Kirill V. Larin, Ph.D. - US grants

DESCRIPTION (provided by applicant): Abnormalities in the development of the cardiovascular system are among the most common congenital human birth defects. The mouse has proven to be an excellent model to study aberrant cardiovascular development, with the range of phenotypes observed in the mouse rivaling those encountered in humans. In many cases it is clear that complex phenotypes arise because impaired function causes an array of secondary defects that mask the original defect. Better tools are needed to characterize mutations that primarily affect cardiovascular function at early time points so primary defects in function are not missed. Here we will develop tools for using Optical Coherence Tomography as a routine method for studying cardiovascular abnormalities in mouse embryos. This method has a higher spatial resolution than currently used Ultrasound methods. The goal of this study is to show that OCT can be used to obtain sensitive measurements of heart function and blood flow, rivaling those that we have obtained in very early embryos using high-speed confocal microscopy. Our long term goal is to develop turn-key OCT systems for the routine phenotyping of mutant mice produced by large-scale screens. PUBLIC HEALTH RELEVANCE: 7. Project Narrative This proposal will develop novel technology and methods to study congenital birth defects in mouse models. Approximately 35,000 children are born each year with a congenital defect in the cardiovascular system and this is the number one cause of birth-defect related deaths. Although there has been much progress in defining genes that are required for normal cardiovascular development, we need new methods to understand how mutations in these genes cause specific alterations in cardiac and endothelial cell morphogenesis that result in birth defects.

PROJECT SUMMARY The fundamental physical properties of the outer tunic of the eye determine the structural characteristics of the ocular globe and may be altered in several devastating disease states including axial elongation in myopia, pathological deformation in keratoconus, and iatrogenic keratoectasia following corneal refractive surgery. These biomechanical tissue characteristics not only influence our clinical interpretation of diagnostic tests, e.g. measurement of intraocular pressure, but have been implicated as important factors in the development of glaucoma. Currently, there is no available reliable method to perform quantitative measurement of corneal elasticity in vivo. Here we will develop novel method for the assessment of corneal elastic properties that could potentially be used for routine clinical diagnostic and treatment. This method will take advantages of highly localized air pressure stimulation and ultra-sensitive detection and analysis of the pressure waves propagation on corneal posterior and anterior surfaces with a line-field Optical Coherence Tomography to reconstruct volumetric biomechanical properties of the cornea. Our previous work has made fundamental advances in the understanding of corneal biomechanics through a novel approach with potentially impactful applications in other disciplines (e.g. cataract surgery, LAISK, corneal cross-linking, and tissue transplants with personalize treatments). The proposed studies will accelerate transition of this technology into clinics, influence our selection and application of corneal surgical treatments and will help us to understand the structural consequences of corneal disease and wound healing: Aim 1. Develop a line-field OCE (LF-OCE) system for ultrafast 3D clinical imaging. Aim 2. In vivo studies with rabbits. Aim 3. Preliminary clinical studies in humans. Aim 4. Refine numerical (FEM) and Artificial Intelligence (AI) models of the depth-dependent nonlinear viscoelastic properties of the cornea.

? DESCRIPTION (provided by applicant): The overall objective of this study is to develop an optical coherence tomography (OCT) based high- resolution mouse embryonic brain imaging and analysis approach, and to use this method in correlation with molecular analysis to understand the interplay between ethanol (EtOH) and nicotine (NIC) effects on embryonic brain development. Maternal exposures to these substances are linked to fetal growth retardation and neurotoxicity, and these toxins often co-abused during pregnancy. However, studies on the combined fetal effects of NIC and EtOH are very limited and their combined effects on molecular mechanisms of fetal development, particularly the brain, are poorly understood. Therefore, there is a critical need to understand the interplay between the effects of EtOH and NIC via development of high-resolution imaging technique capable of live longitudinal analysis of developing brain. Intriguingly, our recent studies suggested that EtOH and NIC exert mutually antagonistic effects on fetal neuronal stem cells development. In this proposal, we will investigate if these toxins have indeed antagonistic or synergetic effects on embryonic brain development in live mouse embryos with implementation of an innovative higher-resolution embryonic brain imaging and dynamic quantitative analysis approaches, which we develop. We have pioneered OCT-based methodology for live in utero imaging and longitudinal phenotypic analysis of mouse fetuses in utero. Here we propose to further develop both the technology and the methodology for longitudinal brain imaging and analysis. The study is focused on the second trimester- equivalent period of development, when the neuronal stem cells give rise to most of the neurons of the adult brain. The central hypothesis of this proposal is that EtOH and NIC have partially antagonistic effects in fetal brain development. By successful accomplishment of the proposal, we will establish a live mouse embryonic brain imaging approach, will develop a set of protocols and detailed assessments to quantitatively characterize dynamic embryonic brain development with cellular resolution, and will investigate if EtOH and NIC synergize to disrupt the brain development or exhibit partially antagonistic effects. We will also assess the feasibility of using nicotinic receptor antagonists and agonists t prevent the individual and combined effects of EtOH and NIC. Therefore, studying this effect is highly significant from both fundamental biology and teratogenic points of view since it may have particular significant impact for the development of novel and innovative therapies for reversing teratology.

This R01 application will investigate a novel signaling pathway, the Hippo pathway, in mammalian heart regeneration. The long-term goal is to develop new treatments for patients with heart failure by generating new cardiomyocytes in the adult heart. The objectives of this application are to gain insight into biomechanical properties of cardiac tissue that are regulated by Hippo signaling. The central hypothesis is that Hippo signaling is a negative regulatory pathway that prevents cardiomyocyte regeneration in the adult mammalian heart by promoting a more fetal-like elasticity profile of cardiac muscle. The specific aims are to define the Hippo regulated biomechanical properties and cardiac aging, to determine dynamic changes in biomechanical properties after myocardial infarction, and to determine the functional characteristics of regenerated cardiomyocytes in Hippo mutant hearts. The project is both conceptually and technically innovative. The concepts to be tested are new ideas in cardiomyocyte biology and new and cutting edge imaging technologies are used to address hypotheses. The significance is high because there are no treatments for heart failure due to cardiomyocyte loss. Devising ways to generate new cardiomyocytes is highly significant.

PROJECT ABSTRACT (Reduced to fit in < 30 lines) The objective of this proposal is to develop a non-contact, all-optical imaging technology to map elastic moduli and forces involved in critical aspects of embryonic development with high 3D resolution. The proposed technology is based on combined Brillouin spectroscopy and Optical Coherence Tomography (OCT), which will be used to gain fundamental understanding of biomechanical factors involved during neural tube closure (NTC) in normal and pathological cases using established and well validated murine neural tube defect (NTD) models. NTDs are the second most common structural birth defect in humans, affecting upwards of 500,000 pregnancies worldwide and ~ 2400 pregnancies each year in the United States alone. NTC comprises a complex series of processes that involve tissue motion, thus are driven by forces. However, the biophysics of NTC, namely the interplay between tissue forces and stiffness, remains poorly understood, mostly because of sub-optimal measurement techniques. In the past few years, our groups have developed advanced imaging technologies; OCT for structural/functional imaging of developing embryos and Brillouin microscopy for mechanical mapping of tissues, that, when combined, can be transformative to elucidate the biomechanics underlying the development of NTDs. Our long-term goal is to elucidate how mechanical properties controlling NTC in developing embryos can be manipulated to ensure proper neural development in at risk embryos. Our central hypothesis is that failure of NTC leading to NTDs in genetically predisposed embryos is mediated by mechanical alterations and abnormal forces at the edge of the fusing neural folds that can be imaged with Brillouin-OCT multimodality. To test this central hypothesis, our objective is to combine OCT, Brillouin microscopy and analytical modeling to establish a platform technology to map elastic moduli and forces in developing mouse embryos. The research premise of filling a significant data gap in our understanding of NTC biomechanics is supported by strong preliminary data. The proposal is developed with high research rigor: our Aim 1 will focus on the advanced development of Brillouin microscopy to measure live embryonic tissue. A combined Brillouin/OCT instrument will be developed and tested in Aim 2. Finally, in Aim 3 we will test the hypothesis that mechanical properties and forces critically mediate genetically predisposed or teratogen-induced NTDs. To accomplish our objective, we have assembled a multidisciplinary team with expertise in OCT (Larin), Brillouin technology (Scarcelli), biomechanical modeling (Aglyamov), and developmental biology and NTD disorders (Finnell). The successful completion of the proposed research program will produce a unique platform technology, which will enable studies where a mechanical phenotype is correlated with gene and protein expression profiles developed globally, in order to provide mechanistic understanding of the entire developmental spectrum of events leading to NTDs and potentially other complex congenital malformations.

Abstract Systemic sclerosis (SSc-scleroderma) is an autoimmune disease leading to widespread fibrosis in skin and internal organs. Skin involvement is a prominent source of distress and morbidity in this disease. The modified Rodnan skin score (mRSS) as the current gold standard for assessment of skin thickening has limited accuracy, high inter-observer variability, requires extensive training which have cumulatively contributed to the fact there are currently no FDA approved medications for skin involvement in SSc. Therefore, an objective and accurate tool for assessment of skin fibrosis can be paradigm shifting for clinical trial design and patient management in SSc by improving our ability to track treatment response and disease progression. Our preliminary data indicate that the imaging by optical coherence tomography (OCT) and elasticity measurement by optical coherence elastography (OCE) can provide a safe, rapid, and accurate assessment of SSc skin fibrosis. Specifically, OCT/OCE is the first objective dermal assessment method showing criterion validity and outperforming mRSS for correlation with the histological dermal thickness in the forearm area. As the OCE primarily assesses tissue stiffness, its performance is weaker in some other body areas such as fingers, where the skin can be tethered to the underlying tissue. On the other hand, an improved high-resolution OCT technology with accompanying optical density measurement that can image deeper layers of dermis would provide an additional objective assessment of dermal fibrosis. This is especially relevant in SSc as fibrosis primarily occurs in deeper layers of dermis (reticular dermis). Our hypothesis is that an improved OCT based structural imaging that can capture the deeper dermal layers in combination with the OCE based stiffness assessment will improve our ability to accurately measure dermal fibrosis in SSc. The primary goal of this project to develop and validate a deep OCT/OCT based tool for assessment of SSc skin thickness. To accomplish this goal, the following Specific Aims will be pursued during the R61 phase: Aim 1: To enhance signal-to-noise ratio (up to 120 dB) and imaging depth (up to 2 mm) of the currently available OCE/OCT system for more accurate assessment of SSc skin. Aim 2: To determine the accuracy of combined OCE and deep dermal OCT imaging for assessment of skin fibrosis and for monitoring response to treatment in bleomycin induced dermal fibrosis mouse model. Aim 3: To characterize the accuracy and reliability of a combination of deep OCT/OCE for assessing skin fibrosis in SSc patients. Building on the above studies, we will characterize the longitudinal changes in the OCE/OCT assessment of skin fibrosis and determine its sensitivity to change in SSc patients during the R33 phase. This project can lead to development of a safe and quantitative tool for objective assessment of dermal fibrosis which can be paradigm shifting by facilitating approval of novel treatments for SSc skin involvement. Moreover, it can aid clinicians to accurately track disease progression and response to treatment and ultimately lead to improved monitoring and treatment strategies in this potentially devastating disease.

Prenatal alcohol exposure (PAE) is an established cause of brain-based disability, though co- exposure to other drugs, i.e., poly-substance use, can exacerbate adverse PAE-associated infant outcomes. The practice of Simultaneous Alcohol and Cannabinoid (SAC) use, i.e., co- ingestion, is an emerging trend among adolescents and adults of child-bearing age. SAC is motivated and maintained because combined use of alcohol and cannabinoids amplifies each drug?s psychological effect. Cannabinoids are known contributors to teratogenicity. However, we know virtually nothing about potential consequences of SAC for birth outcomes. The premise of the proposed studies is informed by the published literature, which indicates that PAE effects are, in part, mediated by activation of cannabinoid receptor signaling pathways and that PAE and prenatal cannabinoids engender similar fetal developmental outcomes. Moreover, recently published data shows developmental synergy between sub-teratogenic doses of cannabinoids and ethanol in non-mammalian vertebrate models. Based on these, as well as our own preliminary data we plan to address two questions: Firstly, ?is SAC more damaging to fetal development than either alcohol or cannabinoids alone??; Secondly, and importantly, ?will cannabinoid antagonists protect against effects of PAE & SAC??. Our studies will focus on the effects of SAC on neurogenesis and vasculogenesis, the complementary growth of vasculature that supports fetal brain growth. We plan to use in vivo and ex vivo mouse PAE models in combination with molecular assays and behavioral assays for hyperactivity and conditioned place preference, as well as state-of-the-art optical imaging (optical coherence tomography and light-sheet microscopy) and high-resolution ultrasound imaging, to assess the effects of SAC on, (Aim #1) brain and behavior, and (Aim #2) on vasculogenesis and cerebrovascular blood flow. Our overarching goal, to identify and minimize the contribution of factors, including poly-drug use, that contribute to increased risk for brain disabilities due to PAE, is consistent with the goals of the Collaborative Research on Addiction at NIH (CRAN) initiative. The rapid spread of recreational cannabis use means that SAC is an important emerging mode of drug consumption, and a potential contributor to the severity of PAE effects. As an outcome of these studies, we will acquire evidence to guide human studies on SAC birth outcomes, and to assess the efficacy of novel pharmacological intervention strategies targeted to cannabinoid receptors as a means to prevent or reverse effects of PAE.

PROJECT ABSTRACT Presbyopia, the progressive age-related loss of near visual function, is associated with a stiffening of the crystalline lens. There are currently several investigational approaches for presbyopia treatment that rely on lens softening or lens replacement with softer materials. Lens softening approaches are expected to have a transformative impact on the field because they are non-invasive and they preserve the anatomical relationship between the lens and other tissues involved in accommodation. They have therefore the potential to restore the natural dynamic accommodative function. However, one of the fundamental roadblocks towards the development of lens softening procedures is that there is currently no method available to directly measure lens stiffness and thus assess the efficacy of lens softening procedures in vivo. The goal of the project is to develop new technology capable of precise spatially-resolved non-destructive, noninvasive and depth-resolved quantitative measurements of the lens mechanical properties in a clinical setting. The technology will combine Brillouin microscopy, Optical Coherence Tomography (OCT), and Optical Coherence Elastography (OCE) - BOE. The instrument will be used to generate the first age-dependent data on lens mechanical properties quantified in vivo as well as quantitatively assess therapeutic procedures aimed to restore accommodation. Our overall hypothesis is that the novel BOE technology can acquire absolute measurements of the lens stiffness gradient with the accuracy and precision required to detect both age-related changes and changes induced by lens softening treatments. The ability to quantify lens softening in vivo will have a major impact on pre-clinical and clinical testing, validation and optimization of lens softening procedures. The project has three specific aims: Aim 1: Develop a combined BOE imaging device for depth-resolved quantitative lens elastography. Aim 2: Validate BOE measurements in animal and human lens ex vivo and animal lens in vivo. Aim 3: Quantify the mechanical properties of the human lens in vivo To accomplish our objective, we have assembled a multidisciplinary team with expertise in optical coherence tomography and elastography (Larin), Brillouin technology (Scarcelli), biomechanical modeling (Aglyamov), clinical ophthalmic instrumentation and crystalline lens physiology (Manns, Parel, Ruggeri, Yoo).