Early identification of umbilical blood flow restriction and maternal placental hypoperfusion with photoacoustic imaging

Purpose: To investigate the relations of neuropeptide Y (NPY) and heme oxygenase-1 (HO-1) expressions with fetal brain injury in rats with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty rats pregnant for 15 days were randomly divided into experimental and control groups. The ICP model was established in experimental group. On the 21st day, the blood biochemical test, histopathological examination of pregnant rat liver and fetal brain tissues and immunohistochemical analysis of fetal rat brain tissues were performed. Results: On the 21st day, the alanineaminotransferase, aspartate aminotransferase and total bile acid levels in experimental group were significantly higher than control group (P<0.01). Compared with control group, there was obvious vacuolar degeneration in pregnant rat liver tissue and fetal brain tissue in experimental group. NPY expression in fetal brain tissue was negative in control group and positive in experimental group. HO-1 expression in fetal brain tissue was strongly positive in control group and positive in experimental group. There was significant difference of immunohistochemical staining optical density between two groups (P<0.01). Conclusion: In fetal brain of ICP rats, the NPY expression is increased, and the HO-1 expression is decreased, which may be related to the fetal brain injury.

Prenatal ethanol exposure (PEE) can lead to structural and functional abnormalities in fetal brain. Although neural developmental deficits due to PEE have been recognized, the immediate effects of PEE on fetal brain vasculature and hemodynamics remain poorly understood. One of the major obstacles that preclude the rapid advancement of studies on fetal vascular dynamics is the limitation of the imaging techniques. Thus, a technique for noninvasive in-vivo imaging of fetal vasculature and hemodynamics is desirable. In this study, we explored the dynamic changes of the vessel dimeter, density and oxygen saturation in fetal brain after acute maternal ethanol exposure in the second-trimester equivalent murine model using a real-time photoacoustic tomography system we developed for imaging embryo of small animals. The results indicate a significant decrease in fetal brain vessel diameter, perfusion and oxygen saturation. This work demonstrated that PAT can provide high-resolution noninvasive imaging ability to monitor fetal vascular dynamics.

Photoacoustic Imaging (PAI) is an emerging technology with strong potential for broad clinical applications from breast cancer detection to cerebral monitoring due to its ability to compute maps of blood oxygen saturation (SO 2 ) distribution in deep tissues using multispectral imaging. However, no well-validated consensus test methods currently exist for evaluating oximetry-specific performance characteristics of PAI devices. We have developed a phantombased flow system capable of rapid SO 2 adjustment to serve as a test bed for elucidation of factors impacting SO 2 measurement and quantitative characterization of device performance. The flow system is comprised of a peristaltic pump, membrane oxygenator, oxygen and nitrogen gas, and in-line oxygen, pH, and temperature sensors that enable real-time estimation of SO 2 reference values. Bovine blood was delivered through breast-relevant tissue phantoms containing vessel-mimicking fluid channels, which were imaged using a custom multispectral PAI system. Blood was periodically drawn for SO 2 measurement in a clinical-grade CO-oximeter. We used this flow phantom system to evaluate the impact of device parameters (e.g.,wavelength-dependent fluence corrections) and tissue parameters (e.g. fluid channel depth, blood SO 2 , spectral coloring artifacts) on oximetry measurement accuracy. Results elucidated key challenges in PAI oximetry and device design trade-offs, which subsequently allowed for optimization of system performance. This approach provides a robust benchtop test platform that can support PAI oximetry device optimization, performance validation, and clinical translation, and may inform future development of consensus test methods for performance assessment of photoacoustic oximetry imaging systems.

Neuregulin 1 (NRG1) is a multifunctional neurotrophin that mediates neurodevelopment and schizophrenia risk. The NRG1 gene undergoes extensive alternative splicing, and association of brain NRG1 type IV isoform expression with the schizophrenia-risk polymorphism rs6994992 is a potential mechanism of risk. Novel splice variants of NRG1-IV (NRG1-IVNV), with predicted unique signaling capabilities, have been cloned in fetal brain tissue. The authors investigated the temporal dynamics of transcription of NRG1-IVNV, compared with the major NRG1 isoforms, across human prenatal and postnatal prefrontal cortical development, and they examined the association of rs6994992 with NRG1-IVNV expression. NRG1 type I-IV and NRG1-IVNV isoforms were evaluated with quantitative real-time polymerase chain reaction in human postmortem prefrontal cortex tissue samples at 14 to 39 weeks gestation and postnatal ages 0-83 years. The association of rs6994992 genotype with NRG1-IVNV expression and the subcellular distribution and proteolytic processing of NRG1-IVNV isoforms were also determined. Expression of NRG1 types I, II, and III was temporally regulated during prenatal and postnatal neocortical development. NRG1-IVNV was expressed from 16 weeks gestation until age 3. Homozygosity for the schizophrenia risk allele (T) of rs6994992 conferred lower cortical NRG1-IVNV levels. Assays showed that NRG1-IVNV is a novel nuclear-enriched, truncated NRG1 protein resistant to proteolytic processing. To the authors' knowledge, this study provides the first quantitative map of NRG1 isoform expression during human neocortical development and aging. It identifies a potential mechanism of early developmental risk for schizophrenia at the NRG1 locus, involving a novel class of NRG1 proteins.

Photoacoustic (PA) Imaging can estimate the spatial distribution of oxygen saturation (sO 2 ) and total hemoglobin concentration (HbT) in blood, and be co-registered with B-Mode ultrasound images of the surrounding anatomy. This study will focus on the development of a PA imaging mode on a commercially available array based micro-ultrasound ( US) system that is capable of creating such images. The syst em will then be validated in vivo against a complementary technique for measuring partial pressure of oxygen in blood (pO 2 ). The pO 2 estimates are converted to sO 2 values based on a standard dissociation curve found in the literature. Finally, the system will be used for assessing oxygenation in a murine model of ischemia, both during injury and recovery. Keywords: Photoacoustics; Micro-ultrasound; Small animal imaging; Oxygen saturation; Hemoglobin; Ischemia. 1. INTRODUCTION Photoacoustic (PA) Imaging is sensitive to differences in optical absorption from biological tissues but detects these signals with ultrasound. PA Imaging exploits the photoacoustic effect, whereby an acoustic wave is generated from an object that is illuminated by pulsed electromagnetic radiation. By illuminating tissue, a thermoelastic expansion can occur, and is dependent on the optical absorption at the excitation wavelength of the light. This expansion creates an ultrasound wave that can be detected with an ultrasound transducer. The most commonly accepted PA scanners use either a tomographic (PAT) [1] or a plan ar geometry with a linear array trans ducer [2],[3]. The tomographic approach offers a large effective aperture for data collection, but suffers from a low frame rate, due to the need for hundreds to thousands of laser pulses per frame. The use of a linear array allows a 2-D frame to be acquired with just a few laser pulses, providing much higher frame rates. Because PA Imaging is dependent on op tical absorption, it may be used for discriminating blood from tissue signals. Specifically, the oxygenation of hemoglobin (Hb), the blood pr otein that carries oxygen to tissues, can be assessed with PA Imaging. Hemoglobin with bound oxygen (HbO

Photoacoustic imaging has the potential to provide real-time, non-invasive diagnosis of numerous prevalent diseases, due to the technology’s unique ability to visualize molecular changes deep within living tissue with spatial resolution comparable to ultrasound. Photoacoustic imaging is a hybrid imaging technique that combines the contrast capabilities and spectral sensitivities of optical imaging with the resolution and tissue penetration capabilities of ultrasound. During the photoacoustic imaging process, materials absorb light energy, and convert the light to heat via non-radiative relaxation. When materials heat, they expand in size due to their thermoelastic properties, which generates a pressure wave. These pressure waves can propagate through the surrounding environment to be detected at the surface. This effect is familiar to everyone who has experienced a summer thunderstorm—lightning rapidly heats the air, resulting in the air expanding and generating audible thunder. In general, the heating which induces the expansion of the material (e.g., the thermoacoustic effect) could be caused by many forms of energy transfer, but the term “photoacoustic” specifies the conversion of light into heat, resulting in the generation of characteristic sound waves. The photoacoustic effect was first discovered by Alexander Graham Bell in 1880.1 His experiments deduced that an intermittent bright light could heat optically absorbing materials, causing expansion of the material in a way that generated audible vibrational waves. Bell demonstrated that darker fibers produced louder sounds than lighter fibers, a principle which is consistent with the general photoacoustic relationship in use today—the amplitude of the generated photoacoustic signal is proportional to the amount of absorbed light. Bell also showed, by separating white light with a prism, certain color combinations of light and fibers could generate a louder sound. Today, multiwavelength photoacoustic imaging uses this same principle, changing the wavelength of the light and correlating the amplitude of the photoacoustic response to the absorption spectra of the materials being imaged. A modern application of the photoacoustic effect is the generation of medical images of biological chromophores typically present in tissue, which can absorb light energy resulting in the generation of photoacoustic transients. The photoacoustic pressure waves can be received by ultrasound transducers at the external surface of the tissue, making photoacoustic imaging a non-invasive, non-ionizing medical imaging method capable of resolution similar to ultrasound, at significant tissue depth. Photoacoustic medical imaging was first proposed in the mid-1990s,2,3 and initial reports of using the photoacoustic effect to image live animals were published nine years later.4 Today, many in vivo demonstrations of photoacoustic imaging of biomedical applications relevant to medical diagnostics exist, including cancer,5,6 brain vasculature and function,7–9 cardiovascular,10 and tissue engineering scaffolds,11,12 prompting translational advances in clinical photoacoustic imaging.13 While existing medical imaging methods, including ultrasound, are capable of producing remarkable images of what lies beneath our skin, most of these imaging methods provide contrast between anatomical features within tissue—for example, the difference in acoustic impedance between soft tissue and a tumor provide contrast within an ultrasound image. Though the anatomy is critical to understanding the image, in many diseases the anatomy alone cannot be used to indicate a particular diagnosis conclusively. Instead, the physiological and biochemical properties of the system influence the disease progression, and therefore the prognosis of the patient. Functional imaging capabilities are required to provide physiological information, while biochemical information can be provided by molecular imaging. In comparison to ultrasound, photoacoustic imaging provides improved capabilities for functional and molecular imaging. For example, the blood oxygen saturation, an important functional property relevant to many disease processes, can be assessed using photoacoustics.7 Photoacoustic imaging can also provide molecular information through the use of a probe or tracer, which can be used to generate the needed contrast to produce an image.14 Because of the potential to perform real-time, non-invasive in vivo functional and molecular imaging, photoacoustic imaging is increasingly being applied as both a clinical and preclinical method aimed at improving medical diagnostics.

Cardiac remodeling following myocardial infarction (MI) leads to structural and vascular changes in the myocardium. To better understand the mechanisms involved, we used photoacoustic ECG‐gated Kilohertz Visualization imaging (PA EKV) to measure oxygen saturation in the myocardium. Photoacoustic imaging is a noninvasive imaging technique that uses near‐infrared laser light to optically excite molecules in vivo. Oxygenated hemoglobin absorbs laser light at a different wavelength (850nm) from non‐oxygenated hemoglobin (750nm) which allows for the detection of the relative oxygen saturation (sO2) of a tissue and the amount of hemoglobin present (HbT). In this study, photoacoustic imaging was combined with EKV imaging for continuous measurement of sO2 and HbT throughout the contraction cycle.Imaging was done on C57Bl/6J mice (F, 4–5 months of age) to measure baseline sO2 levels in the anterior and posterior myocardium. An MI was induced by ligation of the left anterior descending coronary artery. Mice were imaged at days 1, 7, and 14 after MI to measure changes in myocardial sO2. All data presented here are from diastole of the long axis orientation and similar results were obtained from short axis measurements. Oxygen saturation on the anterior myocardial wall decreased from 74.7% pre‐MI to 19.9% 1 day after MI (p&lt;0.0001). Oxygen saturation showed recovery at 7 (54.9%, p=0.023) and 14 (49.9%, p=0.007) days post infarct, yet remained significantly reduced from pre‐MI levels. Oxygen saturation was lower on the posterior myocardium (57.2%) compared to the anterior myocardium but did not change following MI.Our results demonstrate oxygen saturation begins to recover 7 days after MI as the heart adapts to ligation and neoangiogenesis is stimulated. This study validates the use of PA EKV for the measurement of oxygen saturation in the infarcted myocardium.Support or Funding InformationWe acknowledge funding from National Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505. The UNMC Ultrasound Core is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institution of Health under grant P30 GM127200.

Assessment of Oxygen Saturation in Breast Lesions Using Photoacoustic Imaging: Correlation With Benign and Malignant Disease

VisualSonics has recently developed a preclinical photoacoustic (PA) imaging system called the VevoLAZR that combines the sensitivity of optical imaging and the high resolution of micro-ultrasound. The system incorporates a 40 MHz (centre frequency) ultrasound transducer linear array probe (LZ550) and a tuneable 680-970 nm nanosecond pulsed-laser. We used this system to study in vivo changes in tumor oxygen saturation and haemoglobin density caused by exposure to radiation therapy (RT). For this, DsRed-Me180 human cervical tumors were grown in a nude mouse dorsal skinfold window chamber model until they reached 2.5 mm in diameter. Specifically, we investigated the system's sensitivity and dynamic range to measure relative changes in oxygen saturation in tumor and surrounding healthy tissues 10 days after treatment. Tumors (∼2.5 mm diameter) were focally irradiated with a single dose of 30 Gy using a small animal microirradiator (XRAD225, Precision XRay Inc., North Branford, CT). To measure the dynamic range and stability of our setup for measuring oxygen saturation in vivo, we altered the anesthetised animal's inhaled oxygen from 100% to 7% for 1 min during PA imaging. This test showed that blood oxygen saturation in the healthy dorsal skinfold tissue decreased from 82% to 8% and confirmed the linearity of the measurement technique. Furthermore, we compared vascular morphology obtained by photoacoustic imaging and intravital fluorescent microscopy using FITC-Dextran (2 MDa, injected 20 mins prior). This comparison showed good correlation and confirms that PA imaging can provide important structural information of vascularity. Photoacoustic imaging was performed before and 10 days after irradiation to assess changes in tumour volume, relative blood oxygen saturation, relative tissue oxygen saturation, and relative hemoglobin density. Ten days after irradiation, PA imaging showed that the tumour volume increased from 5.7 to 14.2 mm3, relative blood oxygen saturation decreased from 75.3 to 48.2%, relative tissue oxygen saturation decreased from 40.7 to 0.1%, and hemoglobin density decreased from 18457 to 3253 a.u. These data illustrate the capability of PA imaging to simultaneously measure multiple radiobiological response metrics from a single imaging scan. Pilot results demonstrate: i) the compatibility of the VisualSonics small animal VevoLAZR photoacoustic imaging system with intravital murine tumor models, ii) the sensitivity of the system to detect RT-induced changes in tumor vascular oxygen saturation non-invasively, in real time and in vivo, and iii) a new preclinical application of PA imaging for longitudinal monitoring of tumor response to RT in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4338. doi:1538-7445.AM2012-4338

Photoacoustic imaging holds potential in diagnosis and treatment monitoring of breast cancer, but clinical translation of this technology has often been hindered by bulky and expensive excitation sources. In this work, the potential of a portable, dual-mode multispectral LED-based photoacoustic and ultrasound system (AcousticX) in breast imaging is investigated for the first time. The AcousticX system comprises a linear array ultrasound probe (7 MHz) and two dualwavelength LED arrays (750/850 nm) placed on both sides of the probe. Two experiments were performed to investigate the potential of the system in imaging the breast. In the first instance, interleaved photoacoustic and ultrasound imaging was performed on a semi-anthropomorphic multi-layered 3D breast phantom with possibility to tune oxygen saturation in blood vessel structures. In the second experiment, vasculature of a healthy human breast was imaged in vivo. Skin and multiple vascular features along with its relative oxygen saturation are visualized using photoacoustic imaging and the ultrasound images offered valuable structural information including fat, fibroglandular tissue and pectoral muscles. In human in vivo experiments, we achieved an imaging depth of around 1.7 cm at a display frame rate of 10 Hz, the highest in vivo imaging depth reported in LED-based photoacoustic imaging using a 7 MHz probe. With the capability of providing real-time structural and functional information, AcousticX holds clinical translation potential in non-invasive breast imaging.

The tumor vasculature and its hypoxic microenvironment are constantly undergoing changes. These alterations are key attributes associated with aggressive cancer phenotypes, raising the need for non-invasive methods to track these changes. Similarly, in many cases, various cancer treatments also affect tumor vasculature, and preferably – should be monitored. Dynamic contrast-enhance ultrasound (DCEUS) and photoacoustic (PA) imaging are two promising candidates. DCEUS has the ability to measure functional tissue perfusion, whereas multispectral PA imaging can be used to evaluate tissue oxygenation related parameters. This study investigates the relationship between blood perfusion, oxygen saturation levels and hemoglobin concentration in two hind-limb tumor models, and evaluates the ability of these two modalities to image vascular structures and functions. Xenograft tumors were induced in SHO mice using either LS174T human colorectal cancer cells (n = 6), or PC3 human prostate cancer cells (n = 6). Tumors were grown to a depth of 4-6 mm before imaging was performed using a laser integrated high-frequency ultrasound system (Vevo®LAZR, VisualSonics Inc.). Contrast enhanced images were collected after a 50μL bolus injection of MicroMarker ultrasound contrast agents (VisualSonics Inc.) using non-linear contrast imaging. Perfusion parameters were quantified after applying wavelet denoising to the DCEUS clips. PA images were acquired using a 21MHz linear array transducer with fiber optical bundles integrated to each side, used to deliver light from a 680-970 nm tunable laser. Oxygen saturation levels and hemoglobin concentration were estimated from the PA measurements using spectral un-mixing. Tumor vascularity and hypoxia were confirmed with immunohistochemistry staining for CD31 and CA9. Reasonable correlations were found between corresponding pixels in the DCEUS perfusion maps and oxygen saturation maps (R = 0.63 and R = 0.5 for LS174T and PC3 respectively). In contrast, the correlation between blood perfusion and hemoglobin concentration was nil for LS174T tumors (R = -0.1), and low for PC3 tumors (R = 0.34). This discrepancy was explained by the presence of blood pools in LS174T tumors, observed in tumor histology. The presence of hemoglobin inside regions of hemorrhage together with the limited capability to separate hypoxic and necrotic regions, impeded the ability of PA imaging to detect blood vessels inside tumors. Compared to PA imaging, DECUS provides better detection of functional vasculature and enables the visualization of single blood vessels around the tumor core, without including blood pools. This study demonstrates that a multi-modality imaging scheme combining DCEUS and PA imaging can provide both distinctive and complementary information on tumor microenvironment in experimental animal studies. Citation Format: Melissa Yin, Avinoam Bar-Zion, Dan Adam, Stuart Foster. Combined contrast enhanced ultrasound and photoacoustic imaging reveals both functional flow patterns and dysfunctional vascular pooling in tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4197.

Photoacoustic (PA) imaging is an emerging technology that combines structural and functional imaging of tissues using laser and ultrasound energy. We evaluated the ability of PA imaging system to measure real-time systemic and microvascular mean oxygen saturation (mSAO2) in a rat model of hypoxic shock. Male Sprague Dawley rats (n = 6) underwent femoral artery catherization and were subjected to acute hypoxia by lowering the fraction of inspired oxygen (FiO2) from 1.0 to 0.21, and then to 0.08. PA measurements of mSaO2 were taken in the femoral artery near the catheter tip using the Vevo 2100 LAZR at each FiO2 and compared to co-oximetry on blood removed from the femoral catheter. Both co-oximetry and PA imaging measured a similar stepwise decline in femoral artery mSaO2 as FiO2 was lowered. We also measured mSaO2 in the feed arteriole of the rat spinotrapezius muscle and adjacent microvessels (n = 6) using PA imaging. A significant decrease in mSaO2 in both the feed arteriole and adjacent microvessels was recorded as FiO2 was decreased from 1.0 to 0.08. Moreover, we detected a rapid return toward baseline mSaO2 in the feed arteriole and microvessels when FiO2 was increased from 0.08 to 1.0. Thus, PA imaging is noninvasive imaging modality that can accurately measure real-time oxygen saturation in the macro and microcirculation during acute hypoxia. This proof-of-concept study is a first step in establishing PA imaging as an investigational tool in critical illness.

Photoacoustic (PA) imaging of blood flow can provide label free and non-invasive assessment of red blood cell (RBC) aggregation and the oxygen saturation (sO 2 ). Our group has previously demonstrated that the interrelationship between RBC aggregation and the sO 2 during a pulsatile blood flow could be potentially assessed using PA imaging. The pulsatile blood flow yields spatiotemporal changes in RBC aggregation, affecting PA imaging. A simple particle motion model was developed based on the blood flow velocity measured from the human radial artery (RA). The positions of randomly distributed, identical circular particles (2.7 um radius) in the lateral-axial plane (20 mm by 2 mm) were traced at each time step of an experimentally measured velocity profile. At each step, the time dependent PA power $(P_{PA})$ from each single cell (or particles interacting to form aggregates) was computed by modeling and accounting for the directivity of a 21 MHz (9.2 to 32.8 MHz bandwidth) linear array. In-vivo PA images of the RA of healthy volunteers were acquired using the VevoLAZR equipped with a 21 MHz linear-array probe. The measured PA images were compared to the simulated PA images. The aggregates formed a parabolic front along the axial direction and were driven to the right-hand side along the lateral direction as the simulation propagated in time. The $P_{PA}$ was also large at the parabolic front, and was also driven to the right-hand side for every time step. The spatiotemporal distribution of the computed $P_{PA}$ was comparable to the experimental $P_{PA}$. Specifically, the $P_{PA}$ increased by 12 dB along the lateral direction. These results can be used to study the label-free, non-invasive assessment of the spatiotemporal distribution of sO 2 in vivo. Furthermore, the improved particle model can provide insights into the mechanism of PA wave generation from RBC aggregation during in vivo blood flow.

Photoacoustic (PA) imaging is an imaging modality that integrates anatomical, functional, metabolic, and histologic insights. It has been a hot topic of medical research and draws extensive attention. This review aims to explore the applications of PA clinical imaging in human diseases, highlighting recent advancements. A systemic survey of the literature concerning the clinical utility of PA imaging was conducted, with a particular focus on its application in tumors, autoimmune diseases, inflammatory conditions, and endocrine disorders. PA imaging is emerging as a valuable tool for human disease investigation. Information provided by PA imaging can be used for diagnosis, grading, and prognosis in multiple types of tumors including breast tumors, ovarian neoplasms, thyroid nodules, and cutaneous malignancies. PA imaging facilitates the monitoring of disease activity in autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic sclerosis, arteritis, and inflammatory bowel disease by capturing dynamic functional alterations. Furthermore, its unique capability of visualizing vascular structure and oxygenation levels aids in assessing diabetes mellitus comorbidities and thyroid function. Despite extant challenges, PA imaging offers a promising noninvasive tool for precision disease diagnosis, long-term evaluation, and prognosis anticipation, making it a potentially significant imaging modality for clinical practice.

Activities of branched-chain amino acid transaminase were assayed in maternal skeletal muscle, liver and fetal skeletal muscle, cardiac muscle, liver, kidney and placenta obtained from fed and 5-day-fasted late gestation ewes. Very high activities were found in placenta; fetal skeletal muscle also had high activity. Fetal brain had intermediate activity, followed by cardiac muscle and kidney. Fetal liver possessed negligible activity. Activities were low in both maternal liver and skeletal muscle. Trends were seen for fasting to increase activities in fetal placenta, skeletal muscle, brain, kidney, heart and maternal liver, but these changes were statistically significant only for fetal brain and placental tissue. Fetal skeletal muscle activity was 100 times that of maternal skeletal muscle. These data imply differences in the metabolism of the branched-chain amino acids by fetal and adult ruminants and expand the thesis that branched-chain amino acids are important to the metabolism of the ovine fetus.