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Towards a standardization of the murine ferric chloride‐induced carotid arterial thrombosis model

2011; Elsevier BV; Volume: 9; Issue: 9 Linguagem: Inglês

10.1111/j.1538-7836.2011.04287.x

1538-7933

A. Philip Owens, Yi Lü, Herbert C. Whinna, Christian Gachet, William P. Fay, Nigel Mackman,

Acute Ischemic Stroke Management

Mouse models of thrombosis have played a vital role in understanding the pathological process of thrombus formation in vivo [1Day S.M. Reeve J.L. Myers D.D. Fay W.P. Murine thrombosis models.Thromb Haemost. 2004; 92: 486-94Crossref PubMed Scopus (80) Google Scholar, 2Sachs U.J. Nieswandt B. In vivo thrombus formation in murine models.Circ Res. 2007; 100: 979-91Crossref PubMed Scopus (130) Google Scholar, 3Westrick R.J. Winn M.E. Eitzman D.T. Murine models of vascular thrombosis.Arterioscler Thromb Vasc Biol. 2007; 27: 2079-93Crossref PubMed Scopus (156) Google Scholar]. The most commonly used model is ferric chloride (FeCl3) injury of the carotid artery. The goal of this report from the Animal Models Subcommittee of the International Society of Thrombosis and Hemostasis is to standardize this model. FeCl3 is applied to the exterior surface of vessels and triggers vascular wall injury and denudation of the endothelium via a mechanism involving the generation of reactive oxygen species [4Wollard K.J. Sturgeon S. Chin‐Dustin J.P.F. Salem H.H. Jackson S.P. Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride‐induced vascular injury.J Biol Chem. 2009; 284: 13110-8Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 5Eckly A. Hechler B. Freund M. Zerr M. Cazenave J.‐.P. Lanza F. Mangin P. Gachet C. Mechanism underlying FeCl3‐induced arterial thrombosis.J Thromb Haemost. 2011; 9: 779-89Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar]. Exposure of collagen triggers platelet activation and exposure of tissue factor activates the coagulation cascade [5Eckly A. Hechler B. Freund M. Zerr M. Cazenave J.‐.P. Lanza F. Mangin P. Gachet C. Mechanism underlying FeCl3‐induced arterial thrombosis.J Thromb Haemost. 2011; 9: 779-89Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 6Wang L. Miller C. Swarthout R.F. Rao M. Mackman N. Taubman M.B. Vascular smooth muscle‐derived tissue factor is critical for arterial thrombosis after ferric chloride‐induced injury.Blood. 2009; 113: 705-13Crossref PubMed Scopus (92) Google Scholar]. A flow monitoring system, including a small animal vascular flowmeter, Powerlab system with software, dissecting microscope, flow probe, heating pad and rectal probe to maintain the body temperature around 37 °C, an interfacing computer and FeCl3 are required. Several factors can affect the occlusion time, including the genetic background of the mice, the anesthesia utilized, method of blood flow measurement, method of FeCl3 application, concentration of the FeCl3 solution, time the FeCl3 is in contact with the artery wall, and definition of occlusion. Performing the mouse carotid artery FeCl3 model is relatively simple because the carotid artery can be easily exposed and injured with FeCl3 (Fig. 1). Anesthesia is delivered through either parenteral injections (subcutaneous, intraperitoneal or intravenous) or via inhalation. A small piece of filter paper saturated with a solution of FeCl3 is applied to the adventitial surface of the carotid artery for several minutes (3 min is the most commonly used). The size of the filter paper varies from 0.5 × 1 to 1 × 2 mm. FeCl3 concentrations typically range from 2.5% (0.15 M) to 20% (1.2 M). One piece of filter paper is placed on the ventral surface of the carotid artery, although some studies use two pieces of filter paper. The artery should be carefully dried and isolated from surrounding tissue prior to applying the filter paper in order to avoid dilution of the FeCl3 and 'wicking' of the solution away from the surface of the artery. After the filter paper is removed, the vessel is thoroughly washed with PBS. Eckly et al.[5Eckly A. Hechler B. Freund M. Zerr M. Cazenave J.‐.P. Lanza F. Mangin P. Gachet C. Mechanism underlying FeCl3‐induced arterial thrombosis.J Thromb Haemost. 2011; 9: 779-89Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar] found that the size of the thrombus was increased dose‐dependently from 2.5% to 10% FeCl3. Wang and Xu[7Wang X. Xu L. An optimized murine model of ferric chloride‐induced arterial thrombosis for thrombosis research.Thromb Res. 2005; 115: 95-100Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar] found that 2.5% FeCl3 produced occlusion of the mouse carotid artery in 10 min with the application of two pieces of filter paper. Hypercoagulable states, such as hyperlipidemia, produce a faster occlusion time and require shorter times (1–2 min) of exposure to FeCl3 [8Ghosh A. Li W. Febbraio M. Espinola R.G. McCrae K.R. Cockrell E. Silverstein R.L. Platelet CD36 mediates interactions with endothelial cell‐derived microparticles and contributes to thrombosis in mice.J Clin Invest. 2008; 118: 1934-43PubMed Google Scholar]. Interestingly, a difference in occlusion time between factor IX−/− mice and wild‐type mice was only observed using 5% FeCl3, and not with 10% FeCl3 [9Wang X. Cheng Q. Xu L. Feuerstein G.Z. Hsu M.Y. Smith P.L. Seiffert D.A. Schumacher W.A. Ogletree M.L. Gailani D. Effects of factor IX or factor XI deficiency on ferric chloride‐induced carotid artery occlusion in mice.J Thromb Haemost. 2005; 3: 695-702Crossref PubMed Scopus (225) Google Scholar]. Thrombosis is most commonly monitored by measuring blood flow at the site of injury using a microvascular ultrasonic flow probe. Thrombosis is represented as the 'time to occlusion' after removal of the filter paper. Prior to occlusion, blood flow may fluctuate due to the formation of unstable thrombi. It can sometimes be challenging to determine precisely when blood flow falls to zero, as low levels of flow (e.g. < 10% of baseline flow, occurring when the artery is nearly occluded by thrombus) can be difficult to differentiate from background signal. However, reliable and reproducible occlusion times are typically obtained with practise. The definition of an occlusive clot in the FeCl3 model has differed between studies. Some studies define the initial time point that flow falls to 0 mL min−1 as the occlusion time, while others define occlusion as the time required for the flow to fall to 0 mL min−1 for 30–60 s. We believe that the latter is more accurate. Another study defined an artery as 'occluded' when flow fell to < 25% of baseline for at least 3 min [6Wang L. Miller C. Swarthout R.F. Rao M. Mackman N. Taubman M.B. Vascular smooth muscle‐derived tissue factor is critical for arterial thrombosis after ferric chloride‐induced injury.Blood. 2009; 113: 705-13Crossref PubMed Scopus (92) Google Scholar]. Therefore, it is not too surprising that occlusion times vary widely in the literature due to differences in techniques, experimental set‐up and the genetic background of the mice. When 10% FeCl3 or more is used, occlusive times are typically ≤10 min. However, a range of times between 8 and 26 min has been reported in four different studies [10Konstantinides S. Schafer K. Thinnes T. Loskutoff D.J. Plasminogen activator inhibitor‐1 and its cofactor vitronectin stabilize arterial thrombi after vascular injury in mice.Circulation. 2001; 103: 576-83Crossref PubMed Scopus (154) Google Scholar, 11Iwatsuki Y. Kawasaki T. Hayashi K. Moritani Y. Nii T. Miyata K. Combined effects of a factor Xa inhibitor YM466 and a GPIIb/IIIa antagonist YM128 on thrombosis and neointima formation in mice.Thromb Haemost. 2004; 92: 1221-8Crossref PubMed Scopus (14) Google Scholar, 12Zhu Y. Carmeliet P. Fay W.P. Plasminogen activator inhibitor‐1 is a major determinant of arterial thrombolysis resistance.Circulation. 1999; 99: 3050-5Crossref PubMed Scopus (159) Google Scholar]. Two recent studies have analyzed the kinetics and structure of thrombus formation in the model [5Eckly A. Hechler B. Freund M. Zerr M. Cazenave J.‐.P. Lanza F. Mangin P. Gachet C. Mechanism underlying FeCl3‐induced arterial thrombosis.J Thromb Haemost. 2011; 9: 779-89Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 13Cooley B. In vivo fluorescence imaging of large‐vessel thrombosis in mice.Arterioscler Thromb Vasc Biol. 2011; 31: 1351-6Crossref PubMed Scopus (60) Google Scholar]. In our laboratory, we use 6–8‐week‐old male C57BL/6J mice. Mice are initially sedated in an anesthesia chamber supplying 2% isoflurane and maintained under anesthesia via a facemask supplying 1–1.5% isoflurane. We use one piece (1 mm2) of filter paper soaked in FeCl3 solution and apply this to the ventral surface of the carotid artery. We use a microvascular ultrasonic flow probe to detect blood flow and define occlusion as the cessation of blood flow (0 mL min−1) for 30 s. We found that exposure of mouse carotid arteries to 5% FeCl3 for 3 min produced an occlusion time of 23.8 ± 1.6 (mean ± standard deviation, n = 6) from the time the filter paper was removed. While this model offers many advantages, there are some limitations. The aggressive oxidative stress induced by FeCl3 may denature or alter circulating and membrane‐bound proteins. The use of occlusion time as the main endpoint of thrombosis experiments is somewhat crude and may not detect subtle yet significant effects of genetic modifications and therapeutic interventions on thrombosis. While there are drawbacks to any model, there are many benefits of analyzing thrombosis in vivo. The murine FeCl3 carotid artery thrombosis model has yielded important insights into the pathological process of thrombus formation in vivo.