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. L., Bartkowski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion:+evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.</a> Stroke. 17 (3), 472-476 (1986).</li><li>Henninger, N., Bouley, J., Br&#229;tane, B. 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. 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=Animal+models+of+focal+brain+ischemia.">Animal models of focal brain ischemia.</a> Exp Transl Stroke Med. 1 (7), (2009).</li><li>Florian, B., 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=Long-term+hypothermia+reduces+infarct+volume+in+aged+rats+after+focal+ischemia.">Long-term hypothermia reduces infarct volume in aged rats after focal ischemia.</a> Neurosci Lett. 438 (2), 180-185 (2008).</li><li>Noor, R., Wang, C. 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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.

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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...

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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 ...

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26.8K 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

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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 ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

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 ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.