This study is a part of a larger project aiming to evaluate the neurobiological mechanisms underlying the relationship between sleep and pain in people with non-specific chronic low back pain. Specifically, this study aims to evaluate the neurobiological mechanisms underlying the relationship between chronic sleep disturbances and pain sensitivity in people with non-specific chronic low back pain and chronic insomnia.

Sleep disturbances, and especially insomnia, reflect one of the most common comorbidities in people with chronic pain, including people with chronic low back pain. Previous research has furthermore demonstrated that a lack of sleep is associated with an increased pain sensitivity. The goal of this study is to evaluate the role of brain neuroinflammation in the relationship between chronic sleep disturbances and both clinical and experimental pain sensitivity in people non-specific chronic low back pain and/or chronic insomnia.

A cross-sectional study will be conducted across four age- and sex-matched study groups: healthy controls with good sleep habits (Group 1); people with non-specific chronic low back pain and good sleep habits (Group 2); pain-free individuals with chronic insomnia (Group 3); and people with non-specific chronic low back pain and comorbid chronic insomnia (Group 4). The study will be performed over nine consecutive days. All participants will arrive at the lab at a standardized time, relative to their habitual bedtime, in the afternoon of the first study day. There, they will first fill out a number of baseline questionnaires, after which a battery of quantitative sensory test, utilizing both manual and computerized pressure algometry, will be performed to assess experimental pain sensitivity. After being provided a standardized dinner, the participants will then perform a clinical polysomnography (i.e., sleep study) during an over-night stay at the hospital sleep center (UZ Leuven, Belgium) to acquire data on sleep time, stability, and efficiency, as well as to identify participants with combined sleep-disorder-comorbidity profiles (e.g. insomnia + apnea). Directly upon awakening in the morning of the second day, all participants will first fill in a few additional questionnaires, and then be provided a standardized low-fiber breakfast alongside a bolus of pure fiber. Next, the participants will be escorted to the magnetic resonance imaging facility and perform two blocks of brain scanning separated by an approximate 10-min break. During the first block, magnetic resonance spectroscopy and diffusion-weighted magnetic resonance imaging will be performed to measure markers of neuroinflammation, whereas the second block utilizes functional magnetic resonance imaging during an evoked-low-back-pain task to acquire data on back pain-evoked brain activity. After the two scanning blocks, the second study day ends by the collection of a venous blood sample to assess systemic levels of inflammation (via highly sensitive C-reactive protein) and short chain fatty acids. During the following seven days, all participants will complete a sleep diary once per day, provide momentary ratings of pain, sleepiness, fatigue, and affect eight times per day, and wear an Actigraph at all times (except during heavy water contact). During this seven-day period, the participants will also be instructed to collect a stool sample at a time of their own convenience, but preferably within the first three days, to be used for gut microbiota composition analyses. Continuous dietary intake will therefore also be recorded during the first three days of the seven-day period, while participants who are not able to collect their stool sample during any of these three days will continue to record their dietary intake until a stool sample has been collected.

General inclusion criteria (all groups):

• 18-60 years old.
• Ability to speak and understand the Dutch Language.
• Body mass index <30.
• No smoking or nicotine use.
• Low caffeine use (≤3 cups of coffee/day, ≤1 energy drink/day).
• Low alcohol use (≤9 alcoholic units/week).
• No use of neuro- and/or psychopharmacological treatments and/or immunosuppressive agents (including NSAIDs) with a suspected influence on neuroinflammation unless terminated within a sufficient time frame before the start of the study, or unless falling under the Pharmacological exceptions criteria (see below).
• Willing to (try to) adhere to the acute pre-test restrictions, including refraining from analgesics (unless stable use), caffeine, alcohol, and/or strenuous physical activity (>3 METs) in the 24 hours preceding the main test day.

General exclusion criteria (all groups):

• Shift worker.
• Use of blood thinners.
• (History of) Substance dependence/abuse.
• Chronic pain conditions (≥3 months) other than chronic low back pain.
• Severe intrinsic sleep disorders (assessed during a one-night, laboratory-based polysomnography).
• Restless leg syndrome
• Central or peripheral neurological disorder/condition (e.g., epilepsy, multiple sclerosis, peripheral neuropathy).
• History of spinal surgery.
• Major medical (e.g., cardiac disease, cancer) and/or psychiatric disorder/disease (e.g., major depressive disorder, bipolar disorder).
• Claustrophobia.
• Contraindications for magnetic resonance imaging (e.g., metal/cochlear implants, pacemaker).
• Pregnancy or being <12 months post-natal.

Pharmacological exceptions criteria:

- Participants with chronic low back pain and/or (comorbid) insomnia will be allowed to be on a stable low dose of weak pain and/or sleep agents (e.g., Tramadol, Zolpidem) if medically prescribed specifically for their low back pain and/or insomnia, and if approved by the national medicine register of Belgium (FAMHP, Federal Agency for Medicine and Health Products). These participants will also be allowed stable and/or occasional use of low-to-moderate doses of over-the-counter medications for pain and/or sleep that are suspected to have no or minimal impact on neuroinflammation (e.g., paracetamol, melatonin). Habitual use of medications according to the above-stated criteria should be maintained across the entire study period, with the only exception being analgesics taken on an occasional basis. Specifically, because of the extensive battery of pain tests that will be utilized in the study, all analgesics that are only taken occasionally should be refrained from during the 24 hours preceding the main test day (pre-test instructions).

Specific inclusion criteria for "pain-free" groups:

• Currently pain free, defined as having no pain that affects daily living/functioning and/or which has led to a consultation with a healthcare practitioner.
• No episode of low back pain that interfered with daily functioning/living and/or which led to a consultation with a healthcare practitioner within 12 months from the time of inclusion.
• No history of chronic low back pain.

Specific inclusion criteria for "chronic low back pain" groups:

• Low back pain reported on most days (≥50%), every week, for more than 3 months.
• Low back pain-related emotional disress OR functional disability rated as ≥3 out of 10.

Specific inclusion criteria for "good sleeper" groups:

• Stable sleep phase within 10 PM and 10 AM (assessed using the Pittsburgh Sleep Quality Index).
• Average habitual total sleep time between 7 and 9 hours (assessed using the Pittsburgh Sleep Quality Index).
• A Pittsburgh Sleep Quality Index ≤5.
• An Insomnia Severity Index ≤7.
• An Epworth Sleepiness Scale ≤10.
• Sleep efficiency ≥85% during a one-night, laboratory-based polysomnography screening.
• No episode of sleep disturbances that interfered with daily functioning/living and/or which led to a consultation with a healthcare practitioner within the preceding 12 months from the time of inclusion.
• No history of chronic insomnia.

Specific inclusion criteria for "chronic insomnia" groups:

• Chronic insomnia according to the ICSD-3-TR criteria (assessed using an ICSD-3-TR-adapted version of the Pittsburgh Sleep Quality Index).
• An Insomnia Severity Index ≥15.

Participants across all study groups will be recruited from the broad area around Leuven, Belgium, via local flyers and social media. Participants with non-specific chronic low back pain and/or chronic insomnia will also be recruited from the pain and/or sleep center of UZ Leuven, Belgium, as well as via primary care units and patient support groups targeting people in the Leuven area. Participants will be recruited and stratified according to sex and age to enable matching to the primary group of interest (chronic low back pain and comorbid insomnia), and a majority of females (~two thirds) will be recruited to better represent the sex-distribution within the population representative of this group.

Individual participant data (IPD) that underlie results in a publication will be made available to other researchers with all identifying information removed.

The data will become available upon publication with no time limitations.

The data will be publicly available.