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>
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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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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 height="40" style="height:40px;">4% paraformaldehyde in PBS</td>
			<td>Alfa Aesar</td>
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			<td height="40" style="height:40px;">10% neutral buffered formalin&#160;</td>
			<td>Richard-Allan Scientific</td>
			<td>5701</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>
			<td></td>
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			<td height="40" style="height:40px;">ADP/Collagen cartridges</td>
			<td>Siemens Diagnostics</td>
			<td>B417021A</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>
			<td>BD 369714</td>
			<td></td>
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			<td height="40" style="height:40px;">4.5 ml lithium heparin vacutainer&#160;</td>
			<td>Becton Dickinson</td>
			<td>BD 368056</td>
			<td></td>
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			<td height="40" style="height:40px;">EDTA K3 vacutainers&#160;</td>
			<td>Becton Dickinson</td>
			<td>BD455036</td>
			<td></td>
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			<td height="40" style="height:40px;">Doppler flow probe</td>
			<td>Transonic Systems Inc</td>
			<td>MA2.5PSL</td>
			<td></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>
			<td></td>
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			<td height="40" style="height:40px;">LabChart Software</td>
			<td>ADInstruments Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Prisma Fit 3 tesla (3T) magnet</td>
			<td>Siemen&#39;s Diagnostics</td>
			<td></td>
			<td></td>
		</tr>
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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>
			<td></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>
			<td></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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