Name: phase contrast magnetic resonance imaging in the rat common carotid artery
Uploaded: 2018-09-05T15:30:00
Description: Watch this Scientific Journal Video about Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery at JoVE.com

This work was supported by grants from Ministry of Science and Technology, Taiwan, under grant number of MOST-105-2314-B-039-044-MY2.

The Institutional Care and Use Committees (IACUC) of the China Medical University approved all procedures.
1. Animal Preparation and Monitoring
<ol>
	<li>Leave all magnetically susceptible objects such as wallets, keys, credit card, etc. outside the scanner room prior to commencing animal preparation for MRI scanning.</li>
	<li>Initially anesthetize the rat (2-month-old male Sprague-Dawley (SD) rat, 280&#8211;350 g) in an induction box using a mixture of 5% isoflurane (ISO) and oxyg...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+2D+and+3D+phase+contrast+MRI:+optimized+analysis+of+blood+flow+and+vessel+wall+parameters.">Quantitative 2D and 3D phase contrast MRI: optimized analysis of blood flow and vessel wall parameters.</a> Magn Reson Med. 60 (5), 1218-1231 (2008).</li><li>Dall'Armellina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+quantification+of+regional+cardiac+function+in+mice+using+phase-contrast+MRI.">Improved method for quantification of regional cardiac function in mice using phase-contrast MRI.</a> Magn Reson Med. 67 (2), 541-551 (2012).</li><li>Peng, S. L., Ravi, H., Sheng, M., Thomas, B. 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PC-MRI is a comprehensive approach for non-invasive and longitudinal evaluation of blood flow. We present a protocol for performing PC-MRI of the rat CCA. This procedure is easy to perform in any animal MRI scanner and demonstrates good reproducibility.
The PC-MRI technique has gained increasing popularity in human 10,11 as well as animal studies 4,12. Among these studies, resu...

Magnetic resonance imaging (MRI) is a versatile approach that provides detailed information on internal body structures and physiology, and is increasingly being used for clinical diagnosis and in preclinical animal studies. Animal models are vital for better understanding of considerable clinical implication 1. As animal models considerably differ from humans with respect to anesthesia requirements and physiological parameters, optimization of MRI procedures for such animals assumes importance.
Phase contrast MRI (PC-MRI) is a specialized type of MRI that uses the velocity of flowing spins to quantify flow-related parameters such as blood flow. With PC-MRI, mapping flow patterns in the major arteries using animal models can help shed light on cardiovascular pathologies 2. Moreover, PC-MRI can non-invasively monitor the inherent alternations in blood flow in pathophysiological conditions 3. These observations suggest that PC-MRI is a valuable approach that can be used in animal models of human cardiovascular diseases.
In this report, we describe a method for the quantification of blood flow in the common carotid artery (CCA) of rats. The two CCAs supply the head and neck with oxygenated blood, and the carotid artery disease is a major cause of stroke. Therefore, detecting the early pathology in the CCA is pivotal. This procedure has a duration of approximately 15 min and can be potentially applied to conditions with hemodynamics alterations, such atherosclerosis or stroke.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">7T small animal MRI system</td>
			<td style="width:123px;">Bruker</td>
			<td style="width:344px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane&#160;</td>
			<td>Baxter</td>
			<td>1001936040</td>
			<td>anesthetic</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ECG lead&#160;</td>
			<td>3M</td>
			<td>2269T</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matlab</td>
			<td>MathWorks</td>
			<td></td>
			<td>sofeware for image processing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Monitoring and gating system</td>
			<td>SA instruments, Inc</td>
			<td>Model 1030</td>
			<td style="width:193px;"></td>
		</tr>
	</tbody>
</table>

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.

Correct slice geometry is pivotal for ensuring the success of the PC-MRI experiment. The accurate image plane positioning yields a &#34;round&#34; artery shape (Figure 3a), and as angulation increases, i.e., when it is less perpendicular to the artery, the resulting artery geometry becomes ovoid, leading to larger partial volume effects (Figure 3b). Severe partial volume effects would lead to the overestimation of blood ...

Watch this Scientific Journal Video about Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery at JoVE.com

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

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 ...

This method can answer key question in cardiovascular field relating to blood flow abnormality. The main advantage of this technique is that it is noninvasive. It can be done in any small animal MRI system.The implication of this technique extend toward diagnosis of cardiovascular diseases because it can shed light on the hemodynamic alterations. Visual demonstration of this method is critical, as triggered scan are important for successful phase contrast MRI scan, but are difficult to learn and are not commonly used in routine scans. Demonstrating the procedure will be Chiu Shao-Chieh and Hsu Shih-Ting, research assistant from our center.Prior to performing this technique, remove all magnetically susceptible objects, such as wallets, keys, and credit cards, and leave them outside of the scanner room. Following the initial dose of anesthesia, place the rat in the MRI bed in a head-first prone position, and deliver two to 3%isoflurane through a nose cone to maintain anesthesia. Monitor the animal's breathing by placing a respiratory pillow sensor under the animal's torso, and connect the sensor to a monitoring system.Check that the rat's respiration rate is between 40 and 50 beats per minute before proceeding. For cine-gated PC-MRI acquisition, place one electrode on the right forepaw and one on the left hindpaw. Twist the electrocardiography cables together, and ensure that they are connected to the monitor.Next, position the rat in a head holder with ear bars and a bite bar to secure the animal and to restrict its head movement. Then, turn on the warm air heating system or use gauze pads to help the rat maintain its body temperature while in the MRI. Finally, ensure that the R-wave is clear on the ECG monitor, and place the animal in the scanner.Once the animal is found to be physiologically stable, start the MRI scans. Select the localizer sequence from the console monitor of the MRI scanner, and acquire scout images along all three orientations using any fast image acquisition sequence, such as the fast spin echo, to create coronal, axial, and sagittal images. Look at the scout images to ensure that the center of the animal's head and neck is at the center of the magnet.If necessary, adjust the animal's position until the correct position is reached. If the animal is repositioned, repeat the scout image scans. Once the rat is properly positioned, select the time-of-flight angiogram sequence from the console monitor of the MRI scanner, and acquire a 2D time-of-flight angiogram first to ascertain the precise anatomical location of the common carotid artery.Ensure that the saturation band is on and is placed on the top to avoid interference from venous signals. The saturation band usually comes with time-of-flight sequence. If the saturation band does not show on monitor, please notify the service person.After locating the common carotid artery using the time-of-flight angiogram, target the image plane of the phase contrast magnetic resonance imager to the center of the same artery, and orient it such that the slice is perpendicular to the direction of blood flow. Ensure that both respiration and ECG gating are connected to the MRI system, showing the clear signal on the monitor computer, and set the trigger module to be on in the trigger mode from the console monitor of the MRI scanner. At this point, again confirm that the animal's physiological responses are stable, and verify that the gating selections are on in both the monitor computer and the console monitor of the MRI scanner.Please remember to make sure that the gating selections are on in both monitor computer and the monitor one of MRI scanner. Next, select the sequence of PC-MRI sequence from the console monitor of the MRI scanner, and perform the gated PC-MRI scans. Repeat this process for each region of interest.Once all of the regions have been imaged, remove the animal from the scanner and return it to its cage. Warm the animal with a heating lamp to maintain body temperature. Keep the lamp at least 15 centimeters away from the animal to prevent overheating until the animal starts to move and exhibits a response to a tail or toe pinch.Finally, be sure to save the MRI data, and process it as described in the accompanying text protocol. A correct slice having round geometry is pivotal for ensuring the success of the PC-MRI experiment. As angulation increases, the resulting artery geometry becomes ovoid, leading to larger partial volume effects and the overestimation of blood flow.If the slice on the right was observed, the scan should be repositioned and repeated. When correctly positioned, intra-scan reproducibility of changes in blood flow within one cardiac cycle should be high. As can be seen, blood flow reaches its maximum during the systolic phase and returns to baseline during the diastolic phase.The blood flow in the common carotid artery in rats is age-dependent, highlighted here, in the longitudinal scans by the difference in the peak systolic flow between a two-month-old rat and one that is four months old. After watching this video, you should have a good understanding of how to apply PC-MRI to noninvasively measure the blood flow in the rat common carotid artery in under 30 minutes if performed properly. While attempting this procedure, it is important to remember to connect the gating system properly.After its development, this technique paved the way for research in the field to explore the mechanism behind some cardiovascular disease.

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Research

JoVE Journal

Medicine

Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF. Current technologies for CMR are time consuming and difficult to implement in clinical practice. Feature tracking is a technology that can lead to more automization and robustness of quantitative analysis of medical images with less time consumption than comparable methods.
Methods: An automatic or manual input in a single phase serves as an initialization from which the system starts to track the displacement of individual patterns representing anatomical structures over time. The specialty of this method is that the images do not need to be manipulated in any way beforehand like e.g. tagging of CMR images.
Results: The method is very well suited for tracking muscular tissue and with this allowing quantitative elaboration of myocardium and also blood flow.
Conclusions: This new method offers a robust and time saving procedure to quantify myocardial tissue and blood with displacement, velocity and deformation parameters on regular sequences of CMR imaging. It therefore can be implemented in clinical practice.

An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF.

Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that ...

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

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 ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...