Synergistic interaction of nerve growth factor and glial cell-line derived neurotrophic factor in muscular mechanical hyperalgesia in rats

Key points Nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are essential for neuronal development and survival in embryo. However, after birth they play pivotal roles in the generation of hyperalgesia in many painful conditions. Both factors are believed to act on different groups of primary afferents, but interaction between them has not yet been studied. Here we show a synergism between the two factors. Intramuscular injection of a mixture of both factors at a low concentration, each of which alone had no effect, induced a significant muscular mechanical hyperalgesia in rats. We show that synergism occurs in the primary afferent neurons and find that about 25% primary afferents innervating the muscle express both TrkA (NGF receptor) and GFR alpha 1 (GDNF receptor). We show by pharmacological means that afferent neurons with TrkA and GFR alpha 1 express both TRPV1 and ASICs. Our data establish a basis for synergism between NGF and GDNF. In some inflammatory conditions both nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are upregulated and play pivotal roles in inducing hyperalgesia. However, their interaction has not been studied. We examined whether and where interaction between both neurotrophic factors occurs in SD rats. Intramuscular injection to gastrocnemius muscle (GC) of a mixture of NGF (0.1 mu m) and GDNF (0.008 mu m), which alone had no effect, induced a significant mechanical hyperalgesia (F-(6,F-30) = 13.62, P = 0.0001), demonstrating synergism between the two factors. Phosphorylated extracellular signal-regulated kinase (pERK) immunoreactivity in dorsal root ganglia (DRGs) induced by compression of GC increased after injection of the mixture (P = 0.028, compared with PBS); thus the interaction of NGF and GDNF could occur at the primary afferent level. An in situ hybridization study (n = 4) demonstrated that 23.7-29.2% of GC-innervating DRG neurons coexpressed TrkA (NGF receptor) and GFR alpha 1 (GDNF receptor). The cell size of the coexpressing GC DRG neurons showed no skewing towards the small size range but was distributed widely from the small to the large size ranges. Therefore, some of the coexpressing neurons with thin axons are thought to contribute to this mechanical hyperalgesia. The hyperalgesia was reversed by both amiloride (F-(1,F-13) = 5.056, P = 0.0425, compared with PBS) and capsazepine (F-(1,F-10) = 8.402, P = 0.0159, compared with DMSO), suggesting that the primary afferents sensitized by the mixture express both TRPV1 and ASICs. These results showed a basis of synergism between NGF and GDNF.