Name: in vivo gene transfer to the rabbit common carotid artery endothelium
Uploaded: 2018-05-06T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

We thank AdVec, Inc. for permission to use HDAd reagents, Julia Feyk for administrative assistance, and the Department of Comparative Medicine veterinary services for surgical advice and support. This work was supported by HL114541 and the John L. Locke, Jr. Charitable Trust.

All methods described here were approved by the University of Washington Office of Animal Welfare and associated Institutional Animal Care and Use Committee (IACUC), and were completed in accordance and compliance with all relevant regulatory and institutional guidelines.
Note: Gene transfer to rabbit common carotid arteries is performed on rabbits by a surgeon with the aid of an anesthesiologist or assistant.
1. Gene transfer to rabbit common carotid arteries: Pre-operation
<ol>
	<li>Anesthetize the rabb...

Certain aspects of surgical technique merit particular attention. Full exposure and mobilization of the common carotid artery via careful dissection will facilitate gene transfer and arteriotomy repair. However, during the dissection, direct manipulation of the carotid artery should be minimized&#160;to prevent vasospasm. In addition, any bleeding adjacent to the artery should be stopped by applying light pressure with gauze and extravasated blood should be cleaned up immediately by rinsing the area with normal saline. I...

The goal of this method is to introduce a transgene into the endothelium of rabbit common carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product both in normal arteries and in rabbit models of human arterial disease. Overexpression of the transgene in disease models can reveal whether the transgene (and its protein product) show promise as therapeutic agents1,2,3,4. Inclusion of cis-acting regulatory elements in the transgene expression cassette enables assessment of the activity of these elements in arterial endothelium in vivo5,6. Knowledge of the activity of specific cis-acting regulatory elements can be used to design more-active expression cassettes and to probe mechanisms of gene regulation in large artery endothelium in vivo7.
Rabbits are a valuable model for various aspects of human vascular physiology and disease. Rabbits share many vascular features with humans. For example, baseline hematological values, hemostatic regulation, and vascular longitudinal tension are similar between rabbits and humans8. Rabbit models of vascular diseases replicate key features of many human diseases including: aneurysms (similar geometric and flow characteristics)9, vasospasm (similar response to endovascular treatment)10,11, and atherosclerosis (intimal plaques with similar features including a core rich in lipid, macrophages, and smooth muscle cells in a fibrous cap)12,13. Accordingly, rabbit models have been developed for many vascular diseases such as thrombosis, vasospasm, aneurysm, diabetes, vascular graft stenosis, and atherosclerosis8,13,14,15,16.
For researchers choosing among animal models of vascular physiology and disease, the rabbit has several advantages. Compared to rodents, the larger vessels of rabbits allow easier surgical manipulation, use of endovascular devices, and a larger amount of tissue for quantitative measurements. Rabbits are much closer phylogenetically to primates than are rodents17, and the greater genetic diversity of outbred rabbits better approximates the genetic variability of humans. Genetic diversity is particularly important for preclinical studies, which-by their nature-aim to develop therapies that can be applied to the genetically diverse human population. As with many if not all other model species, rabbit genes are easily cloned or synthesized because the rabbit genome has been sequenced with high coverage (7.48x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway?db=oryCun2]. Compared to other large animal models (such as dogs, pigs, or sheep), rabbits are relatively inexpensive to purchase and house and they are easier to breed and handle. Specific vascular disease models in rabbits each have their own advantages and shortcomings as models of human disease that are beyond the scope of this manuscript8,12,18. An investigator should review these advantages and shortcomings to determine if the rabbit is the best model for answering a specific experimental question.
Introduction of deoxyribonucleic acid (DNA) regulatory sequences into endothelial cells in vivo enables investigation of the activity of these sequences in a complex physiologic environment. In vitro studies in transfected endothelial cells can be useful for the initial assessment of DNA regulatory sequences; however, expression levels in tissue culture models are sometimes not reproduced when the studies are repeated in vivo5,19,20. In vitro systems can also be useful for exploring basic pathways of protein signaling and endothelial physiology as well as communication between cultured vascular cells; however, more-complex pathways or regulatory networks that are influenced by complex populations of neighboring vascular cells or the immune system are best studied in an in vivo system6,20. The method described herein provides a platform for exploring regulation of transgene expression in the endothelium within the context of an intact vessel, with or without disease. The in vivo system also permits investigation of physiological and pathological cellular crosstalk and identification of contributions of the immune system to regulation of gene expression6.
Germ-line transgenesis (especially in mice) is an alternative approach for directing transgene expression to endothelial cells. This approach can provide life-long transgene expression, with endothelial targeting mediated by specific promoter or regulatory regions21,22. However, the generation of transgenic mice is time-consuming and expensive, several transgenic lines must be often tested to ensure targeting of the transgene to the desired cell type and achievement of adequate transgene expression levels, and experimental results in murine systems can be strain-dependent. Murine transgenic models with endothelial-targeted transgenes have many advantages: there is no need to perform surgery on every experimental animal in order to achieve transgenesis, experimental mice can be bred with numerous other available transgenic mice in order to test genetic and phenotypic interactions, and there is a wide selection of antibodies that react with murine proteins, facilitating characterization of phenotypes. However, targeting of transgenes to the endothelium via the germ line typically results in transgene expression throughout the vasculature,22 making it difficult to determine the site at which the transgene product is acting. This is especially true when the transgene product is secreted, because a transgene product secreted by endothelial cells throughout the vasculature could have biological activity at any number of sites within an animal. Although the method described in this manuscript requires technical expertise and specialized facilities, it can be less time consuming and less expensive than developing an endothelial-specific transgenic mouse line. It allows for the assessment of the function of a protein selectively in endothelial cells of a segment of large artery, and it permits use of the contralateral common carotid as a paired control (eliminating systemic factors that can vary among experimental animals-for example, blood pressure or cholesterol levels-as uncontrolled variables).
Gene therapy is a promising approach for the treatment of vascular diseases, particularly chronic diseases, because a single application can provide sustained or possibly life-long expression of a therapeutic gene23. The therapeutic promise of gene therapy has been explored in animal models of somatic gene transfer, often targeting the liver24,25, which is a relatively easy target because many blood-borne viral vectors are hepatotropic. However, to have an effect on vascular disease, gene therapy targeted to the liver must achieve systemic overexpression of proteins. This typically requires large doses of vector, which can be toxic or even fatal26. Moreover, increased systemic levels of a protein raise the risk of off-target side effects, which could complicate or even obscure interpretation of experimental results. Local gene therapy targeting vascular endothelium as described in this manuscript could avoid systemic side effects because the infused vector is not widely disseminated beyond the transduced arterial segment, and local vascular effects can be achieved without changes in systemic plasma levels of protein.27 In addition, a far lower amount of vector is needed to transduce an arterial segment than is needed to achieve robust hepatic transduction. Transgene expression from the liver has been reported to decline over time, probably due to cell turnover, requiring repeated dosing if high-level transgene expression is to be maintained.28 In contrast, the low turnover rate of the endothelium provides stable expression for at least 48 weeks in chow-fed rabbits and for at least 24 weeks in atherosclerotic lesions of cholesterol-fed rabbits.1,27
To determine if this method of gene transfer to rabbit common carotid endothelium is appropriate, the advantages and disadvantages (Table 1) should be considered in the context of the specific research goals. Advantages of this method include: outbred rabbits are better representative of human genetic diversity than are inbred mice (important for preclinical work); rabbits provide larger vessels for easier manipulation and more tissue for analysis; the method can achieve endothelium-targeted transgene expression far more quickly than does germ-line endothelial targeting in transgenic mice; vector dose can be easily adjusted to model variable levels of transgene expression; processes specific to large-artery endothelium can be investigated; and local vascular transgenesis allows the opposite carotid in the same animal to be used as a control, eliminating systemic factors as uncontrolled variables. Disadvantages include: special facilities and expertise are required; fewer genetically modified backgrounds on which to experiment are available in rabbits than in mice; and there is a less extensive selection of antibodies to rabbit versus mouse proteins (for immunodetection of transgene protein and other antigens that may be important in interpreting experimental results).

<table border="0" cellpadding="0" cellspacing="0" style="width:888px;" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Disposables</td>
			<td style="width:181px;"></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">3mL syringe with 24G needle</td>
			<td>Becton Dickinson</td>
			<td>309571</td>
			<td style="width:199px;">2x for gene transfer surgery; 3x for harvest surgery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">1mL syringe with 27G needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td style="width:199px;">6x for gene transfer surgery; 1x for harvest surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">20mL syringe, luer lock</td>
			<td>Nipro Medical Corp</td>
			<td>JD+20L</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheters, 24G x 3/4&#34;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">19G needle</td>
			<td>Becton Dickinson</td>
			<td>305187</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">21G needle</td>
			<td>Becton Dickinson</td>
			<td>305165</td>
			<td style="width:199px;">For 20 mL syringe of saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Gauze 4&#34; x 4&#34;</td>
			<td>Dynarex</td>
			<td style="width:149px;">3242</td>
			<td style="width:199px;">~10-15 per surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-244</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-182</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8648P</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>398A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alcohol swabs</td>
			<td>Covidien Ltd.</td>
			<td>6818</td>
			<td style="width:199px;">For placement of I.V. line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheter plug</td>
			<td>Vetoquinol</td>
			<td>411498</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Ketamine HCl, 100 mg/mL</td>
			<td>Vedco Inc.</td>
			<td>05098916106</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Xylazine, 100 mg/mL</td>
			<td>Akorn Inc.</td>
			<td>4821</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Lidocaine HCl, 2%</td>
			<td>Pfizer</td>
			<td>00409427702</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Bupivacaine HCl, 0.5%</td>
			<td>Pfizer</td>
			<td>00409161050</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Beuthanasia D-Special</td>
			<td>Intervet Inc.</td>
			<td>NDC 00061047305</td>
			<td style="width:199px;">Harvest surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Buprenorphine HCl, 0.3 mg/mL&#160;</td>
			<td>Patterson Veterinary</td>
			<td>12496075705</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Saline IV bag, 0.9% sodium chloride</td>
			<td>Baxter</td>
			<td>2B1309</td>
			<td style="width:199px;">2x for gene transfer surgery; can use vial of sterile saline in place of one</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Heparin&#160; (5000 U/mL)</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-047-10</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Fentanyl patch, 25 mcg/hr&#160;</td>
			<td>Apotex Corp.</td>
			<td>NDC 60505-7006-2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:359px;">Gene transfer vector</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;">Dilute 350 &#181;L per artery; 2 x 1011 vp/mL for adenovirus; gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Instruments</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Metzenbaum needle holder 7&#34; straight</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7900</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Operating scissors 6.5&#34; straight blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6828</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Needle holder /w suture scissors</td>
			<td>Miltex</td>
			<td style="width:149px;">8-14-IMC</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo needle holder, 5.75&#34; straight with lock</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6412</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Stevens scissors 4.25&#34; curved blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5943</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alm retractor 4&#34; 4X4 5mm blunt prongs</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6514</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Backhaus towel clamp 3.5&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">4x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Micro clip setting forceps 4.75&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6496</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Micro vascular clips, 11 mm</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">2x for gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surg-I-Loop</td>
			<td>Scanlan International</td>
			<td style="width:149px;">1001-81M</td>
			<td style="width:199px;">5 cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Bonaccolto forceps, 4&#8221; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5210</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Dumont #3 forceps Inox tip size .17 X .10mm</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5042</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Graefe forceps, 4&#8221; (10 cm) long serrated straight, 0.8mm tip</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5280</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; straight, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7110</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; curved, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7111</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Jacobson mosquito forceps 5&#34; curved extra delicate, 0.9 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7117</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Kantrowitz forceps, 7.25&#34; 90 degree delicate, 1.7 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7305</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Tissue forceps 5&#34;, 1X2 teeth, 2 mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-8162</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11092-12</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Adson forceps, 12 cm, serrated, straight</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11006-12</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery handpiece and electrode</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">HPAC-1; R-F11</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Suite Equipment</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Circulating warm water blanket and pump</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Forced air warming unit</td>
			<td>3M</td>
			<td style="width:149px;">Bair Hugger Model 505</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">IV infusion pump</td>
			<td>Heska</td>
			<td style="width:149px;">Vet IV 2.2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane vaporizer and scavenger</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary multi-parameter monitor</td>
			<td>Surgivet</td>
			<td style="width:149px;">Surgivet Advisor</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery unit</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">MV-9</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical microscope</td>
			<td style="width:181px;">D.F. Vasconcellos</td>
			<td style="width:149px;">M900</td>
			<td style="width:199px;">Needs ~16x magnification</td>
		</tr>
	</tbody>
</table>

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.

To implement this method with confidence, preliminary experiments are necessary to establish that the operator achieves efficient and reproducible gene transfer, with transgene expression primarily in luminal endothelial cells. In our experience, this is most easily assessed using a vector that expresses &#946;-galactosidase. 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal) staining of common carotid segments removed 3 days after vector infusion, as well as measurement of &#9...

Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

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.

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 ...

The overall goal of this surgical procedure is to introduce a transgene into the endothelial cells of rabbit carotid arteries. This method helps answer questions in vascular biology and gene therapy about the roles of arterial wall transgene products and the roles of DNA-regulatory sequences in controlling transgene expression. This technique allows the efficient expression of transgenes within a focal segment of the vascular endothelium, allowing the site-specific study of the biological roles of endothelial-expressed genes.Generally, individuals new to this method will struggle as new investigators often puncture back wall of the artery and do not tie off all of the small side branches. After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and shave both ears and the left-rear middle toe. Insert a 24 gauge IV catheter into the left ear vein and apply a fentanyl patch to the right ear.Shave the anterior neck from the sternal notch to the edge of the mandible. Place the rabbit in the supine position on the operating table with a rolled neck support towel just below the animal's head. Gently extend the rabbit's neck until it is straight and approximately horizontal.To begin the surgery, use electrocautery to cut the skin approximately seven to nine centimeters along the midline. Clamp the tissue open with towel clamps and make a short lateral cut through the fascia with the electrocautery. Insert large scissors into the cut to bluntly dissect the fascia along the midline and cut through the dissected fascia with the electrocautery device.Beginning on the right side, use small dissecting scissors to dissect between the V-shaped sternocephalic muscles and the sternohyoid muscle over the trachea to expose the common carotid arteries. Dissect the right common carotid artery from the surrounding tissues from the base of the neck caudally to the crossing of the pharyngeal nerve cranially and use surgical silicon loops to aid in the retraction of the artery. Ligate any branches coming off of each side of the common carotid artery with 5-0 silk sutures.Then cut each branch distally one to two millimeters away from the sutures to free a four to five centimeter segment of the carotid. Next, move a surgical microscope into position and drape a sterile towel over the microscope to allow manipulation of the microscope without contaminating the sterile field. Place two silk ties, each with a loose overhand knot, around the midportion of the mobilized carotid segment.Place a vascular clip at the cranial end of the isolated artery segment to allow the artery to fill, then place a vascular clip at the caudal end of the artery. Using a 19 gauge needle bent just above the bevel at an approximately 80-degree angle, puncture the artery just cranial to the caudal vascular clip, taking care not to puncture the back or side walls of the vessel. Advance the needle tip into the lumen, withdrawing the tip twice to make sure the arteriotomy completely traverses the artery wall before carefully withdrawing the needle completely.Next, bend an IV catheter about four millimeters from the tip to an approximately 75-degree angle and loosely attach the IV catheter to a syringe filled with sterile tissue culture medium. Insert the catheter into the artery up to the bend point and fill the artery with 250 microliters of medium, then gently press with a gloved finger along the artery from just caudal to the cranial vascular clip to the arteriotomy to flush out the medium. After a second wash as just demonstrated, tighten the two silk sutures around the catheter tip to seal the lumen and attach a syringe containing the virus solution to the catheter.Gently depress the syringe plunger to infuse 250 microliters of the virus until the artery is distended to its physiological caliber and gently lay the syringe on a nest of gauze for support. Place a 7-0 polypropylene suture in the common carotid adventitia just caudal to the cranial vascular clip to mark the cranial boundary of gene transduction. After 20 minutes, replace the virus-containing syringe with an empty syringe and gently aspirate the virus-containing solution until the vessel collapses.Then remove the syringe, silk ties, and catheter. Using 7-0 polypropylene suture, make a loose X-pattern across the arteriotomy. Very briefly release the cranial vascular clip to flush the artery of any air or residual virus and tighten the suture.Release the cranial and caudal vascular clips using light pressure and gauze to stop any bleeding and close the midline fascia with a continuous suture, then close the skin with an intradermal pattern with buried knots at both ends. To harvest the transduced carotid arteries at the appropriate experimental endpoint, use 3-0 silk suture to ligate the common carotid artery cranially and caudally to the segment infused with the vector. The 7-0 suture placed in the adventitia marks the cranial end and the arteriotomy marks the caudal end.Excise the carotid segment between the ligations and flush the lumen with saline. Then trim away any excess adventitia and cut the transduced carotid segment into smaller pieces for the appropriate experimental endpoint analyses. En face images of the lumenal surfaces of rabbit common carotid arteries infused with adenovirus expressing beta-galactosidase demonstrate a robust endothelial staining with X-gal.Axial images of the lumenal surfaces of intact carotid rings reveal X-gal staining primarily on the lumenal surface. X-gal staining is also observed primarily in the endothelial cells of axially-opened vessels, although a small amount of staining is found in the adventitial layer. Analysis of RNA extracted from the vessel segments reveals substantial over-expression of transgenes relative to their endogenous levels, with considerable intra-and inter-artery variability in transduction efficiency and transgene expression observed among arteries transduced by experienced operators.Once mastered, the rabbit common carotid artery gene transfer surgical procedure can be completed in less than two hours. Following this procedure, RT-PCR, immunohistochemistry, Western blotting, or other methods can be performed to answer questions about the role of DNA-regulatory sequences in altering transgene expression and the role of transgene products within the artery wall. After its development, this technique paved the way for the study of gene therapy in treatment of vascular diseases such as arterial atherosclerosis and of vein graft disease.Don't forget proper bio-safety precautions should always be taken when working with viral vectors and sharp objects.

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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 ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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 ...

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 ...

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 ...

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, ...

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 ...