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: Procedures are in compliance with NIH guidelines and have been approved by UC Irvine Animal Care and Use Committee. Experimental subjects are 295-400 g male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA) and the following anesthesia procedure should be used:
	<ol>
		<li>Inject rat intraperitoneally with a sodium pentobarbital bolus (55 mg/kg b.w.) followed by an intramuscular injection of atropine (0.05 mg/kg, b.w.) in the hind leg, and administered 3.0 cc of 5% dextrose in water subcutaneously.</li>
		<li>Supplement sodium pentobarbital (27.5 mg/kg, b.w.) injections as necessary. Administer an ophthalmic antibiotic ointment to the eyes to protect the corneas during the following procedures. Administer 5% dextrose (3 ml) and atropine (0.05 mg/kg, b.w.) every six hours decrease respiratory secretions during anesthesia. Measure body temperature via a rectal probe, and maintain body temperature at 37 &deg;C by a self-regulating thermal blanket.</li>
	</ol>
	</li>
	<li>Locate the MCA by either:
	<ol class="lalpha">
		<li>Thinning a 2 x 2 mm imaging/visualizing window over the somatosensory cortex using a size HP 3 drill bit until the skull is nearly transparent and then thinning to full transparency using a size HP 2 drill bit. MCA's location can then be seen through this window and its proximal trajectory used to approximate the location of the initial segment. MCA will generally run diagonally across this window in a rostral to caudal/ventral to dorsal direction (for example, left to right/bottom to top when viewing left hemisphere from surgeon's point of view). The surgical window can then be created above where the observer estimates the M1 segment (proximal to cortical branching) to be located based on the distal branches visible through the first window. In order to minimize the amount of skull that is removed in order to gain access to MCA, the imaging/visualizing window should be positioned close to, but separate from the surgical window. 
		Or</li>
		<li>A small surgical window should be positioned approximately 3 mm anterior and 1 mm lateral to the foramen ovale or the mandibular nerve, close to the arch rostrum 30,33,34. In order to effectively access the stem of the MCA (also known as the M1 segment), the temporalis muscle is temporarily reflected away from the skull surface. (Note: In the case of long-term survival surgeries, our lab's experience has been that by allowing the temporalis muscle to remain attached at its anchor, the muscle will re-anneal to the skull surface, allowing for healthy eating behavior and effective maintenance of body weight.</li>
	</ol>
	</li>
	<li>Follow MCA to the rostral, ventral corner of the imaging window (if using this as a reference) in order to estimate where its initial cortical branch lies.</li>
	<li>Create a new thin-skull region (we refer to this as the surgical window) slightly rostral and ventral to the imaging window (if using this as a reference) where the M1 segment (pre-cortical branching) of MCA should be. IMPORTANT NOTE: Leave approximately a 2 mm gap between the imaging window (if using this as a reference) and the surgical window.</li>
	<li>Locate the stem of the MCA (also known as the M1 segment) just before cortical branching of the artery as shown in Figures 1A and 1B.</li>
	<li>Using a size HP-3 drill bit, thin the skull above the estimated M1 segment location. When the skull becomes somewhat transparent, switch to the more delicate size HP-2 drill bit and thin the skull until it is completely transparent. Confirm visually as surgical window area becomes thin enough to view vasculature, and assess the location of M1 at this point and complete the window such that there is 2-3 mm on either side of the length of the M1 segment (this allows room for insertion and exit of the suture needle on either side of MCA).</li>
</ol>
IMPORTANT NOTE: Stop thinning when the thickness of the skull is similar to that of plastic wrap. The vessel will rupture if the drill breaks through the skull and dura. If the skull is not thin enough on the other hand, removing it for the occlusion will be difficult and could result in damage to the cortex or artery.
<ol start="7">
	<li>Take a 30 gauge (30 G) hypodermic needle and bend the tip of the needle, using serrated tweezers.</li>
	<li>Use the 30 G needle to puncture the skull carefully in an area not directly above an artery. Use this puncture hole to allow tweezers to grasp skull and carefully remove the thinned area of the surgical window.</li>
	<li>Take a new 30 G need, bend its tip as in Step 6, and carefully remove the dura.</li>
</ol>
NOTE: Cutting the dura will cause it to peel back and MCA will become more prominent, as a result of reduced pressure.
3. Occluding the MCA
<ol>
	<li>Use wire cutters to trim a half-curve reverse cutting suture needle (round 3/8, 16 mm suture needle) down to about 3-5 mm.</li>
	<li>Thread the trimmed suture needle as shown in the picture in Figure 4E. IMPORTANT NOTE: It is important that the needle is threaded so both ends of the suture thread are of equivalent length. This enables the pulling of both thread ends under M1 at the same time, the needle can then be cut free leaving two lengths of thread to tie the two knots around MCA.</li>
	<li>Use the serrated the tweezers slip the suture needle under M1. Insert with about 0.5-1 mm distance from MCA, staying as shallow as possible so as to minimize damage to the cortex but avoiding too much strain on MCA as well.</li>
	<li>When the suture needle comes out the other side such that it is under MCA, use a fine tip tweezer (as shown below) to pull the tip of the suture needle from the opposite side while continuing to feed or pushing the other end of the suture needle with the serrated tip tweezers.</li>
	<li>Once the suture needle is completely passed under MCA and has been pulled out, continue to pull on the suture needle or thread until the length of the thread is equal on either side of MCA. Pressing down on the thread as it is fed through to minimize strain on MCA can be helpful to prevent rupture as the thread passes under the artery.</li>
	<li>Cut the thread close to the suture needle.</li>
	<li>Use both fine point tweezers to untangle the two resulting suture threads so that there are two independent threads strung under MCA that are not touching. Ideally the threads will be about 1 mm apart where they pass under MCA.</li>
	<li>Use both fine point tweezers to tie two separate knots (two ligatures) with the threads around MCA attempting to maintain that ~1 mm of space between the knots to allow room for transection.</li>
</ol>
NOTE: If an internal sham control is desired, prepare the occlusion leaving the occlusion knots loose so that they do not constrict MCA at all and collect data prior to tightening the knots and cutting the vessel. Trim the thread to prevent it catching on anything prior to occlusion but leave enough thread to allow tightening of the knots later. This way, any baseline imaging or data collection can be performed with all of the same surgical invasion as the occlusion and the knots tightened at the appropriate time point with little delay.
<ol start="9">
	<li>Once the knots have been pulled tight, use the micro scissors to transect M1 in between the two knots.</li>
	<li>In the case of long-term, survival studies:
	<ol class="lalpha">
		<li>Suture incised scalp flap back in place with sterile surgical thread or secure the tissue using sterile wound clips.</li>
		<li>Administer antibiotics locally to the wound area (such as bacitracin ointment) and systemically by prophylactic injection of ampicillin (150 mg/kg IM).</li>
		<li>While the subject is still anesthetized administer an ophthalmic antibiotic ointment to the eyes.</li>
		<li>Administer supplemental atropine (0.05 mg/kg IM) to decrease respiratory secretions during anesthesia. </li>
		<li>Inject flunixin meglumine (1.1 mg/kg) subcutaneously at the conclusion of surgery and again the following morning (~12 hr later) for pain control.</li>
		<li>Place the animal on a dry, warm, slanted surface such that the animals nose is above its tail on the incline (this facilitates breathing until the animal is awake).</li>
		<li>Monitor the animal until it is awake and moving safely on its own.</li>
		<li>Once the animal is back in the vivarium, the animal's activity, appearance, vocalization, and feeding and drinking behavior should be monitored daily.</li>
	</ol>
	</li>
</ol>
4. Euthanasia
<ol>
	<li>At the conclusion of each experiment, rats should be euthanized with sodium pentobarbital (2-3 ml, intraperitoneally).</li>
</ol>

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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></ol>

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 
 No.:510461</td>
 <td></td>
 </tr>
 <tr>
 <td>30 gauge needle, &frac12;" length</td>
 <td>Fine Science Tools</td>
 <td>NC9867376 
 
 No.:ZT-5-030-5-L/COL</td>
 <td></td>
 </tr>
</table>

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

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JoVE Journal

Medicine

14.8K 次观看.  University of California, Irvine. 我们描述了一个高度可重复性的方法永久啮齿类动物的主要脑血管闭塞。这种技术可以完成与周损伤非常少，最小的失血，长期生存的一种高速率，和一致的梗死体积与人类的临床人口。

视频: Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

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

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

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

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

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.