Analgesia induced by dietary restriction is mediated by the kappa-opioid system

Abstract

Progress in the control and treatment of pain may be facilitated by a better understanding of mechanisms underlying nociceptive processing. Here we show that mice subjected to an intermittent fasting diet (IFD) display markedly reduced responses in models of thermal and visceral pain compared with mice fed ad libitum (AL). Pharmacological analyses suggest that a change in the endogenous kappa-opioid system underlies IFD-induced analgesia. The levels of prodynorphin mRNA and kappa-opioid receptors in the spinal cord are higher in IFD than in AL mice. Furthermore, in spinal cord nuclear protein extracts, the activity of the transcriptional repressor DREAM (downstream regulatory element antagonist modulator), the main regulator of prodynorphin expression, is lower in IFD than in AL mice. Finally, c-Fos expression in dorsal spinal cord after noxious stimulation is significantly lower in IFD than in AL animals, indicating that dynorphin could block nociceptive information at the spinal cord. These results suggest that dietary restriction together with administration of kappa-opioid agonists could be useful as a new therapeutic approach for pain relief.

Figure 3.

Prodynorphin and κ-opioid receptor expression are increased in the spinal cord of IFD mice. A, Basal expression of pdyn mRNA in the spinal cord of AL and IFD mice, as assessed by semiquantitative RT-PCR. gapdh mRNA served as control. Graphs represent the relative abundance of prodynorphin-specific PCR products in the two animal groups (open circles, IFD mice; filled circles, AL mice). B, Competition of DREAM binding from lumbar spinal cord nuclear extracts of AL mice to DRE using 30-fold excess of wild-type, DRE, CRE (cAMP response element), and AP-1 (activator protein-1) cold sequences (lanes 1-4). The basal activities of DREAM protein from lumbar spinal cord of AL and IFD mice are also shown (lanes 5, 6). The histogram represents the relative abundance of DREAM activity in the two groups. C, Western blot analyses of DREAM and actin expression in the lumbar spinal cord of AL and IFD mice. The histogram represents the relative abundance of DREAM protein in the two groups, expressed in arbitrary units. A total of 6-10 animals was used for each experimental group. Animals were collected from three independent experiments.

Figure 4.

κ-Opioid receptor expression is increased in the spinal cord of IFD mice. A, Basal expression of kor mRNA in AL and IFD mice spinal cord as assessed by semiquantitative RT-PCR. gapdh mRNA served as control. Graphs represent the relative abundance of kor-specific PCR products for AL (open circles) and IFD (filled circles) mice. B, Photomicrographs and immunohistochemical analyses of the expression patterns of κ-opioid receptor in the dorsal horn of lumbar spinal cord of AL (i) and IFD (ii) mice. The histogram represents the number of positive neurons in laminas I-II and laminas III-V in each group (filled and open bars represent AL and IFD mice, respectively). We used 6-10 animals per experimental group. These animals were obtained from three independent experiments. The statistical analysis was performed by one-way ANOVA and two-tailed Student's t tests. Asterisks indicates statistical significance of the same treatments in groups AL and IFD. ^***p ≤ 0.001.

Figure 1.

Weight and motor activity do not change in IFD animals. A, Weights (in grams) of AL and IFD animals after a 3 month diet. B, Locomotor activity of AL and IFD groups in nonstressful conditions. Activity is indicated as broken beams per minute. Filled bars represent AL mice, and open bars represent IFD mice. Mean values for weighs and motor activities were computed from ≥50 animals per group. The statistical analysis was performed by two-tailed Student's t test.

Figure 2.

Analgesia induced by IFD is mediated by activation of κ-opioid receptors. A, B, Effects of naloxone hydrochloride (1 mg/kg, s.c., 15 min before each pain test) on the hot-plate test (A) and visceral pain in response to acetic acid (B) (0.6%) injection intraperitoneally. In the hot-plate test (A), values represent the mean latency (in seconds) to paw licking, whereas in visceral pain (B), values represent the number of abdominal stretches (writhes). C, D, Pharmacological analysis of IFD-induced analgesia in the hot-plate test (C) and after acetic acid injection (D). Injections of 2 mg/kg nor-BNI, 7 and 1 mg/kg of a naloxonazine and 3-methoxynaltrezone mixture, or 3 mg/kg naltrindole, were given subcutaneously 15 min before the pain test. In all of the tests, n ≥10 animals per group. Experiments were repeated at least three times, and the number of mice per group in each experiment ranged from four to six. The statistical analysis was performed by one-way ANOVA and two-tailed Student's t tests. ⁺ indicates statistical significance of the same treatments in groups AL and IFD; ^* indicates statistical significance of the different treatments with respect to vehicle in the same group; ⁺⁺,^**p ≤ 0.01; ⁺⁺⁺,^***p ≤ 0.001.

Figure 5.

The κ-opioid system blocks neuron activation, as indicated by c-Fos expression, after nociceptive stimulation of the spinal cord in IFD mice. A, Photomicrographs and immunohistochemical analyses of the expression patterns of c-Fos protein in the dorsal lumbar spinal cord of AL (i, ii) and IFD (iii, iv) mice 90 min after nociceptive treatment (ii, iv) or nontreatment (i, iii). The graph represents the number of positive neurons in each group. In the graph, filled bars represent nociceptive treatment, and open bars represent nontreated animals. B, Effect of nor-BNI on the c-fos mRNA transcription in thoracic and lumbar spinal cord after acetic acid injection in AL and IFD mice. gapdh mRNA served as control. We used six animals for each experimental group. These animals were obtained from three independent experiments. The statistical analysis was performed by two-tailed Student's t test. ⁺ indicates statistical significance of the same treatments in groups AL and IFD; ^* indicates statistical significance of the different treatments with respect to vehicle in the same group; ⁺⁺⁺,^***p ≤ 0.001.

Figure 6.

Absence of neuronal activation after noxious stimulation in brainstem nuclei of IFD mice. A, B, Photomicrographs and immunohistochemical analyses of the expression patterns of c-Fos protein in the parabrachial nucleus (A; PBn, black), a relay station of the ascending pain pathway, and in the dorsal raphe (B; DR, black) and gray periaquedultal substance (PAG; gray), components of the descending pain (modulatory) pathway of AL and IFD mice, 90 min after nociceptive treatment (ii is for AL, and iii is for IFD mice) or nontreatment (i). Histograms represent the number of positive neurons in each group. Because nontreated AL and IFD mice showed equivalent c-Fos-positive neurons, both groups are considered here as the same group (open bars); black bars are for AL animals, and dotted bars are for IFD animals treated with acetic acid. A total of five animals per group, obtained from two independent experiments, were used. The statistical analysis was performed by one-way ANOVA and two-tailed Student's t tests. ^*p ≤ 0.05; ^**p ≤ 0.01; ^***p ≤ 0.001.

Figure 7.

Peripheral mechanisms are not regulating anti-nociception induced by IFD. The effects of naloxone methiodide (2 mg/kg, s.c.) on chemical visceral pain test were checked in AL and IFD mice. A total of eight animals per group was used. The statistical analysis was performed by two-tailed Student's t test. ⁺ indicates statistical significance of the same treatments between groups AL and IFD; ^* indicates statistical significance of the different treatments with respect to vehicle in the same group; ⁺⁺,^**p ≤ 0.001; ⁺⁺⁺,^***p ≤ 0.001.