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>

<ol>
	<li>Zhu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+local+inflammation+contributes+to+the+neuroprotective+effect+of+ginsenoside+Rb1+in+rats+with+cerebral+ischemia.">Suppression of local inflammation contributes to the neuroprotective effect of ginsenoside Rb1 in rats with cerebral ischemia.</a> Neuroscience. 202, 342-351 (2012).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-1906,+a+Novel+Regulator+of+Toll-Like+Receptor+4,+Ameliorates+Ischemic+Injury+after+Experimental+Stroke+in+Mice.">MicroRNA-1906, a Novel Regulator of Toll-Like Receptor 4, Ameliorates Ischemic Injury after Experimental Stroke in Mice.</a> Journal of Neuroscience. 37, 10498-10515 (2017).</li><li>McBride, D. W., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+Stroke+Animal+Models:+the+Permanent+MCAO+Model+Should+Be+the+Primary+Model,+Not+Transient+MCAO.">Precision Stroke Animal Models: the Permanent MCAO Model Should Be the Primary Model, Not Transient MCAO.</a> Translational Stroke Research. , (2017).</li><li>Liu, F., McCullough, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+model+in+rodents:+methods+and+potential+pitfalls.">Middle cerebral artery occlusion model in rodents: methods and potential pitfalls.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 464701 (2011).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+with+less+damage:+intranasal+delivery+of+tetracycline-inducible+replication-defective+herpes+simplex+virus+type-1+vector+to+brain.">A new approach with less damage: intranasal delivery of tetracycline-inducible replication-defective herpes simplex virus type-1 vector to brain.</a> Neuroscience. 201, 96-104 (2012).</li><li>Lopez, M. S., Vemuganti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Transient+Focal+Ischemic+Stroke+in+Rodents+by+Intraluminal+Filament+Method+of+Middle+Cerebral+Artery+Occlusion.">Modeling Transient Focal Ischemic Stroke in Rodents by Intraluminal Filament Method of Middle Cerebral Artery Occlusion.</a> Methods in Molecular Biology. 1717, 101-113 (2018).</li><li>Pais-Roldan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+assessment+of+recovery+from+coma+in+a+rat+model+of+diffuse+brainstem+tegmentum+injury.">Multimodal assessment of recovery from coma in a rat model of diffuse brainstem tegmentum injury.</a> NeuroImage. 189, 615-630 (2019).</li><li>Merwick, A., Werring, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+circulation+ischaemic+stroke.">Posterior circulation ischaemic stroke.</a> The British Medical Journal. 348, 3175 (2014).</li><li>Nogueira, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombectomy+6+to+24+Hours+after+Stroke+with+a+Mismatch+between+Deficit+and+Infarct.">Thrombectomy 6 to 24 Hours after Stroke with a Mismatch between Deficit and Infarct.</a> The New England Journal of Medicine. 378, 11-21 (2018).</li><li>Wilkinson, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+recanalization+of+basilar+artery+occlusion+in+a+previously+healthy+17-month-old+child.">Late recanalization of basilar artery occlusion in a previously healthy 17-month-old child.</a> Journal of Neurointerventional Surgery. 10, 17 (2018).</li><li>Huang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+Subtypes+and+Topographic+Locations+Associated+with+Neurological+Deterioration+in+Acute+Isolated+Pontine+Infarction.">Stroke Subtypes and Topographic Locations Associated with Neurological Deterioration in Acute Isolated Pontine Infarction.</a> Journal of Stroke and Cerebrovascular Diseases: The Official Journal of National Stroke Association. 25, 206-213 (2016).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-stent+restenosis+after+vertebral+artery+stenting.">In-stent restenosis after vertebral artery stenting.</a> International Journal of Cardiology. 187, 430-433 (2015).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographic+location+of+unisolated+pontine+infarction.">Topographic location of unisolated pontine infarction.</a> BMC Neurology. 19, 186 (2019).</li><li>Banerjee, G., Stone, S. P., Werring, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+circulation+ischaemic+stroke.">Posterior circulation ischaemic stroke.</a> The British Medical Journal. 361, 1185 (2018).</li><li>Wojak, J. C., DeCrescito, V., Young, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basilar+artery+occlusion+in+rats.">Basilar artery occlusion in rats.</a> Stroke: A Journal of Cerebral Circulation. 22, 247-252 (1991).</li><li>Namioka, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+infusion+of+mesenchymal+stem+cells+for+protection+against+brainstem+infarction+in+a+persistent+basilar+artery+occlusion+model+in+the+adult+rat.">Intravenous infusion of mesenchymal stem cells for protection against brainstem infarction in a persistent basilar artery occlusion model in the adult rat.</a> Journal of Neurosurgery. , 1-9 (2018).</li><li>Jiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+brain-derived+neurotrophic+factor+protects+brain+from+ischemic+insult+via+modulating+local+inflammation+in+rats.">Intranasal brain-derived neurotrophic factor protects brain from ischemic insult via modulating local inflammation in rats.</a> Neuroscience. 172, 398-405 (2011).</li><li>Schaar, K. L., Brenneman, M. M., Savitz, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessments+in+the+rodent+stroke+model.">Functional assessments in the rodent stroke model.</a> Experiments in Translational and Stroke. 2, 13 (2010).</li><li>Wu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keep+warm+and+get+success:+The+role+of+postischemic+temperature+in+the+mouse+middle+cerebral+artery+occlusion+model.">Keep warm and get success: The role of postischemic temperature in the mouse middle cerebral artery occlusion model.</a> Brain Research Bulletin. 101, 12-17 (2014).</li><li>Wen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+behavioural+tests+for+the+prediction+of+outcomes+in+mouse+models+of+focal+middle+cerebral+artery+occlusion.">Optimization of behavioural tests for the prediction of outcomes in mouse models of focal middle cerebral artery occlusion.</a> Brain Research. 1665, 88-94 (2017).</li></ol>

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 th...

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 border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="663">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">4-0 sucture</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td style="width:167px;">Surgical instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Adhesive tape</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Animal anesthesia system</td>
			<td style="width:121px;">RWD</td>
			<td style="width:161px;"></td>
			<td>Wear mask when using the system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bone cement</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Cured clamp</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">General tissue scissors</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">IndoPhors</td>
			<td style="width:121px;">Guoyao of China</td>
			<td style="width:161px;"></td>
			<td>Sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isoflurane</td>
			<td style="width:121px;">RWD</td>
			<td align="right" style="width:161px;">217181101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Lesion Making Device</td>
			<td style="width:121px;">Shanghai Yuyan</td>
			<td style="width:161px;"></td>
			<td>Making a lesion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MRI system</td>
			<td style="width:121px;">Bruker Biospin</td>
			<td style="width:161px;"></td>
			<td>Confirmation of infarction in vivo</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Needle holder</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicilin</td>
			<td style="width:121px;">Guoyao of China</td>
			<td style="width:161px;"></td>
			<td>Infection Prevention</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Probe</td>
			<td style="width:121px;">Anke</td>
			<td style="width:161px;"></td>
			<td>Need some modification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Q-tips</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Shearing scissors</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Stereotaxic apparatus</td>
			<td style="width:121px;">RWD</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Suture needle</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Tissue holding forcepts</td>
			<td style="width:121px;">Shanghai Jinzhong</td>
			<td style="width:161px;"></td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">TTC</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">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...

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

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

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 ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

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 ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Massive Pontine Hemorrhage by Dual Injection of Autologous Blood

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred microliters of autologous blood was taken from the tail vein and stereotaxically injected into the pons. The injection process was divided into 2 steps: First, 10 &#181;L of blood was injected into a specific location, anteroposterior position (AP) -9.0 mm; lateral (Lat) 0 mm; vertical (Vert) -9.2 mm, followed by a second injection of the residual blood located at AP -9.0 mm; Lat 0 mm; Vert -9.0 mm with a 20-minute interval. The balance beam test, limb placement test, and the modified Voestch neuroscore were used to evaluate neurological function. Magnetic Resonance Imaging (MRI) was used to assess the volume of hemorrhage in vivo. The symptoms of this model were in line with patients with massive pontine hemorrhage.

We present a protocol to establish a massive pontine hemorrhage model in a rat via dual injection of autologous blood.

We provide a protocol to establish a massive pontine hemorrhage model in a rat. Rats weighing about 250 grams were used in this study. One hundred ...

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the patient&#39;s pathophysiology. Despite the long history of DBS, its mechanism of action and appropriate protocols remain unclear, highlighting the need for research aiming to solve these enigmas. In this sense, evaluating the in vivo effects of DBS using functional imaging techniques represents a powerful strategy to determine the impact of stimulation on brain dynamics. Here, an experimental protocol for preclinical models (Wistar rats), combined with a longitudinal study [18F]-fluorodeoxyclucose positron emission tomography (FDG-PET), to assess the acute consequences of DBS on brain metabolism is described. First, animals underwent stereotactic surgery for bilateral implantation of electrodes into the prefrontal cortex. A post-surgical computerized tomography (CT) scan of each animal was acquired to verify electrode placement. After one week of recovery, a first static FDG-PET of each operated animal without stimulation (D1) was acquired, and two days later (D2), a second FDG-PET was acquired while animals were stimulated. For that, the electrodes were connected to an isolated stimulator after administering FDG to the animals. Thus, animals were stimulated during the FDG uptake period (45 min), recording the acute effects of DBS on brain metabolism. Given the exploratory nature of this study, FDG-PET images were analyzed by a voxel-wise approach based on a paired T-test between D1 and D2 studies. Overall, the combination of DBS and imaging studies allows describing the neuromodulation consequences on neural networks, ultimately helping to unravel the conundrums surrounding DBS.

In vivo Positron Emission Tomography to Reveal Activity Patterns Induced by Deep Brain Stimulation in Rats

We describe a preclinical experimental method to evaluate metabolic neuromodulation induced by acute deep brain stimulation with in vivo FDG-PET. This manuscript includes all experimental steps, from stereotaxic surgery to the application of the stimulation treatment and the acquisition, processing, and analysis of PET images.

Deep brain stimulation (DBS) is an invasive neurosurgical technique based on the application of electrical pulses to brain structures involved in the ...