Name: inducing ischemia via middle cerebral and common carotid artery occlusion in a rat model
Uploaded: 2025-03-31T15:00:44.000+00:00
Duration: 1 min 19 s
Description: Source: Wayman, C. et al., Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with ...

inducing ischemia via middle cerebral and common carotid artery occlusion in a rat model

Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model

Incise the skin at the midpoint between the right orbit and the external auditory canal. Next, blunt dissect the temporalis muscle to reveal the skull. Then, using up to five elastic 3 millimeter hook retractors, retract and pin the skin to the cork board. Then, place a thumb wheel adjusted gravity-driven saline drip above the open site at the highest point of the skull near the ear, and set up an aspirator system with a nozzle at the lowest point to remove any bone debris and to clear any minor bleeding from the exposed site.
Adjust the wheel to visualize the source of bleeding as necessary. Now, use a dental drill with a coarse 1.6-millimeter diamond coated drill burr at approximately 8,000 RPM to perform a 5 millimeter by 5 millimeter craniotomy on the exposed region, taking care to apply circular and lateral but not downward pressure during the drilling. When it becomes completely transparent, use forceps to remove the bone. Then, using a homemade dural hook, carefully open the dura, taking care to avoid rupturing the large surface blood vessels.
Next, use a pair of angled jeweler diathermy forceps with 0.25-millimeter pointed tips to coagulate the MCA from where the inferior cerebral vein crosses to the point of the artery bifurcation and then along the caudal branch of the MCA, until the vessels are fully occluded. Then to confirm the occlusion is complete, use microvascular scissors to cut the section of the occluded MCA that lies beneath where the inferior cerebral vein crosses the MCA. Now, cover the exposed area with a saline-soaked gauze pad, and return the rat to the supine position. Reopen the loosely tied suture on the neck to re-expose the carotid arteries, and tie a knot in the suture around the artery on the same side as the occluded MCA to permanently ligate the vessel.

inducing-ischemia-via-middle-cerebral-common-carotid-artery-occlusion

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JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Surgery
<ol>
	<li>Use female elderly Lister-Hooded rats at 16-18 months (250 to 400 g) and induce anesthesia with 5% isoflurane in 1.5 L/min O2. Following induction of anesthesia, reduce the level of isoflurane and maintain it at an adequate but minimized depth for surgery (e.g., 1.5-2%). Deliver anesthesia to the animal by a face mask, and use a scavenging system to limit the surgeon&#39;s exposure to isoflurane.
	<ol>
		<li>Perform all procedures up to, but not including craniotomy on sham animals, as this procedure can produce behavioral deficits. Consider experimental goals when designing middle cerebral artery occlusion (MCAO) experiments, and decide whether or not a sham group to control for craniotomy should be included.</li>
	</ol>
	</li>
	<li>Administer pain relief pre- or peri-operatively (Carprieve, 0.25 mg/kg, s. cut).
	<ol>
		<li>Add 1 ml to 19 ml sterile 0.9% saline to make the stock solution and inject 0.6 ml per 300 g rat. The use of Carprieve is preferred over Carprofen because the former is stable at room temperature (RT).</li>
	</ol>
	</li>
	<li>Shave the fur on the ventral neck region and the temporal region on the right hemisphere to expose the skin. Disinfect the surgical sites using ethanol swabs.
	<ol>
		<li>Perform this step away from the operating area to minimize the amount of fur at the incision site.</li>
	</ol>
	</li>
	<li>Place the rat on a corkboard covered with a sterile drape in the supine position on a heating pad. Apply lidocaine cream to the shaved regions of the head and neck. Insert a rectal probe to monitor and maintain the animal&#8217;s temperature between 36.5-37.5 &#176;C with a homeothermic system.
	<ol>
		<li>Put lacrilube ointment on the eyes to prevent drying. Give the rat an injection of atropine sulfate solution (0.05 ml of a 600 &#181;g/ml solution, subcutaneously) to reduce tracheal secretions. 
		NOTE: Researchers should consider measuring physiological variables such as blood gases and pressure.</li>
	</ol>
	</li>
	<li>Before beginning surgery, check the hindpaw pinch withdrawal and blink reflexes to confirm full anesthesia.</li>
	<li>Under a dissecting microscope, make a 2 cm central midline incision on the exposed neck using a scalpel. Move the salivary glands gently lateral to the trachea on both sides.</li>
	<li>Loop non-absorbable silk suture around the skin that overlies the common carotid artery on one side. Gently pull away the skin from the site using the silk suture and stick these down to the corkboard using surgical tape to reveal the common carotid artery. Carefully blunt dissect the arteries free from the surrounding fascia and the vagal nerves using fine forceps.
	<ol>
		<li>Be careful not to damage the muscle or vagus nerves during this step, as this will impair feeding, swallowing, and breathing.</li>
	</ol>
	</li>
	<li>Once the common carotid artery is exposed, use a non-absorbable silk suture (5/0) to isolate the carotid, first separating the carotid from the vagus nerve by reverse dissecting with fine forceps and being careful not to make contact with the nerve. Once the vessel has been looped (but not tied) with the suture, tape the ends of the suture together using surgical tape to prevent this step from being undone. Remove the sutures holding back the overlying skin.</li>
	<li>Repeat for the other side.</li>
	<li>Place a sterile saline-soaked gauze into the wound to keep tissues moist during the remainder of the surgery. Loosely suture the skin and place an additional saline-soaked gauze over the area to prevent further dehydration.</li>
	<li>Place rats in a lateral position and make an incision in the skin at the midpoint between the right orbit and the external auditory canal. Retract the skin using up to five elastic 3 mm hook retractors pinned to the corkboard and then blunt dissect the temporalis muscle to reveal the skull.</li>
	<li>Place a thumb wheel-adjusted gravity-driven saline drip above the open site (flow rate approximately 2 ml/min) and set up an aspirator system to remove bone debris and to clear minor bleeding from the exposed site throughout this phase of the surgery.
	<ol>
		<li>Place the saline delivery nozzle at the highest point of the skull near the ear, and the aspirator nozzle at the lowest point. Adjust the wheel during bleeds to supply more saline to the area to better visualize the source of any bleed for cautery of the vessel, reducing the amount of time the animals are under anesthesia.</li>
	</ol>
	</li>
	<li>Perform a craniotomy on the exposed region (approximately 5 mm x 5 mm) using a dental drill with a coarse 1.6 mm diamond coated drill burr at approximately 8,000 rpm, ensuring the application of circular and lateral pressure and not downward pressure whilst drilling the exposed area. Once the bone is thin enough that it looks completely transparent, remove using forceps.</li>
	<li>Use a homemade dural hook, made by bending one tip of fine forceps approximately 180&#176; to form an arc, to carefully open the dura, being cautious to avoid the large surface blood vessels, as they are delicate and easy to rupture. 
	NOTE: The exposed area of the brain will reveal the middle cerebral artery (MCA); the desired segment measures approximately 2 mm in length (see Figure 1).</li>
	<li>Coagulate the MCA from where the inferior cerebral vein crosses, to the point of artery bifurcation and then along the caudal branches of the MCA using a pair of diathermy forceps until fully occluded. Use angled Jeweler diathermy forceps with 0.25 mm pointed tips.
	<ol>
		<li>When occluding the artery, alter the saline flow as necessary to keep this area cool, preventing the coagulating forceps from adhering to the blood vessel. When occluded, the blood vessel appears black and no sign of blood flow should be present; blood flow can be seen in partially occluded vessels.</li>
		<li>Cut the MCA at this point to confirm complete occlusion. Using microvascular scissors, cut beneath where the inferior cerebral vein crosses the MCA.</li>
		<li>Cover the exposed area with a saline-soaked gauze pad before continuing to the next step.</li>
	</ol>
	</li>
	<li>Turn the rat back to a supine position and reopen the loosely tied suture on the neck to re-expose the carotid arteries. Ligate the carotid artery on the same side as the occluded MCA (right) permanently by tying a knot in the silk suture around the carotid, whilst the left carotid artery is occluded transiently using a 13 mm stainless steel artery clip with 125 g pressure for 60 min. Loosely suture up the incision in the neck during this time and place a sterile saline-soaked gauze on top to prevent dehydration.
	<ol>
		<li>Note that the animal&#39;s right-hand side will be on the surgeon&#39;s left-hand side, given the supine positioning.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carprofen</td>
			<td>Norbrook</td>
			<td>Vm No; 02000/4229</td>
			<td>give 0.25 mg/kg</td>
		</tr>
		<tr>
			<td>Atropine Sulfate</td>
			<td>AmTech</td>
			<td>RXATRINJ-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>UHS</td>
			<td>20021</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine cream (Emla)</td>
			<td>AstraZeneca</td>
			<td>12901</td>
			<td>Apply a pea sized drop to the shaved neck and temporal regions</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Harvard Instruments</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11019-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk sutures</td>
			<td>Harvard Apparatus</td>
			<td>723288</td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery system</td>
			<td>Eschmann</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 mm Jeweler caurtery forceps</td>
			<td>Eschmann</td>
			<td>8330349</td>
			<td></td>
		</tr>
		<tr>
			<td>fine Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thumb driven saline drip system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacusafe aspirator system</td>
			<td>INTEGRA BIOSCIENCES</td>
			<td>158320</td>
			<td></td>
		</tr>
		<tr>
			<td>16 mm coarse diamond coated Steel burrs</td>
			<td></td>
			<td>K801 104 016</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld dental drill</td>
			<td>NSK</td>
			<td>NSKVMAXVRE (Handpiece NSKEX6B)</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular scissors</td>
			<td>World Precision Instruments</td>
			<td>501790</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Vicryl sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clip and applicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Operating microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Anaesthesia System Isoflurane K/F Single Gas</td>
			<td>VetTech Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel Scalpel blades No. 10</td>
			<td>Swann-Morton</td>
			<td>201</td>
			<td></td>
		</tr>
		<tr>
			<td>25g needles</td>
			<td>Terumo</td>
			<td>NN-2525R</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1 ml and 5 ml)</td>
			<td>Terumo</td>
			<td>SS+01T1 / SS*05SE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (Sodium Chloride 0.9%)</td>
			<td>Fresenius Kabi</td>
			<td>Pl 08828/0178</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td>Johnson and Johnson</td>
			<td>5.00021E+12</td>
			<td>sterilize before use</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td></td>
			<td></td>
			<td>sterilize before use</td>
		</tr>
	</tbody>
</table>

Source: Wayman, C. et al., <a href="https://app.jove.com/v/53106">Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure of performing permanent distal middle cerebral artery occlusion, or MCAO, with the common carotid artery, or CCA ligation, in aged rats to model ischemic stroke. This blocks blood flow to the brain, leading to neuronal death and generating a model to study stroke mechanisms and potential treatments.

<img alt="Figure 1" src="/files/ftp_upload/23115/23115_Figure1.jpg" /> 
Figure 1. Surgical setup of the permanent distal middle cerebral artery occlusion model. The equipment used in the setup for the rat craniotomy is shown for the right hemisphere, and inset, the positioning of the aspirator, and saline drip around the craniectomy site. Also shown are the key features of the vasculature; the middle cerebral artery (red) and inferior cerebral vein (blue) are shown, ...

Source: Wayman, C. et al., Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with ...

Begin with a restrained, anesthetized rat with a loosely sutured right common carotid artery, or CCA, in the neck region.
This CCA branches into the middle cerebral artery, or MCA,&#160; supplying oxygenated blood to the brain.
Reposition the rat laterally and make an incision between the right eye and ear.
Retract the skin and dissect the muscle to expose the skull. Position a saline dripper and aspirator for washing.
Drill over the skull and remove the bone flap. Next, cut the protective layer to expose the MCA.
Using diathermy forceps, apply heat to coagulate the right MCA at multiple points, then cut it at the vein crossing point.
Coagulation causes arterial occlusion, reducing oxygenated blood flow to the brain, induces ischemia, and leads to neuronal death.
Cover the exposed brain.
Re-expose the neck, then tightly ligate the right CCA. This prevents the permanent blood flow to the MCA and intensifies the ischemia.

17 Views. Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model

Video: Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model - Experiment

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

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Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

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The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

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Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

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Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model

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Inducing Ischemia via Middle Cerebral and Common Carotid Artery Occlusion in a Rat Model