The surgical technique was originally acquired in the lab of Dr. William D. Hill at the Medical College of Georgia. The authors would also like to thank Dr. David A. Rempe and Landa Prifti for the use of the dissection camera. This research was supported by NIH NS041744, NS051279, F31 NS064700 and AHA 30815697D.

This protocol was approved by the University of Rochester committee devoted to the ethical use of animals in research (UCAR). Aseptic techniques should be followed during the protocol. The use of sterile gloves and a mask is required.
All the equipment, materials, chemicals, and tools that are used during the protocol are described in Table 1. 
1. Pre-surgical Preparation
<ol>
<li>Inject mice subcutaneously with buprenorphine (0.05mg/kg) 2 h before surgery, immediately after the surgery and then every 3-5 h during the first 24 h post-operative period.</li> 
<li>Sterilize all the surgical instruments, gauze sponges, and cotton tip applicators by autoclave. Keep surgical tools in 70% ethanol during the surgery and air dry on sterile gauze right before use. Spray the work area with 70% ethanol. </li>
<li>Anesthetize the mouse with a 3% isofluorane-20% oxygen gas mixture by using an anesthetic vaporizer. Adjust the oxygen amount with the flow meter. Test the level of anesthesia by toe or tail pinching (they should be unresponsive). Maintain the level of anesthesia with 2% (v/v) isofluorane.</li>
<li>Apply artificial tears to the mouse&#x2019;s eyes and use caution to avoid damaging the eye during the surgical procedure. Place the mouse on its right side in a lateral position.</li>
<li>Shave the area on the left side between the left eye and the base of the left ear using the animal clippers. Cleanse the area by alternating between a betadine solution and 70% ethanol using cotton tip applicators and lightly rubbing the area. Repeat as necessary.</li> 
<li>Place the mouse on to a heating pad connected to a rectal probe to maintain body temperature at 37&deg;C. Insert the rectal probe by using mineral oil. </li>
<li>Position the mouse on its right side under the microscope and secure with tape. Cut a window in a gauze sponge and cover surgical area.</li>
</ol>
2. Surgical Procedure and MCA Ligation
<ol>
<li>Make a vertical incision between the left eye and the base of the left ear by using fine straight scissors. Use curved hemostats to keep the surgical area open.</li>
<li>Make a horizontal incision on the temporal muscle by using spring scissors and slightly separate the temporal muscle from the skull by slowly pulling with forceps. </li>
<li>Make a small subtemporal craniotomy at the junction of zygomatic arch and squamosal bone by using an 18G needle while holding the jawbone with curved forceps.</li>
<li>Expose the MCA by removing the small pieces of skull with bone rongeurs. The zygomatic arch and orbital contents should not be damaged during this process. If excessive drying of tissue occurs during this process, apply sterile PBS with cotton tip applicators. </li>
<li>Ligate the distal portion of the MCA by using a small vessel cauterizer. </li>
<li> Place the temporal muscle back into its original position and close the incision site with surgical 5-0 nylon sutures. </li>
<li> Discontinue anesthesia and remove the rectal probe. Inject the mouse with a 2nd dose of buprenorphine (0.05mg/kg) subcutaneously. Return the mouse to the cage which was kept at 37&deg;C with a heating panel.</li> 
<li>Closely monitor the mouse for the next 24 h for any discomfort (decreased appetite/water consumption, hunched posture, increased respiration, pilo-erected hair). Inject the mouse with buprenorphine subcutaneously every 3-5 h post-surgery up to 24h. Provide the mouse with recovery gel during this period. </li>
</ol>
3. TTC Staining and Determination of Stroke Volume
<ol>
<li>Twenty-four hours after the surgery deeply anesthetize the mouse, transcardially perfuse the mouse with a 4% TTC (w/v) in phosphate buffered saline (PBS) for 15 min and then with a 4% paraformaldehyde solution for 10 min by using a mini peristaltic pump at medium flow rate. Remove the brain and place it into the 4% paraformaldehyde solution overnight. </li>
<li> Position the brain into the sectioning block. Slice the brain into 1mm thick slices by using razor blades. </li>
<li> Place the slices next to a millimeter scale ruler. Photograph the slices by using a digital camera connected to the dissecting microscope.</li>
<li> Calculate the stroke area by subtracting the non-infarcted area of the ipsilateral site from the total area of the contralateral site using Image J Software (http://rsbweb.nih.gov/ij/). Calculate the stroke volume by stacking the stroke area of the slices 22.</li> 
</ol>
4. Directions to use Image J software 
<ol>
<li> Open the image file to be analyzed in Image J software by clicking on the file menu. </li>
<li> Click on the &#x2018;straight line&#x2019; tab in the program. Draw a straight line between the two margins on the ruler. Click on &#x2018;analyze&#x2019; menu and select &#x2018;set scale&#x2019;. Set the known distance as 1, unit of length as mm.</li>
<li> Click on &#x2018;freehand circle&#x2019; tab. Draw a circle to outline the contralateral hemisphere. Click on &#x2018;analyze&#x2019; menu and select &#x2018;measure&#x2019;. The calculation window will pop up showing the area of the circle drawn. </li>
<li> Draw another circle around the ipsilateral hemisphere excluding stroke (white) area. Measure the area as indicated in step 4.3. The new value will be added to the calculation window. The difference between the 1st and 2nd values represents the area of the infarct which is indicated as A in the formula below. </li>
<li> Calculate the stroke area in each slice by repeating steps 4.2-4.4. Take summation of the stroke area calculated in each slice (&sum;An). This represents stroke volume knowing the fact that the thickness of each slice is 1mm. 
&sum;An x 1mm (thickness of each slice) = Stroke volume</li>
</ol>
5. Representative Results: 
The infarcts obtained by permanent MCA ligation in the mouse are mostly cortical. However, it is possible to obtain subcortical lesions if the MCA is ligated proximal to the lenticulostriate branch. The stroke volumes after MCA ligation might vary from 10mm&sup3; to 35mm&sup3; 19, 23. The stroke volumes calculated with TTC staining in Moyanova et al. 19 are between 10mm&sup3; to 22mm&sup3; while the stroke volumes determined from MRI images in Filiano et al 23 are between 20mm&sup3; to 35mm&sup3;. The possible reason for these differences maybe the exact location of the ligation and the different methods used to measure the stroke volumes. 
In Figure 1, a TTC stained brain 24 hr post permanent MCA ligation in a wild type C57/BL6 mouse is shown. The stroke site is white in appearance (Fig. 1A, B). Figure 1A and B illustrate the infarct site from different angles. Figure 2 shows 1mm thick slices of the TTC stained stroke brain from anterior to posterior (Fig. 2A-G). Infarct volume was calculated 24 h post-surgery. The volume of the infarct is &tilde;23mm&sup3;. 

<img src="/files/ftp_upload/3039/3039fig1.jpg" alt="Figure 1"/> Figure 1. Representation of stroke site. A-B, TTC stained whole brain images, 24 h after MCA ligation. White area represents the infarct. 
<img src="/files/ftp_upload/3039/3039fig2.jpg" alt="Figure 2"/> Figure 2. Representative results. (A&gt;G), TTC stained 1mm brain sections, 24 h after MCAL. Slices are aligned from anterior to posterior. White area represents the infarct.

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No conflicts of interest declared.

The permanent MCA ligation method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available. The ease and the short duration (&tilde;30 min) of the procedure make it even more practical. The method is widely used in both mice and rats.
This technique requires an invasive surgery under a stereomicroscope. Therefore, experience in operating under a microscope and perfecting a successful craniotomy is essential. It is best to establi...

<table border="1">
 	<tbody>
 	<tr>
 	<th>NAME</th>
 <th>COMPANY AND CATALOGUE NUMBER</th>
 </tr>
 <tr>
 	<td>A. Solutions and Chemicals</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Povidone-Iodine Solution, Surgical Scrub </td>
 <td>Aplicare, 82-209</td>
 </tr>
 <tr>
 	<td>70% Ethanol</td>
 <td>Koptec, V1001</td>
 </tr>
 <tr>
 	<td>2,3,5 Triphenyltetrazolium chloride (TTC)</td>
 <td>Sigma, T8877</td>
 </tr>
 <tr>
 	<td>4% Paraformaldehyde </td>
 <td>Electron Microscopy Sciences, 159SP</td>
 </tr>
 <tr>
 	<td>Phosphate Buffered Saline, pH:7.4</td>
 <td>Made and sterilized in the lab.</td>
 </tr>
 <tr>
 	<td>Mineral Oil </td>
 <td>PML Microbiologicals, R6570</td>
 </tr>
 <tr>
 	<td>Isofluorane</td>
 <td>The Butler Company, 029405</td>
 </tr>
 <tr>
 	<td>Buprenorphine (0.3mg/ml)</td>
 <td>Hospira, NDC 0409-2012-32</td>
 </tr>
 <tr>
 	<td>Artificial tears</td>
 <td>The Butler Company, 007312</td>
 </tr>
 <tr>
 	<td>Recovery Gel</td>
 <td>Clear H2O, 72-01-5022</td>
 </tr>
 <tr>
 	<td>B. Surgical Materials and Equipment</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>C57/BL6 mouse, 10-12 weeks old</td>
 <td>The colony was bred in the university vivarium</td>
 </tr>
 <tr>
 	<td>100% oxygen tank</td>
 <td>Airgas</td>
 </tr>
 <tr>
 	<td>Anesthetic Vaporizer and Flow meter</td>
 <td>Surgivet, Model 100</td>
 </tr>
 <tr>
 	<td>Heating Pad with Rectal Probe</td>
 <td>Fine Science Tools, TR200</td>
 </tr>
 <tr>
 	<td>Dissecting microscope</td>
 <td>Zeiss, Stemi 2000</td>
 </tr>
 <tr>
 	<td>Heating panel</td>
 <td>Petco Services Ltd. 0307-013</td>
 </tr>
 <tr>
 	<td>Small Vessel Cauterizer Set</td>
 <td>Fine Science Tools, 18000-00</td>
 </tr>
 <tr>
 	<td>Mini Peristaltic Pump</td>
 <td>Harvard Apparatus, MPII</td>
 </tr>
 <tr>
 	<td>1ml syringe</td>
 <td>BD, 329650</td>
 </tr>
 <tr>
 	<td>18G needle</td>
 <td>Becton Dickinson, 305196</td>
 </tr>
 <tr>
 	<td>Surgical 5.0 Nylon Suture</td>
 <td>Ethilon, 698H</td>
 </tr>
 <tr>
 	<td>Sectioning Block</td>
 <td>Kent Scientific, 1mm</td>
 </tr>
 <tr>
 	<td>Single Edge Razor Blades</td>
 <td>Electron Microscopy Sciences, 71962</td>
 </tr>
 <tr>
 	<td>Label Tape</td>
 <td>Scienceware, Bel-art Products F13463-2075</td>
 </tr>
 <tr>
 	<td>Gauze Sponges</td>
 <td>Kendall Curity 2252, Johnson and Johnson 6415</td>
 </tr>
 <tr>
 	<td>Cotton Tip Applicators</td>
 <td>Puritan 806-WCL</td>
 </tr>
 <tr>
 	<td>C. Surgery Tools</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Surgical Spring Scissors</td>
 <td>Fine Science Tools 15003-08</td>
 </tr>
 <tr>
 	<td>Microdissecting forceps angled</td>
 <td>Fine Science Tools 5/45</td>
 </tr>
 <tr>
 	<td>Microdissecting forceps curved</td>
 <td>Fine Science Tools 5 Ti</td>
 </tr>
 <tr>
 	<td>Fine straight scissors</td>
 <td>Fine Science Tools 14568-12</td>
 </tr>
 <tr>
 	<td>Curved bone rongeur</td>
 <td>Fine Science Tools 16021-14</td>
 </tr>
 <tr>
 	<td>Hartman Hemostat Curved</td>
 <td>Fine Science Tools 13003-10</td>
 </tr>
 </tbody>
</table>

Table 1. The list of materials, tools and chemicals used for the MCA ligation procedure. A. Solutions and Chemicals, B. Surgical Materials and Equipment, C. Surgery Tools.

the application of permanent middle cerebral artery ligation in the mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

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The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

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19.5K vues.  University of Rochester. L'artère cérébrale moyenne (MCA) ligature est une technique pour étudier l'ischémie cérébrale focale chez des modèles animaux. Dans cette méthode, l'artère cérébrale moyenne est exposée par craniotomie et ligaturée par cautérisation. Cette méthode donne les volumes d'infarctus hautement reproductible et une augmentation des taux de survie post-opératoires par rapport aux autres méthodes disponibles.

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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 ...

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 ...

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 ...

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 ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

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, ...

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

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

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

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

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 ...