inducing thrombotic stroke through hypoxia and ischemia in a mouse model

Inducing Thrombotic Stroke Through Hypoxia and Ischemia in a Mouse Model

Place a 10 to 13-week-old, 22 to 30-gram male C57 black 6 mouse onto the surgical bed. After confirming anesthesia by toe pinch, apply ointment to the animal&#39;s eyes. Stretch the four limbs along the neck roll and secure them with medical tape.
Then, using a depilatory cream, remove the hair on the animal&#39;s neck and clean the surgical site. Now move the surgical bed under a dissecting microscope and make a 0.5-centimeter right cervical incision in the skin along the midline. Using a pair of fine forceps, pull apart the trachea and muscle to expose the right common carotid artery and place two pre-cut 5-0 silk sutures underneath the artery. Be careful to separate the carotid artery from the vagus nerve.
Live knot to pre-cut releasable 5-0 silk sutures on the RCCA. Then use 4-0 nylon monofilament sutures to close the skin, and quickly transfer the mouse to the hypoxia system.
Place the face mask over the animal&#39;s nose and mouth and a rectal probe, so that a 37.5 degrees Celsius body temperature is maintained throughout the hypoxia delivery. Adjust the heat lamps.

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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. Setup
<ol>
	<li>Prepare the surgical bed on a warming pad connected to a heat pump at 37 &#176;C for at least 15 minutes before the surgery. Place a neck roll using the barrel of a 3 ml syringe on the surgical bed. Prepare the anesthesia gas with 2% isoflurane in medical air.</li>
	<li>Prepare autoclaved forceps, scissors, micro needle holders, a hemostat, cotton swabs, and sutures. Also, prepare tissue glue and eye ointment.</li>
	<li>Set up the hypoxia system and temperature controllers with a heating lamp and rectal probe. Prepare hypoxia gas with 2% isoflurane in 7.5% O2 balanced by 92.5% N2.</li>
	<li>One hour before surgery, mice are analgesized by subcutaneous injection of a slow-release Meloxicam (4.0 mg/kg).</li>
</ol>
2. Transient Cerebral Hypoxia-ischemia (Figure 1B)
<ol>
	<li>Anesthetize 10-13 week-old male C57BL/6 mice weighing 22 to 30 g in the anesthesia induction chamber with 3% isoflurane until the animal is unresponsive to foot squeeze, and then remove the hair on the right neck using an electronic shaver.</li>
	<li>Place mice on the surgical bed connected with 2% isoflurane in medical air at a flow rate of 2 L/min. Secure forelimbs stretched out along neck roll at sides using medical tape.</li>
	<li>Clean the surgical site for incision with betadine followed by alcohol and then cotton swabs.</li>
	<li>Under a dissecting microscope, make a 0.5 cm right-cervical incision using straight forceps and micro scissors about 0.2 cm lateral from the midline skin.</li>
	<li>Use a pair of finely serrated forceps to pull apart the fascia and tissue to expose the right common carotid artery (RCCA). Carefully separate the RCCA from the vagal nerve using a pair of fine smooth forceps.</li>
	<li>Live knot two precut 5-0 silk sutures (releasable) on the RCCA, and then sew up the skin using 4-0 Nylon monofilament sutures (Figure 1C).</li>
	<li>Apply eye ointment on both eyes to prevent dryness.</li>
	<li>Quickly transfer the mice to the hypoxia system and put their noses and mouths in a face mask with 2% isoflurane in 7.5% O2 at a flow rate of&#160;&#160; 0.5-1 L/min for 30 min.
	<ol>
		<li>During hypoxia, use temperature controllers with heating lamps to control the rectal temperature at 37.5 &#177; 0.5 &#176;C. Monitor the respiratory rate at 80-120 breaths/min. Maintaining the body temperature above 37 &#176;C during hypoxia is important to create consistent brain infarction. Low respiratory rate usually happens after 20 minutes of hypoxia. Remove the face mask and allow normal air supply if the respiratory rate drops below 40. This takes 1-2 minutes and does not count toward the 30-minute hypoxia duration</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6</td>
			<td>10~13 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Mobile Laboratory Animal Anesthesia System</td>
			<td>VetEquip</td>
			<td>901807</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Medical air (Compressed) air tank</td>
			<td>Airgas</td>
			<td>UN1002</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-25</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Multi-Station Lab Animal Anesthesia System</td>
			<td>Surgivet</td>
			<td>V703501</td>
			<td>Hypoxia system</td>
		</tr>
		<tr>
			<td>7.5% O2 balanced by 92.5% N2 tank</td>
			<td>Airgas</td>
			<td>UN1956</td>
			<td>Hypoxia system</td>
		</tr>
		<tr>
			<td>Temperature Controller with heating lamp</td>
			<td>Cole Parmer</td>
			<td>EW-89000-10</td>
			<td>Temperature controllers</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>Cole Parmer</td>
			<td>NCI-00141PG</td>
			<td>Temperature controllers</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td>SZ40</td>
			<td>Surgical setup</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td>Surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>Surgical supplies</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>Surgical supplies</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>Surgical supplies</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063</td>
			<td>Eye dryness prevention</td>
		</tr>
	</tbody>
</table>

Source: Sun, Y. Y., et al., <a href="https://app.jove.com/v/52978">A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia.</a> J. Vis. Exp. (2015).`
This video demonstrates the procedure of replicating a thrombotic stroke in a mouse model by blocking the right common carotid artery and exposing the mouse to a low-oxygen environment. This process reduces blood and oxygen flow to the brain, causing blood clot formation and brain damage.

<img alt="Figure 1" src="/files/ftp_upload/23210/23210_Figure1.jpg" /> 
Figure 1: Procedure of transient cerebral hypoxia-ischemia (tHI) insult in adult mice. (A) Virchow&#39;s triad that propels thrombosis includes stasis of blood flow, endothelial injury, and hypercoagulability of the blood. (B) A schematic diagram of the tHI stroke procedure. Two releasable knots were tied onto the right common carotid artery (CCA), and followed by delivery of 7.5% oxygen via a no...

Source: Sun, Y. Y., et al., A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia. J. Vis. Exp. (2015).`
This video demonstrates the ...

Begin with an anesthetized and secured mouse.
Remove hair from the mouse&#39;s neck and disinfect the surgical site.
Make an incision on the neck and retract the underlying muscle and trachea to expose the right common carotid artery.
The carotid artery supplies the oxygenated blood to the brain.
Tie two knots around the artery to restrict the upward blood flow.
Suture the incision and transfer the mouse to a low-oxygen chamber. Insert a rectal probe to monitor body temperature, then adjust the heat lamp to maintain the appropriate temperature.
The low oxygen in the chamber reduces the mouse&#39;s blood oxygen levels, creating a hypoxia condition.
With the carotid artery sutured, the blood and oxygen supply to the brain are further reduced. This leads to ischemia, which causes neuronal damage.
Gradually, blood components adhere to the vessel wall, forming a clot or thrombus.
This obstructs the blood flow, increasing neuronal damage and inducing a thrombotic stroke.

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비디오: Inducing Thrombotic Stroke Through Hypoxia and Ischemia in a Mouse Model - 실험

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

철 염화물에 의한 경동맥 및 장간막 용기에 혈전증 마우스 모델

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

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Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

철 염화물에 의한 쥐 혈전증 모델

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

Inducing Thrombotic Stroke Through Hypoxia and Ischemia in a Mouse Model

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Inducing Thrombotic Stroke Through Hypoxia and Ischemia in a Mouse Model