Two-Dimensional Super-Resolution Visualization of Rat Brain Microvasculature Using Ultrasound Localization Microscopy

Summary

Here, we describe a protocol for ultrasound localization microscopy (ULM), which achieves 12.5 µm spatial resolution to image the brain microvasculature in rats. It enables detailed visualization of blood flow direction and velocity, offering a powerful tool for advancing studies of cerebral circulation and vascular disorders.

Abstract

The cerebral microvasculature forms a complex network of vessels essential for maintaining brain function. Diseases such as stroke, Alzheimer's disease, gliomas, and vascular dementia can profoundly disrupt the microvascular system. Unfortunately, current medical imaging modalities offer only indirect observations at this scale. Inspired by optical microscopy, ultrasound localization microscopy (ULM) overcomes the classical trade-off between penetration depth and spatial resolution. By localizing and tracking individual injected microbubbles (MBs) with sub-wavelength precision, vascular and velocity maps can be generated at the micrometer scale. Here, we present a robust protocol for super-resolution imaging of the brain microvasculature in vivo in rats using a commercial ultrasound platform. This method achieves 12.5 µm spatial resolution, reconstructing the microvascular architecture and providing detailed information on blood flow direction and velocity, greatly enhancing our understanding of cerebral microcirculation. The protocol can be extended to rat disease models, offering a powerful tool for the early diagnosis and treatment of neurovascular diseases.

Introduction

The cerebral microvasculature, comprising capillaries, arterioles, and venules, is essential for maintaining brain function by facilitating nutrient delivery, oxygen exchange, and waste removal^1,2. Disruptions in this network are implicated in neurological disorders such as stroke³, Alzheimer's disease⁴, gliomas⁵, and vascular dementia⁶, leading to impairments in brain physiology. Microvascular changes frequently precede the onset of clinical symptoms, making them a critical target for diagnostic and therapeutic int....

Protocol

All animal experiments performed in this work are approved by the Ethics Committee of Fudan University (Approval Number: 2022JS-004). The protocol strictly follows the animal care guidelines of Fudan University to ensure the humane treatment of animals. Prior to experimental initiation, rats must be allowed a 1-week period for environmental acclimatization, during which they are provided with sufficient feed and water. The photoperiod is carefully regulated in accordance with their biological rhythms to ensure the maintenance of normal physiological states. At the end of the experiment, euthanasia is performed using an overdose of inhaled isoflurane.

Results

Figure 1 illustrates the detailed setup for in vivo brain microvascular ULM imaging in rats, with each element carefully designed to minimize experimental variability and ensure accurate data acquisition for reliable super-resolution imaging results.

Figure 2A displays the ULM-reconstructed structure of the microvasculature in the rat brain, positioned at -1 mm from the Bregma point, with an imaging depth nearing 12 mm. The e.......

Discussion

This protocol successfully utilized ULM to perform super-resolution imaging of in vivo rat brain microvasculature. Compared to other imaging modalities, ULM simultaneously accommodates both spatial resolution and penetration depth. The exposed rat brain was imaged rather than through the skull, avoiding attenuation and distortion caused by the presence of bone. Under a transducer with a central frequency of 15.625 MHz, vascular structures at a depth of approximately 12 mm were captured, with a spatial resolution of up to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alcohol	DICHANG	https://www.dehsm.com/goods-17187.html	75%
Beamforming program	Institute of Biomedical Engineering at the University of Montreal	Matlab Ultrasound Toolbox 3.4 version
Body temperature maintenance device	RWD Life Science Co., Ltd.	69026
Brain stereotaxic instrument	RWD Life Science Co., Ltd.	71000-R	Adaptable to breathing mask
Cranial Microinjection Surgical Instrument Kit	RWD Life Science Co., Ltd.	SP0005-R
Digital microscope	RWD Life Science Co., Ltd.	DOM-1001
Drug delivery catheter	RWD Life Science Co., Ltd.	https://www.rwdls.com/product-solutions/life-sciences/administration/draw-blood
Erythromycin ointment	Renhe Pharma	H36020018	1% x 15 g
Gas anesthesia machine	RWD Life Science Co., Ltd.	R500IE	Includes breathing mask
Handheld electric clipper	GUAZHOUMU	MJD-DTJ02
Handheld mini cranial drill	RWD Life Science Co., Ltd.	78001
Indwelling needle	Kindly EnterpriseDevelopment Group Co., LTD	Positive Pressure Model	26 G
Iodine solution	HYNAUT	https://www.hainuocn.com/index/detail/524.html	4.5–5.5 g/L
IQ demodulation program	Institute of Biomedical Engineering at the University of Montreal	Matlab Ultrasound Toolbox 3.4 version
Isoflurane	RWD Life Science Co., Ltd.	R510-22-10
MATLAB software	MathWorks	Version R2021a
Microinjection pump	RWD Life Science Co., Ltd.	R462
Sodium chloride injection	SHENG'AO animals pharmaceutical Co., Ltd.	270071460	0.90%
SonoVue	Bracco	https://www.bracco.com/en-se/product/sonovue
Spherical drill bit	RWD Life Science Co., Ltd.	HM1027/HM1010
Supporting Positioning Software	RWD Life Science Co., Ltd.	V2.0.0.30400
Syringe	Kindly EnterpriseDevelopment Group Co., Ltd.	RWLB	1 mL
Tracking program	Jean-Yves Tinevez	2016 version
Ultrasound gel	Junkang Medical Equipment Co., Ltd.	Model DS-1
Ultrasound probe	VERASONICS, INC.	L22-14vX LF
Verasonics Ultrasound System	VERASONICS, INC.	Vantage-256	ultrasound platform