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.

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			<td height="21" style="height:21px;width:327px;">1 mL Syringe</td>
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			<td style="width:119px;">14955450</td>
			<td style="width:183px;"></td>
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			<td height="21" style="height:21px;">1 mL Syringe with needle</td>
			<td style="width:148px;">BD</td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 French Fogarty Balloon Embolectomy Catheter</td>
			<td style="width:148px;">Edwards LifeSciences</td>
			<td style="width:119px;">120602F</td>
			<td></td>
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			<td style="width:119px;">J214H</td>
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			<td style="width:148px;">Ethicon Inc</td>
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			<td>for veterinary use</td>
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			<td height="21" style="height:21px;">Gauze</td>
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			<td style="width:119px;">2252</td>
			<td></td>
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			<td height="21" style="height:21px;width:327px;">IHC-Tek Diluent (pH 7.4)</td>
			<td style="width:148px;">IHC World</td>
			<td style="width:119px;">IW-1000</td>
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			<td height="21" style="height:21px;">Insufflator</td>
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			<td style="width:119px;">IN4130</td>
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			<td height="21" style="height:21px;">Iodine solution</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Lubricating Eye Ointment</td>
			<td style="width:148px;">Dechra</td>
			<td style="width:119px;"></td>
			<td>for veterinary use</td>
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			<td height="21" style="height:21px;">Mayo Scissors</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">14010-15</td>
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			<td height="21" style="height:21px;">Micro Serrefines</td>
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			<td style="width:119px;">18055-05</td>
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			<td height="21" style="height:21px;">Microdissection Scissors</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">15004-08</td>
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			<td height="21" style="height:21px;">Micro-Serrefine Clamp Applying Forceps</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">18057-14</td>
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			<td height="21" style="height:21px;">Needle Holder</td>
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			<td style="width:119px;">12003-15</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pluronic-127 (diluted in sterile water)</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2443</td>
			<td style="width:183px;">25% prepared</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Rabbit Anti-CD31</td>
			<td style="width:148px;">Abcam</td>
			<td style="width:119px;">ab28364</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Retractor</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td style="width:183px;">Bent paper clips work well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rimadyl (Carprofen)</td>
			<td style="width:148px;">Zoetis Inc</td>
			<td style="width:119px;"></td>
			<td>for veterinary use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline solution</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Forceps</td>
			<td style="width:148px;">Fine Science Tools</td>
			<td style="width:119px;">11006-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Drape</td>
			<td style="width:148px;">Dynarex</td>
			<td style="width:119px;">4410</td>
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			<td height="21" style="height:21px;">T-Pins</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></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 ...

a-rat-carotid-artery-pressure-controlled-segmental-balloon-injury

Research

JoVE Journal

Medicine

4.9K Views.  University of North Carolina at Chapel Hill. 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.

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

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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

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

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...