Name: a rat carotid balloon injury model to test anti-vascular remodeling therapeutics
Uploaded: 2016-09-19T12:30:00
Description: Watch this Scientific Journal Video about A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics at JoVE.com

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

NOTE: the use of rat balloon injury model and associated procedures including injection of recombinant sRAGE and ultrasound sonographic studies have been approved by the Animal Care and Use Committee (ACUC) of National Institute on Aging, NIH.
1. Pre-surgery Preparations
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	<li>Instrument, Surgical Platform, and Personal Protection Equipment.
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		<li>See Materials and Equipment&#39;s Table for all the surgical instruments and reagents used in this pro...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+post-intervention+arterial+remodelling.">Mechanisms of post-intervention arterial remodelling.</a> Cardiovasc Res. 96, 363-371 (2012).</li><li>Khan, R., Agrotis, A., Bobik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+role+of+transforming+growth+factor-beta1+in+intimal+thickening+after+vascular+injury.">Understanding the role of transforming growth factor-beta1 in intimal thickening after vascular injury.</a> Cardiovasc Res. 74, 223-234 (2007).</li><li>Schillinger, M., Minar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restenosis+after+percutaneous+angioplasty:+the+role+of+vascular+inflammation.">Restenosis after percutaneous angioplasty: the role of vascular inflammation.</a> Vasc Health Risk Manag. 1, 73-78 (2005).</li><li>Katz, G., Harchandani, B., Shah, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-eluting+stents:+the+past,+present,+and+future.">Drug-eluting stents: the past, present, and future.</a> Curr Atheroscler Rep. 17, 485 (2015).</li><li>Jain, M., Singh, A., Singh, V., Barthwal, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+vivo+evidence+demonstrating+neointimal+migration+of+adventitial+fibroblasts+after+balloon+injury+of+rat+carotid+arteries.">Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries.</a> Circulation. 101 (12), 1362-1365 (2000).</li><li>Noda, 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=New+endoplasmic+reticulum+stress+regulator,+gipie,+regulates+the+survival+of+vascular+smooth+muscle+cells+and+the+neointima+formation+after+vascular+injury.">New endoplasmic reticulum stress regulator, gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury.</a> Arterioscler Thromb Vasc Biol. 35 (5), 1246-1253 (2015).</li><li>Deuse, 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=Dichloroacetate+prevents+restenosis+in+preclinical+animal+models+of+vessel+injury.">Dichloroacetate prevents restenosis in preclinical animal models of vessel injury.</a> Nature. 509 (7502), 641-644 (2014).</li><li>Guo, 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=p55gamma+functional+mimetic+peptide+N24+blocks+vascular+proliferative+disorders.">p55gamma functional mimetic peptide N24 blocks vascular proliferative disorders.</a> J Mol Med (Berl). , (2015).</li><li>Oh, C. 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=Dimethylfumarate+attenuates+restenosis+after+acute+vascular+injury+by+cell-specific+and+Nrf2-dependent+mechanisms.">Dimethylfumarate attenuates restenosis after acute vascular injury by cell-specific and Nrf2-dependent mechanisms.</a> Redox Biol. 2, 855-864 (2014).</li><li>Tae, H. 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=The+N-glycoform+of+sRAGE+is+the+key+determinant+for+its+therapeutic+efficacy+to+attenuate+injury-elicited+arterial+inflammation+and+neointimal+growth.">The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth.</a> J Mol Med (Berl. 91 (12), 1369-1381 (2013).</li><li>Zhou, Z., 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=Receptor+for+AGE+(RAGE)+mediates+neointimal+formation+in+response+to+arterial+injury.">Receptor for AGE (RAGE) mediates neointimal formation in response to arterial injury.</a> Circulation. 107 (17), 2238-2243 (2003).</li><li>Lindner, V., Fingerle, J., Reidy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+arterial+injury.">Mouse model of arterial injury.</a> Circ Res. 73 (5), 792-796 (1993).</li><li>Le, V., Johnson, C. G., Lee, J. D., Baker, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+femoral+artery+wire+injury+with+implantation+of+a+perivascular+drug+delivery+patch.">Murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch.</a> J Vis Exp. (96), e52403 (2015).</li><li>Iannaccone, P. M., Jacob, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats!+.">Rats! .</a> Dis Model Mech. 2 (5-6), 206-210 (2009).</li><li>Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., Joung, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods Mol Med. 139, 1-30 (2007).</li><li>Zhang, W., Trebak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+balloon+injury+and+intraluminal+administration+in+rat+carotid+artery.">Vascular balloon injury and intraluminal administration in rat carotid artery.</a> J Vis Exp. (94), (2014).</li><li>Tae, H. 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=Vessel+ultrasound+sonographic+assessment+of+soluble+receptor+for+advanced+glycation+end+products+efficacy+in+a+rat+balloon+injury+model.">Vessel ultrasound sonographic assessment of soluble receptor for advanced glycation end products efficacy in a rat balloon injury model.</a> Curr Ther Res Clin Exp. 76, 110-115 (2014).</li><li>Tae, H. 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There have been two methods used to inflate the balloon in order to generate the injury that removes the mural endothelium in the carotid arterial lumen. One is to fill the attached syringe with liquid20, and the other is to use air pressure21. We prefer to use liquid-filled syringe because the exact liquid volume (0.02 ml) will be used for each procedure. This renders a precise and reproducible inflation of the balloon, leading to a similar level of injury in each animal undergoing the procedure. U...

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

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			<td height="21" style="height:21px;width:287px;">2 F Fogarty balloon embolectomy catheter&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Edwards Lifesciences&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Standard scalpel</td>
			<td style="width:163px;">Fine Science Tools</td>
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			<td height="40" style="height:40px;width:287px;">Small&#160; curved forceps (Large radius Dumont#7shanks curved)&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Large, medium and small micro-scissors</td>
			<td style="width:163px;">Roboz</td>
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			<td height="21" style="height:21px;width:287px;">Needles (20 G)&#160;&#160;</td>
			<td style="width:163px;">TycoHealthcare</td>
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			<td height="41" style="height:41px;width:287px;">Micro-surgery forceps with micro-blunted atraumatic tips</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Atraumatic straight small arterial clamps&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="41" style="height:41px;width:287px;">Retractor&#160; with maximum spread 5.5 cm long blunt teeth</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Silk suture (4.0 and&#160; 6.0 )</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Syringe (1.0 ml)&#160;</td>
			<td style="width:163px;">BD&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Curity gauze sponges</td>
			<td style="width:163px;">AllegroMedical</td>
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			<td height="21" style="height:21px;width:287px;">Cotton tip applicators sterile and non-sterile</td>
			<td style="width:163px;">Puritan Medical Products</td>
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			<td height="21" style="height:21px;width:287px;">Compact hot bead sterilizer</td>
			<td style="width:163px;">Fine Science Tools</td>
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			<td height="21" style="height:21px;width:287px;">Self-regulating heating pad</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">ADS200 anesthesia system/ventilator</td>
			<td style="width:163px;">Paragon Medical</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoflurane (forane), liquid form</td>
			<td style="width:163px;">Baxter</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium chloride 0.9% (Saline)&#160;</td>
			<td style="width:163px;">Hospira</td>
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			<td height="41" style="height:41px;width:287px;">Buprenex (buprenorphine)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Reckitt Benckiser Healthcare (UK) Ltd.&#160;</td>
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			<td height="21" style="height:21px;width:287px;">70% alcohol</td>
			<td style="width:163px;">Fisher</td>
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			<td height="21" style="height:21px;width:287px;">1: 10 Betadine</td>
			<td style="width:163px;">Fisher</td>
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a rat carotid balloon injury model to test anti-vascular remodeling therapeutics

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

Two weeks after the balloon injury, the rat is euthanized and the carotid arteries are isolated for histo-morphological analysis. Both operated left carotid artery and un-operated right artery are cross-sectioned, processed, and paraffin-embedded. The paraffin samples are then further thin-sectioned, and stained with hematoxylin-eosin (H &#38; E). Histo-morphological analyses are performed using a digital imaging analysis system. The details of artery harvest and histo-morphological analy...

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

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

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

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

The overall goal of this Rat Carotid Balloon Injury procedure is to mimic clinical angioplasty and its potential consequences. This message can help answer key questions in the cardiovascular research field, about the cardiovascular surgical injury elicited neointimal hyperplasia which can significantly compromise the benefits of the surgery. The main advantage of this technique is that the result can be obtained within two weeks, allowing a quick evaluation of the therapeutic reagents administered during the treatment period.Demonstrating the procedure will be Dr.Natalia Petrasheskatya from the laboratory of cardiovascular sciences and at the National Institute on Aging. Before beginning the surgery, autoclave all of the surgical instruments and use 70%ethanol to disinfect the operating platform surface. Put on the proper personal protective equipment, placing a box of sterilized gloves near the surgical platform for additional use.Next, fill a 1 milliliter syringe with sterile water and attach the syringe to a sterile, water-filled, two-way stopcock. Gently push the water through the stopcock to fill the lure-lock portion of the balloon catheter, and remove any air bubbled from the opening of the catheter. Then, test the balloon inflation to confirm that the balloon can be inflated with 0.02 milliliters of water.Now, check the pedal withdrawal reflex of an anesthetized 400 to 450 gram male Wistar rat. If the rat does not withdraw its foot, transfer the animal to the surgical platform. Cover the rat with a sterile surgical sheet, and cut the sheet to expose the neck region.Then adjust the animal into the supine position on a headed pad with the head toward the surgeon and tape the limbs into place. To isolate the left carotid artery, us a number 14 scalpel to make a straight incision from below the chin to the top of the sternum, just below the ribcage. Using a 7S forceps, blunt dissect under the skin and through the salivary gland tissues to expose the underlying muscle layer.Separate the muscle tissues to gain access to the carotid vasculature and the vagus nerve in the neck. Continue to blunt dissect the tissues surrounding the carotid artery, carefully separating the vagus nerve and the vascular fascia without damaging either tissue, until the bifurcation of the common carotid artery and the internal and external carotid branches are clearly visible. When the left common carotid artery has been exposed all the way to the sternum, place a 4-0 silk stay suture behind the artery as close to the sternum as possible.Permanently ligate the smaller arteries with the sutures, along the external carotid artery, including the ascending pharyngeal, the occipital, and the superior thyroid to avoid arterial leakage. Place another 4-0 silk stay suture immediately distal to the bifurcation, behind the external carotid artery as far from the bifurcation as possible. Then gently retract the overlying carotid artery to the right to visualize the internal carotid artery and place a 4-0 silk stay suture around the artery to avoid significant retrograde blood-loss.To introduce the balloon catheter, retract the proximal suture on the common carotid artery, and temporarily stop the blood flow with an arterial clamp. Make a small incision on the external carotid artery as distal to the suture as possible, and gently insert the uninflated balloon catheter into the incision. Advance the catheter into the lumen until it is close to the arterial clamp on the common carotid artery.Then remove the clamp and advance the catheter to the aortic arch, approximately 35 to 40 millimeters from the incision. Now use the syringe of sterile water to manually inflate the catheter to a 0.02 milliliter volume, and turn the stopcock to the locked position. Slowly withdraw the catheter while rotating to produce the injury.When the instrument is close to the arterial incision, deflate the balloon and retract the catheter back to its original position. After repeating the balloon injury procedure three times, carefully remove the catheter from the arterial lumen and close the arteriotomy with size 5-0 silk stay sutures. Release the internal carotid artery to restore the blood flow and check for arterial leakage.Finally, remove the stay sutures and clamps and close the glandular tissue with 6-0 silk sutures. Then, close the skin and monitor the animal until it is fully recovered. Two weeks after the balloon injury, the carotid arteries can be isolated for histomorphological analysis.The balloon injury elicits neointimal growth, or thickening of the vessel wall. If left untreated or treated with a saline control, compared to a non-operated vessel section from the same animal. By contrast, the vessel section from a rat that received a therapeutic reagent that blocks neointimal growth demonstrates a significantly reduced thickening in the vessel wall.The effects of the balloon injury and therapeutic treatment can also be evaluated in vivo, by ultrasound sonography. And these data collaborate well with the results from histomorphological analysis. When mastered, this techniques can be completed in 30 minutes, if it is performed properly.While attempting this procedure, it is important to remember to prepare and test the balloon catheter for leaks prior to the surgery. After watching this video, you should have a good understanding of how to perform the rat carotid balloon injury procedure, for evaluating tracks and therapeutic reagents that block neointimal growth and to study the mag-ni-sen of injury elicited neointimal growth. Don't forget that working with animals requires an approved protocol by your institutional AICUC.You should also consult with your institutional laboratory animal veterinarians to establish the best practices for post-surgical care, and monitoring of the animals.

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Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

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 ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

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 ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

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, ...

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

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

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

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

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...