This work was supported by the American Heart Association Predoctoral Fellowship 788808-41910, the NIH-NINDS NS-066001 and NS-055832, and The Center for Hearing Research NIH Training Grant 1T32DC010775-01.

1. Getting Started: Required Surgical Instruments
See Figure 4
<ol>
	<li>Dental drill (Kavo Dental Equipment, Model: UMXL-TM), 2-bit drill, and 3-bit drill</li>
	<li>Two ~30 gauge hypodermic needles</li>
	<li>Serrated tweezers, curved tip optional (can be helpful but not essential)</li>
	<li>Two fine tip tweezers</li>
	<li>wire cutters</li>
	<li>Suture thread</li>
	<li>Micro scissors</li>
</ol>
2. Creating the Surgical Window
<ol>
	<li>Anesthesia: P...

<ol>
	<li>Caplan, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Caplan's Stroke, A Clinical Approach. , (2009).</li><li>Blumenfeld, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy Through Clinical Cases. , (2002).</li><li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2011+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2011 Update: A Report From the American Heart Association.</a> Circulation. , (2011).</li><li>Dirnagl, U., Iadecola, C., Moskowitz, M. 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Blood Flow Metab. 1, 53-60 (1981).</li><li>Dittmar, M., Spruss, T., Schuierer, G., Horn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+carotid+artery+territory+ischemia+impairs+outcome+in+the+endovascular+filament+model+of+middle+cerebral+artery+occlusion+in+rats.">External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats.</a> Stroke. 34, 2252-2257 (2003).</li><li>Bederson, J. B., Germano, I. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+without+craniectomy+in+rats.+Which+method+works+best?.">Middle cerebral artery occlusion without craniectomy in rats. Which method works best?.</a> Stroke. 24, 294-297 (1993).</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>Schmid-Elsaesser, R., Zausinger, S., Hungerhuber, E., Baethmann, A., Reulen, H. 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The authors have nothing to disclose at this time.

This protocol was developed in order to induce ischemia within the rodent cortex, and to do so with minimal peripheral impact to experimental subjects. The double occlusion and transection method allows for visual confirmation that the vessel has been permanently occluded, and may be performed without excessive invasion or tissue damage, and with a high survival rate. This occlusion protocol may be applied to any cortical vessel that can be accessed via craniotomy in order to induce ischemia within a specific cortical do...

In order to induce ischemic conditions that effectively mimic human ischemic stroke, several animal stroke models are widely employed, with varying volumes of infarct resulting. In the photothrombotic model, the brain is irradiated through the intact skull using laser illumination after intravenous injection of a photosensitive substance (such as rose-bengal), resulting in photochemical coagulation, blockage of the irradiated vessels, and ischemia within the surrounding tissue 6,7. Photothrombosis can result in very small, isolated regions of infarct and is typically used as a means of modeling "mini-strokes", or "micro-strokes".
The more widely adopted technique for inducing ischemic stroke, particularly in middle cerebral artery (MCA), is the intraluminal monofilament model 8, in which a filament is surgically introduced into the external carotid artery and advanced until the tip occludes the base of MCA. A primary challenge of intraluminal filament occlusion is the high mortality rate (70% when MCA is occluded for 3 hr, a relevant time point for stroke research) 9. Other issues with the method included possible subarachnoid hemorrhage, incomplete occlusion, and variable infarct volume 10,11. This model results in an extensive degree of infarct both in the cortex and subcortically 12, and models a massive human stroke.
Although both micro and massive stroke models are important, human strokes are typically somewhere in between. In large clinical studies, stroke infarct ranges in size from 28-80 cm3, which translates to 4.5-14% of the ipsi-ishemic hemisphere 9. In comparison, our rat pMCAO infarct size ranges from approximately 9-35 mm3, which constitutes 3 to 12% of the ipsi-ishemic hemisphere. Our pMCAO model, therefore, closely resembles human ischemic stroke infarct volumes by percentage of brain volume.
In addition to modeling the structural damage of stroke, pMCAO results in functional and behavioral deficits similar to the human condition. At minimum, an effective model of stroke results in movement deficits contralateral to stroke damage 13-15, loss or disruption of motor and sensory function 16,17, loss or disruption of evoked neuronal activity 16,18, reductions in cerebral blood flow 19,20, and infarct 21,22. Accordingly, our pMCAO models a serious occlusion of MCA resulting in physical disability, loss of function within the sensory cortex (and neighboring cortices), disruption of neuronal activity, a severe reduction in MCA blood flow, and infarct- hallmark attributes of ischemic stroke 23-25, therefore serving as an effective model of human stroke.
Procedurally, pMCAO involves a small craniotomy in which we carefully remove the skull and dura from a 2 x 2 mm "surgical window" over the initial (M1) segment of MCA, just prior to the primary bifurcation of MCA into the anterior and posterior cortical branches (Figures 1A and 1B). We pass a half-curve reverse cutting suture needle and thread (6-0 silk) through the pial layer of the meninges, below MCA and above the cortical surface (see Table of specific reagents and equipment for the surgical supplies necessary to carry out pMCAO). We then tie a double ligature, tighten the two knots around MCA, and transect the vessel between the two knots. The double ligature and transection through M1 occurs just distal to the lenticulostriate branching, such that only the cortical branches of MCA are affected- thus only cortical infarct (no subcortical damage) occurs 26,27 (Figure 2). Although human stroke often involves subcortical infarct, modeling this in rodents requires increased invasiveness (occluding cerebral vessels prior to cortical branching requires accessing arteries via the carotid artery in the neck and necessitates additional occlusions) in technique and increased variability in infarct size. The model described here cannot be performed more proximally as access to earlier branches of MCA is not possible via a simple craniotomy. While it may be surgically possible to induce a subcortical infarct via pMCAO, occlusion would entail an extremely invasive procedure and is therefore not ideal.
Effectiveness of occlusion may be confirmed via laser Doppler, or laser speckle imaging 12,24,25 (Figure 3), or histologically post-mortem (Figure 2). It should be noted that previous research has shown that sensory stimulation can play a major role in the evolution and outcome of infarct; conferring protection from damage when administered within 2 hr of pMCAO and causing an increase in stroke damage when administered at 3 hr post pMCAO 24,25,28. We have confirmed that at 5 hr post-pMCAO, stimulation no longer has an effect on outcome (unpublished data). Therefore, sensory stimulation of subjects should be minimized for 5 hr following pMCAO to obtain infarct volumes with minimal variability. Accordingly, our group runs "untreated controls" of this type by keeping rats anesthetized for 5 hr post-pMCAO, in the dark, with minimal sensory stimulation, and expressly no whisker stimulation.
It should be further noted that occasional variation in MCA structure, including excessive branching, multiple primary segments, or the absence of communicating arteries can occur at a frequency of 10 to 30% in male adult Sprague Dawley rats 29,30. If abnormalities in MCA are observed, it is advisable not to use that particular subject as adding animals with such vascular abnormalities will increase infarct variability.
Additionally, there are several practical aspects of our procedure that make this occlusion method advantageous for stroke investigation. First, sutures may be placed around the artery but not tightened in order to collect a baseline assessment, followed by post-ischemic assessment after ligature and transection. In this manner, surgical preparation necessary for the occlusion is effectively controlled for, within subjects. Because subjects may remain stationary or within a stereotaxic frame throughout occlusion, it is possible to conduct experimental assessment of each subject prior to, during, and after occlusion without moving the subject or disturbing any experimental equipment in use 25,28. Furthermore, this procedure results in a very low mortality rate, even within aged rodent subjects 21-24 months of age (equivalent to an elderly human) 31, and may therefore be used to evaluate stroke treatments in rats that more closely model the most common age bracket of stroke sufferers 25,28. Vessel transection also serves several practical purposes. The absence of bleeding after transection confirms that the vessel was completely occluded at both ligature sites. Additionally, transection ensures a permanent disruption of blood flow. Finally, transection ensures that any blood flow detected in the distal portions of the occluded vessel must come from an alternate source.
Finally, although we specifically describe this occlusion technique for MCA in this manuscript and video, the same double ligature transection technique may be applied to any cerebral vessel that can be accessed via craniotomy. Our laboratory, for example, utilized pMCAO in conjunction with several additional permanent occlusions of distal MCA branches in order to block both primary, and collateral blood flow 24 in a manner to similar to techniques designed to selectively induce ischemia within the primary somatosensory cortex 32.
In conclusion, this method for permanent occlusion as applied to MCA closely models three primary facets of human ischemic stroke: the most common location (MCA), type (ischemia), and degree of damage (infarct) associated with the human clinical literature of stroke. Furthermore, this method of occlusion may be applied to single or multiple occlusion sites throughout the brain, and may be conducted in aged subjects with a high rate of survival. Given the dynamic, permanent, and relatively noninvasive nature of this occlusion, this technique represents an additional tool for preclinical researchers evaluating novel approaches for the protection from and treatment of stroke.

<table border="1">
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Extra Fine Graefe Forceps - 0.5 mm Tips Slight Curve (1)</td>
 <td>Fine Science Tools </td>
 <td>11151-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Ceramic Coated Dumont #5 Forceps (2)</td>
 <td>Fine Science Tools </td>
 <td>11252-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Extra Fine Bonn Scissors, straight (1)</td>
 <td>Fine Science Tools </td>
 <td>14084-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Round 3/8 (16 mm) Suture Needles</td>
 <td>Fine Science Tools</td>
 <td>12050-02</td>
 <td></td>
 </tr>
 <tr>
 <td>6-0 Braided Silk Suture</td>
 <td>Fine Science Tools</td>
 <td>NC9071061 
 Harvard Apparatus 
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 <td>30 gauge needle, &frac12;" length</td>
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 No.:ZT-5-030-5-L/COL</td>
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permanent cerebral vessel occlusion via double ligature and transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Successful occlusion of a vessel can be confirmed using laser speckle imaging (LSI) among other blood flow imaging techniques. Blood flow in the major cortical branches of MCA should drop to ~25% of baseline or less following occlusion depending on the level of noise in the recording system and sensitivity of the technique. See Figure 3 for a representative LSI image of a segment of a cortical branch of MCA before and after MCA occlusion. When the described occlusion technique is applied...

Watch this Scientific Journal Video about Permanent Cerebral Vessel Occlusion via Double Ligature and Transection at JoVE.com

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

permanent-cerebral-vessel-occlusion-via-double-ligature

Research

JoVE Journal

Medicine

14.7K Görüntüleme.  University of California, Irvine. Biz bir kemirgen büyük bir beyin damar daimi tıkanıklığı için son derece tekrarlanabilir yöntem açıklanmaktadır. Bu teknik çok az çevresel zarar, en az kan kaybı, uzun süreli sağkalım oranının yüksek ve insan klinik nüfusu ile orantılı tutarlı infarkt hacmi ile gerçekleştirilebilir.