Name: Murine PAUSAT Imaging
Uploaded: 2023-06-02T12:20:00
Description: Watch this Scientific Journal Video about Murine PAUSAT Imaging at JoVE.com

murine pausat imaging

缺血性卒中的无创综合 PAUSAT 全脑成像

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>

作者聚焦：用于缺血性卒中的无创全脑成像的集成光声、超声和血管造影断层扫描 （PAUSAT）  

集成光声、超声和血管造影断层扫描 （PAUSAT） 用于缺血性卒中的无创全脑成像

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.

Watch this Scientific Journal Video about Murine PAUSAT Imaging at JoVE.com

Murine PAUSAT Imaging  

鼠PAUSAT成像

要设置成像系统，首先将 532 纳米激光器打开 15 分钟以预热。接下来，将定制的斜坡连接到成像膜旁边的手动可调载物台上。将带有连接呼吸管的鼠标牙架连接到坡道上，并将加热垫固定在坡道表面上。激光预热后，在光纤束输入前面放置一个近红外探测器卡，并通过确保激光进入束来检查激光对准。通过用电动剃须刀从眼睛附近到耳朵后面修剪麻醉小鼠头部的头发来开始动物准备。在头顶涂抹脱毛霜。保持五分钟后，用湿棉签擦去乳霜。重复应用，直到头部皮肤完全脱毛。在将动物转移到系统平台之前，使用27号针头以原液浓度回轨道注射100微升微气泡。现在，将一滴护目化妆水滴在眼睛上。通过在成像窗口表面填充蒸馏水以进行声耦合来开始PAUSAT成像。接下来，将鼠标的头部固定在牙齿支架中，并确保适当的麻醉和气流。使用加热灯和连接到温度控制器的直肠探头，将动物的体温保持在 37 摄氏度。打开成像应用程序并导航到 B 模式超声波。使用实时超声窗口手动将鼠标的头部调整到所需位置，并相应地调整载物台高度。将 B 模式下的超声传输频率值调整为 16 兆赫兹。然后，在映像应用程序中输入保存目录信息。接下来，使用浮动框选择所需的脑部扫描区域。图像采集完成后，按下采集静态按钮并检查扫描结果，以确保所需的区域成像。要执行声学血管造影成像，请返回“图像采集”，然后将模式更改为“声学血管造影”。接下来，输入所需的扫描协议参数，将帧间距设置为0.2毫米，每个位置10帧。在图像分析下检查扫描结果以确保图像质量。要使用光声断层扫描成像，请打开光学参量振荡器或 OPO 应用程序，并将其设置为 756 纳米。然后，手动将线性阵列探头转换为先前确定的坐标，以自动共同配准摆动体积和线性阵列体积。打开激光应用程序并打开 532 纳米激光器。将扫描参数设置为0.4毫米步长、20毫米扫描长度和每个位置平均10帧。现在，打开超声数据采集系统MATLAB程序，然后按运行按钮。接下来，在扫描应用程序中按开始。扫描完成后，打开 MATLAB 保存程序。将保存名称更改为所需的文件名，然后按运行。然后将OPO波长更改为798纳米并重复扫描。声学血管造影或AA成像显示，在未受伤的大脑中，两个半球的血管分布相似。在两种不同波长的光声图像中观察到类似的结果。在电灼小鼠的AA图像中观察到右外侧皮层的信号显着减少。同一区域也显示组织氧合减少，提示缺血。PAUSAT成像可以通过识别血流供应减少和氧饱和度降低的上皮层区域，基于光血栓性卒中模型识别卒中区域。小鼠年龄会影响成像，因为头骨变厚并导致不同的声阻抗，从而导致老年小鼠的成像深度降低。

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Neuroscience

Murine PAUSAT Imaging

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 ...