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
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	<li>Freshly prepare 50 mL of ferric chloride (F...

<ol>
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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>
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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>
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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>
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			<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, ...

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