Name: Author Spotlight: Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke
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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.

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&#39;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.
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Non-Invasive Integrated PAUSAT Whole-Brain Imaging of Ischemic Stroke

<ol>
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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&#39;s head during imaging can often block the ultrasound from travelling to or from the transdu...

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 stroke and identify and test new treatments is the use of small-animal models in the lab. Treatments discovered in the lab aim to be translated to clinical use and improve patients&#39; lives. However, the use of animals in biomedical research needs to be carefully evaluated according to Russell and Burch&#8217;s 3Rs principles: replacement, reduction, and refinement3. The objective of the reduction component is to reduce the number of animals without compromising data collection. With this in mind, being able to longitudinally evaluate the lesion evolution via noninvasive imaging allows a great advantage in reducing the number of animals required, as well as maximizing the information obtained from each animal4.
Photoacoustic tomography (PAT) is a hybrid imaging modality that combines optical absorption contrast with ultrasound imaging spatial resolution5. The imaging mechanism of PAT is as follows. An excitation laser pulse is illuminated on the target being imaged. Assuming the target absorbs light at the wavelength of the excitation laser, it will increase in temperature. This quick increase in temperature results in a thermoelastic expansion of the target. The expansion causes an ultrasound wave to propagate out from the target. By detecting the ultrasound wave at many positions, the time required for the wave to propagate from the target to the detectors can be used to create an image through a reconstruction algorithm. The ability of PAT to detect optical absorption in deep tissue regions differentiates PAT from ultrasound imaging, which detects boundaries of differing acoustic impedances of tissues5. In the visible and near-infrared spectra, the primary highly absorbing biomolecules that are abundant in organisms are hemoglobin, lipids, melanin, and water7. Of particular interest in the study of stroke is hemoglobin. Since oxyhemoglobin and deoxyhemoglobin have different optical absorption spectra, PAT can be used with multiple excitation laser wavelengths to determine the relative concentration of the two states of the protein. This allows the oxygen saturation of hemoglobin (sO2), or blood oxygenation, to be quantified in and outside of the infarct region8,9. This is an important measure in ischemic stroke, as it can indicate the level of oxygen in the damaged brain tissue following ischemia.
Acoustic angiography (AA) is a contrast-enhanced ultrasound imaging method that is particularly useful for imaging the morphology of vasculature in vivo10. The method relies on the use of a dual-element wobbler transducer (a low frequency element and a high frequency element) in conjunction with microbubbles injected into the circulatory system of the imaging subject. The low-frequency element of the transducer is used for transmitting at the resonant frequency of the microbubbles (e.g., 2 MHz), while the high-frequency element is used to receive the super harmonic signals of the microbubbles (e.g., 26&#160;MHz). When excited at a resonant frequency, the microbubbles have a strong nonlinear response, resulting in the production of super harmonic signals that surrounding body tissues do not produce11. By receiving with a high-frequency element, this ensures that only the microbubble signals are detected. Since the microbubbles are confined to the blood vessels, the result is an angiographic image of blood vessel morphology. AA is a powerful method for imaging ischemic stroke, as the microbubbles that flow through the circulatory system are not be able to flow through blocked vessels. This allows AA to detect regions of the brain that are not perfused due to ischemic stroke, which indicates the infarct region.
Preclinical ischemic stroke research generally relies on the use of histology and behavioral testing to assess the location and severity of the stroke. Triphenyltetrazolium chloride (TTC) staining is a common histological analysis used to determine the stroke infarct volume. However, it can only be used at an endpoint, since it requires the animal to be euthanized12. Behavioral tests can be used to determine motor function impairment at multiple time points, but they cannot provide quantitative anatomical or physiological values13. Biomedical imaging provides a more quantitative approach to studying the effects of ischemic stroke noninvasively and longitudinally9,14,15. However, existing imaging technologies (such as small-animal magnetic resonance imaging [MRI]) can come at a high cost, be unable to provide concurrent structural and functional information, or have limited penetration depth (as most optical imaging techniques).
Here, we combine photoacoustic, ultrasound, and angiographic tomography (PAUSAT; see system diagram in Figure 1), which allows for complementary structural and functional information of blood perfusion and oxygenation following ischemic stroke16. These are two important aspects in assessing the severity of injury and monitoring the recovery or response to treatments. Using these integrated imaging methods can increase the amount of information obtained by each animal, reducing the number of animals required and providing more information in the study of potential treatments for ischemic stroke.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65319/65319fig01.jpg" /> 
Figure 1: PAUSAT diagram. (A) Complete schematic of the PAUSAT system, including the laser and OPO used for PAT. (B) Inside view of the PAUSAT system, including two ultrasound transducers. The dual-element wobbler transducer is used for both B-mode ultrasound and AA, and the linear-array transducer is used for PAT. Both transducers are mounted on the same 2D motorized stage, allowing for scanning to generate volumetric data. This figure has been modified from16. <a href="https://www.jove.com/files/ftp_upload/65319/65319fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 GA catheter</td>
			<td>BD Insyte Autoguard Winged</td>
			<td>381534</td>
			<td>For mouse intubation</td>
		</tr>
		<tr>
			<td>2,3,5-Triphenyltetrazolium chloride</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>Necessary for TTC-staining brain for validation</td>
		</tr>
		<tr>
			<td>532nm Laser</td>
			<td>Quantel</td>
			<td>Q-smart 850</td>
			<td>Laser used to pump the OPO for PAT</td>
		</tr>
		<tr>
			<td>Automatic Ventilator Rovent Jr.</td>
			<td>Kent Scientific</td>
			<td>RV-JR</td>
			<td>To keep mice under anesthesia during surgical procedure</td>
		</tr>
		<tr>
			<td>Black braided silk 4-0 USP</td>
			<td>Surgical Specialties</td>
			<td>SP116</td>
			<td>Used for sutures on the neck for pMCAO surgery</td>
		</tr>
		<tr>
			<td>Bupivacaine</td>
			<td>Hospira</td>
			<td>0409-1159-18</td>
			<td>Used prior to closing wounds during surgical procedure</td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Jackson Lab</td>
			<td>#000664</td>
			<td>Mice used for studying ischemic stroke (2-6 month old male/female)</td>
		</tr>
		<tr>
			<td>Clear suture</td>
			<td>Ethicon</td>
			<td>8606</td>
			<td>Used for closing wound (PT stroke and pMCAO). A clear suture won&#39;t interfere with PAT</td>
		</tr>
		<tr>
			<td>Cold Light LED</td>
			<td>Schott</td>
			<td>KL 1600</td>
			<td>Needed to create PT stroke</td>
		</tr>
		<tr>
			<td>Disposable Razor Blade</td>
			<td>Accutec Blades</td>
			<td>74-0002</td>
			<td>For sectioning mouse brain</td>
		</tr>
		<tr>
			<td>Electric drill</td>
			<td>JSDA</td>
			<td>JD-700</td>
			<td>Used to expose MCA during pMCAO procedure</td>
		</tr>
		<tr>
			<td>Electrocauterization tool</td>
			<td>Wet-Field</td>
			<td>Wet-Field Bipolar-RG</td>
			<td>Stops blood flow after drilling during pMCAO procedure</td>
		</tr>
		<tr>
			<td>Hair removal gel</td>
			<td>Veet</td>
			<td>8282651</td>
			<td>Used to remove hair from mouse prior to imaging</td>
		</tr>
		<tr>
			<td>High Temperature Cautery Loop Tip</td>
			<td>BOVIE Medical Corporation</td>
			<td>REF AA03</td>
			<td>Used to avoid bleeding when separating the temporal muscle from the skull</td>
		</tr>
		<tr>
			<td>IR Detector Card</td>
			<td>Thorlabs</td>
			<td>VRC5</td>
			<td>Used to ensure light path is aligned</td>
		</tr>
		<tr>
			<td>Laser Power Meter</td>
			<td>Ophir</td>
			<td>StarBright, P/N 7Z01580</td>
			<td>Can be used to calibrate the laser energy prior to imaging</td>
		</tr>
		<tr>
			<td>Laser Speckle Imaging System</td>
			<td>RWD Life Science Co.</td>
			<td>RFLSI-III</td>
			<td>Can be used to validate stroke surgery success</td>
		</tr>
		<tr>
			<td>Lubricant Eye Ointment</td>
			<td>Soothe</td>
			<td>AB31336</td>
			<td>Can be used to avoid drying of the eyes</td>
		</tr>
		<tr>
			<td>Manually adjustable stage</td>
			<td>Thorlabs</td>
			<td>L490</td>
			<td>Used with custom ramp for multiple focal depth AA imaging</td>
		</tr>
		<tr>
			<td>Modified Vega Imaging System</td>
			<td>Perkin Elmer</td>
			<td>LLA00061</td>
			<td>System containing both B-mode/AA and PAT transducers</td>
		</tr>
		<tr>
			<td>Optical Parametric Oscillator</td>
			<td>Quantel</td>
			<td>versaScan-L532</td>
			<td>Allows for tuning of excitation wavelength in a large range</td>
		</tr>
		<tr>
			<td>Programmable Ultrasound System</td>
			<td>Verasonics</td>
			<td>Vantage 256</td>
			<td>Used for PAT part of system</td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>330000</td>
			<td>Necessary to induce PT stroke</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>LOOK</td>
			<td>SP116</td>
			<td>Used for permanent ligation of CCA</td>
		</tr>
		<tr>
			<td>Temperature Contoller</td>
			<td>Physitemp</td>
			<td>TCAT-2</td>
			<td>Used to maintain stable body temperature of mice during procedures</td>
		</tr>
		<tr>
			<td>VesselVue Microbubbles</td>
			<td>Perkin Elmer</td>
			<td>P-4007001</td>
			<td>Used for acoustic angiography (2.43 &#215; 10^9 microbubbles/mL)</td>
		</tr>
	</tbody>
</table>

integrated photoacoustic, ultrasound, and angiographic tomography (pausat) for noninvasive whole-brain imaging of ischemic stroke

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.

Author Spotlight: Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke  

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

There are a few experimental challenges. For example, the presence of hair in the scalp can cause artifact in the acquired images. The age of the animal also plays a key role since the thickness of the skull is currently a limitation for the detection of deeper signals.Many purely optical imaging techniques require an invasive procedure and can only image about one millimeter deep. With PAUSAT, we combine the advantages of both ultrasound and optical imaging. We can get both structural and functional information surrounding ischemic stroke in a completely safe and non-invasive way.Being able to evaluate the evolution of an injury with or without treatment from the same animal at different time points allows us to collect very valuable information. And also to reduce the number of animals required for different studies. We are constantly working on ways to improve the imaging speed, depth, resolution and contrast for both photoacoustic and ultrasound imaging.With better imaging systems, we can answer more fundamental biological questions and evaluate new experimental treatments.

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...

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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 ...