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, 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.
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.
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
2. Canine Carotid Artery Occlusion
3. Canine Angiography
NOTE: This is shown in Figure 2 and is done during the surgery at time points of interest.
4. Magnetic Resonance- Diffusion Weighted Imaging (DWI) and T2 weighted imaging (T2WI) of canine brain
NOTE: This is shown in Figure 3A-3B.
5. Hematoxylin and Eosin (H&E) staining of the canine brain
NOTE: This is shown in Figure 3D.
6. Hematoxylin and Eosin (H&E) staining of canine carotids
NOTE: This is shown in Figure 1 (Left).
7. 2,3,5-triphenyl-2H-tetrazolium Chloride (TTC) Staining of the Canine Brain
NOTE: This is shown in Figure 3C.
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 model. Figure 1B provides an example of both the contralateral (top) and injured (bottom) canine carotid sections stained with H&E that verifies the re-canalization status at the time of sacrifice. A multitude of software programs are available to analyze the perfused area of the vessel by tracing the blood vessel (without thrombus) which can be divided by total area of the vessel to arrive at a percent of canalization with each treatment. Figure 2 shows several examples of carotid artery angiography detailed in this canine imaging protocol which can be used to determine if the thrombus is occlusive. In addition, using the square flow probe as a marker, investigators can determine the length of the thrombus at each time point the angiogram is taken. Although, here we have presented images before injury, 60 min after vehicle treatment, and at the time of sacrifice, the investigator can tailor imaging to their needs. Figure 3 is the result of the magnetic imaging parameters applied to the canine brain ~ 4 h after carotid artery occlusion immediately before sacrifice, utilizing both diffusion weighted (Figure 3A) and T2 weighted imaging (Figure 3B) detailed in section 4 of this protocol. Although we only show one photo of the entire array taken at different levels in the brain, the size of both hemorrhage and stroke volume can be identified in different levels and areas of the brain and quantitated at each time point desired by the investigator. Quantitation is completed using the specific MRI machine software specific to the investigator's imager. The TTC staining results as shown in Figure 3C can be used to delineate brain tissue in which the cells are still metabolically active in addition to those that are not. TTC will be enzymatically reduced to result in red stained live cells whereas dead cells will not retain the TTC and thus will not be red. Lastly, the hematoxylin and eosin staining technique demonstrated in canine brain in Figure 3D will result in staining red blood cells bright red which can be used to verify areas where hemorrhage has occurred.
Figure 1: Monitoring of carotid blood flow. (A) Representative carotid artery blood velocity (mL/min) recorded from the Doppler probe from baseline before injury through sacrifice. (B) Representative H&E staining of both occluded carotid artery (bottom) and contralateral control (top). Magnification is at 20X. Please click here to view a larger version of this figure.
Figure 2: Monitoring of the carotid blood flow by angiography. Representative angiography view of right canine carotid artery. Images were taken at baseline before vehicle infusion (A), 60 min after vehicle infusion (B), and at time of sacrifice, 4.5 h after occlusion (C). Red arrows indicate the location on the vessel of the FeCl3-induced injury where the umbilical tape was placed. Please click here to view a larger version of this figure.
Figure 3: Representative images of canine brain after vehicle treatment. Magnetic Resonance Imaging (MRI) performed ~4 h after occlusion, immediately before sacrifice, utilizing diffusion weighted imaging (DWI, (A) and T2 weighted imaging (B). TTC staining of medial section at the time of sacrifice to delineate live from dead tissue (C). H&E staining of medial section, immediately proximal to TTC section, at time of sacrifice to delineate tissue which are metabolically active (red) from those that are not TTC is reduced to a red product when taken up by live cells and therefore, will result in sections that can be quantified with a multitude of software to trace live vs dead regions. (D). Please click here to view a larger version of this figure.
Experimental Parameter | T2-weighted | Diffusion weighted |
Repetition time (TR) ms | 4000 | 4600 |
Echo time (TS) ms | 75 | 86 |
Flip angle, degrees | 180 | 90 |
Acquisition matrix | 320 x 256 | 231 x 257 |
Number of averages | 2 | 4 |
In plane image resolution (pixels/mm) | 2.4615 | 0.9333 |
Table 1: Magnetic Resonance Imaging (MRI) parameters. MRI parameters that were developed for canine T2- and Diffusion-weighted images to maximize for assessment of stroke and hemorrhage volume measurement in canine carotid artery thrombosis model.
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 studied stabilized occlusive thrombi at the site of injury and therefore cannot utilize angiography and histology of the carotid artery to assess the extent of re-canalization for each prophylactic or thrombolytic treatment. In addition to the advantages of a large animal which include ample tissue for analysis (plasma, urine, organs for toxicity studies, etc.), the large animal model much more closely mimics the heart rate, blood pressure, and coagulation cascade in humans than rodent models. Adequate size of both the brain and carotid arteries, result in histological and biological material that can be used for a plethora of inflammatory and biochemical investigations on each experimental animal. Another advantage of modifying the FeCl3-induced vascular model into a canine is that a much larger blood volume can be extracted during the experiment without modifying platelet reactivity or thrombus formation such that blood gas and complete blood counts can be monitored throughout the injury and drug infusion. In addition, the ferric chloride injury has been attributed to the oxidant damage; therefore, it will mimic atherosclerotic damage that precedes clinical stroke and myocardial infarction much more closely than the other types of published arterial injury models7. Lastly, mechanical interventions such as thrombectomy, which are routinely used clinically, can follow pharmacological treatment so that re-stenosis and the status of the endothelial wall health can be addressed with ample histological tissue for multiple investigations on the same canine.
Limitations of this model are few but need to be considered. First, although the time point, and treatment chosen in this publication (sacrifice 4.5 h after carotid artery injury, vehicle) did not result in a measurable stroke or hemorrhage, this method is clinically relevant for the investigation of novel anti-thrombotic and thrombolytic agents to determine efficacy and dosing. Indeed, both stroke and/or hemorrhage with the initial thrombotic insult or with pharmacological treatment (i.e., rTPA) do occur with this model. Secondly, canine costs for purchasing, shipping, genetic manipulation, and per diem are quite high and cost prohibitive until a drug is well-characterized in rodent models.
Critical steps in the protocol center on platelet activation, aggregation and adhesion, the crux of thrombosis. Since the Platelet Function Analyzer (PFA-100) can be performed with 1600 µL of whole blood in duplicate, this method is simple and easy to track platelet reactivity throughout the procedure without affecting blood homeostasis. Further ex vivo studies into platelet activity using impedance or lumi-aggregometry can be performed before vascular injury without affecting experimental thrombosis as long as the last blood draw is >1 week before surgery in addition to after injury and treatment at time of sacrifice. As previously discussed, additional applications of umbilical tape soaked in 50% FeCl3 may be necessary depending on gender, breed, or age of each canine. We allowed 30 minutes after injury for occlusion and re-applied fresh 50% FeCl3 for another 15 min if necessary. This process did not result in a significant difference in thrombolytic deviation with age or gender using adult beagles. In this study, we have elucidated the critical and state-of-the-art diffusion weighted imaging (DWI), a MR imaging based on measurement of Brownian motion (random diffusion of particles) of water molecules within the tissue voxel. This technique is useful in detection of acute ischemic stroke among other pathologies such as tumors by showing diffusion in hyper-cellular tissues or those with cellular swelling is low with higher diffusion coefficient17,18,19. Diffusion maps vary with the diffusion of water molecules in the brain tissue17,18,19. The B-value measures the gradient for diffusion of H2O molecules. In the ischemic injury site the free water experiences strongest signal attenuation at higher B-values19.
In addition to use of this canine protocol for studies in dosing, toxicity, and efficacy of prophylactic and thrombolytic agents, the size of a canine makes experiments in mechanical thrombectomy clinically relevant and easily achievable. Carotids can be easily processed, stained, and assessed utilizing protocols for human histology for study of immune cell infiltration after clot retrieval. These studies will be our next detailed protocols.
The authors have nothing to disclose.
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.
1/8” umbilical tape | Jorgensen Laboratories Inc., | #J0025UA | for ferric chloride application |
4% paraformaldehyde in PBS | Alfa Aesar | AAJ61899AP | |
10% neutral buffered formalin | Richard-Allan Scientific | 5701 | |
2% 2,3,5-triphenyltetrazolium chloride (TTC in PBS, pH 7.4) | Sigma Aldrich | T8877 | |
ADP/Collagen cartridges | Siemens Diagnostics | B417021A | |
4.5 ml 3.2% sodium citrate blood vacutainer | Becton Dickinson | BD 369714 | |
4.5 ml lithium heparin vacutainer | Becton Dickinson | BD 368056 | |
EDTA K3 vacutainers | Becton Dickinson | BD455036 | |
Doppler flow probe | Transonic Systems Inc | MA2.5PSL | |
Hematoxylin 560 | Surgipath | 3801570 | |
Eosin | Surgipath | 3801602 | |
LabChart Software | ADInstruments Inc. | ||
Prisma Fit 3 tesla (3T) magnet | Siemen's Diagnostics | ||
Sodium heparin for injection (to coat blood gas syringe) | NovaPlus | 402525D | |
HUG-U-VAC positioning system | DRE Veterinary | 1320 |