Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

The protocol was reviewed and approved by the Institution Animal Care and Use Committee of The Second Affiliated Hospital of Guangzhou Medical University, an institution accredited by AAALACi. The rats were provided by the Animal Center of Southern Medical University.

1. Animal

1. Use adult male Sprague-Dawley rats weighing 250 ± 10 g.
2. Upon transport, house the rats for at least 1 week before surgery under controlled environmental conditions with an ambient temperature of 25 °C, relative humidity of 65%, and 12 h/12 h light/dark cycle.
3. Provide food and water ad libitum.  

2. Establishment of infarction in the pons

1. Weigh the rats prior to surgery and assess neurological performance according to the behavioral tests described below (section 3).
2. Preheat the heating pad immediately before anesthesia.
3. Attach the skull drill to the holder on the stereotaxic frame.
4. Intraperitoneally inject the rats with 50 mg/kg ketamine and 5 mg/kg xylazine. Check for the lack of toe-pinch response.
5. Mount the rat onto the stereotaxic frame in 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.
6. Maintain anesthesia by isoflurane (100% oxygen, 2.5% isoflurane) via a stereotaxic nose cone attachment for rats with inlet and outlet ports. Maintain the temperature at 37 °C using a heating pad and monitor the temperature throughout the procedure.
7. Use eye ointment to prevent corneal drying. Use forceps to slightly pinch the paws to ensure that there is no pain response.
8. Shave the hair of the skull with a micro-shaver. Apply chlorhexidine surgical scrub in a circular fashion starting at the surgical incision site and rotating outward.
9. 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 should be marked by a surgical marker pen.
10. Use a cotton swab to remove an blood.
11. Place a piece of surgical tape placed on each side of the skin flap to expose the scalp (Figure 1).
12. 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.
13. Identify the bregma. Chose the central point of the bregma as the origin point and mark it using a fine-tip black surgical marker pen.
14. Place a drill at 6.0 mm AP, 2.0 mm ML (range from 0.5–3.0 mm, Figure 2A).
15. Perform craniotomy (1 mm diameter) using an automatic drill. Proceed carefully, because this point is close to the venous sinus.
16. Remove the drill from the stereotaxic frame.
17. Place a 22 G probe with an insulated sheath in the stereotaxic frame (Figure 3A). The tip of the probe should be placed 2 mm above the proximal end of the sheath (Figure 3A,B; Figure 2B).
18. Ensure that the sheath enters the brain 7 mm (7 mm DV, Figure 2B; Figure 1C).
19. 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).
20. 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).
21. 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.
22. Leave the probe in position for 5 min after stimulation.
23. Remove the probe from the brain (Figure 1F).
24. Use bone cement to cover the craniotomy. Allow the cement to dry before wound suturing.
25. Suture the wound with 4-0 polyamide suture filaments. After three or four stiches, tie 2-1-1 standard surgical knots.
26. Inject the rats with penicillin (0.25 mL, 80 IU diluted in 4 mL of saline) intraperitoneally to prevent infection.
27. Inject the rats subcutaneously with meloxicam at a dose of 2 mg/kg and then repeat it every 24 h.
28. 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 hands. Maintain sterile suture material within the sterile field at all times. 

3. Behavioral tests

1. Longa score¹⁷
   1. Place the rats on the surface of the table.
   2. Record scores as follows: 0 = no neurologic deficit; 1 = failure to fully extend contralateral forepaw, a mild focal neurologic deficit; 2 = circling to the left, a moderate focal neurologic deficit; 3 = falling to the left, a severe focal deficit; 4 = no spontaneous walking and a depressed level of consciousness.
2. Berderson score¹⁸
   1. Hold the rat by the tail and let the forelimbs reach out for a table. Record the scores as follows: 0 = both limbs reached the table; 1 = only one limb reaches the table.
   2. Place the animal on a rough surface. Record the scores as follows: 0 = a strong grasp on the rough surface with good resistance when pushed; 1 = a slight resistance only seen in one paw; 2 = no resistance when pushed in one direction.
   3. Place the rat in an enclosed area (18 in × 36 in) and allow it to roam freely. Record the scores as follows: 0 = walk the entire length of the enclosure without circling; 1 = walk the entire length of the enclosure with circling; 2 = cannot walk the length of enclosure but can circle; 3 = cannot move much. Use the sum of the assessment scores from each task as the final assessment score.
3. Balance beam test¹⁹
   1. Ensure that the apparatus consists of a 3 cm wide and 70 cm long beam and is 20 cm above the floor. Place a darkened box at the far end of the beam with a narrow entryway.
   2. Place a white noise generator and bright light source at the start of the beam. The noise and light were used to motivate the rat to traverse the beam and enter the goal box.
   3. Terminate the stimuli when the animals enter the darkened box. Record the latency to reach the goal box (in seconds) and hindlimb performance of the rat when traversing the beam.
   4. Record the scores for each performance as follows: 0 = balances with steady posture; 1 = grasps side of beam; 2 = hugs beam and 1 limb falls off of beam; 3 = hugs beam and two limbs fall off of beam, or spins on beam after >60 s; 4 = attempts to balance on beam but falls off after >40 s; 5 = attempts to balance on beam but falls off after >20 s; and 6 = falls off, no attempt to balance or hang onto beam after <20 s.
4. Adhesive removal somatosensory test²⁰
   1. Place the rats in a clear plexiglass box and allow them to explore the new environment for 2 or 3 min.
   2. Place a 10 mm diameter green color adhesive label on the inside surface of each forelimb above the thumb and on the wrist.
   3. Return the rats to the plexiglass box.
   4. Record the time for the rat to remove the first label and all other labels, respectively. Allow a maximum of 3 min. The test should be conducted 2x in training.
5. Limb placement test
   1. Hold the rats in a horizontal position and prevent movement.
   2. Once the rat loses contact with table surface (passive limb movement), apply tactile and proprioceptive stimuli to the paw with the table edge.
   3. Assess the placement of the paw (success or failing) onto the table edge.
   4. Record the scores as follows: 0 = no placement; 0.5 = unfinished and/or delayed placement; 1 = immediate and complete placement.

4. Infarct confirmation by MRI

1. Perform the MRI scan 24 h after surgery.
2. Anesthetize the rat by isoflurane (5% for induction, 1%–1.5% for maintenance).
3. Secure the rat head in a rat brain array coil and combined with a transmit-only volume coil.
4. Place the coil and the rat in the MRI scanner. Secure the rat within the cradle using the tooth and ear bars.
5. Maintain body temperature at 37 °C ± 0.5 °C during the MRI scanning procedure using a closed-circuit thermal jacket.
6. Use a pilot sequence to ensure correct geometry.
7. Collect T2-weighted scans using a fast-spin echo sequence: echo time (TE) = 33 ms; repetition time (TR) = 8,000 ms; field of view = 30 mm x 30 mm; acquisition matrix = 512 × 512; 50 slices; 0.4 mm thick.
8. Collect a four-shot spin-echo planar imaging DWI scans: echo time = 30.5 ms; repetition time = 8000 ms; matrix = 96 × 96; field of view = 25 mm x 25 mm; three directions = x, y, z; B values = 0 1,000 s/mm² and 1,000 s/mm²; 50 contiguous axial slices; 0.4 mm thick.
9. Return the rats to the cage.

5. Infarct confirmation by TTC staining

1. Sacrifice the rats at the timepoint according to the experimental design. In this experiment, we sacrificed the rats 24 h after surgery.
2. Prepare a 2% TTC solution before the sacrifice. Add 0.2 g TTC powder to the 10 mL 0.01 M PBS (pH 7.4). Transfer the dilution to a 10 cm dish covered with silver paper and prewarmed to 37 °C in a water bath. 
3. Expose the rat to 5% isoflurane until loss of consciousness. Then, expose the rat to CO₂ (20%–30% of the volume of the cage per min) until breathing has stopped, then maintain 2 min of CO₂ exposure.
4. Use the following signs to confirm death: no rising and falling of chest, no palpable heartbeat, poor mucous membrane color, no response to toe pinch, color change or opacity in eyes.
5. Perform cervical dislocation.
6. Secure the animals by taping the paws on a sterile platform. Create a midline incision from the clavicle to the hypogastrium and a lateral incision from the xiphoid to the left along the ribcage. Make a cut in the diaphragm also along the ribcage and a thorax midline incision to expose the heart.
7. Connect the tip of a needle (27 G) to a perfusion pump containing 0.01 M PBS at 4 °C in the left ventricle.
   NOTE: Advance the tip along the left edge of the ventricle to avoid entering the atrium. Turn on the perfusion pump to ensure the tip is in the left ventricle and cut the right atrium. If fluid drains out of the nostril, the tip is in the atrium and needs to be adjusted or reinserted.
8. Use approximately 100 mL of 0.01 M PBS, maintained at 4 °C for perfusion. Turn off the perfusion pump until the liver turns white.
9. Decapitate the rats and dissect the whole brains using the scissors and forceps. Remove any water from the brain surface with blotting paper.
10. Store the whole brain at -80 °C for 1 min (cutting brain sections is easier after freezing).
    NOTE: This step can be skipped if the brain sections can be cut well without freezing.
11. Place the brain into the matrix with the dorsal side up.
12. Identify the hole in the surface of the brain as shown in Figure 1G and insert a stainless steel 0.21 mm thick blade. Usually, the largest area of infarction is in the plane of the probe; thus, one blade should be inserted in this region.
13. Insert the other blades at an interval of 2 mm.
14. Simultaneously remove the blades, all at once, from the matrix and place the whole brain with the blades in the TTC solution in the dish. Remove the blades carefully.
    NOTE: Here, the brain sections were not easily removed from the liquid because some residual pia mater in the basis cranii interfered with sectioning. If any sections remain in the matrix, use a small spatula to transfer them to the dish.
15. Place the dish with TTC solution and brain sections in a water bath at 37 °C.
16. Check the dish every 5 min and ensure no overlap of sections.
17. Add 10 mL of 4% paraformaldehyde solution to the dish to terminate the TTC reaction.
18. Orient the sections from the rostral to caudal and take pictures.

6. Statistics

1. Use statistical analysis software (e.g., GraphPad Prism) to perform a Student’s t-test.
   NOTE: All data are expressed as mean ± SE. Differences between groups are determined with two-tailed Student’s t-tests (p < 0.05 defined as statistical significance).