Effects of Spinal Manipulative Treatment on Inflammatory Markers in Low Back Pain Patients
Condition: Low Back Pain
MeSH terms: Back Pain; Low Back Pain
Study type: Interventional

Summary: 
      It is expected that mechanical low back pain (LBP) is associated with inflammatory changes
      localized to the affected tissues. Could such changes be detected in cells involved in the
      inflammatory process in an in vitro model? The investigators wish to test such a model to
      compare inflammatory markers in acute and chronic LBP patients and also examine the effect of
      spinal manipulative treatment (SMT) on changing the level of selected key inflammatory
      markers. The investigators hypothesize that:

        1. Proinflammatory markers will be elevated while antinflammatory markers will be reduced
           in acute LBP patients relative to chronic back pain patients as well as in healthy study
           participants who have no LBP or any inflammatory conditions (controls).

        2. SMT will cause a reduction in the production of proinflammatory markers while
           anti-inflammatory markers will increase relative to baseline levels as well as relative
           to controls
    

Detailed description: 
      Background: Spinal manipulative treatment(SMT) may work by reducing mechanical irritation to
      joint tissues (1) and thereby diminishing local inflammation. Evidence for an
      anti-inflammatory effect of instrument-assisted SMT has been observed in an animal model (2).
      These findings are consistent with the recently proposed hypothesis that the origin of all
      pain may be associated with inflammation and augmented production of inflammatory mediators
      (cytokines), principally TNFα (3). TNFα plays a major role in the pathophysiology of
      neuropathic pain-associated inflammation (4) and has been implicated in spinal pain
      syndromes, including intervertebral disc-related low back pain (5, 6) and sciatica (7,8). Our
      recent studies demonstrated that production of inflammatory mediators is elevated in patients
      with chronic cervical spine pain, both in vitro and in vivo, and accompanied by up-regulation
      of chemokine (CC) synthesis (9).

      Recent reports on clinical (10), animal (2) and human in vitro models (11) suggest that SMT
      may exert an anti-inflammatory effect. Thus, Song et al. (2) found that SMT reduced
      inflammatory neuropathic pain. Teodorczyk-Injeyan et al. (11) demonstrated a significant
      attenuation of pro-inflammatory cytokine production in vitro, and no changes in serum
      substance P (SP) levels following SMT. Other studies from our laboratory showed that SMT may
      enhance both the production of and the response to the immunomodulatory cytokine, IL-2, and
      IL-2-dependent antibody synthesis (12, 13). Reduction in serum TNFα levels has been reported
      for cervicogenic headache patients (n=2, case studies) (14, 15). These observations suggest
      that SMT effects may be transduced into cellular components of the immune system.

      Thus far, the clinical relevance of the effect of SMT as a modulator of inflammatory mediator
      production is unknown. Despite evidence of inflammatory pathophysiology of spinal pain (16),
      including a subtype of non-specific spinal pain (17), only one clinical study (18) evaluated
      the correlation between serum TNFα levels, pain intensity and back function.

      In this proposed study we intend to use a clinical model to investigate the baseline levels
      of proinflammatory cytokines in acute and chronic low back pain patients and explore the
      potential anti-inflammatory effect of SMT following a course of manipulative treatments.
      Thus, Aim 1 is to determine baseline pro-inflammatory cytokine levels in individuals
      experiencing acute or chronic lower spinal pain of mechanical etiology, and compare them with
      asymptomatic controls'. Aim 2 is to explore the relationships between SMT, pain level and
      functional impairment, and the production of inflammatory mediators relative to baseline.

      Anti-inflammatory cytokines (IL-10 and IL-1ra) have been found to be produced alongside, and
      in parallel, with their respective pro-inflammatory counterparts (TNFα and IL-1β), and act in
      concert to sustain/restore homeostasis (19). The proposed study will include determinations,
      in addition to pro-inflammatory cytokine (IL-1, TNFα, IL-6) levels, of the levels of IL-1
      receptor antagonist (IL-1ra) and IL-10. Assessment of IL-10 synthesis is particularly
      relevant as this cytokine up-regulates the production of IL-1ra, which competes with active
      IL-1 for binding to IL-1 receptors, and which acts as a potent natural anti-inflammatory
      protein (19, 20).

      Study design; Subject Recruitment: Subjects (volunteers) of both sexes, between the ages 20
      and 60 years, experiencing acute (less than 4 weeks in duration) or chronic (12 weeks or
      longer in duration) mechanical low back pain (experienced between spinal levels L1- L5, with
      or without sacroiliac [SI] joint involvement) will be recruited by Canadian Memorial
      Chiropractic College (CMCC) personnel and posters from CMCC's outpatient clinics, and from
      the general public through newspaper advertisements . CMCC is in the Greater Toronto Area
      (GTA), where the population is a diverse ethnic mix (21). Potential participants who have
      presented to one of the CMCC clinics for the purpose of treatment will be
      encouraged/recruited by interns/clinicians prior to commencement of treatments. Others who
      present to participate in the research study will first be assigned to one of the clinic pods
      for initial assessment prior to entering the study. In no case, however, will participants
      have received SMT 4 weeks prior to commencement of the study.

      Candidates will be interviewed in order to determine eligibility (see exclusion criteria,
      Appendix 1). They will complete the research intake form (Appendix 2) and be given detailed
      explanations about the research protocol (Appendix 3). A cohort of matching healthy
      asymptomatic subjects, recruited from the general population, will serve as controls for the
      determination of baseline cytokine levels.

      Sample size determination: Data published for TNFα levels in chronic neck pain patients
      versus asymptomatic controls (9) were used to calculate a sample size estimate for this
      study. The study is powered for the primary outcome measure related to Question 1, looking at
      the difference between symptomatic and asymptomatic low back pain subjects at baseline (see
      Statistical Analyses). From Cohen's table (22), based on a power of 0.8, a two-tailed test
      with a p value < 0.05, the sample size was estimated to be 17 per group. In order to account
      for drop-outs and errors that may arise in the blood cultures, the sample size will be
      increased to 20 per group. As a result, there will be 40 symptomatic subjects and 20
      asymptomatic controls, which should provide a more than adequate sample to test the primary
      outcome.

      Subject assessment and group assignment: Qualified subjects will be scheduled. They will be
      greeted by one of two investigators (depending on recruitment site) , who will brief them and
      review the intake/eligibility form (Appendix 3) to confirm their eligibility before asking
      them to sign the informed consent form (Appendix 4). All subjects will then be asked to
      indicate their pain intensity level on the 10-point visual analogue scale (VAS) and complete
      the Oswestry functional disability questionnaire. They will then be assessed with standard
      chiropractic, orthopaedic and neurological tests by their respective chiropractic interns and
      supervising clinicians, who will assign them to the acute or chronic LBP groups, and who will
      formulate and explain a treatment plan.. For the duration of the study, treatments will
      consist of manual SMT and, where needed, manual (not instrument-assisted) soft tissue work
      provided by the clinician. Other treatment modalities will not be used.

      Patients will receive 6 treatments over the course of 2 weeks. (comprised of manipulation of
      one lumbar or sacroiliac articulation, with or without soft tissue therapy). SMT will consist
      of a single high velocity, low amplitude (HVLA) thrust intended to cavitate and restore
      mobility to the joint. If at initial presentation a manipulative segment could not be
      identified, the patient will be excluded from the study. If on the other hand a manipulable
      segment is not found at subsequent visits, the clinician will limit the treatment to
      palpation and some soft tissue work as indicated . In all cases a blood sample will be drawn
      (see below) after initial assessment and prior to commencement of treatments of patients, in
      order to establish a baseline level of cytokines for each participant (primary outcome). At
      their 7th visit (at least 2 days after last treatment), a blood sample will be taken prior to
      commencement of any further treatment, and they will be asked to complete the exit
      questionnaire including a VAS (Appendix 5). Should a patient recover following a few
      treatments, as assessed by subjective feedback from the patient and a VAS result of less than
      3/10, then a blood sample will be taken earlier and such will be duly noted. Should a patient
      require continued treatments beyond the 6 stipulated by the study, s/he will be free to do so
      under the direction of the clinician in charge of their case.

      Interventions: Manipulation: As noted above, each spinal manipulation will consist of a HVLA
      thrust to an affected segment (23). A clinician will deliver treatments according to his/her
      assessment findings on a given day.

      Venipuncture: On the day of admission into the study and at the completion of SMT therapy, an
      experienced phlebotomist will perform venipuncture using standard procedures (antecubital
      fossa, 21gauge needle) in the seated position. Heparinized blood samples (10 ml each) will be
      collected and transferred (at room temperature) to the laboratory within an hour of
      collection for the preparation of whole blood cultures as described below .

      Laboratory studies

        1. Induction of inflammatory cytokines To assess cytokine production in vitro, a whole
           blood (WB) culture system will be utilized (19). Briefly, multiple sets of WB cultures
           representing different treatment/culture conditions for each subject will be prepared.
           Cultures will be stimulated at initiation with 10 μg/ml lipopolysaccharide (LPS) for the
           induction of TNFα and IL-1β production. Phytohemagglutinin (PHA, 10 µg/ml) alone and in
           combination with LPS will be used to induce production of the anti-inflammatory
           cytokine, IL-10 and 2 chemokines, CCL2 and CCL3 . Cultures will be maintained at 37C
           (Celsius) in a humidified 5% C02 incubator. As clinical conditions involving
           inflammatory responses may cause a time shift in the capacity for cytokine production
           (19, 27), the levels of the studied mediators (TNFα, IL-1β, IL-1ra, IL-10, CCL2 and
           CCL3) will be examined at 24 h intervals in culture supernatants harvested between
           24-72h post-initiation. Aliquots of the supernatants will be stored at -76C (Celsius)
           until tested. This model will allow investigation of the relation between the releases
           of pro- and anti-inflammatory mediators.

        2. Determinations of cytokine levels by enzyme-linked immunosorbent assay The levels of
           TNFα, IL-1β , and IL-10 and IL-1ra in supernatants from whole blood cultures will be
           determined by specific enzyme-linked immunosorbent assays (ELISA) using DuoSet ELISA
           development system for natural and recombinant human cytokines (R&D Systems,
           Minneapolis, MN) as described previously (9, 11), and Quantikine Immunoassay Kits will
           be used for CCL2 and CCL3 determinations. All immunoassay procedures, including reagent
           and sample diluent preparations, will be carried out as per manufacturer's
           recommendations. Each of the studied samples will be tested at least twice at 2-4
           different dilutions.

      Statistical analyses All of the studied mediators will be measured for the LBP patients and
      asymptomatic subjects at baseline and at the completion of the treatment period. For Question
      1 (the primary outcome), baseline comparison for each TNFα and IL-1β will be compared between
      symptomatic subjects (acute and chronic) versus asymptomatic control subjects. Two unpaired
      t-tests will be used to test the associated hypothesis. For Question 2, the mean pre-post
      difference scores will be compared for each of TNFα and IL-1β between the three groups
      (acute, chronic and asymptomatic) using an ANOVA. If baseline values are significantly
      different between groups (as hypothesized from Question 1), then ANCOVA will be used to
      account for this difference. It is anticipated that the sample size is sufficiently large to
      accommodate this analysis. However, after determining the results for Question 2, a power
      analysis will be completed. If the study is insufficiently powered for this question, then
      another sample size estimate will be calculated to inform future work.

      For other pro-inflammatory and anti-inflammatory cytokines measured in this study, regression
      modeling will be used in an attempt to predict cytokine responses. It is understood that any
      model created during this process will need to be confirmed in a future investigation. Both
      models created and descriptive data derived from the investigation will be used to inform
      future work.

      Time frame Based on experience, up to 5 subjects per week can be tested. However, based on
      condition prevalence, we project that patient recruitment and sample collection will take
      8-12 months. Cytokine level determinations in cell cultures derived from the three groups of
      participants will lag behind by 3-4 months, and data analysis will be completed within
      another 2 months. Preparation of manuscripts will require an additional 4-6 months. Thus we
      anticipate completing this project within 18 -24 months of initiation of the study.
    

Intervention name(s): Spinal manipulation
Intervention type(s): Other
Intervention description(s): Spinal manipulation will consist of a single high velocity low amplitude thrust to a hypomobile vertebral segment determined by the treating clinician to contribute to the problem.
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: Single (Outcomes Assessor)
Primary purpose: Treatment
Keywords: Spinal manipulation; Tumor necrosis factor; Interleukin 1; Interleukin 10; Inflammatory cytokines