Figure legends
Figure 1. Schematic representation of experimental design. Exposure to unpredictable sound stress occurred over 3 days. Animals were placed 4-5 per cage and the cage placed 25 cm from a speaker that emitted 4 pure tones (5, 11, 15, and 19 kHz), whose amplitudes varied through time independently from 20−110 dB sound pressure level at random times each minute, lasting 5 or 10 seconds. Non-stressed (NST) animals were placed in the sound chamber for 30 minutes but without exposure to the sound stimulus. Following sound or non-stress, mice were returned to their home cages in the animal care facility. Firstly, baseline grimace scale, periorbital and hind paw thresholds to von Frey filaments were recorded for each animal. After post-stress induction the behavioral experiments were evaluated on days 1, 3, 7, 10, and 14 days. Subsequently, on the nociceptive peak after stress induction (7 days) the treatment protocol with CGRP antagonist or the different plasma or trigeminal ganglion analyses were performed.
Figure 2. Unpredictable sound stress evokes periorbital mechanical allodynia (PMA), hind paw mechanical allodynia and grimacing pain behavior in mice. Hind paw mechanical allodynia was measured in (A) male and in (B) female mice at different time points after stress (ST group) or control (non-stressed group, NST). (C) Comparison of hind paw mechanical allodynia between male and female mice at day 7 after stress or in NST group. PMA was measured in (D) male and in (E) female mice. (F) Comparison of PMA between male and female mice at day 7 after stress or in NST group. Grimacing pain behavior were evaluated in (G) male and in (H) female at different time points after stress (ST group) or control (non-stressed group, NST). (I) Comparison of grimacing pain behavior male and female mice at day 7 after stress or in NST group. Baseline measurements (described as B in the graph) were taken before induction. Spontaneous nociception was analyzed by mouse grimace scale (MGS). Data are expressed as mean ± S.E.M. (n = 8). #P < 0.05 when compared to NST group, and between days (1, 7, and 14 post-stress) comparing the ST group and NST group. *P < 0.05, when compared between the sexes within ST group [Repeated measures two-away ANOVA followed by Bonferroni’s post hoc test].
Figure 3. Anxiety-like symptoms induction and decrease exploratory activity after unpredictable sound stress induction. Time spent (A) grooming, (B) sniffing, (C) rearing, (D) number of crossings, (F) time in the peripheral zone were observed in the open field test on days 1, 7, and 14 post-stress (ST group) or in non-stressed group (NST). (G) Representative image of crossings and time in the peripheral zone represented by heat map. Data are expressed as mean ± S.E.M. (n = 8).* P < 0.05, when compared between the sexes within the ST group. # P < 0.05 when compared to the NST group. [Repeated measures two-away ANOVA followed by Bonferroni’s post hoc test].
Figure 4. Unpredictable sound stress induces an increase in IL-6 (Interleukin-6), TNF-α TNF-α (Tumor necrosis factor-alpha) and CGRP calcitonin gene-related peptide levels in plasma. Plasmatic levels of (A and B) TNF-α, (C and D) IL-6 (Interleukin-6), and (E and F) CGRP detected at 7 days after stress (ST group) or in non-stressed group (NST). Data are expressed as mean ± S.E.M. (n = 12) in the graphs (A,C and D) and n = 6 in the graphs (B, D and F) #P < 0.05, when compared to NST group in (A, C, and E) [nonparametric Student’s t-test], and (B, D, and F) [Two-way ANOVA followed by Bonferroni’s post hoc test]. * P < 0.05 when compared between the sexes within the ST group.
Figure 5. CGRP antagonist reduced migraine-like behaviors induced by unpredictable stress model in mice. BIBN4096BS (olcegepant, 100 mg/kg) intraperitoneal (i.p.) administration reduced the (A and B) hind paw mechanical allodynia, (D and E) periorbital mechanical allodynia (PMA), and (G and H) grimacing pain behavior in male and female stressed (ST) mice when compared to vehicle (Veh) treated group. Antinociceptive effect of BIBN4096BS at 1 hour after treatment for (C) hind paw mechanical allodynia, (F) PMA, and (I) grimacing pain behavior. Baseline measurements (described as B in the graph) were observed before induction. Time 0 represents the measures taken 7 days after ST or in NST group. Spontaneous nociception was analyzed by mouse grimace scale (MGS). Data are expressed as mean ± S.E.M. (n = 8). * P< 0.05, when compared vehicle/stressed group (ST) or between the sexes within the ST group. # P < 0.05 when compared to the NST group § P< 0.05 when compared to the vehicle (Veh) treated group [Repeated measures two-away ANOVA followed by Bonferroni’s post hoc test].
Figure 6. CGRP antagonist revert the anxiety-like symptoms and increased exploratory activity after unpredictable sound stress. BIBN4096BS (olcegepant, 100 mg/kg) intraperitoneal (i.p.) administration reverted the alterations of different behavioral parameters: Time spent (A) grooming, (B) sniffing, (C) rearing, (D) number of crossings, (F) time in the peripheral zone were observed in the open field test at day 7 post-stress (ST group) or in non-stressed group (NST) 1 hour- post BIBN4096BS administration. (G) Representative image of crossings and time in the peripheral zone represented by heat map. Data are expressed as mean ± S.E.M. (n = 8). * P < 0.05, when compared between the sexes within the ST group. # P< 0.05 when compared to the NST group. §P < 0.05 when compared to the vehicle (Veh) treated group [Repeated measures two-away ANOVA followed by Bonferroni’s post hoc test].