Equipment to Support Advance Research in Basic Mechanism of Oxidative Stress
Abstract
We propose to purchase a Seahorse XFe96 instrument to enhance our ONR-funded research and support advanced research into the basic mechanism of cell bioenergy fluctuation in Noise Induced Hearing Loss (NIHL). This system integrates our ongoing studies utilizing the normoxia, hypoxia and NIR platforms. The requested system will enable us to effectively test the numerous responsive pathways under investigation in the metabolic response of cells to oxidative stress and parse the key regulatory components driving NIHL susceptibility. Proteomic research currently underway linking metabolism, endocytosis and molecular transport within the cell will be fundamentally enhanced by accelerated throughput and improved resolving power of the requested instrumentation. Additionally, integrated injection ports, sequentially delivering up to 4 compounds will allow dose response, agonist or antagonist and pathway perturbation analysis in real time, enabling us to understand the energy balance in the control of transport of molecules into the cells, paving the way to drug delivery manipulations. Our overall goal of the research is to investigate hypoxia toxicity in cell metabolism and release of exosome metabolic protein biomarkers within cochlear cell microenvironments. Ongoing research conducting metabolic and proteomic NIHL evaluation of hypoxic cellular response under oxidative and inflammatory stress triggered by hypoxia and ototoxic factors relevant for the microenvironment of the inner ear will benefit from this instrument. We investigate the release of exosomes using hypoxia or gentamicin-treated cells and study exosomal proteomic content in relation to metabolic proteins as potential biomarkers for cell therapeutic intervention. Bioenergetic manipulation associated with proteome analysis of cochlear pericyte-derived exosomes in normoxic and hypoxic conditions are then revealed. The hypoxic response of the cochlear microvasculature has been shown to potentiate NIHL. Harmful noise causes transient blood stop inducing ischemia/reperfusion damages through oxidative stress, free radical formation and hypoxia. In order to respond to decreased O2 availability mitochondria switch from respiration to glycolysis. Long periods of hypoxia induce turnover in cytochrome C, with implications for the metabolism and bioenergetic balance of cells. Pathological changes in the cell and mitochondria, and increase in mitochondrial free radical leaking ultimately induce damage and cell death. Our results show that in conditions of intermittent hypoxia, pericytes of the blood brain barrier of the inner ear microvasculature undergo a significant decrease in ATP production in treated cells. In hypoxic conditions, ATP reduction is exacerbated at 24 hours by simultaneous treatment with gentamicin. Furthermore, our data show that total cell number did not decrease after 24 hours of gentamicin and hypoxia treatment but the number of released exosomes did. We have found fewer exosomes recovered after gentamycin treatment or after hypoxia, relative to their respective controls. Changes in cell metabolism and bioenergy may play a role in this shift. Exosomes are capable of instigating cell analogous response in target cells. Treatment with exosomes obtained from metabolically healthy cells may provide promising alternative for inner ear cells protection, metabolic stabilization of bioenergetic pathways and even regeneration. This Seahorse instrument will allow us to measure oxygen consumption at the cellular level in context with exosomes under normoxic and hypoxic conditions. This system will complement the already acquired Hypoxia chamber Xvivo system (Biospherix) operationally active in our research facility which is seeking a non-toxic, enabling treatment strategy suitable to cross the blood brain barrier to protect and increase the endurance of the cochlea against NIHL experienced by military personnel.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 10, 2018
- Source ID
- N000141812234
Entities
People
- Rick Rogers
Organizations
- Office of Naval Research
- President and Fellows of Harvard College
- United States Navy