This work was supported by funding from NIH grants HL87012 (HJ), HL75209 (HJ), UO1HL80711 (DH, DG, HJ) and a World Class University Project (HJ) from the Ministry of Science, Technology and Education of S. Korea.

1. Partial ligation of the left carotid artery
 <ol>
 <li>Mice are used at ~ 8 weeks of age. The weight of the mice is recommended to be at least 18 grams.</li>
 <li>Sterilize the instruments for 30 second in bead instrument sterilizer and allow to cool down</li>
 <li>Induce anesthesia with Ketamine (80mg/kg) and Xylazine (10mg/kg) intraperitoneal (i.p.) injection or alternative anesthesia as approved by IACUC. For i.p. injection, inject mouse in the left low quadrant of abdomen.</li>
 <li>Place mouse in a warming chamber until completely anesthetized. Verify depth of anesthesia with response to toe pinch.</li>
 <li>Place mouse on a surgical stage, in supine (belly side up) position. Tape the fore-paws palms up and the hind-paws sole down to the stage.</li>
 <li>Apply liberal amount of depilatory agent (e.g. Nair) to neck between the mandible and the sternum. Gently massage until all hair is removed.</li>
 <li>Clean off the depilatory agent and apply liberal amount of Betadine to depilated area.</li>
 <li>Prepare 1 inch-long sections of 6-0 silk suture for ligations. Each mouse will need two pieces of suture.</li>
 <li>Using a sharp small scissors, make a 4-6mm vertical incision in the middle of the neck. Both the skin and the underlying fascia need to be cut.</li>
 <li>Move the left parotid gland from the midline to the left and start bluntly dissecting just right of the trachea. You should be able to identify the pulsating left common carotid easily.</li>
 <li>Follow the common carotid caudally until you locate the bifurcation.</li>
 <li>Bluntly dissect the bifurcation to expose the following four distal branches off the left common carotid artery: external carotid artery, internal carotid artery, occipital artery and superior thyroid artery. These vessels, especially superior thyroid, can be fragile. Be very careful with your dissection.</li>
 <li>Tie off the external carotid ABOVE the superior thyroid using 6-0 silk suture. To do this, pass one forceps underneath the artery and grab a piece of precut suture with the help of your other forceps. Gently pull the suture to pass underneath the artery. Tie firmly avoiding any surrounding tissue being caught in the knot. If you tie below the take off of the superior thyroid artery, by the end of the procedure you will effectively have performed a complete ligation which is a substantially different model.</li>
 <li>Tie off the internal carotid and occipital arteries with one knot using the same technique.</li>
 <li>Verify position of sutures and patency of superior thyroid artery.</li>
 <li>Approximate the skin and close skin using a small amount of Tissue-Mend.</li>
 <li>Place mice in a warming chamber until recovery. Recovery typically takes 30 to 60 minutes.</li>
 <li>A single dose of Buprenorphine (0.1mg/kg) should be administered immediately post surgery if the anesthesia used does not provide prolonged pain relief (ie. inhaled isoflurane) or if animal is in distress within the first 24 hours. In our experience, the Ketamine dose administered for anesthesia provides sufficient analgesia when surgery is performed by experienced surgeon and no further analgesia is needed.</li>
 </ol>
2. Carotid ultrasound examination
 <ol>
 <li>To validate whether the partial ligation induced disturbed flow, carotid flow should be examined one day post-ligation</li>
 <li> Induce anesthesia with isoflurane - anesthesia induction box could be used if needed</li>
 <li>Place anesthetized mice on imaging stage, belly side up</li>
 <li>Tape arms and legs to ECG sensor on imaging stage</li>
 <li>Defoliate neck by liberal application of Nair (this step is only needed if not already done for ligation procedure)</li>
 <li>Insert rectal thermometer to record temperature</li>
 <li>Apply Echo gel to neck</li>
 <li>Start in B-mode.</li>
 <li>Imaging plane should be 90 degrees (knob on probe should point toward the nose of mouse) </li>
 <li>Lower probe on to mouse neck until image is obtained.</li>
 <li>Manipulate stage left and right until trachea (mid-line structure) is identified</li>
 <li>Identify left common carotid artery</li>
 <li>Tilt imaging stage so that an angle is created between imaging plane and the carotid. The greater this angle, the stronger the Doppler signal will be.</li>
 <li>Place pulse wave Doppler in middle of left common carotid.</li>
 <li>Use angle correction as needed.</li>
 <li>Repeat measurements on the right common carotid.</li>
 <li>Successful partial carotid ligation will result in overall reduction in flow (~80-90%) in the left common carotid compared with the right side, with reversal of flow towards aortic inlet during diastole.</li>
 <li>Once satisfactory images have been obtained, turn off anesthesia, wipe echo gel off mice and free mice from tape restraints. Place mice in warmed recovery chamber.</li>
	</ol>
3. Intimal RNA isolation from carotid
 <ol>
 <li>Sacrifice mice by CO2 inhalation according to the institutional IACUC protocol</li>
 <li>Tape the paws to a paper towel.</li>
 <li>Cut the skin of the mouse from the abdomen to the top of the thorax.</li>
 <li>Open the abdominal wall below the ribcage with a sharp pair of scissors.</li>
 <li>Lift the sternum with forceps and cut the diaphragm, then cut away the ribcage to expose the heart.</li>
 <li>Cut the vena cava with scissors.</li>
 <li>Pressure perfuse (120 mmHg) for 2 to 3 min with normal saline containing 10 Units/mL heparin through the left ventricle until the lungs and liver become pale.</li>
 <li>Cut the skin of the neck and remove all the fat, muscles, and connective tissues until the carotid arteries are exposed.</li>
 <li>Place the mouse under a dissecting microscope.</li>
 <li>Puncture a hole right below the ligation sites in the left carotid for the second perfusion.</li>
 <li>Pressure perfuse again for ~ 1 min with normal saline containing 10 Units/mL heparin through the left ventricle, making sure the left carotid is well perused.</li>
 <li>Using a fine tip forceps and small spring scissors, carefully remove the peri-adventitial tissues surrounding the carotids. Be careful not to squeeze or stretch the carotids during this cleaning step.</li>
 <li>Cut the left carotid artery between the aortic arch and the ligation points above the carotid bifurcation.</li>
 <li>Cut the right carotid artery between the right subclavian artery branching point and the carotid bifurcation.</li>
 <li>Transfer the carotids to a 35 mm culture dish, containing ice-cold HBSS. If needed, carefully remove any remaining peri-adventitial tissue.</li>
 <li>Prepare an insulin syringe (3/10 ml syringe) with a 29g needle by filling with 150 &mu;l QIAsol lysis buffer (Qiagen) per each carotid.</li>
 <li>Carefully insert tip of needle into one end of the carotid.</li>
 <li>While holding the carotid and the tip of the needle with a forceps, quickly (~ 1 sec) flush the QIAsol lysis buffer (150 &mu;l) into a 1.5ml tube (intima eluate). </li>
 <li>Rinse the carotid leftover (media+adventitia) once in HBSS, put it in a 1.5 ml tube, and snap-freeze in liquid nitrogen. </li>
 <li>Intima eluate and the frozen leftover are then used for intimal or media+adventitia RNA isolation using the miRNeasy mini kit (QIAGEN) according to the manufacturer's instructions.</li>
 </ol>
4. Representative Results:
We partially ligated mouse left carotid arteries and ultrasound examination was carried out one day following the procedure. As shown in Figure 1, successful partial ligation reduces blood flow velocity and reverses flow (disturbed flow) in the left common carotid artery during diastole. Partial ligation of ApoE knockout (KO) mice accompanied by a high fat diet induces robust atherosclerosis in the left common carotid artery(LCA), but not in the contralateral right carotid, by two weeks (Figure 2). RNA collected from the carotid intima contains endothelial marker genes such as PECAM-1, while free of medial smooth muscle cell marker genes such as a-SMA (Figure 3).
<img src="/files/ftp_upload/1861/1861fig1.jpg" alt="Figure 1" /> Figure 1: Schematic of carotid anatomy and ligations: Three branches of the left common carotid artery (LCA) [external carotid artery (ECA), internal carotid artery (ICA), and occipital artery (OA)] were ligated, while leaving the superior thyroid artery (STA) open.
<img src="/files/ftp_upload/1861/1861fig2.jpg" alt="Figure 2" /> Figure 2: The ultrasound images show flow velocity profiles and reveal that partial ligation induces flow reversal (indicated by arrows) in LCA during diastole. Flow in RCA remains unchanged after ligation.
<img src="/files/ftp_upload/1861/1861fig3.jpg" alt="Figure 3" /> Figure 3: Partial ligation and high-fat diet rapidly induces atherosclerosis in LCA of ApoE KO mice. ApoE KO mice were partially ligated and fed the high-fat diet for 1 to 3 weeks. Frozen sections from LCA were stained with Oil-Red-O.
<img src="/files/ftp_upload/1861/1861fig4.jpg" alt="Figure 4" /> Figure 4. Method of intimal RNA preparation. Intimal RNA and medial+adventitial (m+a) RNA were obtained from sham-operated RCA and LCA in C57BL/6 mice. RNAs were analyzed by semi-quantitative RT-PCR (A) and qPCR (B, C) for PECAM-1 and &alpha;-SMA using 18s as an internal control. Bar graphs are mean &plusmn; SEM, n=3.

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li></ol>

In this procedure, proper identification of all the branches of the common carotid including the external carotid, internal carotid, occipital artery and superior thyroid artery is essential. It is also important to verify the flow pattern using ultrasound as demonstrated. In our experienced hands, our success rate of partial ligation surgeries based on the ultrasound studies of more than 500 mice is better than 90%. For the carotid intimal RNA preparations, it is important to pay attention to the appearance of the caro...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Normal Saline (0.9% sodium chloride)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>2B1323</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe (3/10 ml 29g syringe)</td>
<td>&nbsp;</td>
<td>Tyco Health care</td>
<td>8881600145</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>21-023</td>
<td>&nbsp;</td>
<tr>
<td>miRNeasy mini kit (including, QIAzol)</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>217004</td>
<td>&nbsp;</td>
<tr>
<td>Vevo 770 High-Resolution Micro-Imaging System</td>
<td>&nbsp;</td>
<td>Visualsonics</td>
<td>a.Visualsonics 770 system b.Imaging stage c.Vevo integrated rail system d.RMV scan head for mice</td>
<td>or similar system</td>
</tr>
<tr>
<td>6-0 Silk Suture, sterile</td>
<td>&nbsp;</td>
<td>Covidien</td>
<td>s-1172 c2</td>
<td>&nbsp;</td>
<tr>
<td>Small scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5675</td>
<td>or similar</td>
</tr>
<tr>
<td>Dissecting Forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5005</td>
<td>or similar</td>
</tr>
<tr>
<td>Tissue Mend II</td>
<td>&nbsp;</td>
<td>Webster Vet Supply</td>
<td>07-856-7946</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

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

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 ...

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K Views.  Emory University. 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.

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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 ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

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 ...

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, ...