Name: establishment of acute pontine infarction in rats by electrical stimulation
Uploaded: 2020-08-27T13:00:00
Description: Watch this Scientific Journal Video about Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation at JoVE.com

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 ...

<ol>
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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.

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	<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 ...

We provide a protocol to establish infarction in a pons of rat. This pontine infarction rat model was easily performed, easily reproduced, and highly successful model. The electrical stimulation could be used to make infarction in any other brain area like thalamus.People who want to perform this model should learn the pontine autonomy before the experiment. Before the surgery, weigh the rats and assess their neurological performance according to the behavioral tests as described in the accompanying text. Preheat the heating pad to 37 degrees Celsius immediately before anesthesia and attach the skull drill to the holder on the stereotaxic frame.Confirm a lack of response to pedal reflex in the first anesthetized rat and mount the rat onto the stereotaxic frame in the prone position on the heating pad. Position the ear bars above the ear canal to secure the head, taking care that the skull is kept horizontal to avoid any skewing of the injection. Apply ointment to the animal's eyes and use a micro shaver to remove the hair from the skull.Use a marker to draw a three centimeter line along the midline of the skull from the line of the bilateral lateral canthus to 0.5 centimeters behind the posterior fontanelle and use a scalpel to make a midline incision along the marked line. Use a cotton swab to remove any blood and place a piece of surgical tape on each side of the skin flap to expose the skull. Use a new swab soaked with 0.9%sodium chloride to gently remove any connective tissue from the skull and locate the bregma.Use a fine tip marker pen to indicate the central point of the bregma as the origin point, place the drill at six millimeters anterior posterior, two millimeters medial lateral. Use the drill to carefully perform a one millimeter diameter craniotomy. Next, replaced the drill with a 22 gauge probe with an insulated sheath with the tip placed two millimeters above the proximal end of the sheath.Insert the sheets seven millimeters into the brain and advance the probe along the sheath until the top of the probe is nine millimeters below the surface of the brain. Connect the electrodes to an electrical stimulator and connect the anode to the probe. Then connect to the cathode to the ear of the rat.Next, turn on the electrical stimulator and set the single pulse width to 4, 050 milliseconds, the voltage to 50 volts and the current to four milliamps. Leave the probe in position for five minutes after stimulation before removing the device from the brain. Cover the craniotomy with bone cement and allow the cement to dry before using 4.0 polyamide suture filaments to close the wound.After three or four stitches, tie 2-1-1 standard surgical knots. Then intraperitoneally inject the rat with 250 microliters of penicillin and monitor the rat every 15 minutes until fully awake before returning the animal to its cage. In this analysis, six animals were subjected to the surgery as demonstrated and six animals were treated as controls.The brain slices in these images were derived from one rat per group. MRI scanning revealed that the infarction was located in the basis of the pons mimicking anterolateral infarctions in human patients. Because an insulated sheath was used, the infarction was not observed in the cortex, cerebellum, or midbrain.Diffusion weighted images also revealed the acute pontine infarction. TTC staining was used to confirm the infarction 24 hours post-surgery. As expected, the infarction volume was significantly higher in the surgery group compared to the control group.Behavioral scores were measured before and after surgery. Compared to the control group, rats with a pontine infarction circled to the left. Significant differences were observed in the Longa, Berderson, limb placement, beam balance, and adhesive removal somatosensory test scores between rants with pontine infarction and control group rats.Any specific brain area are going to be destroyed by the electrical stimulation.

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