Early Ultrafast Ultrasound Imaging of Cerebral Perfusion correlates with Ischemic Stroke outcomes and responses to treatment in Mice

Abstract

In the field of ischemic cerebral injury, precise characterization of neurovascular hemodynamic is required to select candidates for reperfusion treatments. It is thus admitted that advanced imaging-based approaches would be able to better diagnose and prognose those patients and would contribute to better clinical care. Current imaging modalities like MRI allow a precise diagnostic of cerebral injury but suffer from limited availability and transportability. The recently developed ultrafast ultrasound could be a powerful tool to perform emergency imaging and long term follow-up of cerebral perfusion, which could, in combination with MRI, improve imaging solutions for neuroradiologists. Methods: In this study, in a model of in situ thromboembolic stroke in mice, we compared a control group of non-treated mice (N=10) with a group receiving the gold standard pharmacological stroke therapy (N=9). We combined the established tool of magnetic resonance imaging (7T MRI) with two innovative ultrafast ultrasound methods, ultrafast Doppler and Ultrasound Localization Microscopy, to image the cerebral blood volumes at early and late times after stroke onset and compare with the formation of ischemic lesions.Results: Our study shows that ultrafast ultrasound can be used through the mouse skull to monitor cerebral perfusion during ischemic stroke. In our data, the monitoring of the reperfusion following thrombolytic within the first 2 h post stroke onset matches ischemic lesions measured 24 h. Moreover, similar results can be made with Ultrasound Localization Microscopy which could make it applicable to human patients in the future. Conclusion: We thus provide the proof of concept that in a mouse model of thromboembolic stroke with an intact skull, early ultrafast ultrasound can be indicative of responses to treatment and cerebral tissue fates following stroke. It brings new tools to study ischemic stroke in preclinical models and is the first step prior translation to the clinical settings.

Keywords: Ischemic stroke; Outcome; Thrombolysis; Ultrasound Imaging; Ultrasound Localization Microscopy.

Figure 1

Transcranial ultrafast ultrasound imaging to monitor cerebral perfusion before, during and after stroke A. Experimental setup with an ultrasound probe connected to an ultrafast ultrasound acquisition system (Inserm Accelerator of Technological Research) and mounted on a 4 axis-motor for 3D scanning over the whole mice brain by steps of 0.3 mm. A volume over the whole brain is reconstructed every 40 s. Ultrafast Ultrasound monitors cerebral perfusion during the early phase of the ischemic episode, before, during and after onset, including treatment with the gold standard recombinant-tPA (rtPA). At 24 h MRI reveals the final ischemic lesion. B. Ultrafast Doppler reveals hypoperfusion in the ipsilateral cortices subjected to thrombin injection (clot formation) in the middle cerebral artery (MCA) and reperfusion of the corresponding territory after injection of the thrombolytic, rtPA. C. Registration and comparison with MRI reveal tight relationships between cerebral perfusions at the early phase of stroke and the final lesion volumes. Scale bar 1 mm.

Figure 2

In the absence of treatment, the final volume of lesion corresponds to the early hypoperfused area. A. Experimental timeline for ultrasound monitoring of cerebral blood volumes after MCAo and comparison with MRI. B. Differences between ultrafast Doppler imaging performed before and after occlusion of the middle cerebral artery (MCAo) reveal hypoperfusion in the corresponding ipsilateral cortices. C. Differences between ultrafast Doppler imaging just after MCAo and 2 h later show no sign of reperfusion in the corresponding ipsilateral cortices. D. Monitoring of blood flows in the MCA on ultrafast Ultrasound over β+1 mm shows permanent occlusion with no clear recanalization occurring during the first 2 h. E. Monitoring of cerebral perfusion in the hypoperfused volume shows no sign of reperfusion. F. Patterns of hypoperfusions directly after MCAo reveal large hypoperfused volumes in the cortex. G. Patterns of hypoperfusions 2 h after MCAo reveal similar hypoperfused volumes. H. Patterns of the final ischemic lesions measured at 24 h after MCAo revealed by T₂ MRI.

Figure 3

Early injection of rtPA causes arterial recanalization, tissue reperfusion and reduces the volume of lesion. A. Differences between ultrafast Doppler before and after MCAo reveal hypoperfusion in the corresponding ipsilateral cortices. B. Differences between ultrafast Doppler just after MCAo and 2 h later show reperfusion in the hypoperfused part of the corresponding cortices. C. Corresponding MRIs reveal the formation of smaller lesions. D. Monitoring of flows in the MCA on ultrafast ultrasound images over β+1 mm shows rapid and effective recanalization. E. Monitoring of cerebral perfusion in the hypoperfused volumes show some tissue reperfusions. F. Patterns of hypoperfusions directly after MCAo revealed by ultrafast ultrasound imaging. G. Patterns of hypoperfusions 2 h after MCAo revealed by ultrafast ultrasound imaging. H. Patterns of the ischemic lesions 24 h after MCAo revealed by MRI in rtPA treated animals.

Figure 4

Differences in reperfusion patterns revealed by ultrafast ultrasound imaging correspond to the formation of ischemic lesions. A. For a typical Grade 0/1 mouse, the reperfusion curves in three different parts of the hypoperfused areas show no reperfusion. B. Hypoperfused profiles immediately post MCAo and after 2 h show no significant evolution of the formation of the ischemic lesions co-localized with hypoperfusions. C. Ischemic lesions on the corresponding areas. D. For a typical Grade 2 mouse, the reperfusion curves in three different parts of the hypoperfused areas show inhomogeneous reperfusions. E. Hypoperfused profiles immediately post MCAo and after 2 h show significant evolutions of the formation of the ischemic lesions co-localized with the remaining hypoperfusions at 2h. F. Ischemic lesions on the corresponding areas. G. For a typical Grade 3 mouse, the reperfusion curves in three different parts of the hypoperfused areas show early and effective reperfusions. H. Hypoperfused profiles immediately post MCAo and after 2 h show significant reductions of hypoperfusions associated with the formation of reduced ischemic lesions. I. Ischemic lesions on the corresponding areas.

Figure 5

Prediction of lesions and responses to treatment based on early Transcranial ultrafast Doppler imaging. A, B, E = Unpaired t test Two-tailed (n=10; 9); C, D = Pearson correlation (n=10; 9); F = Sidak's multiple comparisons test (n=10; 9), *Intergroup differences; $ Intragroup differences; £ significant compared to T^0-. A. Final ischemic lesions determined at 24 h indicate reduction of lesions in animals treated with rtPA. B. State of reperfusion 2 h after MCAo shows improved reperfusions in the rtPA treated group. C. Directly after MCAo, the hypoperfused volume in ultrafast Doppler imaging is a marker of final ischemic lesion volumes for the NaCl treated animals (red) with the lesions that correspond to the complete hypoperfused volumes, aligned around the diagonal. For the rtPA treated group (blue), the lesions are smaller than the volumes at risk defined directly after occlusion because of effective tissue reperfusions. D. After rtPA treatment, the remaining hypoperfused volumes are predictive of the final lesion volumes in both groups. E. Effectiveness of the prediction of lesions based on reperfusion using the ultrafast ultrasound based TICI score. F. Evolutions of perfusions in the volumes at risk at the early time after stroke onset (0 and 2 h) and at 24 h show that after a therapeutic window of 2 h, perfusion is no longer predictive of the lesion.

Figure 6

Predicting the outcomes and evaluating responses to treatment with transcranial ULM. A. Timeline for ULM acquisitions. B. For mice treated with NaCl, a large lesion can be seen in ULM just after ischemia and very little reperfusion can be observed 2 h after the onset. In ultrafast Doppler, the hypoperfused area is the infarcted lesion seen in MRI. C. On rtPA treated mice, the reperfusion can be evaluated with ULM and predicts the formation of the lesion. Scale bar 1 mm.