Published: June 2nd, 2023
This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.
Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.
Blood transports oxygen (via the hemoglobin protein) and other important nutrients to tissues in our bodies. When the flow of blood through tissues is interrupted (ischemia), severe damage to the tissues can occur, the most immediate effects of which are due to a lack of oxygen (hypoxia). Ischemic stroke is the result of interrupted blood flow to a certain region of the brain. The brain damage resulting from an ischemic stroke can occur within minutes of a vessel blockage, and can often have debilitating and lasting effects1,2. A highly valuable strategy to evaluate the physiopathology after ischemic ....
All animal procedures were approved by the Duke University Medical Center Animal Care and Use Committee and were conducted in accordance with the United States Public Health Service's Policy on Humane Care and Use of Laboratory Animals. Male and female C57BL/6J mice (see Table of Materials) were used for these studies. A minimum of three animals were imaged per stroke model group. See Figure 2 for the workflow followed in this protocol.
Imaging of blood vessel morphology in the brain
AA generates blood vessel morphology images by exciting microbubbles in the circulatory system at their resonant frequency and receiving the super harmonic response of the microbubbles. By using the customized ramp (Figure 2C) attached to a manually adjustable stage, we can image the mouse brain with AA mode at two different focal depths. When deeper regions are targeted, more superficial regions (such as the cerebral cor.......
There are a few vital aspects of this method that, if done incorrectly, can lead to significantly decreased image quality and quantitative analysis. The most commonly occurring result of user-error in PAUSAT images is either a lack of signal or very low signal strength, both of which can occur for a variety of reasons. One such reason is a problem with the acoustic coupling. Large air bubbles in the water surrounding the mouse's head during imaging can often block the ultrasound from travelling to or from the transdu.......
The authors would like to acknowledge the engineering team at SonoVol Inc. for their technical support. This work was partially sponsored by the American Heart Association Collaborative Sciences Award (18CSA34080277), to J. Yao and W. Yang; The United States National Institutes of Health (NIH) grants R21EB027981, R21 EB027304, RF1 NS115581 (BRAIN Initiative), R01 NS111039, R01 EB028143; The United States National Science Foundation (NSF) CAREER award 2144788; the Chan Zuckerberg Initiative Grant (2020-226178), to J. Yao; and NIH grants R21NS127163 and R01NS099590 to W. Yang.....
|20 GA catheter
|BD Insyte Autoguard Winged
|For mouse intubation
|Necessary for TTC-staining brain for validation
|Laser used to pump the OPO for PAT
|Automatic Ventilator Rovent Jr.
|To keep mice under anesthesia during surgical procedure
|Black braided silk 4-0 USP
|Used for sutures on the neck for pMCAO surgery
|Used prior to closing wounds during surgical procedure
|Mice used for studying ischemic stroke (2-6 month old male/female)
|Used for closing wound (PT stroke and pMCAO). A clear suture won't interfere with PAT
|Cold Light LED
|Needed to create PT stroke
|Disposable Razor Blade
|For sectioning mouse brain
|Used to expose MCA during pMCAO procedure
|Stops blood flow after drilling during pMCAO procedure
|Hair removal gel
|Used to remove hair from mouse prior to imaging
|High Temperature Cautery Loop Tip
|BOVIE Medical Corporation
|Used to avoid bleeding when separating the temporal muscle from the skull
|IR Detector Card
|Used to ensure light path is aligned
|Laser Power Meter
|StarBright, P/N 7Z01580
|Can be used to calibrate the laser energy prior to imaging
|Laser Speckle Imaging System
|RWD Life Science Co.
|Can be used to validate stroke surgery success
|Lubricant Eye Ointment
|Can be used to avoid drying of the eyes
|Manually adjustable stage
|Used with custom ramp for multiple focal depth AA imaging
|Modified Vega Imaging System
|System containing both B-mode/AA and PAT transducers
|Optical Parametric Oscillator
|Allows for tuning of excitation wavelength in a large range
|Programmable Ultrasound System
|Used for PAT part of system
|Necessary to induce PT stroke
|Used for permanent ligation of CCA
|Used to maintain stable body temperature of mice during procedures
|Used for acoustic angiography (2.43 × 10^9 microbubbles/mL)
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