| Zinc may have an important, causative role in neural injury that occurs in a variety of clinically important central nervous system pathologies, including stroke, epilepsy, and force trauma. The goal of this thesis was to examine the mechanisms by which elevated intracellular free zinc ([Zn2+] i) kills neurons. To this end, we used dissociated cultures of forebrain neurons and mitochondria isolated from rat brain. These models presented simplified and inexpensive systems that allowed us to ask focused questions with minimal interference from confounding factors.;The first phase of this thesis was directed primarily at characterizing Zn2+-induced death in neurons and glia (Chapter 2). We found that neurons were much more sensitive than glia to the toxic effects of elevated [Zn2+]i. Because both cell types saw equal [Zn 2+]i loads, the heightened sensitivity of neurons must be due to some intrinsic vulnerability such as a decreased capacity for buffering and/or extruding excess Zn2+. We then established that elevated [Zn2+]i disrupted neuronal pHi homeostasis (Chapter 3). Increased [Zn2+]i caused a gradual and long-lasting intracellular acidosis. Elevated [Zn2+] i also impaired neuronal recovery from reduced pHi. Disruption of proton homeostasis represents a possible mechanism by which Zn2+ could contribute to neuronal injury. Another set of experiments revealed that pathophysiologically relevant [Zn2+] causes widespread impairment of isolated brain mitochondria (Chapter 4). Neurons are highly active cells and are particularly reliant on mitochondria for energy production. Therefore, Zn2+-mediated mitochondrial dysfunction could have neurotoxic consequences. In a final, related study, we identified important limitations in the current practice of using live-cell fluorescent indicators to determine precise concentrations of intracellular zinc (Appendix).;In conclusion, this thesis identifies several candidate mechanisms by which Zn2+ could contribute to the death of neurons. Additionally, we have established important technical considerations critical for proper usage of Zn2+-sensitive fluorophores in livecell fluorescent microscopy. |
