Figure 4.

CREB antisense prevents (A), but does not reverse (B), hyperalgesic priming induced by intraganglion (i.gl.) injection of 8-bromo cAMP. A, Rats were treated with daily spinal intrathecal injections of ODN AS (black bars) for CREB mRNA, for 3 consecutive days, which decreases its levels in the sensory neuron, and prevents its activation by the priming inducer 8-bromo cAMP (10 μg, injected into the DRG, i.gl.), injected on the fourth day. Control animals were treated, following the same protocol, with ODN MM (white bars). To prevent the possibility that prolonged activation of a signaling pathway might produce priming following the administration of 8-bromo cAMP, the ODN treatment continued through day 5, when the mechanical nociceptive paw-withdrawal thresholds were not significantly (NS) different from pre 8-bromo cAMP baseline values (t₍₅₎ = 2.445; p = 0.0583, NS, for the MM group; t₍₅₎ = 0.1117; p = 0.9154, NS, for the AS group; paired Student's t test). The presence of hyperalgesic priming was assessed by intradermal injection of PGE₂ (100 ng) into the dorsum of the hindpaw. Mechanical hyperalgesia was evaluated 30 min and 4 h later, by the Randall–Sellitto paw-withdrawal test. Average paw-withdrawal thresholds before the injection of 8-bromo cAMP and before the injection of PGE₂ (1 d later) were as follows: 119.0 ± 2.7 and 114.3 ± 2.0 g, respectively, for the CREB MM-treated group; and 118.0 ± 2.0 and 118.3 ± 2.0 g, respectively, for the AS-treated group. Two-way repeated-measures ANOVA followed by Bonferroni post-test showed significant mechanical hyperalgesia induced by PGE₂, measured 30 min after injection, in both groups. However, while in the MM-treated group the magnitude of PGE₂ hyperalgesia was still significant at the fourth hour, in the AS-treated group it was strongly attenuated (***p < 0.001 when the hyperalgesia in those groups was compared at that time point). When tested again for priming with PGE₂ 1 week after the last treatment with ODN AS or MM, the prolongation of PGE₂-induced hyperalgesia was still attenuated (at the 4 h time point) in the ODN AS-treated group, but not in the ODN MM-treated group, indicating a role of CREB in the induction of hyperalgesic priming by i.gl. injection of 8-bromo cAMP (***p < 0.001 when the MS- and the AS-treated groups are compared; N = 6 paws per group). Of note, no difference in the mechanical thresholds was observed at this time point, when compared with prepriming stimuli baseline thresholds: 119.0 ± 2.7 and 116.3 ± 3.1 g, respectively, for the CREB MM-treated group (t₍₅₎ = 2.169; p = 0.0822, NS), and 118.0 ± 2.0 and 115.3 ± 2.2 g, respectively, for the AS-treated group (t₍₅₎ = 1.019; p = 0.3548, NS; paired Student's t test). B, Rats that were treated with i.gl. injection of 8-bromo cAMP (10 μg) 5 d before were treated with ODN AS (black bars) for CREB mRNA for 3 consecutive days, to decrease the levels of CREB in the nociceptor. Control animals were treated, following the same protocol, with MM (white bars). On the fourth day, PGE₂ (100 ng) was injected intradermally into the dorsum of the hindpaw, and the mechanical threshold was evaluated 30 min and 4 h later. The average paw-withdrawal thresholds before the injection of 8-bromo cAMP and before the injection of PGE₂ were 118.3 ± 3.0 and 120.3 ± 3.6 g, respectively, for the CREB MM-treated group (t₍₅₎ = 0. 0000; p = 1.0000, NS), and 123.3 ± 3.6 and 122.6 ± 2.1 g, respectively, for the AS-treated group (t₍₅₎ = 0.6742; p = 0.5301, NS). Paired Student's t test showed no significant (NS) difference between these two values. Two-way repeated-measures ANOVA followed by Bonferroni post-test showed no difference in the magnitude of the PGE₂-induced hyperalgesia at 30 min and 4 h between the ODN AS- and MM-treated groups (p > 0.05, NS), indicating that CREB does not play a role in the maintenance of hyperalgesic priming induced by 8-bromo cAMP (N = 6 paws per group).