Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

Secure an anesthetized rat in a stereotaxic unit.

Remove the fur from the scalp and sanitize the incision site. Make a midline incision and retract the skin to expose the skull.

Remove the connective tissue to avoid interference with the drill. Identify the bregma as a reference point to determine the coordinates of the pons, a structure located in the brainstem.

Drill a hole in the skull.

Insert a probe through the hole to reach the pons.

Next, connect the electrodes to an electrical stimulator, attaching the anode to the probe for brain stimulation and the cathode to the rat's ear to complete the circuit.

Deliver an electrical pulse to the pons, inducing localized tissue damage or infarction.

After stimulation, leave the probe in place briefly to stabilize the stimulation effect.

Then, slowly remove the probe and seal the surgical site with bone cement.

Suture the incision and return the rat to its cage for recovery and neurological analysis.

Preheat the heating pad to 37 degrees Celsius immediately before anesthesia, and attach the skull drill to the holder on the stereotaxic frame. Confirm a lack of response to pedal reflex in the first anesthetized rat, and mount the rat onto the stereotaxic frame in the prone position on the heating pad. Position the ear bars above the ear canal to secure the head, taking care that the skull is kept horizontal to avoid any skewing of the injection.

Apply ointment to the animal's eyes, and use a micro-shaver to remove the hair from the skull. Use a marker to draw a 3-centimeter line along the midline of the skull from the line of the bilateral lateral canthus to 0.5 centimeters behind the posterior fontanel, and use a scalpel to make a midline incision along the marked line. Use a cotton swab to remove any blood, and place a piece of surgical tape on each side of the skin flap to expose the skull.

Use a new swab soaked with 0.9% sodium chloride to gently remove any connective tissue from the skull, and locate the bregma. Use a fine-tipped marker pen to indicate the central point of the bregma as the origin point. Place the drill at 6 millimeters anterior-posterior, 2 millimeters medial-lateral. Use the drill to carefully perform a 1-millimeter diameter craniotomy. Next, replace the drill with a 22-gauge probe with an insulated sheath, with the tip placed 2 millimeters above the proximal end of the sheath.

Insert the sheath 7 millimeters into the brain, and advance the probe along the sheath until the top of the probe is 9 millimeters below the surface of the brain. Connect the electrodes to an electrical stimulator, and connect the anode to the probe. Then, connect the cathode to the ear of the rat. Next, turn on the electrical stimulator, and set the single pulse width to 4,050 milliseconds, the voltage to 50 volts, and the current to 4 milliamps.

Leave the probe in position for five minutes after stimulation, before removing the device from the brain. Cover the craniotomy with bone cement, and allow the cement to dry before using 4-0 polyamide suture filaments to close the wound. After three or four stitches, tie 2-1-1 standard surgical knots. Then, intraperitoneally inject the rat with 250 microliters of penicillin, and monitor the rat every 15 minutes until fully awake before returning the animal to its cage.