All procedures described in this article were performed in accordance with the guidelines and regulations of the Landesamt f&uuml;r Gesundheit und Soziales, Berlin, Germany. 
Pre-surgical procedures
1.	Preparation of Monofilaments 
<ol>
 <li>For preparation of intraluminal filaments, 8-0 nylon monofilament sutures were used and were cut to a length of 13 mm each.
 </li>
 <li>For coating of the monofilaments, Xantopren M mucosa, (Heraeus Kulzer GmbH, Hanau, Germany) was used activated by adding a drop of activator solution (Activator, Universal plus, Heraeus Kulzer GmbH, Hanau, Germany). </li>
 <li>The monofilament was slowly pulled through the silicone until it was completely covered with it. By doing so, the monofilament would be evenly covered with silicone. Avoid waiting too long because the activated coating material will become firm and dry quickly. </li>
 <li>Before the silicone starts to solidify, it is recommended to strip off excessive silicone from the filament by pulling it over a sheet of paper. By doing so, the silicone layer can be thinned out to an approximate thickness of 200 &mu;m.
 </li>
 <li>The base of the silicone-covered monofilament was stuck into modeling clay in order to let it dry overnight. </li>
</ol>
Comment: Ideally, only sterilized or at least disinfected monofilaments should be used to ensure sterility at the surgical site. In practice, sterilization or disinfection of the hand-made monofilaments is very difficult because the quality of the monofilaments may worsen 9. Therefore, it might be preferable to use commercially available sterile monofilaments instead. 
2.	Preparation of Surgical Instruments and of the Animals
<ol>
 <li>All surgical instruments were kept in 70% ethyl alcohol. Additionally, prior to each new surgical procedure, all surgical instruments were carefully cleaned and disinfected using 70% ethyl alcohol. </li>
 <li>The surgical area was thoroughly disinfected using 70% ethyl alcohol in all animals before transection. The disinfected sites were allowed to completely dry before performing the incisions. Additional clipping of the fur was not performed as this may cause microabrasions and subsequent skin inflammation 9. </li>
</ol>
Surgical procedures
3.	Positioning of the LDF Probe and LDF Monitoring
<ol>
 <li>For induction of anesthesia, mice were exposed to a gaseous mixture consisting of 30% oxygen, 70% N2O and 2.5% isoflurane using a vaporizer. For maintenance of anesthesia, isoflurane concentration was reduced to 1.5%. Mice were breathing spontaneously via breathing mask throughout the surgical procedure. </li>
 <li>A rectal temperature probe was inserted and mice were fixed to the stereotactic frame exposing the parietal head. During surgery, mice were resting on a thermostat-controlled heating pad, ensuring a constant core temperature of 37.0 &plusmn; 0.5 &deg;C. A sagittal midline incision (~1 cm length) was performed to expose the parietal skull. </li>
 <li>The left side of the skull was carefully dissected and a fiberoptic probe was fixed directly to the skull 2 mm caudal and 5 mm lateral from bregma using instant glue. Afterwards, wound edges were carefully adapted to avoid brain hypothermia as well as infections. </li>
 <li>The mouse was turned around to expose the ventral side of the neck. The fiberoptic probe was connected to the LDF device (PeriFLUX System 5000, Perimed, J&auml;rf&auml;lla, Sweden) via connecting probe adaptor (Master Probe 418-1, Perimed; J&auml;rf&auml;lla, Sweden). </li>
 </ol>
 Comment: While turning the mouse around, the fiberoptic probe should be bent with care in order to avoid that it breaks off from the skull.
 <ol start="5">
 <li>The LDF device was connected to a laptop computer. Continuous recording of LDF values was initiated using PeriSoft for Windows (Version 2.50, J&auml;rf&auml;lla, Sweden). Figure 1 demonstrates the mean CBF course obtained from ten LDF recordings during BCCAO and reperfusion. </li>
</ol>
4.	Bilateral Common Carotid Artery Occlusion
<ol>
 <li>Anesthesia was maintained as described in 3.1. Mouse was placed on its back. The animal's tail and paws were fixed to the heating pad using adhesive tape. A sagittal ventral midline incision (~ 1cm length) was performed. </li>
 <li>Both salivatory glands were carefully separated and mobilized to visualize the underlying CCA's.</li>
 <li>Both CCA's were carefully separated from the respective vagal nerves and accompanying veins without harming these structures. Manipulations of the vagal nerves might lead to transient or permanent dysfunction of the parasympathetic nerve system, which has the potential for the occurrence of significant cardiac arrhythmia or even irreversible cardiac arrest. Therefore, it is crucial to avoid any manipulations of the vagal nerves. A loose 5-0 silk suture loop was made around each CCA. 
 </li>
 <li>A small silicon tubing (diameter 2 mm) was inserted in between the CCA and the silk sutures on each side. This tubing was used as a splinting of the CCA's in order to avoid damaging of the arterial walls when the sutures were tightened (Figure 4). 
 </li>
 <li>Both CCA's were occluded for one minute by tightening the silk sutures. Only mice, in which the LDF signal was reduced to &lt;10% of the baseline signal were included in the study. After reopening of both CCA's, a five minutes reperfusion period was initiated. By LDF a complete reperfusion was ensured. Animals with incomplete reperfusion (i.e. &lt;90 % recovery of CBF compared to baseline CBF) were excluded. The sequence of one minute of BCCAO and five minutes of reperfusion was repeated twice for a total BCCAO duration of 3 min. Figure 1 shows the mean CBF course obtained from ten LDF recordings during BCCAO and reperfusion. During the procedure, it is recommended to keep the neck wound wet using normal saline. 
 </li>
 <li>After the last reperfusion, all tubing and sutures as well as the fiberoptic probe attached to the skull were removed, the wounds were sutured and lidocaine ointment (Xylocain 2%; AstraZeneca GmbH, Wedel, Germany) was repeatedly applied to the wounds as needed for analgetic management.
 </li>
 <li>Mice were transferred to a pre-heated (30 &deg;C) recovery box (MediHEAT. PecoServives Ltd. Brough, Cumbria, UK) until they had regained complete consciousness. Thereafter, mice were returned to their animal cages. </li>
</ol>
5.	Middle Cerebral Artery Occlusion 
<ol>
 <li>Mice were re-anesthetized, a rectal temperature probe was inserted and mice were positioned on a heating pad to expose the ventral side of the neck. Afterwards, they were fixed as described in the BCCAO section. </li>
 <li>A permanent ligature using a long 7-0 silk suture was made around the left proximal CCA and the thread was gently pulled caudally using a mosquito clamp (Figure 4). </li>
 <li>Another permanent ligature was made around the left external carotid artery (ECA) and the thread was pulled towards the right side of the animal (Figure 4). </li>
 <li>Connective tissue surrounding the left ICA was carefully mobilized and the hypoglossal nerve was gently lifted to get a good overview of the distal course of ICA. The left ICA bends to the left while the pterygopalatine artery (PPA) follows the course of the proximal ICA to the right (Figure 4). </li>
 </ol>
 Comment: There is another artery to be identified which turns to the right in caudal direction. This is the occipital artery, which variably originates either from the CCA bifurcation or from the proximal part of the ECA (Figure 4). 
<ol start="5">
 <li>A loose suture loop was made around the proximal ICA using a 5-0 silk thread. </li>
 <li>Using a small microvascular clamp, the ICA was temporarily occluded distally to the 5-0 loose suture loop (Figure 4). </li>
 <li>A small incision was made in the distal part of CCA just before the CCA bifurcation using microvascular scissors (Figure 4). </li>
 <li>A preselected silicone-covered monofilament was introduced into the CCA via the small incision and pushed into the ICA until it stopped at the microvascular clamp (Figure 4). </li>
 <li>The microclamp was carefully removed from the ICA, while the monofilament was advanced deeper into the ICA until it stopped (Figure 4).
 Comment: At this point, flip the forceps in the left hand so that you do not hold it like a pen. This helps to grab the filament with the forceps more easily. The vascular clamp-applicator should be held in the right hand. It might be necessary to nearly tighten the loose 5-0 suture around the ICA in order to prevent bleeding from the incision side.</li>
 <li>The exact location of the monofilament tip was identified to check if it was advanced into the ICA and not into the PPA. If the monofilament tip was introduced into the proximal PPA, the filament was gently pulled back just before the ICA/PPA bifurcation. It was attempted to advance the monofilament into the ICA again, while gently pulling the proximal ICA a bit to the right side. By doing so, the natural course of the distal ICA was simulated making it easier to access the ICA. </li>
 <li>After the monofilament was advanced into the ICA almost completely, the loose 5-0 suture loop around the proximal ICA was tightened in order to hold the filament in place. Afterwards, all silk threads were shortened and the skin wound was sutured. </li>
 <li>The fluid loss during surgery was replenished by injecting 1.0 ml of normal saline solution i.p. Afterwards, the mouse was transferred to the pre-heated recovery box until it regained complete consciousness.</li>
 <li>For reperfusion, mice were re-anaesthetized and the monofilament was removed after carefully loosening the 5-0 silk suture around the proximal ICA. Afterwards, the 5-0 suture was tightened again carefully in order to prevent bleeding. </li>
 <li>After confirmation of complete hemostasis, the neck wound was sutured. </li>
 <li>Lidocaine ointment was repeatedly applied locally to the skin as needed for pain relief and the mouse was transferred to the pre-heated (30 &deg;C) recovery box until it regained complete consciousness. </li>
 <li>As a first hint for a successful MCAO procedure, a clockwise circling of the animal was observed. The severity of the functional deficit was quantified using the Bederson score modified for mice 10. According to this modified Bederson score, neurological scores were graded as follows: 0 (normal motor function), 1 (flexion of torso and contralateral forelimb when mouse was lifted by the tail), 2 (circling to the contralateral side when mouse was held by the tail on a flat surface, but normal posture at rest), 3 (leaning to the contralateral side at rest), and 4 (no spontaneous motor activity or dead). </li>
</ol>
6.	BCCAO Sham Surgery
For BCCAO sham procedures, mice were anaesthetized for the duration of a real BCCAO PC procedure, the salivation glands were mobilized and the CCA's were exposed. It is crucial that direct manipulations to the carotid arteries or vagal nerves are avoided during sham procedure. Manipulations of the CCA can damage its endothelial layer, which can result in a release of vasoactive compounds from endothelium or induce local thrombosis within the arterial lumen. As a consequence, thromboembolic events might occur after sham surgery. This would be a major confounder of the procedure. On the other hand, manipulations of the vagal nerves might lead to transient or permanent dysfunction of the parasympathetic nerve system, which has the potential for the occurrence of significant cardiac arrhythmia or even irreversible cardiac arrest. Therefore, it is crucial to avoid any manipulations of the vagal nerves during BCCAO.
Post-surgical procedures
7.	Infarct Volume Measurements 
For assessment of infarct sizes, mice survived for 72 hr after MCAO. Afterwards animals were deeply anesthetized, brains were removed, snap-frozen in methylbutan, sectioned and stained with hematoxylin (using Papanicolau's protocol) for infarct volume measurements. Brain sections were collected at 600 &mu;m-intervals and infarct volumes were determined using an image analyzer (MCID core 7.0 Rev.2.0, GE Healthcare Niagara Inc) and corrected for edema according to the method of Lin et al. 7,11.
8.	Inclusion Criteria
We included only animals which had a LDF-measured CBF reduction by more than 90% during each of the three BCCAO episodes. Ideally, LDF monitoring should be performed in both cerebral hemispheres during BCCAO. For practicability, we performed LDF monitoring only in the left hemispheres, which will be subjected to MCAO later on. This approach has been described before 6,12. Reperfusion after each BCCAO episode was required to be complete (i.e. return to baseline values). For reasons of reducing time duration mice were kept under anesthesia, we did not monitor CBF during MCAO surgery. In a pilot study with a separate group of animals however, we monitored CBF by LDF during MCAO surgery. In this group of mice, we consistently measured a reduction in CBF by 90% compared to baseline before CCA occlusion. Additionally, after removal of monofilaments CBF recovered to &gt;90% of the respective baseline values after CCA occlusion. These LDF criteria are established in the literature and result in consistent infarct volumes 7. We only included animals which had a Bederson score of at least of 1 after recovery from anesthesia after MCAO. Furthermore, only animals which survived for 72 hr after MCAO and which showed no signs of intracranial hemorrhage were included in the study.

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We have nothing to disclose.

In our study, we have shown that three sequences of BCCAO, each lasting for one minute and followed by reperfusion for five minutes, are an adequate ischemic PC stimulus to induce early IT. 
Furthermore, we have demonstrated that the preconditioning protocol used is a safe and minor invasive procedure with no noteworthy postoperative morbidity, no signs of apoptosis in brain and no mortality. Several different preconditioning protocols have been described previously 6-7,12. Others ...

Ischemic stroke is a disease with a high mortality and an enormous socio-economic burden 1. Despite intensive experimental and clinical scientific efforts throughout recent decades, treatment options for acute ischemic stroke patients remain very limited 2. In contrast to that, increase in the percentage of elderly people in developed countries will dramatically increase incidences and prevalences of patients with ischemic stroke in the next decades 3. Therefore, there is a pressing need for new treatment strategies in patients with ischemic stroke. 
One approach is to gain further understanding in mechanisms of endogenous adaptation of brain to cope with a damaging stimulus. This brain-derived neuroprotection is also known as ischemic preconditioning (PC) or ischemic tolerance (IT) and describes a phenomenon, in which a non-damaging noxious stimulus applied to the brain, induces a transient resistance against a subsequent damaging ischemic insult 4. IT occurs in two different time windows: early IT, which occurs within minutes to a few hours after PC and delayed IT, which needs a latency of a couple of hours to occur 4.
So far, research on IT in brain has focused on delayed IT. Much less is known on the mechanisms of early IT. The objective of this study is to establish bilateral common carotid artery occlusion (BCCAO) which is an established stimulus to induce delayed IT, as an adequate PC stimulus to induce early IT to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.
During both surgical procedures (BCCAO and MCAO), monitoring of cerebral blood flow (CBF) by laser Doppler flowmetry (LDF) was performed. 
BCCAO was performed in anesthetized and spontaneously breathing mice. Both common carotid arteries (CCA) were exposed and occluded for 60 sec followed by 5 min of reperfusion. This BCCAO/reperfusion sequence was repeated twice. After surgery, mice recovered quickly and showed no signs of functional impairment. In order to rule out, that the BCCAO protocol described above did not result in delayed cell death in the brain, we performed a TdT-mediated dUTP-biotin nick end labelling (TUNEL) staining 72 hr after BCCAO (or sham surgery) in a separate group. 
MCAO is a widely established model for induction of focal cerebral ischemia in rodents 5-7. The surgical procedures were carried out using standardized operating procedures (SOP) 8. After exposing left CCA, a silicon-covered monofilament was introduced into the distal CCA, advanced via the internal carotid artery (ICA) into the Circle of Willis until the anterior cerebral artery (ACA) and thereby, the origin of the middle cerebral artery (MCA) was occluded for a defined period of time. In this study, we used 45 min of MCAO, which typically results in an ischemic lesion in the MCA territory involving striatal and cortical brain areas. 
In our study, we analyzed the temporal profile of early IT using BCCAO as the PC stimulus. Our data indicated, that the optimal time delay between BCCAO and MCAO to induce early IT is 30 min.
In this video, we will give a demonstration of both surgical procedures, i.e. BCCAO and MCAO.

<table border="1">
<tbody>
<tr>
	<td>Name of the instrument</td>
 <td>Company</td>
 <td>Catalogue number/model</td>
</tr>
<tr>
	<td>Binocular surgical microscope</td>
 <td>Zeiss</td>
 <td>Stemi 2000 C</td>
</tr>
<tr>
	<td>Light source for microscope</td>
 <td>Zeiss</td>
 <td>SteREO CL 1500 ECO</td>
</tr>
<tr>
	<td>Heating pad with rectal probe</td>
 <td>FST</td>
 <td>21061-10</td>
</tr>
<tr>
	<td>Stereotactic frame</td>
 <td>David Kopf</td>
 <td>Model 930</td>
</tr>
<tr>
	<td>Scissors</td>
 <td>FST</td>
 <td>91460-11</td>
</tr>
<tr>
	<td>Dumont forceps #5</td>
 <td>FST</td>
 <td>11251-10</td>
</tr>
<tr>
	<td>Dumont forceps #7</td>
 <td>FST</td>
 <td>11271-30</td>
</tr>
<tr>
	<td>Mircovascular clamp</td>
 <td>FST</td>
 <td>00398-02</td>
</tr>
<tr>
	<td>Clamp applicator</td>
 <td>FST</td>
 <td>00072-14</td>
</tr>
<tr>
	<td>Springscissors</td>
 <td>FST</td>
 <td>15372-62</td>
</tr>
<tr>
	<td>Needle holder</td>
 <td>FST</td>
 <td>12010-14</td>
</tr>
<tr>
	<td>Needles	</td>
 <td>Feuerstein, Suprama	</td>
 <td>BER 562-20</td>
</tr>
<tr>
	<td>5-0 silk suture</td>
 <td>Feuerstein, Suprama	</td>
 <td></td>
</tr>
<tr>
	<td>7-0 silk suture</td>
 <td>Feuerstein,Suprama	</td>
 <td></td>
</tr>
<tr>
	<td>8-0 silk suture</td>
 <td>Feuerstein, Suprama	</td>
 <td></td>
</tr>
</tbody>
</table>

bilateral common carotid artery occlusion as an adequate preconditioning stimulus to induce early ischemic tolerance to focal cerebral ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

Our BCCAO protocol resulted in immediate and profound (&gt;90%) CBF reductions when both CCA's were occluded (Figure 1). Reopening of the CCA's led to complete reperfusion as monitored by LDF (Figure 1). After PC, animals showed no signs of functional disability. Furthermore, there was no mortality. TUNEL staining performed 72 hr after BCCAO in a separate group of animals showed no signs of delayed cell death in the brain after BCCAO (data not shown). 
When an...

Watch this Scientific Journal Video about Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia at JoVE.com

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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JoVE Journal

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28.9K Views.  Charité - Universitätsmedizin Berlin, Germany. Esistono evidenze, che il precondizionamento ischemico (PC) - una sfida ischemica non-dannoso per il cervello - conferisce una protezione transitoria a un successivo insulto ischemico dannoso. Abbiamo stabilito bilaterale dell'arteria carotide comune occlusione (BCCAO) come stimolo di precondizionamento per indurre la tolleranza ischemica precoce (IT) di ischemia cerebrale focale transitoria (indotta da occlusione dell'arteria cerebrale, MCAO) in C57Bl6 / J topi.

Video: Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

Mouse Model of Middle Cerebral Artery Occlusion

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

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

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

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

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

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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, ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...