Sound-induced analgesia cannot always be observed in adult mice

Abstract

Music seems promising as an adjuvant pain treatment in humans, while its mechanism remains to be illustrated. In rodent models of chronic pain, few studies reported the analgesic effect of music. Recently, Zhou et al. stated that the analgesic effects of sound depended on a low (5 dB) signal-to-noise ratio (SNR) relative to ambient noise in mice. However, despite employing multiple behavioral analysis approaches, we were unable to extend these findings to a mice model of chronic pain listening to the 5 dB SNR.

Findings

Sound-including music and noise can relieve pain in humans, which has been proved by cumulative human brain imaging studies^.1–4 Zhou et al. delivered consonant sound to complete Freund’s adjuvant (CFA) injected mice through a closely positioned audio speaker and used von Frey filaments to assess mechanical sensitivity ⁵ . They found that delivery of consonant, dissonant sound, or white noise at a 5-dB signal-to noise ratio (SNR) elevated the inflamed hind paw nociceptive threshold. This effect lasted for at least 2 days after repeated exposure to 5-dB SNR sound for 3 days (40 min per day). Behavioral tests, including open field, light-dark box, and elevated plus maze (EPM), showed that neither 5-dB nor 15-dB SNR white noise evoked anxiety-like behaviors in mice under acute pain conditions (3 days after CFA injection), and neither sound reduced anxiety under chronic pain conditions (14 days after CFA injection).

Using similar approaches, our group failed to repeat the 5-dB sound-induced analgesia in mice of the CFA model. In 10 mice that 10 μL CFA has been injected into the plantar surface of their left hind paws, the mechanical nociceptive threshold of the left hind paw was significantly decreased 3 days after the injection compared to the sham group (n = 10 mice for the CFA group, n = 8 for the sham group, two-way ANOVA, F_{(3, 64)} = 13.78, p < 0.001; Post hoc Turkey’s test, CFA 3D vs Sham 3D, p = 0.01). However, after listening to the 55-dB SNL white noise with the 50-dB ambient noise for 40 min, there are no significant changes in mechanical threshold (two-way ANOVA, F_(1,64) = 0.17, p = 0.68; Post hoc Turkey’s test, CFA 3D vs Sham 3D, p = 0.14, Figure 1(a)). In mice suffering inflammatory pain for 2 weeks, 5-dB SNR white noise did not rescue the decrease of mechanical nociceptive threshold (CFA 2W vs Sham 2W, p = 0.006 for control group, p = 0.002 for 5 dB SNR group, Figure 1(a)). The thermal nociceptive threshold was detected by the hot-plate (55°C) test and tail-flick test. The 5 dB SNR had neither affected the chronic CFA-treated mice nor the acute ones (n = 10 mice for the CFA group, n = 8 for the sham group, two-way ANOVA, F_(1,64) = 0.0045, p = 0.95, Figure 1(b)). There are no significant changes in the tail-flick latency before and after the 5-dB SNR white noise in CFA mice (n = 10 mice for the CFA group, n = 8 for the sham group, two-way ANOVA, F_(1,64) = 0.02, p = 0.88, Figure 1(c)). In all the experiments above, the 5-dB SNR does not affect the nociceptive behavior in sham mice.

Figure 1.

No evidence of a sound-induced analgesic effect in the CFA model of mice. (a) Comparison of the mechanical nociceptive threshold. There was no significant difference in hind paw withdrawal to von Frey filaments before and after being exposed to 5-dB SNR white noise (n = 10 for the CFA group and n = 8 for sham, *p < 0.05, **p < 0.01, compared with the sham group). (b) The 5-dB SNR white noise had no significant analgesic effect on the latency of hot plate test at 55°C. (n = 10 for the CFA group and n = 8 for sham, **p < 0.01 compared with the sham group). (c) Effect of 5-dB SNR white noise in the tail-flick test. 5-dB SNR white noise did not affect the spinal nociceptive tail-flick reflex.

We also observed the anxiety-like behavior by open-field test after listening to the 5-dB SNR white noise for 40 min. Only in the chronic model (2 weeks after CFA injection), both the time in the center area and the total travel distance was slightly decreased by 5-dB SNR sound exposure (n = 10 mice for the CFA group, n = 8 for the sham group, for the time in the center area, two-way ANOVA, F_(1,64) = 0.44, p = 0.51; for total travel distance, F_(1,64) = 3.18, p = 0.079, Figure 2(a) and (b)). In summary, we observed neither an analgesic effect nor an anxiolytic effect by the 5-dB SNR white noise. On the contrary, the sound may promote thermal pain and anxiety-like behavior. The mechanism remains to be further investigated. The discrepancy between our results and Zhou et al.’s maybe due to the protocol used for detection. The duration of the sound is longer than that in Zhou’s study in order to make sure the animal have adapted to the sound. Besides, we did not use the same sound facilities as Zhou et al. used. The sound level meter we used is a more common device and the sound was played by a laptop. We believe that if the sound analgesia can be used as a therapeutic approach, the condition of application is supposed to be broad.

Figure 2.

5-dB SNR white noise reduced the activity of CFA mice in the open field. (a) Representative traces showing the movement of sham, 3 days after CFA injection and 2 weeks after CFA injection group in the open field for 30 min. (b) The time spent in the center area of the CFA 2W group was slightly decreased after being exposed to 5-dB SNR white noise for 40 min. (c) The total travel distance of the CFA 2W group slightly decreased after exposure to 5-dB SNR white noise (n = 10 for the CFA group and n = 8 for sham, **p < 0.01 compared with the sham group).

In cortical regions related to pain sensation, the anterior cingulate cortex (ACC) plays a vital role in chronic pain and its related emotional disorders.^6–8 Activation of the ACC facilitated nociception and pain-related aversion. On the contrary, inhibition of the ACC caused an analgesic effect. ⁹ Previous studies have proved that there are reciprocal projections between the auditory cortex and the ACC^10–12(Figure 3). And these connections are likely to be excitatory. If so, activation of ACC excitatory neurons by auditory inputs may enhance pain, while activation of interneurons may also contribute to inhibition of pain. In the ACC, interconnections between the two semi-spheres have been founded and their roles in pain perception have been investigated (Li et al. Unpublished data). For the descending modulation, direct projection from the ACC to the spinal dorsal horn neurons has been observed and its facilitatory effect on the spinal excitatory transmission in animal models of chronic pain has been investigated. ¹³ Therefore, the auditory cortex-ACC-thalamus pathway can be another candidate for the sound-induced modulation to the nociception in addition to the auditory cortex-thalamus pathway, and their effect could be opposite or biphasic. It also remains to be investigated if different types of music may activate different populations of neurons in the brain. There, such music induced modulation of pain may be specific for human brains. That may be the reason that sound-induced analgesia may not be a common phenomenon in mice.

Figure 3.

Connections of the ACC, auditory cortex, and subcortical structures. Sound information has been conveyed to the auditory cortex from the Cochleo-vestibular nerve. The auditory cortex sends reciprocal projections to the ACC. The auditory cortex projects to the thalamus to modulate the sound-induced analgesia. The ACC also processes the nociceptive information conveyed by the ipsilateral ACC or the thalamus.

Materials and methods

Animals

In this study, 8–10 weeks old male and female C57BL/6J mice (purchased from Charles River or Jackson Laboratories) were used. These mice were housed, 3–5 per cage in a colony, in a stable environment (23°C–25°C ambient temperature) with ad libitum access to standard lab. mouse pellet food and water on a 12 h light/12 h dark cycle (lights on from 07:00 to 19:00). All husbandry and experimental procedures in this study were approved by the Animal Care Committee of Qingdao International Academician Park.

Complete Freund’s adjuvant injection

Inflammatory pain was induced by injecting 50% complete Freund’s adjuvant (CFA, diluted by sterile saline, 10 μL, Sigma) into the plantar surface of the left hindpaw of each mouse under brief isoflurane anesthesia.

Auditory stimuli

The noise level of the environment was measured in decibels (dB) using a Sound Level Meter (DL333201, Deli, China). White noise was played by a laptop (Redmi, China).

Mechanical withdrawal measurement

The mechanical hypersensitivity was determined using the up-down method with von Frey filaments (Stoelting; Wood Dale, Illinois) applied perpendicularly to the plantar surface as previously reported. ¹⁴ Mice were individually placed into a plastic cage with wire mesh floors and allowed to acclimate for 30 min before testing. A series of filaments (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, 1.4, 2.0 g) with various bending forces were applied to the plantar surface of the hindpaw until it was bended bent slightly and held for 3 s. Licking, biting, and sudden withdrawal of the hindpaw was was defined as positive responses. An initial filament force of 0.4 g was applied to test if the mouse was sensitive to this force. If the positive response occurred, the filament force was incrementally decreased until a negative result was obtained with an interval of 3–5 min between two tests. If the mouse was insensitive to 0.4 g filament force, a stronger filament force was applied until a positive response was obtained. The paw withdrawal thresholds were finally determined using the up-down method until the positive/negative responses crossed five times.

Open field test

The open-field test was performed as previously described. ¹⁴ The open field consisted of an opaque cube (40 × 40 × 30.5 cm) and was divided into a center zone (20 × 20 cm) and an outer zone as the periphery. A single mouse was placed into the arena center and allowed to explore freely for 15 min with dim illumination. The movement traces were tracked using the tracking master v3.0 system and all measurements (total distance, time in center, entries) were quantified relative to the mouse body.

Hot plate test

The mouse was placed on a hot plate set at 50 ± 1°C or 55 ± 1°C. And the latency time was recorded when the reaction of the hind paw (licking, shaking, or lifting) first appeared. The cut-off times (40 s for 50°C and 20 s for 55°C) were used to avoid tissue damage. Mice were measured a total of three times with an inter-trial interval of 30 min. The average of three repeated measurements was calculated as the final latency time.

Tail flick test

The tail-flick (TF) reflex was measured using a 50 W projector lamp which produced noxious radiant heat. The TF latencies to reflexive removal of the tail from the heat were recorded for three repeated measurements with an inter-trial interval of 30 min. The cut-off time of 10 s was used to avoid heat damage to the tail.

Statistical analysis

All data were reported as means ± S.E.M. OriginPro 2021 and SPSS 22.0 softwares were separately used for plotting figures and data analysis. Statistical significance was assessed using the two-way ANOVA. In all cases, p < 0.05 was considered to be the threshold for statistical significance.