inducing acute pons infarction in a rat model using electrical stimulation

Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

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&#39;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.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Establishment of infarction in the pons
<ol>
	<li>Preheat the heating pad immediately before anesthesia.</li>
	<li>Attach the skull drill to the holder on the stereotaxic frame.</li>
	<li>Intraperitoneally inject the rats with 50 mg/kg ketamine and 5 mg/kg xylazine. Check for the lack of toe-pinch response.</li>
	<li>Mount the rat onto the stereotaxic frame in the prone position. Position the ear bars above the ear canal to secure the head. Ensure that the skull is kept horizontal to avoid any skewing of the injection.</li>
	<li>Maintain anesthesia by isoflurane (100% oxygen, 2.5% isoflurane) via a stereotaxic nose cone attachment for rats with inlet and outlet ports. Use a heating pad to maintain the temperature at 37 &#176;C and monitor it throughout the procedure.</li>
	<li>Use eye ointment to prevent corneal drying. Use forceps to pinch the paws to ensure no pain response slightly.</li>
	<li>Shave the hair of the skull with a micro-shaver. Circularly apply chlorhexidine surgical scrub starting at the surgical incision site and rotating outward.</li>
	<li>Make a 3 cm midline incision with a scalpel from the line of the bilateral lateral canthus to 0.5 cm behind the posterior fontanelle, which a surgical marker pen should mark.</li>
	<li>Use a cotton swab to remove blood.</li>
	<li>A piece of surgical tape should be placed on each side of the skin flap to expose the scalp (Figure 1).</li>
	<li>Gently remove the connective tissues from the skull bone with a cotton swab dipped in 0.9% NaCl. If not removed, the connective tissues will get caught in the drill.</li>
	<li>Identify the bregma. Choose the central point of the bregma as the origin point and mark it using a fine-tip black surgical marker pen.</li>
	<li>Place a drill at 6.0 mm AP, 2.0 mm ML (range from 0.5&#8211;3.0 mm, Figure 2A).</li>
	<li>Perform craniotomy (1 mm diameter) using an automatic drill. Proceed carefully because this point is close to the venous sinus.</li>
	<li>Remove the drill from the stereotaxic frame.</li>
	<li>Place a 22 G probe with an insulated sheath in the stereotaxic frame (Figure 3A). The probe tip should be placed 2 mm above the proximal end of the sheath (Figure 3A, B; Figure 2B).</li>
	<li>Ensure that the sheath enters the brain 7 mm (7 mm DV, Figure 2B; Figure 1C).</li>
	<li>Advance the probe along the sheath (Figure 1D) until the tip of the probe is 9 mm below the surface of the brain (Figure 2D).</li>
	<li>Connect the electrodes to an electrical stimulator (Figure 3C). Connect the anode to the probe as shown in Figure 1D. Connect the cathode to the rats (usually to the ear of the rats).</li>
	<li>Turn the electrical stimulator on and set up the following parameters: single pulse width = 4,050 ms; voltage = 50 V; and current = 4 mA (Figure 3C). During electrical stimulation, the rat will exhibit trembling. In this study, the device was not turned on for control group rats used for the behavioral tests, MRI, and TTC.</li>
	<li>Leave the probe in position for 5 min after stimulation.</li>
	<li>Remove the probe from the brain (Figure 1F).</li>
	<li>Use bone cement to cover the craniotomy. Allow the cement to dry before wound suturing.</li>
	<li>Suture the wound with 4-0 polyamide suture filaments. After three or four stiches, tie 2-1-1 standard surgical knots.</li>
	<li>Inject the rats with penicillin (0.25 mL, 80 IU diluted in 4 mL of saline) intraperitoneally to prevent infection.</li>
	<li>Inject the rats subcutaneously with meloxicam at a dose of 2 mg/kg and then repeat it every 24 h.</li>
	<li>Monitor the rats every 15 min until fully awake and return them to the cage with a heating pad. Provide free access to food and water until sacrifice. 
	NOTE: All procedures should follow the aseptic surgical principles. Before the surgery, put on a scrub top, surgical mask, and sterile gloves after a surgical scrub of the hands. Always maintain sterile suture material within the sterile field.</li>
</ol>

Source: Luo, M., et al. <a href="https://app.jove.com/v/60783">Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation.</a> J. Vis. Exp. (2020)
This video demonstrates a procedure to induce tissue damage or infarction in the pons of an anesthetized rat using electrical stimulation.

<img alt="Figure 1" src="/files/ftp_upload/23001/23001_Figure1.jpg" /> 
Figure 1: Infarction establishment. (A) A hole made in the skull. (B) The sheath is moved to the hole. (C) Injection of the sheath. (D) Injection of the probe. (E) The anode (red arrow) is connected. (F) The probe is removed. (G) Hole (red arrow) left in the brain surface.
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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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&#39;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.

10 Views. Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

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