Platelet rich clots are resistant to lysis by thrombolytic therapy in a rat model of embolic stroke

Abstract

Background: Early recanalization of occluded vessels in stroke is closely associated with improved clinical outcome. Microbubble-enhanced sonothrombolysis is a promising therapy to improve recanalization rates and reduce the time to recanalization. Testing any thrombolytic therapy requires a model of thromboembolic stroke, but to date these models have been highly variable with regards to clot stability. Here, we developed a model of thromboembolic stroke in rats with site-specific delivery of platelet-rich clots (PRC) to the main stem of the middle cerebral artery (MCA). This model was used in a subsequent study to test microbubble-enhanced sonothrombolysis.

Methods: In Study 1 we investigated spontaneous recanalization rates of PRC in vivo over 4 hours and measured infarct volumes at 24 hours. In Study 2 we investigated tPA-mediated thrombolysis and microbubble-enhanced sonothrombolysis in this model.

Results: Study 1 demonstrated stable occlusion out to 4 hours in 5 of 7 rats. Two rats spontaneously recanalized at 40 and 70 minutes post-embolism. Infarct volumes were not significantly different in recanalized rats, 43.93 ± 15.44% of the ischemic hemisphere, compared to 48.93 ± 3.9% in non-recanalized animals (p = 0.7). In Study 2, recanalization was not observed in any of the groups post-treatment.

Conclusions: Site specific delivery of platelet rich clots to the MCA origin resulted in high rates of MCA occlusion, low rates of spontaneous clot lysis and large infarction. These platelet rich clots were highly resistant to tPA with or without microbubble-enhanced sonothrombolysis. This resistance of platelet rich clots to enhanced thrombolysis may explain recanalization failures clinically and should be an impetus to better clot-type identification and alternative recanalization methods.

Keywords: Embolic Stroke; Microbubbles; Platelet Rich Clot; Rat; Sonothrombolysis; Thrombolysis; Ultrasound.

Figure 1

Visualisation of vascular filling and clot presence (Study 2). Vessels were perfused post-mortem with Microfil (yellow) to visualise the vasculature and clot presence (black). All animals had clot in the major cerebral vessels after treatment. (A) Shows the vasculature from the view of the Circle of Willis, (B) shows the lateral surface of the right hemisphere. Vessels labelled are: middle cerebral artery (MCA), anterior cerebral artery (ACA) and internal carotid artery (ICA). Images of all brains can be viewed in the Additional file 1.

Figure 2

Laser Doppler flowmetry and infarction following middle cerebral artery occlusion with platelet rich clots (Study 1). A total of seven rats had successful embolization of the MCA. (A) Five rats remained occluded for the duration of LDF observation (mean ± SD). One rat died overnight and was not included in TTC assessment of infarct. (B) Two rats recanalized at 40 min (black trace) and 70 min (grey trace) (raw data).

Figure 3

Laser Doppler flowmetry (LDF) of regional cerebral blood flow in treatment groups (Study 2). Animals underwent embolization of the middle cerebral artery (MCA) with platelet rich clot. LDF confirmed occlusion in all animals and indicated no recanalization post-treatment. Data represents the mean of n = 10 per group. There were no significant differences between groups. (U/S = ultrasound; BR38 = microbubbles).