Name: ferric chloride-induced canine carotid artery thrombosis: a large animal model of vascular injury
Uploaded: 2018-09-07T14:45:00
Description: Watch this Scientific Journal Video about Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury at JoVE.com

We would like to thank the Center for Cognitive and Behavioral Brain Imaging at The Ohio State University for their financial and scientific support to develop and perform canine magnetic resonance imaging.

The investigations described conform to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by The Ohio State University Institutional Animal Care and Use Committee (#2015A00000029). All surgical manipulations were performed under deep anesthesia and the animals did not experience pain at any stage during the procedure. All experiments described were non-recovery.
1. Preparation
<ol>
	<li>Freshly prepare 50 mL of ferric chloride (F...

<ol>
	<li>Adams, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke:+a+vascular+pathology+with+inadequate+management.">Stroke: a vascular pathology with inadequate management.</a> Journal of Hypertension Supplement. 21 (5), S3-S7 (2003).</li><li>Lansberg, M. G., Bluhmki, E., Thijs, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+tissue+plasminogen+activator+3+to+4.5+hours+after+acute+ischemic+stroke:+a+metaanalysis.">Efficacy and safety of tissue plasminogen activator 3 to 4.5 hours after acute ischemic stroke: a metaanalysis.</a> Stroke. 40 (7), 2438-2441 (2009).</li><li>Nagel, S., 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=Therapy+of+acute+basilar+artery+occlusion:+intraarterial+thrombolysis+alone+vs+bridging+therapy.">Therapy of acute basilar artery occlusion: intraarterial thrombolysis alone vs bridging therapy.</a> Stroke. 40 (1), 140-146 (2009).</li><li>Ciccone, A., Motto, C., Abraha, I., Cozzolino, F., Santilli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+IIb-IIIa+inhibitors+for+acute+ischaemic+stroke.">Glycoprotein IIb-IIIa inhibitors for acute ischaemic stroke.</a> The Cochrane database of systematic reviews. 3 (3), (2014).</li><li>The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Plasminogen+Activator+for+Acute+Ischemic+Stroke.">Tissue Plasminogen Activator for Acute Ischemic Stroke.</a> New England Journal of Medicine. 333 (24), 1581-1588 (1995).</li><li>Leadley, R., Chia, L., Rebellob, S., Gagnon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+in+vivo+models+of+thrombosis+to+the+discovery+and+development+of+novel+antithrombotic+agents.">Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents.</a> Journal of Pharmacological and Toxicological Methods. 43 (2), 101-116 (2000).</li><li>Bodary, P. F., Eitzman, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Thrombosis.">Animal Models of Thrombosis.</a> Current Opinion In Hematology. 16 (5), 342-346 (2009).</li><li>Sachs, U. J. H., Nieswandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+thrombus+formation+in+murine+models.">In vivo thrombus formation in murine models.</a> Circulation Research. 100 (7), 979-991 (2007).</li><li>Bonnard, T., Hagemeyer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+Chloride-induced+Thrombosis+Mouse+Model+on+Carotid+Artery+and+Mesentery+Vessel.">Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel.</a> Journal of Visualized Experiments. (100), e52838 (2015).</li><li>Kurz, K. D., Main, B. W., Sandusky, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+arterial+thrombosis+induced+by+ferric+chloride.">Rat model of arterial thrombosis induced by ferric chloride.</a> Thrombosis Research. 60 (4), 269-280 (1990).</li><li>Karatas, H., Erdener, S. E., 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=Thrombotic+distal+middle+cerebral+artery+occlusion+produced+by+topical+FeCl(3)+application:+a+novel+model+suitable+for+intravital+microscopy+and+thrombolysis+studies.">Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies.</a> Journal of Cerebral Blood Flow and Metabolism. 31 (6), 1452-1460 (2011).</li><li>Li, W., McIntyre, T. M., Silverstein, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+murine+carotid+arterial+injury:+A+model+of+redox+pathology.">Ferric chloride-induced murine carotid arterial injury: A model of redox pathology.</a> Redox Biology. 1 (1), 50-55 (2013).</li><li>Vilahur, G., Padro, T., Badimon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis+and+Thrombosis:+Insights+from+Large+Animal+Models.">Atherosclerosis and Thrombosis: Insights from Large Animal Models.</a> Journal of Biomedicine and Biotechnology. 2011, 1-12 (2011).</li><li>Coller, B. S., Folts, J. D., Smith, S. R., Scudder, L. 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Correlation with Bleeding Time, Platelet Aggregation, and Blockade of GPIIb/IIIa Receptors.</a> Circulation. 80 (6), 1766-1774 (1989).</li><li>Folts, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+Model+of+Experimental+Arterial+Stenosis,+Intimal+Damage,+and+Periodic+Thrombosis.">An in vivo Model of Experimental Arterial Stenosis, Intimal Damage, and Periodic Thrombosis.</a> Circulation. 83 (6 Suppl), (1991).</li><li>Yasuda, 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=A+canine+model+of+coronary+artery+thrombosis+with+superimposed+high+grade+stenosis+for+the+investigation+of+rethrombosis+after+thrombolysis.">A canine model of coronary artery thrombosis with superimposed high grade stenosis for the investigation of rethrombosis after thrombolysis.</a> Journal of the American College of Cardiology. 13 (6), 1409-1414 (1989).</li><li>Schob, S., 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=Correlation+Between+Aquaporin+4+Expression+and+Different+DWI+Parameters+in+Grade+I+Meningioma.">Correlation Between Aquaporin 4 Expression and Different DWI Parameters in Grade I Meningioma.</a> Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging. 19 (1), 138-142 (2017).</li><li>Schob, S., 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=Diffusion-Weighted+Imaging+Using+a+Readout-Segmented,+Multishot+EPI+Sequence+at+3+T+Distinguishes+between+Morphologically+Differentiated+and+Undifferentiated+Subtypes+of+Thyroid+Carcinoma-A+Preliminary+Study.">Diffusion-Weighted Imaging Using a Readout-Segmented, Multishot EPI Sequence at 3 T Distinguishes between Morphologically Differentiated and Undifferentiated Subtypes of Thyroid Carcinoma-A Preliminary Study.</a> Translational Oncology. 9 (5), 403-410 (2016).</li><li>Schob, S., 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=Diffusion-Weighted+MRI+Reflects+Proliferative+Activity+in+Primary+CNS+Lymphoma.">Diffusion-Weighted MRI Reflects Proliferative Activity in Primary CNS Lymphoma.</a> Public Library of Science One. 11 (8), e0161386 (2016).</li></ol>

The FeCl3 induced vascular injury model is widely used to study thrombosis in small animals and is easy to translate into a large animal, pre-clinical model with a multitude of advantages. Slight modifications to adapt the protocol into a canine allow the utilization of both magnetic resonance imaging to assess stroke and hemorrhage volumes after a pharmacological intervention and angiography to assess vessel canalization before, during, and after treatment. Other thrombotic large animal models have not studie...

Stroke therapy is largely modeled after coronary artery disease treatment, mainly because interventions in cardiovascular disease have responded well to drug therapy and endovascular interventions1. These treatments, however, have not successfully translated to cerebral infarction. The difficulties with the current stroke treatment are that the recombinant tissue plasminogen activator (rTPA) cannot be reversed, and that administration carries a significant 6.4% risk of hemorrhagic conversion2,3,4. The resulting morbidity and mortality limits its use to a small, often unattainable window5. Also, restenosis and occlusion occur often after initial thrombolysis, reversing initial neurological improvement. In summary, there is a narrow temporal window to administer rTPA that excludes the large majority (~90%) of patients who suffer ischemic cerebrovascular insults.
The role of intravenous antiplatelet therapy has shown promise in treating ischemic stroke with improved vessel recanalization, survival and outcome2. Unfortunately, these drugs have a predictable side-effect of intra-cranial and extra-cranial hemorrhage, largely because there is no way to adequately reverse or control their activity2. While effective in preventing platelet aggregation, the risk of hemorrhage and the inability to reverse their activity have precluded their use in the routine care of stroke patients. A need, therefore, exists for potent antithrombotic drugs that act alone or in combinations to prevent and lyse clots yet have a safety profile that will allow the use in a closed, low volume space such as the brain, where hemorrhage is poorly tolerated.
Understanding the mechanism of arterial thrombosis and re-stenosis, and evaluating thrombolytics and drugs that prevent re-stenosis, requires both small and large animal models as a part of pre-clinical drug development. Ferric chloride-induced vascular injury is a widely utilized technique to rapidly and accurately induce the formation of thrombi in exposed blood vessels of mice, rats, guinea pigs, and rabbits6,7,8,9,10,11,12. These smaller species offer several advantages including ease of genetic manipulation, inexpensive animal purchase, and low per diem housing costs. Unfortunately, small animal experiments negate multiple blood draws during the surgery to access platelet reactivity, blood gas analysis, and inflammatory response. More importantly, large animals much more closely mimic human platelet physiology6,13. The FeCl3 carotid artery injury model has played a predominant role in the study of the pathophysiology of thrombosis, in the validation of novel anti-platelet and anti-coagulant drugs, and in the discovery of potential thrombolytics6,7,8,9,10,11,12. Previous models in mice, rats, guinea pigs, and rabbits have provided ease and flexibility for the genetic manipulation, but translatable pre-clinical models are critical to patient dosing and toxicity studies of potential therapeutics6,13. Although several models of thrombotic disorders have been developed in mice, large animal models of thrombosis that are applicable to the peripheral vascular disease, stroke and myocardial infarction are few and far. The first thrombosis models in monkeys, dogs, and pigs focused on stenosis, applying hemostats and later cylinders to vessels, commonly resulting in cyclic flow reductions14,15,16. Instead of an occlusive thrombus at the site of the endothelial damage as in the ferric chloride model, the thrombus in these models resulted in cyclic thrombosis, distal embolization and return to normal blood flow. In comparison, the ferric chloride model modified here in a large animal, results in an occlusive thrombus at the injury site and is stabilized and verified by angiography before thrombolytic treatment. Provided that the investigator has ample funds for per diem and purchase of canines and adequate surgical expertise, we detail here a large canine model of vascular injury to allow laboratories to study thrombosis utilizing surgical, imaging and histological techniques.

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			<td height="40" style="height:40px;width:397px;">1/8&#8221; umbilical tape&#160;</td>
			<td style="width:195px;">Jorgensen Laboratories Inc.,&#160;</td>
			<td style="width:344px;">#J0025UA&#160;</td>
			<td style="width:201px;">for ferric chloride application</td>
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			<td>Richard-Allan Scientific</td>
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			<td height="40" style="height:40px;">&#160;2% 2,3,5-triphenyltetrazolium chloride (TTC in PBS, pH 7.4)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
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			<td height="40" style="height:40px;">ADP/Collagen cartridges</td>
			<td>Siemens Diagnostics</td>
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			<td height="40" style="height:40px;">4.5 ml 3.2% sodium citrate blood vacutainer&#160;</td>
			<td>Becton Dickinson</td>
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			<td height="40" style="height:40px;">4.5 ml lithium heparin vacutainer&#160;</td>
			<td>Becton Dickinson</td>
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			<td height="40" style="height:40px;">EDTA K3 vacutainers&#160;</td>
			<td>Becton Dickinson</td>
			<td>BD455036</td>
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			<td height="40" style="height:40px;">Doppler flow probe</td>
			<td>Transonic Systems Inc</td>
			<td>MA2.5PSL</td>
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			<td height="40" style="height:40px;">Hematoxylin 560&#160;</td>
			<td>Surgipath</td>
			<td>3801570</td>
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			<td height="40" style="height:40px;">Eosin</td>
			<td>Surgipath</td>
			<td>3801602</td>
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			<td height="40" style="height:40px;">LabChart Software</td>
			<td>ADInstruments Inc.</td>
			<td></td>
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			<td height="40" style="height:40px;">Prisma Fit 3 tesla (3T) magnet</td>
			<td>Siemen&#39;s Diagnostics</td>
			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">Sodium heparin for injection (to coat blood gas syringe)</td>
			<td>NovaPlus</td>
			<td>402525D</td>
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			<td height="40" style="height:40px;">HUG-U-VAC positioning system &#160;</td>
			<td>DRE Veterinary</td>
			<td>1320</td>
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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.

Following the detailed procedures herein will result in the development of a model that can be used for prophylactic or thrombolytic assessment of occlusive arterial interventions. Figure 1A shows baseline flow velocity and the resulting blood flow velocity before, during, and after treatment recorded by a commercial software. Data from this recording can be used to determine the percent of re-perfusion with carotid artery injury and treatment in this canine ...

Watch this Scientific Journal Video about Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury at JoVE.com

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

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, ...

This method can help answer key questions in the thrombosis field, allowing the translation of therapeutic treatments in a large animal model. The main advantage of this technique is that the large animal model allows for monitoring of physiological parameters relevant to humans, such as complete blood count and platelet function. This experimental technique allows us to study thrombosis and the disorders that result in thrombosis.The advantages include that it is reproducible, it can be monitored by angiography, we can capture histology, and perform serial blood sampling throughout the procedure. Before inducing the injury to determine the extent of canalization of the carotid artery and the length of the occlusive thrombus, feel for a pulse in the right inguinal region of an anesthetized adult canine and make a three-to five-centimeter ventral sagittal incision. Use blunt dissection to expose and separate the femoral artery from the surrounding tissues, and use a right-angle hemostat to place zero-silk sutures around the proximal and distal ends of the exposed artery.Tie the distal zero-silk suture and clamp the loose zero-silk suture ends to the skin with hemostats to apply slight tension to the artery. Use an 18-gauge introducer needle to puncture the artery midway between the proximal and distal zero-silk suture ties and allow the insertion of a guide wire into the artery. Put an assembled 6 French sheath and dilator onto the guide wire, grasping the wire as it exits from the assembly.Advance the dilator and sheath over the guide wire, taking care to maintain a firm grip on the wire as it protrudes from the dilator. After full insertion, tie the proximal zero-silk suture around the sheath to maintain hemostasis at the arterial puncture site. Then simultaneously remove the dilator and the guide wire, and open the one-way valve to verify the presence of pulsatile blood flow.Flush the valve with three to five milliliters of normal saline, and connect the access sheath one-way valve to a fluid-filled extension line attached to a pressure transducer to monitor and record invasive blood pressure measurements. Preload a 0.35-inch guide wire into a 4 French angiographic catheter, and connect the catheter to the contrast injection manifold set with a two-way Y adapter. Pull the tip of the guide wire just inside the distal tip of the catheter, and flush the entire assembly with saline.Insert the angiographic catheter into the hemostatic valve of the sheath, and advance the guide wire about five centimeters beyond the distal catheter tip. Under fluoroscopic guidance and using a retrograde transaortic approach, slowly advance the catheter into the aortic arch. Inject two to three milliliters of contrast agent diluted one to one with normal saline to identify the takeoff of the brachiocephalic trunk.Use the guide wire to direct the catheter into the brachiocephalic trunk and selectively into the right common carotid artery. Remove the guide wire and inject two to three milliliters of diluted contrast to verify that the catheter is placed within the carotid. Then, inject three to four milliliters of undiluted contrast agent while recording angiographic runs using both digital subtraction and standard angiographic techniques.Make an eight-to 10-centimeter incision of the skin directly on top of the right common carotid artery region, and dissect the fascia to isolate the vessel from the surrounding tissue. Carefully use forceps to separate the carotid artery and the vagus nerve. Place the Doppler flow probe around the carotid, allowing ample space to wrap the umbilical tape around the carotid without touching the flow probe, and record the baseline blood velocity for about five minutes before using a hemostat to place a piece of prepared ferric chloride umbilical tape below the flow probe.After 15 minutes, remove and discard the tape and stabilize the occlusive thrombus for 45 minutes, adding additional saline to the probe as necessary to maintain signal. To determine the stroke or hemorrhage volume, in addition to the downstream thromboembolic events that may result from pharmacological interventions, the canine can be transported to a magnetic resonance imaging machine. While still under a deep surgical plane of anesthesia, collect whole blood as appropriate for later analyses.After exsanguination, rinse the heart with PBS and flash-freeze the samples in liquid nitrogen if desired. Before removing the carotids, measure and record the clot length, and place a tissue marker on the middle of the clot. Mark the contralateral carotid at the same length, and remove the entire length of both vessels for fixation in 10%formalin.Flash-freeze half of the contralateral carotid artery in liquid nitrogen for later analysis. Rinse the brain in PBS, and use a sharp scalpel to laterally remove two four-millimeter sections from the middle of the brain. Then place one four-millimeter section into 2%TTC for ischemic demarcation, and place the other tissue sample in 10%formalin for seven days for subsequent hematoxylin and eosin staining.Data from the baseline flow velocity recording can be used to determine the percent of re-perfusion with carotid artery injury and treatment in this canine model. Staining of the contralateral and injured canine carotid sections can be used to verify the recanalization status at the time of sacrifice. Carotid artery angiography as demonstrated can be used to determine if the thrombus is occlusive.In addition, using the square flow probe as a marker, the length of the thrombus can also be determined at each time point that the angiogram is obtained. As demonstrated in these diffusion and T2-weighted magnetic resonance images, the size of both the hemorrhage and the stroke volume can be identified in different levels in areas of the brain for quantification at each time point of interest. Further, TTC staining can be used to delineate brain tissue in which the cells are still metabolically active, while hematoxylin and eosin staining can be used to identify areas where the hemorrhage has occurred.This technique, once mastered, should take approximately eight to 10 hours when properly performed. If imaging is going to be included as part of this procedure, please make sure to consider anesthesia as part of the transport to MR or CT for analysis. Using this protocol, procedures like endovascular thrombectomies can be utilized to look at the effect of recanalization or even the effect of the vascular device on the endothelial surface.After watching this video, you should have a good understanding of how to perform a ferric chloride acid-induced injury model in a large animal. Remember, that when performing the angiography portion of this protocol that one, it's iodine-based dyes that are used, and so if there's an allergy present, one should be very careful. Secondly, the angiography utilizes radiation, so everybody in the room should have lead on.

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Research

JoVE Journal

Medicine

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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 ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

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 ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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 ...