Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

<p>Abstract</p> <p>Background</p> <p>Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion.</p> <p>Methods</p> <p>For this investigation we use Second Harmonic Generation (SHG) imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties.</p> <p>Results</p> <p>We find the SHG emission attributes (directionality and relative intensity) and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen.</p> <p>Conclusions</p> <p>Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.</p

Region-Specific Microstructure in the Neonatal Ventricles of a Porcine Model

© 2018, Biomedical Engineering Society. The neonate transitions from placenta-derived oxygen, to supply from the pulmonary system, moments after birth. This requires a series of structural developments to divert more blood through the right heart and onto the lungs, with the tissue quickly remodelling to the changing ventricular workload. In some cases, however, the heart structure does not fully develop causing poor circulation and inefficient oxygenation, which is associated with an increase in mortality and morbidity. This study focuses on developing an enhanced knowledge of the 1-day old heart, quantifying the region-specific microstructural parameters of the tissue. This will enable more accurate mathematical and computational simulations of the young heart. Hearts were dissected from 12, 1-day-old deceased Yorkshire piglets (mass: 2.1–2.4kg, length: 0.38–0.51m), acquired from a breeding farm. Evans blue dye was used to label the heart equator and to demarcate the left and right ventricle free walls. Two hearts were used for three-dimensional diffusion-tensor magnetic resonance imaging, to quantify the fractional anisotropy (FA). The remaining hearts were used for two-photon excited fluorescence and second-harmonic generation microscopy, to quantify the cardiomyocyte and collagen fibril structures within the anterior and posterior aspects of the right and left ventricles. FA varied significantly across both ventricles, with the greatest in the equatorial region, followed by the base and apex. The FA in each right ventricular region was statistically greater than that in the left. Cardiomyocyte and collagen fibre rotation was greatest in the anterior wall of both ventricles, with less dispersion when compared to the posterior walls. In defining these key parameters, this study provides a valuable insight into the 1-day-old heart that will provide a valuable platform for further investigation the normal and abnormal heart using mathematical and computational models

Microscale characterization of prostate biopsies tissues using optical coherence elastography and second harmonic generation imaging

© 2018 USCAP, Inc All rights reserved. Photonics, especially optical coherence elastography (OCE) and second harmonic generation (SHG) imaging are novel high-resolution imaging modalities for characterization of biological tissues. Following our preliminary experience, we hypothesized that OCE and SHG imaging would delineate the microstructure of prostate tissue and aid in distinguishing cancer from the normal benign prostatic tissue. Furthermore, these approaches may assist in characterization of the grade of cancer, as well. In this study, we confirmed a high diagnostic accuracy of OCE and SHG imaging in the detection and characterization of prostate cancer for a large set of biopsy tissues obtained from men suspected to have prostate cancer using transrectal ultrasound (TRUS). The two techniques and methods described here are complementary, one depicts the stiffness of tissues and the other illustrates the orientation of collagen structure around the cancerous lesions. The results showed that stiffness of cancer tissue was ∼57.63% higher than that of benign tissue (Young's modulus of 698.43±125.29 kPa for cancerous tissue vs 443.07±88.95 kPa for benign tissue with OCE. Using histology as a reference standard and 600 kPa as a cut-off threshold, the data analysis showed sensitivity and specificity of 89.6 and 99.8%, respectively. Corresponding positive and negative predictive values were 99.5 and 94.6%, respectively. There was a significant difference noticed in terms of Young's modulus for different Gleason scores estimated by OCE (P-value<0.05). For SHG, distinct patterns of collagen distribution were seen for different Gleason grade disease with computed quantification employing a ratio of anisotropic to isotropic (A:I ratio) and this correlated with disease aggressiveness

Order versus Disorder: in vivo bone formation within osteoconductive scaffolds

In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration

Imaging Immune and Metabolic Cells of Visceral Adipose Tissues with Multimodal Nonlinear Optical Microscopy

Visceral adipose tissue (VAT) inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs) and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO) microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface

In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography, Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model

Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4–8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success

Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds

In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis

Light-evoked Somatosensory Perception of Transgenic Rats That Express Channelrhodopsin-2 in Dorsal Root Ganglion Cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain