Beverly A. Rzigalinski - US grants

DESCRIPTION (adapted from applicant's abstract): Traumatic brain injury (TBI) induces activation of microglia, the immune effector cells of the brain. The role of microglia in TBI is one of double-edged sword, having both neuroprotective and neurodegenerative effects. Therefore, a treatment goal would be to minimize negative, neurotoxic microglial actions while maximizing neuroprotection. Dissection of microglial interactions with other brain cell types and identification of biochemical pathways involved in microglial activation after TBI is difficult to accomplish in vivo. Many of the current models of in vitro microglial injury do not reproduce the major component of TBI, that being tissue strain or stretch. The goal of this proposal is to examine the signal transduction pathways responsible for TBI-induced microglial activation and determine the consequences of activation on neuronal injury, using a well-characterized in vitro stretch-injury model. Our recent studies demonstrate that microglia are not directly activated by stretch injury, but are activated by soluble factors released from stretch-injured astrocytes. Activated microglia had morphological alterations, increased arachidonic acid release, and enhanced intracellular calcium signaling consistent with macrophage activation. For the first time, we have identified upregulation of a glutamate-mediated calcium-signaling pathway in activated microglia, which may constitute a signaling network between microglia and injured astrocytes and neurons. We have also found that stretch-injured astrocytes and severely injured neurons release ATP into the extracellular space. We hypothesize that glutamate and ATP released by traumatically injured astrocytes and neurons initiates microglial chemotaxis activation. In the present proposal, we will continue to dissect the signaling pathways involved in stretch-induced microglial activation by examining arachidonic acid release, glutamate-mediated calcium signaling, morphological changes, proliferation, major histocompatibility antigen expression, phagocytosis, and chemotaxis. We will also determine the mechanisms through which stretch-activated microglia alter neuronal calcium signaling or exert neurotoxic effects. Using inhibitors of the arachidonic acid cascade, calcium signaling, purinergic and glutamatergic receptors, we will attempt to counteract microglial neurotoxicity. In vitro dissection of the mechanisms involved in the neuroprotection and neurotoxicity of microglia after trauma could permit the development of more effective strategies aimed at reduction of secondary injury after TBI by shifting the balance of microglial effects from neurotoxic to neuroprotective.

DESCRIPTION (provided by applicant): The field of engineering has made substantial advances in nanotechnology, particularly in materials science and the molecular construction of nanoscale devices. In this proposal, the PI (Rzigalinski) and co-PI (Seal) have merged the studies of cell biology and nanoscale materials science to intervene in a common biomedical pathology, that being free radical cell damage and aging. We have engineered cerium oxide nanoparticles, 2-20 nm, by a novel non-agglomeration modified sol-gel process and assessed the activity of these particles in a tissue culture model using rat brain cells. Our preliminary data suggests that cerium oxide nanoparticles prolong brain cell longevity in culture, by 2-3 fold. Aged neurons in nanoparticle treated cultures maintained functional synaptic connections and had normal intracellular calcium signaling. Further, cerium oxide nanoparticles reduced hydrogen peroxide (H2O2) and UV light - induced cell injury by over 65%. We hypothesize that the unique structure of cerium oxide nanoparticles, with respect to valence structure and oxygen defects, promotes cell longevity and decreases toxic insults by scavenging free radicals. In this proposal we will synthesize and further characterize the physical and chemical properties of cerium oxide nanoparticles, and determine their behavior in physiologically relevant fluids and in the intracellular environment. We will further dissect the role of nanoparticles in cell longevity and determine their mechanism of action. Using microarray technology, alterations in gene transcription in control and nanoparticle treated cells will be during their lifespan. Lastly we will examine the ability of cerium oxide nanoparticles to increase longevity in the fruit fly. These studies will provide substantial insight into the pathology of aging and age-associated disorders and initiate a nanotechnological approach to pharmacotherapy.

DESCRIPTION (provided by applicant): Free radicals are thought to play a key role in the pathophysiology of traumatic brain injury (TBI). The production of free radicals and the ensuing state of oxidative stress may contribute to the chemical destruction of neuronal membranes, damage to intracellular constituents and ion channels, and apoptotic processes. The reduction of free radical activity thus remains an important avenue of treatment for TBI, yet traditional free radical scavengers may be limited by poor brain penetration, extensive dosing requirements, or both. However, new developments in the field of nanomedicine may provide treatment options not possible with traditional pharmacological approaches. This proposal is designed to determine if cerium oxide nanoparticles (CeONP) improve functional outcome and reduce oxidative stress following TBI. Recent studies suggest that CeONP are highly efficient free radical scavengers with excellent brain penetration, and CeONP have been shown to prevent neurodegeneration in response to several tissue culture models of oxidative stress. Moreover, the physicochemical properties of CeONP suggest that they are regenerative free radical scavengers that, unlike traditional antioxidants, require limited dosing. Motivated by these recent findings, we reasoned that the application of nanomedicine to the treatment of TBI would represent a novel therapeutic approach that offers unique possibilities not available with traditional pharmacological approaches. One hypothesis of this proposal is that TBI induces the production of damaging free radicals that overwhelm innate cellular defenses, resulting in a state of oxidative stress. Oxidative stress in turn results in neuronal death or dysfunction via several different pathways, ultimately resulting in poor functional outcome. Thus, the central hypothesis of this proposal is that reducing free radical activity and damage with administration of CeONP has the potential to improve functional outcome following TBI. Our preliminary studies indicate that CeONP are neuroprotective in an in vitro model of TBI. Furthermore, our preliminary work indicates that pre-injury administration of CeONP improves functional outcome following experimental TBI in rats. Thus, the specific aims of this proposal are: 1) to test the hypothesis that post-injury administration of CeONP improves functional outcome following TBI by reducing free radical damage, and 2) to test the hypothesis that delayed, post-injury administration of CeONP improves functional outcome following TBI by reducing free radical damage. The long-term objectives of these studies are to expand our knowledge of the role of free radicals in the pathophysiology of TBI, provide information on the role of oxidative stress in functional outcome following TBI, and provide novel information on the potential application of nanomedicine to the treatment of brain injury and other disease conditions involving oxidative stress. PUBLIC HEALTH RELEVANCE: Traumatic brain injury is a leading cause of morbidity and mortality throughout the world, yet there is currently no accepted treatment for TBI. Thus, we believe that studies designed to investigate novel therapeutic treatments for TBI are consistent with the mission of the NIH. We expect that our findings will increase understanding of the role of free radicals in the pathophysiology of TBI and their role in poor functional outcome. Furthermore, because numerous disease states are associated with free radical production and oxidative stress, we expect that our findings will be of interest to the broad community of neuroscientists.