We are grateful to Dr. Clowes for first developing and describing this method. We are also thankful to Dr. Tulis for his detailed protocol which has been fundamentally helpful to our previous, current and future work. This work was supported by grants R01HL097111 and R01HL123364 from the NIH to M.T., and by American Heart Association grant 14GRNT18880008 to M.T.
We would like to thank Rachel Newton for her expert technical support and for her valuable help during the filming process.

NOTE: The use of animals for the following experiments has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
1. Preoperative Procedures
<ol style="margin-left: 40px;">
	<li>Sterilize surgical instruments before use.
	<ol>
		<li>Autoclave all surgical instruments 24 hr or less before surgery. If multiple surgeries are performed on the same day, sterilize the instruments by a dry bead sterilizer in between surgeries.</li>
	</ol>
	</li>
	<li>Filter-sterilize the saline solution before use.</li>
	<li>Weigh the rat and calculate the dose of anesthetic drugs (ketamine 80 mg/kg and xylazine 7 mg/kg).</li>
	<li>Administer the anesthetic drugs intra-peritoneally (i.p.).
	<ol>
		<li>Verify the adequacy of sedation by toe pinch in 5-10 min. Administer an additional small dose of drugs (ketamine 7 mg/kg and xylazine 0.6 mg/kg) if sedation is not complete.</li>
		<li>Make sure the needle is deep enough to delivery drugs intra-peritoneally because failure to deliver the entire drug solution into the peritoneal cavity will cause inadequate sedation. 
		NOTE: Ulcers and hair loss in the skin at the injection site will be visible several days after surgery, due to inadvertent subcutaneous (s.c.) drug solution injection.</li>
	</ol>
	</li>
	<li>Inject 3 ml sterile saline solution subcutaneously (s.c.). Using a sterile cotton swab, apply a small amount of ophthalmic ointment to both eyes to prevent the corneas from drying out.</li>
	<li>Prepare the heating equipment. Pre-warm heat pads by microwave or water bath.</li>
	<li>Put the animal supinely on the surgical platform.
	<ol>
		<li>Remove the hair in the ventral neck region. Apply the hair remover with a cotton swab, wait 30 sec and wipe off completely with gauze.</li>
		<li>Swab the neck with Povidone-Iodine scrub and 70% ethanol.</li>
	</ol>
	</li>
	<li>Put on personal protective equipment, including gown, hair cover, surgical mask, and glasses. Put on sterile surgical gloves at the end before handling the sterile surgical instruments and supplies.</li>
	<li>Drape the rat with a sterile surgical sheet with only the neck region exposed.</li>
</ol>
2. Surgical Procedures
<ol style="margin-left: 40px;">
	<li>During the surgical procedure, check sedation depth of animal by toe pinch in every 15 min. If animal responds to toe pinch, add additional small dose (10% of the initial dose) of ketamine and xylazine.</li>
	<li>Dissect left common carotid artery (CCA)
	<ol>
		<li>Use a scalpel to make a straight longitudinal incision in the middle of neck.&#160;The approximate length of skin incision is 1.5-2 cm, for the purpose of isolating a 1.5-2 cm portion of artery. The length may vary depends on the purpose of study.</li>
		<li>Bluntly dissect the connective tissue from the skin. Keep the forceps tips up and make sure not to puncture the skin or the underlying tissue.</li>
		<li>Dissect muscle layers longitudinally along the left side of trachea.</li>
		<li>When opening up the muscle layer, visualize the left CCA with the vagus nerve closely attached. Bluntly dissect alongside the left carotid artery with extreme caution to separate the vagus nerve with minimal stretching.</li>
		<li>Dissect CCA distally till the bifurcation. Carefully dissect the bifurcation and two branches - internal carotid artery (ICA) and external carotid artery (ECA).</li>
		<li>Keep dissecting around the CCA, until approximately a portion of 1.5-2 cm of the artery is isolated from the surrounding tissues.</li>
	</ol>
	</li>
	<li>Balloon injury
	<ol>
		<li>Permanently make a ligature on the ECA at approximately 5 mm away from bifurcation. Permanently ligate the occipital branch of ECA which is close to the bifurcation of ECA and ICA. Also permanently ligate other branches, if any, locating between the bifurcation and ECA ligature &#8211; for example, the superior thyroid branch. Suture that used for all ligatures is 4-0 black silk. Clip on the proximal end of CCA and the distal end of ICA. 
		NOTE: Now, blood flow has been stopped either permanently through ligation (on ECA) or temporarily through clipping (on CCA and ICA). Luminal contents in the bifurcation area have been isolated from the systemic circulation.</li>
		<li>Make an arteriotomy incision on ECA by small-micro scissors. Ensure that the incision is close to the distal suture knot. Clean blood with saline and cotton swabs.</li>
		<li>Insert the uninflated 2F balloon catheter into the ECA lumen. Advance the balloon catheter proximally to the CCA lumen. Keep advancing the catheter proximally until its tip reaches where the clip stays.</li>
		<li>Connect the balloon inflation device to a female luer lock on a 3-way stopcock and connect the male luer lock of the 3-way stopcock to the balloon catheter.</li>
		<li>Slowly inflate the balloon with approximately 1.5 atm pressure, in order to distend the carotid artery to 1.5-fold of the diameter.</li>
		<li>Gently pull the balloon rotationally back to the bifurcation.</li>
		<li>Deflate the balloon and advance it back to the proximal end. Inflate it again and repeat the pulling-back procedure two more times.</li>
		<li>Withdraw the balloon catheter from the artery lumen.</li>
	</ol>
	</li>
	<li>Intraluminal administration of reagents (e.g., siRNA, drugs) 
	NOTE: Here, the reagent used was a solution containing lentiviral particles encoding either shRNA targeting Stromal Interaction Molecule 1 (STIM1) or non-targeting shRNA control. STIM1 is a single transmembrane endoplasmic reticulum (ER) protein that is an ER Ca2+ sensor controlling the activation of plasma membrane Ca2+ channels and is up-regulated during vascular smooth muscle dedifferentiation into a proliferative migratory phenotype12,30-33.
	<ol>
		<li>Attach an intravascular over-the-needle catheter to a syringe (24 G, 1.6 cm). Aspirate 30 &#181;l solution of the testing reagents. Insert the catheter into the same incision on ECA.</li>
		<li>Advance the catheter tip into the CCA and tie a piece of suture with a single knot on the ECA to fix the catheter and close the incision temporarily.</li>
		<li>Inject the testing reagent solution into the lumen of CCA. Keep the solution in the vessel lumen for 30 min. Keep the exposed tissue moist with saline and cover it with a piece of wet gauze.</li>
		<li>After incubation, aspirate the remaining solution. Loosen that single knot and withdraw the catheter.</li>
		<li>Tie the ECA with a piece of suture proximally to the arteriotomy incision. Make the knot as close as possible to the bifurcation.</li>
	</ol>
	</li>
	<li>Close up the wound.
	<ol>
		<li>Since the balloon injury and catheter introduction may cause a leak or puncture of the blood vessel, remove the clip on the ICA and check if there is any leak. If bleeding is observed, apply a piece of gauze and apply gentle pressure to stop bleeding.</li>
		<li>Remove the other clip on CCA.</li>
		<li>Make sure there are no signs of bleeding, and then remove all clamps and other surgical instruments. Cut off excess sutures.</li>
		<li>Close wound using skin sutures (4-0 black silk). Swab on all sides of the closed wound with the Povidon-Iodine or other anti-septic/bactericide/virucide agent. Inject 3 ml of sterile saline s.c.</li>
	</ol>
	</li>
</ol>
3. Postoperative Procedures
<ol style="margin-left: 40px;">
	<li>Keep rat on heat pad after surgery. Administer one dose of 0.05 mg/kg buprenorphine via intramuscular injection (i.m.) to the rat. During the recovery procedure, keep animals&#8217; eyes and mouth moist. Monitor the animal until it is awake and ambulant.</li>
	<li>Place the animal in a clean cage without any bedding until it is fully recovered. After recovery, the animal will be returned to the animal room and housed individually.</li>
</ol>

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The authors declare that they have no competing financial interests.

The rat carotid balloon injury has been well described by Tulis in 200734. It has been comprehensively discussed all the details of this procedure by Dr. Tulis. The readers who are interested in performing this procedure are highly recommended to read Tulis&#8217; protocol. However, there is one thing we are not agreeing with Dr. Tulis: Instead of inflating the balloon with saline or any kind of liquid, we suggested to inflate it with air. According to our personal experience, inflating with liquid can hardly avoid air bubbles. In addition, it is more difficult to flexibly adjust the pressure during the injury procedure and may cause extra stress and injury to the artery. Another&#160; technical tip is to use a small &#8220;pillow&#8221; (made of paper towel or gauze) to support animal&#8217;s neck during surgery.
Based on previous reports and several years of experience with rat balloon injury procedure, the authors have generated a slightly modified and simplified protocol35. In addition, the intraluminal infusion of therapeutics right after injury has been demonstrated. This has roughly doubled the time of this survival surgery procedure and thus requires additional hands-on experience. The major concern during the prolonged time period is the anesthetic plane. Initial dose of anesthetic drug can maintain rat&#8217;s sedation for 30-45 min and so the animal should be frequently checked by toe-pinch, especially during the 30 min infusion time. The reason to use injectable anesthetic drugs instead of inhalant anesthesia for this specific surgery is majorly due the orientation of rat&#8217;s head. According to the authors&#8217; experience, balloon insertion into carotid artery is much easier to perform when rat&#8217;s lying with its head towards surgeon. While performing surgery, it is highly recommended to do it under a fume hood (to avoid potential allergy) or a biosafety cabinet when lentivirus presents. In this case, the bulky inhalation cone and tubing would disturb the airflow of hood, and also make the surgical area harder to access. However, it is still highly recommended to use inhalant anesthesia if the surgeon can well perform the surgery with the opposite orientation of animals&#8217; head.
During infusion, please note to avoid air bubbles in the vessel lumen.
Like any other rodent surgery, hypothermia is the major concern throughout the entire procedure. Use proper heating equipment to avoid animal suffering from hypothermia, which can lead to death. Meanwhile, over-heating/hyperthermia should also be avoided. When using the heat-pad, towels are recommended to be placed between the heat-pad and animal body to prevent the animal from over-heating.
Two solutions, saline and lidocaine hydrochloride (1%), are highly recommended to be applied on exposed tissues when necessary during the surgical procedure. Tissues that are exposed during surgery should be kept moist by sterilized saline. Surgical stretch frequently causes muscular spasm and vessel contraction of the carotid artery. Insertion of the balloon catheter into a contracted artery is prone to fail; when balloon insertion is successful under these conditions, it will cause severe stretching or damage on the artery. Lidocaine as a topical anesthetic drug can be used to relax and dilate the vessel.
The balloon is inflated with approximately 1.5 atm pressure and proper adjustment is required for each surgery, due to the change in plasticity of the aged balloon (if reused) and the variation of CCA diameter. In addition, due to stiffness of some arteries the balloon may be over-inflated but cannot be seen. In this case, when the balloon is inflated, the surgeon should slightly pull the catheter to check the resistance of artery and adjust balloon pressure accordingly. Robust pulling will cause severe damage or rupture of artery, blood leakage, and failure of the experimental model. The balloon catheter can be reused several times as long as the balloon still works well. Disinfect balloon catheter using Cidex for the purpose of reusing. The materials that made of balloon catheter, the natural rubber latex and polyethylene, have been approved to be compatible with Cidex. The detailed protocols for disinfecting medical devices using Cidex have been described37. It is important for the surgeon to check the leakage of balloon every time before use.
The continuous suture pattern is usually not recommended to use for skin closure. Instead, wound clips and interrupted suture pattern are usually recommended. However, when the incision is at the highly active and sensitive body part, the neck, this may not be the case. According to our experience, wound clips and interrupted suture pattern ended up with higher rates of closure failure. Clips lost or sutures broken by animal scratching happened very often, which was most likely due to more itching caused by the metal clips or multiple suture ends. When using continuous suture pattern, the failure rate was only 1% in our lab with hundreds of rats. In addition, the best method to close skin is probably the intradermal suture pattern, although we were not able to show it in the video.
There are a variety of methods available for histological staining of tissue sections. H&#38;E staining is the most commonly used one. Readers are referred to a comprehensively discussed article36, by Tulis, for further reading. In order to gain more accurate information of laminar structures, Verhoeff&#8217;s Elastic Tissue Stain with Van Gieson Counterstain (VVG staining) is highly recommended.

Balloon catheters are medical devices used in the procedure of angioplasty, for the purpose of widening obstructed site(s) of atheroma or thrombus in a blood vessel. The narrowed vessel lumen is forced to open up by the inflated balloon and blood supply would be restored sequentially to relieve downstream ischemia symptoms, such as angina, myocardial infarction, and leg pain. Nevertheless, the great success of angioplasty has been diminished by post-operative complications such as results from force causing vascular barotrauma (balloon injury), namely vessel wall remodeling and in many cases re-narrowing of the vessel lumen (restenosis)1.
A number of animal models have been developed mimicking the angioplasty procedure to help investigators understand mechanisms underlying the balloon-injury-related vessel wall remodeling2. Among all the animal species utilized for modeling, rat is the most frequently used one. Compared to rabbits, dogs and swine, the advantages of rats are their low cost, their relative ease of use and the current knowledge of rat physiology. Although mice have an added advantage in a wide range of genetically manipulated strains, the mice vessel is too small to insert a balloon catheter. Over the past three decades, experimental rats have allowed researchers to gain better understanding of the molecular and cellular mechanisms underpinning neointima formation and vascular remodeling3-6. Beyond balloon injury, vascular remodeling are also involved in most major vascular diseases, such as atherosclerosis7,8, hypertension9, and aneurysm10. Thus, knowledge gained through the balloon injury model is in general beneficial to overall vascular wall disease studies.
The overall goal of the rat balloon injury model is not only to further understand vascular diseases but also to test the potency of novel agents for disease control11,12. Current clinical drug treatment to restenosis is applied by drug-eluting stents placed via the vessel lumen right after angioplasty. In animal models, an efficient yet more economical way for new agent testing is a well-developed local intraluminal perfusion method. Candidate agents that have been tested through this method include small molecule drugs13,14, cytokine or growth factors15,16, gene manipulating agents (cDNA clones, siRNA, etc.)17-20, and novel pharmaceutical formulations21,22.
So far, the rat balloon injury model remains one of the most useful models for studying vascular diseases/disorders. It is the fundamental step from bench to bedside, usually as the first step moving from in vitro to in vivo, but it should not be the last one. The outcome of rat experiments needs to be deliberated and further characterized before translation into human clinical use, due to the difference in vascular beds and vessel anatomy as well as the intrinsic species differences between human and rat23-26. Nevertheless, it is still an essential tool in translational medical research. While such research used to be limited by the lack of genetically modified rats, it has no longer been an issue since novel genomic approaches such as zinc-finger nucleases27, TALENs28 and CRISPR-Cas29 have made knockout rats easily accessible.

<table><tbody><tr><td>Fogarty balloon embolectomy catheters, 2 French&amp;nbsp;</td><td>Edwards Lifesciences, Germany</td><td>&amp;nbsp;120602F</td><td></td></tr><tr><td>Deltaphase Operating Board - Includes 2 Pads &amp;amp; 2 Insulators</td><td>Braintree Scientific, Inc.</td><td>39OP</td><td></td></tr><tr><td>LED light source</td><td>Fisher Scientific</td><td>12-563-501&amp;nbsp;</td><td></td></tr><tr><td>Hartmann Mosquito Forceps 4&amp;rdquo; curved</td><td>Apiary Medical, Inc. San Diego, CA</td><td>gS 22.1670</td><td></td></tr><tr><td>Crile Retractor 4&amp;rdquo; double ended</td><td>Apiary Medical, Inc.</td><td>gS 34.1934</td><td></td></tr><tr><td>Other surgical instruments</td><td>Roboz Surgical Instrument Company, Inc., Gaithersburg, MD</td><td></td><td></td></tr><tr><td>Peripheral Intravenous (I.V.) Cannula, 24 G</td><td>BD</td><td>381312</td><td></td></tr><tr><td>Ketamine HCl, 100 mg/ml, 10 ml</td><td>Ketaset- Patterson Vet</td><td>07-803-6637&amp;nbsp;</td><td></td></tr><tr><td>Xylazine (AnaSed), 20 mg/ml, 20 ml</td><td>Ketaset- Patterson Vet</td><td>07-808-1947</td><td></td></tr><tr><td>Buprenex, 0.3 mg/1 ml (5 Ampules/Box)</td><td>Ketaset- Patterson Vet</td><td>07-850-2280</td><td></td></tr><tr><td>Nair Baby Oil Hair Removal Lotion - 9 oz</td><td>Amazon/Walmart/CVS</td><td>N/A</td><td></td></tr><tr><td>Inflation Device</td><td>Demax Medical</td><td>DID30</td><td></td></tr><tr><td>D300 3-way Stopcock</td><td>B.Braun Medical Inc.</td><td>4599543</td><td></td></tr><tr><td>Artificial Tears Ointment&amp;nbsp;</td><td>Rugby Laboratories, Duluth, GA</td><td>N/A</td><td></td></tr></tbody></table>

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.

Two weeks after injury, carotid arteries are harvested, sectioned and subjected to morphological analysis. Arteries are cross-sectioned and stained with H&#38;E (Figures 1, 2B, C and 3). Rat carotid artery wall contains four layers of elastic lamina, which appear as pink lines. The area between the outermost lamina, external elastic lamina (EEL) and the innermost lamina, internal elastic lamina (IEL) is the media smooth muscle layer (Figure 1). The area inside of the IEL is the intima, a monolayer of endothelial cells in intact vessels; or neointimal hyperplasia in injured vessel. In the injured carotid artery, the media is thicker than in control vessels due to proliferation of smooth muscle cells. The thickness of neointima is similar to or greater than the thickness of the media in the same artery. Adventitia is also thickened with robust collagen deposition (Figure 1B, D). For the artery treated with the reagent tested for its ability to inhibit neointima formation (in this case, STIM1 shRNA), the neointimal area of the cross section is smaller compared to the control injured artery (Figure 2). The validity of STIM1 knockdown with shRNA was evident as the STIM1 expression level largely decreased in the injured vessel neointima and media, compared to the control injured vessel.
As will be mentioned in Discussion section, surgeons should be cautious not to over-inflate the balloon and injure the vessel excessively. This would cause the vessel wall to rupture, which will cause blood leakage and robust thrombus formation both in the lumen and on the outer surface of artery, as shown in Figure 3.
<img alt="Figure 1" src="/files/ftp_upload/52045/52045fig1highres.jpg" /> 
Figure 1: Rat carotid artery cross sections stained with Hematoxylin &#38; Eosin (H&#38;E). (A) Morphology of the normal/intact (right) CCA. (B) Morphology of the injured (left) CCA, showing neointima formation and adventitia/media thickening. (C) Structure of the normal rat carotid artery wall. The intima is a monolayer of endothelial cells lining on the internal elastic lamina (IEL). Media is the smooth muscle cells and elastic tissues between IEL and external elastic lamina (EEL). Adventitia is the outer layer. (D) Rat carotid artery wall robust thickening 2 weeks after injury. Smooth muscle cell proliferation and migration lead to neointima formation and media thickening. Typical concentric neointima formatted and media/adventitia thickened, showing successful generation of balloon injury phenotype. The red (eosin) staining is enhanced in adventitia, due to robust collagen synthesis. Scale bars are 100 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/52045/52045fig2highres.jpg" /> 
Figure 2: Cross sections of rat carotid arteries with treatment of shRNA-STIM1 or shRNA control, two weeks post injury. (A) Immunofluorescent staining of STIM1 and DAPI staining of nuclei on cross sections from both groups. The attenuated expression of STIM1 and neointima formation exhibit in the shRNA-STIM1 treated artery section. (B) H&#38;E staining of the shRNA-STIM1 artery section. C. Digital tracing of the neointima border, IEL and EEL, for the purpose of measuring areas of lumen, neointima and media. Scale bar are 100 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/52045/52045fig3highres.jpg" /> 
Figure 3: Example of sub-optimal or failure to generate neointimal modeling. (A) Eccentric instead of concentric neointima formed due to improper ballooning. (B) Excessive injury (by an over-inflated balloon) caused severe damage to the vessel and the rupture of the vessel wall, which is evident by the discontinuous elastic laminas. Severe injury caused thrombus, which has blocked the entire vessel lumen and expanded out into the adventitia. Furthermore, enhanced adhesion occurred between the adventitia and the surrounding fat tissue. Scale bar are 100 &#181;m.

Watch this Scientific Journal Video about Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery at JoVE.com

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid 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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

16.4K Views.  State University of New York College of Nanoscale Science and Engineering (SUNY CNSE). 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.

Video: Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

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 ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

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 ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

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

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

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

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

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of ...

Rodent Venoplasty Balloon Model for Deep Vein Treatment Studies

Rodent Inferior Vena Cava Venoplasty Balloon Model

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

Author Spotlight: Utilizing Venoplasty Balloon Model in Rodents to Simulate Surgical Interventions for Deep Veins

The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.