The work was supported by the intramural research program of the NIH, National Institute on Aging, and by a Priority Research Centers Program grant from the National Research Foundation (NRF-2009-0093812) funded by the Ministry of Science, Information and Communication Technology, &amp; Future Planning, the Republic of Korea (H.T.). We thank Dr. Han-sol Park for putting "material" part together for the manuscript.

NOTE: the use of rat balloon injury model and associated procedures including injection of recombinant sRAGE and ultrasound sonographic studies have been approved by the Animal Care and Use Committee (ACUC) of National Institute on Aging, NIH.
1. Pre-surgery Preparations
<ol>
	<li>Instrument, Surgical Platform, and Personal Protection Equipment.
	<ol>
		<li>See Materials and Equipment&#39;s Table for all the surgical instruments and reagents used in this procedure. Autoclave all surgical instruments prior to surgical procedure. 
		NOTE: For multiple surgeries performed at the same day, use a pre-heated glass bead sterilizer to sterilize the instruments between surgeries.</li>
		<li>Sterilize the surface of operating platform with 70% alcohol. Put on surgical gown, hair cover, surgical mask, safety glasses, and gloves. Leave a box of sterilized gloves near the surgical platform for additional use.</li>
	</ol>
	</li>
	<li>Preparation of the Balloon Catheter.
	<ol>
		<li>Fill 1 ml syringe with sterile water and attach the syringe to a sterile water-filled two-way stopcock. Gently push the water through the stopcock to fill the Luer-lock portion of the balloon catheter, and remove air bubbles from the opening of the catheter.</li>
		<li>Test balloon inflation to assure the balloon can be inflated by 0.02 ml of water. Store the tip of the balloon in a container of sterilized water before the surgery to avoid desiccation and contamination.</li>
	</ol>
	</li>
	<li>Anesthesia and Animal Preparation.
	<ol>
		<li>Weigh male Wistar rats (400-450 g) and check the ear tag to confirm the ID. Place the rat in an induction chamber, then turn on the vaporizer and adjust the isoflurane level to 3.5-4.0% and the oxygen level to 1.5-2.0 L. Check the depth of sedation by checking pedal withdrawal reflex such as pinching the toe or tail of the animal.</li>
		<li>After reaching the anesthesia (i.e. the animal lacks pedal withdrawal reflex), transfer the animal to the surgical platform and hook the rat with a nose cone that connects to an anesthesia vaporizer. Adjust isoflurane concentration to 1.5-2.0 and oxygen to 0.6-1.0 L. Periodically check the depth of sedation by pinching the toe or tail of the animal to assure that the rat is completely relaxed and immobile.</li>
		<li>After reaching the surgical level of anesthesia, shave the ventral neck region from the chin down to just above the sternum with animal hair clippers and swab the shaved skin with alternating 1:10 dilution of povidone-iodine and 70% ethanol 3 times.</li>
		<li>Place ophthalmic solution on the open eyes of the animal with a cotton swab to prevent them from drying. Moisten the tongue and mouth of the animal with drinking water to avoid drying during the operation.</li>
		<li>Cover the rat with a sterile surgical sheet and cut the sheet to expose the neck region. Place the animal in supine position on a heated pad on the surgical platform with the head toward the surgeon. Fix the limbs of the animal to the surgical platform using tape.</li>
	</ol>
	</li>
</ol>
2. Surgical Procedures
<ol>
	<li>Dissection and Isolation of the Left Carotid Artery
	<ol>
		<li>Use a scalpel to make a straight incision below the chin in a direction toward the tail all the way to the top of the sternum just above the rib cage. Use a 7S forceps to bluntly dissect the salivary glandular tissues under the skin to expose the muscular layer. Separate muscular tissues to gain access to carotid vasculature and the vagus nerve in the neck region.</li>
		<li>Bluntly dissect tissues surrounding the carotid artery, and carefully separate the vagus nerve and vascular fascia to avoid any damage. Continually dissect till the carotid artery bifurcation to expose the internal and external branches and the common carotid artery down to the sternum.</li>
	</ol>
	</li>
	<li>Preparation of Arteriotomy
	<ol>
		<li>For retraction and hemostasis, place a ligature at the most proximal site on the left common carotid artery, and another ligature at the location immediate distal to the bifurcation.</li>
		<li>Place a 4.0 silk suture at the external carotid artery as far from the bifurcation as possible to retract the artery toward the head. Permanently ligate small arteries using 4.0 silk sutures along the external carotid artery including the ascending pharyngeal, the occipital and the superior thyroid to avoid arterial leak.</li>
		<li>Place a 4.0 silk suture around the internal carotid artery to avoid a significant retrograde blood loss. Usually, the available length of the internal branch is minimal. To visualize the internal carotid artery, expose the internal carotid artery as much as possible by gently retracting the overlying carotid artery to the right.</li>
	</ol>
	</li>
	<li>Introduction of the Balloon Catheter
	<ol>
		<li>Retract the proximal suture on the common carotid artery and place an arterial clamp on the vessel to temporarily stop the blood flow. Make a small (1/3 to 1/4 of the circumference of the artery) incision on the external carotid artery at a location as distal to the suture as possible.</li>
		<li>Gently insert an un-inflated balloon catheter into the incision and then advance the catheter in the lumen close to the arterial clamp on the common carotid artery. Remove the clamp, and further advance the balloon catheter to the aortic arch, approximately 35-40 mm from the incision.</li>
	</ol>
	</li>
	<li>Balloon Injury
	<ol>
		<li>Manually inflate the catheter to a volume of 0.02 ml via the attached syringe and then lock the stopcock between the syringe and the catheter. Slowly withdraw the catheter with rotations. When the catheter is close to the arteriotomic incision, deflate the balloon. Advance the catheter back to original location.</li>
		<li>Repeat 2.4.1 for a total 3 times. Carefully remove the catheter from the artery lumen.</li>
	</ol>
	</li>
	<li>Close Up the Surgical Wound
	<ol>
		<li>Close the arteriotomy with sutures. Release the internal carotid artery to restore the blood flow. Check for arterial bleeding or leak. If bleeding occurs, apply a piece of gauze with a bit pressure to stop the bleeding.</li>
		<li>Remove the rest of sutures and clamps used for the procedure. Close the glandular tissue with 6.0 sutures. Close the skin with skin sutures or wound clips. Turn off isoflurane and leave the animal with pure oxygen for a few min.</li>
	</ol>
	</li>
</ol>
3. Administration of Therapeutics, Analgesics, Post-surgery Care, and Euthanasia
<ol>
	<li>Administration of Therapeutics and Analgesics.
	<ol>
		<li>Following the surgery, inject buprenorphine (0.03 mg/kg) intramuscularly. Inject 5 ml pre-warmed sterile saline subcutaneously to compensate the blood loss during the surgery. Inject sRAGE (0.5 ng/g) (therapeutics) into the peritoneum.</li>
		<li>Moisten the eyes again with ophthalmic ointment. Place the rat on the heat pad and monitor the animal until it regains complete consciousness to maintain sternal recumbency.</li>
		<li>Return the rat to a cage with clean bedding and bring the cage back to the vivarium. Place a special observation card on the cage to alert the attending veterinarians and caretakers.</li>
	</ol>
	</li>
	<li>Post-surgery Cleanup
	<ol>
		<li>Put used surgical material including pads, swabs, needles et al either into the trash or a special disposal container. Disinfect the surgery platform and clean the surgical instruments.</li>
	</ol>
	</li>
	<li>Post-surgery Care
	<ol>
		<li>Administer buprenorphine (0.03 mg/kg) twice a day (b.i.d.) during the 48 hr post-surgery period. Monitor the animal closely during the 48 hr post-surgery period for the common signs of distress such as immobility, inability to feed, hutched posture, ruffled fur, and grimace. Monitor wound conditions for redness, swelling, dehiscence, and infections. 
		NOTE: Additional treatments may be prescribed according to attending veterinarian&#39;s recommendation.</li>
		<li>Remove the suture or wound clips according to attending veterinarian&#39;s recommendation.</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>Two weeks after the surgery, euthanize the rats to isolate the carotid arteries for post-mortem histo-morphological analyses22. Place the rat in a chamber containing 5-20% isoflurane saturated air for at least 2 min till complete cessation of respiration. Verify the death prior to dissection of the organs and tissues.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

There have been two methods used to inflate the balloon in order to generate the injury that removes the mural endothelium in the carotid arterial lumen. One is to fill the attached syringe with liquid20, and the other is to use air pressure21. We prefer to use liquid-filled syringe because the exact liquid volume (0.02 ml) will be used for each procedure. This renders a precise and reproducible inflation of the balloon, leading to a similar level of injury in each animal undergoing the procedure. Using liquid in the syringe indeed requires removal of air bubbles in the liquid. This can be achieved by pushing the bubbles out from the opening of the catheter. After removing a majority of air bubbles, any residual, tiny air bubbles will dissipate in 1-2 hr. We suggest that researchers make pre-surgery materials ready 1-2 hr prior to the procedure, and check the degree of the balloon inflation before each individual surgery to ensure the appropriate inflation.
For the surgery, the surgeon and his/her assistant(s) must wear all necessary protection gears including face masks, sterile gloves, and surgical gowns to protect both animals and researchers from infection. It is important to use fully grown, male rats with similar bodyweights (e.g. between 380-450 g) to ensure the proper size of carotid artery for the introduction of standard 2F Fogarty balloon embolectomy catheter used for this procedure. Before the surgery, check the level of isoflurane in the vaporizer to assure sufficient amount of the anesthetic that covers the entire surgery, and refill the vaporizer if necessary before each surgery starts. Check the pressure gauge of the oxygen tank to ensure sufficient oxygen for the surgery. To ensure a proper depth of anesthesia is reached, one can also monitor the respiratory rate of the rat. A normal rat has a respiratory rate of ~85 breath/min; an increase in respiratory depth and regular rhythm, and a decrease in respiratory rate signifies surgical anesthesia.
During the surgery, anesthesia must be continuously monitored and maintained by checking respiration, pedal reflex, and the response of the animal to the surgical stimulus. Of note, the surgeon should change gloves if his/her hands touch non-sterile parts of the animal during the surgery such as checking pedal reflex to prevent potential contamination and infection.&#160;&#160;In addition, the surgeon and the assistant should also carefully monitor the vital signs of the animal during the operation to prevent anesthetic overdose and take quick corrective actions to avert the situation if it occurs by turning off the isoflurane and increase oxygen level to 100%. The signs of anesthetic overdose include slow and shallow respirations, and weak and irregular pulse. We also suggest a good preparation of arteriotomy before the balloon injury including a complete separation of carotid artery sections from their adjacent tissues. This will help to avoid unexpected bleedings during the operation.
The animal under anesthesia, including during the post-surgical recovery phase, should never be left unattended. As an alternative to the regular buprenorphine (0.03 mg/kg b.i.d.) during the 48 hr post-surgery period, sustained-release buprenorphine (buprenorphine SR) can also be used. Injection of buprenorphine SR (1.0-1.2 mg/kg) immediately after the surgery will cover the post-surgical period up to 72 hr. In general, postsurgical complications are not anticipated; but a careful monitory practice ensures the welfare of the animals and is a policy by the institutional ACUC.
Because it is most important to reproduce the same level of the balloon injury in the study group of animals (8-15), adequate surgical skills and familiarity with the procedure are highly recommended. For researchers new to this procedure, we suggest first to practice the entire procedure including the preparation of arteriotomy, the balloon injury and the wound closure on rat carcasses that may be available in the institutional animal facility. Practicing the surgical procedure with veterinarians or researchers familiar with the procedure also ensures to obtain reproducible and consistent balloon injury results.
In our view, drugs and/or therapeutics that negate neointimal growth are best administered immediately after balloon injury in order to offset the inflammation at early stage. Some reagents may need to be administered continually during the subsequent days of recovery to achieve the effect. An early intervention helps to block or reduce the ensuing vascular remodeling13,22,24.

Angioplasty is an endovascular procedure used to widen the narrowed or obstructed arteries resultant from pathological conditions such as atherosclerosis. One common complication of angioplasty is the post-operational neointimal hyperplasia, or restenosis, which occurs owing to surgical injuries and the subsequent inflammation-induced vascular remodeling. These conditions lead to proliferation of vascular smooth cells, and multiple pathophysiological consequences1-3. Neointimal hyperplasia re-thickens the vessel, and occurs in up to 60% post-angioplasty patients within the first year. Therefore, restenosis is a major setback of the widely used angioplasty procedure4. Although the implantation of the drug-elution stent may help to prevent restenosis, only selected candidates can undergo this costly procedure5.
Both animal and clinical studies have established that chronic inflammation generated by vascular injury and /or surgical wound serves as the main stimulus for post-angioplasty neointimal growth2,4. The rat carotid balloon injury model mimics the clinical situation and therefore serves as a valuable model system to identify cellular factors that involve in the vascular remodeling and vascular cell proliferation6-9. This model system is also a highly useful tool to evaluate and/or screen for drugs and therapeutic reagents that suppress neointimal growth in pre-clinical translational studies10-14.
Compared to the murine carotid wire injury model15 and the murine femoral artery wire injury model16, the rat carotid balloon injury model has the advantages of being sufficiently large in size for the ease of surgical procedure that facilitates reproducibility of the inflicted injury. It can provide a larger number of primary cells (e.g. vascular smooth muscle cells, endothelial cells) for additional in vitro studies to delineate the molecular mechanism governing vascular remodeling. Importantly, compared to mice, rats are also known to be a better model for physiological and toxicological studies17. Although a disadvantage or limitation of the rat model is the lack of genetic modified and gene knockout models, this disadvantage can be overcome by the availability of the rat genomic sequence and the recent development of powerful genomic editing tools such as CRISPR-CAS technology that renders possible manipulation of vast ranges of genomic sequences in different model systems18,19.
Although the rat balloon injury model has been used by multiple labs and various comprehensive protocols have been published20,21, this protocol aims at providing more details at pre-surgery preparations and may guide researchers new to this procedure to set up this surgical practice. We also emphasize the post-surgery care of the animals that allows not only post-mortem pathological and histomorphological analyses of the therapeutic effects on arterial remodeling, but also ultrasound sonographic studies in live animals13,22.

<table><tbody><tr><td>2 F Fogarty balloon embolectomy catheter&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Edwards Lifesciences&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Standard scalpel</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Small&amp;nbsp; curved forceps (Large radius Dumont#7shanks curved)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Large, medium and small micro-scissors</td><td>Roboz</td><td></td><td></td></tr><tr><td>Needles (20 G)&amp;nbsp;&amp;nbsp;</td><td>TycoHealthcare</td><td></td><td></td></tr><tr><td>Micro-surgery forceps with micro-blunted atraumatic tips</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Atraumatic straight small arterial clamps&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Retractor&amp;nbsp; with maximum spread 5.5 cm long blunt teeth</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Silk suture (4.0 and&amp;nbsp; 6.0 )</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Syringe (1.0 ml)&amp;nbsp;</td><td>BD&amp;nbsp;</td><td></td><td></td></tr><tr><td>Curity gauze sponges</td><td>AllegroMedical</td><td></td><td></td></tr><tr><td>Cotton tip applicators sterile and non-sterile</td><td>Puritan Medical Products</td><td></td><td></td></tr><tr><td>Compact hot bead sterilizer</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Self-regulating heating pad</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>ADS200 anesthesia system/ventilator</td><td>Paragon Medical</td><td></td><td></td></tr><tr><td>Isoflurane (forane), liquid form</td><td>Baxter</td><td></td><td></td></tr><tr><td>Sodium chloride 0.9% (Saline)&amp;nbsp;</td><td>Hospira</td><td></td><td></td></tr><tr><td>Buprenex (buprenorphine)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Reckitt Benckiser Healthcare (UK) Ltd.&amp;nbsp;</td><td></td><td></td></tr><tr><td>70% alcohol</td><td>Fisher</td><td></td><td></td></tr><tr><td>1:10 Betadine</td><td>Fisher</td><td></td><td></td></tr></tbody></table>

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.

Two weeks after the balloon injury, the rat is euthanized and the carotid arteries are isolated for histo-morphological analysis. Both operated left carotid artery and un-operated right artery are cross-sectioned, processed, and paraffin-embedded. The paraffin samples are then further thin-sectioned, and stained with hematoxylin-eosin (H &#38; E). Histo-morphological analyses are performed using a digital imaging analysis system. The details of artery harvest and histo-morphological analysis have been described13,23. The balloon injury-elicited neointimal growth, or the thickening of the vessel wall, if left untreated (or treated with saline as the placebo), is evident as shown in Figure 1B, compared to non-operated vessel section from the same rat (Figure 1A). The vessel section from the rat received therapeutic reagent (in this case, sRAGE) that blocks neointimal growth showed significantly reduced thickening of the vessel wall (Figure 1C). The effects of the balloon injury as well as the therapeutic treatment can also be evaluated in vivo using ultrasound sonography22, which collaborate well with the results from histo-morphological analysis (Figure 2). The details for the evaluation of the efficacy and the administration window of therapeutic sRAGE in the rat balloon injury model have been described in previous publications13,22. To evaluate therapeutic reagents that reduce or block the injury-elicited vessel wall remodeling, a group of rats (n = 8-15) are to be operated in order to obtain statistically meaningful conclusions.
<img alt="Figure 1" src="/files/ftp_upload/53777/53777fig1.jpg" /> 
Figure 1: Representative results from the rat carotid balloon injury model. H &#38; E staining of the carotid artery cross-sections (A) the non-operated right carotid artery section; (B) the balloon injured and placebo-treated left carotid artery section (arrows indicate the neotimal areas); (C) the balloon injured and sRAGE-treated left carotid artery section. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53777/53777fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53777/53777fig2.jpg" /> 
Figure 2: Correlation of ultrasound sonography and histology. sRAGE (0.5 ng/g) treated carotid vessels shown in scatter plot of data from (A) lumen diameter and (B) vessel wall thickness at 2 weeks post-injury (the non-operated, injured with sRAGE, and placebo-treated, n = 12 of each group). (C) Representative sonographic and histologic (100X) images. (Figure from reference22, with permission from the publisher). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53777/53777fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

8.7K Views.  National Institute on Aging. 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. 

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

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 ...

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

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 ...

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 ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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 ...

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 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 ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

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

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