This work was supported by grants from the American Heart Association Greater Southeast Affiliate (09GRNT2060421), the American Medical Association, and from the University of Florida Clinical and Translational Science Institute. Adam Mecca is a NIH/NINDS, NRSA predoctoral fellow (F30 NS-060335). Robert Regenhardt received predoctoral fellowship support from the University of Florida Multidisciplinary Training Program in Hypertension (T32 HL-083810).

This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and is in compliance with the "Guide for the Care and Use of Laboratory Animals" (eighth edition, National Academy of Sciences, 2011).
Materials
<ol>
 <li>Animals: Eight-week-old, male, Sprague Dawley rats (Charles River Farms, Wilmington, MA, USA) weighing 250-300 g at the time of surgery. </li>
 <li>Anesthesia
 <ol class="lalpha">
 <li>Inhalation anesthesia system (VetEquip Inc., Pleasanton, CA, USA) </li>
 <li>Isoflurane anesthetic (Baxter Pharmaceutics, Deerfield, IL, USA) </li>
 </ol>
 </li>
 <li>Stereotaxic system (David Kopf Instruments, Tujunga, CA, USA)
 <ol class="lalpha">
 <li>Small animal stereotaxic system </li>
 <li>Non-rupture ear bars for rats </li>
 <li>Gas anesthesia head holder for rats </li>
 </ol>
 </li>
 <li>Temperature regulation
 <ol class="lalpha">
 <li>BAT-12 microprobe thermometer (World Precision Instruments, Inc., Sarasota, FL, USA) </li>
 <li>T/PUMP, TP600 Thermal blanket (Gaymar Industries, Inc., Orchard Park, NY, USA) </li>
 </ol>
 </li>
 <li>Surgical instruments
 <ol class="lalpha">
 <li>Metzenbaum scissors, iris forceps, bulldog clamp retractors, 10 &mu;l syringe with 26 gauge beveled needle, Bovie cautery kit (World Precision Instruments, Inc., Sarasota, FL, USA) </li>
 <li>Micromotor drill (Stoelting, Wood Dale, IL, USA) </li>
 <li>0.8 mm round drill bur (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA) </li>
 <li>STORZ Bonn Suturing Forceps (Bausch and Lomb, Inc., Rochester, NY, USA) </li>
 </ol>
 </li>
 <li>Surgical Supplies
 <ol class="lalpha">
 <li>3.0 nylon suture (Oasis, Mettawa, IL, USA)</li>
 <li>Cotton swabs, Puralube eye ointment (Fisher Scientific, Pittsburg, PA, USA)</li>
 <li>Surgical points and strips (Medtronic Xomed, Inc., Jacksonville, FL, USA)</li>
 <li>Electric hair clippers (Oster, Providence, RI, USA) </li>
 </ol>
 </li>
 <li>Chemicals
 <ol class="lalpha">
 <li>Endothelin-1 (American Peptide, Sunnyvale, CA, USA) </li>
 <li>Chlorhexidine 2% (Agrilabs, St. Joseph, MO, USA) </li>
 </ol>
 </li>
 <li>Visualization Equipment
 <ol class="lalpha">
 <li>Surgical microscope (Seiler Instrument and Manufacturing; St. Louis, MO, USA) </li>
 <li>Sony Handycam HDR-SR12 (Sony, Minato, Tokyo, Japan) </li>
 <li>Fiber Optic illuminator (TechniQuip Corp., Livermore, CA, USA)</li>
 </ol>
 </li>
 <li>Measurement of vessel diameter
 <ol class="lalpha">
 <li>VLC media player (Paris, France) </li>
 <li>Image J software (ImageJ 1.42q software, U.S. National Institutes of Health, Bethesda, MA, USA) </li>
 </ol>
 </li>
</ol>
1. Pre-surgical Steps
<ol>
 <li>Prior to surgery, the rats are housed under a 12:12 light/dark cycle with free access to water and rodent chow. </li>
 <li>Anesthesia is induced with 4% isoflurane in 100% O2 gas mixture in an induction chamber. </li>
 <li>The crown of the head is shaved with electric hair clippers. </li>
 <li>The rat is placed in a prone position on an absorbent pad lying on a temperature-controlled operating surface (thermal blanket) and the head is placed in the stereotactic apparatus starting with placement of the gas anesthetic face mask.</li>
 <li>Next, the ear bars are inserted and tightened.</li>
 <li>During the procedure anesthesia is maintained with 2% isoflurane in 100% O2 gas mixture.</li>
 <li>Opthalmic lubricant ointment is applied to both eyes, and the eyelids are closed to prevent eye desiccation during the surgical procedure. </li>
 <li>A rectal temperature probe is inserted to maintain a constant animal core temperature of 37&plusmn;0.5 &deg;C. </li>
 <li>With the head of the anesthetized rat held firmly in the stereotaxic device the surgical area is cleansed with alternating chlorhexidine and saline three times. </li>
</ol>
2. Pre-cranial Window Preparation
Prior to creating a cranial window, the rat should be prepared for any experiments by implantation of other required hardware and should receive any required surgical procedures. For this protocol, we have previously implanted a guide cannula for endothelin-1 (ET-1) induced MCAO as shown in a companion publication titled "Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat."
 
3. Creation of the Cranial Window
After placement of a guide cannula or equipment required for experimentation, a cranial window is created to directly visualize proximal portions of the middle cerebral artery during a stroke procedure. 
<ol>
 <li>First, scissors are used to incise the skin overlying the temporalis muscle starting medially and working laterally. </li>
 <li>The temporalis muscle is bisected using electrocautery and then retracted using 3.0 nylon suture to visualize the squamous portion of the temporal bone. </li>
 <li>An approximately 3-4 mm square is drawn on the squamous portion of the temporal bone caudal to the orbit and superior to the base of the zygomatic process as it reflects off of the temporal bone. </li>
 <li>A drill is used to gradually cut the outlined piece of bone free from the temporal bone. Care should be taken to avoid applying too much pressure to the drill since it is possible to damage the dura or cerebral cortex.</li>
 <li>Frequent rinses with sterile saline are performed to improve visualization of the surgical field and prevent overheating of the skull.</li>
 <li>Starting at a loose corner, the piece of temoporal bone is carefully removed using fine rat-toothed forceps while making sure not to tear any vessels associated with the dura. </li>
 <li>The dura is left intact and debris is rinsed away using sterile saline. </li>
</ol>
4. Recording of Cerebral Artery Constriction
To demonstrate how to capture images in real time, a rat that is undergoing ET-1 induced MCAO is used for this protocol.
<ol>
 <li>Prior to application of vasoactive compounds, a baseline video should be recorded for at least 1 min. For ET-1 induced MCAO, the baseline recording is made once the needle has been lowered into the brain parenchyma but prior to the ET-1 injection. </li>
 <li>The syringe pump is started for injection and recorded for 1 hr or until the desired endpoint. The needle is left in place during video recording in order to prevent disturbances of the focal plane. </li>
 <li>The rat should be deeply anesthetized and euthanized according to an approved protocol following this procedure. </li>
</ol>
5. Image Analysis
Vessel diameter can be determined for any part of the visualized MCA. As an example, we will use one branch of the MCA to measure vessel diameter at time-points before and after ET-1 injection. Still frames from the video are captured at 1 min intervals using VLC media player (VideoLAN).
<ol>
 <li>VLC player is installed and opened. </li>
 <li>By selecting tools and then preferences, the setting is changed to "All" under "Show settings." </li>
 <li>The "video menu" is expanded in the left side bar and "output modules" is then expanded. "Scene filters" is selected to bring up the scene video filter menu. </li>
 <li>"Scene" is typed into the box for filename prefix.</li>
 <li>A directory path prefix is defined. This is where still frames are saved.</li>
 <li>A recording ratio is selected based on the desired rate of still frame capture. If a video was captured at 29 frames per sec, then "1749" (29 frames/sec x 60 sec/min) should be entered in the box to save one still frame every 1 min. All changes are then saved.</li>
 <li>The desired video is then opened in VLC player to automatically save still frames once every 1 min. </li>
 <li>Vessel diameter is then measured using these images. First, ImageJ software (NIH) is opened. </li>
 <li>Then the still frames that you will measure are opened in ImageJ. </li>
 <li>By selecting the "analyze" menu, "set measurements" is then opened and all boxes are unchecked. </li>
 <li>Next, the straight line tool is selected.</li>
 <li>The keyboard shortcut "Ctrl +" is used to zoom in as needed and a line is placed perpendicular to the vessel path to measure vessel diameter. </li>
 <li>Finally, vessel length is obtained by selecting the "analyze" menu and then "measure" (ctrl+M) to obtain vessel length. </li>
 <li>This process is repeated at least three times and averaged for each measured vessel measured in each still frame. </li>
 <li>Vessel diameter at each time point is normalized to the baseline vessel diameter so that comparisons can be made using multiple rats. To do this, use the formula current diameter/baseline diameter x 100% to calculate the % baseline diameter of each vessel. </li>
</ol>

<ol>
	<li>Levasseur, J. E., Wei, E. P., Raper, A. J., Kontos, A. A., Patterson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+description+of+a+cranial+window+technique+for+acute+and+chronic+experiments.">Detailed description of a cranial window technique for acute and chronic experiments.</a> Stroke. 6, 308-317 (1975).</li><li>Baumbach, G. L., Dobrin, P. B., Hart, M. N., Heistad, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+cerebral+arterioles+in+hypertensive+rats.">Mechanics of cerebral arterioles in hypertensive rats.</a> Circ. Res. 62, 56-64 (1988).</li><li>Regrigny, O., 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=Effects+of+melatonin+on+rat+pial+arteriolar+diameter+in+vivo.">Effects of melatonin on rat pial arteriolar diameter in vivo.</a> Br. J. Pharmacol. 127, 1666-1670 (1999).</li><li>Mecca, A. P., O'Connor, T. E., Katovich, M. J., Sumners, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candesartan+pretreatment+is+cerebroprotective+in+a+rat+model+of+endothelin-1-induced+middle+cerebral+artery+occlusion.">Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion.</a> Exp. Physiol. 94, 937-946 (2009).</li><li>Mecca, A. P., 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=Cerebroprotection+by+angiotensin-(1-7)+in+endothelin-1-induced+ischaemic+stroke.">Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke.</a> Exp. Physiol. 96, 1084-1096 (2011).</li></ol>

No conflicts of interest declared.

In summary, this cranial window preparation technique is very versatile as it can be altered to meet the needs of many experiments with minor modifications4, 5. For example, we have successfully monitored cerebral blood flow in specific MCA branches using laser doppler flowmetry to focus directly on a cerebral artery visualized through a cranial window (Mecca AP 2009 and 2011). In addition, a similar preparation with the dura incised can be used with topical administration of vasoactive compounds to create an in vivo vascular reactivity bath3. Several factors should be taken into consideration when preparing a cranial window in order to decrease the failure rate for this technique. Many of these factors are related to obtaining good visualization of cerebral arteries. First, care must be taken when creating the craniectomy so that the dura or blood vessels overlying it are not disrupted with the drill bit. This is best accomplished by frequent washes with sterile saline to clear debris and cool the skull. Second, the bone fragment should be lifted gently when it is removed. If the fragment does not pull away easily, then the drill bit should be used to cut away more bone. Lastly, small amounts of blood or CSF can easily alter the appearance of the cranial window during this procedure. The craniectomy performed provides an opening in the skull that is larger than required for visualization. Therefore, it is easy to place several absorbent sponges in the dependent portion of the surgical site to prevent fluid from accumulating. These sponges can be changed as needed if care is used not to obstruct the window with surgical tools.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Inhalation anesthesia system</td>
 <td> VetEquip Inc., Pleasanton, CA, USA</td>
 <td>901806</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane anesthetic</td>
 <td>Baxter Pharmaceutics, Deerfield, IL, USA</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Small animal stereotaxic system </td>
 <td>David Kopf Instruments, Tujunga, CA, USA</td>
 <td>900</td>
 <td></td>
 </tr>
 <tr>
 <td>Non-rupture ear bars, rat</td>
 <td>David Kopf Instruments, Tujunga, CA, USA</td>
 <td>957</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat gas anesthesia head holder </td>
 <td>David Kopf Instruments, Tujunga, CA, USA</td>
 <td>1929</td>
 <td></td>
 </tr>
 <tr>
 <td>BAT-12 microprobe thermometer</td>
 <td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
 <td>BAT-12</td>
 <td></td>
 </tr>
 <tr>
 <td>T/PUMP, Thermal blanket</td>
 <td>Gaymar Industries, Inc., Orchard Park, NY, USA</td>
 <td>T/PUMP, TP600</td>
 <td></td>
 </tr>
 <tr>
 <td>Metzenbaum Scissors</td>
 <td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
 <td>501254</td>
 <td></td>
 </tr>
 <tr>
 <td>Iris forceps</td>
 <td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
 <td>15915</td>
 <td></td>
 </tr>
 <tr>
 <td>Bulldog clamp retractors</td>
 <td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
 <td>14119-G</td>
 <td></td>
 </tr>
 <tr>
 <td>10 &mu;l syringe 26-gaugue</td>
 <td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
 <td>SGE010RNS</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovie, high temperature cautery kit</td>
 <td>World Precision Instruments, Inc., Sarasota, FL, USA</td>
 <td>500392</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat tooth forceps 0.12</td>
 <td>Stotz</td>
 <td>E1811</td>
 <td></td>
 </tr>
 <tr>
 <td>Micromotor drill</td>
 <td>Stoelting, Wood Dale, IL, USA</td>
 <td>51449</td>
 <td></td>
 </tr>
 <tr>
 <td>0.8 mm round drill bur</td>
 <td>Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA</td>
 <td>RS-6280C-1</td>
 <td></td>
 </tr>
 <tr>
 <td>STORZ Bonn suturing forceps</td>
 <td>Bausch and Lomb, Inc., Rochester, NY, USA</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon Suture, size 3.0</td>
 <td>Oasis, Mettawa, IL, USA</td>
 <td>MV-663</td>
 <td></td>
 </tr>
 <tr>
 <td>Cotton swabs</td>
 <td>Fisher Scientific, Pittsburg, PA, USA</td>
 <td>22-029-488</td>
 <td></td>
 </tr>
 <tr>
 <td>Puralube eye ointment</td>
 <td>Fisher Scientific, Pittsburg, PA, USA</td>
 <td>NC0138063</td>
 <td></td>
 </tr>
 <tr>
 <td>Electric hair clippers </td>
 <td>Oster, Providence, RI, USA</td>
 <td>78005-301</td>
 <td></td>
 </tr>
 <tr>
 <td>ET-1 diluted to 80 &mu;M concentration in PBS </td>
 <td>American Peptide, Sunnyvale, CA, USA</td>
 <td>88-1-10A</td>
 <td></td>
 </tr>
 <tr>
 <td>Chlorhexidine, 2% </td>
 <td>Agrilabs, St. Joseph, MO, USA</td>
 <td>1040, Rev. 6-06, NAC No.: 10580322</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical microscope </td>
 <td>Seiler Instrument and Manufacturing, St. Louis, MO, USA</td>
 <td>Evolution xR6</td>
 <td></td>
 </tr>
 <tr>
 <td>Sony Handycam</td>
 <td>Sony, Minato, Tokyo, Japan</td>
 <td>HDR-SR12</td>
 <td></td>
 </tr>
 <tr>
 <td>Fiber optic illuminator </td>
 <td>TechniQuip Corp., Livermore, CA, USA</td>
 <td>FO1&#x2013;150</td>
 <td></td>
 </tr>
 <tr>
 <td>VLC media Player</td>
 <td>(Paris, France)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Image J software</td>
 <td>U.S. National Institutes of Health, Bethesda, MA, USA</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Still images taken from the captured video show that the change in cerebral artery diameter after ET-1 injection can be readily appreciated using this cranial window technique (Figure 2). Within minutes of ET-1 injection, the vessel will begin to constrict. Eventually the vessels will be difficult to visualize and the brain tissue will become pale. After about 20 min the effects of ET-1 will diminish and the vessels will begin to dilate, gradually returning to baseline diameter after about 45 min. In addition to the more obvious vasoconstriction that occurs, the cortical surface becomes paler after ET-1 administration. It is possible to calculate the absolute change in vessel diameter with a calibrated microscope reticle if desired. For comparison between multiple rats we calculate the relative change in vessel diameter that occurs during a procedure. These measurements are performed using ImageJ software (NIH). Then a graph representing the relative changes in vessel diameter over time can then be constructed (Figure 3). 
<img src="/files/ftp_upload/50015/50015fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50015/50015fig1highres.jpg"/> 
Figure 1. Diagram of the location of temporal craniectomy. This diagram depicts the skeletal anatomy of the rat skull with anterior oriented to the left. The temporalis muscle has its origin along the lateral skull ridge. This muscle must be detached from this ridge and bisected in order to visualize the squamous portion of the temporal bone. An approximately 3-4 mm craniectomy can be performed at this location just posterior to the orbit and superior to the base of the zygomatic process as it reflects off of the temporal bone. The large arrow indicated the location to perform the craniectomy. The 3 small arrows indicate the MCA and its branches. All arteries in this location will be branches of the MCA and arteries can be distinguished from veins by both their non-tortuous appearance and sensitivity to vasoactive compounds. 
<img src="/files/ftp_upload/50015/50015fig2.jpg" alt="Figure 2" /> 
Figure 2. Cranial window before ET-1 injection, after ET-1 injection, and after reperfusion. Starting at the left, a representative image of MCA branches as viewed through a cranial window is shown. Arteries can be identified by their morphology. The relatively straight MCA enters the field at the lower left and has one major branch point in this image. Other vessels in these pictures are cerebral veins which can be identified by their deeper tone and tortuous appearance. During occlusion arteries will rapidly constrict and the tissue will become pale. Slowly, the artery will dilate and return to baseline diameter. 
<img src="/files/ftp_upload/50015/50015fig3.jpg" alt="Figure 3" /> 
Figure 3. Representative vessel diameter over time for a single rat. Percent baseline diameter can be calculated over time using the simple formula, current diameter/baseline diameter x 100%. This can be done with any vasoactive compound.

Watch this Scientific Journal Video about Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion at JoVE.com

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

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 ...

utilizing-cranial-window-to-visualize-middle-cerebral-artery-during

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14.9K Views.  University of Florida. 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.

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

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 ...

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.

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 ...

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 ...

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 ...

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.

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.

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

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.