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

Ikke-invasiv integreret PAUSAT helhjernebilleddannelse af iskæmisk slagtilfælde

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>

Forfatter Spotlight: Integreret fotoakustisk, ultralyd og angiografisk tomografi (PAUSAT) til ikke-invasiv helhjernebilleddannelse af iskæmisk slagtilfælde  

Integreret fotoakustisk, ultralyd og angiografisk tomografi (PAUSAT) til ikke-invasiv helhjernebilleddannelse af iskæmisk slagtilfælde

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  

Billeddannelse af murin PAUSAT

For at konfigurere billedbehandlingssystemet skal du starte med at lade 532 nanometerlaseren være tændt i 15 minutter for at varme op. Placer derefter en tilpasset rampe, der er fastgjort til et manuelt justerbart trin ved siden af billedmembranen. Fastgør en musetandholder med et tilsluttet åndedrætsrør til rampen, og fastgør en varmepude på rampeoverfladen.Når laseren er varmet op, skal du placere et nær IR-detektorkort foran fiberbundtindgangen og kontrollere laserjusteringen ved at sikre, at laserlyset kommer ind i bundtet. Begynd dyreforberedelse ved at trimme håret på den bedøvede musens hoved med en elektrisk barbermaskine fra nær øjnene til bag ørerne. Påfør en hårfjerningscreme på toppen af hovedet.Når du har ladet det sidde i fem minutter, skal du tørre cremen af med en våd vatpind. Gentag applikationen, indtil hovedets hud er helt depileret. Lige før du overfører dyret til systemets platform, skal du bruge en 27 gauge nål til retro-orbitalt at injicere 100 mikroliter mikrobobler ved lagerkoncentration.Sæt nu en dråbe af øjenbeskyttelseslotionen på øjnene. Start PAUSAT imaging ved at fylde billedvinduet med destilleret vand på overfladen for akustisk kobling. Fastgør derefter musens hoved i tandholderen og sørg for korrekt anæstesi og luftstrøm.Brug en varmelampe og en rektal sonde forbundet til en temperaturregulator til at holde dyrets kropstemperatur på 37 grader Celsius. Åbn billedbehandlingsprogrammet, og naviger til ultralyd i B-tilstand. Brug live ultralydsvinduet til manuelt at justere musens hoved til den ønskede position og scenehøjden i overensstemmelse hermed.Juster værdien af ultralydstransmissionsfrekvensen i B-tilstand til 16 megahertz. Indtast derefter de gemte mappeoplysninger i billedbehandlingsprogrammet. Vælg derefter det ønskede område af hjernescanningen ved hjælp af den flydende boks.Tryk på knappen Hent statisk, og kontroller scanningsresultaterne, når billedoptagelsen er afsluttet for at sikre den ønskede regionbilleddannelse. Hvis du vil udføre akustisk angiografibilleddannelse, skal du vende tilbage til Billedoptagelse og derefter ændre tilstanden til Akustisk angiografi. Indtast derefter de ønskede scanningsprotokolparametre, indstil rammeafstanden til 0,2 millimeter og 10 billeder pr. Position.Kontroller scanningsresultaterne under billedanalyse for at sikre billedkvaliteten. For at afbilde med fotoakustisk tomografi skal du åbne den optiske parametriske oscillator eller OPO-applikationen og indstille den til 756 nanometer. Oversæt derefter manuelt den lineære array-transducer til de tidligere bestemte koordinater for automatisk at registrere wobblervolumenerne og de lineære array-volumener.Åbn laserprogrammet, og tænd for 532 nanometerlaseren. Indstil scanningsparametrene som 0,4 millimeter trinstørrelse, 20 millimeter scanningslængde og 10 billeder i gennemsnit pr. position. Åbn nu ultralydsdataindsamlingssystemet MATLAB-programmet, og tryk på kørselsknappen.Tryk derefter på start i scanningsprogrammet. Når scanningen er afsluttet, skal du åbne MATLAB-lagringsprogrammet. Skift gemnavnet til det ønskede filnavn, og tryk på Kør.Skift derefter OPO-bølgelængden til 798 nanometer, og gentag scanningen. Akustisk angiografisk eller AA-billeddannelse viste, at begge halvkugler i den uskadte hjerne præsenterer en lignende fordeling af blodkar. Lignende resultater observeres i de fotoakustiske billeder ved to forskellige bølgelængder.Bemærkelsesværdig signalreduktion i højre laterale cortex blev observeret i AA-billederne af elektrokauteriserede mus. Den samme region viste også reduceret iltning af væv, hvilket tyder på iskæmi. PAUSAT-billeddannelse kan identificere slagtilfældeområder baseret på fototrombotisk slagtilfældemodel ved at identificere øvre cortex-region med reduceret blodgennemstrømningsforsyning og nedsat iltmætning.Musens alder kan påvirke billeddannelsen, da kraniet bliver tykkere og resulterer i forskellig akustisk impedans, hvilket resulterer i nedsat billeddybde for ældre mus.

Research

JoVE Journal

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 ...