Name: mouse model of intraluminal mcao: cerebral infarct evaluation by cresyl violet staining
Uploaded: 2012-11-06T12:00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining at JoVE.com

This study was supported by a CIHR TEAM grant # CTP 82946, a CIHR grant # MOP 102741, as well as a Canada chair # 216684 and a Strauss Foundation. E.R. was supported by a fellowship from the Canadian Heart &amp; Stroke Foundation.

Transient Middle Cerebral Artery Occlusion (MCAO)
 1. Surgery Procedure (Figure 1)
 Transient middle cerebral artery occlusion (tMCAO) is performed on 2- to 3-month old male C57Bl/6 mice (22-28g). This protocol was approved by the IRCM bioethics committee animal care. Surgical tools were sterilized by autoclaving (121 &deg;C at 15 psi for 60 min). Between each animal, they were sterilized using the hot bead sterilizer (15 sec). Surgery table and other equipment are sanitized using 70% ethanol. 
 Two hours before surgery, mice were analgesized with buprenorphine (0.03 mg/kg b.w. i.p.). 
 Figure 1A. 
 <ol>
 <li>Deeply anaesthetize mice with isoflurane 5% and then maintain the anesthesia at 2.5% isoflurane. Body temperature of the mice is maintained constant during surgery with a heating pad.</li>
 <li>Disinfect the fur and skin with 70% ethanol or Betadine. Since shaving produces hair fragment release, microabrasions and inflammation that may impact on stroke pathophysiology, we do not shave mouse fur.</li>
 <li>Make a midline neck incision and gently pull apart the soft tissues.</li>
 <li>Under a stereo microscope (Stemi 2000, Zeiss), a blunt dissection is performed to expose the trachea and retract the muscles to locate the carotid artery.</li>
 <li>The left common carotid artery (CCA) is carefully dissected from surrounding tissue and the CCA is temporarily occluded by a temporary suture (1) using 5-0 silk suture cut into 20 mm segments. Great care should be exercised not to harm the vagal nerve.</li>
 <li>Separate the bifurcation of the left internal common carotid artery (ICA) and external common carotid artery (ECA). A permanent suture (2) is placed around the ECA, as distally as possible, and another temporary suture slightly tight (3) is placed on the ECA distal to the bifurcation.</li>
 <li>Clip the left ICA (4) using a reverse-action tweezers to avoid bleeding. Great care should be exercised not to harm the vagal nerve.</li>
 <li>Cut a small hole into ECA (5) between permanent (2) and temporary (3) sutures.</li>
 </ol>
 Figure 1B. 
 <ol start="9">
 <li>Introduce a 12 mm-long 6-0 silicon-coated (about 9-10mm is coated silicon) monofilament suture (Doccol Corporation) into the ECA (6), completely cut the ECA distal to the permanent suture and invert the occluder into the ICA. The suture is tightly tied around the monofilament to prevent bleeding and the reverse-action tweezers are removed. </li>
 <li>The occluder is introduced to occlude the origin of the MCA in the circle of Willis. Stop its insertion around 9-10 mm beyond the bifurcation of ECA and CCA. The occluder is blocked and cannot move anymore. Care must be exercised not to penetrate the pterygopalatine artery. The suture (3) on the ECA is tightly tied to fix the monofilament in position.</li>
 <li>Close the skin with an autoclip wound closing system.</li>
 <li>Inject 1 ml of saline solution subcutaneously and place mice under an infrared heating lamp during all the post-occlusion period (60 min).</li>
 </ol>
 2. Restoration of Middle Cerebral Artery Blood Flow
 Before reanesthesia, the neuroscore can be checked to evaluate the success of the surgery.
 <ol>
 <li>Anesthetize the mice as previously described and remove the autoclips.</li>
 <li>Slightly open the suture (3) onto the ECA to allow monofilament withdrawal and blood reperfusion.</li>
 <li>Permanently tie off the temporary suture on the ECA to prevent blood loss. The monofilament is kept for reuse.</li>
 <li>Remove the temporary suture (1) onto the CCA to allow blood recirculation.</li>
 <li>Close the wound. The mice should receive another 1 ml saline solution subcutaneously.</li>
 <li>Place the mice under the infrared heating lamp for 1 hr. After checking that the animal regains mobility, the mouse is returned to its cage and left under the heating pad and towels for 72 hr. Note that only half of the cage is placed on the heating pad to allow mice to choose their environment. Because post-surgical weight loss is generally observed, mashed food is placed in a Petri dish to encourage eating. 
 Twelve hours post-surgery, mice received another dose of buprenorphine (0.03 mg/kg b.w. i.p).</li>
</ol>
 3. Sham Operation
 For sham operations, all procedures are identical except that the occluder is not inserted.
 4. Neuroscore
 Neurological deficits allow the evaluation of the success of tMCAO just after reperfusion and later the estimation of the degree of severity of the injury. Neurological deficits are scored as previously described 5 and performed at 1, 24, 48 and 72 hr post-reperfusion. An expanded six-point scale is used:
 <ul>
 <li>0: normal.</li>
 <li>1: mild circling behavior with or without inconsistent rotation when picked up by the tail, &lt;50% attempts to rotate to the contralateral side.</li>
 <li>2: mild consistent circling, &gt;50% attempts to rotate to the contralateral side.</li>
 <li>3: consistent strong and immediate circling, the mouse holds a rotation position for more than 1-2 sec, with its nose almost reaching its tail.</li>
 <li>4: severe rotation progressing into barreling, loss of walking or righting reflex.</li>
 <li>5: comatose or moribund.</li>
 </ul>
 5. Cresyl Violet Staining (Figure 2)
 Following PBS solution perfusion, brains are quickly frozen in isopentane and stored at -80 &deg;C. Mouse brains may be also perfused with 4% paraformaldehyde (PFA) depending on the planned immunohistochemistry studies. Cryostat-cuts of coronal brain sections (17 &mu;m) are performed. One section out of every 30 is collected on the same slide to have a representative cerebral injury. For volume quantification, 2 slides are stained by cresyl violet staining and an image analysis system (Scion Corporation, Frederick, MD) was used to evaluate the lesion. The injury volume was calculated in arbitrary units (pixels), and expressed as a percentage of the contralateral non-lesioned area for each section. Other slides are kept at -80 &deg;C for immunohistochemistry studies. Alternatively, PFA-fixed brain can be cut at 30-50 &mu;m using vibratome and the infarct volume measured as recommended by Han et al. (2009)6.

<ol>
	<li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema,+I:+a+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Jpn. J. Stroke. 8, 1-8 (1986).</li><li>Chan, P. H., Kamii, H., Yang, G., Gafni, J., Epstein, C. J., Carlson, E., Reola, L. B. r. a. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=infarction+is+not+reduced+in+SOD-1+transgenic+mice+after+a+permanent+focal+cerebral+ischemia.">infarction is not reduced in SOD-1 transgenic mice after a permanent focal cerebral ischemia.</a> Neuroreport. 5, 293-296 (1993).</li><li>Yang, G., Chan, P. H., Chen, J., Carlson, E., Chen, S. F., Weinstein, P., Epstein, C. J., Kamii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+copper-zinc+superoxide+dismutase+transgenic+mice+are+highly+resistant+to+reperfusion+injury+after+focal+cerebral+ischemia.">Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia.</a> Stroke. 25, 165-170 (1994).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+stroke+model+guidelines+for+preclinical+stroke+trials+(1st+edition.">Rodent stroke model guidelines for preclinical stroke trials (1st edition.</a> J. Exp. Stroke Transl. Med. 2, 2-27 (2009).</li><li>Jiang, S. X., Lertvorachon, J., Hou, S. T., Konishi, Y., Webster, J., Mealing, G., Brunette, E., Tauskela, J., Preston, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlortetracycline+and+demeclocycline+inhibit+calpains+and+protect+mouse+neurons+against+glutamate+toxicity+and+cerebral+ischemia.">Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia.</a> J. Biol. Chem. 280, 33811-33888 (2005).</li><li>Han, R. Q., Ouyang, Y. B., Xu, L., Agrawal, R., Patterson, A. J., Giffard, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postischemic+brain+injury+is+attenuated+in+mice+lacking+the+beta2-adrenergic+receptor.">Postischemic brain injury is attenuated in mice lacking the beta2-adrenergic receptor.</a> Anesth. Analg. 108, 280-287 (2009).</li><li>Rousselet, E., Marcinkiewicz, J., Kriz, J., Zhou, A., Hatten, M. E., Prat, A., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCSK9+reduces+the+protein+levels+of+the+LDL+receptor+in+mouse+brain+during+development+and+after+ischemic+stroke.">PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke.</a> J. Lipid Res. 52, 1383-1391 (2011).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J. Cereb. Blood Flow Metab. 1, 53-60 (1986).</li><li>Chen, Y., Ito, A., Takai, K., Saito, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+pterygopalatine+arterial+blood+flow+decreases+infarct+volume+variability+in+a+mouse+model+of+intraluminal+suture+middle+cerebral+artery+occlusion.">Blocking pterygopalatine arterial blood flow decreases infarct volume variability in a mouse model of intraluminal suture middle cerebral artery occlusion.</a> J. Neurosci. Methods. 174, 18-24 (2008).</li><li>Tsuchiya, D., Hong, S., Kayama, T., Panter, S. S., Weinstein, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+suture+size+and+carotid+clip+application+upon+blood+flow+and+infarct+volume+after+permanent+and+temporary+middle+cerebral+artery+occlusion+in+mice.">Effect of suture size and carotid clip application upon blood flow and infarct volume after permanent and temporary middle cerebral artery occlusion in mice.</a> Brain Res. 970, 131-192 (2003).</li><li>Tureyen, K., Vemuganti, R., Sailor, K. A., Dempsey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infarct+volume+quantification+in+mouse+focal+cerebral+ischemia:+a+comparison+of+triphenyltetrazolium+chloride+and+cresyl+violet+staining+techniques.">Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques.</a> J. Neurosci. Methods. 139, 203-207 (2004).</li></ol>

Production and Free Access to this article is sponsored by Zeiss, Inc.

Different stroke models have been developed to mimic stroke consequences in patients. The choice of the stroke model depends on the biological question. The intraluminal MCAO model mimics one of the most common types of ischemic stroke in patients and is less invasive and more consistent than the Tamura model 4,7. It is a really interesting model for neuroprotection, neurorepair and cell death analyses. The success of the intraluminal model depends on many factors such as animal sex, age and weight, temperature, anesthesia and the time of surgery, which have to be controlled. The physical proprieties of the occluder (tip diameter, length, shape and fexibility) are critical for the consistency of the MCAO 4. Herein, we use the 12 mm-long 6-0 monofilament coated with silicon on 9-10 mm from Doccol Corporation. The great advantage of this monofilament is to reduce subarachnoid hemorrhages and because its length covered all the ICA length, residual blood flow, which comes from the anterior and posterior communicating arteries of the Circle of Willis and PPA, is prevented and the variability in the infarct volume decreases (15% of variability in our hands) 8. The suture of CCA also decreases the variability of the infarct volume 9.
	The extent of brain injury following tMCAO can be assessed by different ways. Neurological deficits can be measured as previously mentioned. However, it is difficult to have an efficient measure by this way. The extent of the infarct is commonly performed by TTC staining. More recently, magnetic resonance techniques are also used, a particularly interesting technology for neuroprotective treatments. To understand the underlying cellular mechanisms involved in stroke such as neurorepair, cell death or cell proliferation, the study of different markers by immunohistochemistry is critical and very informative. Thus, our protocol of brain section preparations has the great advantage of allowing these analyses, and minimizes the number of mice used. Moreover, Tureyen et al. 10 have reported that there is good correlation between cresyl violet staining and TTC.

<table border="1">
 <tr>
 <td>Description</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Stereo microscope </td>
 <td>Zeiss </td>
 <td>Stemi 2000</td>
 </tr>
 <tr>
 <td>Fiber-Light High Intensity Illuminator </td>
 <td>Dolan-Jenner Industries Inc. </td>
 <td></td>
 </tr>
 <tr>
 <td>Anesthesia system for isoflurane</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>CDMV</td>
 <td>108737</td>
 </tr>
 <tr>
 <td>Heating blanket</td>
 <td>Gaymar Medsearch</td>
 <td>TP22G</td>
 </tr>
 <tr>
 <td>Infrared heating lamp</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ultra fine tweezers, style 5</td>
 <td>Electron Microscopy Sciences</td>
 <td>78320-5TI</td>
 </tr>
 <tr>
 <td>Vannas Scissors 3 mm Cutting Edge, Straight</td>
 <td>Electron Microscopy Sciences</td>
 <td>72932-01</td>
 </tr>
 <tr>
 <td>Style LA-1 and MPF-1 All smooth blades</td>
 <td>Electron Microscopy Sciences</td>
 <td>77926-5S</td>
 </tr>
 <tr>
 <td>Negative action tweezers, style KOPINC 5</td>
 <td>Electron Microscopy Sciences</td>
 <td>72850-F</td>
 </tr>
 <tr>
 <td>Dissecting scissors 5 1/2" Straight</td>
 <td>Cedarlane</td>
 <td>72960</td>
 </tr>
 <tr>
 <td>Autoclip starter set</td>
 <td>Harvard apparatus </td>
 <td>34-0557</td>
 </tr>
 <tr>
 <td>BD Autoclip Wound Closing Syst., 9mm long,100/pk</td>
 <td>Fisher</td>
 <td>01-804-5</td>
 </tr>
 <tr>
 <td>5-0 suture</td>
 <td>Harvard apparatus</td>
 <td>517763</td>
 </tr>
 <tr>
 <td>Suture material PDS II monofilament violet, 5-0, RB-1; Z303H</td>
 <td>CDMV</td>
 <td>6167</td>
 </tr>
 <tr>
 <td>12 mm-long 6-0 silicon-coated monofilament suture</td>
 <td>Doccol Corporation</td>
 <td>60SPRePK5</td>
 </tr>
 <tr>
 <td>Seringes 1ml </td>
 <td>Fisher</td>
 <td>14-823-2F</td>
 </tr>
 <tr>
 <td>Needles 26G1/2</td>
 <td>Fisher</td>
 <td>BD-305111</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>IRCM</td>
 <td></td>
 </tr>
 </table>

mouse model of intraluminal mcao: cerebral infarct evaluation by cresyl violet staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The neuroscore evaluation confirms the success of tMCAO post-stroke and determines the efficiency of the tMCAO post-reperfusion. In our hands, all mice subjected to the intraluminal procedure presented at least a mild consistent curling (neuroscore 2) and the neurological deficits are generally stable up to 72 hr. Most mice exhibit a mild consistent curling (neuroscore 2). Mice showing absence of curling are excluded from the study. However, the neuroscore evaluation does not allow us to distinguish between mice with an injured hippocampus and those with an intact hippocampus.
	No mortality was observed during the surgery day, suggesting that subarachnoid hemorrhages did not occur. When subarachnoid hemorrhage is identified in a mouse, this one is systematically excluded from analysis. The use of silicon-coated monofilament from Doccol Corporation, which is smoother than home-prepared monofilaments, increases the success of tMCAO and reduces subarachnoid hemorrhages. Mortality between 24 hr and 72 hr is 14% in this model, as generally reported for 60 min of occlusion. The mortality observed is probably due to a large infarct volume in this mouse strain. A strong lesion reproducibility has also been observed (standard deviation is 15%), which is very interesting to study neuroprotection molecules where their effect(s) could be hidden by the variability of the model.
	To evaluate the extent of brain injury following tMCAO, we opted to use cresyl violet staining (Figure 2) rather than TTC in order to have a lot of materials to test relevant markers6. The extent of the lesion is relatively consistent. However, we noticed that in some mice, the hippocampus was injured (around 30% of mice). It is interesting to note that cresyl violet staining can be applied up to 1 week later. In the literature, the percentage of brain infarct varies from one study to the other. It depends on the choice of mouse strain, the anesthesia, the thickness of brain sections, the monofilament used, or the staining used 6, 7.
	<img src="/files/ftp_upload/4038/4038fig1.jpg" alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/4038/4038fig1highres.jpg" /> 
Figure 1. Scheme of the occlusion of the middle cerebral artery using silicon-coated intraluminal monofilament. A. Simplified scheme of mouse brain and cerebral arteries showing successive sutures and clip to prepare the introduction of silicon-coated monofilament. B. The position of monofilament through the circle of Willis is represented. The monofilament is introduced into ICA via ECA to occlude the base of the MCA. ACA, anterior cerebral artery; BA, basilar artery; CCA, common carotid artery; C. Willis, Circle of Willis; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; PCA, posterior communicating artery; PPA, pterygopalatine artery.
	<img src="/files/ftp_upload/4038/4038fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative coronal sections of mouse brain stained with cresyl violet after 72 hr post-reperfusion. Infarct area (mainly striatum, cortex and adjacent brain areas) appears in white (unstained by cresyl violet). The injury volume (white part, right hemisphere) was delimited and expressed as a percentage of the contralateral non-lesioned area (left hemisphere).

Watch this Scientific Journal Video about Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining at JoVE.com

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

mouse-model-intraluminal-mcao-cerebral-infarct-evaluation-cresyl

Research

JoVE Journal

Medicine

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 ...

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 ...

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 ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...