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B. Kim, D. M. Grzybowski, A. M. Mahmoud, P. A. Weber, C. Roberts; Heat Shock Protein Expression Following Micropulse and Continuous Wave Diode Laser Irradiation of Cultured Human Trabecular Meshwork Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1632.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the perfusion response and associated expression of heat shock proteins in trabecular meshwork (TM) endothelial cells exposed to both micropulse and continuous wave diode laser irradiation.
The cultured TM cell monolayers on filter inserts were placed in a specialized perfusion apparatus at a pressure of 5mm Hg. In a steady-state condition, the entire monolayer was irradiated under one of two energy delivery profiles: continuous wave (CW) diode laser (n= 3) at 0.4286J/cm2 (1200mW and 1.5s exposure) and micropulse (MP) diode laser (n= 3) at 2000mW and 2.0s (50% duty cycle). Data collection started 15 minutes prior to irradiation, and continued for 1hour post irradiation. Each irradiated monolayer was run simultaneously with a non-irradiated (Control) monolayer under the same conditions. Post-perfusion viability tests were performed. To investigate cellular mechanisms, cultured TM cells were subjected to one of three "heat treating" conditions, plus a non-treated Control: CW (n=3) and MP irradiation (n=3), both using the same parameters as the perfusion studies, as well as a water bath (WB) at 44oC for 15minutes (n=3). After heat treatment or Control, proteins were extracted at 1, 3, 6, 9, 12 and 24 hours in 37.5oC post incubation. The expression levels of heat shock protein (HSP) 27, which is a major protein to prevent apoptosis and protein aggregation and maintain thermo tolerance, were detected by western blot.
MP irradiation produced a statistically significant 12% increase in hydraulic conductivity compared to Controls, at 1 hour post-irradiation and CW irradiation produced a statistically significant 25% increase relative to Controls (p <0.05). HSP27 was significantly increased after WB at 9, 12, and 24 hours post treatment, and after MP irradiation at 24 hours post treatment (p <0.05). Although HSP27 showed an increasing trend after CW irradiation, it was not statistically significant due to variability. Controls did not show a statistical change from baseline at all time points.
Laser irradiation produces rapid, nonlethal heating on a short time scale (seconds), compared to a WB which produces slow heating on a much longer time scale (minutes). Yet, HSP expression occurred after both forms of "heat treatment". The expression of HSPs was associated with an increase in hydraulic conductivity of perfused TM cell monolayers, and may be involved in the increase in flow facility after laser trabeculoplasty.
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