This work was supported by National Institutes of Health grants HL087990 (G.L.).

Ethics Statement: Male adult Sprague-Dawley rats (weighing 330-380 g) were used in this protocol. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the LSU Health Science Center-Shreveport and is in compliance with the &#39;Guide for the Care and Use of Laboratory Animals&#39; (eighth edition, National Academy of Sciences, 2011).
1. Preparation of Modified PE-50 Tube
<ol>
	<li>Hold a 30 cm long PE-50 tube above a gas fire by hands, gradually soften the tube. Gently stretch the tube.</li>
	<li>Select a point on the stretched tube using a digital caliper (Figure 1), mark it, and cut the modified PE-50 tube into a 25 cm long segment with a 1 cm long tip (outside diameter: 0.30-0.34 mm).</li>
</ol>
2. Preparation of Homologous Blood Clots
<ol>
	<li>Anesthetize the rat with isoflurane (5% for induction, 2-3% for maintenance) in 70% N2O and 30% O2 by a face mask prior to blood collection. Confirm anesthesia by a toe pinch.</li>
	<li>Perform femoral arterial cannulation of a donor rat with published method4 and transfer femoral artery blood directly into a 20 cm long PE-50 tube. Place the tube for 2 hr at room temperature to clot blood, and then retain the tube for 22 hr at 4 &#176;C. Note: Cannulation is performed using aseptic technique as described in step 3.1. The donor rat is recovered for further sampling.</li>
	<li>Cut the PE-50 tube into 50 mm segments and flush out the clot from the tube into a sterile Petri dish containing normal saline.</li>
	<li>Transfer the 50 mm long clot into a 60 mm long PE-10 tube and connect each end of the PE-10 tube to a 20 cm long PE-50 tube connected to 1 ml syringe containing normal saline with 23 G needle (Figure 2).</li>
	<li>Shift the clot by continuous alternating movement from one syringe to the other for 5 min, which can remove blood cells from the clot and minimize the fragile of extravascular coagulated clot, without disrupting the fibrin core.</li>
	<li>Cut the clot into a 40 mm long segment and transfer the clot segment into a modified PE-50 catheter as described in step 1.2 under aseptic conditions. Note: All PE tubing is sterilized with ethylene oxide. </li>
</ol>
3. Embolic Middle Cerebral Artery Occlusion (Figure 3A,B)
<ol>
	<li>Sterilize all surgical tools by autoclaving (minimum 121 &#176;C, 15 PSI, for 15 min). Sanitize the surgery table and associated surgical equipment using 70% ethanol.</li>
	<li>Anesthetize the rat with isoflurane as described in step 2.1. Test the depth of anesthesia by performing a toe pinch on both rear feet, and any observed movement (withdrawing the paw) indicates that the animal is not sufficiently anesthetized to do surgery. Apply a small amount of vet ointment on both eyes to prevent dryness while under anesthesia. Shave the fur on the ventral neck and head regions with electric clippers to expose the skin areas.</li>
	<li>Place the rat in the supine position on a heating pad. Insert a rectal probe, control and maintain body temperature between 36.5-37.5 &#176;C using a homeothermic blanket control unit.</li>
	<li>Disinfect the shaved skin and surrounding fur with 10% povidone-iodine followed by 70% ethanol.</li>
	<li>Make a 2-cm-long midline incision on the neck under a dissecting microscope. Use retractors to expose the surgical field and dissect the right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA), and pterygopalatine artery (PPA) free from surrounding nerves and fascia (Figure 3A). Note: Before surgery, change sterile gloves if no assistant helps to prepare the animal.</li>
	<li>Dissect the CCA free from the surrounding nerves (without harming the vagal nerve) and place a sterile 5-0 silk suture under the artery. Tie a slip knot (#1) and grasp the suture using a small hemostat and pull taught toward the body.</li>
	<li>Dissect the ECA and its two branches, the occipital artery (OA) and the superior thyroid artery (STA). Coagulate two branches using a veterinary electrosurgical unit. Place two pieces of sterile 6-0 silk sutures under the ECA.</li>
	<li>Separate the two silks placed under the artery, one piece towards the head (distal end) and the other towards the body. Tie a tight ligature (#2) on the side closest to the head, grasp the silk with small hemostats and pull taught toward the head. Prepare a loose knot (#3) with the other silk to use later.</li>
	<li>Dissect the ICA and PPA from the surrounding nerves (without harming the vagal nerve). Tie the PPA with a 6-0 suture (#4). Make a slip knot with 6-0 suture around the ICA (#5). Alternatively, clip the ICA using a microvascular clip.</li>
	<li>Cut a small hole (1/2 thru) into the ECA between the tight (#2) and loose (#3) ligatures with a Vannas-style spring scissors. Insert the modified PE-50 tube containing blood clot into the incision and advance to the bifurcation of the CCA. Tighten the loose ligature (#3) around the lumen just enough to secure preserve mobility of the in-dwelling tube.</li>
	<li>Cut off the ECA at the site of small hole to free the stump and position the stump below the bifurcation of the ECA and ICA; this will allow the modified tube easily slide into the ICA. Open the ICA and gently advance the tube from the lumen of the ECA into the ICA until the tip of the catheter reaches the origin of the MCA (~17 mm from the bifurcation). Note: Before insert the tip of tube into the ECA, sterile the outer surface of tube with 70% ethanol, and then wash with sterile normal saline.</li>
	<li>Inject the clot through the modified PE-50 catheter along with 10 &#956;l of saline over 10 sec using a 100 &#956;l Hamilton syringe (Figure 3B).</li>
	<li>Withdraw the catheter from the ECA 5 min later. Tie the ECA and reopen CCA. Suture the incision on the neck.</li>
</ol>
4. Monitoring Regional Cerebral Blood Flow (rCBF)
<ol>
	<li>Prior to MCA occlusion, make a 1.2 cm long midline incision in the scalp to expose the skull bone. Remove the tissues on the skull bone with a dental scraper and sterile cotton swaps. Note: Before surgery, the exposed scalp area and surrounding fur are disinfected with 10% povidone-iodine followed by 70% ethanol.</li>
	<li>Drill a 1.5 mm diameter burr hole located at 2 mm posterior and 5 mm lateral to the bregma using a 0.7 mm spherical stainless steel burr (Figure 4). 
	NOTE: Keep the dura intact.</li>
	<li>Place the probe 0.5 mm above the dura surface. Monitor the rCBF at 0 (baseline), 5, 15, 30, 60, 90, and 120 min after embolization and continue at 5, 15, 30, 45, and 60 min after intravenous administration of tPA. After the last measurement of rCBF, the scalp incision is closed by suture. Note: Prior to neck incision we measure rCBF as baseline before MCA occlusion. Post MCA occlusion we measure rCBF after suture closure of neck incision. Thus, the neck incision is kept sterile. </li>
</ol>
5. Post-operative Care
<ol>
	<li>Inject 2.5 ml of saline solution subcutaneously to prevent dehydration and inject Buprenex (0.05 mg/kg, SC) immediately after the surgery and every 6-12 hr as needed for pain relief.</li>
	<li>Stop isoflurane anesthesia. Place the rat in a 37 &#176;C veterinary recovery chamber (keep animal warm) and keep observation. It generally takes 10 min for the rat to recover from anesthesia. Then, put the animal into a sterilized cage, place some wetted food in a Petri dish in the cage, and return the cage to animal sterilization room.</li>
	<li>24 hr after stroke, euthanize the rat with an overdose of sodium pentobarbital (100 mg/kg body weight, IP).</li>
</ol>
6. Neurological Deficit Score
<ol>
	<li>Perform the Bederson score before and at 2 hr after embolization. Use a grading scale of 0-3 as previously described5: 0, raising the rat by tail, the animal extending both forelimbs to the floor and exhibiting no other deficits (normal movement); 1, raising the rat by tail, flex the contralateral forelimb; 2, decreased resistance to lateral push (and forelimb flexion) without circling; 3, the same behavior as grade 2 with circling. 
	NOTE: Rats showing Bederson score = 0 (no deficit) at 2 hr after embolization are excluded from further study.</li>
</ol>

<ol>
	<li>Overgaard, K., Sereghy, T., Boysen, G., Pedersen, H., H&#248;yer, S., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+reproducible+cerebral+infarction+using+thrombotic+blood+clot+emboli.">A rat model of reproducible cerebral infarction using thrombotic blood clot emboli.</a> J Cereb Blood Flow Metab. 12 (3), 484-490 (1992).</li><li>Zhang, R. L., Chopp, M., Zhang, Z. G., Jiang, Q., Ewing, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+focal+embolic+cerebral+ischemia.">A rat model of focal embolic cerebral ischemia.</a> Brain Res. 766 (1-2), 83-92 (1997).</li><li>Dinapoli, V. A., Rosen, C. L., Nagamine, T., Crocco, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+MCA+occlusion:+a+precise+embolic+stroke+model.">Selective MCA occlusion: a precise embolic stroke model.</a> J. Neurosci. Methods. 154 (1-2), 233-238 (2006).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> J Vis Exp. 59 (3496), (2012).</li><li>Bederson, J. B., Pitts, L. H., Tsuji, M., Nishimura, M. C., Davis, R. 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T., Bastan, B., Shea, M., Fisher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Doppler+flowmetry+predicts+occlusion+but+not+tPA-mediated+reperfusion+success+after+rat+embolic+stroke.">Laser Doppler flowmetry predicts occlusion but not tPA-mediated reperfusion success after rat embolic stroke.</a> Exp Neurol. 215 (2), 290-297 (2009).</li><li>Zhu, H., 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=Annexin+A2+combined+with+low-dose+tPA+improves+thrombolytic+therapy+in+a+rat+model+of+focal+embolic+stroke.">Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke.</a> J Cereb Blood Flow Metab. 30 (6), 1137-1146 (2010).</li><li>Cheng, T., 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=Activated+protein+C+inhibits+tissue+plasminogen+activator-induced+brain+hemorrhage.">Activated protein C inhibits tissue plasminogen activator-induced brain hemorrhage.</a> Nat Med. 2 (11), 1278-1285 (2006).</li><li>Taniguchi, H., Andreasson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxic-ischemic+encephalopathy+model+of+perinatal+ischemia.">The hypoxic-ischemic encephalopathy model of perinatal ischemia.</a> J Vis Exp. 21 (955), (2008).</li><li>Sicard, K. 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We do not have any potential conflicting interests to disclose.

In this study, we demonstrated a standard method for performing an embolic MCAO stroke model in the rat, in which the origin of the MCA is occluded by a fibrin-rich clot. The major advantage of this model is: the occlusion of the stem of MCA with a fibrin-rich blood clot is similar to thromboembolic stroke in humans, the embolic stroke model is suitable for performing preclinical investigation of fibrinolytic therapy, and that this model can develop a reproducible and predictable infarct volume within the territory suppl...

A correction was made to <a href="/video/51956">Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats</a>. The institution information was updated.
The institution "Louisiana State University Health Science Center" was changed to "Louisiana State University Health Science Center, Shreveport".

A correction was made to <a href="/video/51956">Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats</a>. The institution information was updated.

Erratum: Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Stroke is the third leading cause of death in the United States, but treatment options for acute stroke remain limited. At present, intravenous infusion of recombinant tissue plasminogen activator (tPA) to dissolve the blood clots is the most efficient therapy for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of intracerebral hemorrhage. Therefore, a model of stroke suitable for thrombolytic research is urgently needed.
The middle cerebral artery (MCA) is the artery most often occluded in stroke in humans. Focused on this artery, many animal models of ischemic stroke have been established. At present, two major types of rodent focal ischemia models by occluding MCA have been developed: suture MCAO model and embolic MCAO model. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic human stroke, as up to 80% of human strokes are caused by thrombosis or embolism. However, embolic stroke model using blood clots closely mimics human stroke and is considered suitable for thrombolytic study. This embolic model was first developed in rats by&#160;Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972 and by Dinapoli et al. in 20063. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories.
In this article, we demonstrate a standard method for producing embolic MCAO with homologous blood clots in the adult rat, which can develop a predictable infarction within the MCA territory. The techniques presented in this article should help investigators to overcome technical problems for establishing this model for stroke research.

<table><tbody><tr><td>rh-tPA</td><td>Chemical</td><td>Genentech</td><td></td></tr><tr><td>2,3,5-triphenyltetrazolium chloride</td><td>Chemical</td><td>Sigma</td><td>T8877</td></tr><tr><td>Anesthesia vaporizer</td><td>Equipment</td><td>Soma Technology</td><td>Drager Vapor 19.1&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Rechargeable high speed micro drill</td><td>Equipment</td><td>Fine Science Tools</td><td>18000-17</td></tr><tr><td>Curved scissors</td><td>Equipment</td><td>Fine Science Tools</td><td>14117-14</td></tr><tr><td>Dumont forceps (Medical #7)</td><td>Equipment</td><td>Fine Science Tools</td><td>11270-20</td></tr><tr><td>Dumont forceps (Medical #5)</td><td>Equipment</td><td>Fine Science Tools</td><td>11251-35</td></tr><tr><td>Vannas-style spring scissors</td><td>Equipment</td><td>Fine Science Tools</td><td>15000-03</td></tr><tr><td>Veterinary recovery chamber&amp;nbsp;</td><td>Equipment&amp;nbsp;</td><td>Peco Services&amp;nbsp;</td><td>V1200&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Genie plus syringe pump</td><td>Equipment</td><td>Kent Scientific Corporation</td><td></td></tr><tr><td>Rat brain matrix</td><td>Equipment</td><td>Kent Scientific Corporation</td><td>RBMA-310C</td></tr><tr><td>Digital caliper&amp;nbsp;</td><td>Equipment</td><td>World Precision Instruments</td><td>501601</td></tr><tr><td>Dissecting microscope</td><td>Equipment</td><td>World Precision Instruments</td><td>PZMTIII-BS-LWD</td></tr><tr><td>Hamilton syringe</td><td>Equipment</td><td>Hamilton</td><td>model 710</td></tr><tr><td>Homeothermic blanket control unit</td><td>Equipment</td><td>Harvard Apparatus</td><td></td></tr><tr><td>Electric clipper</td><td>Equipment</td><td>Braintree Scientific</td><td>CLP-9931</td></tr><tr><td>Veterinary electrosurgical unit</td><td>Equipment</td><td>MACAN Manufacturing Company</td><td>MV-9</td></tr><tr><td>Blood flowmeter</td><td>Equipment</td><td>Adinstruments</td><td></td></tr><tr><td>PowerLab 4/30</td><td>Equipment</td><td>Adinstruments</td><td></td></tr><tr><td>LabChart 7.2</td><td>software</td><td>Adinstruments</td><td></td></tr><tr><td>1 ml syringe</td><td>Consumable</td><td>Becton, Dickinson and Company</td><td>309659</td></tr><tr><td>23 G needle</td><td>Consumable</td><td>Becton, Dickinson and Company</td><td>305143</td></tr><tr><td>30 G needle</td><td>Consumable</td><td>Becton, Dickinson and Company</td><td>305106</td></tr><tr><td>PE-50 tubing</td><td>Consumable</td><td>Becton, Dickinson and Company</td><td>427517</td></tr><tr><td>PE-10 tubing</td><td>Consumable</td><td>Becton, Dickinson and Company</td><td>427400</td></tr><tr><td>6-0 Silk suture</td><td>Consumable</td><td>Harvard apparatus</td><td>723287</td></tr><tr><td>5-0 Silk suture</td><td>Consumable</td><td>Harvard Apparatus</td><td>517607</td></tr></tbody></table>

embolic middle cerebral artery occlusion (mcao) for ischemic stroke with homologous blood clots in rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Laser Doppler flowmetry (LDF) was used to monitor rCBF during induction of cerebral ischemia6,7. Many laboratories including our laboratory have been using rCBF to identify animals with successful MCA occlusion, but the thresholds of baseline varied between laboratories which are related to the site of measurement. The probe of the LDF is positioned at 2 mm posterior and 5 mm lateral to the bregma as described previously6. rCBF was monitored at 0, 5, 15, 30, 60, 90, and 120 min after injection of th...

Watch this Scientific Journal Video about Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats at JoVE.com

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

embolic-middle-cerebral-artery-occlusion-mcao-for-ischemic-stroke

Research

JoVE Journal

Medicine

26.9K Views.  Louisiana State University Health Science Center, Shreveport. In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke. 

Video: Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

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

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

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

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

Neuroscience

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

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

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

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

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

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

Immunofluorescence Staining to Study Cell Death in Rat MCAO Stroke Models

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

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

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

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

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

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Chapters in this video

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

Mouse Model of Middle Cerebral Artery Occlusion

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

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

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

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