The importance of clot constitution in a rat embolic stroke model

Background and aims: This study was conducted to compare white and red autologous blood clots in their ability to induce experimental embolic stroke in rats, which can be successfully dissolved by thrombolytic therapy. Methods: Eight male Wistar rats (347+/-13.2 g) were used. Autologous blood was withdrawn from the tail vein. To prepare white emboli, blood was mixed with a 10% volume of 4% sodium citrate and centrifuged. 200 μl of plasma was separated and mixed with 0.5 μl of the pellet and coagulation was initiated with 20 μl of 3% calcium chloride. Red emboli were prepared without separating plasma. All emboli were allowed to coagulate spontaneously during 15-20 h within a PE-50 tube (inner diameter: 0.58 mm), without the use of thrombin. Experimental stroke was induced by injecting a 25-mm long embolus into the right internal carotid artery through a PE-50 tube connected to a syringe filled with 1 mg/ml rat albumin in phosphate-buffered saline. Two hours following stroke induction, rats received rtPA (10 mg/kg, i.v.). Serial MRI was done on a 9.4 T MR system. Lesion volume was measured with diffusion- and T2-weighted MRI at 1-2 h, 24 h, and 7 or 8 days after stroke induction. In addition, blood-brain barrier leakage was assessed with T1-weighted MRI before and 30 min after Gd-DTPA injection (0.2 mmol/kg, i.v.), at 24 h and 7/8 days after stroke induction. Neurological deficit scores (NDS: 0-21) were taken at 1, 3 and 7/8 days after stroke. Results: Rats that had received white emboli (n=3) had no or a small lesion in the middle cerebral artery (MCA) territory (apparent diffusion coefficient (ADC) lesion volume: 0+/-0 mm3 at 1-2 h (i.e., pre-rtPA); T2 lesion volume: 4.6+/-5.5mm3 at 24 h post-stroke (i.e., post-rtPA)). Also, neurological deficiency was minor (NDS: 1.3+/-0.6 at 3 days post-stroke). Visual analysis of white clots showed that these were irregularly shaped and compressible, as described by Kirchof (ref1). Occlusion of the MCA with red emboli (n=4) resulted in larger lesions depending on the quality of the injected red clot. Emboli with a relatively low amount of erythrocytes entrapped in the fibrin network induced smaller lesions (ADC lesion volume: 1.3+/-1.8 mm3 at 1-2 h (n=2); T2 lesion volume: 2.6 mm3 at 24 h post-stroke (n=1)). Emboli closely packed with erythrocytes induced large lesions (ADC lesion volume: 99.2+/-101.5 mm3 at 1-2 h; T2 lesion volume: 163.3+/-70.2 mm3 at 24 h post-stroke (n=2)), clear Gd-DTPA leakage and increased NDS (8.5+/-2.1 at 3 days post-stroke). Conclusions: Our findings suggest that close packing of erythrocytes within the fibrin network is of importance for the preparation of stable, spontaneously coagulated emboli that can effectively occlude the MCA in rat embolic stroke models. Suitable red emboli can be prepared without additional thrombin, which has been shown to affect efficacy of thrombolytic treatment (ref.2). However, experimental conditions should allow rapid coagulation to prevent sedimentation of erythrocytes.