Susan Sheps Margulies, Ph.D. - US grants

9702088 Margulies The primary objective of this integrated research and educational plan is to develop research and educational programs that span the interdisciplinary chasm between the level of the cell and the organ, with an emphasis on the effects of applied physical forces on function and structure. The research plan is a first step towards the long-term goals of understanding the deformations of the epithelial lining of the lung in vivo, and determining the effects of these deformations on the lung's adverse effects. Barotrauma, or the presence of extra-alveolar air after rupture of over-distended alveoli, is the consequence of a sustained mechanical ventilation with large tidal volumes. The overall hypothesis is that the alveolar epithelial lining of the lung is vulnerable to injury from large static and dynamic deformations during mechanical ventilation. The research plan relates mechanical deformation to disruption of the epithelial surfactant-producing and barrier properties of the lung using in vitro cell culture studies, mathematical models, and in vivo experimental studies. The long-term goal of the educational component of the career development plan is to create Bioengineering graduate courses that integrate fundamental engineering theories with biomedical applications. Three courses will be developed: 1) a course in integrated cardiothoracic physiology that brings together basic knowledge and recent research findings from the molecular, cellular and organ levels to correlate cellular and molecular mechanisms with macroscopic functional responses; 2) a course in viscoelastic tissue behavior and large strain measurement techniques and analysis; and 3) experimental design and analysis, and scientific responsibility. The dual mission of the integrated research and educational career development plan is to meet the growing need for research and educational opportunities that cut across disciplines to answer fundamental scientific questions. ***

DESCRIPTION (provided by applicant): The broad long-term objective of our research is to reduce the incidence of ventilator-induced lung injury (VILI), characterized by pneumothorax, progressive impairment in pulmonary mechanics, alveolar cell dysfunction, and profound changes in lung fluid balance and blood-gas barrier permeability. In the previous funding periods we identified cyclic and tonic deformation magnitudes above which we measured acute alterations in epithelial cell viability and barrier properties. Moreover, the injurious deformation magnitudes we applied in vitro correlated well with tidal volumes and positive end-expiratory pressures used clinically that have been associated with clinical morbidity in ventilator induced lung injury (VILI). These findings underscore the potential relevance of using experimental models with carefully controlled conditions to identify the mechanisms responsible for stretch-induced disruption of the alveolar epithelial barrier, and to identify opportunities for injury intervention. The in vitro model for VILI that we established during the first 6 years of funding is limited to healthy rat alveolar epithelial cell monolayers exposed to 1 hr periods of deformation. In this competitive renewal our major objective is to determine specific stretch-induced mechanical and molecular signals that modulate alveolar epithelial permeability during clinically relevant conditions - including chronic continuous cycling and ARDS. In Aim 1 we hypothesize that the environmental stressors of sepsis, high cycling rates and long stretch durations each compromise epithelial monolayer barrier properties in both primary cell monolayers and intact lungs. In Aim 2 we hypothesize that stretch induces barrier alterations by activation of mitogen- activated protein kinases, which initiate nuclear factor kappa B dependent cytokine expression and release to ultimately increase monolayer permeability. In Aim 3 we will test the hypotheses that acute (<1 hr) stretch reorganizes the cytoskeleton, which increases monolayer permeability directly by dissociating tight junctional (TJ) proteins from actin, and indirectly by initiating kinase-mediated TJ phosphorylation and TJ complex disassembly. Thus, we build on the foundation established during previous funding periods - both to deepen our understanding of basic mechanisms and to enhance the clinical translation of our findings - and progress towards our goal of reducing acute VILI due to epithelial over-distension.

Funds are requested for the purchase of a multi-photon microscope to be conveniently located near two of the major Penn Institutes whose faculty will share the equipment. Some 14 users from the School of Engineering and Applied Science, the School of Medicine, and several campus institutes will use the equipment for biomedical and biomaterial research investigations. The new equipment offers significant advantages over existing equipment in imaging penetration depth, sensitivity, photodamage, and the ability to observe multiple fluorophores simultaneously. Specific studies include the effect of environmental stimuli on cells, the microscopic basis of biomaterial behavior, polymer behavior at interfaces, etc. The facility will be made available to researchers at nearby Universities.

DESCRIPTION (provided by applicant): Traumatic brain injury is the most common cause of death in childhood, yet etiology and treatment of pediatric head injuries remain controversial. Previously, we established an interdisciplinary research paradigm using acute animal experiments, biomechanical tissue tests, retrospective clinical studies, anthropomorphic "doll" studies, and computational simulations. Our data show that children do indeed have injury mechanisms that are distinct from the adult, and require age-specific injury prevention strategies and treatments. In this competitive renewal, we build on the foundation we have established, both to deepen our understanding of basic injury mechanisms and to enhance the clinical relevance of our findings. We will supplement our current platforms with novel preparations - survival studies, porcine behavioral outcomes, cerebral blood flow (CBF) measures, post-injury respiratory insufficiency, and injury treatment studies - to enhance translation of research findings from the laboratory to the clinical setting. Our overall hypothesis is that rapid rotations of the immature brain without impact produce brain injury via both mechanical and biochemical signals, resulting in sustained functional and histological abnormalities. We will compare long-term outcomes after single and multiple head injuries to determine if injury interval modulates injury severity and if axonal injury is reduced with folate supplementation (Aim 1). We will use human computational models to extend our animal studies to cyclic shaking motions to estimate contribution of harmonic amplification to injury risk (Aim 2). Using animal experiments and computer models, we will identify cerebral strains associated with rapid regional decreases in CBF and brainstem deformations associated with loss of cerebral autoregulation (Aim 3). We will modulate regional deformations by altering rotation direction, and endothelial response with hypertonic saline to validate acute mechanical and biochemical signaling pathways. In piglets with respiratory insufficiency after head injury (Aim 4), we hypothesize that resuscitation with 100% FiO2 results in exacerbated neuropathology mediated by free radical release, and we will compare outcomes with room air and 100% FiO2, as well as with and without free radical scavengers to verify functionality. The proposed studies address our long-term goal of elucidating injury mechanisms and potential treatment strategies for traumatic brain injuries in children.

DESCRIPTION (provided by applicant): PROJECT ABSTRACT/SUMMARY Traumatic brain injury (TBI) is the most common cause of death in childhood. The predominant etiologies of TBI in young children are motor vehicle accidents, firearm incidents, falls, and child abuse. Unfortunately, falls are the leading cause of non-inflicted head injury in infants less than a year old, and are also the most common history provided by caretakers suspected of child abuse. Some cases are easy to diagnose, but in cases of uncertainty accurate diagnosis of inflicted trauma is hindered by a lack of clarity regarding specific head injury mechanisms for young infants. The objective of this research proposal is to provide clinicians with a biomechanics-based tool to aide in the diagnosis of inflicted or non-inflicted trauma with a history of a low- height fall. In Aim 1, biomechanical tolerances of extra-axial hemorrhage (subdural and subarachnoid hemorrhage) are determined by using a porcine computational model to simulate non-impact rapid head rotation experiments in 3-5 day old piglets. Mechanical responses from the model (cortical displacement relative to skull and peak cortical tissue strain) are statistically correlated with the actual occurrence of extra- axial hemorrhage (EAH) on the piglets'cortex to identify biomechanical tolerances associated with 10, 50 and 90 percent probability of EAH. To validate this tolerance and determine probability thresholds with the greatest specificity and sensitivity to predicting EAH in cases of well-witnessed falls, a human infant computational model is developed in Aim 2 to predict the brain and skull response to impact. Simulating well-witnessed cases of falls in infants, the biomechanical tolerance for EAH in Aim 1 and a biomechanical tolerance for skull fracture previously published from our lab are used to determine the probability of skull fracture and EAH most predictive of the injuries in each of the case simulations. To measure loads in common household fall settings, a 1 1/2 month old human infant biofidelic surrogate is used in Aim 3 to recreate falls from 1, 2, and 3 feet onto carpet, concrete, hardwood, tile, and linoleum to simulate common household fall settings. Impact force and angular accelerations for ten repetitions of each height, surface, and primary head impact location (parietal or occipital) are obtained, creating a load corridor for each fall scenario. Combining the biomechanical loads from the surrogate experiments, existing pediatric large animal TBI data, and a new biofidelic computational model of the brain and skull of a human infant, a predictive tool to is created in Aim 4 to determine the plausibility of skull fracture and extra-axial hemorrhages (EAH) in infants following low height falls. Validated with real-word clinical data, this biomechanical data will advance the understanding of injury thresholds in common non- inflicted scenarios that will ultimately improve the accuracy in detection of inflicted and non-inflicted head trauma. National objectives from Healthy People 2010 calls for a reduction in child maltreatment, a reduction in fatalities caused by child maltreatment, a reduction in unintentional injury and a reduction in deaths from falls. Developing a clinical tool that is biomechanically sound and informs clinical practice will directly contribute to the fulfillment of these national health and welfare priorities. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Our proposed research plan will advance the understanding of injury thresholds in common non-inflicted scenarios that will ultimately improve the accuracy in detection of inflicted and non-inflicted head trauma. There is no topic greater than pediatric abusive head trauma that evokes controversial discussion focusing on the emotional, medical, and legal aspects of misdiagnosis. Our plan is consistent with the national objectives from Healthy People 2010, designed to contribute to a reduction in child maltreatment, a reduction in fatalities caused by child maltreatment, a reduction in unintentional injury, and a reduction in deaths from falls.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Traumatic brain injury (TBI) is a leading cause of childhood morbidity and mortality in the United States, with an annual rate of approximately 200 per 100,000 children requiring hospitalization. The increasing monetary costs result not only from the acute intensive care for these patients but also from long-term care that is often necessary. Brain injuries in these patients result from both the primary trauma and secondary insults such as reductions in cerebral blood flow, derangements in cerebral metabolism, and the ensuing ischemic injuries that continue to progress well after the traumatic event. Optimal therapeutic efficacy thus requires knowledge of cerebral blood flow, oxygen supply, and oxygen consumption in patients after TBI.

DESCRIPTION (provided by applicant): We propose to use our immature large animal models of traumatic brain injury (TBI) to accelerate basic science therapeutic discoveries to clinical trials for TBI in children. Because mitochondria play a key role in many primary and secondary pathologic pathways in TBI, we will use cyclosporin A (CsA) to rescue mitochondrial function, reduce cell death and improve neurofunction. Due to its safety profile in humans, pleiotropic effects, and success in multiple preclinical adult rodent TBI models, CsA (CsA) has exciting potential as a therapy for pediatric TBI. Because CsA is also off-patent and already in use in children for other indications, the results of the proposed preclinical therapy development plan can be translated rapidly to clinical trial. We determine the optimal dose of CsA for the spectrum of moderate TBI in the child, using our established immature porcine TBI models: focal lesions from controlled cortical impact and diffuse brain injury from rapid nonimpact head rotation. In addition, we include optimization at 1 hr after TBI to determine dosing strategies in the field, and at 6 hrs after TBI for hospital-based strategies. To enhance translation, we include clinically relevant physiological monitoring and current critical care management strategies. Furthermore, we use both histological and neurofunctional endpoints to identify agents that reduce brain injury acutely, and have sustained cognitive benefits. In Aim 1, we will evaluate short-term dose response using short term terminal outcomes. For each start time and injury type, the most effective and the lowest dose with significant effect will continue to Aim 2. In Aim 2 we test these dosing strategies for efficacy in neurocognitive outcomes, measured 6 days after injury, to identify the optimal dosing strategy to evaluate in pediatric TBI clinical trials. In Aim 3 we will identify sex-specific cognitive recovery and toxicology responses to the optimal dose of CsA. In Aim 4 we design the clinical trial from the porcine data and published human studies, and submit an IND application to the FDA. This state-of-the-art, innovative preclinical study design can be applied to future evaluations of other therapies longer treatment windows, other ages, other brain injuries, and to other agents that promote neurorecovery or repair.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is the most common cause of death in childhood. Extra-axial hemorrhage (EAH, which includes subdural and subarachnoid hemorrhage) carries additional medical-legal significance, as it is often used to distinguish between accidental and abusive injury etiologies in young children. Although EAH is generally thought to occur from tearing of bridging veins (BVs) and/or cortical vessels, there is a paucity of data about the age dependence of BV properties, as well as the relationship between number of vessels ruptured and extent of EAH. In this proposal to investigate TBI mechanisms specific to young children, our hypotheses are that rapid rotations of the immature head can produce BV failure, that the biomechanical BV properties of the infant will reveal lower BV rupture stress and strain than the adult, conferring a vulnerability for BV rupture to the infant. n addition, we hypothesize that cyclic elongation will soften the BVs, rendering them more fragile with repeated head rotations than single events. With expertise in biomechanical testing and finite element analysis, and archived data from patient studies, animal experiments and anthropomorphic doll simulations of accidental and abusive TBI events, we are uniquely poised to determine the mechanisms associated with EAH in TBI. In this exploratory R21 grant application, our 2-year goal is to leverage these previous discoveries with new experiments to identify the mechanisms of traumatic EAH in children. In just two years, we will be the first to determine mechanical properties of pediatric BVs (Aim 1); integrate them into computational finite element models (FEMs) for EAH prediction, and validate the predictive capabilities of our procedures (Aim 2); and use the FEMs to identify EAH potential in single and cyclic head movements (Aim 3). Taken together, the results of this basic research plan will provide the scientific foundation for the development of new strategies for injury prevention, clinical diagnosis, and injury mitigation to reduce the number of children with new debilitating TBIs each year.

NRT-IGE: Penn Pathfinders

The United States has led scientific advancement and education in biomedical science and engineering for the past 50 years empowered by a stellar scientific workforce. As non-traditional career paths continue to expand, the majority of science and engineering PhD graduates increasingly choose careers outside academia, such as teaching, entrepreneurship, policy, and research in industry or government. However, most graduate programs train students for academic careers, thereby failing to adequately acquaint students with alternative career paths, or provide them with the basic tools and experiences to succeed in multiple professional sectors. This National Science Foundation Research Traineeship (NRT) award in the Innovations of Graduate Education (IGE) Track to the University of Pennsylvania (Penn Pathfinders) will focus on a pilot training program that will provide trainees in biomedical sciences and engineering with career development opportunities and investigate its effectiveness in comparison to two non-trainee cohorts. The pilot training program appropriately targets preparing graduate students for a wider range of career pathways through professional skills development and increased awareness of and preparation for non-academic career pathways.

The objectives of this innovative testbed project are to foster career path awareness via multiple platforms, to provide longitudinal professional path mentoring, and to devise and enact individualized development plans for acquiring workplace skills and career path-specific competencies. The pilot will involve a carefully selected diverse cohort to be trained in making informed decisions about career paths and in acquiring the essential competencies to be successful in pursuing their career objectives. Data will be collected on trainee demographic profiles and inclusion, program participation, program quality, satisfaction, time-to-degree, and effectiveness of program content in enhancing career development-ultimately via longitudinal career tracking. Using comprehensive comparisons between trainees and non-trainees, this three-year pilot program will be considered successful if significant differences are identified between cohorts in improving the awareness of and satisfaction with preparation for their preferred career path, with the ultimate goal of enhancing the broad impact and engagement of scientists and engineers in society. The critical elements that contribute significantly to improved career path awareness and preparedness will be identified and these features will be incorporated into larger-scale graduate student training programs at Penn and other institutions. The study outcomes will be disseminated in both local and global settings to inform and improve career advising of biomedical scientists and engineers.

The NSF Research Traineeship (NRT) Program is designed to encourage the development and implementation of bold, new, potentially transformative, and scalable models for STEM graduate education training. The Innovations in Graduate Education Track is dedicated solely to piloting, testing, and evaluating novel, innovative, and potentially transformative approaches to graduate education.

SUMMARY In the US, there are more than four sports-related traumatic brain injuries every minute. Sports-related concussion (SRC) in youth has received heightened attention due to emerging evidence that SRCs can affect academics, behavior, and neurocognitive processes, such as working memory, concentration, processing speed, and eye and motor function. A recent Institute of Medicine report on SRCs in youth revealed how little is known about concussion in the young brain, and called for urgent attention to determine the incidence of SRCs in boys and girls by sport and demographic; research to identify unbiased, sensitive prognostic and diagnostic metrics/markers; longitudinal studies to determine outcomes; and to delineate age- and sex-related biomechanical determinants of injury risk. This innovative hypothesis-driven Bioengineering Research Grant will generate objective diagnostic tools for use in concussion (Aim 1), new technologies to translate human outcome metrics to animals to provide a human-like platform to develop and test injury treatments in the future (Aim 2), and new knowledge regarding high-risk sports settings for youth (Aim 3) that will drive safety equipment design. The most innovative feature of the study is the integration across Aims to use BOTH male and female high school students and piglets in a deliberately parallel study design to determine optimal SRC assessments and identify mechanistic relationships between sex, loading conditions, and SRC symptoms. The integration of human and animal studies which employ similar neuro-functional assessments leverages the strengths of each approach: human studies ensure the study of biofidelic physiologic processes, and animal studies allow application of specific loading conditions and outcomes not easily measured in living humans, such as neuropathology. Extensive pilot data establish feasibility and sample sizes in all Aims. In Aim 1 an unbiased numerical assessment suite for SRC will be developed and independently validated to establish ?95% sensitivity, and determine if these metrics are predictive of days-to-clearance for sports. Because the Aim 1 objective metrics are nonverbal and effort-independent, they have been ?translated? to animals and reveal human-like physical, cognitive, and sleep symptoms of SRC in animals after rapid controlled head rotations. In Aim 2, single head rotations and multiple sub-concussive rotations are computationally scaled from teens to an immature large animal model of mild TBI, to identify the effects of sex and load frequency, magnitude and direction on neuro-function, biomarkers and neuropathology. Aim 2 will identify biomechanical settings of greatest risk for the young brain. The biomechanical insights from pigs in Aim 2 are translated to teens in Aim 3, where head impact sensors are used to quantify biomechanical load exposure by sport and sex, and the relationships between load exposure and neuro-functional metrics. The proposed studies in animals and humans will have broad impact ? by reducing the healthcare burden of SRC, enhancing accurate and objective diagnosis, and identifying gender-specific prevention and intervention strategies.