A canine thromboembolic model of anterior circulation large vessel occlusion stroke

Abstract

Purpose: To develop a large animal preclinical model of thromboembolic stroke with stable, protracted large vessel occlusion (LVO) utilizing an autologous clot.

Materials and methods: A reproducible canine model of large vessel occlusion stroke was established by endovascular placement of an autologous clot into the middle cerebral artery (MCA) of six adult hounds and confirmed using digital subtraction angiography (DSA). Infarct volume and evidence of hemorrhage were determined by magnetic resonance imaging (MRI) 7 h after occlusion and Thrombolysis in Cerebral Infarction scale (TICI) was assessed before and after clot placement and at 1, 6, 7, and 9 h after middle cerebral artery occlusion (MCAO). Heart rate (HR) and blood pressure (BP) were monitored continuously and invasively through an arterial sheath throughout the procedures and complete blood count and blood gas analysis completed at time of sacrifice. Histopathological findings at time of sacrifice were used to confirm stroke volume and hemorrhage.

Results: MCAO with resulting TICI 0 flow was observed in all six animals, verified by serial DSA, and lack of collateral flow persisted for 9 h after clot placement until time of sacrifice. The mean infarct volume was 47.0 ± 6.7% of the ipsilateral hemisphere and no events of spontaneous recanalization or clot autolysis were observed.

Conclusion: We demonstrate a thromboembolic canine model of MCAO that is both feasible and results in consistent infarct volumes to generate a clinically relevant LVO. This model is important to evaluate treatment of LVO in acute ischemic stroke (AIS) outside the established 4.5 h recombinant tissue plasminogen activator (rTPA) therapeutic window utilizing a prolonged occlusive thrombus.

Keywords: Canine model; Middle cerebral artery occlusion; Stroke.

Fig. 1

Posterior access MCAO canine occlusion model. A. Animal positioning and views of arterial blood supply and microcatheter path (inset, blue) for autologous clot placement to the left middle cerebral artery. Mean (B), systolic (C), and (D) diastolic arterial blood pressure from clot placement to sacrifice in each of six hounds. E. Heart rate from clot placement to sacrifice in each of six hounds. F. Mean values ± SD of all blood gas parameters measured at time of sacrifice, 9 h after MCAO initiation. G. White blood count at baseline (6.33 ± 1.49 × 103 cells/ul) and time of sacrifice (13.24 ± 3.48 × 103 cells/ul) showed significant increase 9 h after MCAO (n = 6, p < .0013). H. Platelet count at baseline (243.8 ± 67.7 × 103 cells/ul) and time of sacrifice (181.2 ± 57.1 × 103 cells/ul) were insignificant (n = 6, p = .1137). I. Representative autologous clot H&E staining at 10× magnification (left) and insets of immunofluorescent staining for fibrinogen (green), platelets (yellow), and von Willebrand Factor (purple), 60 min after autologous whole blood draw (n = 4).

Fig. 2

Serial representative digital subtraction angiography (DSA) of intracranial arteries in MCAO model canine study. A. Baseline image demonstrating cerebrovascular anatomy of the hound. B. Location of microcatheter tip (arrow) immediately before clot placement. C. MCAO 3 min after clot placement with no collateral flow to MCA territory (arrow). D. Persistent localized lack of flow to MCA territory 6 h after occlusion. E. Last DSA demonstrating stable MCAO with no reflow immediately before transport to MRI facility 7 h after MCAO induction. F. Final DSA after MRI and return transport to angiography suite verifying stable MCAO 9 h after ischemic start. Microcatheter and contrast injection is at the bottom of the images to visualize the entire field of blood perfusion whereas it is placed at the MCA in B to visualize occlusion.

Fig. 3

Representative Canine MCAO Imaging and Histopathology. (A) TOF-MRA image in dorsal orientation of a representative hound with relevant arteries and collaterals demonstrating occlusion of middle cerebral artery (red arrow). (B,C) T2 weighted images in grayscale and pseudocolor demonstrating hyperintense lesion formation in the left MCA territory. (D,E) Final DWI in grayscale and pseudocolor showing an MCA territory infarct. (F,G) Corresponding hypointense lesions on ADC in grayscale and pseudocolor maps. (H) TTC stained coronal brain section identifying areas in red that are still metabolically active compared to white inactive regions 9 h after MCAO. (I) Hematoxylin and eosin stained coronal brain section wth no evidence of hemorrhage.