(L-463) Hyperexcitable Hippocampal Activity Associates with Knee Pain and Poor Sleep

Introduction:: Debilitating chronic pain affects 50-100 million people in the US, impairing physical and emotional health¹, and consuming more than $250billion in healthcare costs. Chronic pain patients commonly report related sleep dysfunction which can exacerbate pain experience. Moreover, chronic pain and poor sleep can independently alter central nervous system (CNS) functions, further aggravating mental health comorbidities. This triumvirate is poorly understood as few studies include assessment of all three: pain, sleep, and CNS measures. Managing pain is complex, expensive, and often ineffective and this knowledge gap hinders development of better therapies.

While many CNS changes occur in response to chronic pain and poor sleep, the hippocampus is especially sensitive. The hippocampus is a delicate brain structure largely controlling learning and memory. Sleep deprivation and chronic pain can degrade the hippocampus, affecting memory and its ability to adapt in response to stimuli (neuroplasticity). Neuroplasticity is essential for responding to life experiences and can take two primary forms: long-term potentiation (LTP), which strengthens synaptic connections, and long-term depression, which weakens those connections. While chronic pain and hippocampus LTP relationships are largely studied using neuropathic pain models, this relationship is poorly understood in other models of pain, such as inflammatory joint pain, which may have different interactions between sleep, pain, and hippocampal activity. Thus, the objective of this work is to leverage a preclinical model of inflammatory knee pain to characterize pain experience, sleep quality, and hippocampal LTP.

Materials and Methods:: To model inflammatory knee pain, adult c57b/6 mice were prepared for aseptic surgery, anesthetized (2-3% isoflurane), and received a 10µL intra-articular injection of either monoiodoacetate (MIA, n=5 or saline control (n=4). Hind paw mechanical sensitivity, 3-stage sleep (wake, non-rapid-eye-movement (REM) sleep, and REM sleep), resting respiration, and homecage motor behaviors (eating, drinking, grooming, rearing, exploring) were measured before and weekly for 6 weeks after injection. Mechanical sensitivity was assessed using Chaplan’s up-down filament protocol, while sleep, respiration, and motor behaviors were measured using novel, noninvasive electric field motion sensors developed and validated by our group.

Terminal electrophysiology experiments evaluated left and right hippocampus LTP. Animals were deeply anesthetized with isoflurane prior to euthanasia. Brains were dissected, chilled in Ca₂⁺ free artificial cerebrospinal fluid (ACSF) bubbled with 95%O²/5%CO², mounted, coronally sectioned (350µm, Bregma -1.94 mm), then stabilized for 120 minutes in ACSF at 35ºC. Sections were immobilized on an 8x8 microelectrode array (MEA) (MED-A64MD1, Alpha Med Scientific) and imaged to confirm placement. Visual confirmation of evoked field excitatory postsynaptic potentials (fEPSP) dictated the best test stimulation (-40 µA, 10s duration) site to assess hippocampal LTP. Baseline responses were recorded for 30 minutes prior to being tetanized (10-30µA bursts, 100 pulses for 1 second, 100 Hz, 50% amplitude). fEPSP responses were observed for 60 minutes to determine early LTP responses and fEPSP slope was calculated for each waveform and normalized to baseline. Mean differences were analyzed using students t-tests and correlations were calculated using a general linear regression model.

Results, Conclusions, and Discussions:: Results: MIA animals displayed heightened mechanical sensitivity (p< 0.001) and reduced duration of sleep events (p< 0.038) compared to sham controls 6 weeks post-injection. Relative to sham controls, percent time in REM sleep (p< 0.099) tended to be elevated and REM latency (p< 0.056) tended to be reduced. MIA animals exhibited hyperexcited fEPSP slopes compared to saline controls immediately post-tetanus (p< 0.023, Figure 1). There were no group differences at early LTP, though 3/5 MIA animals and 1/2 saline animals were able to maintain plasticity.

Mechanical sensitivity correlated with time spent awake (p< 0.027, R²=0.66), number of REM events (p< 0.011, R²=0.76), and nearly significantly with post-tetanus fEPSP slope hyperexcitability (p< 0.079, R²=0.49). Normalized post-tetanus fEPSP slope hyperexcitability correlated with time spent awake (p< 0.018, R²=0.70, Figure 2) and time spent in REM sleep (p< 0.044, R²=0.59) while trending with several other measures of fragmented sleep. We anticipate these findings to be strengthened upon further analysis and addition of the respiration and motor behavior data.

Discussion: Chronic pain, poor sleep, and altered hippocampal LTP associate in many chronic pain conditions, but this is one of the first studies to explore that relationship in chronic inflammatory knee pain. MIA-induced inflammatory knee pain largely affected duration of sleep and REM sleep measures suggesting premature arousal possibly due to activated somatic pathways. Hippocampal hyperexcitability associated with more sleep measures, especially those indicating fragmented sleep, including REM sleep. As REM sleep plays and important role in neural health, impaired REM sleep may be an indicator or predictor of hippocampal health. While the initial findings from this work reinforce the association between sleep, pain, and altered CNS plasticity, further work is needed to explore temporal causal relationships for inflammatory pain specifically.

Conclusions: Unveiling the intricate relationship between chronic pain, sleep, and altered higher brain functions can help develop more effective treatments to improve patient outcomes, potentially to develop new pain management techniques such as chronotherapy

Acknowledgements (Optional): :

References (Optional): : 1. Gaskin DJ, Richard P. The Economic Costs of Pain in the United States. In: Institute of Medicine (US) Committee on Advancing Pain Research, Care, and Education. Relieving Pain in America: A Blueprint for Transforming Prevention, Care, Education, and Research. Washington (DC): National Academies Press (US); 2011. Appendix C. Available from: https://www.ncbi.nlm.nih.gov/books/NBK92521/