Spinal PI3K-AKT involvement in carrageenan-induced hyperalgesia in rats

Abstract

The spinal PI3K/AKT pathway is thought to participate in central sensitization following peripheral injection of formalin or capsaicin, possibly due to phosphorylation of AMPA receptors. This work investigates the role of pAKT in paw-carrageenan induced hyperalgesia. Rats were implanted with intrathecal catheters ending just rostral to the lumbar enlargement. Five days post-surgery, mechanical thresholds on the plantar paw were measured using von Frey filaments and the up-down method. Measurements were taken before and at 1, 2, 3, and 4 hrs after intraplantar injection of carrageenan (100 &mug, 2%). Prior to injection, rats were administered spinal drug or vehicle. In other experiments, rats were anesthetized at various time points after carageenan and dorsal lumbar enlargements and DRGs harvested for Western blotting. One-way ANOVA with Tukey's post-hoc tests were used. Behavioral data indicate that pretreatment with PI3K/AKT antagonists (Wortmannin (50-100 &mug), LY294002 (1-5 &mug) and Akt inhibitor IV (0.6-3.0 &mug), as well as etanercept (TNF antagonist- 100-300 &mug) dose dependently blocked carrageenan induced hyperalgesia relative to their respective vehicles (N = 6-8/gp). These differences were maximal 2-3 hrs post-carrageenan for most agents. Wortmanin pre-treatment resulted in the longest duration anti-hyperalgesia (p ≤ 0.01). Western Blots using antibodies to either pAkt (Ser473) or (Thr 308) demonstrated increased phosphorylation as soon as 1 hr post injection with peaks in the 2-3 hr period. Preliminary data indicate that pAkt (Ser473) was reduced by etanercept pre-treatment. These data indicate that activation of the PI3K/Akt pathway contributes to central sensitization. This activation appears to be downstream of spinal TNF release. Experiments are under way to determine if increased pAkt is TNF receptor mediated and if either TNF or pAkt inhibition blocks AMPA receptor phosphorylation.