Rapid and delayed death of cultured trabecular meshwork cells after selective laser trabeculoplasty

Abstract

<h4>Background and objective</h4>Selective laser trabeculoplasty (SLT) is becoming increasingly employed to reduce elevated intraocular pressure in glaucoma patients. SLT is known to target the ocular trabecular meshwork (TM), but the exact response mechanisms to this treatment have not been clearly delineated. The aim of the present study, therefore, was to investigate the modes of death of cultured bovine TM cells subjected to SLT in vitro.<h4>Materials and methods</h4>Bovine TM cell cultures were established, pigmented with exogenous melanin and irradiated with a Q-switched, frequency doubled, Nd:YAG laser, at different energy settings (0.05-1.0 mJ). Influences on cells were determined for up to 10 days post-treatment by trypan blue exclusion, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and by morphological assessment. Furthermore, homogeneous mixtures of pigmented and non-pigmented TM cells were irradiated to ascertain selectivity of laser effects.<h4>Results</h4>At higher energy levels (1.0, 0.75 mJ), immediate loss of cells was detected at the irradiated site. Trypan blue exclusion analysis showed that necrotic cell death subsequently occurred up to 8 hours following irradiation, peaking at 60 minutes. This was followed by delayed cell death peripheral to the irradiated area which was characteristic of apoptosis and which peaked at 2-3 days post-treatment. When mixed cultures were tested, laser treatment selectively killed pigmented cells at an energy level equivalent to the lower cell killing threshold in the initial studies (0.2 mJ) but at the higher laser energy of 0.35 mJ, all cells were non-selectively killed.<h4>Conclusions</h4>SLT treatment killed pigmented TM cells in culture by a variety of processes (instant vaporization, rapid necrosis, delayed apoptosis), depending on the magnitude of the energy used and the distance from the center of the irradiated zone. These data may assist in the elucidation of the mechanism of action of the SLT procedure on TM cells in situ.