Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

We provide a protocol to establish infarction in a pons of rat. This pontine infarction rat model was easily performed, easily reproduced, and highly successful model. The electrical stimulation could be used to make infarction in any other brain area like thalamus.

People who want to perform this model should learn the pontine autonomy before the experiment. Before the surgery, weigh the rats and assess their neurological performance according to the behavioral tests as described in the accompanying text. 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 three centimeter line along the midline of the skull from the line of the bilateral lateral canthus to 0.5 centimeters behind the posterior fontanelle 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 tip marker pen to indicate the central point of the bregma as the origin point, place the drill at six millimeters anterior posterior, two millimeters medial lateral. Use the drill to carefully perform a one millimeter diameter craniotomy. Next, replaced the drill with a 22 gauge probe with an insulated sheath with the tip placed two millimeters above the proximal end of the sheath.

Insert the sheets seven millimeters into the brain and advance the probe along the sheath until the top of the probe is nine millimeters below the surface of the brain. Connect the electrodes to an electrical stimulator and connect the anode to the probe. Then connect to 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 four 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. In this analysis, six animals were subjected to the surgery as demonstrated and six animals were treated as controls.

The brain slices in these images were derived from one rat per group. MRI scanning revealed that the infarction was located in the basis of the pons mimicking anterolateral infarctions in human patients. Because an insulated sheath was used, the infarction was not observed in the cortex, cerebellum, or midbrain.

Diffusion weighted images also revealed the acute pontine infarction. TTC staining was used to confirm the infarction 24 hours post-surgery. As expected, the infarction volume was significantly higher in the surgery group compared to the control group.

Behavioral scores were measured before and after surgery. Compared to the control group, rats with a pontine infarction circled to the left. Significant differences were observed in the Longa, Berderson, limb placement, beam balance, and adhesive removal somatosensory test scores between rants with pontine infarction and control group rats.

Any specific brain area are going to be destroyed by the electrical stimulation.