Abstract
Purpose: :
Current study examined if enhancement of Δ-opioidergic activity opposes the production of TNF-α within the optic nerve against glaucomatous injury. Moreover, studies also determined if TNF-α-induced secretion of MMP-2, MMP-3, or NF-kB expression in optic nerve head (ONH) astrocytes is blocked by Δ-opioid-receptor activation.
Methods: :
Brown Norway rats were used to elevate intraocular pressure (IOP) by injecting 50µL of 2M hypertonic saline into the circumferential limbal veins. IOP was recorded as the average of 6-8 consecutive measurements prior to surgery (baseline IOP) and weekly after treatment, using a calibrated Tonolab tonometer. Immediately after saline injections, animals were treated daily with Δ-opioid-receptor agonist, SNC-121 (1mg/kg; i.p.), for 7 days. Retinas were collected at 3, 7, and 42 days post injury. Primary cultures of human ONH astrocytes were isolated and purified by immunopanning. Cells were treated with SNC-121 (1μM), 15 minutes prior to TNF-α (25 ng/mL) treatment for 6 hours. The expression of TNF-α, MMP-2, MMP-3, and NF-kB was determined by Western blotting.
Results: :
In ocular hypertensive eyes, expression of TNF-α was significantly (P<0.05) up-regulated in the optic nerve when measured between 3-42 days post injury. TNF-α production was completely inhibited when animals were treated with the selective Δ-opioid-receptor agonist, SNC-121. To dissect out the TNF-α-induced signaling mechanisms, ONH astrocytes were treated with TNF-α for 6 hours in the presence or absence of 1μM SNC-121, followed by analysis of MMP-2, MMP-3, and NF-ΚB expression. TNF-α treatment increased the secretion of MMP-2 and MMP-3 by 870±215 and 588±168%, respectively, at 6 hours when compared with controls. The MMP-2 and MMP-3 secretion was reduced to 171±8 and 238±48%, respectively, at 6 hours, when cells were pre-incubated with 1μM SNC-121. Additionally, TNF-α increases NF-kB (p65) expression by 61±5%, which was inhibited significantly (P<0.05) in the presence of SNC-121.
Conclusions: :
These data provide evidence that TNF-α is produced from glial cells in the early phase of glaucomatous injury, and remain significantly elevated during disease progression. Mechanistic data in ONH astrocytes provide clues that Δ-opioid-receptor activation opposes the secretion of MMP-2 and MMP-3 by inhibiting the upstream regulator transcription factor, NF-kB (p65).
Keywords: astrocytes: optic nerve head • optic nerve • signal transduction: pharmacology/physiology