Attenuation of the Diffuse Noxious Inhibitory Controls in Chronic Joint Inflammatory Pain Is Accompanied by Anxiodepressive-Like Behaviors and Impairment of the Descending Noradrenergic Modulation

Abstract

The noradrenergic system is paramount for controlling pain and emotions. We aimed at understanding the descending noradrenergic modulatory mechanisms in joint inflammatory pain and its correlation with the diffuse noxious inhibitory controls (DNICs) and with the onset of anxiodepressive behaviours. In the complete Freund's adjuvant rat model of Monoarthritis, nociceptive behaviors, DNICs, and anxiodepressive-like behaviors were evaluated. Spinal alpha2-adrenergic receptors (a2-AR), dopamine beta-hydroxylase (DBH), and noradrenaline were quantified concomitantly with a2-AR pharmacologic studies. The phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) were quantified in the Locus coeruleus (LC), amygdala, and anterior cingulate cortex (ACC). DNIC was attenuated at 42 days of monoarthritis while present on days 7 and 28. On day 42, in contrast to day 28, noradrenaline was reduced and DBH labelling was increased. Moreover, spinal a2-AR were potentiated and no changes in a2-AR levels were observed. Additionally, at 42 days, the activation of ERKs1/2 was increased in the LC, ACC, and basolateral amygdala. This was accompanied by anxiety- and depressive-like behaviors, while at 28 days, only anxiety-like behaviors were observed. The data suggest DNIC is attenuated in prolonged chronic joint inflammatory pain, and this is accompanied by impairment of the descending noradrenergic modulation and anxiodepressive-like behaviors.

Keywords: anxiodepressive comorbidities; chronic joint inflammatory pain; descending noradrenergic pain modulation; diffuse noxious inhibitory controls; locus coeruleus; pERK1/2.

Figure 1

Evaluation of the development of inflammation and nociceptive behavior in control and monoarthritic rats. (A) Inflammation scores; (B) movement-induced allodynia, assessed by the ankle bend test; (C,D) paw withdrawal threshold observed in the Randall–Selitto test in the ipsilateral (C) and contralateral (D) paws. For the inflammation score and ankle bend test, only the ipsilateral paw values are shown in the graphs. For the contralateral paws, the values obtained for these parameters were null and, therefore, statistical tests could not be applied. Values expressed in Mean ± SEM. Six animals per group. *** p < 0.001. Two-way ANOVA with repeated-measures test followed by the Tukey’s post hoc test for multiple comparisons between the monoarthritic and control groups. BL = baseline; D = day.

Figure 2

Effects of intrathecal cumulative doses of clonidine on the nociceptive responses in the ipsilateral (A) and contralateral paws (B) of control and monoarthritic rats. (A) The cumulative doses of clonidine induced a significant increase of the mechanical withdrawal threshold of the ipsilateral paw in monoarthritic rats, but not in control rats. (B) In the contralateral paw, no significant differences were found between the monoarthritis and control groups. Values expressed in Mean ± SEM. Six animals per group. Two-way ANOVA with repeated-measures test followed by Tukey’s multiple comparisons post hoc test. ** p < 0.01; *** p < 0.001, for comparison between the monoarthritis and control group; ⁺ p < 0.05; ⁺⁺ p < 0.01; ⁺⁺⁺ p < 0.001, for comparisons between each clonidine dose and the baseline before the first clonidine intrathecal injection. BL= baseline before the first clonidine intrathecal injection.

Figure 3

Effects of chronic inflammatory joint pain on a2A-AR protein levels in the spinal cord of control and monoarthritic rats quantified by immunofluorescence and western blot. Immunofluorescence labelling for a2A-AR in the spinal cord dorsal horn (laminae I-VI) of L4 (A) and L5 (B) segments in control (left) and monoarthritic (right) rats. (C,D) Percentage of a2A-AR-positive pixels in spinal cord segments L4 (C) and L5 (D). No significant changes were detected between monoarthritic and control animals. (E) Relative expression of a2A-AR in the spinal segments L4 and L5, normalized against alpha-tubulin levels. No significant difference was found in a2A-AR levels between monoarthritic and control groups. (F) Representative blot showing a2-AR (59 kDA) and alpha-tubulin (52 kDA, loading control) bands for the ipsilateral and contralateral sides of the spinal cord of a control (left) and a monoarthritic (right) animal. Values expressed in Mean ± SEM. Unpaired t-test for comparisons between the monoarthritic and control group in the immunofluorescence; five animals per group. Mann–Whitney non-parametric test for comparisons between the monoarthritic and control group in the western blot; four animals per group.

Figure 4

Effects of chronic inflammatory joint pain on the DBH labelling and noradrenaline concentration in the spinal cord of control and monoarthritic rats at 28 and 42 days after intraarticular injection, quantified by densitometric analysis and high-performance liquid chromatography (HPLC), respectively. (A,B) At 42 days, the monoarthritic rats (right) show significantly more DBH immunolabelled fibers in superficial laminae of L4 (A) and L5 (B) spinal cord segments than controls (left), but the same is not observed at 28 days. (C,D) Monoarthritis induced a significant increase in percentage of DBH positive pixels in spinal cord segments L4 (C) and L5 (D) at 42 days after intraarticular injection, as compared to controls (n = 5, for both experimental groups). No significant differences were found between groups at 28 days (n = 6, for both experimental groups); (E) At 42 days of monoarthritis, but not at 28 days, significant differences were found in the levels of noradrenaline on spinal segments L4 and L5 (pmol/mg) between control and monoarthritic animals groups, pointing to a decrease in the concentration of this neurotransmitter in the monoarthritic rats in comparison to the non-diseased group (n = 10 for controls and n = 9 for monoarthritis at 42 days; n = 6 for both experimental groups at 28 days). Values expressed in Mean ± SEM. * p < 0.05; One-way ANOVA followed by the Tukey’s post hoc test for multiple comparisons between groups.

Figure 5

Effects of the diffuse noxious inhibitory control (DNIC) stimulation on the nociceptive responses of the ipsilateral and contralateral paws of control and monoarthritic rats on day 7 (A,B), 28 (C,D), and 42 (E,F) post intraarticular injection of complete Freund’s adjuvant solution (CFA)-vehicle or CFA. For all time points, the values of force needed for paw withdrawal (g) before intraarticular injection (BL) and at pre- and post-DNIC stimulation are presented. Post-DNIC stimulation significantly increased the withdrawal threshold on the ipsilateral hind paw of monoarthritic rats at all time points, in comparison to pre-DNIC and to the same paw in the control group. The DNIC magnitude was significantly reduced on day 42 compared to days 7 and 28 (G). Values expressed in Mean ± SEM. Six rats per group. Two-way ANOVA with repeated-measures test followed by Tukey’s multiple comparisons post hoc test; ** p < 0.01; *** p < 0.001, for comparisons between the monoarthritic and control group. ⁺ p < 0.05; ⁺⁺ p < 0.01; ⁺⁺⁺ p < 0.001, for comparisons between pre- and post-DNIC withdrawal thresholds in the monoarthritic rats. One-way ANOVA followed by Tukey’s multiple comparisons post hoc test; # p < 0.05; §§ p < 0.01, for comparisons between days 7, 28, and 42. BL = baseline before intraarticular injection; D = day.

Figure 6

Anxiodepressive-like behaviors in monoarthritic rats at 28 and 42 days post intraarticular CFA injection. (A) MB: The monoarthritic group hid more marbles than the control group, showing anxiety-like behavior at both time points; (B) EZM: At 28 and 42 days of monoarthritis, monoarthritic rats spent less time in the open arms in comparison to the control group, which is another indication of anxiety-like behavior. (C) No differences in the distance travelled in the EZM test were found between groups. (D–G) FST: At 42 days, the monoarthritic rats spent more time immobile (D) and less time swimming (E) in comparison to the control group, therefore showing pain-induced depressive-like behaviors. No significant differences were found on climbing (F) and latency to immobility (G) behaviors. At 28 days, no significant differences were found between groups in any of the analyzed parameters. Values expressed in Mean ± SEM. Six and five animals per group at 28 and 42 days of monoarthritis, respectively. One-way ANOVA followed by the Tukey’s post hoc test for multiple comparisons between groups; * p < 0.05; ** p < 0.01; *** p < 0.001 for comparisons between the monoarthritic and control groups.

Figure 7

Activation of ERK1/2 in the Locus coeruleus (LC), anterior cingulate cortex (ACC), basolateral amygdala (BLa), and medial amygdala (Me) of monoarthritic and control rats at 42-days post-intraarticular injection was assessed by quantification of the immunolabeling for its phosphorylated form. (A,C,E,G) Immunoreaction for pERK1/2 in the LC (A), ACC (C), BLa (E), and Me (G) of control (left) and monoarthritic (right) rats. (B) Monoarthritis induced a significant increase in percentage of pERK1/2 positive pixels in the LC. (D,F,H) The monoarthritic rats show a higher number of pERK1/2-IR cells in the ACC (D) and BLa (F) but not in the Me (H); Values expressed in Mean ± SEM. 5 animals per group. ** p < 0.01; Unpaired t-test for comparisons between the monoarthritic and control groups. The black dashed lines are delineating the areas of interest. ACC = anterior cingulate cortex; BLA = Basolateral amygdala (anterior part); BLV = basolateral amygdala (ventral part); Ce = central amygdala; LaDL = lateral amygdaloid nucleus (dorsolateral part); LC = Locus coeruleus; Me = medial amygdala.

Figure 8

Pharmacological studies timeline. Three consecutive injections of saline or of increasing cumulative doses of clonidine hydrochloride (1, 5, and 10 μg) were administered through the catheter. All injections were separated by 20-min intervals. The Randall–Selitto test was used to evaluate the effects of each administration and was performed before the first intrathecal administration to collect a baseline value and 30 min after each injection.