Cytokine & MMP Production After Laser Irradiation in Responsive vs Non-Responsive Cultured Human TM EC

Abstract

Purpose:: To assess the short-term thermally-mediated response of permeability (Lp) responsive and non-responsive human Trabecular meshwork endothelial cells (TMEC) using control, continuous wave laser irradiated, Micropulse laser irradiated, and slowly heated (water bath) TM cells at 24 and 48 hours by quantifying the expression of IL-1α, IL-1ß, TNF-α, MMP3 and MMP11 using real time RT-PCR.

Methods:: Cultures of human TM cells from three donors were grown, identified, and perfusion tested to determine their permeability responsiveness to laser irradiation. Responsive and non-responsive cells were then seeded into 12-well culture plates. Two wells were harvested for each treatment group for analysis at each time point. Four treatment groups were used: Control, water bath (44^oC for 15 minutes), IRIDEX OcuLight SLX continuous wave diode laser (=810 nm) (1200mW for 1500ms), and IRIDEX Micropulse (2000mW, 2000ms, 50% duty cycle). Before Laser irradiation, the media was changed to PBS (37^oC) to remove the phenol red pigment. The plate to be irradiated is placed on a black background to absorb all laser irradiation that passes through the cells. Sham control cells simply follow the timing and placement of the corresponding experimental group. Cells were harvested at both post-treatment time points and total RNA was extracted from the harvested cells, reverse transcribed and amplified using primers specific to IL-1α, IL-1ß, TNF-α, MMP3 and MMP11.

Results:: Responsive TM cell cultures had an increase in permeability (Lp) of 0.17 µl/min/mm Hg/cm2 while the non-responsive group had a decrease in Lp of -0.057 µl/min/mm Hg/cm². Real time RT-PCR analysis showed a statistically significant negative correlation between thermally-mediated permeability responsiveness and mRNA expression levels of IL-1ß (p=0.0182), MMP3 (p=0.0001), and TNF-α (p=0.0143).

Conclusions:: It was shown that the thermally-mediated induced expression of IL-1ß, TNF-α and MMP3 are statistically different in Lp responsive cultured human TM EC within 24 hours of treatment. These cytokines are known to subsequently stimulate TM cells to increase production of stromelysin and increase the permeability of cultured human Schlemm’s canal EC. These differences may contribute to the clinical responsiveness of Laser Trabeculoplasty.

Keywords: outflow: trabecular meshwork • cytokines/chemokines • laser