N.E.B. was supported by a training grant from the National Institute of Environmental Health Sciences (5T32ES007126-35, 2018), and an American Heart Association pre-doctoral fellowship (20PRE35120321). E.S.M.B. was a KL2 scholar partially supported by the UNC Clinical and Translational Science Award-K12 Scholars Program (KL2TR002490, 2018), and the National Heart, Lung, and Blood Institute (K01HL145354). The authors thank Dr. Pablo Ariel of the UNC Microscopy Services Laboratory for assisting with LSFM. Light Sheet Fluorescence Microscopy was performed at the Microscopy Services Laboratory. The Microscopy Services Laboratory, Department of Pathology and Laboratory Medicine, is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill.
1. Preoperative procedures
<ol>
	<li>Sterilize surgical instruments. Autoclave all surgical instruments before surgery. If performing multiple surgeries on the same day, sterilize instruments between surgeries using a dry bead sterilizer.</li>
	<li>Prepare therapeutic in 25% Pluronic-127 gel (diluted in sterile distilled water).</li>
	<li>Set up a 2F Fogarty balloon catheter to the insufflator and place the balloon end of the catheter in a 1 mL syringe filled with saline solution.</li>
	<li>Induce anesthesia by placing the rat in a chamber with 5% isoflurane.
	<ol>
		<li>Remove the rat from the chamber and record the rat&#8217;s weight. Use hair clippers to shave fur on the ventral neck region.</li>
		<li>Place the rat back into the chamber with 5% isoflurane to ensure induction of anesthesia.</li>
	</ol>
	</li>
	<li>Place the rat supine on a surgical platform, inserting the face into the nose cone so that the rat face is toward the surgeon.
	<ol>
		<li>Reduce inhalational anesthesia to 1.5% isoflurane. Verify the depth of anesthesia by a toe-pinch reflex on all four feet.</li>
		<li>Tape all four legs down to the surgical platform.</li>
	</ol>
	</li>
	<li>Turn on the heat lamp.</li>
	<li>Inject Atropine (0.01 mg/kg) subcutaneously to reduce airway secretions.</li>
	<li>Inject Carprofen (5 mg/kg) subcutaneously for prophylactic pain management. If anti-inflammatory drugs are not acceptable for the experiment refer to steps 3.2.2 and 3.2.3.</li>
	<li>Apply lubricant eye ointment to both eyes using a sterile cotton swab to prevent corneas from drying during surgery.</li>
	<li>Swab the neck three times in a circular motion alternating between 70% ethyl alcohol followed by Betadine from the center of the shaved region outwards to sterilize the incision site.</li>
	<li>Infiltrate the incision line s.c. with 0.25% bupivacaine.</li>
	<li>Put on sterile surgical gloves before handling sterile surgical instruments and supplies.</li>
	<li>Lay out all autoclaved surgical instruments on a sterile surgical sheet.</li>
	<li>Cut three independent 1 inch strands of sterile 7-0 Prolene suture.</li>
	<li>Place cotton swabs and gauze on surgical sheet.</li>
	<li>Drape the rat with a sterile surgical sheet that only exposes the sterilized neck region.</li>
	<li>Cut an additional small opening in sheet that exposes part of the nose cone. This will be the site for taping down the balloon catheter during injury.</li>
</ol>
2. Operative procedures
<ol>
	<li>During the entire surgical procedure, assess depth of anesthesia by monitoring the respiratory rate (rate should be consistent and deemed normal) as well by toe pinch every 15 min. If respiratory rate increases or there is a response to the toe pinch, then pause surgical manipulation and increase isoflurane up to 2.5%.</li>
	<li>Expose the common carotid artery (CCA).
	<ol>
		<li>Make a superficial, straight, longitudinal neckline incision between the jaw bones of the rat. Incision will be approximately 1.5-2 cm in length.</li>
		<li>Make an incision through the connective tissue under the skin until the muscle layer is exposed. Displace the salivary glands underneath the skin to access the muscle tissue.</li>
		<li>Bluntly separate the connective tissue from the muscle by inserting closed scissors between the muscle layer and connective tissue and gently opening the scissor while pulling the skin upward.</li>
	</ol>
	</li>
	<li>Dissect the two visible muscles (sternohyoid and sternomastoid) longitudinally along the left side of the trachea until a third muscle (omohyoid) that runs perpendicular to the two superficial muscles is observed.</li>
	<li>Use forceps to create a window separating this perpendicular muscle (omohyoid) from the longitudinal muscle (sternohyoid) running atop the trachea. Gently perform this separation to prevent blunt trauma to the trachea.</li>
	<li>Reach forceps underneath the perpendicular muscle and cut to separate the two longitudinal muscles and expose the CCA.</li>
	<li>Dissect the CCA.
	<ol>
		<li>Dissect the CCA near the bifurcation until the internal carotid artery (ICA) and external carotid artery (ECA) are exposed.</li>
		<li>Dissect the superior thyroid artery (STA), which branches from the ECA.</li>
		<li>Using the pre-cut Prolene sutures, ligate the STA and the ECA near their respective bifurcation. Leave the majority of the suture to one side of the knot and grab each suture with a curved hemostat.</li>
		<li>Finish dissecting around the ICA, reach forceps underneath and around the ICA, and use a non-crushing vascular clamp to achieve distal control. Clamp the occipital artery together with the ICA.</li>
		<li>Dissect the CCA proximal to the bifurcation, ensuring to separate the vagus nerve from the CCA.</li>
		<li>Reach forceps underneath and around the CCA and use a non-crushing vascular clamp to achieve proximal control. Place clamp at least 5 mm from the bifurcation.</li>
	</ol>
	</li>
	<li>Perform balloon injury.
	<ol>
		<li>Maneuver the curved hemostats holding each ligated artery branch to expose the bifurcation between the ECA and superior branch.</li>
		<li>Gently dissect tissue at the bifurcation and then make an arteriotomy incision between the ECA and superior branch using microdissection scissors.</li>
		<li>Use a cotton swab to push all blood out of the CCA and clean up the arteriotomy site.</li>
		<li>Insert the uninflated balloon catheter through the arteriotomy and advance into the CCA until the proximal end of the balloon is past the bifurcation.</li>
		<li>Tape catheter to the nose cone so the balloon does not slip out of the artery during inflation.</li>
		<li>Slowly inflate the balloon to 5 atmospheres of pressure and leave in the CCA for 5 min to induce arterial injury. Ensure that the pressure stays constant for the entire 5 min.</li>
		<li>After 5 min, deflate balloon and gently remove from the CCA through the arteriotomy.</li>
		<li>Flush the CCA by gently squeezing on the clamp at the CCA. Do not remove the clamp.</li>
		<li>Ligate the ECA proximal to the arteriotomy and then remove the clamps from the CCA and ICA to restore blood flow through the CCA to the ICA. Ensure there is no visible bleeding around the arteriotomy and that the CCA is pulsating.</li>
	</ol>
	</li>
	<li>Apply 100 &#181;L of therapeutic or Pluronic gel vehicle alone periadventitially along the injured CCA. Do so by applying 50 &#181;L to the left side of the CCA and then 50 &#181;L to the right side of the CCA to ensure even coating of the injured artery.</li>
	<li>Close the wound site.
	<ol>
		<li>Cut excess Prolene sutures.</li>
		<li>Close the wound using interrupted 4-0 or 6-0 vicryl layers along the connective tissue.</li>
		<li>Finish closing the wound using running 4-0 nylon suture along the skin.</li>
	</ol>
	</li>
</ol>
3. Postoperative procedures
<ol>
	<li>Place the rat alone in a clean cage with half the cage under a heating lamp and monitor until rat regains sufficient consciousness to maintain sternal recumbency. Keep the rat in a separate cage until the animal is fully alert and mobile before transferring back to their original cage.</li>
	<li>Monitor the rat daily for the next three days and then three times per week until euthanasia. Euthanize using isoflurane overdose followed by bilateral thoracotomy as described below.
	<ol>
		<li>Utilize the National Centre for the Replacement Refinement &#38; Reduction of Animals in Research (NC3Rs) grimace scale to identify postoperative pain levels. If any animal appears to be experiencing pain or develop any neurologic compromise, sacrifice immediately.</li>
		<li>For animals that do not receive carprofen, administer acetaminophen 6 mg/mL in their drinking water 24 h prior to surgery through 48 h post-surgery. Acetaminophen provides analgesia with minimal anti-inflammatory effects.</li>
		<li>Alternatively, other analgesia strategies with minimal anti-inflammatory effects can be used, such as buprenorphine or buprenorphine extended release. Consult the veterinary team at your institution.</li>
	</ol>
	</li>
</ol>
4. Tissue harvest and imaging
<ol>
	<li>Two weeks after surgery, euthanize the rat by overdose of anesthesia (5% isoflurane). Alternatively, euthanize rats at an earlier time point to analyze the various aspects of the arterial injury response.
	<ol>
		<li>Once breathing stops perform bilateral thoracotomy as a secondary method of euthanasia.</li>
	</ol>
	</li>
	<li>Make a lateral incision through the abdomen, and then cut upwards, through the diaphragm and ribs, exposing the thoracic cavity.</li>
	<li>Perfuse and fix the arteries.
	<ol>
		<li>Insert an 18 G cannula attached to a gravitational perfusion-fixation system through the left ventricle. Maintain equivalent pressure between rats by marking the height of the perfusion system relative to the benchtop (120 cm elevation, equivalent to 91 &#177; 3 mmHg).</li>
		<li>Clamp the cannula together with the ventricle using a curved hemostat.</li>
		<li>Make a cut in the right atrium, opening the vascular circuit, and begin perfusion with PBS followed by 2-4% paraformaldehyde (about 250 mL each).</li>
		<li>Prepare paraformaldehyde diluted in PBS the day of sacrifice, or at most the night before sacrifice. If preparing on the day of sacrifice, ensure paraformaldehyde has cooled to room temperature before beginning the perfusion. Store paraformaldehyde at 4 &#176;C.</li>
	</ol>
	</li>
	<li>After fixation, extract the left and right carotid arteries and store at 4 &#176;C for 2 h in 2-4% paraformaldehyde.</li>
	<li>Transfer arteries to 30% sucrose and store overnight at 4 &#176;C.</li>
	<li>After 16-24 h, embed the arteries in optimal cutting temperature (OCT) compound and freeze OCT-embedded artery blocks.
	<ol>
		<li>Condition arteries in OCT at room temperature for 10 min. Place the artery parallel to the plane of the cryomold filled with OCT, marking the side of the cryomold to which the arterial bifurcation is facing. Snap-freeze in liquid nitrogen.</li>
		<li>Store frozen blocks long-term at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Section frozen blocks using a cryostat.
	<ol>
		<li>Collect six 5 &#181;m thick arterial cross sections per slide, with slide 1 starting at the bifurcation.</li>
		<li>Section frozen blocks until hyperplasia no longer visible (around 100 slides).</li>
	</ol>
	</li>
	<li>Hematoxylin &#38; eosin (H&#38;E) stain slides18
	<ol>
		<li>Find the area of injury by staining one in every ten slides along the entire artery starting from the bifurcation (e.g., slides 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100).</li>
		<li>Stain additional slides around the site of injury to find the slide with peak occlusion (e.g., slides 20, 30, and 40 had visible hyperplasia, thus stain slides 15, 25, 35, and 45).</li>
		<li>Stain and quantify the slide with peak occlusion and equidistant slides before and after the peak occlusion slide (e.g., peak occlusion found at slide 35, then stain and quantify slides 25, 45, etc.) for a total of 3-10 slides per rat.</li>
	</ol>
	</li>
	<li>For light sheet fluorescence microscopy imaging, store arteries overnight at 4 &#176;C after fixation in step 4.4.
	<ol>
		<li>Probe artery with 1:500 dilution of rabbit anti-CD31 primary antibody in diluent (pH 7.4) for 3 days. Then counterstain artery with 1:500 dilution of anti-rabbit Alexa Fluor 647 secondary antibody for 2 days19.</li>
		<li>Clear the artery using iDISCO+20.</li>
		<li>Image the artery using a light sheet fluorescence microscope21. Render images using software (e.g., Imaris)19.</li>
	</ol>
	</li>
	<li>Quantify neointimal hyperplasia. Perform quantification in a blinded manner if possible.
	<ol>
		<li>Use ImageJ software to trace the perimeter of the intima, internal elastic lamina (IEL), and external elastic lamina (EEL) of an artery on each of the 3-10 slides determined above (step 4.8.3).</li>
		<li>Quantify the area of each traced region in ImageJ and export these values. The intima trace yields the lumen area, the IEL trace yields the IEL area, and the EEL trace yields the EEL area.</li>
		<li>Average the values obtained from the 3-10 slides to get the average injury (% occlusion, intima:media (I:M) ratio, neointimal hyperplasia) per rat carotid artery. 
		Neointimal Hyperplasia = IEL area - Lumen area 
		<img alt="Equation 1" src="/files/ftp_upload/60473/60473eq1.jpg" /> 
		<img alt="Equation 2" src="/files/ftp_upload/60473/60473eq2.jpg" /></li>
	</ol>
	</li>
</ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effect+of+a+short-acting+NO+donor+for+the+prevention+of+neointimal+hyperplasia.">Beneficial effect of a short-acting NO donor for the prevention of neointimal hyperplasia.</a> Free Radical Biology and Medicine. 44 (1), 73-81 (2008).</li><li>Cao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100B+promotes+injury-induced+vascular+remodeling+through+modulating+smooth+muscle+phenotype.">S100B promotes injury-induced vascular remodeling through modulating smooth muscle phenotype.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1863 (11), 2772-2782 (2017).</li><li>Madigan, M., Entabi, F., Zuckerbraun, B., Loughran, P., Tzeng, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+inhaled+carbon+monoxide+mediates+the+regression+of+established+neointimal+lesions.">Delayed inhaled carbon monoxide mediates the regression of established neointimal lesions.</a> Journal of Vascular Surgery. 61 (4), 1026-1033 (2015).</li><li>Khurana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis-dependent+and+independent+phases+of+intimal+hyperplasia.">Angiogenesis-dependent and independent phases of intimal hyperplasia.</a> Circulation. 110 (16), 2436-2443 (2004).</li><li>Tsihlis, N. D., Vavra, A. K., Martinez, J., Lee, V. R., Kibbe, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+is+less+effective+at+inhibiting+neointimal+hyperplasia+in+spontaneously+hypertensive+rats.">Nitric oxide is less effective at inhibiting neointimal hyperplasia in spontaneously hypertensive rats.</a> Nitric Oxide. 35, 165-174 (2013).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neointimal+hyperplasia+in+the+rat+carotid+artery+injury+model+by+a+HMGB1+inhibitor.">Inhibition of neointimal hyperplasia in the rat carotid artery injury model by a HMGB1 inhibitor.</a> Atherosclerosis. 224 (2), 332-339 (2012).</li><li>Mano, T., Luo, Z., Malendowicz, S. L., Evans, T., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+GATA-6+downregulation+promotes+smooth+muscle+differentiation+and+inhibits+intimal+hyperplasia+in+balloon-injured+rat+carotid+artery.">Reversal of GATA-6 downregulation promotes smooth muscle differentiation and inhibits intimal hyperplasia in balloon-injured rat carotid artery.</a> Circulation Research. 84 (6), 647-654 (1999).</li><li>Becher, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+Provides+Detailed+Atherosclerotic+Plaque+Morphology+and+Reveals+Angiogenesis+after+Carotid+Artery+Ligation.">Three-Dimensional Imaging Provides Detailed Atherosclerotic Plaque Morphology and Reveals Angiogenesis after Carotid Artery Ligation.</a> Circulation Research. 126 (5), 619-632 (2020).</li></ol>

The authors declare that there are no conflicts of interest regarding the publication of this manuscript.

The rat carotid artery balloon injury is one of the most extensively used and studied restenosis animal models. Both the original balloon injury model3 and the modified pressure-controlled segmental injury variation10 have informed many aspects of the arterial injury response that also occurs in humans, with the few limitations being that fibrin-rich thrombus rarely develops and local inflammation is minimal compared to other injury models such as in hypercholesterolemic ra...

Cardiovascular disease (CVD) remains the leading cause of death worldwide1. Atherosclerosis is the underlying cause of most CVD-related morbidity and mortality. Atherosclerosis is the build-up of plaque inside arteries that results in a narrowed lumen, hindering proper blood perfusion to organs and distal tissues2. Clinical interventions for treating severe atherosclerosis include balloon angioplasty with or without stent placement. This intervention involves advancing a balloon catheter to the site of plaque, and inflating the balloon to compress the plaque to the arterial wall, widening the luminal area. This procedure damages the artery, however, initiating the arterial injury response3. Prolonged activation of this injury response leads to arterial restenosis, or re-narrowing, secondary to neointimal hyperplasia and vessel remodeling. During angioplasty the intimal layer is denuded of endothelial cells leading to immediate platelet recruitment and local inflammation. Local signaling induces phenotypic changes in vascular smooth muscle cells (VSMC) and adventitial fibroblasts. This leads to the migration and proliferation of VSMC and fibroblasts inwards to the lumen, leading to neointimal hyperplasia4,5. Circulating progenitor cells and immune cells also contribute to the overall volume of restenosis6. Where applicable, drug-eluting stents (DES) are the current standard for inhibiting restenosis7. DES inhibit arterial re-endothelialization, however, thus creating a pro-thrombotic environment that can result in late in-stent thrombosis8. Therefore, animal models are integral for both understanding the pathophysiology of restenosis, and for developing better therapeutic strategies to prolong the efficacy of revascularization procedures.
Several large and small animal models9 are utilized for studying this pathology. These include balloon-injury3,10 or wire-injury11 of the luminal side of an artery, as well as partial ligation12 or cuff placement13 around the artery. The balloon and wire injury both denude the endothelial layer of the artery, mimicking what occurs clinically after angioplasty. In particular, balloon-injury models utilize similar tools as in the clinical setting (i.e., balloon catheter). The balloon injury is best performed in rat models, as rat arteries are an appropriate size for commercially available balloon catheters. Herein we describe a pressure-controlled segmental arterial injury, a well-established, modified version of the rat carotid artery balloon injury. This pressure-controlled approach closely mimics the clinical angioplasty procedure, and allows for reproducible neointimal hyperplasia formation two weeks after injury14,15. Additionally, this pressure-controlled arterial injury results in complete endothelial layer restoration by 2 weeks after surgery16. This directly contrasts the original balloon injury model, described by Clowes, where the endothelial layer never returns to full coverage3.
After surgery, therapeutics may be applied to or directed towards the injured artery through several approaches. The method described herein uses periadventitial application of a small molecule embedded in a Pluronic gel solution. Specifically, we apply a solution of 100 &#956;M cinnamic aldehyde in 25% Pluronic-F127 gel to the artery immediately after injury to inhibit neointimal hyperplasia formation15. Pluronic-F127 is a non-toxic, thermo-reversible gel able to deliver drugs locally in a controlled manner17. Meanwhile, arterial injury is local, hence local administration allows for testing an active principle while minimizing off-target effects. Nevertheless, effective delivery of a therapeutic using this method will depend on the chemistry of the small molecule or biologic used.

<table><tbody><tr><td>1 mL Syringe</td><td>Fisher</td><td>14955450</td><td></td></tr><tr><td>1 mL Syringe with needle</td><td>BD</td><td>309626</td><td></td></tr><tr><td>2 French Fogarty Balloon Embolectomy Catheter</td><td>Edwards LifeSciences</td><td>120602F</td><td></td></tr><tr><td>4-0 Ethilon (Nylon) Suture</td><td>Ethicon Inc</td><td>662H</td><td></td></tr><tr><td>4-0 Vicryl Suture</td><td>Ethicon Inc</td><td>J214H</td><td></td></tr><tr><td>7-0 Prolene Suture</td><td>Ethicon Inc</td><td>8800H</td><td></td></tr><tr><td>70% ethyl alcohol</td><td></td><td></td><td></td></tr><tr><td>Anti-Rabbit Alexa Fluor 647</td><td>Thermo Fisher Scientific</td><td>A21245</td><td></td></tr><tr><td>Atropine Sulfate</td><td>Vedco Inc</td><td></td><td>for veterinary use</td></tr><tr><td>Cotton Swabs</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Curved Hemostats</td><td>Fine Science Tools</td><td>13009-12</td><td></td></tr><tr><td>Fine Curved Forceps</td><td>Fine Science Tools</td><td>11203-25</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Gauze</td><td>Covidien</td><td>2252</td><td></td></tr><tr><td>IHC-Tek Diluent (pH 7.4)</td><td>IHC World</td><td>IW-1000</td><td></td></tr><tr><td>Insufflator</td><td>Merit Medical</td><td>IN4130</td><td></td></tr><tr><td>Iodine solution</td><td></td><td></td><td></td></tr><tr><td>Lubricating Eye Ointment</td><td>Dechra</td><td></td><td>for veterinary use</td></tr><tr><td>Mayo Scissors</td><td>Fine Science Tools</td><td>14010-15</td><td></td></tr><tr><td>Micro Serrefines</td><td>Fine Science Tools</td><td>18055-05</td><td></td></tr><tr><td>Microdissection Scissors</td><td>Fine Science Tools</td><td>15004-08</td><td></td></tr><tr><td>Micro-Serrefine Clamp Applying Forceps</td><td>Fine Science Tools</td><td>18057-14</td><td></td></tr><tr><td>Needle Holder</td><td>Fine Science Tools</td><td>12003-15</td><td></td></tr><tr><td>Pluronic-127 (diluted in sterile water)</td><td>Sigma-Aldrich</td><td>P2443</td><td>25% prepared</td></tr><tr><td>Rabbit Anti-CD31</td><td>Abcam</td><td>ab28364</td><td></td></tr><tr><td>Retractor</td><td></td><td></td><td>Bent paper clips work well</td></tr><tr><td>Rimadyl (Carprofen)</td><td>Zoetis Inc</td><td></td><td>for veterinary use</td></tr><tr><td>Saline solution</td><td></td><td></td><td></td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Sterile Drape</td><td>Dynarex</td><td>4410</td><td></td></tr><tr><td>T-Pins</td><td></td><td></td><td></td></tr></tbody></table>

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.

Figure 1 shows all of the materials and surgical tools used to perform this surgery. Hematoxylin &#38; eosin (H&#38;E) staining of two-week injured arterial cross sections allows for clear visualization of neointimal hyperplasia. Figure 2 shows representative images of H&#38;E-stained arterial cross-sections of a healthy, injured, and treated artery. Figure 2 also outlines how to quantify the level of neointimal hyperplasia in an in...

Watch this Scientific Journal Video about A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application at JoVE.com

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

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 ...

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4.9K Views.  University of North Carolina at Chapel Hill. De rat carotis slagader ballon letsel bootst de klinische angioplastiek procedure uitgevoerd om de bloedstroom in atherosclerotische vaten te herstellen. Dit model induceert de arteriële letselrespons door de arteriële wand te verwijderen en de intimale laag endotheelcellen te ontnuderen, wat uiteindelijk remodellering en een intimale hyperplastische respons veroorzaakt.

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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

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

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

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

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

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

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

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

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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 ...

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 ...

Transvenous Embolization of Carotid Cavernous Fistula through Inferior Petrosal Sinus with Detachable Coils and Ethylene Vinyl Alcohol Copolymer

Carotid cavernous fistula (CCF) is a rare disease caused by abnormal communications between the internal carotid artery (direct fistula) or meningeal branches of the external carotid artery (indirect fistula) and the cavernous sinus (CS). Trauma is the most common cause of CCF. The clinical presentation of CCF is closely related to the venous drainage pattern. Orbital and neuro-ophthalmological symptoms are the most common clinical presentation of CCF with drainage through the superior ophthalmic vein (SOV). Endovascular embolization by arterial or venous approaches is the most common management of CCF. Transvenous embolization using detachable coils and ethylene-vinyl alcohol copolymer (EVOH) is an alternative method for the treatment of CCF. Endovascular embolization offers different options to treat CCF by minimally invasive approach decreasing morbidity and residual fistulas. The purpose of this article is to report our treatment experiences via the inferior petrosal sinus (IPS), and immediate-term outcomes of endovascular embolization of CCF by using detachable coils and EVOH.

The purpose of the article is to present our experience in endovascular treatment via the inferior petrosal sinus of the carotid cavernous fistula with detachable coils and ethylene-vinyl alcohol copolymer.

Carotid cavernous fistula (CCF) is a rare disease caused by abnormal communications between the internal carotid artery (direct fistula) or meningeal ...

Complete and Partial Resuscitative Endovascular Balloon Occlusion of the Aorta for Hemorrhagic Shock

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for hemorrhage control. With the advent of purpose-built devices, REBOA has become increasingly common in civilian trauma and acute care settings. Currently available REBOA catheters were designed as complete aortic occlusion devices. However, the therapeutic window for complete aortic occlusion is time-limited due to ischemia-reperfusion injury. The partial procedure allows blood flow past the level of occlusion while maintaining targeted proximal pressure, which has been shown to reduce distal ischemia and adjunctive resuscitation requirements in preclinical studies with prolonged occlusion times as compared to traditional complete occlusion.
pREBOA-PRO is the first catheter designed to enable partial and complete aortic occlusion and is currently in limited market release at seven Level I trauma centers in North America. This paper will focus on procedural considerations for REBOA, including patient selection criteria and a comparison of complete and partial aortic occlusion in a simulator, along with highlighting critical steps to improve clinical outcomes. Additionally, this paper reviews a contrast-enhanced CT scan from a trauma patient that shows distal perfusion after 2 h of partial aortic occlusion using this newly designed catheter and discusses representative results from the limited market release to highlight the profound effect of technological innovation on outcomes in vascular emergencies.

A commercial catheter was designed to facilitate true partial resuscitative endovascular balloon occlusion of the aorta (REBOA) and address the complications associated with complete aortic occlusion. Initial clinical reports indicate that partial REBOA improves transition to reperfusion, reduction of distal ischemia, and extension of safe occlusion time compared to complete occlusion.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

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

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