(E-190) Glucose levels affect neuronal calcium response to laser-induced shockwave

Introduction:: Low and high glucose mediums can affect neuronal cells differently. Hyperglycemia and hypoglycemia demonstrate drastic fluctuations of glucose within the human body. Non-physiological high levels of glucose are shown to induce changes in neuronal viability and cause neuronal death by disturbing regular glutamate transmission. We utilize a laser microscope system to produce a laser-induced shockwave (LIS) within the dish to investigate the calcium response between cortical cells in low and high glucose mediums. The system continuously monitors the changing levels of calcium within each cortical cell before and after the LIS. LIS is used to simulate traumatic brain injury. By causing an immediate disturbance of neuronal activity, we are able to study how cortical cells react differently based on the medium they are in. The results of this investigation allow us to analyze the change in amplitude, full width half max (FWHM), and mean rise rates of calcium within a cell according to distance of the cell from the shockwave.


Materials and Methods:: Cortical cells from p0 mice were dissected according to established protocols. Cells were grown in a medium containing high glucose(25mM) or low glucose(3mM). After 4 days in vitro, cells were loaded with the calcium indicator Fluo-4 AM 40 minutes prior to experiments. To produce a LIS a single pulse from a nanosecond 1030 nm laser was focused through a 40x, 1.3 NA oil immersion objective (Zeiss). Images of the dish were taken every 3 seconds over a time period of 30 minutes. After collecting the photos, we measured the intensities and distances through a self-coded program run through ImageJ. The range of interest (ROI) of the selected cells within each FOV was then analyzed through a Calcium Analysis GUI. The amplitude, FWHM, and mean rise rates of each FOV were organized separately and then the distances of each cell within an FOV were compared to their own amplitude, FWHM, and mean rise rates in PRISM.


Results, Conclusions, and Discussions:: Our results show that cells in low glucose had a greater calcium transient amplitude. However, the duration of the elevation(FWHM) and rise rate were statistically insignificant when compared to high glucose. Interestingly, there was a correlation between distance and amplitude, FWHM, and mean rise rate in high glucose but no correlation in low glucose. In conclusion, glucose levels affect the sensitivity of neurons to shockwave distance, where cells in high glucose have a different response to shockwave according to distance but cells in low glucose do not.


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