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Development of a 4D Cerebral Microvascular Imaging Platform for Mouse Stroke Model
Yoshihisa Kaneko, Moe Kumai, Hiroyuki Igarashi, Daisuke Ando, Kuniyasu Niizuma, Hidenori Endo, Yoshifumi Saijo, Takuro Ishii
TL;DR
Cerebral hemodynamic changes during ischemia and reperfusion remain incompletely understood despite their association with stroke outcomes. The study develops a four-dimensional ultrasound platform that continuously scans a mouse brain during tMCAO, and demonstrates visualization of local and whole-brain responses, with each volumetric acquisition completed in 5 s.
Problem
Whole-brain cerebral hemodynamic alterations during ischemia and reperfusion remain incompletely understood despite their association with tissue viability and stroke outcomes.
Method
The platform extends rapid three-dimensional ultrasound imaging with continuous mechanical scanning and section-wise SVD processing during surgically induced ischemia and controlled reperfusion in a mouse.
Results
The platform visualized local and whole-brain hemodynamic responses throughout tMCAO, including decreased flow during occlusion and increased flow after reperfusion.
Takeaways & Limitations
The platform demonstrated feasibility for rapid and continuous whole-brain cerebral hemodynamic assessment during experimentally induced ischemia and reperfusion.
Abstract
from arXiv · showhide
In ischemic stroke, changes in cerebral hemodynamics during both the ischemic and reperfusion phases strongly influence stroke outcomes. However, these hemodynamic changes remain incompletely understood. To address this challenge, we devised an imaging platform that enables time-resolved ultrasound microvascular imaging during the experimental induction of ischemia and reperfusion in a mouse model. The platform leverages our previous ultrasound imaging framework combined with continuous mechanical scanning, which acquires whole-brain blood-flow signals within 5 s. The experiments demonstrated that the proposed platform can visualize both local and whole-brain hemodynamic responses to the induction of ischemia and reperfusion, suggesting its potential for rapid and continuous whole-brain hemodynamic assessment in small-animal models.
I. INTRODUCTION
Whole-brain hemodynamic changes during ischemia and reperfusion remain incompletely understood, motivating imaging that combines broad coverage, microvascular detail, and rapid volumetric acquisition. This study develops and evaluates such a four-dimensional ultrasound platform in a mouse stroke model.
- I. INTRODUCTION: Cerebral blood-flow changes can extend beyond the ischemic territory, and their evolution during ischemia and reperfusion is associated with tissue viability and stroke outcomes.Whole-brain hemodynamic alterations remain incompletely understood.
- I. INTRODUCTION: Existing modalities trade off broad hemodynamic coverage against vessel-scale visualization, field of view, or imaging depth.fMRI and fUS provide broad coverage but limited vessel-scale visualization, whereas two-photon microscopy offers high resolution but limited field of view and depth.
- I. INTRODUCTION: An imaging approach combining brain-wide coverage, microvascular resolvability, and rapid volumetric acquisition would support investigation of dynamic cerebrovascular responses.
- I. INTRODUCTION: The study extends a previous rapid three-dimensional imaging framework into a four-dimensional platform for ultrasound imaging during surgically induced ischemia and controlled reperfusion.The platform was evaluated by capturing whole-brain hemodynamic responses in a mouse stroke model.
A. Animal Preparation for Imaging
The mouse tMCAO preparation combines transient MCA occlusion with a cranial acoustic window and fixed head mount, enabling ultrasound imaging during controlled ischemia and reperfusion.
- A. Animal Preparation for Imaging: Transient cerebral ischemia was induced by inserting an occluding monofilament through the ICA toward the MCA origin, then withdrawing it after 60 min to initiate reperfusion.The procedure is illustrated schematically in Fig. 1.
- A. Animal Preparation for Imaging: A craniotomy and 9 × 11 mm head-mount opening provided an acoustic window for coupling the transducer to the exposed brain surface.A gel pad and acoustic gel were placed within the opening for acoustic coupling.
- A. Animal Preparation for Imaging: The 9-week-old C57BL/6 mouse received dexamethasone before craniotomy and was transferred to the imaging setup after head-mount preparation.
- A. Animal Preparation for Imaging: Animal preparation and acquisition were performed under continuously monitored isoflurane anesthesia with body temperature maintained by a heating pad.The study followed NIH animal-care guidelines and received approval from Tohoku University’s ethics committee.
B. Imaging System Setup and Imaging Sequence
The imaging setup preserves surgical access to the ventral neck while continuously scanning the brain from below, acquiring a 7 × 8 × 5 mm volume in 5 s.
- B. Imaging System Setup and Imaging Sequence: The mouse was fixed supine so the neck remained accessible for tMCAO surgery while the transducer contacted the cranial window from underneath the head.A stereotaxic fixation device secured the custom head mount.
- B. Imaging System Setup and Imaging Sequence: Continuous mechanical translation scanned the transducer elevationally at 1 mm/s for 5 s using a 30 MHz linear array.
- B. Imaging System Setup and Imaging Sequence: The synchronized scan and ultrasound sequence acquired a predefined 7 × 8 × 5 mm axial, lateral, and elevational volume.An Arduino trigger synchronized translation with transmit-receive timing.
- B. Imaging System Setup and Imaging Sequence: Plane-wave compounding used ten steered raw frames at ±5° and 5 kHz PRF, producing 500 Hz compounded frames.Each 5-s scan yielded 2,500 compounded B-mode frames, with 2 ms per frame and 2 μm translation between frames.
C. Data Acquisition Protocol
Ultrasound volumes were acquired at eight defined stages spanning baseline, 60 min of MCA occlusion, and early reperfusion, with microbubbles administered before each acquisition.
- C. Data Acquisition Protocol: The protocol sampled one control point, three ischemia points at 1, 15, and 60 min, and four reperfusion points at 1, 15, 20, and 30 min.The 60-min ischemic acquisition preceded filament withdrawal and reperfusion.
- C. Data Acquisition Protocol: One minute before each acquisition, 100 μL of microbubble suspension was injected through the contralateral ICA as an ultrasound contrast agent.
D. Signal and Image Processing Pipeline
The pipeline reconstructs 3D microvascular images by applying section-wise SVD filtering to compounded volumetric data, then aligns time-series images for vessel-level hemodynamic assessment.
- Signal extraction: Each volumetric acquisition is divided into 49 overlapping sections, and SVD filtering independently extracts blood-flow signals from each section.The 2,500 compounded frames are grouped into 100-frame sections with 50-frame overlap.
- Threshold selection: Common SVD cutoff indices are selected from four representative sections and applied across all sections within each volume.Threshold selection is performed independently for each of the eight volumetric acquisitions before generating Power Doppler images.
- Temporal alignment: 3D registration using mutual information spatially aligns the time-series vascular images to quantify hemodynamic changes in the same vessels.
III. RESULTS AND DISCUSSION
The platform produced fixed-animal, whole-brain 4D microvascular images throughout ischemia and reperfusion, capturing regional signal loss and recovery while preserving broad spatial coverage.
- Imaging capability: The platform enabled four-dimensional imaging at all eight time points without repositioning the mouse while preserving surgical access for ischemia and reperfusion.The displayed images are maximum intensity projections of elevational sections covering the MCA territory.
- Hemodynamic responses: Blood-flow signals decreased in cortical and deep MCA-territory regions during occlusion, while deep signals reappeared after reperfusion.In the right cortical region, intensity decreased by 6.7 dB during ischemia and increased by 12.2 dB after reperfusion.
- Spatial coverage: Whole-brain coverage extended from cortical surfaces to deep and basal regions, enabling repeated assessment of the same brain during ischemia and reperfusion.
- Temporal sampling: Each volumetric acquisition required 5 s, although the study used 1–45 min intervals and high-frequency repeated imaging remains to be developed.The authors state that intervals as short as 10 s would be technically feasible, but protocol timing limited the present sampling.
IV. CONCLUSION
The study developed a non-repositioning platform for four-dimensional whole-brain microvascular imaging during tMCAO surgery and demonstrated feasibility for assessing ischemia- and reperfusion-related hemodynamics.
- Conclusion: The platform enables four-dimensional whole-brain microvascular imaging during tMCAO surgery without repositioning the animal.
- Conclusion: The experiment demonstrated feasibility for rapid and continuous assessment of cerebral hemodynamics during experimentally induced ischemia and reperfusion.