Tissue Plasminogen Activator for preclinical stroke research: Neither “rat” nor “human” dose mimics clinical recanalization in a carotid occlusion model

www.nature.com/scientificreports OPEN received: 27 March 2015 accepted: 08 October 2015 Published: 02 November 2015 Tissue Plasminogen Activator for preclinical stroke research: Neither “rat” nor “human” dose mimics clinical recanalization in a carotid occlusion model Amelia J. Tomkins1,2, Rebecca J. Hood1,2, Christopher R. Levi2,3,4 & Neil J. Spratt1,2,3 Tissue plasminogen activator (tPA) is the only approved thrombolytic therapy for acute ischemic stroke, yet many patients do not recanalize. Enhancing thrombolytic efficacy of tPA is a major focus of stroke research. Traditionally, a “rat dose” of 10 mg/kg has been used in rodent models. Recent studies suggested that the clinical “human” dose (0.9 mg/kg) may better mimic clinical recanalization. These studies only compared the rat and clinical doses, and so we aimed to test recanalization efficacy of multiple tPA doses ranging from 0.9 to 10 mg/kg in a model of endothelial injury and vessel stenosis. The common carotid artery of rats was crushed and stenosed to allow insitu occlusive thrombus formation (Folt’s model of ‘physiological’ thrombus). Intravenous tPA was administered 60 minutes post-occlusion (n = 6-7/group). Sustained recanalization rates were 0%, 17%, 67% and 71%, for 0.9, 1.8, 4.5, and 10 mg/kg, respectively. Median time to sustained recanalization onset decreased with increasing dosage. We conclude that 10 mg/kg of tPA is too effective, whereas 0.9 mg/kg is ineffective for lysis of occlusive thrombi formed in situ. Neither dose mimics clinical tPA responses. A dose of 2x the clinical dose is a more appropriate mimic of clinical tPA recanalization in this model. Tissue plasminogen activator (tPA) is the only approved thrombolytic therapy for acute ischemic stroke. Early recanalization of occluded vessels is associated with improved clinical outcome, yet less than 50% of all stroke patients treated intravenously with tPA will successfully recanalize1. In the setting of carotid artery occlusion, tPA is even less effective, with recanalization rates of 10–30%2,3. There is a great need for improved therapies for stroke and one approach has been to enhance thrombolysis with adjuvant therapies such as sonothrombolysis4. For any new or adjuvant thrombolytic therapy, rigorous preclinical testing should occur and aim to mimic the clinical conditions of ischemic stroke and tPA efficacy. There has been controversy regarding what a “human equivalent” dose of tPA is for preclinical research—particularly in rodents, in which most such studies are performed. Traditionally, a dose of 10 mg/kg tPA has been used for rodents. This “rat dose” was based on an in vitro study from the early 1980’s that demonstrated that the fibrinolytic system of rats is 10-fold less sensitive than humans5. Two recent comparisons of rat and clinical doses in rats6 and mice7 both 1 School of Biomedical Sciences & Pharmacy, Medical Sciences Building, University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia. 2Hunter Medical Research Institute, Lot 1 Kookaburra Circuit, New Lambton Heights, NSW, 2305, Australia. 3Hunter New England Local Health District, Department of Neurology, Lookout Road, New Lambton Heights, NSW, 2305, Australia. 4School of Medicine and Public Health, University of Newcastle, Newcastle, NSW, Australia. Correspondence and requests for materials should be addressed to N.J.S. (email: ) Scientific Reports | 5:16026 | DOI: 10.1038/srep16026 1 www.nature.com/scientificreports/ Recanalization Rate (%) 100 75 50 25 0 0 30 60 90 120 150 180 210 Minutes Post tPA bolus Figure 1. Recanalization rates at varying doses of tPA. Rats with carotid artery occlusion were administered intravenous tPA at the clinical dose (0.9 mg/kg, ● ), 2x the clinical dose (1.8 mg/kg, ▲ ), 5x the clinical dose (4.5 mg/kg, ■), and the rat dose (10 mg/kg, ♦ ). Treatment began 60 minutes post-occlusion. n =  6 per group for 0.9, 1.8 and 4.5 mg/kg, n =  7 for 10 mg/kg. Data presented as the percentage of animals with sustained recanalization per group. Fisher’s exact test, p =  0.015. indicated that the clinical dose was a better mimic of the clinical situation6,7. Additional doses were not compared in these studies. The typical method for determining a “human equivalent” dose of any therapeutic utilizes a conversion based on body surface area of humans to the target species8. For rats, this conversion requires multiplication of the human dose (0.9 mg/kg) by 6.2, indicating a “human equivalent” dose of 5.58 mg/kg tPA for rats. However, this conversion does not take in to account additional factors that may affect the fibrinolytic process other than body size, and is not generally used when converting doses of tPA for stroke research. Multiple methods of forming experimental thrombi exist for preclinical stroke models. Formation of thrombi in situ or ex vivo and the presence or absence of added pro-thrombotic factors, such as thrombin and/or CaCl2, leads to variability of final thrombus composition. All of these factors play a key role in the overall thrombolytic susceptibility of the thrombus and therefore the variability of tPA efficacy between studies9,10. Ideally, a preclinical model for testing stroke thrombolytics and thrombolytic enhancers should use thrombi that closely mimic human stroke thrombi and have similar recanalization rates. For this study, we developed a method of physiological thrombus formation by endothelial injury and stenosis of the carotid artery in rats. To determine which tPA dose best reflects clinical recanalization rates in this model, we aimed to investigate sustained recanalization rates of varying doses of tPA ranging from the clinical dose to the traditional rat dose. Time to sustained recanalization was a secondary outcome. Results To test the thrombolytic efficacy of varied doses of tPA on a physiological thrombus we used a rat model of carotid occlusion with a mild underlying stenosis. Recanalization was monitored every 30 minutes post-tPA delivery to 4.5 hours post-occlusion. Sustained recanalization, defined as recanalization without reocclusion, was observed in 0% of 0.9 mg/kg treated rats (0/6), 17% of 1.8 mg/kg treated rats (1/6), 67% of 4.5 mg/kg treated rats (4/6), and 71% of 10 mg/kg treated rats (5/7) (Fisher’s exact test, p =  0.015, Fig. 1). Recanalization/reocclusion was observed in 2 animals, both in the 10 mg/kg dosage group. In pilot experiments we found that recanalization was easily confirmed (as in Fig. 3B). However accurate quantification of the degree of recanalization was not possible because coupling of the flow probe to the vessel with saline caused fluctuations in the baseline of the flow trace (data not shown). We did not see evidence of major changes in flow once vessels did recanalize. Therefore, in the interests of accuracy, we chose sustained recanalization as the marker of recanalization success based on dose, rather than attempting to quantify percentage recanali (...truncated)