Age-Dependent Sexual Dimorphism in Susceptibility to Develop Chronic Pain in the Rat

Abstract

Neonatal pain has been suggested to contribute to the development and/or persistence of adult pain. Observations from animal models have shown that neonatal inflammation produces long-term changes in sensory neuron function, which can affect the susceptibility of adults to develop persistent pain. We used a preclinical model of transition to chronic pain, hyperalgesic priming, in which a previous inflammatory stimulus triggers a long-lasting increase in responsiveness to pro-algesic mediators, prototypically prostaglandin E₂ (PGE₂), to investigate if post-natal age influences susceptibility of adult rats to develop chronic pain. Priming was induced by tumor necrosis factor alpha (TNFα), in male and female rats, 1, 2, 3, 4, 5 or 7weeks after birth. When adults (8weeks after birth), to evaluate for the presence of priming, PGE₂ was injected at the same site as TNFα. In males that had received TNFα at post-natal weeks 1, 2 or 3, priming was attenuated compared to the 4-, 5- and 7-week-old treated groups, in which robust priming developed. In contrast, in females treated with TNFα at post-natal week 1, 2, 3, or 4, but not at 5 or 7, priming was present. This age and sex difference in the susceptibility to priming was estrogen-dependent, since injection of TNFα in 3-week-old males and 5-week-old females, in the presence of the estrogen receptor antagonist ICI 182,780, did produce priming. These results suggest that estrogen levels, which vary differently in males and females over the post-natal period, until they stabilize after puberty, impact pain as an adult.

Keywords: chronic pain; estrogen; hyperalgesia; hyperalgesic priming; nociceptor; rat.

Figure 1. Age dependence for induction of hyperalgesic priming by TNFα in male and female rats

Male (left panel) and female (right panel) rats received an intradermal injection of TNFα (100 ng in 5 μl) on the dorsum of the hind paw 1, 2, 3, 4 or 5 weeks after birth (white bars). The control group (adults, black bars) received TNFα at the 7^th week after birth. On the 8^th week after birth, PGE₂ (100 ng) was injected at the same site as TNFα and, the mechanical hyperalgesia evaluated by the Randall-Selitto paw-withdrawal test. The detection of hyperalgesia 4h after PGE₂ injection indicated the presence of priming. We found that, in the male rats, the induction of priming by injection of TNFα 1, 2 or 3 weeks after birth was significantly attenuated, when compared to the control group and the groups treated at the 4^th and 5^th post-natal week, in which priming was fully developed (F_5,31 = 18.86; ****p < 0.0001, when the 1-, 2- and 3-week-old groups are compared to the control and the 4- and 5-week-old groups, one-way ANOVA followed by Bonferroni post-hoc test). On the other hand, while injection of TNFα in 5-week-old and adult females did not induce priming, when injected in 1-, 2-, 3- or 4-week-old females, it did produce priming (F_5,29 = 22.74; ****p < 0.0001, when the 1-, 2-, 3- and 4-week-old groups are compared to the control and 5-week-old groups). (males, control, TNFα/4 weeks old, and TNFα/5 weeks old groups: N = 6 paws; TNFα/1 week old, and TNFα/2 weeks old groups: N = 5 paws; TNFα/3 weeks old group: N = 9 paws; females, control and TNFα/1 week old group: N = 5 paws; TNFα/2 weeks old, TNFα/4 weeks old, and TNFα/5 weeks old groups: N = 6 paws; TNFα/3 weeks old group: N = 7 paws)

Figure 2. Estrogen attenuates the induction of priming by TNFα in 3-week-old male and 4-week-old female rats

The non-selective estrogen receptor antagonist ICI 182,780 (ICI, 10 μg/day, gray bar) was injected, subcutaneously on the back of the neck, once a day for 8 consecutive days, in male (left panel, starting 5 days before completing the third postnatal week) and female (right panel, starting 5 days before completing the 5^th post-natal week) rats. The priming stimulus, TNFα (100 ng in a volume of 5 μl), was injected intradermally on the dorsum of the hind paw on the 5^th day of the ICI treatment. When rats were 8 weeks old, PGE₂ (100 ng) was injected at the same site as TNFα. Mechanical nociceptive threshold was evaluated before and at the 4^th h after PGE₂ injection. Left panel: The ICI-treated males were compared with groups shown in Figure 1 (left panel), that had received TNFα alone, at the 7^th post-natal week (black bar), in which priming had fully developed, or at the 3^rd post-natal week (control, white bar), in which the induction of priming was significantly attenuated, as indicated by the smaller magnitude of the PGE₂-induced hyperalgesia 4h after injection (t₁₃ = 6.641; p < 0.0001, when the hyperalgesia in the 3- and the 7-week-old groups is compared, unpaired Student’s t-test). We found that, when 3-week-old rats received TNFα in the presence of ICI 182,780 (gray bar), the induction of priming was robust, since the magnitude of the hyperalgesia induced by PGE₂ at the 4^th h was significantly higher when compared to the 3-week- old + TNFα alone group (t₁₂ = 3.106; ** p = 0.0091, when both groups are compared), indicating an inhibitory effect of estrogen in the induction of priming in young male rats. (TNFα/7 weeks old group: N = 6 paws; TNFα alone/3 weeks old group: N = 9 paws; TNFα + ICI 182,780/3 weeks old group: N = 5 paws); Right panel: The PGE₂-induced hyperalgesia in the rats treated with ICI + TNFα was compared with control groups, shown in Figure 1 (right panel), which did not develop priming when received TNFα alone at the 5^th (white bar), or at the 7^th post-natal week (black bar). In contrast to the controls, the PGE₂-induced hyperalgesia was present at the 4^th h in the group that received TNFα in the presence of ICI 182,780 (t₉ = 9.297; t₁₀ = 8.258; **** p < 0.0001 for both, when the PGE₂-induced hyperalgesia in the 3-week-old TNFα + ICI 182,780 group is compared, respectively, with the groups that received TNFα alone 7 and 5 weeks after birth, unpaired Student’s t-test), indicating that priming had developed. This result suggests that estrogen has an inhibitory effect on the induction of priming in 5-week-old female rats. (TNFα/7 weeks old group: N = 5 paws; TNFα alone/5 weeks old and TNFα + ICI 182,780/5 weeks old groups: N = 6 paws)