Poster CC5 - Longitudinal Sex-Specific Differences in Sleep and Pain Pathogenesis in a Mouse Model of Joint Inflammation

Introduction: Females are disproportionately affected by chronic joint disease, suffering worse pain and outcomes (Overstreet 2023, IJERPH). Furthermore, females have higher lifetime risk of developing sleep disorders (Tobias 2021, Clin Chest Med). Sleep and pain have a well-established, negatively reinforcing relationship in which chronic pain can disrupt sleep and poor sleep exacerbates pain experience (Finan 2013, J Pain). Though sleep and pain have strong associations, little is understood about direct mechanistic interactions, especially in chronic joint disease which likely impacts patient response to pain management techniques and, we posit, potentially contributes to sex disparities in joint disease outcomes. Sleep has recently been implicated in contributing to joint disease onset and pathogenesis (Dai 2020, J. Rheumatol), but few studies have investigated sex-specific differences in sleep and pain relationships across joint disease development. While many hormonal, genetic, and psychosocial factors influence pain experience, they do not fully explain the relationship. Preclinical models of joint disease are well-suited to explore these relationships across the whole pathogenic timeline. Behavioral pain assessment in preclinical models is widely used, but sleep architecture is rarely included missing key insights to mechanistic relationships between sleep and pain in joint disease. Here, we examine longitudinal sex-specific changes in pain experience and sleep architecture in a mouse model of inflammatory joint pain to gather key insights for pathogenic differences and potential therapeutic targets and timelines in pain and sleep changes.

Materials and

Methods: Adult C57B/6J mice were pair-housed under standard 12:12 hour light-dark cycles with ad libitum food and water. Animals received intra-articular knee injection of either 1-2% monoiodoacetate (MIA, Male n=2 analyzed of 10, Female n=9 analyzed of 10) or phosphate-buffered saline (Saline, Male n=6 analyzed of 10, Female n=4 analyzed of 10) as control. Baseline hind paw mechanical sensitivity (Chaplan’s up-down protocol, Chaplan 1994, J Neurosci Methods), resting respiration, and 3-state sleep (wake, Rapid eye movement [REM] sleep, and non-REM sleep) were recorded before injection and then bi-weekly for 6 weeks post-injection to model chronic injury. Homecage respiration and 3-state sleep were noninvasively collected using electric field sensors during the 12 hour dark cycle. Sleep measures calculated were: microarousal index (# brief arousals/hour asleep), sleep fragmentation index, average sleep bout duration, percentage time spent in each state (wake, non-REM sleep, REM sleep), percentage sleep events with REM sleep, and REM sleep latency. Respiration measures were collected during non-REM sleep: average respiration rate and respiration rate variability (i.e. breath to breath consistency).
Post-euthanasia, knee tissue was collected and processed for histological sectioning and stained with toluidine blue. These sections were used to measure synovial thickness of ipsilateral knee joint with thicker synovia indicating joint inflammation. Data means across sexes, injuries, and timepoints were analyzed using multifactorial ANOVAs with Tukey’s HSD post-hoc. Correlations were assessed using general linear regression models.

Results, Conclusions, and Discussions: By week 6, female MIA animals develop ipsilateral hind paw mechanical hypersensitivity compared with respective contralateral paw (p < 0.01). In females, increasing mechanical sensitivity correlated with more time spent awake (p=0.002, R2=0.17, Figure 1A) and longer durations of REM sleep events (p < 0.05, R2=0.14). In females, more erratic breathing correlated with reduced microarousals (p < 0.01, R2=0.18), longer sleep bout duration (p < 0.05, R2=0.12), and increased REM sleep latency (p < 0.05, R2=0.10). In males, more time spent awake correlated with shorter REM sleep duration (p < 0.05, R2=0.22) and less time spent in REM sleep (p < 0.05, R2=0.15). There were no significant differences between groups in synovial thickness nor did it correlate with sleep, pain, or respiration changes. Temporally, sleep and pain changes developed asynchronously in males, but together in females (Figure 1B).
This is the first study to examine sex-specific sleep changes across time in a model of mouse inflammatory joint pain. We expect group-level significance upon completion of analysis, however early findings here show important sex-specific relationships between pain and sleep. In females, increasing pain reorganized REM sleep distribution. Furthermore, erratic breathing during sleep can give insight into deteriorating autonomic health which correlated with sleep consolidation in females, likely due to increased sleep pressure. Males appeared to be more protected from pain-related sleep changes and exhibited an uncoupling of sleep and pain, highlighting for the first time longitudinal sex-specific behavioral differences between individual pain and sleep relationships. Males also have some sleep changes in the opposite direction to females (Figure 1B) even with similar pain, indicating more comprehensive measures of behavior (e.g. sleep) are need to distinguish preclinical pathological pain experiences between sexes beyond single modality behavior assays (e.g. mechanical sensitivity).
Pain and sleep are exacerbated in females with joint disease, but pathogenesis is poorly understood and few animal models exist to examine mechanistic interactions. Only recently have sex-differences been explored in pain-sleep interactions. Our work provides sensitive, noninvasive approaches to assess joint pain and sleep in orthopedic models identifying new sex differences in pain-sleep experiences and physiology. Ultimately, our work will identify potential therapeutic targets and timelines to develop personalized pain management therapies.

Acknowledgements (Optional):