Abstract
Purpose: :
Primary open-angle glaucoma (POAG) is a neurodegenerative disease causing retinal ganglion cell death which is usually associated with increased intra-ocular pressure (IOP). Previous studies have implicated an inflammatory component in causing both neurotoxicity and increased IOP, and recently this laboratory discovered that low-molecular-weight hyaluronic acid (LMW-HA) fragments can initiate this pathway, interacting with toll-like receptor 4 (TLR4) to activate the innate immune system. Therefore, finding a viable inhibitor to the TLR4 pathway is of critical importance for neuroprotection in POAG. This study examines viability of trabecular meshwork (TM) and naïve PC-12 cells when challenged with LMW-HA in the presence and absence of naloxone, a known TLR4-pathway inhibitor in non-ocular cells.
Methods: :
Cells were grown in 10% FBS in Eagle's medium until confluent, plated at a density of 40,000 cells per well in full media for 24 hours, and then were placed in 0.1% FBS and challenged with 0.2, 2.0, and 20.0 uM concentrations of purified LWM-HA (50 kD; Sigma) in the presence or absence of 10.0 uM naloxone (naloxone hydrochloride dihydrate, Sigma). The cells were treated for 24 hours (n=6), harvested, and stained with trypan blue. Cell viability was determined by two researchers without knowledge of experimental conditions.
Results: :
Naloxone showed significant protective properties in both TM and PC-12 cells. TM cells in the naloxone+LMW-HA condition were significantly more viable than those treated with only LMW-HA, with an average viability increase of 19% (P<0.02). Naloxone exhibited its greatest protection at higher concentrations of LMW-HA; the 20 uM LMW-HA condition showed a 38.6% increase in viability (P<0.005). Protection of PC-12 cells was even more dramatic, with an average viability increase of 64% (P<0.005) in the naloxone+LMW-HA conditions relative to LMW-HA alone.
Conclusions: :
These findings demonstrate that naloxone prevents the cytotoxic effects of LMW-HA in vitro, confirming that LMW-HA acts through TLR-4 to induce these effects. These findings are significant in that they provide promising leads for protection of TM and retinal ganglion cells, a critical step in the treatment of POAG.
Keywords: neuroprotection • optic nerve • trabecular meshwork