This project is partially supported by NIH T32 GM008440 to Espen Walker, R01 NS27713 to William L.Young, P01 NS44155 to William L.Young&#160; and Hua Su, R21 NS070153 to Hua SU and by the American Heart Association&#160; AHA 10GRNT3130004 to Hua Su. Dr Ana Rodr&#237;guez-Hern&#225;ndez is supported by a grant from &#8220;Obra Social La Caixa&#8221;

1. Preparing the Mouse
<ol>
	<li>Induce general anesthesia in the mouse with isoflurane gas. Inject 0.15 ml of intraperitoneal brupenorphine for pain management. Before proceeding, check if the anesthesia level is satisfactory by pricking the mouse&#39;s paws.</li>
	<li>Put the mouse in dorsal recumbency&#160;with the four limbs fixed by adhesive tape. Remove the hair of the neck and the upper chest with scissors. Via subcutaneous injection, administer 0.2-0.4 ml of 0.9% saline to keep the mouse hydrated during the surgical procedure.</li>
	<li>Prepare the operative field following a strict sterile method. The area of the skin incision should be cleaned with 90% alcohol.</li>
</ol>
2. Dissecting the Common Carotid Artery and the External Jugular Vein
<ol>
	<li>Make a horizontal midline cervical incision across the lower neck area of the mouse. After deepening the wound, elevate the salivary glands and cervical soft tissue by using a traction suture (Figure 1). Expose the right external jugular vein (EJV) lateral to the sternocleidomastoid muscle (SCM). This step should be performed under the microscope, since excessive traction over the vein might damage it and induce its thrombosis.</li>
	<li>Carefully dissect the right EJV along its course from the clavicle to the skull base. Usually electric bipolar forceps, coagulate and divide any branches to prepare an adequate length for the later temporary clip placement and anastomosis.</li>
	<li>Lateral to the trachea and medial to the SCM, explore the common carotid artery (CCA). It should be carefully exposed from the clavicle to just beyond its bifurcation into the external and internal carotid arteries. During this step, attention should be given again to avoid excessive traction to the EJV that could later compromise the patency of the anastomosis.</li>
</ol>
3. Preparing the Anastomosis
<ol>
	<li>Ligate the CCA with 10-0 Nylon just proximal to its bifurcation. Next, apply a temporary clip over the proximal CCA as close to the clavicle as possible.</li>
	<li>Once the flow has been interrupted, transect the artery just under the bifurcation ligature and irrigate it with saline to wash out any remaining blood inside the lumen. Avoid bipolar coagulation in this step, since thermal injury to the artery wall could put the future anastomosis in jeopardy.</li>
	<li>In order to improve the visibility of the edges of the EJV, the medial wall is marked with a blue marking pen along the course of the planned venotomy. Once the EJV is marked, use a 10-0 suture to ligate the distal end as caudal as possible and apply a temporary vascular clip on the proximal end as cranial as possible.</li>
	<li>With a fine 30 G needle and 0.5 ml syringe, make an initial aperture over the marked area of the EJV and immediately irrigate the lumen with saline to avoid thrombus formation. Next, extend the venotomy with the microscissors until the length is about 2-3 times the diameter of the CCA. Avoid violent stretching and pay attention to keep a sharp and neat cutting edge.</li>
	<li>Approximate the end of the CCA to the EJV. Make a side-cut incision in the donor end of the CCA to adjust the diameter to the length to the venotomy size (Figure 2).</li>
</ol>
4. End-to-side Anastomosis
<ol>
	<li>Use a 11-0 monofilament nylon suture for the CCA-to-EJV end-to-side anastomosis. Suturing the medial wall of the anastomosis in a craneo-caudal direction is the initial step. Every stitch should be placed from outside-in the venous wall first (Figure 3) and from inside-out the arterial wall (Figure 4) next. This will keep the knot on the outside surface of the anastomotic vessels at all times (Figure 5). Either interrupted or continuous sutures can be used, but all continuous sutures should be tightened as the final step.</li>
	<li>Once the medial wall has been sutured, repeat the procedure from caudal to cranial with the lateral wall. Now every stitch should be placed from outside-in the arterial wall first and from inside-out the venous wall next. Saline irrigation will help keep the lumen of the anastomosis visible at all times during the procedure.</li>
	<li>After finishing all the steps of the anastomosis, remove the temporary clip from the vein first and from the artery next. The arterial blood will flow into the EJV with no or little oozing from the anastomosis (see videos VH model and VH model2). The minimal bleeding should stop without using cotton compression over the anastomosis. This must be avoided in order to prevent thrombosis of the delicate vein.</li>
	<li>Once pulsatile flow through the anastomosis is confirmed and no apparent bleeding is observed, irrigate the surgical field with saline and close the cervical incision with a 6-0 nylon suture. Finally, administer 0.15 ml more of intraperitoneal brupenorphine for postoperative pain management and 0.2-0.4 ml of subcutaneous 0.9% saline to replenish any blood loss during the surgery.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonischemic+cerebral+venous+hypertension+promotes+a+pro-angiogenic+stage+through+HIF-1+downstream+genes+and+leukocyte-derived+MMP-9.">Nonischemic cerebral venous hypertension promotes a pro-angiogenic stage through HIF-1 downstream genes and leukocyte-derived MMP-9.</a> J. Cereb. Blood Flow Metab. 29, 1482-1490 (2009).</li><li>Kim, H., Su, H., Weinsheimer, S., Pawlikowska, L., Brain Young, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=arteriovenous+malformation+pathogenesis:+a+response-to-injury+paradigm.">arteriovenous malformation pathogenesis: a response-to-injury paradigm.</a> Acta Neurochir. Suppl. 111, 83-92 (2011).</li><li>Lawton, M. T., Arnold, C. M., Kim, Y. J., Bogarin, E. A., Stewart, C. L., Wulfstat, A. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transgenic+arteriovenous+fistula+in+the+rat:+an+experimental+model+of+gene+therapy+for+brain+arteriovenous+malformations.">The transgenic arteriovenous fistula in the rat: an experimental model of gene therapy for brain arteriovenous malformations.</a> Neurosurgery. 54, 1463-1471 (2004).</li><li>Schaller, B., Graf, R., Sanada, Y., Tolnay, M., Rosner, G., Wienhard, K., Heiss, W., D, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+changes+after+occlusion+of+the+posterior+superior+sagittal+sinus:+an+experimental+PET+study+in+cats.">Hemodynamic changes after occlusion of the posterior superior sagittal sinus: an experimental PET study in cats.</a> AJNR Am J Neuroradiol. 24, 1876-1880 (2003).</li><li>Zhu, Y., Lawton, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+acquired+arteriovenous+fistulas+in+rats+due+to+venous+hypertension.">Development of acquired arteriovenous fistulas in rats due to venous hypertension.</a> J. Neurosurg. 80, 884-889 (1994).</li><li>Yassari, R., Sayama, T., Jahromi, B. S., Aihara, Y., Stoodley, M., Macdonald, R. 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A., Knudsen, B., Misra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+arteriovenous+fistula+model.">The mouse arteriovenous fistula model.</a> J Vasc Interv Radiol. 20, 946-950 (2009).</li><li>Choi, E. J., Choi, E. J., Walker, E. J., Shen, F., Oh, S. P., Arthur, H. M., Young, W. L., Su, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+Homozygous+Endothelial+Deletion+of+Eng+with+VEGF+Stimulation+Is+Sufficient+to+Cause+Cerebrovascular+Dysplasia+in+the+Adult+Mouse.">Minimal Homozygous Endothelial Deletion of Eng with VEGF Stimulation Is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse.</a> Cerebrovascular diseases. 33, 540-547 (2012).</li><li>Hao, Q., Su, H., Marchuk, D. A., Rola, R., Wang, Y., Liu, W., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tissue+perfusion+promotes+capillary+dysplasia+in+the+ALK1-deficient+mouse+brain+following+VEGF+stimulation.">Increased tissue perfusion promotes capillary dysplasia in the ALK1-deficient mouse brain following VEGF stimulation.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H2250-H2256 (2008).</li><li>Su, H., Hao, Q., Shen, F., Zhu, Y., Lee, C. Z., Young, W. L., Yang, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+cerebral+microvascular+dysplasia+model+in+rodents.">Development of a cerebral microvascular dysplasia model in rodents.</a> Acta Neurochir. Suppl. 105, 185-189 (2008).</li><li>Walker, E. J., Su, H., Shen, F., Degos, V., Jun, K., Young, W. 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The experimental procedures with laboratory animals were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (UCSF).
The authors have no potential conflicts of interest related to the drugs and materials used in this procedure.

Sustained cerebral venous hypertension has been closely related with more severe clinical manifestations and poor prognosis in patients with dural AVFs and brain AVMs3. These effects of CVH have been widely studied in rat models1,2,8. An equivalent model in the mouse would allow the use of genetically modified animals which would ultimately allow the analysis of molecular pathways involved on the pathogenesis of venous hypertension and its relationship with dural AVF and brain AVM.
<p class="jov...

Animal models of cerebral venous hypertension have proved to be a key tool in the understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas1-7. The most widely used is the rat model created through an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV), which provokes a consistent cerebral venous hypertension (CVH) in the rat1,8-10. Equivalent mice models, by opening the possibility of using different transgenic mice strains, would allow further study on not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases. Furthermore, 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.11 However, previous attempts to construct these mice models have demonstrated the difficulties with patency of the fistula due to the diminutive anatomy5,12. Here, we describe our step-by-step protocol for a successful anastomosis of the murine CCA and EJV that translates into a long-term patent fistula and a durable venous hypertension in the mouse.

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>10-0 Sterile Microsuture</td>
			<td>Arosurgical Ic.</td>
			<td>VT5A010Q10</td>
		</tr>
		<tr>
			<td>11-0 Sterile Microsuture</td>
			<td>Arosurgical Ic</td>
			<td>VT4A00N07</td>
		</tr>
		<tr>
			<td>DUROTIP Scissors</td>
			<td>Aesculap</td>
			<td>BC210R</td>
		</tr>
		<tr>
			<td>Micro-Adson Tissue Forceps</td>
			<td>Aesculap</td>
			<td>BD510R</td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Aesculap</td>
			<td>OC496R</td>
		</tr>
		<tr>
			<td>Micro Forceps #5 Jewelers</td>
			<td>Aesculap</td>
			<td>BD331R</td>
		</tr>
		<tr>
			<td>Angled Jewelers Forceps</td>
			<td>Aesculap</td>
			<td>BD329R</td>
		</tr>
		<tr>
			<td>Micro Suture Forceps</td>
			<td>Aesculap</td>
			<td>BD338R</td>
		</tr>
		<tr>
			<td>DUROGRIP Needle Holder</td>
			<td>Aesculap</td>
			<td>BM009R</td>
		</tr>
	</tbody>
</table>

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.

A successful outcome of the model is a patent arteriovenous fistula that induces venous hypertension in the murine brain. To validate the model we initially measured the intracranial venous pressure in the sagital sinus of the mice at 2, 3 and 4 weeks after surgery. 6 different mice were assigned to every time group. The sinus pressure was 8.8 &#177; 1.2 mmHg in the group measured two weeks after the surgery. In the 6 mice measured 3 weeks after surgery, the sinus pressure was 4.7 &#177; 1.4 mmHg. Finally, the 6 mice mea...

Watch this Scientific Journal Video about Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis. at JoVE.com

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

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 ...

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14.0K Views.  University of California, San Francisco. 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.

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

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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.

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

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, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...