Traumatic brain injury (TBI) is a major health and socioeconomic concern in the United States and across the globe. Experimental models of TBI are used to study the mechanisms underlying cell dysfunction and death that result from injury, the functional deficits that result from injury, and the potential of various therapies to treat injury. This thesis explores the fundamental mechanical damage associated with brain trauma, investigating the effects of mechanical deformation on neurons at the molecular, cellular, tissue, and animal levels. First, a novel hydrogel system was developed to support 3-D neuronal cultures, and the cultures were studied in an in vitro model of neuronal injury. The dependence of cell viability on hydrogel stiffness and extracellular matrix ligand concentration revealed a role for molecular interactions in the cellular response to injury. Subsequently, in a rat model of TBI neuronal plasma membrane damage was observed coincidentally with cell death within the hippocampus; however not all permeable cells died, suggesting a complex role for plasma membrane damage in neuronal degeneration. The spatial profile of permeable cells in the hippocampus reveals further heterogeneity of neuronal plasma membrane damage, with populations of cells in certain hippocampal subregions exhibiting an increased vulnerability to plasma membrane damage. These observations support recent finite element model predictions of strains in the brain during injury. Finally a system for measuring locomotor disturbances is used for the first time following brain injury. Continued investigation of how neurons deform and fail mechanically will contribute to the understanding of the pathophysiology of brain injury and may help identify potential therapeutic targets.

Traumatic brain injury (TBI) affects 1.4 million people a year in the United States alone and despite the fact that 96% of people survive a TBI, the health and socioeconomic consequences can be grave, partially due to the fact that very few clinical treatments are available to reduce the damage and subsequent dysfunction following TBI. To better understand the various mechanical, electrical, and chemical events during neural injury, and to elucidate specific cellular events and mechanisms that result in cell dysfunction and death, new high-throughput models are needed to recreate the environmental conditions during injury. This thesis project focuses on the creation of a novel and clinically relevant single-cell injury model of traumatic brain injury (TBI). The implementation of the model requires the development of a novel injury device that allows specialized micro-interfacing functionality with neural micro environments, which includes the induction of prescribed strains and strain rates onto neural tissue, such as groups of cells, individual cells, and cell processes. The device consists of a high-resolution micro-electro-mechanical-system (MEMS) microtweezer microactuator tool that is introducible into both biological and aqueous environments and can be proximally positioned to specific targets in neural tissue and neural culture systems. This microtweezer, which is constructed using traditional photolithography and micromachining processes, is controllable by a custom developed software-automated controller that incorporates a high precision linear actuator and utilizes a luer-based microtool docking interface. The injury studies will include examination of intracellular calcium concentration over the injury time course to evaluate neuronal plasma membrane permeability, which is a significant contributor to secondary injury cascades following initial mechanical insult. Mechanical strain and strain rate input tolerance criteria will also be used to determined thresholds for cellular dysfunction and death.

Biomarker research is of great interest in the field of traumatic brain injury (TBI), since there are numerous potential markers that may indicate central nervous system damage, yet the brain is normally well isolated and discovery is at its infancy. Traditional methods for biomarker discovery include time consuming multi step chromatographic mass spectrometery (MS) techniques or pre-defined serial probing using traditional assays, making the identification of biomarker panels limiting and expensive. These shortfalls have motivated the development of a MS based probe that can be embedded into 3D neural cultures and obtain temporal and spatial information about the release of biomarkers. Using the high sensitivity MS ionization method of nano-electrospray ionization (nano-ESI) with an in-line microdialysis (MD) unit allows us to use MS to analyze low concentrations of TBI biomarkers from within cell cultures with no need for off-line sample manipulation. This thesis goes through the development of the probe by studying the theoretical principles, simulations and experimental results of the probe's capability to sample small local concentrations of a marker within cell culture matrix, the MD unit's sample manipulation capabilities, and the ability to detect markers using in-line MD-nano-ESI MS.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Liver transplantation is the best therapy for patients with end-stage liver diseases and unresectable early hepatocellular carcinoma (HCC). Living donor liver transplantation (LDLT) has been successfully implemented as an alternative to deceased donor liver transplantation (DDLT) and likewise offers comparable excellent survival rate. However, the inferior post-transplant oncological outcomes are found in LDLT recipients with HCC. The liver grafts used in LDLT are usually small-for-size and less effective in coping with shear stress from transient portal hypertension, which results in small-for-size liver graft injury. Acute phase small-for-size liver graft injury may promote late phase tumor recurrence, whereas the underlying mechanism remains unclear. CXCL10, an inflammatory chemokine, initiates liver inflammatory response during hepatic ischemia-reperfusion (IR) injury and may link acute phase small-for-size liver graft injury and late phase tumor recurrence, yet the precise mechanisms remain elusive. Endothelial progenitor cells (EPCs) participate in tissue repair for graft recovery and also provide an angiogenic environment for tumor growth. Myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) can suppress the activation of the immune system and play a critical role in graft rejection and cancer development. We here established the rat orthotopic liver transplantation with whole graft or small-for-size graft model to study the impact of acute phase small-for-size liver graft injury on the mobilization of EPCs, MDSCs and Tregs, and intragraft CXCL10 and its receptor, CXCR3,gene expressions. We further subjected CXCL10-/-mice and CXCR3-/-mice to hepatic IR injury and major hepatectomy to study the role of CXCL10/CXCR3 signaling on the mobilization of EPCs, MDSCs and Tregs. We also investigated the effect of CXCL10 on EPC migration and tube formation in vitroas well as intratumoral microvessel density (MVD) in the rat liver transplantation with tumor growth model and EPCs on tumor growth in nude mice. Key findings: 1. Liver transplantation with small-for-size graft resulted in severe intragraft vascular injury and higher CXCL10 andCXCR3 gene expressions as well as more EPC, MDSC and Treg cell mobilizationin circulation than whole graft. 2. CXCL10-/-mice and CXCR3-/-mice had less circulating EPCs, MDSCs and Tregs than WT mice after hepatic IR injury and major hepatectomy. 3. CXCL10 recruited EPCs in dose-dependent and CXCR3-dependent manners and promoted EPC tube formation in vitro. 4. Higher intratumoral MVD was observed in small-for-size graft than in whole graft in liver transplantation with tumor growth model. 5. Tumor grew more quickly by combining EPC infusionin nude mouse orthotopic liver tumor model. In conclusion, acute phase small-for-size liver graft injury significantly mobilizes EPCs, MDSCs and Tregs after transplantation through CXCL10/CXCR3 signaling. More EPC mobilization and intragraft differentiation after transplantation with small-for-size liver graft may be related to higher intratumoral MVD in small-for-size liver graft after transplantation with tumor development. Therefore, targeting at post-transplant CXCL10/CXCR3 signaling may not only attenuate early phase liver graft injury but also prevent late phase tumor recurrence. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Orthopaedic Surgery / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

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