Optical scatter imaging of cellular and mitochondrial swelling in brain tissue models of stroke.

摘要

The severity of brain edema resulting from a stroke can determine a patient's survival and the extent of their recovery. Cellular swelling is the microscopic source of a significant part of brain edema. Mitochondrial swelling also appears to be a determining event in the death or survival of the cells that are injured during a stroke. Therapies for reducing brain edema are not effective in many cases and current treatments of stroke do not address mitochondrial swelling at all. This dissertation is motivated by the lack of a complete understanding of cellular swelling resulting from stroke and the lack of a good method to begin to study mitochondrial swelling resulting from stroke in living brain tissue. In this dissertation, a novel method of detecting mitochondrial and cellular swelling in living hippocampal slices is developed and validated. The system is used to obtain spatial and temporal information about cellular and mitochondrial swelling resulting from various models of stroke.; The effect of changes in water content on light scatter and absorption are examined in two models of brain edema. The results of this study demonstrate that optical techniques can be used to detect changes in water content.; Mie scatter theory, the theoretical basis of the dual-angle scatter ratio imaging system, is presented. Computer simulations based on Mie scatter theory are used to determine the optimal angles for imaging. A detailed account of the early systems is presented to explain the motivations for the system design, especially polarization, wavelength and light path. Mitochondrial sized latex particles are used to determine the system response to changes in scattering particle size and concentration.; The dual-angle scatter ratio imaging system is used to distinguish between osmotic and excitotoxic models of stroke injury. Such distinction cannot be achieved using the current techniques to study cellular swelling in hippocampal slices. The change in the scatter ratio is then shown to correlate to mitochondrial swelling, as observed with electron microscopy. The system is finally used to study mitochondrial and cellular swelling. Evidence of the susceptibility of certain hippocampal regions, CA1 and the dentate gyrus, to exhibit mitochondrial swelling as the result of oxygen and glucose deprivation is presented. In addition, for the first time, the time course of mitochondrial swelling is seen.; Finally, experiments with scatter imaging and measurement of nitric oxide with carbon fiber electrodes demonstrate a clear link between nitric oxide and cellular swelling. A potential mechanism of the action of nitric oxide is evaluated. Nitric oxide appears to act to cause cellular swelling without the release of glutamate. The use of targeted nitric oxide inhibitors may be useful for the reduction of edema.