Research reportImproved model of thromboembolic stroke and rt-PA induced reperfusion in the rat
Introduction
The purpose of this study was to validate a rat model of thromboembolic stroke that resembles human embolic stroke as closely as possible. For such a model the obstructing emboli should be located in the proximal segment of a large feeder artery, the distal vascular bed should be open and the extent and density of ischemia should be pronounced and consistent enough to allow studies of possible therapeutic interventions. Furthermore, we aimed to ensure full recanalisation of the obstructed artery by fibrinolytic agents, and to investigate reperfusion as induced by thrombolytic therapy.
In the past a suspension of small clot fragments 7, 12, 15has been used for intracarotid embolization of rats. These pioneering experiments were used successfully to test the concept of fibrinolytic therapy. However, the injection of very small clot fragments fills up the arterial tree with clot material and causes additional small and multifocal lesions in several vascular territories of the brain, creating conditions that do not arise in clinical stroke. To overcome this problem, experiments were performed using intracarotid injection of single clots [19]. Unfortunately, this approach does not always produce a sufficient reduction in rCBF. More recently, a model was reported, in which a combination of transient bilateral carotid occlusion and injection of thrombin and clot material close to the MCA origin was used to produce proximal MCA occlusion [21]. However, in this study, rCBF measurements indicated early spontaneous clot lysis.
Our model uses intracarotid injection of 12 medium-sized, fibrin-rich autologous clots to achieve stable proximal MCA occlusion and a good chance of rt-PA induced recanalisation. We validated the model by inspecting the lodging of clots at the MCA origin and measuring the extent of rCBF reduction by iodo[14C]antipyrine autoradiography and the resulting injury by histological (hematoxilin-eosin) and metabolic (triphenyltetrazolium) staining. Furthermore, the frequency of recanalisation after treatment with the recombinant tissue type plasminogen activator (rt-PA) was determined by microscopical inspection of the circle of Willis. Experiments were terminated 3 h after embolisation to ensure that the brains were examined before spontaneous clot lysis could occur.
Section snippets
Surgical procedures and physiological monitoring
All animal handling and surgery was performed in accordance with animal protection guidelines and approved by local authorities.
Male Wistar rats (n=20) were anaesthetised with 1.5% halothane in a 70%/30% mixture of N2O/O2. Catheters were inserted into a femoral artery and vein for blood sampling, blood pressure recording and intravenous infusion of drugs. Animals were tracheotomised and artificially ventilated. They were immobilised with intravenous injections of pancuronium bromide (0.3 mg/kg
General physiological parameters
Table 1 summarises the general physiological parameters before and after MCA occlusion. All variables stayed in the normal range throughout the experiment. rt-PA treatment resulted in a slight decrease in MABP by about 10 mmHg after 2 h, and a reduction of hematocrit by 5% at the end of the experiment. These changes did not differ significantly from those observed in controls.
Histology of clots
Fig. 2A shows the macroscopic appearance of a set of 12 clots, before injection into the carotid artery. The microscopic
Discussion
The rat model presented here produces reliable clot occlusion of the proximal part of the MCA. It resulted in the consistent reduction of rCBF and histological injury in the ipsilateral MCA territory, confirming the absence of spontaneous recirculation. Thrombolysis with intra-arterially applied rt-PA consistently reversed the occlusion and induced recanalisation of the proximal MCA in all cases.
Hill et al. [5]were the first to describe injections of homologous clots for induction of stroke in
Acknowledgements
We thank Mrs. Christiane Stratmann and Mrs. Stephanie Krause for processing the brains for autoradiography and histology. The secretarial help of Mrs. Doris Schewetzky, and the art work prepared by Mrs. Ilka Muehlhoever and Mr. Bertram Huth is also gratefully acknowledged. The project was supported in part by the Deutsche Forschungsgemeinschaft (SFB 194/B1).
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