We thank Beth Rauch and the University of Wisconsin Small Animal Imaging Facility for magnetic resonance imaging. The Department of Neurological Surgery at the University of Wisconsin-Madison provided funding for this work.

MCAO Rat Model
Presurgical Preparations
Aseptic technique should be used for all survival surgical procedures. Disinfect the surgical work surface with commercial disinfectant and prepare sterile surgical packs of instruments, drapes, gauze, swabs, sutures, and scalpel blades by autoclaving. A surgical mask, hair bonnet and sterile gloves should be worn. A Germinator dry bead sterilizer is also used to resterilize surgical instruments between procedures if multiple rat surgeries will be done during one session. Prewarm a water-jacketed homeothermic blanket and place under an absorbent pad in order to prevent hypothermia of the rat during surgery.
<ol>
	<li>Place spontaneously hypertensive rat (or other rat strain of choice) into an induction chamber and induce anesthesia with 5% isoflurane (anesthesia machine should be set to 1.0 L/min O2 and 1.0 L/min N2O). Lower to 1-2% isoflurane to maintain anesthesia.</li>
	<li>Apply Artificial Tears ointment to both eyes.</li>
	<li>Shave the throat and left neck region beyond the prospective incision site using clippers (Oster A5 with #10 blade).</li>
	<li>Apply Betadine to a gauze pad and disinfect the skin starting from the center of the surgical region, spiraling outward. Rinse with sterile gauze pad containing 70% ethanol, moving pad in a similar pattern. Repeat both steps for a total of three cycles.</li>
	<li>Inject 0.2 mL of 0.5% bupivacaine subcutaneously along the prospective incision site.</li>
	<li>Place rat in sterile stockinette or cover with a sterile surgical drape.</li>
</ol>
Transient occlusion of MCA
<ol start="7">
 <li>Under an operating microscope, a ventral midline incision is performed and the superficial fascia is dissected.</li>
 <li>Beneath the superficial fascia, there is glandular tissue to the left and three muscles which form a triangle: the sternohyoid, which lies midline over the trachea; the digastric (easily identified with its shiny white tendinous portion); and lastly, the sternomastoid muscle. </li>
 <li>Careful sharp and blunt dissection is performed within the triangle to identify the carotid artery (exclusive blunt dissection may be preferred by some to minimize accidental tissue damage). The external, internal, and common carotid arteries (ECA, ICA, CCA) are exposed. The carotid artery is large and visibly pulses. The vagus nerve is seen coursing along the lateral aspect of both common and internal carotid arteries and is sharply dissected off the common and internal carotid arteries.</li>
 <li>Two ECA branches are then dissected sharply, the first branch heading medially and the second branch heading laterally. Both branches are cauterized and cut, which allows for greater ease in mobilizing the larger vessels.</li>
 <li>The ECA is now dissected more rostrally. The hyoid bone may be encountered and this will limit the extent of rostral dissection. The external carotid artery is tied off as distally as possible with a 6-0 silk suture. </li>
 <li>Another 6-0 silk suture is placed loosely around the ECA near the bifurcation with the ICA. Make sure not to occlude the vessel as the intraluminal suture to be used for the occlusion will be going through here at a later step.</li>
 <li>Microsurgical clips are placed on common and internal carotid arteries near the bifurcation. </li>
 <li>An initial partial arteriotomy is created between the two silk suture ties on the external carotid artery.</li>
 <li>Introduce a 2.2 -3.0 cm length of 4-0 monofilament nylon suture, with a kink 2.0 cm from its rounded, silicon-coated tip, into the ECA lumen down toward the CCA where the microsurgical clip is located. These sutures are commercially available (Doccol Corp., Redlands, CA, USA). Cut the remaining portion of the ECA (at the site of the partial arteriotomy) to free the stump and position the stump below the bifurcation of the ECA and ICA; this will more easily allow the intraluminal suture to slide into the ICA. </li>
 <li>Tighten the silk suture around the ECA stump to secure the intraluminal nylon suture and prevent bleeding, and then remove the microvascular clip from the ICA. Open the clip slowly before removing it to check for bleeding.</li>
 <li>Continue advancing the nylon suture from the ECA to ICA lumen to the middle cerebral artery (MCA). This length is typically 18-20 mm and is the reason for placing a kink in the suture prior to insertion. After a variable length of nylon suture is inserted, resistance may be felt. If this occurs with much of the nylon suture still present outside of the vessel, it indicates that the suture is likely entering the pterygopalatine artery (see note below). Pull back and curve the suture slightly to continue along the ICA, which will run more medially. Additionally, dissection of the origin of the PPA may be done to better visualize the path of the intraluminal filament. Continue to insert the nylon suture until resistance is felt at and after the 2 cm kinked position. At this point, the intraluminal suture has blocked the origin of the MCA. MCA occlusion can be confirmed by monitoring the reduction in regional cerebral blood flow using a laser Doppler flowmeter (see Materials table for one equipment source). Also note that the origin of the pterygopalatine artery off of the ICA can be directly tied off, if preferred, in order to avoid accidental intubation of this vessel with the intraluminal suture.</li>
 <li>Start the timer and record occlusion start time.</li>
 <li>Remove the microclip from the CCA.</li>
 <li>Close the incision (dermis, panniculus carnosus, subcutaneous tissue layers) quickly with 3-0 silk suture (a simple continuous pattern will facilitate reopening for reperfusion) and carefully place the rat in a recovery cage. Check that the cage floor around the nose and mouth are free of bedding material and monitor recovery from anesthesia.</li>
</ol>
Restoration of MCA blood flow (reperfusion)
<ol start="21">
 <li>Shortly before the occlusion period should end, re-anesthetize the rat, disinfect the incision site with Betadine and 70% ethanol (3 cycles, as before) and reopen the incision by removing the closing sutures.</li>
 <li>Place a microclip on the CCA, as before.</li>
 <li>Withdraw the occluding suture partway from the ICA until the suture end is visible through the ICA. Do NOT fully remove the suture from the ICA/ECA!</li>
 <li>Place a microclip on the ICA above the end of the intraluminal suture.</li>
 <li>Completely remove the occluding suture and tightly tie off the ECA stump. Record end of occlusion (reperfusion start) time.</li>
 <li>Remove the microclip from the ICA.</li>
 <li>Remove the microclip from the CCA.</li>
 <li>Moisten the region with several drops of sterile saline and close the incision layers (dermis, panniculus carnosus, subcutaneous tissue) with 3-0 silk suture using a simple interrupted pattern.</li>
 <li>Administer 0.05 mg/kg buprenorphine (or other appropriate post-operative analgesic following your institutional guidelines) into the peritoneal space.</li>
 <li>Inject 5cc of prewarmed saline intraperitoneally. This will provide hydration during the recovery stage.</li>
 <li>Monitor recovery of rat from anesthesia.</li>
 <li>After the rat has recovered, test for behavioral indication of infarction by holding the rat by the tail and observing whether the rat can turn to both sides. Curling to one side only is expected. Simple scoring scales can be used to record initial post-infarct behavorial functioning (see Discussion).</li>
 <li>Additional injections of buprenorphine (same dosage as above) should be given every 6 to 8 hours for 24 hr of pain relief. Extend analgesia if animal is showing signs of discomfort.</li>
</ol>
Representative Results
<img src="/files/ftp_upload/1978/1978fig1.jpg" alt="Figure 1" /> Figure 1. Occlusion of the middle cerebral artery by endovascular suture. A simplified diagram of the cranial circulatory system of the rat is shown with a silicon-coated intraluminal suture occluding the origin of the MCA. The OA and ST branches off of the left ECA have been ligated and a suture tie around the ECA stump holds the intraluminal suture in place. ACA, anterior cerebral artery; BA, basilar artery; CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; OA, occipital artery; PCA, posterior cerebral artery; PComA, posterior communicating artery; PPA, pterygopalatine artery; ST, superior thyroid artery. Figure adapted from Sasaki et al.4 and Lee3.
<img src="/files/ftp_upload/1978/1978fig2.jpg" alt="Figure 2" /> Figure 2. Representative magnetic resonance imaging of coronal sections of rat brain 24 hours after 1 hour transient MCAO. Brain edema accompanying focal ischemia is visualized in T2-weighted MRI images. The infarct zone appears hyperintense (bright) on T2 images. Similar infarct patterns can be seen with more advanced MRI techniques such as diffusion-weighted imaging and apparent diffusion coefficient mapping5.
<img src="/files/ftp_upload/1978/1978fig3.jpg" alt="Figure 3" /> Figure 3. Representative coronal sections of rat brain stained with TTC 24 hours after 1 hour transient MCAO. TTC staining reveals white (unstained) infarcted regions of cerebral cortex and striatum in the same rat as that shown in Figure 2. Note that the general pattern of focal ischemic lesions is similar to that seen in the MRI T2-weighted images, although the final infarct area determined histologically may be slightly smaller than the T2 lesion.

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	<li>van Gijn, J., Dennis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Issues+and+answers+in+stroke+care.">Issues and answers in stroke care.</a> Lancet. 352, SIII23-27 (1998).</li><li>Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+models+of+focal+cerebral+ischaemia.">New models of focal cerebral ischaemia.</a> British Journal of Clinical Pharmacology. 34, 302-302 (1992).</li><li>Lee, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+cerebral+arteries.">Morphology of cerebral arteries.</a> Pharmacol Ther. 66, 149-173 (1995).</li><li>Sasaki, M., Honmou, O., Kocsis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+middle+cerebral+artery+occlusion+model+and+intravenous+cellular+delivery.">A rat middle cerebral artery occlusion model and intravenous cellular delivery.</a> Methods Mol Biol. 549, 187-195 (2009).</li><li>Br&#229;tane, B. T., Bastan, B., Fisher, M., Bouley, J., Henninger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+lesion+volume+determination+on+diffusion+weighted+images+vs.+apparent+diffusion+coefficient+maps.">Ischemic lesion volume determination on diffusion weighted images vs. apparent diffusion coefficient maps.</a> Brain Res. 1279, 182-188 (2009).</li><li>Chen, S. T., Hsu, C. Y., Hogan, E. L., Maricq, H., Balentine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+focal+ischemic+stroke+in+the+rat:+reproducible+extensive+cortical+infarction.">A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction.</a> Stroke. 17, 738-743 (1986).</li><li>Dogan, A., Ba&#351;kaya, M. K., Rao, V. L., Rao, A. M., 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=Intraluminal+suture+occlusion+of+the+middle+cerebral+artery+in+Spontaneously+Hypertensive+rats.">Intraluminal suture occlusion of the middle cerebral artery in Spontaneously Hypertensive rats.</a> Neurol Res. 20, 265-270 (1998).</li><li>Ogata, J., Fujishima, M., Morotomi, Y., Omae, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+infarction+following+bilateral+carotid+artery+ligation+in+normotensive+and+spontaneously+hypertensive+rats:+a+pathological+study.">Cerebral infarction following bilateral carotid artery ligation in normotensive and spontaneously hypertensive rats: a pathological study.</a> Stroke. 7, 54-60 (1976).</li><li>Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J., Pulsinelli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+brain+ischemia+in+the+rat:+methods+for+reproducible+neocortical+infarction+using+tandem+occlusion+of+the+distal+middle+cerebral+and+ipsilateral+common+carotid+arteries.">Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries.</a> J Cereb Blood Flow Metab. 8, 474-485 (1988).</li><li>Duverger, D., MacKenzie, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantification+of+cerebral+infarction+following+focal+ischemia+in+the+rat:+influence+of+strain,+arterial+pressure,+blood+glucose+concentration,+and+age.">The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age.</a> J Cereb Blood Flow Metab. 8, 449-461 (1988).</li><li>Coyle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+susceptibilities+to+cerebral+infarction+in+spontaneously+hypertensive+(SHR)+and+normotensive+Sprague-Dawley+rats.">Different susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive Sprague-Dawley rats.</a> Stroke. 17, 520-525 (1986).</li><li>Slivka, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension+and+hyperglycemia+in+experimental+stroke.">Hypertension and hyperglycemia in experimental stroke.</a> Brain Res. 562, 66-70 (1991).</li><li>Bouley, J., Fisher, M., Henninger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+coated+vs.+uncoated+suture+middle+cerebral+artery+occlusion+in+the+rat+as+assessed+by+perfusion/diffusion+weighted+imaging.">Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging.</a> Neurosci Lett. 412, 185-190 (2007).</li><li>Schmid-Elsaesser, R., Zausinger, S., Hungerhuber, E., Baethmann, A., Reulen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+reevaluation+of+the+intraluminal+thread+model+of+focal+cerebral+ischemia:+evidence+of+inadvertent+premature+reperfusion+and+subarachnoid+hemorrhage+in+rats+by+laser-Doppler+flowmetry.">A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry.</a> Stroke. 29, 2162-2170 (1998).</li><li>Shimamura, N., Matchett, G., Tsubokawa, T., Ohkuma, H., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+silicon-coated+nylon+suture+to+plain+nylon+suture+in+the+rat+middle+cerebral+artery+occlusion+model.">Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model.</a> J Neurosci Methods. 156, 161-165 (2006).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20, 84-91 (1989).</li><li>Shigeno, T., McCulloch, J., Graham, D. I., Mendelow, A. D., 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=Pure+cortical+ischemia+versus+striatal+ischemia.+Circulatory,+metabolic,+and+neuropathologic+consequences.">Pure cortical ischemia versus striatal ischemia. Circulatory, metabolic, and neuropathologic consequences.</a> Surgical neurology. 24, 47-51 (1985).</li><li>Bederson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion:+evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.</a> Stroke. 17, 472-476 (1986).</li></ol>

The authors have no conflicting interests to disclose. This protocol was approved by the Institutional Animal Care and Use Committee at University of Wisconsin-Madison School of Medicine and Public Health and abides by the National Institutes of Health guidelines for the use of experimental animals.

Rodent models of cerebral ischemia can be classified as global or focal and as reversible or irreversible. We use a reversible focal ischemia model in order to mimic the reperfusion injury that can occur following recanalization in human stroke patients. While the rat shares the advantage of similar cranial circulation to the human, the extensive intracranial collateral circulation due to the Circle of Willis and leptomeningeal anastomoses results in inconsistent infarct volumes6. We use the spontaneously hypertensive rat (SHR) strain because infarct size is greater in SHRs exposed to focal cerebral ischemia compared to normotensive rats7-10. Stroke induction in SHRs is also more consistent and may generate less mortality than in other commonly used strains11. In addition, physiological parameters should be monitored throughout the surgery, because fluctuations can add to the variability in infarct volumes; these parameters include blood pressure, arterial O2 and CO2 levels, hypothermia, and hyperglycemia, which can exacerbate ischemic injury2,12. Hypothermia is of particular concern because it can lessen the degree of neuronal ischemic injury, and therefore, placing the rat on a thermoregulatory heating pad during surgery is essential.
We employ commercially prepared silicon-coated sutures in this procedure in an attempt to increase consistency in the infarct size13-15. Traditionally, intraluminal sutures used to occlude the MCA have been prepared by flame-rounding a 3-0 nylon suture tip or using a hot glue gun to coat the suture tip with a small glue bead16. These procedures produce sutures with varying tip diameters and thus, may require several attempts at occlusion before finding a suture that matches the vessel diameter of a given rat. Additional variations on the MCAO method described in this video include more distal occlusion, sparing the lenticulostriate branches in order to have a pure cortical infarct17, and simultaneous occlusion of the MCA and ipsilateral common carotid artery to reduce collateral blood flow6,9.
The functional effects of stroke can be assessed using a variety of behavioral tests, in which deficits in specific tasks are reflected in a simple scoring scale. For example, Bederson et al.18 developed a neurological score grading rats on the extent of forelimb flexion in an elevated body position, asymmetric resistance to a lateral push, and open field circling behavior. Rats extending both forelimbs to the floor when elevated and exhibiting no other deficits were graded "Normal (0)", whereas rats with contralateral forelimb flexion only were classified with "Moderate deficits (1)". "Severe grade 2" rats displayed decreased resistance on the contralateral side to a lateral force and "Severe grade 3" rats additionally engaged in circling behaviors. Lower grade behavioral deficits were always observed in the next higher grade (e.g., grade 2 and 3 animals also displayed forelimb flexion) and these scores were predictive of differences in infarct size18. Thus, such a simple scoring system allows for a rapid, consistent semi-quantitative assessment of the functional deficits from stroke.
Measurement of the extent of cerebral infarction is commonly accomplished by histological staining for tissue damage using TTC, cresyl violet (Nissl stain) or hematoxylin and eosin. Magnetic resonance imaging techniques, including diffusion-weighted MRI to visualize the infarct at early time points after occlusion, allow for the determination of an infarct volume in each individual rat without the need for harvesting tissue. This is especially valuable in studies aimed at determining the efficacy of a neuroprotective treatment following stroke; imaging the rat after MCAO but prior to treatment administration gives the investigator confidence that all experimental and control animals experienced similar strokes to start, reducing the confounding effects of variability in stroke induction.

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<td> 4-0 Nylon suture</td>
<td>&nbsp;</td>
<td>Doccol Corporation</td>
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<td>2-3 mm silicon coating length, 0.39 mm tip diameter</td>
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<td>Spontaneously hypertensive rats</td>
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<td>Charles River Laboratories</td>
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<td>Gaymar T/Pump thermoregulatory pump and pads</td>
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<td>CellPoint Scientific </td>
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<td>Vasamedics Bpm2Laserflo blood perfusion monitor</td>
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focal cerebral ischemia model by endovascular suture occlusion of the middle cerebral artery in the rat

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

Watch this Scientific Journal Video about Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat at JoVE.com

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

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

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

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79.2K Views.  University of Wisconsin-Madison. Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

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

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

Middle Cerebral Artery Occlusion (MCAO) to Induce Stroke in a Murine Model

To begin, lay an eight to 10 weeks anesthetized C57 black six male mouse on the surgical bench. Using blunt forceps, pull out the tongue and hold it with fingers. Insert a laryngoscope into the mouth and visualize the vocal cord.Stabilize the mouse chin on the laryngoscope and hold the 20 gauge intravenous, or IV catheter with a guide wire inserted in it. Then slightly insert the guide wire into the vocal cord and slowly push the 20 gauge IV catheter into the trachea until its wing part is even with the nose tip. Once the mouse is connected to the ventilator, keep it in a lateral position with the right temporal area facing up.Maintain the rectal temperature at 37 degrees Celsius using a heating pad and a heat lamp. After locating the zygomatic arch and incising the temporal muscle, use two forceps to dissect the underlying zygomatic arch and expose the joint between the maxilla and zygomatic bones. Then using scissors, cut a three millimeter portion of the zygomatic arch and remove it.Finally, separate the masseter muscle from the skull base. Position four small retractors in different directions and expose the cranial skull base with one retractor pulling the trigeminal nerve branches laterally. Apply a saline drop to the skull above the middle cerebral artery, or MCA trunk and proximal to the rhinal cortex branch.Use an electrical grinder to thin the skull until a small fracture is visible. And with the tip of the forceps, remove the thin skull. Place a single-strand loop of black braided silk on top of the MCA.Then insert an 8-O microsurgical needle to lift the MCA trunk and tie the suture under the needle, leaving the needle's two ends on top of the silk thread loop knot. For transient middle cerebral artery occlusion, or MCAO, slightly tighten the silk thread knot under the needle to block arterial blood flow, representing MCAO onset. Use the forceps to hold the suture and slowly remove the needle at the end of the ischemia.The laser speckle contrast imaging confirmed the reduced blood supply in the right MCA. For transient MCAO after suture removal, the cerebral blood flow reperfusion was evident, and further improved after 24 hours. TTC staining of the brain after 24 hours of stroke demonstrated the generation of infarcted tissue in both the cortical and lateral striatum areas in young and aged mice.The infarct size was moderate compared to filament MCAO.

Surgical Procedure for Transient Middle Cerebral Artery Occlusion

To begin, position the anesthetized mouse on the operating table. Maintain body temperature using a rectal probe connected to a heating pad. Make an incision on the skin of the head in the direction of the sagittal suture, starting from the ears to the area between the eyes.Retract the skin and remove the periosteum on the right side of the skull. Then find the coordinates, 2.5 millimeters lateral from bregma. Apply cyanoacrylate to the doppler holder and attach it for laser-doppler flowmetry.After the glue has dried, connect the doppler probe. Then, check for the correct signal readout for assessing the cerebral blood flow. To perform artery occlusion, turn the mouse over to the supine position and fix it to the surgical table with medical tape.Then make a midline incision on the neck. Pull the skin laterally, followed by pulling away the salivary glands using retractors. This exposes the carotid territory.Identify the vascular anatomy of the common carotid artery, or CCA, the ICA, and the ECA, as well as the different arteries derived from them. For easier handling, detach CCA, followed by ECA from the connective tissue. Then cauterize the superior thyroid artery and detach ICA from the connective tissue.Check again to make sure the main arteries are fully detached from the connective tissue. Next, wrap 6-0 silk sutures around the ECA at the maxillary lingual bifurcation. Then suture around the CCA.Place a vascular clip interrupting blood circulation from the ICA. Make a small incision in the ECA close to the area where the tight knot is located. Insert the monofilament until the thick coating has completely entered the arterial lumen.Tighten the second knot to hold the monofilament inside the artery and to prevent the pressure exerted by the blood from pushing it out. Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA. Advance the monofilament via the ICA until the point where the MCA branches out.The occlusion is reflected in an abrupt blood flow drop in the LDF readout. After 40 minutes, anesthetize the mouse again and place it back on the surgical table. Remove the stitches from the neck.After 45 minutes of occlusion, loosen the knot holding the monofilament in place. Pull slowly and gently on the filament and check that tissue recanalization occurs. Then pull out the filament and tighten the knot to prevent blood loss, clean the blood and untie the CCA knot.Ensure that there is no arterial wall damage. Remove the retractors and reposition the muscles, glands, and skin. Suture the skin using a 6-0 suture and apply disinfectant.Disconnect the Doppler probe and detach the holder. Finally, suture and disinfect the skin of the head. The outcome of the transient middle cerebral artery occlusion procedure was evaluated using in-vivo MRI neuroimaging.Lesion evolution assessed at different time points after reperfusion showed that progression of the lesion volume takes approximately two days to complete. Behavioral tests, such as grip strength test, showed a loss of strength in mice after 24 hours of transient middle cerebral artery occlusion. Also, upon stimulation in the corner test, the mice showed a preference to turn to the right side, which was ipsilateral to the lesion, indicating a successful middle cerebral artery occlusion on the right side.

Periorbital Laser Doppler Flowmetry for MCAO-Induced Ischemic Stroke in Rodents

Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion is not guaranteed by even the most skilled surgical hands. Errors primarily occur when the filament is not placed at the correct depth and include instances of either no infarction or vessel perforation, which can cause death. Laser Doppler flowmetry (LDF) is a reliable technique that provides real-time feedback on regional cerebral blood flow (CBF) during the MCAO procedure. Here we demonstrate a rapid technique for periorbital placement of a laser Doppler probe for measurement of CBF in both mice and rats. Our rationale was to simplify LDF implementation, encouraging widespread usage for improved surgical reliability. The technique eliminates the need for skull thinning and specialized equipment, with placement at the periorbital region rather than dorsal placement, promoting efficiency and ease of adoption. The protocol described here encompasses presurgical preparations, periorbital Doppler probe placement, and post-operative care. Representative results include visual depictions of procedural elements along with representative LDF tracings illustrating successful MCAO surgeries, with instances of unsuccessful filament placement leading to complications. The protocol illustrates LDF in confirming proper filament placement and offers a simplified procedure compared to alternative methods.

Author Spotlight: Advancing Stroke Research with Periorbital Doppler Monitoring of Regional Cerebral Blood Flow in Rodent Models

A minimally invasive surgical procedure is shown here, which involves placing the laser Doppler probe onto the skull over the distal region of the middle cerebral artery (MCA), a periorbital location suitable for rats and mice, to assess blood flow during transient MCA occlusion.

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion ...