This study was financially supported by the National Science Foundation of China (81471181 and 81870933) to Y. Jiang and the National Science Foundation of China (No. 81601011), Natural Science Foundation of Jiangsu Province (No. BK20160345) to J. Zhu and by the Scientific Program of Guangzhou Municipal Health Commission (20191A011083) to Z. Qiu.

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
<ol>
	<li>Use adult male Sprague-Dawley rats weighing 250 &#177; 10 g.</li>
	<li>Upon transport, house the rats for at least 1 week before surgery under controlled environmental conditions with an ambient temperature of 25 &#176;C, relative humidity of 65%, and 12 h/12 h light/dark cycle.</li>
	<li>Provide food and water ad libitum. &#160;</li>
</ol>
2. Establishment of infarction in the pons
<ol>
	<li>Weigh the rats prior to surgery and assess neurological performance according to the behavioral tests described below (section 3).</li>
	<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&#160;mg/kg ketamine and 5&#160;mg/kg xylazine. Check for the lack of toe-pinch response.</li>
	<li>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.</li>
	<li>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 &#176;C using a heating pad and monitor the temperature throughout the procedure.</li>
	<li>Use eye ointment to prevent corneal drying. Use forceps to slightly pinch the paws to ensure that there is no pain response.</li>
	<li>Shave the hair of the skull with a micro-shaver.&#160;Apply chlorhexidine surgical scrub in a circular fashion 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 should be marked by a surgical marker pen.</li>
	<li>Use a cotton swab to remove an blood.</li>
	<li>Place a piece of surgical tape 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. Chose 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 tip of the probe 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 hands. Maintain sterile suture material within the sterile field at all times.&#160;</li>
</ol>
3. Behavioral tests
<ol>
	<li>Longa score17
	<ol>
		<li>Place the rats on the surface of the table.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Berderson score18
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Place the rat in an enclosed area (18 in&#8201;&#215;&#8201;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.</li>
	</ol>
	</li>
	<li>Balance beam test19
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#62;60 s; 4 = attempts to balance on beam but falls off after &#62;40 s; 5 = attempts to balance on beam but falls off after &#62;20 s; and 6 = falls off, no attempt to balance or hang onto beam after &#60;20 s.</li>
	</ol>
	</li>
	<li>Adhesive removal somatosensory test20
	<ol>
		<li>Place the rats in a clear plexiglass box and allow them to explore the new environment for 2 or 3 min.</li>
		<li>Place a 10 mm diameter green color adhesive label on the inside surface of each forelimb above the thumb and on the wrist.</li>
		<li>Return the rats to the plexiglass box.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Limb placement test
	<ol>
		<li>Hold the rats in a horizontal position and prevent movement.</li>
		<li>Once the rat loses contact with table surface (passive limb movement), apply tactile and proprioceptive stimuli to the paw with the table edge.</li>
		<li>Assess the placement of the paw (success or failing) onto the table edge.</li>
		<li>Record the scores as follows: 0 = no placement; 0.5 = unfinished and/or delayed placement; 1 = immediate and complete placement.</li>
	</ol>
	</li>
</ol>
4. Infarct confirmation by MRI
<ol>
	<li>Perform the MRI scan 24 h after surgery.</li>
	<li>Anesthetize the rat by isoflurane (5% for induction, 1%&#8211;1.5% for maintenance).</li>
	<li>Secure the rat head in a rat brain array coil and combined with a transmit-only volume coil.</li>
	<li>Place the coil and the rat in the MRI scanner. Secure the rat within the cradle using the tooth and ear bars.</li>
	<li>Maintain body temperature at 37 &#176;C &#177; 0.5 &#176;C during the MRI scanning procedure using a closed-circuit thermal jacket.</li>
	<li>Use a pilot sequence to ensure correct geometry.</li>
	<li>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 &#215; 512; 50 slices; 0.4 mm thick.</li>
	<li>Collect a four-shot spin-echo planar imaging DWI scans: echo time = 30.5 ms; repetition time = 8000 ms; matrix = 96 &#215; 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.</li>
	<li>Return the rats to the cage.</li>
</ol>
5. Infarct confirmation by TTC staining
<ol>
	<li>Sacrifice the rats at the timepoint according to the experimental design. In this experiment, we sacrificed the rats 24 h after surgery.</li>
	<li>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 &#176;C in a water bath.&#160;</li>
	<li>Expose the rat to 5% isoflurane until loss of consciousness. Then, expose the rat to CO2 (20%&#8211;30% of the volume of the cage per min) until breathing has stopped, then maintain 2 min of CO2 exposure.</li>
	<li>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.</li>
	<li>Perform cervical dislocation.</li>
	<li>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.</li>
	<li>Connect the tip of a needle (27 G) to a perfusion pump containing 0.01 M PBS at 4 &#176;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.</li>
	<li>Use approximately 100 mL of 0.01 M PBS, maintained at 4 &#176;C for perfusion. Turn off the perfusion pump until the liver turns white.</li>
	<li>Decapitate the rats and dissect the whole brains using the scissors and forceps. Remove any water from the brain surface with blotting paper.</li>
	<li>Store the whole brain at -80 &#176;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.</li>
	<li>Place the brain into the matrix with the dorsal side up.</li>
	<li>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.</li>
	<li>Insert the other blades at an interval of 2 mm.</li>
	<li>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.</li>
	<li>Place the dish with TTC solution and brain sections in a water bath at 37 &#176;C.</li>
	<li>Check the dish every 5 min and ensure no overlap of sections.</li>
	<li>Add 10 mL of 4% paraformaldehyde solution to the dish to terminate the TTC reaction.</li>
	<li>Orient the sections from the rostral to caudal and take pictures.</li>
</ol>
6. Statistics
<ol>
	<li>Use statistical analysis software (e.g., GraphPad Prism) to perform a Student&#8217;s t-test. 
	NOTE: All data are expressed as mean &#177; SE. Differences between groups are determined with two-tailed Student&#8217;s t-tests (p &#60; 0.05 defined as statistical significance).</li>
</ol>

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No conflict of interest.

The present study provides a protocol for generating an acute pontine infarction rat model. This model can be used for research on prognosis and rehabilitation (including post-stroke chronic pain) in pontine stroke patients.
There are several strengths of this method. First, it provides a rat model of acute pontine infarction for future studies. As mentioned above, pontine infarction is a common stroke subtype that has received less attention. A major shortcoming of stroke research has been the lack of a specific pontine infarction model. Second, in comparison to the existing pontine infarction rat model by ligation of the BA15,16, this model can be adjusted to alter the location and volume of the infarction according to the experimental design. For example, the length of the tip can be changed so that the infarction extends from the surface of the pons, as done here.
Alternatively, a lacunar infarction in the pons may be established by shortening the length of the probe tip (Supplemental Figure 1). Infarctions in different locations of the pons (i.e., anteromedial pontine infarction) and in different planes of the pons (i.e., upper, middle and lower planes) may also be created according to the topographic design. In this model, the upper pontine plane was chosen. Third, this model is easy to establish and possesses a high success rate. Ligation of the BA may not produce infarction due to the potential collateral circulation15, but this model establishes the infarction at a high success rate, which is essential for reliable research models.
There are some limitations of this method. First, the infarction in this model is not a real stroke. Stroke is a result of vascular vessel lesions, disturbance of blood contents, or dysfunction of regulation of cerebral blood flow. The infarction is created by a lesion in the pons that does not spontaneously occur. In other words, this model cannot be used to address why the stroke occurs in the pons. Second, this model requires special equipment, such as the lesion-producing device and stereotaxic apparatus.
In conclusion, the findings prove this model&#8217;s success in establishing an experimental acute pons stroke model. Based on this novel model, the resulting cell loss and prognosis of acute pontine infarction can be further investigated and allow for future therapeutic developments.

Since the 1980s, the middle cerebral artery occlusion (MCAO) model induced by silicone filaments has been widely used in basic stroke research1. Other methods (i.e., suturing of one branch of the MCA2 and photochemically induced focal infarction) have also been used. These models have been termed MCA-based stroke models and have greatly contributed to investigations of the pathophysiological mechanisms underlying stroke and potential therapeutics. Although there are limitations of these experimental models3,4, these methods have been used many labs5,6. MCA-based stroke models represent a stroke in the anterior circulation; however, few reports have investigated models mimicking stroke in the posterior circulation7.
There are significant differences among the etiology, mechanisms, clinical manifestation, and prognosis between anterior and posterior circulation strokes8. Therefore, the results derived from anterior circulation stroke models cannot be applied to posterior circulation stroke. For example, the reperfusion time window for anterior circulation has been extended to 6 h, with a small portion of studies extending to 24 h based on imaging findings9. However, the time window for posterior circulation may be longer than 24 h, according to previous reports10 and our own clinical experiences. This elongated reperfusion time window must be further studied and confirmed in experimental models.
Regarding posterior circulation strokes, pontine infarction is the most common subtype, accounting for 7% of all ischemic stroke cases11,12. According to infarction topography, pontine infarctions are divided into isolated and non-isolated pontine infarctions13. Isolated pontine infarctions are categorized into three types based on the underlying mechanisms: large artery disease (LAD), basilar artery branch disease (BABD), and small artery disease (SAD). Knowledge of the mechanisms, manifestation, and prognosis of pontine infarction has been derived from clinical investigations of cases14. However, a rodent model mimicking pontine infarction has been less investigated.
In previous studies, diffuse brainstem tegmentum injury involving the pons has been explored7. One group attempted to create a pontine infarction model via ligation of the basilar artery (BA)15. Another group used a 10-0 nylon monofilament suture to selectively ligate four points of the proximal BA selectively16. This model mimics LAD, but most pontine infarctions result from BABD and SAD. In addition, selective ligation of the BA is a complicated surgery and has a high death rate.
Provided here is a detailed protocol for an easy-to-perform, easily reproduced, and successful rat model of acute pontine infarction by electrical stimulation.

<table><tbody><tr><td>4-0 sucture</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instruments</td></tr><tr><td>Adhesive tape</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instruments</td></tr><tr><td>Animal anesthesia system</td><td>RWD</td><td></td><td>Wear mask when using the system</td></tr><tr><td>Bone cement</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>Cured clamp</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>General tissue scissors</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>IndoPhors</td><td>Guoyao of China</td><td></td><td>Sterilization</td></tr><tr><td>Isoflurane</td><td>RWD</td><td>217181101</td><td></td></tr><tr><td>Lesion Making Device</td><td>Shanghai Yuyan</td><td></td><td>Making a lesion</td></tr><tr><td>MRI system</td><td>Bruker Biospin</td><td></td><td>Confirmation of infarction in vivo</td></tr><tr><td>Needle holder</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>Penicilin</td><td>Guoyao of China</td><td></td><td>Infection Prevention</td></tr><tr><td>Probe</td><td>Anke</td><td></td><td>Need some modification</td></tr><tr><td>Q-tips</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>Shearing scissors</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>Stereotaxic apparatus</td><td>RWD</td><td></td><td></td></tr><tr><td>Suture needle</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>Tissue holding forcepts</td><td>Shanghai Jinzhong</td><td></td><td>Surgical instrument</td></tr><tr><td>TTC</td><td>Sigma-Aldrich</td><td>BCBW5177</td><td>For infarction confirmation in vitro</td></tr></tbody></table>

establishment of acute pontine infarction in rats by electrical stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided here is a protocol for successfully establishing a rat model of acute pontine infarction. Rats weighing about 250 g are used, and a probe with an insulated sheath is injected into the pons using a stereotaxic apparatus. A lesion is produced by the electrical stimulation with a single pulse. The Longa score, Berderson score, and beam balance test are used to assess neurological deficits. Additionally, the adhesive-removal somatosensory test is used to determine sensorimotor function, and the limb placement test is used to evaluate proprioception. MRI scans are then used to assess the infarction in vivo, and TTC staining is used to confirm the infarction in vitro. Here, a successful infarction is identified that is located in the anterolateral basis of the rostral pons. In conclusion, a new method is described to establish an acute pontine infarction rat model.

Six animals were subjected to the surgery protocol described above. The control group as shown in the Figure 4 consisted of six rats. The brain slices shown in the Figure 4 were derived from one rat per group.
The MRI scanning showed that the infarction was located in the basis of the pons (Figure 4A). Since the probe was injected 2 mm to the left of the midline, the infarction was located laterally. This infarction mimics anterolateral pontine infarctions in patients (Figure 4A). Because an insulated sheath was used, there was no infarction beyond the tip of the probe including the cortex, cerebellum, and midbrain (Figure 4A). DWI images also revealed the acute pontine infarction (Figure 4A).
TTC staining was used to confirm the infarction 24 h post-surgery (Figure 4A). Compared to the control group, the infarction volume was significantly higher (Figure 4B).
Behavioral scores were measured before and after surgery. The scores for the control and infract model groups before and after surgery are presented in Table 1. Due to the lack of a specific behavioral test designed for pontine infarction, the Longa score, Berderson score, and balance beam test were used to assess the neurological deficits. Additionally, the adhesive removal somatosensory test to assess the sensorimotor function as well as limb-placement test to assess the proprioception.
Compared to the control group, the rats with pontine infarction circled to the left (Figure 4A). There were significant differences in Longa score (2.67 &#177; 0.52 vs. 0, p &#60; 0.05, Figure 4C), Berderson score (2.67 &#177; 0.52 vs. 0, p &#60; 0.05, Figure 4D), limb placement test (4.67 &#177; 0.52 vs. 0, p &#60; 0.05, Figure 4E), beam balance test score (118.33 &#177; 2.66 vs. 10.17 &#177; 1.47, p &#60; 0.05, Figure 4F), and adhesive removal somatosensory test score (2.33 &#177; 0.52 vs. 12.0 &#177; 0, p &#60; 0.05, Figure 4G) between rats with pontine infarction and control group rats.&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60783/60783fig1.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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60783/60783fig1large.jpg" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60783/60783fig2.jpg" /> 
Figure 2: Location of probe. (A) Schematic diagram of stereotaxic locations: arrows point to retraction of skin flaps, site of Bregma, and positioning of drill. (B) Schematic diagram of the sheath and probe. (C) Location of tip of sheath placed in the pons. (D) Location of tip of the probe placed in the pons. (E) Experimental design. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60783/60783fig2large.jpg" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60783/60783fig3.jpg" /> 
Figure 3: Lesion-producing device. (A) Separate of sheath and probe. (B) The probe in the sheath. (C) The blue electrode was anode which was connected to the caudal probe; the red electrode was cathode. (D) Electrical stimulator. (E) Surgical instruments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60783/60783fig3large.jpg" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60783/60783fig4.jpg" /> 
Figure 4: Representative results. (A) The infarction was assessed by MRI scanning with T2 and DWI sequence in vivo and was confirmed by TTC staining in vitro 24 h after surgery. Acute pontine infarction located in the right anterolateral pons (dotted line). Behavioral test showed that the rat circled to the contralateral side of lesion. (B) The volume of infarction. (C) Long score. (D) Bederson score. (E) Limb placing test. (F) Balance beam walking test. (G) Adhesive removal somatosensory test. Bars represent mean &#177; SD (p &#60; 0.05 vs. control group). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60783/60783fig4large.jpg" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/60783/60783supfig1.jpg" /> 
Figure S1: Lacunar infarction in the pons. The length of the probe tip is shortening. MRI scanning shows a lacunar infarction in the right pons. (A) T2 image. (B) DWI image. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60783/60783supfig1large.jpg" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td rowspan="2">Rat NO</td>
			<td colspan="2">Longa score</td>
			<td colspan="2">Berderson score</td>
			<td colspan="2">Balance beam test</td>
			<td colspan="2">Adhesive-removal somatosensory test</td>
			<td colspan="2">Limb-placement test</td>
		</tr>
		<tr>
			<td>Pre</td>
			<td>Post-surgery</td>
			<td>Pre</td>
			<td>Post-surgery</td>
			<td>Pre</td>
			<td>Post-surgery</td>
			<td>Pre</td>
			<td>Post-surgery</td>
			<td>Pre</td>
			<td>Post-surgery</td>
		</tr>
		<tr>
			<td>Pontine infarction 1</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
			<td>5</td>
			<td>6</td>
			<td>120</td>
			<td>12</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Pontine infarction 2</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>4</td>
			<td>8</td>
			<td>120</td>
			<td>12</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Pontine infarction 3</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>5</td>
			<td>8</td>
			<td>116</td>
			<td>12</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Pontine infarction 4</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>4</td>
			<td>6</td>
			<td>120</td>
			<td>12</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Pontine infarction 5</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
			<td>5</td>
			<td>7</td>
			<td>114</td>
			<td>12</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Pontine infarction 6</td>
			<td>0</td>
			<td>2</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>5</td>
			<td>7</td>
			<td>120</td>
			<td>12</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Control 1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>9</td>
			<td>11</td>
			<td>12</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Control 2</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>8</td>
			<td>10</td>
			<td>12</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Control 3</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>10</td>
			<td>8</td>
			<td>12</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Control 4</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>7</td>
			<td>11</td>
			<td>12</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Control 5</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>8</td>
			<td>9</td>
			<td>12</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Control 6</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>9</td>
			<td>12</td>
			<td>12</td>
			<td>12</td>
		</tr>
	</tbody>
</table>
Table 1: Behavioral scores.

Watch this Scientific Journal Video about Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation at JoVE.com

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

establishment-acute-pontine-infarction-rats-electrical

Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.9K Views.  The Second Affiliated Hospital of Guangzhou Medical University. Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.

Video: Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Medicine

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Transesophageal Atrial Burst Pacing for Atrial Fibrillation Induction in Rats

Animal studies have brought important insights into our understanding regarding atrial fibrillation (AF) pathophysiology and therapeutic management. Reentry, one of the main mechanisms involved in AF pathogenesis, requires a certain mass of myocardial tissue in order to occur. Due to the small size of the atria, rodents have long been considered 'resistant' to AF. Although spontaneous AF has been shown to occur in rats, long-term follow-up (up to 50 weeks) is required for the arrhythmia to occur in those models. The present work describes an experimental protocol of transesophageal atrial burst pacing for rapid and efficient induction of AF in rats. The protocol can be successfully used in rats with healthy or remodeled hearts, in the presence of a wide variety of risk factors, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of novel prophylactic and/or therapeutic strategies.

The present work describes an experimental protocol of transesophageal atrial burst pacing for efficient induction of atrial fibrillation (AF) in rats. The protocol can be used in rats with healthy or remodeled hearts, allowing the study of AF pathophysiology, identification of novel therapeutic targets, and evaluation of new therapeutic strategies.

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Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Immunofluorescence Staining to Study Cell Death in Rat MCAO Stroke Models

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...

Single-Step Surgery for Inducing Cerebral Ischemic Coma in Rats

Cerebral Ischemic Coma Model Induced by Modified Four-Vessel Occlusion

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model for disease research and drug development. However, the commonly used four-vessel occlusion method mainly involves inserting an electrocoagulation pen into the bilateral pterygoid foramen of the first cervical vertebra behind the neck to electrocoagulate the vertebral arteries. This process carries the risk of incomplete electrocoagulation, bleeding, and damage to the brainstem and spinal cord. Twenty-four hours after surgery, re-anesthetized rats undergo carotid artery ligation in front of the neck. Two surgeries expose the rats to a higher risk of infection and increase the experimental period. In this study, during a single surgical procedure, an anterior cervical incision was used to locate the key site where the vertebral artery penetrates the first cervical vertebra. The bilateral vertebral arteries were electrocauterized under visual conditions, while the bilateral common carotid arteries were separated to place loose knots. When the limbs of the rats began twitching, the bilateral common carotid arteries were quickly ligated to induce ischemic coma. This method can avoid the risk of infection caused by two surgical operations and is easy to perform with a high success rate, providing a useful reference for relevant practitioners.

Author Spotlight: Enhancing Cerebral Ischemia Research with a Simplified Rat Model

This protocol describes the process for inducing a cerebral ischemic coma model using a modified four-vessel occlusion method.

Coma caused by cerebral ischemia is the most serious complication of cerebral ischemia. Four-vessel occlusion can establish a cerebral ischemic coma model ...

Inducing Acute Pons Infarction in a Rat Model Using Electrical Stimulation

Source: Luo, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

Wywoływanie ostrego zawału mostu w modelu szczurzym za pomocą stymulacji elektrycznej

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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