Abstract
Purpose :
Damage to trabecular meshwork (TM) cells seen in glaucoma, is mainly the result of oxidative stress, and causes high intraocular pressure, and ultimately damage to the optic nerve. Reducing damage to the TM is therefore important in preventing ocular hypertension and glaucoma. We hypothesize that opioids will have protective effects on normal human TM (NTM-5) cells exposed to an experimental model of ischemic stress, known to trigger oxidative responses and lead to cell death.
Methods :
Immunocytochemical techniques using antibodies directed against kappa and delta opioid receptors (KOR, DOR) were utilized to determine receptor localization to NTM-5 cells. Receptor protein expression was determined by Western blot. An oxygen/glucose deprivation (OGD) model was used to simulate ischemic conditions. Cytotoxicity and cell viability were evaluated via lactate dehydrogenase (LDH) assay and Guava Viacount system, respectively. Cells were treated with spiradoline (SPR; 0.1 - 10 mM), a highly selective KOR agonist, during and following OGD. Western blot was used to determine the effects of SPR on the apoptotic biomarkers caspases 3 and 9, as well as on the antioxidant, superoxide dismutase (SOD1). PBS controls were included with all experiments. Statistical significance (p < 0.05) was obtained and evaluated using analysis of variance (ANOVA) followed by the Tukey-Kramer and Holm-Sidak multiple comparisons test.
Results :
KORs and DORs were found localized to NTM-5 cells, and protein expression of these receptors was confirmed by Western blot. When compared to controls, NTM-5 cells pre-treated with SPR (24 hrs.), caused concentration-dependent decreases in cytotoxicity (p<0.001). Our data also showed that the protective effects of SPR was more evident with the inclusion of the drug during the OGD process, as well as during the recovery period (p<0.01). Cells pre-treated with SPR (10 μM) also showed increased cell viability when compared to control experiments (p<0.01). Pre-treatment with SPR also caused a reduction in the protein expression of caspases 3 and 9, and an increase in the protein expression of the antioxidant SOD.
Conclusions :
This data provides evidence that a selective KOR agonist reduces apoptotic cell death and protects normal trabecular meshwork cells undergoing stress. Protecting the TM is important in preventing ocular hypertension, and opioids may have a very important role in this process.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.