Accepted Manuscript The Influence of Experimental Anterior Knee Pain During Running on Electromyography and Articular Cartilage Metabolism W. Matt Denning , MS Scott Woodland , MS Jason G. Winward , Michael G. Leavitt , MS Allen C. Parcell , PhD J. Ty Hopkins , PhD Devin Francom , MS Matthew K. Seeley , PhD PII:
S1063-4584(14)01085-1
DOI:
10.1016/j.joca.2014.05.006
Reference:
YJOCA 3149
To appear in:
Osteoarthritis and Cartilage
Received Date: 18 November 2013 Revised Date:
1 May 2014
Accepted Date: 7 May 2014
Please cite this article as: Denning WM, Woodland S, Winward JG, Leavitt MG, Parcell AC, Hopkins JT, Francom D, Seeley MK, The Influence of Experimental Anterior Knee Pain During Running on Electromyography and Articular Cartilage Metabolism, Osteoarthritis and Cartilage (2014), doi: 10.1016/ j.joca.2014.05.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 Title The Influence of Experimental Anterior Knee Pain During Running on Electromyography and Articular Cartilage Metabolism
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Authors
Jason G. Winward Brigham Young University [email protected]
Allen C. Parcell, PhD Brigham Young University [email protected]
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J. Ty Hopkins, PhD Brigham Young University [email protected]
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Michael G. Leavitt, MS Brigham Young University [email protected]
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Scott Woodland, MS Brigham Young University [email protected]
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W. Matt Denning, MS 106 SFH Brigham Young University Provo, UT 84602, USA Phone: 1.801.422.9156 Email: [email protected]
Devin Francom, MS University of California Santa Cruz [email protected]
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Matthew K. Seeley, PhD Brigham Young Univeristy [email protected] Running Title
Experimental knee pain, EMG, and COMP
ACCEPTED MANUSCRIPT 2 Abstract
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Objective: To determine whether anterior knee pain (AKP), during running, acutely affects
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lower-extremity electromyography (EMG) and articular cartilage metabolism.
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Methods: A within-subjects design was used. Each of 12 able-bodied subjects ran on a treadmill
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for 30 minutes for three different sessions: control (no infusion), sham (0.9% NaCl infusion into
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the involved-leg infrapatellar fat pad), and pain (5.0% NaCl infusion into the involved-leg
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infrapatellar fat pad). Bilateral surface EMG was monitored for the vastus medialis (VM), vastus
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lateralis (VL), and gastrocnemius (GA). Serum cartilage oligomeric matrix protein (COMP)
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concentration was determined before, after, and 60 minutes after the run. A functional analysis
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approach was used to compare EMG amplitude, across the entire stance phase, between sessions
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and legs. Mixed-model analysis of covariance was used to compare serum COMP concentration
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between sessions, across time.
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Results: Relative to the uninvolved leg, greater involved-leg VL and GA EMG amplitude existed
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during midstance for the sham and control sessions (p < 0.01). During the painful session,
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however, involved-leg VM, VL, and GA EMG amplitude was 5-10% less than for the
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uninvolved leg. COMP concentration immediately post-run was 14% and 21% greater than pre-
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run (P = 0.01) and 60 minutes post-run concentrations (P < 0.01), respectively. Session,
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however, did not significantly influence COMP.
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Conclusion: During a 30-minute run, AKP acutely alters midstance VM, VL, and GA EMG
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amplitude. AKP during a 30-minute run does not, however, acutely influence articular cartilage
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metabolism.
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Key Words: COMP; Knee Pain; Electromyography; Running
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Introduction Knee pathology and the associated pain is common; 600,000 total knee arthroplasties were performed in the United States in 20101. Cram et al.1 estimated that arthroplasty, alone, cost
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the Unites States 9 billion dollars in 2009, and that 3 million procedures will be performed
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annually by 20302. Although signs and symptoms of knee pathologies vary, pain is a chief
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symptom3. Knee pain has even been described as a musculoskeletal epidemic4. Knee pathology
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and the associated pain are especially common for distance runners. Although distance running
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has not been directly linked to a particular knee injury, as many as 79% of all distance runners
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become injured and 50% of these injuries are knee injuries5. Combining all running injures, 42%
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affect the knee, and anterior knee pain (AKP) is associated with 62% of these knee injuries6.
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AKP is associated with abnormal lower-extremity muscle activation characteristics7-9. Lower-extremity activation characteristics influence knee joint load, and the activation
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characteristics for the vastii and gastrocnemius (GA) are especially influential on knee load10,11.
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AKP is also associated with abnormal lower-extremity kinematics12-14. Small changes in
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kinematics can influence knee load10. Because AKP causes neural alterations, and because
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articular cartilage is sensitive to lower-extremity kinematics15-17 and the resulting mechanical
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conditions18,19, AKP likely affects articular cartilage. It is unclear, however, how long AKP must
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persist before articular cartilage condition is affected. In felines, knee joint degeneration can
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begin to occur, as a direct result of decreased muscle force during cyclical loading, in as little as
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30 minutes20. An experimental AKP model that allows researchers to acutely produce AKP in
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otherwise able-bodied subjects could allow researchers to evaluate potential acute effects of AKP
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on articular cartilage, independent of other potentially confounding knee pathology factors (e.g.,
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knee joint effusion or degradation)14,21.
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Cartilage oligomeric matrix protein (COMP) is a noncollagenous extracellular matrix protein that is synthesized by chondrocytes and helps maintain the collagen matrix integrity by
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binding to type II collagen fibers; COMP also contributes to the mechanical properties of
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articular cartilage and is sensitive to load22-25. COMP is an established biomarker that reflects
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cartilage metabolism in osteoarthritis (OA)26,27 and otherwise injured knee joints28. COMP has
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been used to monitor articular cartilage degradation progress23,29-31. Additionally, COMP
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concentration increases as a result of exercise in able-bodied, asymptomatic individuals, which
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demonstrates that COMP concentration reflects normal articular cartilage turnover15,32-34. Serum
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COMP concentration is influenced by knee load characteristics15,35, and has also been indirectly
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related to muscle activation patterns for lower-extremity muscles during running32.
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The purpose of this study was to determine whether experimental AKP during distance
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running acutely affects lower-extremity muscle activation characteristics and articular cartilage
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metabolism that results from running. We hypothesized that experimental AKP during running
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would decrease muscle activation for the involved leg, relative to the uninvolved leg. Also, we
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hypothesized that experimental AKP during running would enlarge the increase in serum COMP
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that is typically associated with running.
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Methods
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Subjects
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Twelve relatively young, able-bodied volunteers (seven males and five females; mean ± 1
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standard deviation; age = 22 ± 4 years; mass = 72 ± 28 kg; height =1.79 ± 0.27 m; body mass
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index = 22.2 ± 1.9 kg/m2) were recruited from a university setting. It was intended that these
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subjects represent a young able-bodied, asymptomatic population; free from lower-extremity
ACCEPTED MANUSCRIPT 5 pathology and injury. Each subject participated in three different data collection sessions: pain,
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control, and sham. No subject reported a history of lower-extremity injury within six months
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prior to participation or any knee surgery in their lifetime. Because physical activity habits and
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body mass index influence serum COMP concentration before and after physical activity35,
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subjects with similar physical activity levels were recruited. Each subject was also required to (1)
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be participating in moderate physical activity, as defined by the World Health Organization, at
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least three times a week, and (2) have a normal body mass index (between 18.5 and 25). Subjects
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were required to refrain from exercise between 24 hours prior to the first data collection session
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and the completion of the final data collection session. Prior to participation, subjects completed
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an informed consent form that was approved by the appropriate institutional review board. All
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study procedures complied with the Helsinki Declaration. We considered sample size, a priori,
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using pilot EMG data (EMG being the most variable) and a multivariate analysis approach.
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Using this approach, however, the estimated sample size that was required to achieve 80% power
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at a significance level of 0.05 was far beyond the scope of the current study. Consequently, a
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feasible sample size of 12 subjects was chosen.
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Experimental Protocol
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Subjects completed the three different data collection sessions in a counterbalanced
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fashion, 48 hours apart. For each session, subjects ran for 30 minutes on the same treadmill
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(Quinton Cardiology, Bothwell, WA, USA) at a grade of zero. For the control session, subjects
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ran on the treadmill in a typical fashion. For the sham or pain sessions, subjects ran while
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receiving a continuous infusion of isotonic (0.9% NaCl) or hypertonic saline solution,
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respectively, into the right (involved) infrapatellar fat pad, at a rate of 0.216 ml · min-1 (total
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infused volume = 8 ml). This pain model (an identical solution concentration and volume) was
ACCEPTED MANUSCRIPT 6 used previously to induce consistent and satisfactory experimental AKP levels during running14.
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For all data collection sessions, subjects ran in the same pair of their own running shoes, and
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running shorts and shirts that were provided by the investigators.
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At the beginning of each data collection session, subjects rested in a seated position for 30 minutes to limit potential influence of preceding activity on serum COMP concentration.
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Subjects then stood for 10 minutes to allow for body fluid distribution to adjust to the vertical
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posture; electromyography (EMG) surface electrodes were applied to the subject during this
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standing period. Next, the baseline blood sample was drawn. Subjects then completed one of
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three running exercises: control (no infusion), sham (isotonic saline infusion), or pain
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(hypertonic saline infusion). A second blood sample was drawn immediately after the 30-minute
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run. After this blood draw, subjects rested in a seated position for 60 minutes, and a final blood
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sample was then drawn. The general timeline for each session is shown in Figure 1.
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For each data collection session, after the baseline blood sample was collected and EMG sensors were applied, subjects warmed up by running on the treadmill at one of three speeds (3.0,
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3.5, or 4.0 m/s) for 5 minutes. The subjects were instructed to select the greatest of these three
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speeds that they could run at continuously for 30 minutes. Subjects ran at the same speed for all
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data collection sessions. After this 5-minute warm-up, subjects lay supine on a treatment table
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while the isotonic or hypertonic saline infusions were initiated, for the sham or pain sessions,
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respectively. Prior to these infusions, the skin was shaved, if needed, and prepared with iodine
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and an alcohol wipe. The catheter was inserted into the lateral superior infrapatellar fat pad of the
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right leg using a previously described procedure14. After placement, the catheter was secured
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with tape, and used a plastic 0.762-m connection tube to connect the catheter (B. Braun Medical
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Inc., Melsungen, Germany) to a portable syringe pump (Graseby Medical Ltd., Watford,
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ACCEPTED MANUSCRIPT 7 England, UK). Throughout the sham and pain running sessions, this pump was held in a pocket
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that was sewn into a spandex running shirt (Figure 2). Subjects and investigators were blinded
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regarding the saline treatment that was being administered. After the syringe pump was initiated,
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subjects lay supine for three minutes, sat upright for two minutes, and then stood for two
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minutes, in an attempt to familiarize subjects to the experimental AKP and decrease the
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likelihood of syncope. The subjects then ran for 30 minutes at the selected speed while the saline
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was infused. The control session was identical to the sham and pain sessions, except that a
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catheter was not inserted and no infusion was administered. For each session, a 100-mm visual
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analog scale was used to measure subject-perceived AKP immediately prior to and after the
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needle stick, and then every three minutes until 60 minutes after the completion of the run.
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Electromyography
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activation patterns for the gastrocnemius (GA), vastus medialis (VM), and vastus lateralis (VL).
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Previously described recommendations for skin preparation procedures and electrode application
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locations were used36. Thirty seconds of EMG data at two different times of the run (10 and 20
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minutes after beginning the run) were collected, and then processed the EMG data using custom
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algorithms that were written in MatLab (MathWorks, Natick, MA, USA). The DC offset was
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first removed and then a root mean square algorithm (moving window = 50ms) was applied to
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rectify and smooth the EMG signal32. In an effort to minimize the influence of electrode
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placement, the amplitude of the smoothed signal was then normalized to a reference value that
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was the average of ten amplitude peaks from ten different stance phases during the warm-up run.
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Consequently, all EMG amplitude data were reported as a percentage of this mean peak
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amplitude (i.e., from the warm-up run). Also, the EMG signal was time normalized to 100% of
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ACCEPTED MANUSCRIPT 8 the stance phase, for five stance phases from 10 minutes after the beginning of the run and five
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stance phases from 20 minutes after the beginning of the run. Stance phase was manually
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determined using ten high-speed video cameras (200 Hz; VICON, Santa Rosa, CA, USA) that
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completely surrounded each subject, and reflective markers that were placed bilaterally on the
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heel and toe. The video and EMG data were synchronized.
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Serum COMP
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All blood samples (6 ml) were collected from an antecubital vein using a 20 gage
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shielded I.V. catheter (Becton Dickinson & Company, Franklin Lakes, NJ, USA). Immediately
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after catheter placement and every 15 minutes thereafter, the catheter was flushed with 1ml of
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isotonic saline (0.9% NaCl) to prevent clotting. A 1ml waste sample was drawn before each
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blood sample was collected. Blood samples were placed in EDTA vacutainers (Becton
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Dickinson & Company, Franklin Lakes, NJ, USA), centrifuged with an Eppendorf 5403
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refrigerated centrifuge (Hamburg, Germany) for 15 min at 3,000 × gravity, and stored in a
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freezer at -20°C until further analysis. Serum COMP concentration was determined using a
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commercially available enzyme-linked immunosortbent assay kit (ELISA; R&D Systems, Inc.,
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Minneapolis, MN, USA). Serum samples were analyzed in triplicate across each ELISA plate
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according to the manufacturer’s instructions. The present ELISA assay kits had inter- and intra-
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plate coefficients of variation of 13.66% and 2.61%, respectively, for a 273.3 ± 33.8 ng/ml
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sample. Differences due to inter-plate variation were minimized by comparing serum COMP
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concentration within subjects and by testing subject replicates on the same plate.
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Statistical Analysis
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The independent variables for this study were session, time, and leg. The dependent variables were subject-perceived AKP, EMG amplitude, and serum COMP concentration. We
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assumed that the current data would be normally distributed. Prior to statistical analyses, this
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assumption was confirmed via visual inspection. Another assumption that our statistical
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procedure employed, and that we feel is logical, was that the true EMG functions are continuous
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(by true EMG functions we mean that we observed representations of some underlying EMG
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over time, which we would call the true EMG functions). A mixed model repeated measures
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ANOVA was used to compare subject-perceived AKP between sessions every three minutes
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throughout most of each session (Figure 3; α = 0.05). A mixed model repeated measures
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ANOVA was also used to compare serum COMP concentration between sessions across three
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times: (1) baseline (pre-run), (2) immediately post-run, and (3) one hour post-run (Figure 1; α =
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0.05). If a session × time interaction was detected for subject-perceived AKP or serum COMP
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concentration, Tukey’s post hoc comparisons were used to evaluate potential between-time
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differences for each session. Functional linear models were used to evaluate influence of session
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and leg (involved and uninvolved) on EMG amplitude, modeled as polynomial functions, across
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the stance phase of running. This functional analysis evaluated the effect of session and leg
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across all of stance (heel strike to toe-off) in a single statistical test, rather than using multiple
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tests at separate discrete time points within stance, increasing statistical power. For this particular
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analysis, differences were significant if the mean difference and corresponding 99% confidence
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interval crossed zero. All ANOVA analyses were performed using JMP Pro 10 software (SAS
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Institute Inc., Cary, NC, USA). The functional linear models were produced using R statistical
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software.
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Results Average running speed for the present sample was 3.46 ± 0.43 m/s. A significant session × time interaction was observed for subject-perceived AKP (P < 0.01). Throughout the 30-
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minute run, subject-perceived AKP was significantly greater during the pain session (30 mm,
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95% confidence interval (CI): 27, 33), relative to the sham (5 mm, 95% CI: 4, 6) and control
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sessions (0 mm, 95% CI: 0, 0; Figure 3).The subject-perceived AKP significantly influenced
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EMG amplitude at various times within stance for all of the observed muscles. No bilateral EMG
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amplitude differences larger than 10-14%, however, were observed. For the control and sham
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sessions, involved-leg GA EMG amplitude was 5-10% greater than uninvolved-leg GA EMG
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amplitude throughout mid-stance (P < 0.01; Figures 4B, 4C, 4E and 4F). Conversely, for the
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pain session, involved-leg GA EMG amplitude was 5% less than uninvolved-leg GA EMG
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amplitude for a short time around 30% of the stance phase (P < 0.01; Figure 4A and 4D). The
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VM and VL exhibited similar alterations due to AKP. For VM EMG amplitude, bilateral
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symmetry existed for most of the stance phase during the control and sham sessions (Figures 5B,
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5C, 5E, and 5F). For the pain session, however, involved-leg VM EMG amplitude was 5-10%
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less than uninvolved-leg VM EMG amplitude for much of the middle part of stance (P < 0.01;
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Figures 5A and 5D). For VL EMG amplitude, bilateral asymmetries existed for each session, in
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the same direction: VL EMG amplitude for the uninvolved leg was ~14%, ~10%, and ~6%
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greater than for the involved leg during parts of midstance for the pain, sham, and control
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sessions, respectively (P < 0.01; Figures 6A-6F). Time significantly influenced serum COMP
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concentration as a main effect (P < 0.001; Figure 7). Average serum COMP concentration,
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immediately post-run (132 ng·ml-1, 95% CI: 94, 170), was 14% greater than immediately pre-run
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(116 ng·ml-1, 95% CI: 78, 154, P = 0.0092) and 21% greater than 60 minutes post-run (109
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ACCEPTED MANUSCRIPT 11 ng·ml-1, 95% CI: 70, 147; P= 0.0002). Serum COMP concentration was not significantly
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different between pre-run and 60 minutes post-run (P = 0.29). No session × time interaction for
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serum COMP concentration (P = 0.57; Figure 7) was observed. Furthermore, as a main effect,
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session did not significantly affect serum COMP concentration (P = 0.48). When certain subject
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characteristics (running speed, gender, age, and body mass index) were included as covariates,
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all of the reported results remained the same (Appendix A).
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Discussion
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This study was performed to understand whether AKP during distance running acutely
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affects lower-extremity muscle activation and resulting articular cartilage metabolism. It was
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hypothesized that, relative to pain free running, running for 30 minutes with AKP would (1)
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decrease EMG amplitude for the involved leg, relative to the uninvolved leg, and (2) enlarge the
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serum COMP concentration increase that typically results from running. The present results
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supported the first hypothesis, but did not support the second hypothesis: EMG amplitudes for
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the involved leg (for muscles that significantly influence knee joint load) were decreased,
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relative to the uninvolved leg, during AKP running; however, pain free and painful running had
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similar effects on serum COMP concentration.
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The observed EMG alterations are important findings from this study. Although knee
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load was not directly measured, the present decreases for GA, VM, and VL EMG amplitude that
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were due to AKP likely indicate altered knee load: muscle forces significantly and directly
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impact tibial-femoral contact load10,20,37. Specifically, vastii and GA activity influence knee load
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during the stance phase of human locomotion10,11. Although it is admittedly difficult to determine
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whether EMG differences of 10-14% are clinically meaningful, the observed reduction for VM
ACCEPTED MANUSCRIPT 12 EMG amplitude likely contributes to abnormal patellar tracking within the trochlear groove38,39.
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This idea is congruent to the clinical treatment of AKP in young adults that is strengthening the
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oblique VM40. Further, the present statistical analyses of EMG involved confidence intervals,
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which are one way to evaluate clinical significance41. Whenever present confidence intervals did
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not cross the zero line (Figures 4-6), the described differences can be considered as clinically
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significant41.
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Normal knee joint load that (1) results from lower-extremity muscle force and (2) is required to perform everyday function is important to maintain articular cartilage. For example,
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reduced quadriceps strength has been linked to symptomatic and morphological progression of
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knee osteoarthritis42. Additionally, decreased lower-extremity muscle force during cyclical
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loading has been associated with joint degeneration in as little as 30 minutes20. To date, it
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remains unclear if experimental AKP can influence the onset and/or progression of articular
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cartilage degradation. Further research is required to investigate the potential effect of persistent
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experimental AKP (> 30 minutes) on EMG and serum COMP concentration.
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The present findings suggest that AKP alters neuromuscular activation patterns.
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Previously, researchers have theorized that AKP is a result of altered neuromuscular activation
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patterns43,44. The present data indicate that the reverse is also true: altered EMG patterns result
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from AKP. Additionally, bilateral EMG data from the present control session demonstrated
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subtle asymmetries for neural activation for the GA, VM, and VL muscles during able-bodied
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running (Figures 4-6). This fits with previous research documenting lower-extremity EMG
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asymmetries for the GA, VM, and VL muscles during able-bodied walking45,46. While
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attempting to interpret the present EMG data, we were surprised to learn that no one has
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previously evaluated bilateral symmetry for EMG during able-bodied running, especially using
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ACCEPTED MANUSCRIPT 13 the relatively novel statistical approach (functional linear models) that was presently used. Like
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other previously published EMG data, the reliability of the present EMG data is limited due to
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numerous issues that are inherent to EMG (e.g., varying electrode placement, muscle fiber
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length, and contraction velocity). However, efforts were made to minimize such limitations
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where possible (e.g., normalization of EMG amplitude).
The present COMP data and data from previous researchers15,16,33,34, demonstrate that the
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magnitude and duration of serum COMP concentration increase, due to physical activity, is
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dependent upon exercise duration and intensity. For example, serum COMP concentration
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increases 90% and 60% as a result from a 200-km ultramarathon and marathon33, respectively,
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while only a 9.7% serum COMP concentration increase resulted from a less intense 30-minute
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walk35. The present observation of a 14% increase for serum COMP concentration (Figure 7),
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resulting from a 30-minute run, fits with these previously reported data. Additionally, walking
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for 30 minutes resulted in an increased COMP concentration duration of 30 minutes35, while
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running durations of 30 minutes16, 3-4 hours34, and 3-33 hours33 resulted in elevated COMP
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concentration for 1hour, 24 hours, and 6 days, respectively. Fitting with previous results15,16, the
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present COMP concentration returned to baseline levels within 1 hour (Figure 7). It appears that
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exercise sessions involving greater durations and intensities, and greater cumulative knee load,
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elicit greater amounts of COMP released from articular cartilage.
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Interpretation of increased COMP, as a result of exercise, is unclear. Although some
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authors have hypothesized that elevated COMP concentration may indicate detrimental articular
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cartilage degradation15,34, other authors have speculated that increased COMP concentration
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represents typical, healthy articular cartilage turnover16,33. Previously, researchers15,32,35 have
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demonstrated that as serum COMP concentration increases, articular cartilage volume decreases,
ACCEPTED MANUSCRIPT 14 even in a healthy population. Kim et al.33 speculated that running distance and the corresponding
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magnitude and duration of COMP concentration increase may each be positively correlated with
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the degree of articular cartilage damage. It is assumed that the presently observed COMP
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concentration increases reflected healthy articular cartilage turnover. The present subjects ran, on
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average, only 6.2 Km, and COMP levels increased only 14% and returned to pre exercise levels
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in less than 60 minutes (Figure 7). The present data are limited to a single running session, and
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more data are needed to determine the chronic effects of AKP on articular cartilage health.
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cartilage is another novel feature of this study. This pain model was presently used to induce
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consistent experimental AKP during a 30-minute run (Figure 3). This 30-minute duration is
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nearly 70% greater than the previous longest duration14. For this reason, the present results
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further demonstrate that this pain model can be used to study independent effects of AKP during
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various exercises over relatively extended durations. As with any novel method, however, certain
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aspects related to the present AKP model are unclear. Although the magnitude of subject-
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perceived AKP (Figure 3) appears to have been sufficient to represent clinical AKP47, it is
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unclear whether other characteristics of this AKP pain are clinically representative. However,
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previous researchers propose that experimental pain, induced by hypertonic saline, is
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representative of joint pain related to knee pathology21,48-50. The nature, quality, and distribution
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of pain elicited by hypertonic saline has been shown to be similar to clinical AKP21. Also, results
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from animal experiments suggest that group III and IV nociceptive afferents are active due to
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hypertonic saline49,50, providing more evidence that the pathways involved during this
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experimental pain are similar to musculoskeletal pain. Furthermore, hypertonic saline induces
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the release of substance P from C-fiber neurons, which is also consistent with musculoskeletal
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pain48. In conclusion, there are three important findings from this study. First, experimental AKP
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generally caused involved-leg EMG amplitude to decrease, during running, relative to
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uninvolved-leg EMG amplitude (Figures 4-6). Second, experimental AKP did not significantly
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affect systemic serum COMP concentration increase due to running (Figure 4). Third, the
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infusion of hypertonic saline effectively produced consistent subject-perceived AKP throughout
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a 30-minute run (Figure 3).
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Author contributions
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Conception and design: Woodland, Parcell, Hopkins, Seeley
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Acquisition of data: Denning, Woodland, Winward, Leavitt, Parcell, Seeley
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Analysis and interpretation of data: Denning, Woodland, Leavitt, Parcell, Hopkins,
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Drafting and revising of article: Denning, Woodland, Winward, Leavitt, Parcell, Hopkins, Francom, Seeley
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Francom, Seeley
Final approval of the version of the article to be published: Denning, Woodland,
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Winward, Leavitt, Parcell, Hopkins, Francom, Seeley
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Competing Interests Statement
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There are no completing interests for any of the authors.
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Appendix A. Summary of the serum COMP results when running speed, gender, age, and body
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mass index were used as covariates. None of the covariates included in this model were
ACCEPTED MANUSCRIPT 16 statistically significant. Neither running speed (P = 0.55), gender (P = 0.63), age (P = 0.71), nor
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body mass index (P = 0.50) influenced serum COMP concentration. Time was the only
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significant main effect that influenced serum COMP (P < 0.001).
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Table 1. Average (95% CI: lower limit – upper limit) serum COMP concentration (ng·ml-1) for each session and time when running speed, gender, age, and body mass index were used as covariates.
Average
108.7
125.6
97.1
(65.3 – 152.2)
(82.1 – 169.1)
(53.7 – 140.6)
112.9
132.2
109.5
(69.5 – 156.4)
(88.7 – 175.6)
(66.0 – 1 52.9)
120.2
132.5
112.5
(76.7 – 163.6)
(89.1 – 176.0)
113.9
130.1*
(71.1 – 156.7)
(87.3 – 172.9)
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60 Minutes Post Run
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(69.0 – 155.9) 106.4
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Figure 1. A description of the general timeline for each data collection session. EMG #1 and
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EMG #2 indicate the times, within the run, that electromyography data were collected.
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shirt held the portable syringe pump and syringe. A connection tube (B) was used to connect the
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syringe and the catheter (C) that was inserted into the infrapatellar fat pad.
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Figure 3. Means and 95% confidence intervals for subject-perceived pain across time for each
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data collection session (95% confidence intervals are not included for the control session because
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they equaled zero at each time point). Subject-perceived pain significantly increased across time
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during the pain session only (p < 0.01). Asterisks show statistically significant differences when
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the pain session was compared to the sham and control sessions.
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Figure 4. Grand ensembles (99% confidence intervals are only shown for involved (right) leg to
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increase clarity) for gastrocnemius (GA) electromyography (EMG) amplitude (Amp), for the
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pain (A), control (B), and sham (C) sessions. Figures D-F show results of bilateral functional
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comparisons, where mean bilateral differences and corresponding 99% confidence intervals are
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plotted as a function of time. When the shaded area does not overlap with the zero line, a
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bilateral difference (p < 0.01) is indicated. Positive differences indicate that involved-leg (right)
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EMG is greater, while negative differences indicate the opposite.
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Figure 5. Grand ensembles (99% confidence intervals are only shown for involved (right) leg to
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increase clarity) for vastus medialis (VM) electromyography (EMG) amplitude (Amp), for the
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comparisons, where mean bilateral differences and corresponding 99% confidence intervals are
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plotted as a function of time. When the shaded area does not overlap with the zero line, a
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bilateral difference (p < 0.01) is indicated. Positive differences indicate that involved-leg (right)
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EMG is greater, while negative differences indicate the opposite.
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Figure 6. Grand ensembles (99% confidence intervals are only shown for involved (right) leg to
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increase clarity) for vastus lateralis (VL) electromyography (EMG) amplitude (Amp), for the
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pain (A), control (B), and sham (C) sessions. Figures D-F show results of bilateral functional
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comparisons, where mean bilateral differences and corresponding 99% confidence intervals are
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plotted as a function of time. When the shaded area does not overlap with the zero line, a
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bilateral difference (p < 0.01) is indicated. Positive differences indicate that involved-leg (right)
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EMG is greater, while negative differences indicate the opposite.
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Figure 7. Means and 95% confidence intervals for serum cartilage oligomeric matrix protein
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(COMP) concentration at three times for each session. If pooled from each session, serum
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COMP concentration was significantly greater immediately post run, relative to pre run and 60
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minutes post run; this trend was consistent between sessions.
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S1063-4584(14)01085-1
DOI:
10.1016/j.joca.2014.05.006
Reference:
YJOCA 3149
To appear in:
Osteoarthritis and Cartilage
Received Date: 18 November 2013 Revised Date:
1 May 2014
Accepted Date: 7 May 2014
Please cite this article as: Denning WM, Woodland S, Winward JG, Leavitt MG, Parcell AC, Hopkins JT, Francom D, Seeley MK, The Influence of Experimental Anterior Knee Pain During Running on Electromyography and Articular Cartilage Metabolism, Osteoarthritis and Cartilage (2014), doi: 10.1016/ j.joca.2014.05.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 Title The Influence of Experimental Anterior Knee Pain During Running on Electromyography and Articular Cartilage Metabolism
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Authors
Jason G. Winward Brigham Young University [email protected]
Allen C. Parcell, PhD Brigham Young University [email protected]
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J. Ty Hopkins, PhD Brigham Young University [email protected]
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Michael G. Leavitt, MS Brigham Young University [email protected]
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Scott Woodland, MS Brigham Young University [email protected]
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W. Matt Denning, MS 106 SFH Brigham Young University Provo, UT 84602, USA Phone: 1.801.422.9156 Email: [email protected]
Devin Francom, MS University of California Santa Cruz [email protected]
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Matthew K. Seeley, PhD Brigham Young Univeristy [email protected] Running Title
Experimental knee pain, EMG, and COMP
ACCEPTED MANUSCRIPT 2 Abstract
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Objective: To determine whether anterior knee pain (AKP), during running, acutely affects
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lower-extremity electromyography (EMG) and articular cartilage metabolism.
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Methods: A within-subjects design was used. Each of 12 able-bodied subjects ran on a treadmill
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for 30 minutes for three different sessions: control (no infusion), sham (0.9% NaCl infusion into
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the involved-leg infrapatellar fat pad), and pain (5.0% NaCl infusion into the involved-leg
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infrapatellar fat pad). Bilateral surface EMG was monitored for the vastus medialis (VM), vastus
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lateralis (VL), and gastrocnemius (GA). Serum cartilage oligomeric matrix protein (COMP)
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concentration was determined before, after, and 60 minutes after the run. A functional analysis
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approach was used to compare EMG amplitude, across the entire stance phase, between sessions
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and legs. Mixed-model analysis of covariance was used to compare serum COMP concentration
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between sessions, across time.
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Results: Relative to the uninvolved leg, greater involved-leg VL and GA EMG amplitude existed
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during midstance for the sham and control sessions (p < 0.01). During the painful session,
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however, involved-leg VM, VL, and GA EMG amplitude was 5-10% less than for the
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uninvolved leg. COMP concentration immediately post-run was 14% and 21% greater than pre-
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run (P = 0.01) and 60 minutes post-run concentrations (P < 0.01), respectively. Session,
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however, did not significantly influence COMP.
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Conclusion: During a 30-minute run, AKP acutely alters midstance VM, VL, and GA EMG
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amplitude. AKP during a 30-minute run does not, however, acutely influence articular cartilage
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metabolism.
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Key Words: COMP; Knee Pain; Electromyography; Running
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Introduction Knee pathology and the associated pain is common; 600,000 total knee arthroplasties were performed in the United States in 20101. Cram et al.1 estimated that arthroplasty, alone, cost
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the Unites States 9 billion dollars in 2009, and that 3 million procedures will be performed
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annually by 20302. Although signs and symptoms of knee pathologies vary, pain is a chief
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symptom3. Knee pain has even been described as a musculoskeletal epidemic4. Knee pathology
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and the associated pain are especially common for distance runners. Although distance running
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has not been directly linked to a particular knee injury, as many as 79% of all distance runners
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become injured and 50% of these injuries are knee injuries5. Combining all running injures, 42%
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affect the knee, and anterior knee pain (AKP) is associated with 62% of these knee injuries6.
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AKP is associated with abnormal lower-extremity muscle activation characteristics7-9. Lower-extremity activation characteristics influence knee joint load, and the activation
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characteristics for the vastii and gastrocnemius (GA) are especially influential on knee load10,11.
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AKP is also associated with abnormal lower-extremity kinematics12-14. Small changes in
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kinematics can influence knee load10. Because AKP causes neural alterations, and because
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articular cartilage is sensitive to lower-extremity kinematics15-17 and the resulting mechanical
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conditions18,19, AKP likely affects articular cartilage. It is unclear, however, how long AKP must
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persist before articular cartilage condition is affected. In felines, knee joint degeneration can
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begin to occur, as a direct result of decreased muscle force during cyclical loading, in as little as
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30 minutes20. An experimental AKP model that allows researchers to acutely produce AKP in
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otherwise able-bodied subjects could allow researchers to evaluate potential acute effects of AKP
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on articular cartilage, independent of other potentially confounding knee pathology factors (e.g.,
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knee joint effusion or degradation)14,21.
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Cartilage oligomeric matrix protein (COMP) is a noncollagenous extracellular matrix protein that is synthesized by chondrocytes and helps maintain the collagen matrix integrity by
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binding to type II collagen fibers; COMP also contributes to the mechanical properties of
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articular cartilage and is sensitive to load22-25. COMP is an established biomarker that reflects
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cartilage metabolism in osteoarthritis (OA)26,27 and otherwise injured knee joints28. COMP has
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been used to monitor articular cartilage degradation progress23,29-31. Additionally, COMP
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concentration increases as a result of exercise in able-bodied, asymptomatic individuals, which
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demonstrates that COMP concentration reflects normal articular cartilage turnover15,32-34. Serum
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COMP concentration is influenced by knee load characteristics15,35, and has also been indirectly
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related to muscle activation patterns for lower-extremity muscles during running32.
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The purpose of this study was to determine whether experimental AKP during distance
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running acutely affects lower-extremity muscle activation characteristics and articular cartilage
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metabolism that results from running. We hypothesized that experimental AKP during running
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would decrease muscle activation for the involved leg, relative to the uninvolved leg. Also, we
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hypothesized that experimental AKP during running would enlarge the increase in serum COMP
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that is typically associated with running.
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Methods
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Subjects
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Twelve relatively young, able-bodied volunteers (seven males and five females; mean ± 1
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standard deviation; age = 22 ± 4 years; mass = 72 ± 28 kg; height =1.79 ± 0.27 m; body mass
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index = 22.2 ± 1.9 kg/m2) were recruited from a university setting. It was intended that these
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subjects represent a young able-bodied, asymptomatic population; free from lower-extremity
ACCEPTED MANUSCRIPT 5 pathology and injury. Each subject participated in three different data collection sessions: pain,
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control, and sham. No subject reported a history of lower-extremity injury within six months
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prior to participation or any knee surgery in their lifetime. Because physical activity habits and
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body mass index influence serum COMP concentration before and after physical activity35,
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subjects with similar physical activity levels were recruited. Each subject was also required to (1)
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be participating in moderate physical activity, as defined by the World Health Organization, at
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least three times a week, and (2) have a normal body mass index (between 18.5 and 25). Subjects
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were required to refrain from exercise between 24 hours prior to the first data collection session
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and the completion of the final data collection session. Prior to participation, subjects completed
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an informed consent form that was approved by the appropriate institutional review board. All
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study procedures complied with the Helsinki Declaration. We considered sample size, a priori,
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using pilot EMG data (EMG being the most variable) and a multivariate analysis approach.
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Using this approach, however, the estimated sample size that was required to achieve 80% power
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at a significance level of 0.05 was far beyond the scope of the current study. Consequently, a
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feasible sample size of 12 subjects was chosen.
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Experimental Protocol
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Subjects completed the three different data collection sessions in a counterbalanced
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fashion, 48 hours apart. For each session, subjects ran for 30 minutes on the same treadmill
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(Quinton Cardiology, Bothwell, WA, USA) at a grade of zero. For the control session, subjects
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ran on the treadmill in a typical fashion. For the sham or pain sessions, subjects ran while
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receiving a continuous infusion of isotonic (0.9% NaCl) or hypertonic saline solution,
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respectively, into the right (involved) infrapatellar fat pad, at a rate of 0.216 ml · min-1 (total
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infused volume = 8 ml). This pain model (an identical solution concentration and volume) was
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For all data collection sessions, subjects ran in the same pair of their own running shoes, and
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running shorts and shirts that were provided by the investigators.
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At the beginning of each data collection session, subjects rested in a seated position for 30 minutes to limit potential influence of preceding activity on serum COMP concentration.
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Subjects then stood for 10 minutes to allow for body fluid distribution to adjust to the vertical
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posture; electromyography (EMG) surface electrodes were applied to the subject during this
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standing period. Next, the baseline blood sample was drawn. Subjects then completed one of
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three running exercises: control (no infusion), sham (isotonic saline infusion), or pain
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(hypertonic saline infusion). A second blood sample was drawn immediately after the 30-minute
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run. After this blood draw, subjects rested in a seated position for 60 minutes, and a final blood
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sample was then drawn. The general timeline for each session is shown in Figure 1.
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For each data collection session, after the baseline blood sample was collected and EMG sensors were applied, subjects warmed up by running on the treadmill at one of three speeds (3.0,
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3.5, or 4.0 m/s) for 5 minutes. The subjects were instructed to select the greatest of these three
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speeds that they could run at continuously for 30 minutes. Subjects ran at the same speed for all
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data collection sessions. After this 5-minute warm-up, subjects lay supine on a treatment table
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while the isotonic or hypertonic saline infusions were initiated, for the sham or pain sessions,
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respectively. Prior to these infusions, the skin was shaved, if needed, and prepared with iodine
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and an alcohol wipe. The catheter was inserted into the lateral superior infrapatellar fat pad of the
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right leg using a previously described procedure14. After placement, the catheter was secured
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with tape, and used a plastic 0.762-m connection tube to connect the catheter (B. Braun Medical
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Inc., Melsungen, Germany) to a portable syringe pump (Graseby Medical Ltd., Watford,
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ACCEPTED MANUSCRIPT 7 England, UK). Throughout the sham and pain running sessions, this pump was held in a pocket
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that was sewn into a spandex running shirt (Figure 2). Subjects and investigators were blinded
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regarding the saline treatment that was being administered. After the syringe pump was initiated,
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subjects lay supine for three minutes, sat upright for two minutes, and then stood for two
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minutes, in an attempt to familiarize subjects to the experimental AKP and decrease the
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likelihood of syncope. The subjects then ran for 30 minutes at the selected speed while the saline
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was infused. The control session was identical to the sham and pain sessions, except that a
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catheter was not inserted and no infusion was administered. For each session, a 100-mm visual
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analog scale was used to measure subject-perceived AKP immediately prior to and after the
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needle stick, and then every three minutes until 60 minutes after the completion of the run.
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Electromyography
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activation patterns for the gastrocnemius (GA), vastus medialis (VM), and vastus lateralis (VL).
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Previously described recommendations for skin preparation procedures and electrode application
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locations were used36. Thirty seconds of EMG data at two different times of the run (10 and 20
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minutes after beginning the run) were collected, and then processed the EMG data using custom
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algorithms that were written in MatLab (MathWorks, Natick, MA, USA). The DC offset was
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first removed and then a root mean square algorithm (moving window = 50ms) was applied to
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rectify and smooth the EMG signal32. In an effort to minimize the influence of electrode
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placement, the amplitude of the smoothed signal was then normalized to a reference value that
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was the average of ten amplitude peaks from ten different stance phases during the warm-up run.
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Consequently, all EMG amplitude data were reported as a percentage of this mean peak
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amplitude (i.e., from the warm-up run). Also, the EMG signal was time normalized to 100% of
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ACCEPTED MANUSCRIPT 8 the stance phase, for five stance phases from 10 minutes after the beginning of the run and five
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stance phases from 20 minutes after the beginning of the run. Stance phase was manually
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determined using ten high-speed video cameras (200 Hz; VICON, Santa Rosa, CA, USA) that
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completely surrounded each subject, and reflective markers that were placed bilaterally on the
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heel and toe. The video and EMG data were synchronized.
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Serum COMP
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All blood samples (6 ml) were collected from an antecubital vein using a 20 gage
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shielded I.V. catheter (Becton Dickinson & Company, Franklin Lakes, NJ, USA). Immediately
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after catheter placement and every 15 minutes thereafter, the catheter was flushed with 1ml of
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isotonic saline (0.9% NaCl) to prevent clotting. A 1ml waste sample was drawn before each
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blood sample was collected. Blood samples were placed in EDTA vacutainers (Becton
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Dickinson & Company, Franklin Lakes, NJ, USA), centrifuged with an Eppendorf 5403
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refrigerated centrifuge (Hamburg, Germany) for 15 min at 3,000 × gravity, and stored in a
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freezer at -20°C until further analysis. Serum COMP concentration was determined using a
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commercially available enzyme-linked immunosortbent assay kit (ELISA; R&D Systems, Inc.,
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Minneapolis, MN, USA). Serum samples were analyzed in triplicate across each ELISA plate
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according to the manufacturer’s instructions. The present ELISA assay kits had inter- and intra-
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plate coefficients of variation of 13.66% and 2.61%, respectively, for a 273.3 ± 33.8 ng/ml
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sample. Differences due to inter-plate variation were minimized by comparing serum COMP
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concentration within subjects and by testing subject replicates on the same plate.
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Statistical Analysis
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The independent variables for this study were session, time, and leg. The dependent variables were subject-perceived AKP, EMG amplitude, and serum COMP concentration. We
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assumed that the current data would be normally distributed. Prior to statistical analyses, this
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assumption was confirmed via visual inspection. Another assumption that our statistical
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procedure employed, and that we feel is logical, was that the true EMG functions are continuous
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(by true EMG functions we mean that we observed representations of some underlying EMG
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over time, which we would call the true EMG functions). A mixed model repeated measures
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ANOVA was used to compare subject-perceived AKP between sessions every three minutes
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throughout most of each session (Figure 3; α = 0.05). A mixed model repeated measures
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ANOVA was also used to compare serum COMP concentration between sessions across three
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times: (1) baseline (pre-run), (2) immediately post-run, and (3) one hour post-run (Figure 1; α =
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0.05). If a session × time interaction was detected for subject-perceived AKP or serum COMP
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concentration, Tukey’s post hoc comparisons were used to evaluate potential between-time
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differences for each session. Functional linear models were used to evaluate influence of session
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and leg (involved and uninvolved) on EMG amplitude, modeled as polynomial functions, across
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the stance phase of running. This functional analysis evaluated the effect of session and leg
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across all of stance (heel strike to toe-off) in a single statistical test, rather than using multiple
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tests at separate discrete time points within stance, increasing statistical power. For this particular
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analysis, differences were significant if the mean difference and corresponding 99% confidence
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interval crossed zero. All ANOVA analyses were performed using JMP Pro 10 software (SAS
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Institute Inc., Cary, NC, USA). The functional linear models were produced using R statistical
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software.
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ACCEPTED MANUSCRIPT 10 226 227
Results Average running speed for the present sample was 3.46 ± 0.43 m/s. A significant session × time interaction was observed for subject-perceived AKP (P < 0.01). Throughout the 30-
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minute run, subject-perceived AKP was significantly greater during the pain session (30 mm,
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95% confidence interval (CI): 27, 33), relative to the sham (5 mm, 95% CI: 4, 6) and control
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sessions (0 mm, 95% CI: 0, 0; Figure 3).The subject-perceived AKP significantly influenced
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EMG amplitude at various times within stance for all of the observed muscles. No bilateral EMG
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amplitude differences larger than 10-14%, however, were observed. For the control and sham
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sessions, involved-leg GA EMG amplitude was 5-10% greater than uninvolved-leg GA EMG
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amplitude throughout mid-stance (P < 0.01; Figures 4B, 4C, 4E and 4F). Conversely, for the
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pain session, involved-leg GA EMG amplitude was 5% less than uninvolved-leg GA EMG
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amplitude for a short time around 30% of the stance phase (P < 0.01; Figure 4A and 4D). The
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VM and VL exhibited similar alterations due to AKP. For VM EMG amplitude, bilateral
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symmetry existed for most of the stance phase during the control and sham sessions (Figures 5B,
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5C, 5E, and 5F). For the pain session, however, involved-leg VM EMG amplitude was 5-10%
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less than uninvolved-leg VM EMG amplitude for much of the middle part of stance (P < 0.01;
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Figures 5A and 5D). For VL EMG amplitude, bilateral asymmetries existed for each session, in
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the same direction: VL EMG amplitude for the uninvolved leg was ~14%, ~10%, and ~6%
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greater than for the involved leg during parts of midstance for the pain, sham, and control
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sessions, respectively (P < 0.01; Figures 6A-6F). Time significantly influenced serum COMP
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concentration as a main effect (P < 0.001; Figure 7). Average serum COMP concentration,
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immediately post-run (132 ng·ml-1, 95% CI: 94, 170), was 14% greater than immediately pre-run
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(116 ng·ml-1, 95% CI: 78, 154, P = 0.0092) and 21% greater than 60 minutes post-run (109
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ACCEPTED MANUSCRIPT 11 ng·ml-1, 95% CI: 70, 147; P= 0.0002). Serum COMP concentration was not significantly
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different between pre-run and 60 minutes post-run (P = 0.29). No session × time interaction for
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serum COMP concentration (P = 0.57; Figure 7) was observed. Furthermore, as a main effect,
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session did not significantly affect serum COMP concentration (P = 0.48). When certain subject
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characteristics (running speed, gender, age, and body mass index) were included as covariates,
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all of the reported results remained the same (Appendix A).
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Discussion
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This study was performed to understand whether AKP during distance running acutely
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affects lower-extremity muscle activation and resulting articular cartilage metabolism. It was
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hypothesized that, relative to pain free running, running for 30 minutes with AKP would (1)
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decrease EMG amplitude for the involved leg, relative to the uninvolved leg, and (2) enlarge the
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serum COMP concentration increase that typically results from running. The present results
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supported the first hypothesis, but did not support the second hypothesis: EMG amplitudes for
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the involved leg (for muscles that significantly influence knee joint load) were decreased,
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relative to the uninvolved leg, during AKP running; however, pain free and painful running had
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similar effects on serum COMP concentration.
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The observed EMG alterations are important findings from this study. Although knee
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load was not directly measured, the present decreases for GA, VM, and VL EMG amplitude that
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were due to AKP likely indicate altered knee load: muscle forces significantly and directly
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impact tibial-femoral contact load10,20,37. Specifically, vastii and GA activity influence knee load
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during the stance phase of human locomotion10,11. Although it is admittedly difficult to determine
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whether EMG differences of 10-14% are clinically meaningful, the observed reduction for VM
ACCEPTED MANUSCRIPT 12 EMG amplitude likely contributes to abnormal patellar tracking within the trochlear groove38,39.
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This idea is congruent to the clinical treatment of AKP in young adults that is strengthening the
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oblique VM40. Further, the present statistical analyses of EMG involved confidence intervals,
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which are one way to evaluate clinical significance41. Whenever present confidence intervals did
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not cross the zero line (Figures 4-6), the described differences can be considered as clinically
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significant41.
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Normal knee joint load that (1) results from lower-extremity muscle force and (2) is required to perform everyday function is important to maintain articular cartilage. For example,
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reduced quadriceps strength has been linked to symptomatic and morphological progression of
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knee osteoarthritis42. Additionally, decreased lower-extremity muscle force during cyclical
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loading has been associated with joint degeneration in as little as 30 minutes20. To date, it
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remains unclear if experimental AKP can influence the onset and/or progression of articular
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cartilage degradation. Further research is required to investigate the potential effect of persistent
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experimental AKP (> 30 minutes) on EMG and serum COMP concentration.
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The present findings suggest that AKP alters neuromuscular activation patterns.
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Previously, researchers have theorized that AKP is a result of altered neuromuscular activation
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patterns43,44. The present data indicate that the reverse is also true: altered EMG patterns result
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from AKP. Additionally, bilateral EMG data from the present control session demonstrated
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subtle asymmetries for neural activation for the GA, VM, and VL muscles during able-bodied
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running (Figures 4-6). This fits with previous research documenting lower-extremity EMG
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asymmetries for the GA, VM, and VL muscles during able-bodied walking45,46. While
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attempting to interpret the present EMG data, we were surprised to learn that no one has
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previously evaluated bilateral symmetry for EMG during able-bodied running, especially using
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ACCEPTED MANUSCRIPT 13 the relatively novel statistical approach (functional linear models) that was presently used. Like
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other previously published EMG data, the reliability of the present EMG data is limited due to
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numerous issues that are inherent to EMG (e.g., varying electrode placement, muscle fiber
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length, and contraction velocity). However, efforts were made to minimize such limitations
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where possible (e.g., normalization of EMG amplitude).
The present COMP data and data from previous researchers15,16,33,34, demonstrate that the
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magnitude and duration of serum COMP concentration increase, due to physical activity, is
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dependent upon exercise duration and intensity. For example, serum COMP concentration
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increases 90% and 60% as a result from a 200-km ultramarathon and marathon33, respectively,
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while only a 9.7% serum COMP concentration increase resulted from a less intense 30-minute
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walk35. The present observation of a 14% increase for serum COMP concentration (Figure 7),
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resulting from a 30-minute run, fits with these previously reported data. Additionally, walking
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for 30 minutes resulted in an increased COMP concentration duration of 30 minutes35, while
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running durations of 30 minutes16, 3-4 hours34, and 3-33 hours33 resulted in elevated COMP
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concentration for 1hour, 24 hours, and 6 days, respectively. Fitting with previous results15,16, the
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present COMP concentration returned to baseline levels within 1 hour (Figure 7). It appears that
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exercise sessions involving greater durations and intensities, and greater cumulative knee load,
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elicit greater amounts of COMP released from articular cartilage.
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Interpretation of increased COMP, as a result of exercise, is unclear. Although some
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authors have hypothesized that elevated COMP concentration may indicate detrimental articular
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cartilage degradation15,34, other authors have speculated that increased COMP concentration
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represents typical, healthy articular cartilage turnover16,33. Previously, researchers15,32,35 have
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demonstrated that as serum COMP concentration increases, articular cartilage volume decreases,
ACCEPTED MANUSCRIPT 14 even in a healthy population. Kim et al.33 speculated that running distance and the corresponding
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magnitude and duration of COMP concentration increase may each be positively correlated with
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the degree of articular cartilage damage. It is assumed that the presently observed COMP
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concentration increases reflected healthy articular cartilage turnover. The present subjects ran, on
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average, only 6.2 Km, and COMP levels increased only 14% and returned to pre exercise levels
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in less than 60 minutes (Figure 7). The present data are limited to a single running session, and
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more data are needed to determine the chronic effects of AKP on articular cartilage health.
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cartilage is another novel feature of this study. This pain model was presently used to induce
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consistent experimental AKP during a 30-minute run (Figure 3). This 30-minute duration is
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nearly 70% greater than the previous longest duration14. For this reason, the present results
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further demonstrate that this pain model can be used to study independent effects of AKP during
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various exercises over relatively extended durations. As with any novel method, however, certain
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aspects related to the present AKP model are unclear. Although the magnitude of subject-
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perceived AKP (Figure 3) appears to have been sufficient to represent clinical AKP47, it is
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unclear whether other characteristics of this AKP pain are clinically representative. However,
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previous researchers propose that experimental pain, induced by hypertonic saline, is
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representative of joint pain related to knee pathology21,48-50. The nature, quality, and distribution
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of pain elicited by hypertonic saline has been shown to be similar to clinical AKP21. Also, results
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from animal experiments suggest that group III and IV nociceptive afferents are active due to
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hypertonic saline49,50, providing more evidence that the pathways involved during this
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experimental pain are similar to musculoskeletal pain. Furthermore, hypertonic saline induces
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the release of substance P from C-fiber neurons, which is also consistent with musculoskeletal
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pain48. In conclusion, there are three important findings from this study. First, experimental AKP
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generally caused involved-leg EMG amplitude to decrease, during running, relative to
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uninvolved-leg EMG amplitude (Figures 4-6). Second, experimental AKP did not significantly
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affect systemic serum COMP concentration increase due to running (Figure 4). Third, the
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infusion of hypertonic saline effectively produced consistent subject-perceived AKP throughout
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a 30-minute run (Figure 3).
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Author contributions
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Conception and design: Woodland, Parcell, Hopkins, Seeley
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Acquisition of data: Denning, Woodland, Winward, Leavitt, Parcell, Seeley
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Analysis and interpretation of data: Denning, Woodland, Leavitt, Parcell, Hopkins,
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Drafting and revising of article: Denning, Woodland, Winward, Leavitt, Parcell, Hopkins, Francom, Seeley
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Francom, Seeley
Final approval of the version of the article to be published: Denning, Woodland,
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Winward, Leavitt, Parcell, Hopkins, Francom, Seeley
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Competing Interests Statement
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There are no completing interests for any of the authors.
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Appendix A. Summary of the serum COMP results when running speed, gender, age, and body
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mass index were used as covariates. None of the covariates included in this model were
ACCEPTED MANUSCRIPT 16 statistically significant. Neither running speed (P = 0.55), gender (P = 0.63), age (P = 0.71), nor
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body mass index (P = 0.50) influenced serum COMP concentration. Time was the only
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significant main effect that influenced serum COMP (P < 0.001).
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knee to predict in vivo joint contact forces during normal and novel gait patterns. J
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Table 1. Average (95% CI: lower limit – upper limit) serum COMP concentration (ng·ml-1) for each session and time when running speed, gender, age, and body mass index were used as covariates.
Average
108.7
125.6
97.1
(65.3 – 152.2)
(82.1 – 169.1)
(53.7 – 140.6)
112.9
132.2
109.5
(69.5 – 156.4)
(88.7 – 175.6)
(66.0 – 1 52.9)
120.2
132.5
112.5
(76.7 – 163.6)
(89.1 – 176.0)
113.9
130.1*
(71.1 – 156.7)
(87.3 – 172.9)
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Pain
60 Minutes Post Run
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Sham
Post Run
(69.0 – 155.9) 106.4
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Control
Pre Run
(63.6 – 149.2)
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Figure 1. A description of the general timeline for each data collection session. EMG #1 and
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EMG #2 indicate the times, within the run, that electromyography data were collected.
503 Figure 2. The knee saline infusion set-up. A pocket (A) that was sewn into the front of a spandex
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shirt held the portable syringe pump and syringe. A connection tube (B) was used to connect the
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syringe and the catheter (C) that was inserted into the infrapatellar fat pad.
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Figure 3. Means and 95% confidence intervals for subject-perceived pain across time for each
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data collection session (95% confidence intervals are not included for the control session because
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they equaled zero at each time point). Subject-perceived pain significantly increased across time
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during the pain session only (p < 0.01). Asterisks show statistically significant differences when
512
the pain session was compared to the sham and control sessions.
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Figure 4. Grand ensembles (99% confidence intervals are only shown for involved (right) leg to
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increase clarity) for gastrocnemius (GA) electromyography (EMG) amplitude (Amp), for the
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pain (A), control (B), and sham (C) sessions. Figures D-F show results of bilateral functional
517
comparisons, where mean bilateral differences and corresponding 99% confidence intervals are
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plotted as a function of time. When the shaded area does not overlap with the zero line, a
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bilateral difference (p < 0.01) is indicated. Positive differences indicate that involved-leg (right)
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EMG is greater, while negative differences indicate the opposite.
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Figure 5. Grand ensembles (99% confidence intervals are only shown for involved (right) leg to
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increase clarity) for vastus medialis (VM) electromyography (EMG) amplitude (Amp), for the
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comparisons, where mean bilateral differences and corresponding 99% confidence intervals are
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plotted as a function of time. When the shaded area does not overlap with the zero line, a
527
bilateral difference (p < 0.01) is indicated. Positive differences indicate that involved-leg (right)
528
EMG is greater, while negative differences indicate the opposite.
529
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Figure 6. Grand ensembles (99% confidence intervals are only shown for involved (right) leg to
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increase clarity) for vastus lateralis (VL) electromyography (EMG) amplitude (Amp), for the
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pain (A), control (B), and sham (C) sessions. Figures D-F show results of bilateral functional
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comparisons, where mean bilateral differences and corresponding 99% confidence intervals are
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plotted as a function of time. When the shaded area does not overlap with the zero line, a
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bilateral difference (p < 0.01) is indicated. Positive differences indicate that involved-leg (right)
536
EMG is greater, while negative differences indicate the opposite.
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Figure 7. Means and 95% confidence intervals for serum cartilage oligomeric matrix protein
539
(COMP) concentration at three times for each session. If pooled from each session, serum
540
COMP concentration was significantly greater immediately post run, relative to pre run and 60
541
minutes post run; this trend was consistent between sessions.
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