Iratxe Torre

An experimental model of fetal growth restriction based on selective ligature of uteroplacental vessels in the pregnant rabbit.

Introduction: To describe an animal model of growth restriction based on selective ligature of uteroplacental vessels in the pregnant rabbit. Material and Methods: Two experimental protocols (+21 and +25 days of gestation) with three groups were defined: controls, mild (20–30%) and severe (40–50%) uteroplacental vessel ligature. Fetuses were delivered 120 h after the procedure by cesarean section. Biometrical measurements were carried out. Brains were obtained and glial response and cell proliferation were studied by S100β and Ki-67 immunohistochemistry. Results: Mortality rate and biometrical restriction increased across experimental groups according to the time and severity of the procedure. S100β expression was significantly higher in the severe reduction group at 25 days. Ki-67 expression was significantly higher in the mild reduction group at 21 days and in the severe reduction group at 25 days. Discussion: Selective ligature of uteroplacental vessels in the pregnant rabbit results in a gradual model of growth restriction in terms of mortality, biometrical restriction and histological brain changes.

Permanent Cardiac Sarcomere Changes in a Rabbit Model of Intrauterine Growth Restriction

Background Intrauterine growth restriction (IUGR) induces fetal cardiac remodelling and dysfunction, which persists postnatally and may explain the link between low birth weight and increased cardiovascular mortality in adulthood. However, the cellular and molecular bases for these changes are still not well understood. We tested the hypothesis that IUGR is associated with structural and functional gene expression changes in the fetal sarcomere cytoarchitecture, which remain present in adulthood. Methods and Results IUGR was induced in New Zealand pregnant rabbits by selective ligation of the utero-placental vessels. Fetal echocardiography demonstrated more globular hearts and signs of cardiac dysfunction in IUGR. Second harmonic generation microscopy (SHGM) showed shorter sarcomere length and shorter A-band and thick-thin filament interaction lengths, that were already present in utero and persisted at 70 postnatal days (adulthood). Sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR fetal hearts. Conclusion The results suggest that IUGR induces cardiac dysfunction and permanent changes on the sarcomere.

Intrauterine growth restriction is associated with cardiac ultrastructural and gene expression changes related to the energetic metabolism in a rabbit model.

Intrauterine growth restriction (IUGR) affects 7-10% of pregnancies and is associated with cardiovascular remodeling and dysfunction, which persists into adulthood. The underlying subcellular remodeling and cardiovascular programming events are still poorly documented. Cardiac muscle is central in the fetal adaptive mechanism to IUGR given its high energetic demands. The energetic homeostasis depends on the correct interaction of several molecular pathways and the adequate arrangement of intracellular energetic units (ICEUs), where mitochondria interact with the contractile machinery and the main cardiac ATPases to enable a quick and efficient energy transfer. We studied subcellular cardiac adaptations to IUGR in an experimental rabbit model. We evaluated the ultrastructure of ICEUs with transmission electron microscopy and observed an altered spatial arrangement in IUGR, with significant increases in cytosolic space between mitochondria and myofilaments. A global decrease of mitochondrial density was also observed. In addition, we conducted a global gene expression profile by advanced bioinformatics tools to assess the expression of genes involved in the cardiomyocyte energetic metabolism and identified four gene modules with a coordinated over-representation in IUGR: oxygen homeostasis (GO: 0032364), mitochondrial respiratory chain complex I (GO:0005747), oxidative phosphorylation (GO: 0006119), and NADH dehydrogenase activity (GO:0003954). These findings might contribute to changes in energetic homeostasis in IUGR. The potential persistence and role of these changes in long-term cardiovascular programming deserves further investigation.

Automated cardiac sarcomere analysis from second harmonic generation images

Abstract. Automatic quantification of cardiac muscle properties in tissue sections might provide important information related to different types of diseases. Second harmonic generation (SHG) imaging provides a stain-free microscopy approach to image cardiac fibers that, combined with our methodology of the automated measurement of the ultrastructure of muscle fibers, computes a reliable set of quantitative image features (sarcomere length, A-band length, thick–thin interaction length, and fiber orientation). We evaluated the performance of our methodology in computer-generated muscle fibers modeling some artifacts that are present during the image acquisition. Then, we also evaluated it by comparing it to manual measurements in SHG images from cardiac tissue of fetal and adult rabbits. The results showed a good performance of our methodology at high signal-to-noise ratio of 20 dB. We conclude that our automated measurements enable reliable characterization of cardiac fiber tissues to systematically study cardiac tissue in a wide range of conditions.

Automated morphometric characterization of cardiac fibers by second harmonic microscopy imaging

Cardiovascular diseases are one of the main causes of mortality in the world. For this reason it is important to develop techniques that allow for better understanding of cardiac remodelling prior to heart failure. In this line, Second Harmonic Imaging (SHI) provides a label-free microscopy approach to image cardiac fibers. In this work, we automated means for measuring average sarcomere length, intrasarcomeric A-band distance and sarcomere fiber density as such features have been repeatedly correlated with major cardiomyopathies in the literature (previously measured by manual means). We tested our approach on healthy cardiac tissue of rabbits and human samples and compared such results with manual measurements on the same images to validate our methods. We also measured morphometric differences in cardiac fibers from our animal model which undergoes an irregular heart development. We then conclude that our automated measurements enable reliable characterization of cardiac fiber tissues.

Callibration procedure and characterization of a commercial multiphoton microscope to measure Polarization Second Harmonic Generation microscopy

Polarization Second Harmonic Generation enables to quantify some of the underlying molecular details of key biological structures within muscle, axons and collagen at suboptical scales. However, it is only available in dedicated labs. In this work we detail a procedure to adapt and calibrate a commercial multiphoton microscope (Leica TCS-SP5) to acquire PSHG images reliably. For this, two different procedures were developed: (1) using inexpensive starch granules as polarization probe to estimate incident laser polarization within a region, and (2) using a fluorophore in solution to estimate the incident intensity arriving to each pixel of the image. This works might help to broaden the users of PSHG acquisition in microscopy as well as to provide the elements to correct for experimental errors.

OC26.03: Bioinformatic analysis of genes regulating myocardiocyte contractile function in a rabbit model of cardiac dysfunction due to intrauterine growth restriction

Objectives: Non invasive measurement of fetal vascular flow remains an elusive matter, accuracy of current tools is very limited and relies mainly in operator skills. The aim of our study was to develop a tool for indirect flow measurement, accurate and repeatable, by using power Doppler signal, Real time three dimensional ultrasound (4DPD) and mathematical image data simulation and adjustment tools based on artificial neural networks (ANN). Methods: Six pregnant sheep with adequately controlled gestational age of 125 days (near term) were surgically instrumented to access fetal ascending aorta by transventricular catheterization. Cardiac output was measured by Fick thermodilution, as well as by pulsed Doppler. Several sets of 4DPD volumes were taken during the procedure. Measurements were stored in an electronic datasheet. Pearson’s correlation coefficient and simple linear regression were obtained. Linear equation matrix were generated and obtained data was evaluated through an error adjustment process by employing an artificial neural network software (ANN). Results: A total of 30 sets of measurements in controlled conditions were collected during the study period. A mean of six measurements by cardiac cycle were digitally obtained from the velocity curve. A six by six matrix of data was designed for every measurement. Mean velocity at every time was calculated and compared to actual data, intraclass correlation coefficient (95% CI) was 0.9 (0.73–0.99). ANN predicted calculated measurements in 99% of cases. Conclusions: 4DPD might be a reliable, accurate, non invasive tool for fetal vascular flow measurement.

Contrast enhancement in second harmonic imaging: discriminating between muscle and collagen

In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For the whole image we retrieve for every pixel, the effective orientation, θe , of the SHG active structures. As a result a new image is formed which its contrast depends on the values of θe . Collagen presented in the forward direction for a predefined region of interest (ROI), peak distribution of angles θe centered in the region of ~45°, while muscle in the region of ~65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any exogenous labeling or any co-localization with fluorescence imaging.

Myosin helical pitch angle as a quantitative imaging biomarker for characterization of cardiac programming in fetal growth restriction measured by polarization second harmonic microscopy

Fetal growth restriction (FGR) has recently shown a strong association with cardiac programming which predisposes to cardiovascular mortality in adulthood. Polarization Second Harmonic Microscopy can quantify molecular architecture changes with high sensitivity in cardiac myofibrils. In this work, we use myosin helical pitch angle as an example to quantify such alterations related to this high risk population. Importantly, this shows a potential use of the technique as an early diagnostic tool and an alternative method to understand pathophysiological processes.