This experiment was approved by the Institutional Animal Care and Use Committee and was compliant with NIH guidelines for the humane care and use of laboratory animals. Twenty-nine female Sprague-Dawley rats were procured at 4-7 months of age from Charles Rivers (King of Prussia, Pennsylvania), housed and handled until reaching young adulthood (3 months of age at the onset of the experiment). Female rats were used exclusively because: 1) higher incidences of work-related musculoskeletal disorders occur in human females than males [43-45], 2) sex is a potential confounder, and 3) results can be compared with those from our previous interventional studies using female rats [12, 41, 42, 46-48]. Animals were housed individually in standard rat cages (ventilated and with hardwood chip rodent bedding) with free access to water in an AAALAC-accredited animal facility with a 12- hour light: 12-hour dark cycle. Rats were handled at least 3 times per week to reduce investigator-induced stressors, and were provided cage enrichment toys including chew bones. Rats were inspected weekly and postmortem for illnesses and tumors that could contribute to systemic inflammation; none were observed. To reduce the potential for illness-related confounders, additional sentinel rats were examined for presence of illnesses as part of regular veterinary care; none were detected.
All rats included in the study were food-restricted to body weights of no more than 10% less than age-matched free-access-to-food normal controls to encourage involvement in the “food reward-based” lever-pulling task. Normal control rats were used for weight comparisons only, and were not included in the study. All rats in the experiment were weighed twice per week, provided with regular rat chow daily (PicoLab Rodent Diet 5053, Lab Diet, Durham, NC) and food reward pellets (Banana flavored dustless precision pellets; F0024, 45 mg, Bio-Serv, Flemington, NJ) during task performance and allowed to gain weight over the course of the experiment, since they were young adult rats at the onset of the experiment. Food restricted control (FRC) rats that did not perform the task were provided similar amounts of food reward pellets as task rats.
As shown in Figure 1, rats were randomly assigned to one of four groups. Nineteen rats were first trained for 5 weeks (15 min/day, 5 days/week) to learn to pull the lever bar at high force levels, at no specific reach rate, as previously described . Eight of these trained rats then performed a high repetition, high force reaching and lever pulling task for 10 weeks without any intervention (termed hereafter as the 10-wk HRHF group; 2 hrs/day in four 30 min sessions, 3 days/wk), as previously described and depicted , and as described further below. A further five trained rats performed the 10-wk HRHF task in addition to forced treadmill running 1 hr/day, 5 days/wk during the last 6 weeks of the HRHF task (termed hereafter as the 10-wk HRHF+TM group), as described further below. The remaining 6 trained rats did not progress to the reaching and lever pulling task, but instead were engaged only in treadmill running for 6 weeks (TR-then-TM group). Four of the six TR-then-TM rats used both limbs to reach, as did five of the ten 10-wk HRHF rats, and three of the five 10-wk HRHF+TM rats. Therefore, we included data from both reach limbs individually (since task exposures could differ) where appropriate (reflexive grip strength, paw withdrawal thresholds, nerve and muscle macrophage numbers, extraneural fibrosis, and tendon histological assays (ten TR-then-TM, thirteen 10-wk HRHF rats and eight 10-wk HRHF+TM rats). Results were compared to 10 food restricted control rats (FRC group; i.e., no training/HRHF task) that were euthanized at matched time points. FRC rats remained sedentary for the duration of the experiment and underwent handling (3 times/wk), and reflexive sensorimotor testing as per the other animals.
Behavioral Apparatus, Training and Task Regimen
A total of 16 operant rodent chambers were used. Each chamber consisted of a standard open field box placed within a larger sound dampening box (Med Associates, St. Albans, VT) integrated with custom-designed force apparatuses. A metal force lever bar of 15 mm in diameter which task rats were trained to reach and pull on, was placed 2.5 cm outside of each operant chamber wall at the rats’ shoulder height. The lever bar was attached to a miniature tension-compression load cell (LSB200, Futek Advanced Sensor Technology, Irvine, CA) connected with a strain-gauge amplifier (CSG110, Futek). The load cell signal was low pass filtered at 50 Hz and was sampled digitally at 100 Hz by customized Force Lever activity software (ENV-118 M, Product Number SOF-808, Med Associates) that allowed the investigator to select the force level exertion threshold at which the rat received the food reward. Rats were trained to pull the lever at a target force threshold, determined as a percentage of maximum isometric force, for at least 90 ms within a 500 ms auditory cueing window . Training involved learning to reach and pull a lever bar at a force threshold of 60% of the average of all rats’ mean maximum pulling force (MPF, 1.18 Newtons) for 15 min/day, 5 days/wk, for 5 weeks, at no specific reach rate . The HRHF task was a repetitive reaching and lever-pulling task for 10 weeks for a food reward. The specifics of this task were pulling the lever bar at 60% of the rats’ maximum pulling force, a reach rate of 4 reaches/min, for 2 hrs/day, in 30 min intervals (with 1.5 hr break between session), for 3 days/wk. If the lever bar was pulled according to these criteria, a reward light flashed indicating the dispensing of a 45 mg food pellet (Bioserve, NJ) into a trough at floor height . The limb use to reach and grasp the lever bar was tracked for all training and task animals across the course of the experiment.
Forced Treadmill Running
Flat running, either immediately after training (TR-then-TM rats) or during the last 6 weeks of the HRHF task (10-wk HRHF+TM rats), was performed on a treadmill (Columbus Instruments) for 1 hr/day, 5 days/wk, at a speed ramping up to 23 m/min for 20 min just before start of the dark cycle. Electric shock was not used to avoid stressing the rats. Instead, lab staff prompted the animal to continue running with gentle prodding.
Voluntary Task Performance Outcomes
HRHF voluntary reaching and lever pulling outcomes were recorded continuously by the Force Lever Program during each task session, for later calculation of reach performance data via an automated script (MatLab; Mathworks, Natick, MA) and then extraction into Excel [12, 42]. Briefly, grasp force was calculated as the average recordable force (expressed as a percentage of maximum pulling force, assayed in Newtons) applied to the force handle for all reaches on a given day. Grasp time was calculated as the average time (in seconds) spent exerting force on the lever bar for all pulls per day. Reach rate was quantified as the average number of reaches per minute (including partial and full pulls on the lever bar) per day. Success rate was expressed as the percent of successful reaches that resulted in a food reward per day out of all recordable reaches. Duration of voluntary task participation per day was calculated as the amount of time (out of 120 min per day) that the rat spent participating in the task rather than sitting in the chamber not pulling. Grasp time and grasp force were calculated using the interval that started when a reach was detected on the lever bar and ended when the force fell below 2.5% of the minimum required force . These voluntary task outcomes were assessed in each of the 10-wk HRHF and 10-wk HRHF+TM groups on the final day of the 10 week task period and encompassed each of the four HRHF task sessions on that day. These data could not be generated for FRC or TR-then-TM rats as they did not perform the task.
Reflexive Grip Strength and Forepaw/Hindpaw Mechanical Sensitivity Testing
Reflexive grip strength was measured in both forelimbs of all rats using a rat grip strength tester (Stoelting, Wood Dale, IL). The test was repeated 5 times per side. Maximum grip strength of the limbs used to reach was reported for all rats after food restriction, at the end of task week 10 for the 10-wk HRHF and 10-wk HRHF+TM rats, at the end of the 6 week treadmill regimen for the TR-then-TM rats, and at matched time points for FRC rats. The “up-down” von Frey testing method was used for forepaw and hindpaw mechanical sensitivity testing of all rats, bilaterally, as previously described  and as we have previously used [12, 46-48]. Monofilaments (North Coast Medical, Morgan Hill, CA) of different diameters were used to elicit a forepaw withdrawal reflex. The force (in grams) of the smallest-sized filament eliciting a withdrawal reflex was recorded as the paw withdrawal threshold (PWT). The person who carried out these assays was an experienced tester and blinded to group assignment.
Serum and Tissue Analyses
All animals were euthanized and tissues collected at 36 hours after the final task session was completed in task week 10, in order to avoid possible serum cytokine fluctuations induced by exercise [17, 35, 50, 51]. All animals were deeply anesthetized with a terminal dose of sodium pentobarbital (120 mg/kg of body weight). Blood was then collected from all rats using cardiac puncture with a 23-gauge needle. The blood was stored on ice for ~1 hour until it clotted before being centrifuged for 20 minutes at 1000g at 4o C. Serum (the supernatant) was then collected and stored at -80o C until assayed. Custom rat multiplex ELISA kits from Pierce Searchlight were used to assay serum, in duplicate, for: IL-1α and IL-1β, each pro-inflammatory cytokines; IL-6, a proteic cytokine with both pro-inflammatory and anti-inflammatory properties; IL-10, an anti-inflammatory cytokine; and TNFα, a potent pro-inflammatory cytokine. Corticosterone levels were also assayed in the serum (55-CORMS-E01, Alpco, Salem, NH). Array sensitivity of the serum analytes were: 1.5 pg/ml for IL-1α, 6.2 pg/ml for IL-1β, 6 pg/ml for IL-6, 0.8 pg/ml for IL-10, 3.1 pg/ml for TNFα, and 6.1 ng/ml for corticosterone.
Soleus muscles were then collected (prior to perfusion of rats with fixative) for examination of collagen production. Additionally, flexor digitorum muscles were collected from one arm of four of the FRC rats prior to perfusion. Samples were homogenized in phosphate buffered saline containing protease inhibitors, and total protein quantified, as previously described in detail . These samples were prepared for gel electrophoresis by either: 1) boiling and exposing to beta-mercaptoethanol (BME), 2) not boiling the samples before exposing them to BME, 3) or neither boiling nor exposure to BME, in order to detect procollagen, mature collagen or cleavage products, as previously described in detail . All samples were run on a 4-12% Tris-Glycine gel without SDS in the gel, yet with SDS in the sample and loading buffers . After immunoblotting, blots were probed with an antibody against collagen type I (C2456, Sigma-Aldrich, St. Louis, MO). Membranes were stained with Ponceau S prior to antibody probing as a loading control .
After collection of serum and muscles for western blotting (see above), animals were perfused intracardially with 4% paraformaldehyde in 0.1M phosphate buffer using a perfusion pump, before collection of forearm tissues for later histological analyses. The forelimb soft tissue mass (see ) was removed from bones en bloc, fixed in formalin for 3 days, equilibrated in 10% and then 30% sucrose in 0.1M phosphate buffer for 2 days each, before being cryosectioned into 14-micrometer thick longitudinal sections and mounted onto positively charged slides.
Subsets of cryosections containing the median nerve at the level of the wrist were immunostained with an antibody directed against CD68 (a marker of phagocytic macrophages in rats [54-56], Abcam, Massachusetts, United States). After 15 minutes of 0.5% pepsin antigen retrieval at room temperature, sections were incubated for 20 minutes in 4% goat serum in phosphate buffered saline (PBS) and then incubated with the anti-CD68 at a 1:250 dilution in PBS at 4o C overnight. The next day, sections on slides were washed 3 x 15 min each, and then incubated with the secondary antibody, AffiniPure F(ab)2 fragment, conjugated to a red fluorescent cyanine dye (Cy3; Jackson ImmunoResearch, West Grove, PA) at a dilution of 1:100 at room temperature for 2 hours. When cover-slipping, DAPI was used as a nuclear counterstain. Numbers of CD68+ cells per mm2 in the median nerve at the level of the wrist and in the mid-forepaw were quantified, using previously described methods  in three to four non-adjacent sections per nerve, and per rat. Nerves were quantified in ten FRC rat forelimbs, and in ten TR-then-TM rats, thirteen 10-wk HRHF rats, and eight 10-wk HRHF+TM reach limbs. This quantification was performed in 3-4 sections/nerve after batch staining by one individual who was blinded to group assignment. Flexor digitorum muscles were similarly examined for presence of immune cells after hematoxylin and eosin staining, and macrophages after immunostaining with anti-CD68 antibody, then a secondary antibody with a horseradish peroxidase (HRP) tag that was detected with diaminobenzidene (DAB) detection methods, followed by eosin counterstaining .
Epineurium and extraneural connective tissue thickening was quantified in hematoxylin and eosin stained slides containing branches of the median nerve at wrist-level using a digital camera (Retiga 4000R QImaging Firewire Camera, Surry, BC Canada) interfaced with an image analysis system (Life Science, Bioquant Image Analysis Corporation, Nashville, TN). An irregular region of interest (ROI) cursor of 75 micrometers in size was used to outline the median nerve within the epineurium, and then again at micrometers external to that outline . Then a Videocount Area Array option of the software was utilized (defined as the number of pixels in a field that met a user-defined color threshold of staining) to quantify the number of pixels containing dense pink stained connective tissue within the ROI, relative to the total number of pixels in that region . Three to four sections/nerve were quantified by one individual who was blinded to group assignment. Presence of CD68+ macrophages in epitendons was also examined in sections stained for nerve (see above) to determine if the epitendon cellularity was due to only fibroblast proliferation or also more CD68+ macrophages . The latter was qualitatively examined only.
Subsets of sections of forelimb soft tissues containing flexor digitorum tendon sections were stained with hematoxylin and eosin. Tendons were scored using a semi-quantitative method, the modified Bonar scale, using previously described methods . Briefly, using a scale from 0 to 3, 0 represented a normal histological appearance in the epitendon and endotendon (that is, an elongated cell shape, collagen fibers that were aligned with tenocyte cell shape, and even distribution of cells), while 3 represented advanced pathological changes (e.g., rounded cell shape, wavy fibers, and dense distribution of cells). Tendons were quantified in ten FRC rat forelimbs, and in ten TR-then-TM, thirteen 10-wk HRHF rats, and eight 10-wk HRHF+TM reach limbs. The person who performed the scoring was blinded to group assignment.
An a priori power analysis was performed using data from our prior studies on voluntary task outcomes, grip strength and numbers of macrophages in the median nerve [37, 38, 40]. We chose the most conservative sample size needed to detect differences with an alpha level of 0.05 and 80% power. This a priori power analysis indicated that our estimated sample size needed was 5 per group. Since the observed effect sizes were slightly smaller than the expected values, we performed a retrospective power analysis, which determined that median nerve macrophage results was at 77% power and serum TNFα results was at 79% power. Therefore, we increased the sample size for several assays where appropriate (reflexive grip strength, paw withdrawal thresholds, nerve and muscle macrophage numbers, extraneural fibrosis, and tendon histological assays) by including data from each limb used to reach individually (since task exposures could differ from limb to limb) to 8-13 reach limbs/group (Figure 1). This increased the power of median nerve macrophage results to 90%.
Next, both Shapiro-Wilk and Kolmogorov-Smirnov tests of normality were performed, and residuals were inspected. Unpaired, two-tailed t-tests were used to compare voluntary reach outcomes at week 10 between the 10-wk HRHF and 10-wk HRHF+TM groups. One-way ANOVAs were used to compare serum cytokines, grip strength, numbers of macrophages in the median nerve, and numbers of immune cells in muscle, using replicate data for the latter two, between all groups. Tukey’s test was used for post hoc analyses; adjusted p values are reported. As paw withdrawal thresholds and tendon scores were not normally distributed, Kruskal-Wallis nonparametric tests were used to compare data between groups, and post hoc testing using Dunn’s tests for multiple comparisons; adjusted p values are reported. Pearson’s and Spearman’s rank correlation tests, as appropriate for the data, were used to determine correlations between various outcomes. Significance was set at p = 0.05 and results are reported as mean and 95% confidence internals (CI).