N initial spin (15 minutes at 180 g). The platelet-rich plasma layer and buffy coat were collected after a second fast spin (20 minutes at 1500 g). PRC formation was initiated by recalcification of the platelet rich plasma + buffy coat bmjopen-2016-011824 with CaCl2 (20 mM PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14960617 final concentration). The sample was drawn into a 40 cm length of PE-50 tubing (40 cm, i.d. 0.58 mm) and clamped at one end. Using an air filled syringe, pressure was forced into the unclamped end of the catheter to create clots of consistent size. This end was then also clamped to retain the pressure and incubated at 37 for 2 hours. The resultant clot was ejected into a dish of saline and stored at 4 overnight. Clot preparation was the same for both studies, with theTable 1 Experimental protocolsStudy 1 Location Aim Primary Outcome [method] Secondary Outcome(s) [method] Survival post-embolism Treatment groups (n) Laser Doppler Monitoring Newcastle, Australia Develop model, Determine spontaneous recanalization rate Recanalization [Laser Doppler] Study 2 Bad Nauheim, Germany Determine recanalization 2-[(4S)-4,5-Dihydro-4-isopropyl-2-oxazolyl]pyridine rates with microbubble + sonothrombolysis enhancement of tPA Recanalization [Laser Doppler]Infarct Volumes [TTC], Mortality, Neurological Deficit scores Clot lysis [Inspection of the major branches of the cerebral arterial circulation] 24 h No treatment (n = 7) Continuous To 4 h post-embolism 2h Saline (n = 10), tPA (n = 10), tPA + Ultrasound + BR38 microbubbles (n = 10) Discontinuous Pre-embolism, Post-embolism, Pre-treatment, Post-treatmentTomkins et al. Experimental Translational Stroke Medicine (2015) 7:Page 3 ofexception for Study 2 in which the clot was incubated for 5 minutes in Evans blue for visualisation within major branches of the cerebral arterial circulation at sacrifice. The method of site-specific clot delivery was based on the method of MCA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9638577 thread occlusion we use routinely [12-14] with catheter placement and clot delivery as described by DiNapoli et al. [15]. Briefly, a modified catheter (PE-8, o.d. 0.35 mm, connected to silastic tubing) containing a 30 mm length of PRC was inserted into the internal carotid artery via the external carotid artery. It was advanced until a sensation of mild resistance indicated the tip was at the middle cerebral artery (MCA) origin, then retracted 1? mm to restore flow to the MCA allowing site-specific clot injection. The clot was injected with 30?0 l saline and the catheter left in place for 10 minutes to allow the clot to stabilize. The catheter was then completely removed from the vessel and the surgical site sealed.Confirmation of occlusion and recanalization ratesof thrombolysis beyond this time is limited [16]. Animals were then woken and returned to their cages until sacrifice at 24 hours for infarct volume analysis.StudyContinuous monitoring could not be performed during ultrasound treatment for Study 2 due to 4,4,5,5-Tetramethyl-2-(2-methylprop-1-en-1-yl)-1,3,2-dioxaborolane the apparatus set-up, so a discontinuous approach was used [17]. Discontinuous monitoring was performed at 4 time points: pre-embolism (-30 minutes), post-embolism (10 minutes), pre-treatment (50 minutes) and post-treatment (130 minutes), using an Oxyflo2000 Microvascular Perfusion Monitor with Oxyflo XP Probe 17 mm diameter (MNP 100XP-3/ 15, Oxford Optronix, UK). Recordings were made as per Soehle et al. [17]. Animals were sacrificed after final LDF recording for visual inspection of clot presence.Treatment groupsIn both studies, confirmation of occlusion of the MCA after clot injection was made by laser Doppler Flowmetry (LD.