The p75NTR signaling cascade mediates mechanical hyperalgesia induced by nerve growth factor injected into the rat hind paw

Nerve growth factor (NGF) augments the excitability of isolated rat sensory neurons through activation of the p75 neurotrophin receptor (p75NTR) and its downstream sphingomyelin signaling cascade, wherein neutral sphingomyelinase(s) (nSMase), ceramide, and the atypical protein-kinase C (aPKC), protein-kinase M zeta (PKMζ), are key mediators. Here we examined these same receptor-pathways in vivo for their role in mechanical hyperalgesia from exogenous NGF. Mechanical sensitivity was tested by the number of paw withdrawals in response to 10 stimuli (PWF = n/10) by a 4-g von Frey hair (VFH, testing “allodynia”) and by 10 and 15 g VFHs (testing “hyperalgesia”). NGF (500 ng/10 μL) injected into the male rat's plantar hind paw induced long-lasting ipsilateral mechanical hypersensitivity. Mechano-hypersensitivity, relative to baseline responses and to those of the contralateral paw, developed by 0.5-1.5 h and remained elevated at least for 21-24 h, Acute intraplantar pre-treatment with nSMase inhibitors, glutathione (GSH) or GW4869, prevented the acute hyperalgesia from NGF (at 1.5 h) but not that at 24 h. A single injection of N-acetyl sphingosine (C2-ceramide), simulating the ceramide produced by nSMase activity, induced ipsilateral allodynia that persisted for 24 h, and transient hyperalgesia that resolved by 2 h. Intraplantar injection of hydrolysis-resistant mPro-NGF, selective for the p75NTR over the tyrosine kinase (TrkA) receptor, gave very similar results to NGF and was susceptible to the same inhibitors. Hyperalgesia from both NGF and mPro-NGF was prevented by paw pre-injection with blocking antibodies to rat p75NTR receptor. Finally, intraplantar (1 day before NGF) injection of mPSI, the myristolated pseudosubstrate inhibitor of PKCζ/PKMζ, decreased the hyperalgesia resulting from NGF or C2-ceramide, although scrambled mPSI was ineffective. The findings indicate that mechano-hypersensitivity from peripheral NGF involves the sphingomyelin signaling cascade activated via p75NTR, and that a peripheral aPKC is essential for this sensitization.