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. All Sprague-Dawley rats were procured at 4-7 weeks of age from Charles Rivers (King of Prussia, Pennsylvania), housed and handled until they reached young adulthood (2.5 months of age at the onset of the experiment). Animals were housed individually in standard rat cages (ventilated and with hardwood chip rodent bedding) in an AAALAC-accredited animal facility with a 12- hour light: 12-hour dark cycle with free access to water. All rats were handled at least 3 times per week to reduce investigator-induced stressors. Female rats were procured to eliminate sex as a potential confounder, and because females are reported to be more prone to work-related MSDs .
All rats were food-restricted to body weights of no more than 10% less than age-matched normal controls so as to be motivated for food reinforcement provided upon successful completion of the prescribed lever-pulling task. The normal control rats were used for weight comparison purposes only, and were not included in the study. All rats were weighed twice per week, provided regular rat chow daily (PicoLab Rodent Diet #5053, Lab Diet, Durham, NC), in addition to food reward pellets (a mix of Banana sucrose and Chocolate dustless precision pellets; # F0024 and #F0299, 45 mg, Bio-Serv, Flemington, NJ), and allowed to gain weight over the course of the experiment, since they were young adult rats at onset of experiments. Control rats that did not perform the task were provided similar amounts of food reward pellets as task rats, in order to be similarly food restricted. All rats were handled at least twice per week and provided cage enrichment toys that included chew bones, tunnels and paper twists (Diamond Twists, Teklad #7979C.CS, Envigo, South Easton, MA). Rats were inspected weekly and postmortem for illnesses and tumors that could contribute to elevation of serum cytokine; none were observed. To reduce illness-related confounders, additional sentinel rats were examined for presence of illnesses as part of regular veterinary care (none were detected).
Twenty-three young adult female Sprague-Dawley rats were used in this study. Task rats were randomly chosen. Thirteen task rats first trained to perform a high-force task for 5 weeks (15 min/day, 5 days/week) to learn the high force task with no specific reach rate, as previously described . Eight trained rats were randomly chosen to perform a high repetition, high force reaching and lever pulling task for 10 weeks without any intervention (10-week HRHF; 2 hrs/day in four 30 min sessions, 3 days/week), as previously described and depicted  and as described further below. The five remaining trained rats performed the lever pulling task for 10 weeks and ran on a treadmill during their last 6 weeks of task performance (10-week HRHF+TM; 1hr/day, 5 days/week), as described further below. Results were compared to 10 food restricted only control rats (FRC) that went through no training or task performance. FRC rats were euthanized at week 10 as indicated later in the Serum and Tissue Analysis section with a terminal dose of sodium pentobarbital (120 mg/kg of body weight), at matched time points as the 10-week HRHF and 10-week HRHF+TM rats.
A total of 16 operant rodent chambers were utilized. Standard open field boxes were placed within larger sound dampening boxes (Med Associates, St. Albans, VT). The boxes were integrated with custom-designed force apparatuses. The force lever bar, which task rats were trained to reach and pull on, was a metal bar of 1.5 mm in diameter, 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 (Model LSB200, Futek Advanced Sensor Technology, Irvine, CA) connected with a strain-gauge amplifier (Model CSG110, Futek). The load cell signal was low pass filtered at 50 Hz and was sampled digitally at 100 Hz by the 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. A successful lever-pull occurred when the rat recognized the cue provided by the auditory indicator (Med Associates) and pulled on the lever bar at the target force threshold, based on a predetermined percentage of maximum isometric force, within a 90 - 500 ms cueing period. If the lever bar were pulled in the correct time frame to the correct force threshold, a reward light would turn on indicating the dispensing of a 45 mg food pellet (Bioserve, NJ) into a trough at floor height .
Training, Task Regimen and Treadmill Running
All rats were handled and acclimated every day for 1 week upon arrival. A subset of rats was randomly selected as food restricted control rats (FRC, n=10). These FRC rats remained sedentary for the duration of the experiment with weekly handling, and grip strength and von Frey sensitivity testing. The remaining 13 rats were trained 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 10 min/day, 5 days/week, for 5 weeks. Trained rats were then randomly assigned to either a 10-week HRHF (n=8) or a 10-week HRHF+TM group (n=5). These two task groups performed the HRHF task for 2 h/day and 3 days/week for 10 weeks . The daily task was performed in four 30-min sessions, separated by 1.5 h in order to prevent satiation. If the rats could maintain the HRHF 60% MPF pulling force target for between 90 msec and 500 msec after the auditory cue was delivered, a food reward would be delivered and the pull was considered as successful.
The task rats randomly assigned for the treadmill intervention regimen performed flat treadmill running in the last 6 weeks of task performance. These rats ran on the treadmill (Columbus Instruments) for 1 hour/day, 5 days/week, ramping up to 23 m/min in the last 20 min on each day. Electric shock was not utilized to avoid stressing the rats. To be clear, the 10-week HRHF+TM group performed the HRHF task for 10 weeks, in addition to performing the treadmill exercise program in the last 6 weeks of the task.
Voluntary task performance outcomes
HRHF voluntary task reach outcomes were recorded continuously during each task session and later extracted into Excel . For this study, grasp force, grasp time, reach rate, percent success rate, and duration of voluntary participation per task session, were assessed by the Force Lever computer program in 10-week HRHF and 10-week HRHF+TM groups on the last day of task week 10, using previously described methods [31, 32]. These data could not be generated for FRC rats, as they did not perform the task.
Briefly defined, grasp force (in Newtons) was the mean recordable force of all reaches per day; grasp time (in seconds) was the mean time the rat spent exerting force on the lever bar over the total number of pulls per day; reach rate was the mean number of reaches per minute (including partial and full pulls on the lever bar) per day; success rate was the percent of successful reaches that resulted in a food reward per day out of all recordable reaches; and the duration of voluntary task participation per day was 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 which started when a reach was detected on the lever bar and ended when the force fell below 2.5% of the minimum required force .
Reflexive grip strength and forepaw 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-week HRHF and 10-week HRHF+TM rats, and at matched time points for FRC rats.
The “up-down” von Frey testing method was used for forepaw sensory testing of all rats, bilaterally, at similar time points as for grip strength . 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 withdrawal threshold.
The person that carried out these assays was blinded to the rats’ group assignments. Data for only the limbs used to reach were reported.
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. 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 immediately centrifuged at 1000g at 4o C. Serum (the supernatant) was extracted and stored at -80o C until assayed. Custom rat multiplex ELISA kits were used to assay serum, in duplicate, for 6 cytokine and chemokines: (1) CXCL2/MIP2, a macrophage and mast cell secreted, wound-healing signaling inflammatory chemokine; (2 & 3) IL-1alpha and IL-1beta, each pro-inflammatory cytokines; (4) IL-6, a proteic cytokine with both pro-inflammatory and anti-inflammatory properties; (5) IL-10, an anti-inflammatory cytokine; and (6) TNF-alpha, a potent pro-inflammatory cytokine. Array sensitivity of the serum analytes were: 1.5 pg/ml for IL-1alpha, 6.2 pg/ml for IL-1beta, 6 pg/ml for IL-6, 0.8 pg/ml for IL-10, and 3.1 pg/ml for TNF-alpha.
All 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 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 12-micrometer thick longitudinal sections and mounted onto positively charged slides.
Subsets of cryosections containing the median nerve at the level of the wrist and mid-
forepaw were immunostained for CD68, a marker of phagocytic macrophages, using an antibody directed against CD68 (1:500 dilution in phosphate buffered saline (PBS), 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 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 the 10 FRC rats’ forelimbs, and each reach limb of the eight 10-week HRHF rats and five 10-week HRHF+TM rats. This quantification was performed in 3-4 sections/nerve after batch staining by one individual who was blinded to group assignment.
Epineurium and extraneural connective tissue thickening was quantified in hematoxylin and eosin stained slides containing branches of the median nerve at the level of the wrist 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 chosen region of interest, relative to the total number of pixels in that region . This quantification was performed in 3-4 sections/nerve by one individual who was blinded to group assignment.
Subsets 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 the 10 FRC rats’ forelimbs, and each reach limb of the eight 10-week HRHF rats and five 10-week HRHF+TM rats. The person who performed the scoring was blinded to group assignment.
A power analysis from past work was performed and showed a minimum of 5/group was needed [17, 36]. Results are reported as mean and standard error of the mean (SEM). Unpaired t-tests were used to compare voluntary reach outcomes at week 10 between the 10-week HRHF and 10-week HRHF+TM groups. One-way ANOVAs were used to compare differences in grip strength and forepaw mechanical sensitivity between the three groups (FRC, 10-week HRHF and 10-week HRHF+TM) using the maximum grip strength and the smallest-sized filament eliciting a withdrawal reflex, for each individual animal’s reach limb. One-way ANOVAs were used to compare differences in serum cytokines, numbers of macrophages in the median nerve, and various tendon counts between the three groups (FRC, 10-week HRHF and 10-week HRHF+TM) using replicate data from individual animals for each analyte. ANOVAS were followed by Tukey’s Honestly Significant Difference (HSD) tests for multiple comparisons; adjusted p values are reported. All statistical analyses and data visualization were conducted with the aid of GraphPad Prism 8.0.2. Type I error rates were set at 0.05 for all statistical tests.