Supporting Information
Supplementary Table 1. Effects of the acute treatment with N/OFQ or UFP-101 on the locomotor activity and anxiety parameters in reserpine-treated mice.
The values represent the mean ± SEM.
I.C.V. Vehicle Reserpine N/OFQ (1 nmol/site)
Total number entries 20 ± 1.1 8.4 ± 3.6 * 8 ± 1.4 * Entries in open arms 0.8 ± 0.5 1.3 ± 1 1.5 ± 0.8
%Time in open arms 1.6 ± 0.9 12.9 ± 9.6 3.8 ± 1.7
I.C.V. Vehicle Reserpine UFP-101 (0.3 nmol/site) UFP-101 (1 nmol/site)
Total number entries 16.3 ± 3.1 6.7 ± 2.6 * 8.6 ± 2 3.9 ± 1.5 *
Entries in open arms 0.8 ± 0.3 0.4 ± 0.4 1.1 ± 0.5 1.5 ± 0.8
%Time in open arms 1.7 ± 0.5 0.4 ± 0.2 3.5 ± 1.5 14 ± 9.2
I.T. Vehicle Reserpine N/OFQ (0.3 nmol/site) N/OFQ (1 nmol/site) N/OFQ (3 nmol/site) Total number entries 23.2 ± 1.7 11.6 ± 3.1 * 10.5 ± 2.4 * 14.4 ± 3.3 8.1 ± 1.6 *
Entries in open arms 1.1 ± 0.6 2 ± 0.9 1.2 ± 0.4 2.9 ± 1.5 1.9 ± 0.7
%Time in open arms 3.3 ± 1.6 9.2 ± 4.7 3 ± 1.1 6.2 ± 3.8 1.7 ± 0.7
I.T. Vehicle Reserpine UFP-101 (1 nmol/site) UFP-101 (3 nmol/site) UFP-101 (5 nmol/site)
Total number entries 20.2 ± 2.6 11.4 ± 3.2 13.6 ± 3.3 10.5 ± 1.5 13.2 ± 4.2
Entries in open arms 0.5 ± 0.3 1.9 ± 0.9 2 ± 1 0.9 ± 0.4 5.6 ± 1.8
%Time in open arms 1.9 ± 0.9 4.9 ± 2.1 3.1 ± 1.5 4.2 ± 1.8 35.7 ± 12.9
I.P. Vehicle Reserpine N/OFQ (0.3 nmol/kg) N/OFQ (1 nmol/kg) N/OFQ (3 nmol/kg) N/OFQ (5 nmol/kg)
Total number entries 22.5 ± 2 14 ± 2 * 6.4 ± 2.3 * 13.1 ± 2.8 8.4 ± 1.9 * 14.4 ± 1.8
Entries in open arms 2.1 ± 0.5 2.6 ± 0.7 0.9 ± 0.9 1.4 ± 0.6 0.9 ± 0.3 2.8 ± 0.7
%Time in open arms 3.4 ± 1 6.7 ± 1.5 3.2 ± 2.8 4.3 ± 2 2 ± 1 7 ± 2
I.P. Vehicle Reserpine UFP-101 (0.3 nmol/kg) UFP-101 (1 nmol/kg) UFP-101 (3 nmol/kg) UFP-101 (5 nmol/kg)
Total number entries 23.8 ± 2 12.1 ± 2.1 * 13 ± 1.8 4.2 ± 1.5 * 16.2 ± 4 12.7 ± 2.2 *
Entries in open arms 2.4 ± 0.6 2.3 ± 0.7 0.1 ± 0.1 1.2 ± 0.6 3.3 ± 1.3 3.1 ± 0.5
%Time in open arms 4.7 ± 1.4 6.2 ± 1.6 0.4 ± 0.2 8 ± 4.4 9.3 ± 2.9 12 ± 5.4
N/OFQ and UFP-101 were dosed by intracerebroventricular (i.c.v.), intrathecal (i.t.) or intraperitoneal (i.p.) routes, at the fourth day after the onset of fibromyalgia induction, 30 min before the plus maze test. *p < 0.05 when compared to the control vehicle/saline group. Statistical analysis was performed by Kruskal-Wallis followed by Dunn’s post hoc test, or by one-way ANOVA followed by Bonferroni's post hoc test. (N/OFQ i.c.v. treatment) n = 6-8 mice per group; (UFP-101 i.c.v. treatment) n = eight mice per group;
(N/OFQ i.t. treatment) n = 8-10 mice per group; (UFP-101 i.t. treatment) n = 7-10 mice per group; (N/OFQ i.p. treatment) n = 8-17 mice per group; (UFP-101 i.p. treatment) n = 8-18 mice per group.
Supplementary Table 2. Cytokine levels (TNF, IL-1β and IL-10) in brain, spinal cord and masseter muscle (pg/100 mg tissue), or in serum (pg/ml) in the fibromyalgia model induced by reserpine in mice. The values represent the mean ± SEM.
UFP-101 (1 nmol/kg, intraperitoneal) or pregabalin (188 µmol/kg, intraperitoneal) were administered for three consecutive days, 30 min after daily reserpine injection. On the fourth day, mice also received UFP-101 or pregabalin, 30 min before of sample collection. *p < 0.05 when the pregabalin group was compared to the other groups. #p < 0.05 when the pregabalin group was compared to the UFP-101-treated group. The statistical analysis was performed by Kruskal-Wallis followed by Dunn’s post hoc test or by one-
way ANOVA followed by Bonferroni's post hoc test. n = 5-6 mice per group. † ND = not detectable.
Brain TNF IL-10 IL-1β
Vehicle 1573 ± 159.7 1430 ± 195.8 209.4 ± 43.4
Reserpine 1599 ± 142.9 1426 ± 217.6 210.2 ± 58.5
UFP-101 (1 nmol/kg) 1576 ± 233.6 1297 ± 132.8 18 ± 9
Pregabalin (188 µmol/kg) 5707 ± 299.4* 2522 ± 155* 692 ± 103.5#
Spinal cord TNF IL-10 IL-1β
Vehicle 10600 ± 2115 3958 ± 138.9 2934 ± 176
Reserpine 11602 ± 2077 5193 ± 609 2636 ± 381.1
UFP-101 (1 nmol/kg) 13463 ± 1902 4212 ± 371.2 2966 ± 266.1
Pregabalin (188 µmol/kg) 13051 ± 1157 4485 ± 292.2 2338 ± 264.7
Masseter TNF IL-10 IL-1β
Vehicle 22769 ± 2601 4442 ± 556.2 1090 ± 144.6
Reserpine 23173 ± 2745 4207 ± 387.7 1357 ± 225.4
UFP-101 (1 nmol/kg) 20900 ± 2496 4221 ± 393.2 1118 ± 146.8
Pregabalin (188 µmol/kg) 16922 ± 2698 3569 ± 436.8 1189 ± 110
Serum TNF IL-10 IL-1β
Vehicle † ND † ND 2569 ± 151
Reserpine † ND † ND 1856 ± 460.3
UFP-101 (1 nmol/kg) † ND † ND 1767 ± 444
Pregabalin (188 µmol/kg) † ND † ND 2615 ± 65.2
Supplementary Figure 1: Schedule of acute treatment with N/OFQ or UFP-101, administered 15 min (intracerebroventricular, i.c.v. and intrathecal, i.t.) or 30 min (intraperitoneal, i.p.) prior
Experimental sessions Treatment 30
min after reserpine
Treatment 30 min after reserpine Treatment 30
min after reserpine
Treatment 30 min prior tests
1 2 3 4
0
Reserpine (0.25 mg/kg)
Treatment 30 min (i.p.) or 15 min (i.c.v. / i.t.)
prior tests
Experimental sessions
1 2 3 4
Reserpine (0.25 mg/kg) 0
A
B
C
D
behavioural tests, at the fourth day (Panel A). Timeline for the behavioural tests after acute (N/OFQ or UFP-101) or repeated (UFP-101 or SB-612111) treatment (Panel B). Schedule of repeated treatment by i.p. injection of UFP-101 or SB-612111 administered once a day, for 4 consecutive days, 30 min after the onset of reserpine administration (0.25 mg/kg, subcutaneous, once a day for three days) and 30 min prior behavioural tests, at the fourth day (Panel C). Timeline for the fatigue- related tests after the repeated treatment with UFP-101 or SB-612111 (Panel D).
Supplementary Figure 2: Representative DRG images showing the creation of the macro used for the analysis of NOPr immunopositivity by ImageJ Software. (A) An image reference of negative control (vehicle-treated mice) was used for the macro creation. (B) The dark-to-medium brown areas (considered as immunopositive neurons for NOPr) were selected (16 small green multi-points) to determine the pixels for the macro creation. (C) Sampling the range of colours from the selected regions. (D) Determination of colour threshold following the next steps:
(i) Plugins → macros → record; (ii) Image → adjust→ colour threshold. (E) Creation of macro
“MacroNOPr”: Save and install macro in Plugins → macros. (F) Resulting black-and-white image after the analysis of the RGB image. The small window on the right shows the value of the mean grey value of the black regions.The macro “MacroNOPr” was used for analysis of the other images as a reference macro.
Supplementary Figure 3: Immunohistochemistry analysis to confirm the selectivity of the anti- NOPr antibody (A-F). Representative images for NOP receptor immunolabelling in the brain, spinal cord or DRG slides, with (A, C and E) or without (B, D and F) the co-incubation of the internal antigen with the primary antibody. The schematic representations were captured in ×40 magnification. Scale bar (—) represents 100 µm.
Supplementary Figure 4: Effects of N/OFQ alone or after the pre-treatment with naloxone on painful-, depressive- and anxiety-like behaviours in reserpine-treated mice. Effects of N/OFQ (1 nmol/kg) administered acutely, by intraperitoneal (i.p.) route, alone or after the pre-treatment with naloxone (5 µmol/kg, dosed i.p.; 5 min before the agonist treatment), on hind paw mechanical allodynia (A), latency time in response to the hot thermal stimulation (B), immobility time (C), and on plus maze parameters (D-F). All of the tests were performed at the fourth day after the onset of reserpine administration (0.25 mg/kg, subcutaneous, once a day for three days). Each bar represents the mean ± SEM. *p < 0.05 when compared to the control vehicle/saline group; #p < 0.05 when compared to the reserpine/saline group. Statistical analysis was performed by Kruskal-Wallis (E and F), one-way (C and D) or two-way (A and B) ANOVA followed by Bonferroni's post hoc test.
(A-F) n = 6-7 mice per group.
Supplementary Figure 5: Effects of UFP-101 combined with N/OFQ, both dosed i.p., on painful-, depressive- and anxiety-like behaviours in reserpine-treated mice. Effects of UFP-101 (1 nmol/kg) administered acutely, by intraperitoneal (i.p.) route, in combination with N/OFQ (1 nmol/kg or 5 nmol/kg, i.p.), on hind paw mechanical allodynia (A), latency time in response to hot thermal stimulation (B), immobility time (C) and on plus maze parameters (D-F). All of the tests were performed at the fourth day after the onset of reserpine administration (0.25 mg/kg, subcutaneous, once a day for three days). Each column represents the mean ± SEM. *p < 0.05 when compared to the control vehicle/saline group; #p < 0.05 when compared to the reserpine/saline group. Statistical analysis was performed by one-way (C-F) or two-way (A and B) ANOVA followed by Bonferroni’s post hoc test. (A-F) n = eight mice per group.
Supplementary Figure 6: Effects of systemic treatment with UFP-101 combined with N/OFQ (i.c.v. or i.t.) on mechanical and thermal nociception in reserpine-treated mice. Effects of UFP-101 (1 nmol/kg) administered acutely, by intraperitoneal (i.p.) route, in combination with N/OFQ (1 nmol/kg, i.c.v.; A and B) or N/OFQ (1 nmol/kg, i.t.; C and D), on hind paw mechanical allodynia (A and C) and latency time in response to hot stimulation (B and D). All of the tests were performed at the fourth day after the onset of reserpine administration (0.25 mg/kg, subcutaneous, once a day for three days). Each column represents the mean ± SEM. *p < 0.05 when compared to the control vehicle/saline group; #p < 0.05 when compared to the reserpine/saline group. Statistical analysis was performed by two-way (A-D) ANOVA followed by Bonferroni's post hoc test. (A-F) n = eight mice per group.
Supplementary Figure 7: Ultrastructural analysis of the masseter muscle in reserpine-treated mice.
Representative transmission electron microscopic (TEM) images of masseter muscle in vehicle (A), reserpine (B) and UFP-101-treated mice (C). Effects of the repeated administration of UFP-101 (1 nmol/kg, intraperitoneal on mitochondrial area in μm2 (D) or number of mitochondria/field (E) in masseter muscle. Scale bars represent one µm. Original magnification x 8,900. White arrows identify the mitochondria. (A, B) n = four mice per group.
Supplementary Figure 8: Quantitative immunohistochemistry analysis for NOP receptor in the thalamus and masseter muscle of vehicle- and reserpine-treated mice (A-F). The samples were collected at the fourth day after the onset of reserpine administration. Representative images for NOP receptor immunolabelling in the thalamus of the vehicle/saline control (B) or reserpine-treated group (C), and in masseter of the vehicle/saline control (E) or reserpine-treated group (F). The schematic representations of brain and masseter were captured in ×8 and ×400 magnification, respectively. Red continuous lines delimit the region of interest analysed in the brain. Scale bar (—) represents 2 mm for brain and 50 µm for muscle. Statistical analysis was performed by Student t test. (A-C) n = five mice per group; (D-F) n = six mice per group.
Supplementary Figure 9: Substance P levels in brain (A), spinal cord (B) and masseter muscle (C) of vehicle- and reserpine-treated mice. UFP-101 (1 nmol/kg, intraperitoneal) was administered once a day, for four consecutive days, 30 min after the onset of reserpine administration (0.25 mg/kg,
subcutaneous, once a day for three days), and 30 min before the tissue collection, at the fourth day.
Each column represents the mean ± SEM. *p < 0.05 when compared to the control vehicle/saline group. Statistical analysis was performed by Kruskal-Wallis followed by Dunn’s post hoc test (B and C) or by one-way ANOVA (A). (A-B) n = 6 mice per group; (C) n = 5 mice per group.
Supplementary Figure 10: Tissue concentrations of glutathione in brain (A), spinal cord (B) and masseter muscle (C) of vehicle- and reserpine-treated mice. UFP-101 (1 nmol/kg, intraperitoneal) was administered once a day, for four consecutive days, 30 min after the onset of reserpine administration (0.25 mg/kg, subcutaneous, once a day for three days), and 30 min before tissue collection, at the fourth day. Each column represents the mean ± SEM. Statistical analysis was performed by one-way ANOVA. (A-C) n = 5 mice per group.
Supplementary Figure 11: Lactate dehydrogenase (LDH) activity in serum (A) and mitochondrial extracts of masseter (B) of vehicle- and reserpine-treated mice. UFP-101 (1 nmol/kg, intraperitoneal) was administered once a day, for four consecutive days, 30 min after the onset of reserpine administration (0.25 mg/kg, subcutaneous, once a day for three days), and 30 min before
tissue collection, at the fourth day. Each column represents the mean ± SEM. *p < 0.05 when compared to the control vehicle/saline group. #p < 0.05 when compared to the reserpine/saline group. Statistical analysis was performed by Kruskal-Wallis (A) or one-way (B) ANOVA followed by Bonferroni's post hoc test. (A, B) n = eight mice per group.
Supplementary Figure 12: N/OFQ concentration in serum and brain samples of vehicle- and reserpine-treated mice (A), and in saliva of female control subjects and fibromyalgia patients (B).
The levels of endogenous N/OFQ were measured at the fourth day after of the onset of reserpine administration (0.25 mg//kg, subcutaneous, once a day for three days) in mice. Statistical analysis was performed by Student t test. (A) n = five mice per group; (B) n = 10 subjects per group.