An 810-nm diode laser (Iridex Corporation, Mountain View, CA) with a TTT adaptor installed on a slit lamp (model 900; Haag-Streit) was used in a continuous mode. Rats were anesthetized with the same cocktail solution and the coverslip was placed on the cornea with methylcellulose. After that, TTT was performed on the right eyes. A laser beam was focused to the center of the optic nerve head with a spot diameter of 0.5 mm. The gross examination of the optic nerve head was performed before, during, and after the treatment with a slit lamp biomicroscope and a coverslip mounted on the cornea with methylcellulose.
First, various exposure powers (60, 80, 100, 120, 140, 160, 180, and 200 mW) were used with the same exposure duration of 60 seconds. This first step was performed to determine the optimal laser power necessary to induce Hsp70 without tissue damage. The left eyes were used as the control. Immunohistochemical staining was performed to determine Hsp70 expression. In addition, confocal scanning laser ophthalmoscopy (Heidelberg Retina Tomograph; Heidelberg Engineering, Heidelberg, Germany) and scanning electron microscopy (SEM) were performed to evaluate the optic nerve head structure. These two experiments were performed in the selected power group (100, 120, and 140 mW), in which Hsp70 was induced without gross damage of the optic nerve head tissue. Based on the optimal power level determined by the first experiment, various exposure durations of 1, 2, 3, and 5 minutes, respectively, were examined in the second step. Furthermore, immunohistochemical staining and Western blot were performed during the second step of the experiment.