered brain to prevent it from drying and absorb any blood that might be present. Once the gel foam is placed on the brain, swing the manipulator arm into place. Adjust the blade to center over the gel foam.

Then remove the gel foam, and lower the blade until the tip touches the dura without puncturing it. Mark the dorsal-ventral coordinates using the vernier scale on the vertical arm of the stereotaxic frame. Next, using the manipulator arm, slowly lower the blade precisely 3 millimeters into the brain. Allow the blade to stay in place for 5 to 10 seconds.

Then slowly move the stereotaxic arm with the blade in a rostral-caudal direction three times. Allow the blade to reach the rostral and caudal boundaries of the craniotomy before moving to the opposite end. Afterward, slowly raise the stereotaxic arm and remove the blade from the brain.

Place another piece of gel foam on the brain surface immediately to absorb any excess blood or fluid. Meanwhile, remove the stereotaxic arm and dispose of the scalpel blade. Once the bleeding has stopped, remove the gel foam.

Close the wound by suturing the skin with non-absorbable sutures, such as Ethilon or Prolene, and remove the sutures 9 to 10 days after the surgery. Next, administer 0.5 to 1 milliliter of lactated Ringer's solution subcutaneously, to ensure hydration.