This study was funded by the research promotion program &#34;START&#34; of the medical faculty of RWTH Aachen University, Germany (Title: &#34;Die Arterialisation ven&#246;ser Gef&#228;&#223;e&#34; START 690753). We would like to thank Mrs. Mary-Joan Bl&#252;mich for her excellent editing assistance. Dr. Maria Kokozidou was financed from Fresenius 2011_A61.

1. Experimental Animals
<ol>
 <li>Obtain a Local Ethics Committee approval for all experiments which will be performed, in accordance with the animal protection FELASA guidelines. </li>
 <li>Use female Sprague-Dawley rats available to purchase from any commercial breeder (e.g. Charles River Wiga GmbH, Sulzfeld, Germany). We used 21 animals divided in three groups of 7 animals each that were sacrificed at 3, 6 and 12 weeks postoperatively. At the time of purchase, the animals should be 12 weeks of age and weigh 250-300 g.</li>
 <li>Keep the animals in climate-controlled rooms having an ambient temperature of 21 &deg;C, 60% relative humidity and a 12-hr light-dark cycle with free access to food and water. </li>
</ol>
2. Anesthesia and Operative Preparation
<ol>
 <li>Induce general anesthesia by using a Plexiglas box connected to the anesthesia machine, set at an oxygen flow rate of 1 L/min, and an isofluorane vaporizer with 5 % anesthetic gas. </li>
 <li>Maintain the anesthesia with the isofluorane vaporizer at 1.5 % as soon as the deep sensory reflexes of the animals are suppressed. </li>
 <li>Fix the rats in supine position on a body warming plate. </li>
 <li>Prepare the operation region of each animal by removing its fur with a depilation cream, from the jaw angle down to 2 cm below the manubrium sterni. </li>
 <li>Prepare a sterile surgical field by paying attention to the surgical area, the area prepared on the animal and the front of the surgeon: 
 <ul>
 <li>Clean the benchtop with a disinfectant. </li>
 <li>Prepare the incision site by using multiple applications of surgical scrub solution (e.g. Chlorhexidine and 70% isopropyl alcohol). Apply the Chlorhexidine first, the alcohol second and, finally, the scrub solution. </li>
 <li>Wear a disposable surgical gown and surgical gloves, or alternatively, exam gloves that have been previously washed with a surgical disinfectant. </li>
 </ul>
</li>
 <li>Use sterile microsurgery instruments in the operation procedure. </li>
 <li>Use an operation microscope (Wild Heerbrugg M650, Gais, Switzerland) in 10-20-fold magnification. </li>
</ol>
3. Anatomy
Figure 1 depicts the anatomy of the rat's neck:
1a) Situs before transection of the right sternocleidomastoid muscle.
1b) Situs after transection of the right sternocleidomastoid muscle.
4. Cuff Preparation
Cut the cuffs in a length of 3 mm by hand from a plastic tube (Portex, inner diameter 0.75 mm, outer diameter 0.94 mm, Smiths Medical, UK). These cuffs consist of a tubular cuff body including the complete volume of the plastic tube, and a cuff handle consisting of half of the cuff volume. Both the cuff body and the cuff handle should be equally long. The cuff handle is essential for manipulating the cuff during the surgical procedure. After the cuff is slipped over the vessel, a mini Bulldog clamp is placed simultaneously on the cuff handle and the vessel, in order to prevent from bleeding, keep the cuff in position and ensure stability.
5. Cuff Technique
<ol>
 <li>Slip the cuff over the vessel. </li>
 <li>Immediately afterwards, evert the vessel, pull it over the outside of the cuff body and fix it with a 7-0 silk ligature. In this manner, the lumen of the cuff is covered with endothelium. Thereafter, slip the venous graft over the cuff body and fix it with a new 7-0 silk ligature. To prevent thrombus formation, it is essential to fasten the vein graft tie very close to the cuff margin in order to avoid the blood from contacting the tie holding the artery in place. This is crucial for the distal anastomosis due to the blood flow direction. However, according to the authors, it is not considered essential for the proximal anastomosis for the same reason. </li>
</ol>
6. Surgical Procedure
6.1 Preparation of the right external jugular vein
<ol>
 <li>Make an approximate 2.5 cm-long median cervical incision direct above the manubrium sterni.</li>
 <li>Completely separate a 1 cm-long segment of the right external jugular vein from the surrounding tissues </li>
 <li>Ligate the venous tributaries with 7-0 silk sutures. </li>
</ol>
6.2 Preparation of the right common carotid artery
<ol>
 <li>Prepare the sternocleidomastoid muscle and transect it in the middle.</li>
 <li>Prepare the right common carotid artery carefully, making sure not to compress the artery or the Vagus nerve. Mobilize it from the carotid bifurcation down to the brachiocephalic trunk. </li>
 <li>Subsequently, ligate it twice in the middle with 7-0 silk sutures before transection. </li>
 <li>Slip a nylon cuff consisting of a body and a handle (Figure 2a) over the distal end of the common carotid artery, whereby it is important to ensure that the cuff handle is behind the vessel (Figure 2b).</li>
 <li>Clamp the cuffed end of the distal common carotid artery with a mini Bulldog clamp exactly on the cuff handle to ensure stability (Figure 2c). </li>
 <li>Subsequently, cut the artery directly behind the ligature, whereby the resulting arterial stump should be as long as the cuff body.</li>
 <li>Rinse the arterial lumen with heparin-saline solution (20 IU heparin/ ml 0.9% NaCl). Set a ligation loop aside in the proper position for later use. </li>
 <li>Pull the arterial stump up and slip it like a sleeve over the cuff body. </li>
 <li>Subsequently, pull the ligation loop and knot it tightly.</li>
 <li>To prevent the artery from slipping out of its sleeve, you may implement a trick: grasp both the arterial wall and the suture with the right hand simultaneously and with the left-hand forceps (Figure 2d) make the knot (Figure 2e). </li>
 <li>Repeat the same procedure for the proximal arterial stump (Figure 2f). </li>
</ol>
6.3 Venous graft positioning
<ol>
 <li>Excise a 1 cm-long segment of the clamped external jugular vein </li>
 <li>Since the two cuffed arteries are too far apart from each other for the venous interposition, approximate the correct distance for this procedure using a 3-0 Vicryl-suture which you should loop around each mini Bulldog clamp. </li>
 <li>Criss-cross both sutures.</li>
 <li>Subsequently, pull the sutures in opposite directions, enabling the proper positioning of the cuffed arterial stumps (Figure 2g) </li>
 <li>Rinse the vein segment with the heparin-saline solution. </li>
 <li>Slip one end of the previously reversed vein segment over the cuffed distal carotid artery and secure it in the correct position with a silk 7-0 ligature. </li>
 <li>It is important to note here that the circumference of the vein must be completely and evenly grasped. </li>
 <li>Rinse the vein segment with heparin-saline-solution, and slip the other end of the vein segment over the proximal carotid stump. </li>
 <li>Complete the ligation (Figure 2h); </li>
 <li>Remove the mini Bulldog clamps one after the other, first on the distal carotid artery stump and then on the proximal one, thereby allowing unobstructed blood flow and the arterialization of the venous graft (Figure 2i).
 The surgery is considered successful if blood flow and pulsation are observed within the graft and the common carotid artery (assessed visually). </li>
</ol>
6.4. Wound closure
For the subcutaneous running suture, use 4-0 Vicryl. For a cutaneous running suture, use 3-0 Vicryl.
7. Recovery and Postoperative Management
<ol>
 <li>Let the animals recover from anesthesia on a flat paper bedding under an infrared warming lamp. Do not return the operated animals to the animal holding area until all animals appear normal. </li>
 <li>Postoperatively administer Buprenorphine (Temgesic) in the concentration of 0.03-0.05 mg/kg b.w. sc every 8-12 hr for 2 days. Buprenorphine can be administered just before isofluorane induction. </li>
 <li>Monitor animals postoperatively for their general condition and signs of illness or infections. </li>
</ol>
8. Blood Flow Measurement
The postoperative patency of the graft was proved by blood flow measurement, by using a research Transonic blood flow measurement system (Model: TS420, Transonic Systems Europe B.V., Maastricht, The Netherlands) with a perivascular flowprobe (3 mm, MA3PSB). The system is based on the Doppler principle.
9. Representative Results
By faithfully keeping the instructions aforementioned, the mean duration of the surgical operations was about 65 min. The animals presented an excellent physical condition and gained weight as expected throughout the whole experiment: at the starting point of operation the animals weighed 312&plusmn;20.39 g. At the sacrifice time point of 3 weeks post-operatively the animals weighed 325.2&plusmn;28.7 g, at 6 weeks 333&plusmn;39.9 g and at 12 weeks 362.22&plusmn;29 g. 
No infections were observed at the operation site and no sores developed. The operation wounds healed completely at the expected time. The neurological control showed no signs of brain infarction or motoric deficiency. The later was an important point since the animals were operated in the carotid artery and one of the two external jugular veins was removed and used as auto-transplant. No allergic reactions to the postoperative pain alleviation therapy were observed. 
All animals were vigilant and social throughout the whole experiment. Their habits concerning receiving food and water remained unchanged throughout the whole time.
The survival rate of all experiments was 100 % at the time point of sacrifice. The graft patency rate was 60 % for the first 10 operated animals and accordingly 82 % for the remaining 11 animals. The total graft patency was 71 %. The blood flow in the arterialized venous grafts was measured at the time point of sacrifice and was 8&plusmn;3 ml/min without any statistically significant differences between the three sacrifice time points.
<img src="/files/ftp_upload/4124/4124fig1.jpg" alt="Figure 1" /> 
Figure 1. Anatomy of the rat's neck. 1a) Situs before transection of the right sternocleidomastoid muscle. 1b) Situs after transection of the right sternocleidomastoid muscle.
<img src="/files/ftp_upload/4124/4124fig2.jpg" alt="Figure 2" /> 
Figure 2. Intraoperative microscopic pictures: a) cuff consisting of cuff-body and cuff-handle; b) cuff slipped over the distal end of the common carotid artery; c) clamping of the cuffed distal common carotid artery stump; d) eversion of the common carotid artery stump over the cuff; e) securing the everted common carotid artery stump with a ligature; f) situs after eversion of both cuffed distal and proximal common carotid artery stumps; g) criss-cross technique for approximation of the two cuffed common carotid artery stumps; h) situs after venous interposition; i) situs after reperfusion of the venous interposition graft.
<img src="/files/ftp_upload/4124/4124fig3.jpg" alt="Figure 3" /> 
Figure 3. Schematic depiction of the situs after reperfusion of the venous interposition graft. <a href="https://www.jove.com/files/ftp_upload/4124/4124fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Motwani, J. G., Topol, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortocoronary+saphenous+vein+graft+disease:+Pathogenesis,+Predisposition,+and+Prevention.">Aortocoronary saphenous vein graft disease: Pathogenesis, Predisposition, and Prevention.</a> Circulation. 97, 916-931 (1998).</li><li>Bonatti, J., Oberhuber, A., Schachner, T., Zou, Y., Hammerer-Lercher, A., Mittermair, R., Laufer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neointimal+hyperplasia+in+coronary+vein+grafts:+Pathophysiology+and+prevention+of+a+significant+clinical+problem.">Neointimal hyperplasia in coronary vein grafts: Pathophysiology and prevention of a significant clinical problem.</a> The Heart Surg. , E72-E87 (2004).</li><li>Roy-Chaudhury, P., Kelly, B. 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Surg. 25, 538-546 (2011).</li><li>Wang, J., Liu, K., Shen, L., Wu, H., Jing, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+interfering+RNA+to+c-myc+inhibits+vein+graft+restenosis+in+a+rat+vein+graft+model.">Small interfering RNA to c-myc inhibits vein graft restenosis in a rat vein graft model.</a> J. Surg. Res. 169, e85-e91 (2011).</li><li>Schachner, T., Zou, Y., Oberhuber, A., Mairinger, T., Tzankov, A., Laufer, G., Ott, H., Bonatti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+application+of+C-type+natriuretic+peptide+attenuates+neointimal+hyperplasia+in+experimental+vein+grafts.">Perivascular application of C-type natriuretic peptide attenuates neointimal hyperplasia in experimental vein grafts.</a> Eur. J. Cardiothorac. Surg. 25, 585-590 (2004).</li><li>Thomas, A. C., Newby, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+matrix+metalloproteinase-9+knockout+on+vein+graft+remodelling+in+mice.">Effect of matrix metalloproteinase-9 knockout on vein graft remodelling in mice.</a> J. Vasc. Res. 47, 299-308 (2010).</li></ol>

No conflicts of interest declared.

Interposing an external jugular vein graft in the common carotid artery is a sophisticated microsurgery technique. This video helps the novice surgeon master this technique in minimum time with a lower waste of experimental animals.
Inhalative anesthesia is the best option for this operation, since it avoids an excessively deep anesthesia stage, can be well controlled and allows the surgeon enough time to operate. Moreover the rat is an experimental animal that offers a suitable size to practi...

<table border="1">
<tbody>
 <tr>
 <td>Name of 			the reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Braided 			Silk black 7-0 </td>
 <td>Ethicon 			Products</td>
 <td>892100</td>
 <td></td>
 </tr>
 <tr>
 <td>Vicryl 3-0</td>
 <td>Ethicon 			Products</td>
 <td>J285G</td>
 <td></td>
 </tr>
 <tr>
 <td>Vicryl 4-0</td>
 <td>Ethicon 			Products</td>
 <td>J284G</td>
 <td></td>
 </tr>
 <tr>
 <td>Portex non 			sterile nylon tubing 0.75 mm, 0.94 mm Fine-Bore Polyethylene 			(Polythene) Tubing</td>
 <td>Portex UK</td>
 <td>800/200/175/100</td>
 <td></td>
 </tr>
 <tr>
 <td>Isofluorane 			5 %</td>
 <td></td>
 <td></td>
 <td>Any 			genericon</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td></td>
 <td></td>
 <td>Any 			genericon</td>
 </tr>
 <tr>
 <td>0.9% 			saline</td>
 <td></td>
 <td></td>
 <td>Any 			genericon</td>
 </tr>
 <tr>
 <td>Buprenorphine</td>
 <td></td>
 <td></td>
 <td>Any 			genericon</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats at JoVE.com

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

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 ...

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20.2K Views.  University Hospital RWTH Aachen. 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.

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

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 ...

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. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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, ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...