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.

The authors have nothing to disclose.

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. It is also important to avoid damaging the vagus nerve, which runs parallel to the common carotid artery. Trimming the adventitia in the area of the planned arteriotomy will help the operator both to perform a clean and functional arteriotomy and to repair the arteriotomy. Finally, branches off the common carotid artery must be identified and securely ligated to prevent leakage of the vector from the carotid lumen.
There are also critical aspects of vector infusion and arteriotomy repair. During vector infusion, it is important to distend the common carotid to physiological or slightly greater caliber to achieve efficient transduction. When repairing the arteriotomy, the suture should penetrate the vessel wall as close as possible to the edges of the arteriotomy, and should cleanly enter and exit the lumen rather than pass axially through the vessel wall. If only the outer layers of the vessel wall are pulled together because the suture passes axially through the vessel wall rather than passing radially through the wall and entering the lumen, a gap will remain in the intima and the risk of thrombosis will increase. If the suture penetrations are too widely spaced, or if the suture is over-tightened when tied, tissue folds will be created at the arteriotomy site. These folds disrupt normal laminar blood flow, also increasing thrombosis risk. Maintaining consistent flow characteristics is important for experiments performed in animal models of diseases (e.g., atherosclerosis) because altered flow can contribute to the disease process30.
New operators often encounter thrombosed arteries at harvest. Luminal thrombosis is a devastating complication, rendering the artery unusable as an experimental sample. To prevent thrombosis, we routinely administer IV heparin before gene transfer. Thrombosis is also prevented by careful closure of the arteriotomy, as described above. The heparin dosage could be increased to prevent thrombosis, or aspirin could be given postoperatively. However, a higher dose of heparin would also increase the potential for bleeding complications, and aspirin could interfere with experimental end points, especially if inflammation is being studied. Therefore, it is preferable to focus on improved surgical technique as a means of preventing thrombosis.
If manipulated too vigorously, carotid arteries may undergo spasm, which could also contribute to thrombosis by decreasing flow. If vasospasm is encountered, it can be relieved by application of topical papaverine. When vessel harvests are planned for more than 2 - 3 days after transduction, and thrombosis is a concern, transcutaneous ultrasound can be used to noninvasively assess vessel patency. Discovery of thrombosed arteries may lead to euthanasia in order to save on housing costs. Additional animals can also be enrolled promptly, to fill experimental groups. Peri- and post-intervention mortality associated with this method should be &#60;1% (i.e., not different from mortality associated with other surgeries performed on healthy rabbits)31.
After an operator becomes comfortable with the technical aspects of the surgical protocol, an ability to perform efficient gene transfer to the endothelium needs to be verified, using approaches described in the section above on representative results. If issues arise with achieving reproducible gene transfer, the culprit is likely in one of two areas. After the vessel is isolated and the blood is washed from the lumen, it is important to remove the DMEM wash buffer so that the infused vector solution is not diluted during transduction. The other important aspect is to distend the vessel to physiological or slightly greater caliber during vector infusion. Because, in our experience, a carotid artery that is not fully distended or does not remain distended during the 20-minute infusion reliably has low transgene expression, it is likely that distension of the artery to physiologic caliber improves the transduction efficiency. However, we have never studied this systematically. It is possible that increasing the infusion pressure above physiological levels could increase transduction efficiency, and also allow higher levels of transduction of cells in the vascular media. However, disruption of the endothelial barrier would likely damage the vessel and increase the risk of thrombosis. If the vessel does not remain distended for the entire 20-minute vector-incubation period, it is likely that the vector is leaking from branches of the common carotid artery. Be careful during dissection to identify and ligate all branches to prevent leakage of the vector from the carotid lumen.
This method has several limitations that are related to the use of rabbits. Rabbits are less expensive to house and feed than other large animals (dogs, pigs, sheep); however, the costs of purchasing, housing, and feeding rabbits are considerably more than for mice and rats. The operating room facilities and regulatory requirements for rabbit surgeries are also far more extensive than for rodents. In addition, considerable technical expertise is needed to perform the surgical gene transfer protocol effectively. This expertise can be acquired by operators who have had no formal surgical training. At least 2 individuals in our group (including the primary author of this manuscript) have learned the surgical techniques and applied them productively. Nevertheless, meticulous training and a careful validation of an operator&#39;s gene transfer efficiency and reproducibility (as described in the representative results section) are needed before the operator can begin to generate high-quality data.
Confinement of transgene expression almost exclusively to the endothelium with this method29,32,33,34,35,36,37 is useful in that it allows investigation of transgene effects in endothelial cells and measurement of activity of cis-acting DNA regulatory sequences in endothelial cells. A small number of adventitial or medial cells may be transduced; however, the inability of this method to efficiently transduce nonendothelial cells prevents the application of the method to the study of other types of vascular wall cells (such as smooth muscle cells and macrophages). Although overexpression of secreted proteins from transduced endothelial cells can allow investigation of effects of these proteins on other vascular cell types, the roles of non-secreted proteins in these other vascular cell types (e.g. receptors or proteins involved in signal transduction) cannot be investigated with this method.
As a means for testing artery wall-targeted gene therapy, the method differs from other preclinical methods in two major ways. First, the method utilizes a rabbit model for in vivo testing of gene therapy rather than more commonly used rodent models8,12,15,38,39. The use of rabbits allows assessment of transgene expression and the biological role of the transgene product in an outbred animal model, which is more representative of human genetic diversity than are inbred rodents. The model can be used to assess gene therapy in either normal rabbit arteries or in rabbit arteries that have arterial pathology that is similar to that found in diseased human arteries. Rabbit arteries also are closer in size to human arteries than are rodent arteries and provide far more tissue for analysis than is available from rodent arteries. The second major difference is that this method uses a surgical approach to deliver transgene vector to the vascular endothelium. Somatic gene therapy is often delivered systemically, most frequently by targeting the liver with the goal of altering plasma levels of the transgene protein25,40. By targeting vascular endothelium, the method supplies the therapeutic transgene product locally, with its peak concentration within the artery wall, precisely where it is needed for vascular disease treatment. By delivering the transgene only locally, the method also eliminates systemic side effects of the transgene product. Other groups are developing methods using peptides, antibodies, or other capsid modifications for targeting systemically injected vectors to healthy or diseased endothelium to provide local vascular gene therapy41,42,43. However, these targeting methods remain under development, and are still complicated by substantial systemic transduction, especially in the liver42,43,44. Our surgical method provides a precise and efficient introduction of transgenes to the vascular endothelium with minimal - if any - transduction at other locations.1 The method is also a more convenient, efficient, and higher-throughout mean (compared to germ-line transgenesis or systemic injection of endothelial-targeted vectors) for testing the activity of DNA regulatory sequences in the large vessel endothelium, for testing transgene protein function in the artery wall, and for testing vessel-wall-targeted gene therapy.
This method allows investigation of the biological role of any transgene, when it is expressed in the large artery endothelium. If modified to express loss-of-function reagents such as dominant negative receptors or short hairpin RNA, it would permit investigation of the roles of endogenous endothelial proteins and signaling pathways. The method can also be used to measure the transcriptional activity of any cis-acting DNA sequence in large artery endothelial cells5,6,7. In addition, the method allows testing of any type of gene transfer vector for efficiency and safety when delivered locally to endothelium, and will reveal the ability of the vector to achieve durable transgene expression. Finally, the method allows development and testing of combinations of vectors and transgenes that are designed to deliver vascular wall-targeted gene therapy. The method could serve as a screening tool to identify therapeutic genes that might-in the future-be delivered to the endothelium of all large arteries (or all diseased large arteries) by percutaneously injected vascular wall-targeted vectors. Because of pre-existing immunity in humans to adenovirus type 545, it will be challenging to use adenovirus type 5-based vectors in clinical applications. Engineering of the adenovirus 5 capsid, use of alternative adenovirus serotypes46, or use of less-immunogenic vectors such as AAV may be required to bring vascular gene therapy to the clinic. As an experimental tool, however, we are unaware of any vector that can compete with helper-dependent adenovirus 5 in achieving efficient and durable transgene expression in blood vessels1.

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><tbody><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3mL syringe with 24G needle</td><td>Becton Dickinson</td><td>309571</td><td>2x for gene transfer surgery; 3x for harvest surgery</td></tr><tr><td>1mL syringe with 27G needle</td><td>Becton Dickinson</td><td>309623</td><td>6x for gene transfer surgery; 1x for harvest surgery</td></tr><tr><td>20mL syringe, luer lock</td><td>Nipro Medical Corp</td><td>JD+20L</td><td></td></tr><tr><td>Catheters, 24G x 3/4&quot;</td><td>Terumo Medical Products</td><td>SROX2419V</td><td></td></tr><tr><td>19G needle</td><td>Becton Dickinson</td><td>305187</td><td>Gene transfer surgery only</td></tr><tr><td>21G needle</td><td>Becton Dickinson</td><td>305165</td><td>For 20 mL syringe of saline</td></tr><tr><td>Gauze 4&quot; x 4&quot;</td><td>Dynarex</td><td>3242</td><td>~10-15 per surgery</td></tr><tr><td>3-0 silk suture</td><td>Covidien Ltd.</td><td>S-244</td><td></td></tr><tr><td>5-0 silk suture</td><td>Covidien Ltd.</td><td>S-182</td><td>Gene transfer surgery only</td></tr><tr><td>7-0 polypropylene suture</td><td>CP Medical</td><td>8648P</td><td>Gene transfer surgery only</td></tr><tr><td>5-0 polyglycolic acid suture</td><td>CP Medical</td><td>421A</td><td>Gene transfer surgery only</td></tr><tr><td>3-0 polyglycolic acid suture</td><td>CP Medical</td><td>398A</td><td>Gene transfer surgery only</td></tr><tr><td>Alcohol swabs</td><td>Covidien Ltd.</td><td>6818</td><td>For placement of I.V. line</td></tr><tr><td>Catheter plug</td><td>Vetoquinol</td><td>411498</td><td>Gene transfer surgery only</td></tr><tr><td>Ketamine HCl, 100 mg/mL</td><td>Vedco Inc.</td><td>05098916106</td><td></td></tr><tr><td>Xylazine, 100 mg/mL</td><td>Akorn Inc.</td><td>4821</td><td></td></tr><tr><td>Lidocaine HCl, 2%</td><td>Pfizer</td><td>00409427702</td><td></td></tr><tr><td>Bupivacaine HCl, 0.5%</td><td>Pfizer</td><td>00409161050</td><td></td></tr><tr><td>Beuthanasia D-Special</td><td>Intervet Inc.</td><td>NDC 00061047305</td><td>Harvest surgery only</td></tr><tr><td>Buprenorphine HCl, 0.3 mg/mL&amp;nbsp;</td><td>Patterson Veterinary</td><td>12496075705</td><td>Gene transfer surgery only</td></tr><tr><td>Saline IV bag, 0.9% sodium chloride</td><td>Baxter</td><td>2B1309</td><td>2x for gene transfer surgery; can use vial of sterile saline in place of one</td></tr><tr><td>Heparin&amp;nbsp; (5000 U/mL)</td><td>APP Pharmaceuticals</td><td>NDC 63323-047-10</td><td>Gene transfer surgery only</td></tr><tr><td>Fentanyl patch, 25 mcg/hr&amp;nbsp;</td><td>Apotex Corp.</td><td>NDC 60505-7006-2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Gene transfer vector</td><td></td><td></td><td>Dilute 350 &amp;micro;L per artery; 2 x 10&lt;sup&gt;11&lt;/sup&gt; vp/mL for adenovirus; gene transfer surgery only</td></tr><tr><td>&lt;strong&gt;Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metzenbaum needle holder 7&quot; straight</td><td>Roboz</td><td>RS-7900</td><td>Gene transfer surgery only</td></tr><tr><td>Operating scissors 6.5&quot; straight blunt/blunt</td><td>Roboz</td><td>RS-6828</td><td></td></tr><tr><td>Needle holder /w suture scissors</td><td>Miltex</td><td>8-14-IMC</td><td>Gene transfer surgery only</td></tr><tr><td>Castroviejo scissors</td><td>Roboz</td><td>RS-5658</td><td></td></tr><tr><td>Castroviejo needle holder, 5.75&quot; straight with lock</td><td>Roboz</td><td>RS-6412</td><td>Gene transfer surgery only</td></tr><tr><td>Stevens scissors 4.25&quot; curved blunt/blunt</td><td>Roboz</td><td>RS-5943</td><td></td></tr><tr><td>Alm retractor 4&quot; 4X4 5mm blunt prongs</td><td>Roboz</td><td>RS-6514</td><td>2x</td></tr><tr><td>Backhaus towel clamp 3.5&quot;</td><td>Roboz</td><td></td><td>4x</td></tr><tr><td>Micro clip setting forceps 4.75&quot;</td><td>Roboz</td><td>RS-6496</td><td>Gene transfer surgery only</td></tr><tr><td>Micro vascular clips, 11 mm</td><td>Roboz</td><td></td><td>2x for gene transfer surgery only</td></tr><tr><td>Surg-I-Loop</td><td>Scanlan International</td><td>1001-81M</td><td>5 cm length</td></tr><tr><td>Bonaccolto forceps, 4&amp;rdquo; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td><td>Roboz</td><td>RS-5210</td><td></td></tr><tr><td>Dumont #3 forceps Inox tip size .17 X .10mm</td><td>Roboz</td><td>RS-5042</td><td></td></tr><tr><td>Graefe forceps, 4&amp;rdquo; (10 cm) long serrated straight, 0.8mm tip</td><td>Roboz</td><td>RS-5280</td><td></td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; straight, 1.3mm tips</td><td>Roboz</td><td>RS-7110</td><td>2x</td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; curved, 1.3mm tips</td><td>Roboz</td><td>RS-7111</td><td></td></tr><tr><td>Jacobson mosquito forceps 5&quot; curved extra delicate, 0.9 mm tips</td><td>Roboz</td><td>RS-7117</td><td></td></tr><tr><td>Kantrowitz forceps, 7.25&quot; 90 degree delicate, 1.7 mm tips</td><td>Roboz</td><td>RS-7305</td><td></td></tr><tr><td>Tissue forceps 5&quot;, 1X2 teeth, 2 mm tip width</td><td>Roboz</td><td>RS-8162</td><td></td></tr><tr><td>Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td><td>Fine Science Tools</td><td>11092-12</td><td>2x</td></tr><tr><td>Adson forceps, 12 cm, serrated, straight</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Veterinary electrosurgery handpiece and electrode</td><td>MACAN Manufacturing</td><td>HPAC-1; R-F11</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Suite Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Circulating warm water blanket and pump</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Forced air warming unit</td><td>3M</td><td>Bair Hugger Model 505</td><td>Gene transfer surgery only</td></tr><tr><td>IV infusion pump</td><td>Heska</td><td>Vet IV 2.2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane vaporizer and scavenger</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Veterinary multi-parameter monitor</td><td>Surgivet</td><td>Surgivet Advisor</td><td></td></tr><tr><td>Veterinary electrosurgery unit</td><td>MACAN Manufacturing</td><td>MV-9</td><td></td></tr><tr><td>Surgical microscope</td><td>D.F. Vasconcellos</td><td>M900</td><td>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 &#946;-galactosidase mRNA (messenger RNA) with quantitative reverse transcription polymerase chain reaction (qRT-PCR), will reveal efficiency, reproducibility, and location of the transduced cells. For experiments that investigate the effects of transgene expression or measure the activity of cis-acting transcriptional elements, we typically measure transgene mRNA as an initial indication of level and reproducibility of gene transfer.
A new operator in our laboratory sought to establish proficiency with this method by transducing rabbit common carotid arteries with an adenoviral vector (2 x 1011 vp/mL) expressing a &#946;-galactosidase transgene, driven by a cytomegalovirus (CMV) promoter. Transduced arteries were harvested 3 days later and were cut transversely into segments. Individual segments were then either cut open axially or left as intact rings. All of the segments were X-gal stained in microcentrifuge tubes. Segments that had been cut open axially were placed on a horizontal surface and the luminal surface maximally exposed, using pins as needed to prevent the segment from curling up. En face images of the luminal surfaces showed robust endothelial staining with X-gal (Figures 3A and 3B). Axial images of the luminal surfaces of intact carotid rings showed X-gal staining only on the luminal surface (Figures 3C and 3D). The segments that had been opened axially were processed into paraffin, sectioned, and counter-stained with either hematoxylin and eosin or nuclear fast red. Images show X-gal staining primarily in the endothelium, although there is a small amount of staining in the adventitial layer (Figures 3E and 3F). Transduction of adventitial cells could occur via leakage of vector through the arteriotomy site or by leakage via small branches proximal to the sites of side branch ligation.29
In a separate experiment, a new operator sought to establish proficiency in achieving reproducible levels of transgene expression, as a prelude to experiments aimed at investigating biological activities of the transgenes. Rabbit common carotid arteries were transduced with helper-dependent adenovirus (2 x 1011 vp/mL) containing either an apo A-I (HDAdApoAI) or IL-10 (HDAdIL10) transgene, both under control of a CMV promoter. Transduced arteries were harvested 3 days after transduction and cut transversely into segments. RNA was extracted from the vessel segments, and apo A-I and IL-10 mRNA expressions were quantified by qRT-PCR, with normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the same extracts (Figure 4). In HDAdIL10-transduced arteries, only 1 out of 6 arteries had a very low, but detectable apo A-I mRNA signal. Mean expression of apo A-I mRNA was 700-fold greater in the vessels transduced with HDAdApoAI than in vessels transduced with HDAdIL10. Low levels of endogenous IL-10 mRNA were detected in HDAdApoAI-transduced arteries, with mean expression increased 6-fold in HDAdIL10-transduced arteries. Of note, there is considerable intra- and inter-artery variability in transduction efficiency and transgene expression, as shown in Figures 3 and 4, respectively. We find this variability even with experienced operators.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56982/56982fig1.jpg" /> 
Figure 1. Arteriotomy in common carotid artery. Fine forceps are used to grasp the carotid artery adventitia and apply upward traction to the artery. This maneuver expands the vessel lumen and generates a vertical surface into which the bent 19G needle is inserted, thereby minimizing the risk of puncturing the back wall of the artery. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56982/56982fig2.jpg" /> 
Figure 2. Closure of common carotid arteriotomy.&#160;The arteriotomy is closed with a 7-0 polypropylene suture, using an X-pattern. The first pass of the needle and suture enters the artery lumen at bottom right of the arteriotomy (site 1) and exits the lumen at the bottom left (site 2). The needle and suture then cross the arteriotomy and re-enter the lumen at the top right (site 3). The needle then exits the lumen at the top left (site 4). Gentle traction on both ends of the suture closes the arteriotomy. The suture ends (exiting from site 1 and site 4) are tied with 2 square knots. Grey circles represent sites of sutures passing through the vessel wall. Solid blue lines indicate areas where the suture is outside of the vessel wall. Dotted blue lines indicate areas where the suture is within the lumen. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56982/56982fig3.jpg" /> 
Figure 3. Efficient endothelial transgene expression. Rabbit common carotid arteries were transduced with an adenoviral vector expressing a &#946;-galactosidase transgene and harvested 3 days later. Transduced artery segments were X-gal stained either as intact rings or after being opened with an axial cut. (A-B) En face images of the luminal surfaces of carotid rings that were cut open axially. (C-D) Axial views into the luminal space of intact carotid rings. (E-F) X-gal stained, paraffin-embedded carotid segments were sectioned and counter-stained with either (E) hematoxylin and eosin or (F) nuclear fast red. Scale bar = 100 &#181;m. I = intima; M = media; and A = adventitia. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56982/56982fig4.jpg" /> 
Figure 4. Quantification of transgene mRNA expression. Rabbit common carotid arteries were transduced with helper-dependent adenovirus expressing either apo A-I (HDAdApoAI) or IL-10 (HDAdIL10) under control of a CMV promoter. Arteries were harvested 3 days later. mRNA expression of (A) Apo A-I and (B) IL-10 were quantified by qRT-PCR, normalized to GAPDH mRNA in the same artery, and expressed as arbitrary units (AU). Bar indicates mean value; P value is from rank-sum test. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56982/56982fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Advantages</td>
			<td>Disadvantages</td>
		</tr>
		<tr>
			<td rowspan="5">Compared to Rodent Models</td>
			<td>Closer phylogenetically to primates&#160;</td>
			<td>More expensive to purchase and house</td>
		</tr>
		<tr>
			<td>Greater genetic diversity, easing clinical translation&#160;</td>
			<td>More difficult to breed and handle</td>
		</tr>
		<tr>
			<td>Larger vessels allow easier surgical manipulation and provides more tissue for quantitative analysis</td>
			<td>More extensive regulatory requirements</td>
		</tr>
		<tr>
			<td>Allows use of endovascular devices designed for humans</td>
			<td>Fewer genetically modified backgrounds&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>Less-extensive selection of antibodies to rabbit proteins&#160;</td>
		</tr>
		<tr>
			<td rowspan="2">Compared to Larger Animal Models</td>
			<td>Relatively inexpensive to purchase and house&#160;</td>
			<td>Possibly less clinically relevant than other large animal models for some vascular diseases</td>
		</tr>
		<tr>
			<td>Easier to breed and handle</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Compared to Germ-line Transgenesis</td>
			<td>Transgene expressed only in large artery; effect of transgene specifically at site of interest can be determined</td>
			<td>Application of method to cells other than endothelium is difficult</td>
		</tr>
		<tr>
			<td>Can use contralateral carotid as a paired control; eliminates systemic parameters (e.g., blood pressure, cholesterol level) as uncontrolled variables&#160;</td>
			<td>Operating room and surgical expertise required; core facilities likely not available</td>
		</tr>
		<tr>
			<td>Higher throughput for testing DNA regulatory sequence activity in large vessel endothelium</td>
			<td>Transgene cannot be expressed in most vascular beds</td>
		</tr>
		<tr>
			<td>Potentially quicker and less expensive</td>
			<td></td>
		</tr>
		<tr>
			<td>Systemic exposure to transgenic protein unlikely &#8212;minimizes off-target effects</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="5">Compared to Systemic Gene Therapy Approaches 
			(e.g,. Liver Transduction via Peripheral Vein injection)</td>
			<td>More-stable transgene expression than systemic (liver) gene therapy</td>
			<td>Vector delivery requires surgical intervention</td>
		</tr>
		<tr>
			<td>Transgene expressed in artery wall, allowing local delivery of high levels of transgenic protein</td>
			<td>Operating room and surgical expertise required</td>
		</tr>
		<tr>
			<td>Systemic exposure to transgenic protein unlikely &#8212;minimizes off-target effects</td>
			<td>Treatment limited to arteries that are specifically targeted for intervention; does not treat systemic factors (e.g., lipids)</td>
		</tr>
		<tr>
			<td>Can use contralateral carotid as a paired control; eliminates systemic parameters (e.g., blood pressure, cholesterol level) as uncontrolled variables&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Far lower vector dose required</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Advantages and disadvantages of rabbit common carotid artery endothelial-selective gene transfer model.

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 ...

in-vivo-gene-transfer-to-the-rabbit-common-carotid-artery-endothelium

Nanyang Technological University

Research

JoVE Journal

Medicine

9.2K Views.  University of Washington. Deze methode is het introduceren van een transgenic in het endotheel van konijn halsslagaderen. Invoering van de transgenic zodat kan worden nagegaan van de biologische rol van de transgenic product in normale slagaders of ziekte modellen. De methode is ook handig voor het meten van de activiteit van regelgevende opeenvolgingen van DNA.

Video: In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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 ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

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, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Bioengineering

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model

Source: Nishio, H., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
This video demonstrates the interposition of the rabbit carotid artery with the jugular vein to induce venous intimal hyperplasia by subjecting the veins to arterial pressure. This model will help investigate molecular and genetic interventions to attenuate the progression of intimal hyperplasia after revascularization surgeries.

Source: Nishio, H., et al. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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