Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

The overall goal of the following experiment is to trigger reactive astro gliosis in the adult mouse fore brain by creating a mechanical lesion in order to investigate cellular and molecular processes underlying reactive gliosis. This is achieved by performing a craniotomy and subsequently inserting a sterile scalpel blade, three to four millimeters in depth and length into the forebrain to create the lesion. Animals can survive for acute or chronic time points and their tissues are harvested at the conclusion of the experiment.

Next tissue can be processed for histological, biochemical, or molecular biological assays to interrogate the molecular and cellular processes driving reactive astro scoliosis. The results show that this procedure triggers a strong reactive gliosis based on the dramatic upregulation of GFAP and proliferation of reactive astrocytes. The main advantage of this technique over other methods like fluid percussion injury, or controlled cortical impact, is that reactive gliosis can be easily induced and studied without the need of additional equipment.

To begin this procedure, disinfect the surgical table with 70%ethanol, then cover the entire surgical bench with absorbent pads and arrange the surgical instruments on the surgery table. Next, set up the stereotaxic equipment without the manipulator arm. Place the heating pad on the stereotaxic frame and set it to 37 degrees Celsius.

Avoid overheating the animal by placing a small piece of paper towel or surgical pad on the heating pad. Then cut small pieces of gel foam into a sterile Petri dish containing 0.9%sterile saline solution until ready for use. In this step, place a fully sedated mouse in the stereotaxic frame.

Secure its nose in the nose cone, which is connected to iso fluorine. 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 para sagittal 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 one millimeter coddle to the coronal suture, and then mark the left edge of the craniotomy at one millimeter lateral to the sagittal suture. Next, mark the right end coddle borders of the craniotomy at four millimeters from the sagittal and coronal sutures respectively.

Using a 0.5 millimeter 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 roly.

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 three millimeters into the brain. Allow the blade to stay in place for five to 10 seconds.

Then slowly move the stereotaxic arm with the blade in a rostral coddle direction three times allow the blade to reach the rostral and coddled 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 ET or proline, and remove the sutures nine to 10 days after the surgery.

Next, administer 0.5 to one milliliter of lactated ringer solutions subcutaneously to ensure hydration. This figure shows the brightfield immunohistochemistry for GFAP one week following a forebrain stab injury. The lesion site is shown in the ipsilateral hemisphere and the corresponding region in the uninjured contralateral hemisphere is shown here.

Here is the high magnification image of a normal and reactive astrocyte from the contralateral and ipsilateral hemisphere respectively. Note the dramatic hypertrophy of the cell body and processes of the reactive astrocyte in the injured side. This figure shows the proliferation of the reactive astrocytes.

One week following for brain stab injury. Here is the immunofluorescent staining for BRDU and GFAP in the uninjured and injured brains, and the immunofluorescent staining for BRDU and the astrocytic marker. S 100 beta in the uninjured and injured hemispheres is shown here.

Note that many astrocytes are proliferating at the lesion site in the injured cortex, whereas astrocytes in the uninjured cortex are not proliferating Following this surgical procedure. Other methods like biochemistry or molecular biology can be used to examine protein interactions or gene expression in reactive astrocytes.