Michelle LaPlaca - US grants

0093830
LaPlaca
The overall objective of the integrative research and education career plan is to utilize basic engineering principles (cell mechanics and electrical interfacing technology) and biological principles (neurobiology) to optimize neuronal culturing techniques for examining neuropathological conditions and create a interdisciplinary approach to solving problems and advancing the field of neuroengineering. The initial goal of the research component is to further develop three-dimensional (3-D) cultures of the signaling cells of the brain (neurons) by manipulating the cellular support matrix that surrounds the neurons. The optimized 3-D cell cultures will be used to determine how external physical stimuli (from the physiologic to the pathologic/injury range) are transduced from the support matrix to the cellular organelles. In this controlled system, specific mechanisms of mechanically-initiated cell signals will be sorted out in order to develop cellular thresholds and mechanistically-based pharmacologic interventions. In addition, the introduction of 3-D microelectrodes will be incorporated into the cell cultures for purposes of measuring neuronal activity. Educational efforts will be concurrent with the research activities and stem from the research projects. Undergraduate students will be introduced to problem solving and technological approaches in neurobiological interfacing with microsystems through problem-based learning classes. In addition, the PI will expand a graduate course in bioengineering laboratory principles to modules that can be used for undergraduate research experiences and training of high school science teachers.

DESCRIPTION (provided by applicant): Neural transplantation in the injured central nervous system (CNS) has had limited success. It is hypothesized that this hostile environment may require improved cell support through directed extracellular matrix (ECM) protein engineering to improve graft survival and cell function and hence, functional recovery. The overall objective of this research project is to develop minimally-invasive transplantation techniques for optimizing stem/progenitor cell attachment through ECM-based scaffolds and to utilize relevant experimental models (both in vitro and in vivo) for optimization and outcome assessment. This overall goal is divided into 3 interrelated specific aims: (1) To characterize neural stem (NS) cell-ECM-based 3-D constructs in vitro for minimally invasive grafting strategies and maximal cell survival and to elicit a desired degree of proliferation, migration, and differentiation; (2) To determine mechanisms of construct integration by testing NS-ECM constructs in a surrogate hostile in vitro environment; and (3) To analyze the in vivo function of tissue-engineered constructs by transplanting constructs into contused mouse brains and examining post-injury alterations in the host contusion, cell behavior, and cognitive and sensorimotor behavioral outcome. The research proposed is significant because it offers a novel approach to progenitor/stem cell transplant technology with detailed analyses of outcome. This research may have direct application to clinical practice in neurosurgery that would permit therapeutic, cellular replacement in the treatment of traumatic brain and spinal cord injuries and degenerative diseases of the CNS. In addition, this research will provide insight into the mechanisms of CNS regeneration and help to elucidate the necessary cellular environment for neurotransplantation success. By analyzing outcome in well-controlled multi-level systems, this research may also lead to acellular transplantation methodology and establish the requirements necessary for the transplantation of non-embryonic/fetal cell sources.

[unreadable] DESCRIPTION (provided by applicant): Spinal cord injury results in functional deficits that can range from mild to life threatening, but few effective treatments are clinically available. We have observed damage to neuronal cell bodies and axons after traumatic spinal cord injury that is dependent on the severity of the injury. We postulate that this damage is initiated by the mechanical injury and that normal phospholipid homeostasis is disrupted, leading to ongoing damage and ultimately negatively contributes to poor neurological outcome. Our long-term objective is to develop clinically relevant treatments that promote membrane repair following spinal cord injury. The specific objective of this small research program proposal is to assess the effects and mechanisms of plasma membrane repair mediated by citicoline, a membrane stabilizing agent that has been shown to be beneficial in many models of central nervous system disease. The overall hypothesis is that injury-induced membrane damage is detrimental to cell survival and functional outcome, but citicoline treatment can reduce these effects through facilitation of membrane preservation. Specifically, we will use a clinically relevant rodent contusion model to 1) determine a treatment dose of citicoline for spinal cord injury, defined by the ability to reseal compromised plasma membranes in cell bodies and axons and spare tissue, 2) assess possible mechanisms of citicoline-mediated repair, and 3) assess the ability of citicoline to mediate motor recovery. We propose that citicoline leads to membrane resealing by decreasing both lipid peroxidation and phospholipase A2 activity (both of which may contribute to phospholipid breakdown following spinal cord injury) and increasing phospholipid synthesis. These studies are expected to optimize a treatment regimen and begin to determine the mechanisms of membrane damage and repair following spinal cord injury. The proposed studies describe a new research direction and will provide critical data on the effects of targeted membrane repair that will guide future studies. [unreadable] PUBLIC HEALTH RELEVANCE: There is a critical need to develop new treatment strategies for spinal cord injury, which results in functional deficits that can range from mild to life threatening. In this exploratory study, we propose to target cell membrane repair as a possible therapy. We have observed membrane damage after traumatic spinal cord injury and hypothesize that phospholipid breakdown contributes to membrane damage and can be restored by adding citicoline, a membrane stabilizing agent. These studies are directly relevant to public health, as they may lead to novel treatment approaches aimed at neuroprotection for traumatic spinal cord injury and possibly other neurological disorders. [unreadable] [unreadable] [unreadable]

0933506
LaPlaca

The ability to grow and manipulate cells in culture is crucial to understanding basic mechanisms of both normal and disease processes. Three-dimensional thick neural cultures, in particular, mimic brain tissue, but are limited by the lack of a blood supply to deliver nutrients and remove waste. There is a critical need to develop new technology to address this need and produce valid brain tissue models. The intellectual merit of this interdisciplinary research lies in the engineering of a new culture model that includes the major cell types in the brain: neurons, astrocytes, and microglia, together with a highly controllable microfluidic system that perfuses nutrients throughout the culture and permits waste removal and sampling of the cell culture media during periods of both normal conditions and cell injury. Several new innovative elements will be incorporated: 1) include inflammatory cells (microglia) to create a more realistic cell model; 2) introduce a unique, ultrasound-based injury model to produce local injury within the culture; and 3) incorporate microfluidics for perfusion and sampling. Thus, the overall objective is to create a robust and complex neural tissue equivalent that will faithfully represent brain and to investigate the role of microglia following inflammatory triggers. In Task 1, the most appropriate building blocks are chosen to create a novel, complex 3-D neural system for studying inflammation. In addition, microfluidics will be integrated to include perfusion and sampling capabilities. Furthermore, a new traumatic injury model will be developed, providing a means for detailed study of injury mechanisms using highly controllable and tunable methodology. In Task 2, the role of the microglia in the injury response will be tested, as cytokines released from injured microglia are hypothesized to increase cell death. This research is highly significant, as robust culture tools that incorporate multiple cell types and microfluidic perfusion and sampling offer unprecedented levels of spatial and temporal control for determining mechanisms of both normal and injured cells. The broader impact of this research direction will be the development of extremely novel neural tissue equivalents that can be used for numerous applications. It is expected that the next generation of culture systems realized by this approach will revolutionize the way neural cell culturing is done, as the complex interactions among cell types are considered and microcirculation is mimicked through microperfusion. Three-dimensional tissue models with these capabilities will push forward the translation of basic science discoveries for industry, government, and medical breakthroughs. The technical findings will be shared with university, government, and industry researchers with emphasis on collaboration and ultimately having an impact on those affected with traumatic injury or other neurological disorders.

? DESCRIPTION (provided by applicant): A Pre-Clinical Model of TBI Heterogeneity. The majority of traumatic brain injuries are considered mild. Mild TBI (mTBI) is difficult to diagnose, and despite recent progress in public awareness and clinical management, mTBI (or concussion) is a persistent problem in sports, the military, and in the general population. In order to further understand injury mechanisms, identify more sensitive diagnostic tools, and begin to delve into the apparent risk for neurodegeneration, there is a need to improve pre-clinical laboratory studies. This is no easy task given patient heterogeneity and the physiological complexity of TBI. Despite decades of basic research, successful translation of potential treatments from animal to patients is extremely poor. Clinical population heterogeneity is not captured in pre-clinical studies, gravely limiting the ability to validate animal models as reliabl research surrogates. The long-term goal of this research is to improve lab-to-clinic translation using a bi- directional systems approach. The overall goal of this R21 Research Proposal is to reflect the heterogeneity of the mTBI patient population in a pre-clinical animal study and use informatics-based analysis to determine features that contribute to the injury response. The central hypothesis is that systematic institution of animal heterogeneity will result in more reproducible and robust pre- clinical TBI studies and the emergence of clinically relevant risk factors. The experimental aims are: 1) Develop a pre-clinical experimental design for mTBI that selects patient-relevant variables (gender, age, genetic variety, previous mTBI, and chronic stress) and applies them to a heterogeneous rat population using stratified randomization; and, 2) Assess acute neurological response, balance, working memory, and biomarker signature acutely following mTBI using a heterogeneous rat population. Informatics tools will be used in place of traditional multivariate statistics to extract common features of the injury response, classify them, and build predictive knowledge models. Several imbedded hypotheses will be tested to examine the response to mTBI as a function of animal sex, age, strain, previous mTBI, and chronic stress. It is expected that unanticipated relationships among the data will emerge as a result of the robust experimental design and knowledge-based model. The experimental platform presented here is novel and, if successful, can be used as a template for other studies. Addressing patient heterogeneity and clinically relevant acute outcome measures is highly significant and will increase understanding of the complexity of mTBI.

Biopharmaceuticals have revolutionized treatment of cancer, immune, inflammatory and neurological diseases. As these drugs are administered to patients intravenously and then distributed by vasculature to target tissues, their testing in vitro has not been predictive enough. Next, because these drugs are humanized, predicting safety hazards from animal studies has been challenging and volunteers suffered from severe reactions. For these reasons, economics and timeline associated with drug development, biopharmaceutical industry seeks human organ-on-a-chip platforms for more predictive drug testing. Unfortunately, there has not yet been a system that is simple enough for researchers to use, in adequate throughput, and standardized enough to fit into drug screening workflows. The goal of this project is to help solve this problem for researchers by setting a foothold for development of a human organ-on-a-chip platform that is easy for them to use, high-throughput, and in a standard format to readily integrate into their testing routine.

The proposed innovation is a simple tool in a medium throughput, a tool that any researcher can use and be able to answer all the questions that complex commercial perfusion systems do (if all the hardware that services them could be put in one lab and researchers trained on how to use it all). The proposed vascularized organ-on-a-chip platform (PerfusionPal) will be in standard format, high throughput, easy to use and fit into routine pharmaceutical workflows. It will be diagnostic and prognostic; diagnostic to assess target specificity, cross-reactivity with off-target tissues and to identify biomarkers; and prognostic or capable of predicting severe reactions in clinical trials such as cytokine storms, infusion reactions, immune suppression and off-target organ liabilities. PerfusionPal commercialization will triage dangerous drug leads and enable faster and cheaper development of safe drugs from bench to bedside for the benefit of patients, healthcare industry and society.

Project Summary: Lipid Biomarker Efflux from the Brain following TBI, LaPlaca, M.C., Fernández, F.M. Traumatic brain injury (TBI) is a major health problem in the US and worldwide, affecting at least 2.5 million Americans every year. TBI is extremely variable from person to person, making the standardization of injury classification and diagnosis challenging. Biomarkers can potentially provide individualized diagnostic information, yet none are currently approved for clinical use. The objective of the proposed work is to identify novel lipid biomarkers for TBI diagnosis and the routes by which they exit the brain as a function of time and injury severity. The overall hypothesis is that TBI-specific lipids will pass both through the blood brain barrier (BBB) and glymphatic system to the blood post-TBI, and that efflux of biomarkers through different routes will have a variety of post-injury temporal patterns. Lipid biomarkers are promising for several reasons: lipids are abundant in brain, are extremely vulnerable to inflammatory and free radical attack, and may leak out of the brain more readily than proteins, rendering them ideal candidates for peripheral diagnostics. Three mutually- informing aims will be pursued to test the following hypotheses: 1) The absolute magnitude of lipid alterations in brain, blood, cerebrospinal fluid, and lymph will increase with injury severity; 2) More than one route of brain clearance of TBI-generated lipid biomarkers exists and the efflux dynamics depend on lipid size and time post- injury; and 3) Efflux transporters and glymph drainage contribute to post-TBI lipid biomarker efflux in addition to diffusion across brain-to-blood barriers. Preliminary discovery metabolomics data identifying lipids in serum that successfully discriminates between injured and uninjured rats will be expanded to include surveillance in cerebrospinal fluid and lymph out to four weeks post-TBI for mild and moderate TBI in both male and female rats. In addition, different clearance paths will be examined, as efflux routes may change with evolving secondary injury pathology. The route for biomarker clearance from the brain to the blood has been assumed to be primarily via the BBB, however recent identification of dural lymph vessels and a glymphatic route for protein biomarker release after TBI changes the focus from BBB-only transport to one of potentially multiple efflux routes. Given the promise of TBI-specific lipid biomarkers and the unknown dynamics of lipid biomarker release, there is a need to understand lipid clearance routes following TBI. Through this research, we expect to identify novel lipid biomarker panels and determine the major route(s) for their release from the brain. This is significant and novel because, while biomarkers provide a unique window into secondary injury events, changes in efflux patterns directly impact clinical interpretation and implementation.