CFA‐treated mice induce hyperalgesia in healthy mice via an olfactory mechanism

Social interactions with subjects experiencing pain can increase nociceptive sensitivity in observers, even without direct physical contact. In previous experiments, extended indirect exposure to soiled bedding from mice with alcohol withdrawal‐related hyperalgesia enhanced nociception in their conspecifics. This finding suggested that olfactory cues could be sufficient for nociceptive hypersensitivity in otherwise untreated animals (also known as “bystanders”).

The sensory channels through which social context leads to changes in nociception are only now beginning to be defined.Visual cues have been shown to alter nociceptive behaviour in an observer animal, depending on the demonstrator's pain state and the relationship between the interacting animals (Langford et al., 2006;Li et al., 2018;Pitcher et al., 2017).However, given the complexity of social communication, it is likely that multiple senses contribute to social modulation of pain.In rodents, olfactory communication plays an important role in social interactions, and rodents exhibit odour-induced changes in affective and behavioural responses in social encounters (Andraka et al., 2021;Takahashi et al., 2013).Indeed, odours of a stressed rat can produce an antinociceptive effect in a same-sex conspecific (Fanselow, 1985).
Our laboratory recently reported that housing animals in the same room as conspecifics with hyperalgesia associated with withdrawal from alcohol or morphine, or with persistent inflammation, is sufficient to induce hyperalgesia in otherwise experimentally naïve mice (termed "bystander mice", Smith et al., 2016).An analogous phenomenon is seen in prairie voles (Walcott et al., 2018).Because animals in these studies were housed in separate cages, it is unlikely that the relevant cues were visual or auditory (e.g., ultrasonic vocalizations) in nature.Rather, olfactory cues seemed to be important, since exposure to soiled bedding from mice undergoing alcohol withdrawal was sufficient to induce nociceptive hypersensitivity (Smith et al., 2016).This latter finding indicated that odorants emitted by animals undergoing withdrawal from alcohol have the potential to modulate nociception.However, whether olfactory cues contribute to social modulation of pain more generally remained to be determined.
The present study was designed to determine whether hyperalgesic states induced by exposure to conspecifics subjected to persistent inflammation are mediated by olfactory cues, and whether the odorants mediating these olfactory responses modulate nociception by engaging the olfactory sensory system.We therefore tested whether exposure to bedding from C57BL/6J mice with inflammatory pain induced by complete Freund's adjuvant (CFA) would induce hyperalgesia in otherwise untreated mice.Our first experiment tested the effects of long-term (15 days) exposure to bedding on nociceptive behaviours.We next tested effects of an acute bedding exposure, since it was recently demonstrated that a brief interaction with CFA-treated mice can also induce enhanced nociception in same-sex conspecifics (Smith et al., 2021).Then, to determine whether odorants from hyperalgesic mice engage olfactory circuits to modulate nociception, we tested the impact of chemical ablation of the olfactory epithelium on the response to soiled bedding.Finally, to identify possible constituents of the olfactory cue or cues, we used gas chromatography-mass spectrometry (GC-MS) to analyse the volatile compounds in bedding from CFA-treated and control mice to identify changes in the chemical profile following CFA treatment.

| Animals and housing
Adult female and male C57BL6/J mice, 8 weeks of age, were purchased from the Jackson Laboratory (Sacramento, CA).Upon arrival, mice were housed three per cage in standard shoebox cages (18.4 × 29.2 × 12.7 cm) in the Department of Comparative Medicine animal facility at Oregon Health & Science University, provided with 1/2" pelleted cellulose bedding (Biofresh™, Patterson, NY), cotton nestlets and Enviro-Dri for nest building/thermoregulation, as well as access to food (LabDiet 5001; LabDiet) and water ad libitum.They were maintained on a 12-h light: dark cycle (lights on at 0600), with stable temperature and humidity (21 ± 1°C, 32 ± 7%).Experiments were performed between 8:00 and 14:00.The light intensity in the colony cages ranged between 50 and 250 lux during the light phase, depending on position within the cage.Light intensity during the transfer experiments and buried food tests was between 150 and 250 lux during the light phase.Light intensity during testing was 250-350 lux.
After at least 4 days in the facility, mice were transferred to experimental rooms and singly caged for 7 days of habituation.During the entire experiment, animals bedding from mice with inflammatory pain identifies a number of compounds indicative of disease states.These findings demonstrate the importance of olfactory system in influencing pain states.
were housed in shoebox cages with wire tops but without filter lids to allow olfactory communication among neighbouring cages.These cages are placed on three-shelf racks (150 cm width × 61 cm depth × 188 cm height).A total of 150 female and 106 male mice were used in this study.Their body weights were between 20 and 30 g during experimentation.All protocols were approved by the Oregon Health & Science University Animal Care and Use Committee and performed within the National Institutes for Health Guidelines for the Care and Use of Laboratory Animals, as well as the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioural Research.

| Drug treatments
2.2.1 | Localized inflammation CFA (10 μL, 1 mg/mL heat-killed and dried Mycobacterium tuberculosis, Sigma-Aldrich, F5881) was used to induce an inflammatory response.Animals were anaesthetised (3.5% isoflurane), and CFA or saline (10 μL) was injected into the plantar surface of either the left or right hind paw.Mice were then returned to their home cages to recover fully before being tested or interacting with other mice.

| Ablation of olfactory epithelium
Dichlobenil (2,6-dichlorobenzonitrile, Sigma-Aldrich, D57558, 60 mg/mL in DMSO) was used to induce temporary olfactory dysfunction (Bergman et al., 2002;Mori et al., 2000).Dichlobenil (120 mg/kg) or vehicle (DMSO, 2 mL/kg) was injected i.p. 24 h before behavioural testing (Lazarini et al., 2012).Both dichlobenil-and vehicle-treated mice were provided with normal chow softened by water on the bedding to prevent food or water deprivation should they fail to locate these resources on the wire top due to olfactory dysfunction.Animals' body weight and general behaviours were monitored throughout the experiment.

| Von Frey mechanical nociceptive test
The von Frey testing was performed in transparent Plexiglas chambers (9 ×9 × 9 cm) placed on a metal grid sheet elevated 40 cm above the benchtop.The metal grid sheet has round holes of 3 mm diameter with 5 mm spacing (center to center), to allow access to the hind paws with von Frey filaments.
Prior to beginning testing, mice were acclimated in the chambers for 3 h each day for at least three days.On each testing day, mice were placed in the chamber for additional acclimation, until they stopped exploring, approximately 3 h.The "ascending stimulus" method was used (Deuis et al., 2017), with forces ranging from 0.04 to 4 g.Each filament was applied to the center of the mouse's hind paw plantar surface (area surrounded by the toe pads) up to five times.The application of the filament and the delivery of the force was indicated by the bending of the filament.Each application lasted for 6-8 s, with at least 5 s between applications.A positive nociceptive response was noted when the mouse exhibited at least one of the following behaviours: twitching, shaking, stomping, quick withdraw and licking of the hind paw.These behaviours were sometimes accompanied by a brief and sudden opening of the plantar and toes spreading out, as well as guarding/defensive behaviours afterwards.If three stimuli of a given force failed to evoke a response, the filament of the next higher force was then tested.If three out of five stimulations evoked nociceptive responses, then the force of that filament was considered the mechanical nociceptive threshold for that paw, and that paw was not tested further for that data point.

| Hargreaves thermal nociceptive test
The glass platform of the Hargreaves apparatus (IITC Model 400 Heated Base and Series 8 software Model 390G) was maintained at around 32°C throughout the habituation and testing period (Gaskill et al., 2012).The intensity of the radiant light source was kept at 5% in idle and at 22% during stimulation.
Von Frey testing always preceded thermal testing to avoid potential sensory sensitization induced by repeated heat stimuli.In the days leading up to testing, mice were acclimated in the acrylic enclosures (10 × 10 × 12.7 cm) on the Hargreaves heated glass platform for 1 h.On testing days, after completing the von Frey testing, mice were placed in the Hargreaves apparatus until they stopped moving around, approximately 30-45 min.The stimulus was applied at the center of the hind paw plantar surface, in the area surrounded by the toe pads.A timer was started at the initiation of the light stimulation and manually stopped when the mouse exhibited a nociceptive response as defined above.Both left and right hind paws of each mouse were tested four times with at least 30 s between stimulations, and the final three averaged to define response latency for that paw.The first set of the total four sets of latency data were excluded due to inconsistent responses to the initial stimulation.

| Buried food test
To confirm the effect of dichlobenil on olfactory function, animals were tested for their ability to locate buried food.For 2-3 consecutive days before the test, singly housed male and female mice received a piece of palatable food (chocolate chip or dried cranberries, Bio-Serv) in their cages in addition to chow.The cages were checked the following day to verify that the food had been consumed.Female mice were tested with chocolate chips.Later, male mice were tested with dried cranberries, since chocolate chips were not available at that time.
All chow pellets were removed 20 h before testing.On baseline testing day, each mouse was tested alone in a clean cage with fresh bedding in a clean room, without the presence of other mice or food.The mouse was allowed 5 min to explore the testing cage, then briefly removed while the experimenter buried a piece of palatable food 1 cm below the surface of the clean bedding, and then returned to the cage.A filter top was placed to prevent the mouse from escaping.The mouse's behaviour was observed from 2 m away, and the latency for the mouse to find the food recorded.If the mouse did not find the food within 15 min, the latency was recorded as 15 min.Chow pellets were replaced after the test when the animal was returned to the home cage after the test.
Latency to finding the palatable food was assessed at 1, 2, and 8 days after administration of dichlobenil, and compared to baseline latency.To determine whether any failure to locate the palatable food was due to loss of motivation for the food rather than olfactory dysfunction, the buried treat was moved onto the surface of the bedding in the path of the test mouse at the end of the 15-min test.The mouse's consummatory behaviour when they came across the food was documented.

| Prolonged bedding exposure
Female C57BL6/J mice were separated into two rooms for habituation.This experiment was performed using females since it has been shown that males develop mechanical hypersensitivity when cohoused with CFAtreated same sex conspecifics (Smith et al., 2016).In each room, mice were singly caged with wire tops without filter lids and cages placed on three-shelf racks (150 cm width × 61 cm depth × 188 cm height).These mice were divided into six treatment groups: CFA and SAL, mice receiving intra-plantar injection of CFA or saline; BS-CFA and BS-SAL, mice that were "bystanders" of either CFA or SAL mice housed in the same room with the corresponding treated animals; OLF-CFA and OLF-SAL, mice that exposed to the bedding from CFA or SAL mice and housed in different rooms from the above and each other.Cages of BS mice and mice receiving intra-plantar injections were placed next to each other alternately in their designated room.Similarly cages of OLF mice and cages containing the bedding were placed next to each other alternately in their room.There was no additional space between cages.
All six groups and a group of naïve females were tested for mechanical and thermal nociceptive responsiveness.Measurements were taken on 2 consecutive days before any treatment, and the average of the two days used as baseline (BL, Figure 1a).CFA was injected in the left hind paw of the CFA and SAL mice (marked as Tx), and CFA mice and their bystanders were tested on days 1, 3, 5, 7, 9, 11, 13, and 15 post-Tx.At the end of testing day 1, the thickness of the CFA-injected paw was measured as an indicator of the inflammation.Control experiments in which mice receiving saline intraplantar injections and their bystanders were tested were conducted separately on different days.
To expose OLF-CFA and OLF-SAL to bedding from CFA and SAL mice, clean and empty shoe box cages were placed adjacent to home cages of the OLF-CFA and OLF-SAL mice, and were used to hold soiled bedding during the exposure.Soiled bedding (50-60 g in total) was collected from 6 cages of CFA or SAL mice on testing days.Clean bedding was added to replace that removed and ensure consistency in bedding quantity.The soiled bedding from the 6 cages was evenly mixed and distributed into the empty shoe box cages starting on the first testing day of bystander groups (marked as Tx for OLF groups, Figure 1a).Every other day after that, newly collected soiled bedding was added to the existing bedding.Twentyfour h after every addition of soiled bedding, mice in OLF groups underwent nociceptive testing, i.e., OLF mice were tested on days 1, 3, 5, 7, 9, 11, 13, and 15 post-Tx.

| Acute bedding exposure
We next determined whether 1 h exposure to either a conspecific or bedding from a conspecific subjected to CFA treatment was sufficient to induce behavioural hypersensitivity (experimental timeline in Figure 3a).Mice in the BS-CFA and BS-SAL groups were, respectively, allowed to interact with a CFA-or saline-treated conspecific for a period of 1 h.Mice in the OLF-CFA and OLF-SAL groups were respectively exposed to bedding from CFA-and saline-treated conspecifics for a period of 1 h.During the exposure, bedding was placed in the testing chamber but physically separated from mice with Plexiglas dividers.Mechanical threshold (von Frey) was determined prior to exposure (baseline, BL), immediately after interaction or bedding exposure (0 h), and at 1 and 3 h post-exposure.
The effect of dichlobenil (Dich) or vehicle (VEH, DMSO) on nociceptive behaviours in BS and OLF groups was assessed immediately and at 1, 3, and 24 h.The experimenter was blinded to Dich/VEH treatment.
2.8 | SAL and CFA bedding collection for

GC-MS
Soiled bedding from male stimulus mice in the CFA and SAL treatment groups was collected before and 2 days after intra-plantar injection for each of three experimental cohorts.Clean bedding, which contained compounds existing in the bedding material and absorbed from the room atmosphere throughout the experiment, was collected the same time as the soiled bedding and used as a background control.All bedding was from the same batch to maximize the consistency of the samples.Three samples (4 g each) from the CFA-treated and SAL-treated groups were placed in 40 mL VOA vials with Teflon-lined septa (Restek Pure Chromatography, Bellefonte PA).
2.9 | Headspace solid-phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) analysis SPME GC/MS analysis was performed using a Leco Pegasus 4D GC × GC-TOFMS (Leco, St. Joseph, Michigan).The SPME assembly (Sigma-Aldrich, St. Louis, MO) used polydimethylsiloxane/divinylbenzene-coated fibres.Each SPME sampling event was carried out by inserting the fibre into a bedding sample vial for 30 min.The fibre was then inserted into the GC injector (235°C, 10 min), which contained an SPME liner (Restek, Bellefonte, PA) for desorption.The GC oven temperature for each sample was programed as follows: held at the initial 45°C for 3 min, then increased by 10°C/min increment to reach and hold at 200°C for 2 min, followed by 5°C/min increment to 245°C.The GC column (Agilent DB-VRX with 1.4 μm film thickness, 0.25 mm i.d., and 60 m length) interfaced directly into the MS source (200°C).The MS detector was at 1650 V, 50 spectra/s, 35-500 atomic mass units.Samples from each cohort were analysed on one day.The data were processed using the Leco ChromaTOF software with signal noise ratio (S/N) at 30.Compound identification was made by matching spectra to the NIST mass spectral library.

| Von Frey test
Mechanical thresholds for the injected (left) paw are reported for experiments that included CFA-or SALinjected animals.In the acute exposure experiments, in which there were no paw injections, thresholds for the two hindpaws were determined and averaged to define the threshold for that animal.Temporal smoothing was applied by averaging thresholds obtained on two consecutive testing days.Data were analysed with two-way and three-way ANOVA with repeated measures, followed by a post hoc Dunnett's multiple comparisons test within treatment groups comparing to BL, or a post hoc Sidak's multiple comparisons test between treatment groups at individual time points.

| Hargreaves test
Paw withdrawal (PW) latency is reported for the left hindpaw.After temporal smoothing, data were analysed using two-way ANOVA with repeated measures, followed by a post hoc Dunnett's multiple comparisons test within treatment groups comparing to BL, or a post hoc Sidak's multiple comparisons test between treatment groups.

| Buried food test
Latencies to finding the palatable treats before (baseline) and at different time points after administration of dichlobenil or vehicle were analysed with two-way ANOVA with repeated measures on Time, followed by post hoc Dunnett's multiple comparisons test comparing to baseline, and Sidak's multiple comparisons test between treatment groups.

| GC-MS
To semi-quantifiably analyse the volatile chemical composition of the bedding sample, peak area was used to indicate the relative abundance of the compound.Relative abundance (RA) was calculated by averaging the peak areas of the compound from the triplicate samples.Fold change (FC) after CFA or SAL injection was calculated for each compound using the formula: FC for each compound was compared to FC = 1 using a one-sample t-test.
Data are presented as mean ± SEM, and p < 0.05 was considered statistically significant.GraphPad Prism (v.9.4.1) was used to perform statistical analyses and to generate result figures.Detailed statistics are shown in Table 1.Experimental schematics were visualized with BioRender.  | RESULTS

CFA-treated mice and their bedding induced mechanical but not thermal hypersensitivity in otherwise naïve mice
The first experiment determined whether 15-days exposure to bedding from CFA-treated conspecifics (OLF-CFA group) induced hyperalgesia.For comparison, whether "bystander" mice (BS-CFA) housed in the same room as the CFA-treated animals exhibited hyperalgesia was also determined (Figure 1a).CFA induced inflammatory edema in the injected (left) paw (Figure S1) and caused both mechanical and thermal hypersensitivity in the injected paw that lasted for the duration of the experiment (Solid red circles in Figure 1b,d).Both OLF-CFA and BS-CFA mice developed lower mechanical thresholds (OLF-CFA: p < 0.0001 at all time points compared to BL. BS-CFA: p < 0.05 from day 9-15 compared to BL, Figure 1b).However, neither OLF-CFA nor BS-CFA mice showed statistically significant changes in their responses to thermal stimulation (In both groups, p > 0.09 for each time point compared to BL in post hoc Dunnett's test, Figure 1d).
Similar to naïve mice, vehicle control mice receiving intra-plantar saline injection did not show altered nociceptive behaviour in either the von Frey or Hargreaves test (Figure 1c,e; p = 0.08 and p = 0.67, respectively, with one-way ANOVA repeated measure), and there were no changes in mechanical or thermal responses in the BS-SAL or OLF-SAL groups.
Though BS-CFA mice were housed individually, the cages were made with transparent material that would allow visual observation of neighbouring animals' nocifensive behaviours directed towards the injected (left) paw.To examine whether visual cues led to any lateral bias in nociceptive responding in the bystander group, we compared nociceptive thresholds of the left and right hind paws in BS-CFA and CFA groups.In the CFA-treated mice, mechanical and thermal thresholds were significantly reduced in the left compared to the right paw (Figure 2a,c, p < 0.0001 and p < 0.001, respectively).By contrast, the BS-CFA mice showed similar nociceptive thresholds in the two hind paws (Figure 2b,d).This observation indicated that any potential visual communication between CFA and BS-CFA mice in this setting did not result in lateral bias in nociceptive response.We next determined whether an acute bedding exposure would be sufficient to induce enhanced nociception.We examined the von Frey responses of three groups of male and female mice: OLF-CFA, mice who had 1-h exposure to bedding of CFA-treated conspecifics; BS-CFA, mice with 1-h interaction with the same CFA-treated conspecifics, and BS-SAL, control mice with 1-h interaction with SALtreated conspecifics (Figure 3a).During the 1-h interaction with same-sex conspecifics, no fighting was observed in male mice.The von Frey thresholds were measured at 0, 1, and 3 h after bedding exposure or interaction (Figures 3b,c).In both male and female mice, OLF-CFA and BS-CFA groups exhibited significantly lower mechanical thresholds than BS-SAL controls (effect of treatment: male, p < 0.0001 for BS-CFA and p = 0.0035 for OLF-CFA; female, p = 0.024 for BS-CFA and p = 0.0037 for OLF-CFA compared to BS-SAL), and developed mechanical hyperalgesia compared to baseline.Mechanical hypersensitivity was seen in both hind paws in both male and female bystanders and bedding-exposed mice (Figure S2c-f).

| Olfactory ablation inhibited the acute hyperalgesic effect of soiled bedding from same-sex conspecifics
Ablation of the olfactory epithelium (via dichlobenil) was used to determine the contribution of olfactory function to hyperalgesia.The efficacy and duration of olfactory ablation were verified using the buried food test.Compared to vehicle (VEH)-injected control mice, mice receiving dichlobenil (Dich) failed to retrieve the treats buried underneath the bedding within the 15-min cut-off for two days after Dich administration (Figure 4).When the same treat was placed on the surface of the bedding, each mouse located and consumed the treat within 1 min (data not shown).A retest on post-injection day 8 revealed that the ability to locate the treat had recovered to baseline levels.
Based on these results, Dich or VEH was administered to OLF-CFA or BS-CFA mice 24 h before acute bedding exposure or interaction, and von Frey testing was conducted within 2 d after administration (experimental scheme in Figure 5a).VEH-treated mice exhibited significantly lower mechanical thresholds when exposed to bedding from the CFA-treated donors compared to SAL-treated donors (effect of donor group: p = 0.0075 for males, Figure 5b; p = 0.0056 for females, Figure 5d).By contrast, Dichtreated animals did not develop hyperalgesia when exposed to bedding from CFA-treated conspecifics (right panels in Figure 5b,c, main effect of bedding group p < 0.96 for both sexes).We also tested Dich-treated male bystander mice.There was no difference in mechanical nociception between Dich-treated BS-CFA and Dichtreated BS-SAL groups (p > 0.90, Figure 5c, right).By contrast, VEH-treated, olfaction-intact bystander mice showed significant hyperalgesia following a 1 h interaction with CFA-treated animals compared to interaction with SAL-treated animals (BS-CFA versus BS-SAL, p < 0.0001, Figure 5c, left).
These data confirm that a 1-h exposure to bedding from same-sex conspecifics with CFA-induced inflammatory pain induces enhanced mechanical hypersensitivity in both male and female mice.More importantly, they indicate that olfactory function is required for social enhancement of nociception produced by exposure of mice to conspecifics subjected to persistent inflammation.

| treatment altered the profile of volatile compounds in bedding
To identify the volatile odorant compounds in the bedding from CFA-treated animals that have the potential to convey information to conspecifics, we performed GC-MS analysis on bedding from CFA-and SAL-treated donors.GC-MS analysis revealed approximately 200-300 volatile compounds in each sample, which would include compounds secreted by mice and released from the bedding material, as well as other background chemicals in the experimental environment.
CFA or SAL injection did not result in appearance or disappearance of any compound in soiled bedding.There were 110 compounds found in the CFA group before and after injection, and 130 in the SAL group, with 101 of these seen in both groups (Figure 6a, Table 2).Of the shared 101 volatile compounds, 8 were consistently more abundant in clean bedding (2-9.8 fold, indicated by superscript c in Table 2).These likely existed in the environment, but declined in soiled bedding, possibly due to interaction with mice or their excretions.Another 31/101 compounds were found at equivalent levels in both clean and soiled bedding, suggesting that these arose from the bedding itself or the environment, and not from mice.After excluding compounds from the environment and SPME process, the remaining 61 compounds were more abundant in the soiled than clean bedding, both before and after intra-plantar injection, including 22 compounds detected exclusively in soiled bedding.This indicated that mice were likely the source of these 61 compounds, and we focused our analysis on these compounds.
Eight compounds that were less abundant in soiled compared to clean bedding were argon (#1), acetaldehyde (#4), 2-butene (#5), butane (#6), methacrolein (#21), butanal (#23), pentanal (#42), and nonanal (#99).Some of these 8 compounds showed further decrease in abundance in bedding 2 days after SAL or CFA injection (Figure 7b), but no changes were statistically significant.The study expands the growing examples demonstrating social transfer of hyperalgesia between rodents (Lu et al., 2018;Luo et al., 2020;Martin et al., 2014;Mogil, 2015;Smith et al., 2016Smith et al., , 2021)).While rodents housed in individually ventilated cages are unlikely to be influenced by animals in other cages, the social context of animals undergoing nociceptive tests is important.Specifically, here we showed that contact-free presence of soiled bedding from mice subjected to CFA-induced inflammation and hyperalgesia led to mechanical hypersensitivity in  otherwise naïve C57BL/6J mice after both prolonged and acute exposures.Transient partial lesion of the main olfactory epithelium prevented this hypersensitivity in male and female mice, demonstrating that the pro-nociceptive effect required the main olfactory system.Chemical analysis of volatile compounds in the bedding revealed a general increase in the abundance of potential odour molecules in the CFA-treated group, whereas a large proportion of such compounds showed a decrease in the saline-treated group.Thus, behavioural changes in nociception induced by olfactory signals are unlikely to be explained by a change in abundance of one or two molecules.
In both long-term and short-term exposure paradigms, bedding from same-sex conspecifics treated with CFA was sufficient to produce mechanical hypersensitivity.However, while CFA-treated mice exhibited thermal hypersensitivity in Hargreaves test, neither "bystander" mice nor mice exposed to bedding from CFA-treated animals showed significant thermal hyperalgesia.Thermal hypersensitivity has been reported in bystanders using various thermal tests, such as tail immersion test, tail flick and hotplate tests (Mohammadi, Ahmadi-Zeidabadi, et al., 2020;Mohammadi, Kohlmeier, et al., 2020;Smith et al., 2016;Smith et al., 2021) In the present study, olfactory ablation using dichlobenil prevented CFA-bedding-induced nociceptive hypersensitivity.Dichlobenil damages the dorsomedial area of the main olfactory epithelium, an area that contains olfactory sensory neurons (Bergman et al., 2002;Mancuso et al., 1997;Vedin et al., 2004), but spares vomeronasal neurons (Denizet et al., 2017;John & Key, 2003;Piras et al., 2003).Because impairments of olfactory behaviour not necessarily closely match morphological evidence of olfactory damage (Denizet et al., 2017;Genter et al., 1996;Lazarini et al., 2012;Lee et al., 2021;Yoon et al., 2005), we validated dichlobenil's ability to impair olfactory function using the buried food test and tested the effects of bedding exposure within the 2-days window of confirmed olfactory impairment.We also confirmed that dichlobenil treatment did not reduce animals' motivation to consume the treats.The fact that dichlobenil interfered with hypersensitivity induced by exposure to both CFA-treated animals and their bedding strongly argues that olfaction was the primary sensory channel mediating hypersensitivity in these experiments.
Our finding that ablation of the main olfactory epithelium prevented hyperalgesia induced by interaction with a CFA-treated conspecific is inconsistent with an earlier report (Langford et al., 2006) that olfactory lesion by ZnSO 4 did not affect hyperalgesia in mice interacting with a cage mate exhibiting writhing induced by acetic acid.However, mice with acetic acid-induced abdominal pain display prominent spontaneous nociceptive behaviours, by comparison with mice subjected to localized inflammation of a single extremity, and visual observation of spontaneous pain behaviours has often been reported to significantly impact the behaviour of a bystander animal (Langford et al., 2006;Li et al., 2018;Pitcher et al., 2017).Moreover, the effect of a given social encounter may vary, depending on the pain modality (Pitcher et al., 2017).Viewed collectively, these findings suggest that the sensory channel(s) used to assess the health status of conspecifics could depend on particular pain behaviours.
Given olfactory cues present in bedding from CFAtreated animals were sufficient to induce tactile hypersensitivity, we used GC-MS analysis to semi-quantitatively analyse volatile compounds in bedding taken from cages housing the CFA-and SAL-treated animals, before and after treatment.Bedding content reflects the bedding material itself, the housing environment, and compounds released by the animals housed in that cage, and the balance between accumulation (presumably from the animals) and evaporation/degradation over time.That is, a net increase in abundance of a volatile compound indicates that more molecules of this compound were released into the bedding than evaporated, and a net decrease in abundance implies that any release of the compound did not make up for losses from evaporation.CFA and SAL treatment led to differential shifts in abundance of volatile compounds in bedding.While 90% of analysed compounds were increased in abundance in the bedding after CFA treatment, more than 60% of the same compounds were decreased (some significantly) after SAL treatment.
Six volatile compounds were significantly decreased in abundance after injection of SAL but moderately increased after injection of CFA.Several of these compounds are known to be elevated in urine from animals in of stress, aging, and disease.For example, 2-heptanone was elevated in urine from stressed mice and rats (Gutierrez-Garcia et al., 2006;Schaefer et al., 2010), and exposure to 2-heptanone increased immobility in the forced-swim test (Gutierrez-Garcia et al., 2007) and increased activity in amygdala (Contreras et al., 2012).Interestingly, 4-methyl-6-hepten-3-one, significantly reduced post-injection in bedding from SAL-treated animals but elevated in bedding from CFA-treated animals in our study, has been found to be elevated in urine of aged mice and mice treated with bacterial endotoxin, but decreased in the urine of tumour-bearing mice (Hanai et al., 2012), which warrants further and more specific investigation of this compound as a social signal.It thus seems reasonable to conclude that compounds that were decreased in bedding from SAL-treated but not CFA-treated groups could provide a signal to conspecifics, and that activation of olfactory pathways by these compounds, individually or collectively, can contribute to social influences on pain-related behaviours.Though there has not been much research into rodent-sourced volatile compounds modulating pain behaviours, rodents release volatile alarm or appeasing pheromones (Brechbühl et al., 2013;Kiyokawa, 2015;Kiyokawa et al., 2023;Morozov & Ito, 2018).We did not observe any of these known pheromones among the detected volatiles.This observation reciprocates a recent finding that social modulations of pain and fear diverge into different central mechanisms after receiving the respective social signals during an interaction (Smith et al., 2021).
Eight volatile compounds were more abundant in clean than soiled bedding.Most of these compounds are common in industrial manufacturing processes, and hence reasonable to exist in bedding materials.The fact that they decreased in the presence of the animals suggests that mice or mice excreta lead to degradation or increased evaporation of these molecules.However, since there was no difference between the CFA-and SAL-treated groups in the bedding content of these compounds, it is unlikely that the decrease over time contributed to the hyperalgesia seen in the CFA-treated group.It is nonetheless important to acknowledge that environmental factors could modulate nociception, or serve as a background or foundation for socially derived signals.
There were technical limitations in the present study that could be improved in future investigations.While many compounds were decreased in abundance in bedding from the SAL-treated animals compared to CFA-treated animals, relatively few compounds showed statistically significant changes in abundance in either group compared to a pre-injection baseline.This is likely due to the small sample size, a limitation of the manual sampling during GC-MS analysis.Future studies could increase SPME temperature to yield a larger VOC extraction for GC-MS analysis.However, such analysis may also present a somewhat different VOC profile from the olfactory signal that mice received at room temperature.Different types of SPME fibres to extract samples could also be considered.Furthermore, it must still be acknowledged that machine detection may not be as sensitive as the rodent olfactory system.For example, there are reports that various animal species can recognize human diseases via odorant signals (Church & Williams, 2001;Guest et al., 2021;Piqueret et al., 2022;Williams & Pembroke, 1989).During the COVID-19 pandemic, it has been noted that canines are able to identify COVID-positive patients with above 80% accuracy after training, while researchers were not able to identify volatile chemical markers for COVID using GC-MS (Mendel et al., 2021).It would not be surprising if GC-MS analysis was unable to identify all relevant compounds detected by mice during a social interaction or bedding exposure.
It is also worth acknowledging that we did not confirm potential nociceptive effects of the GC-MS-identified compounds in behavioural tests.However, behavioural effects of olfactory cues are frequently contextual (Bourne et al., 2013;Kaur et al., 2014;Ryan et al., 2008), in which case the potential target compounds would only exert a pro-nociceptive effect in the specific social situations.The trigger for the observed bedding-induced pro-nociception is thus likely more complex than simple changes in abundance of a few compounds.A complex experimental design that takes the contextual nature of olfactory social cues into account would be needed to further narrow down or confirm the target compounds without false negative or false-positive identification.
Social modulation of pain in humans is complex and nuanced (e.g., Borsook & MacDonald, 2010;Leonard & Cano, 2006;Saarijärvi et al., 1990).A recent study in humans reported that a volatile compound emitted from human body could affect human aggression in a sex-dependent manner (Mishor et al., 2021).It seems plausible, therefore, that biological mechanisms, including those influenced by olfactory cues, could interact with other varied social factors to influence human pain behaviour.

3. 2 |
Acute exposure to CFA-treated mice or their bedding induced mechanical hypersensitivity in otherwise naïve mice

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Exposure to soiled bedding from CFA-treated conspecifics induced mechanical nociceptive hypersensitivity comparable to that from direct interaction with CFA conspecifics.(a) Experimental design.(b, c) Time course of von Frey thresholds of male (b) and female (c) mice in BS-SAL, BS-CFA, and OLF-CFA groups.Measurements at 0 h were obtained immediately after social interaction or bedding exposure.2-way ANOVA with repeated measures followed by a post hoc pairwise comparison between treatment groups in males and females.*p < 0.05; **p < 0.01; ***p < 0.001 comparing to BS-SAL group.Female sample size: BS-SAL, N = 6; BS-CFA, N = 6; OLF-CFA, N = 8.Male sample size: BS-SAL = 9; BS-CFA, N = 9; OLF-CFA, N = 9.

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I G U R E 4 Partial ablation of olfactory epithelium transiently prevented both (a) male and (b) female mice from finding buried treats.Testing before and at 1, 2, and 8 days after i.p. injection of dichlobenil or vehicle (N = 5 /sex/treatment).2-way ANOVA with repeated measures, followed by a Dunnett's Test for comparison with Day 0. Sidak's multiple comparisons test was performed to compare latencies between treatment groups at the same time points.*p < 0.05; ****p < 0.0001 between VEH and Dich on individual days.#### p < 0.0001 comparing to day 0 within Dich group.F I U R E 5 Olfactory ablation mice in both OLF and BS groups from developing nociceptive hypersensitivity after acute exposure to CFA-bedding or interaction with CFA conspecifics.(a) Experimental timeline.(b-d) Time course of von Frey thresholds of olfaction-intact (VEH, left panel) or ablated (Dich, right panel) mice in male OLF (b), male BS (c), and female OLF (d) groups following bedding or social interaction associated with CFA-(solid red circle) or SAL-treated (blue open squares) conspecifics.Within VEH or Dich treatment groups, data were analysed with 2-way ANOVA (Stimulus [CFA, SAL] × Time [BL, 0, 1, 3, and 24 h]).(b) Male OLF mice: there was main effect of bedding ( ## p = 0.0075) in VEH group; no significant difference was detected in Dich group.Sample size: VEH-CFA, N = 9; Dich-CFA, N = 9; VEH-SAL, N = 9; Dich-SAL, N = 6.(c) Male BS mice: main effect of bedding ( #### p < 0.0001) and time (p = 0.0006) were detected in VEH group, with no interaction (p = 0.20).In Dich group, main effect of time (p = 0.039) but not treatment (p = 0.90).N = 9 in each treatment group.(d) Female OLF mice: main effect of bedding ( ## p = 0.0056) and an effect of time (p < 0.0001), but no interaction (p = 0.30) in VEH group.A main effect of time (p = 0.0012) was detected in Dich group.Sample size: VEH-CFA, N = 8; Dich-CFA, N = 9; VEH-SAL, N = 9; Dich-SAL, N = 8.

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I G U R E 6 GC-MS analysis of volatile compounds in bedding from CFA-and SAL-treated animals.(a) Number of detected compounds categorized based on association with CFA and SAL injection and their relative abundance in soiled vs. clean bedding.(b, c) Fold change (FC) in relative abundance 2 days after stimulus mice received CFA (b) or SAL (c) injection compared to pre-injection for compounds that were richer in soiled bedding than clean bedding.Compounds with significant changes in relative abundance (p < 0.05) are highlighted in red and labelled with numbers corresponding to the numbers of chemicals in

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I G U R E 7 FC in abundance in post-CFA and post-SAL bedding for compounds significantly richer in soiled bedding (a) or in clean bedding (b).(a) Seven compounds that were richer in soiled bedding after CFA (1 compound) or SAL (6 compounds) injection.(b) A total of eight chemicals were richer in clean bedding and showed a modest decrease in abundance after both CFA and SAL injection.

Table 2 .
T A L E 2 Volatile that appeared in analysis results of soiled samples.
, as well as Hargreaves test (Langford VOCs that appeared in analysis of soiled samples, with the retention time (R.T.) in minutes, GC-MS identification, molecular formula, and the mean fold change (FC) in abundance 2 days after mice receiving CFA or SAL treatment.The abundance of VOCs used to generate the mean FC was calculated by subtracting the abundance in clean bedding from that in soiled bedding.p value was calculated from three independent experiments.VOCs without mean FC listed for either CFA or SAL bedding are sometimes more abundant in the soiled bedding, but other times more in the cleaning bedding.Superscript c: VOCs appeared consistently lower in the soiled bedding than clean bedding; Superscript s: VOCs present in the environment or from the SPME fibre material. Note: