Nociceptor subpopulations involved in hyperalgesic priming

Abstract

We have previously developed a model in the rat for the transition from acute to chronic pain, hyperalgesic priming, in which a long-lasting neuroplastic change in signaling pathways mediates a prolongation of proinflammatory cytokine-induced nociceptor sensitization and mechanical hyperalgesia, induced at the site of a previous inflammatory insult. Induction of priming is mediated by activation of protein kinase C(epsilon) (PKC(epsilon)) in the peripheral terminal of the primary afferent nociceptor. Given that hyperalgesic mediator-induced PKC(epsilon) translocation occurs in isolectin B4 (IB4)(+)-nonpeptidergic but not in receptor tyrosine kinase (TrkA)(+)-peptidergic nociceptors, we tested the hypothesis that hyperalgesic priming was restricted to the IB4(+) subpopulation of nociceptors. After recovery from nerve growth factor (NGF)- and GDNF-induced hyperalgesia, a proinflammatory cytokine, prostaglandin E(2) (PGE(2)) induced, PKC(epsilon)-dependent, markedly prolonged hyperalgesia, two features that define the development of the primed state. Thus, hyperalgesic priming occurs in both the IB4(+)-nonpeptidergic and TrkA(+)-peptidergic subpopulations of nociceptive afferents. Of note, however, while attenuation of PKC(epsilon) prevented NGF-induced priming, the hyperalgesia induced by NGF is PKC(epsilon) independent. We propose that separate intracellular pools of PKC(epsilon), in the peripheral terminals of nociceptors, mediate nociceptor sensitization and the induction of hyperalgesic priming.

Figure 1. Hyperalgesic priming induced by intradermal injection of GDNF

(A) Intradermal injection of GDNF (10ng/5μl) into the rat hind paw induces prolonged mechanical hyperalgesia in the rat (one-way repeated measures ANOVA, F(7,77)=41.674, p<0.001). Three weeks later the mechanical threshold returned to the pre-GDNF administration baseline. Testing for the presence of hyperalgesic priming was performed on the 23^rd day. N=12 paws; (B) Test for hyperalgesic priming in GDNF-treated rats: intradermal injection of prostaglandin E₂ (PGE₂, 100ng/5μl) on the 23^nd day after GDNF administration. GDNF-primed rats (black bars) showed decrease in mechanical nociceptive threshold that lasted at least 24 hours after PGE₂ injection (p<0.01 between GDNF+PGE₂ and naïve+PGE₂ groups at 4 and 24 hours after PGE₂ injection). White bars show the effect of PGE₂ injection in naïve animals. N=6 paws.

Figure 2. Hyperalgesic priming induced by intradermal injection of NGF

(A) Intradermal injection of NGF (1μg/5μl) into the rat hind paw induces prolonged mechanical hyperalgesia, which lasted 4 days, in the rat (one-way repeated measures ANOVA, F(4,44)=57.308, p<0.01). On the 6^th day mechanical threshold was not different from the pre NGF administration baseline. Testing for the presence of hyperalgesic priming was performed on the 7^th day. N=12 paws; (B) Test for hyperalgesic priming in NGF-treated rats: intradermal injection of PGE₂ (100ng/5μl, intradermal) on the 7^th day after NGF administration. Rats pretreated with NGF (black bars) show decrease in the mechanical nociceptive threshold, after PGE₂ injection, which lasted at least 24 hours (p<0.01 between NGF+PGE₂ and naïve+PGE₂ groups 4 and 24 hours after PGE₂ injection). Mechanical hyperalgesia was no longer present 4 hours after injection of PGE₂ in the control rats not treated with NGF (grey bars). N=6 paws.

Figure 3. NGF-induced hyperalgesic priming is dependent on PKCε

(A) After return to baseline mechanical threshold NGF-treated rats received spinal intrathecal oligodeoxynucleotide (ODN) antisense (AS) or mismatch (MM) for PKCε mRNA for 3 consecutive days. After this period, the animals were tested for priming with intradermal injection of PGE₂ (100ng/5μl/paw). Four hours later, PGE₂-induced mechanical hyperalgesia was not present in the animals treated with AS (grey bars), while the MM group still demonstrated a decrease in the mechanical nociceptive threshold (black bars). N=6 paws (p<0.001 between antisense and mismatch groups at 240 minutes after PGE₂ injection); (B) Test for hyperalgesic priming in NGF-treated rats three weeks after treatment with PKCε AS or MM: both groups show hyperalgesia that lasts at least 4 hours after intradermal injection of PGE₂. N=6 paws (no difference observed between antisense and mismatch groups).

Figure 4. NGF-induced hyperalgesic priming is prevented by knockdown of PKCε

(A) Treatment with oligodeoxynucleotide (ODN) antisense (AS) or mismatch (MM) for PKCε mRNA starting 3 days before injection of NGF and continuing until the recovery of the mechanical threshold baseline (7^th day after NGF). Mechanical nociceptive threshold in AS- or MM-treated rats was evaluated day 1, 3, 4 and 7 post-NGF administration. N=6 paws (no difference observed between antisense and mismatch groups); (B) Test for priming with intradermal injection of PGE₂ (100ng/5μl/paw) was performed on the 9^th day after NGF injection. In the fourth hour after PGE₂, mechanical hyperalgesia is observed in the mismatch group (black bars), but not in the rats treated with antisense (grey bars). N=6 paws (p<0.001 between antisense and mismatch groups at 240 minutes after PGE₂ injection); (C) NGF-treated rats that were pretreated with AS or MM were also tested for priming 2 weeks after the return to pre-NGF mechanical threshold baseline. Animals treated with AS still did not show hyperalgesic priming (grey bars). N=6 paws (p<0.001 between antisense and mismatch groups at 240 minutes after PGE₂ injection).