Keywords: collagen, cumulative trauma disorder, fibrosis, massage, mobilization, repetitive strain injury, overuse injury, TGF-β1

Subjects

The Temple University Institutional Animal Care and Use Committee approved these experiments in compliance with NIH guidelines for the care and use of laboratory animals. Female Sprague-Dawley rats (3 mo. of age at onset of experiments) were used because human females have a higher incidence of work-related musculoskeletal disorders (Gerr, Marcus et al. 2002,Ratzlaff, Gillies et al. 2007,Srilatha, Bhat et al. 2011,Cote 2012), and to allow comparison to our past studies on same-aged female rats using this model, such as those examining the effectiveness of anti-inflammatory drugs in reducing HRHF-task induced inflammation (Rani, Barbe et al. 2010,Abdelmagid, Barr et al. 2012,Jain, Barr-Gillespie et al. 2014).

All rats were housed in AAALAC-accredited animal facility in separate cages with a 12-hour light:dark cycle with free access to water and environmental enrichment in their home cages (chew toys and tunnels). All rats were handled extensively for 1 week prior to onset of experiments, and then 5 days/week thereafter. All rats were food-restricted to body weights of 5% less than age-matched normal controls (used only for weight comparison) in order to motivate interest in food reward pellets. Rats were weighed weekly and allowed to gain weight across the course of the experiment, as shown previously, since they were young adults. In addition to 45 mg food pellet rewards (a 1:1 mix of purified grain and banana flavored pellets; Bio-Serv, NJ, USA), all rats received Purina rat chow daily (i.e. same food reward and chow rations for all groups).

A power analysis was performed using Statview using prior tissue TGF-β1 and grip strength data (Abdelmagid, Barr et al. 2012,Fisher, Zhao et al. 2015). We chose the most conservative sample size needed to detect differences with an alpha level of 0.05 and 80% power. The power analysis estimated that for ANOVAs of tissue and motor behavioral findings that a sample size of at least n=5/group was needed, per type of tissue analyses (histological versus biochemical). Since not all rats destined for the HRHF task learn the task completely, two additional rats each were added the FRC group and HRHF group. Based on this, we first randomly divided rats into three groups: 1) age-and weight-matched food restricted control rats (FRC, n=7); 2) age-matched rats that were trained for 6 weeks before performing the high-repetition, high-force task (HRHF) task for 12 weeks with no treatment (HRHF-CON, n=6); and 3) age-matched rats that were trained for 6 weeks before performing the HRHF task for 12 weeks that would receive modeled manual therapy (HRHF-MMT, n=6). After training, one rat in the HRHF groups did not train adequately, and were removed from the study, leaving n=6 in the HRHF-CON group and n=5 in the HRHF-MMT group. Later, histological findings of only low inflammation in the 12-week HRHF-CON rats required confirmation; so 5 additional rats per group were added to the FRC and HRHF-CON groups for biochemical analyses. By study end, data from 28 rats were used: FRC rats, n=12; HRHF-CON rats, n=11; HRHF-MMT, n=5.

Behavioral Apparatuses, Training, and Task Regimen

Sixteen custom-designed behavioral apparatuses were used for these experiments, as previously described and depicted (Barbe, Gallagher et al. 2013). Briefly, animals reached through a shoulder height portal and isometrically pulled a force handle attached to a force transducer with a load cell (Futek Advanced Sensor Technology, Irvine, CA) located outside the chamber wall. The load cell was interfaced with custom Force-Lever software (version 1.03.02, Med Associates, St. Albans, VT). Auditory and light indicators cued the reaching rate (defined below). If reach and force criteria (defined below) were met within a 5 second cueing period, a 45 mg food pellet was dispensed into a trough.

All 17 HRHF rats underwent an initial training period for 6 weeks in which they learned the task, as previously described in detail (Barbe, Gallagher et al. 2013). Briefly, rats were initially food-restricted for 7 days to no more than 10% less than their naïve weight, and the weight of age-matched normal control rats with free access to food (used for weight comparisons only, and not included in this study), to initiate interest in food reward pellets. After that week, they were given extra rat chow to gain weight quickly back to only 5% less than the normal control rats. Rats were weighed weekly, maintained at ± 5% less than the normal controls; all were allowed to gain weight during the study, as reported previously (Jain, Barr-Gillespie et al. 2014). The 17 rats underwent operant training to learn the reaching and handle-pulling task during a 5-week period of 10 min/day, 5 days/wk, in which they ramped upwards from naive towards the HRHF task level. All but two of the rats undergoing training reached the HRHF level (described below) during their last week of training. The 16 rats that learned to reach at the HRHF level went on to the next stage of the experiment (HRHF-CON, n=11; HRHF-MMT, n=5).

After the training period, a point equal to week 0 of the HRHF task, all 15 trained rats began the HRHF task regimen for 2 hrs/day, 3 days/wk for up to 12 weeks. The task was divided into 4, 0.5-hr sessions separated by 1.5 hrs in order to avoid satiation. HRHF rats were cued to reach at a rate of 4 reaches/min and to extend their forearm into a portal, grasp a handle, and then exert a target isometric pull for at least 50 milliseconds (ms) at a force effort of 40% ± 5% group maximum (40% maximum pulling load = 77 cN) (SeeSupplemental Fig 1). These criteria had to be met within a 5 second window initiated every 15 seconds, and the handle held with the correct force for 50 ms. If these criteria were met, rats received a food reward deposited into a food trough, and this was considered a successful reach. Rats were allowed to use their preferred limb to reach (the “reach” limb), and data from this limb only is reported.

Modeled Manual Therapy

Because people often seek treatment when they first develop symptoms, we initiated the MMT after training to the high force level, when rats begin to develop symptoms consistent with WMSDs (Rani, Barbe et al. 2010,Abdelmagid, Barr et al. 2012,Barbe, Gallagher et al. 2013,Fisher, Zhao et al. 2015). One group of HRHF rats (HRHF-MMT; n=5) underwent MMT beginning in week 0 after training, and then 5 days/week for the duration of the 12 weeks of HRHF task performance. The FRC and HRHF-CON rats underwent light handling only instead of the MMT treatment for the 12-week duration.

The MMT treatment protocol was developed by an experienced manual therapist (GMB) and taught to the technician (MH) who provided the training, data collection, and care in these and previous studies. The technician had no previous experience with massage therapy delivery. She observed the protocol over a few hours, and then studied video recordings of the initial training sessions across several days; her performance of the MMT was then videotaped for supervision and adjustment as needed by the experienced manual therapist. The protocol, designed to emulate what a massage therapist or other manual therapist might perform, consisted of scaled-down versions of many common modalities. These included (in order) gentle mobilization, skin rolling, and deep strokes (aka “muscle stripping” or “myofascial release”) to the forearm flexor compartment; joint mobilization of the wrist; and stretching combined with long superficial strokes of the entire upper limb (seevideo in Supplemental Information).

Unsedated rats were placed in the lap of the seated operator, and held gently until comfortable (1–2 minutes). The scapula and shoulder were then stabilized with one hand while the other hand delivered the treatment in 5 phases.

1. The thumb, index finger, and middle finger gently compressed the flexor muscles, and mobilized them laterally over the radius and ulna (10 cycles).

2. The skin over the forearm was pinched together and rolled between the thumb and index finger (5 rolls), including all loose skin from the wrist to the elbow.

3. While one hand stabilized the wrist, the index finger of the hand stabilizing the shoulder was used to push the skin distally over the flexor muscles, and then was pressed into the flexor tendons and muscles and pulled proximally. Enough force was used to be able to feel the deeper structures but not enough to cause the rat to withdraw its paw. This provided deep spreading pressure to the muscles and tendons in all but the most proximal part of the flexor compartment. This was repeated 10 times.

4. The distal forearm was stabilized with one thumb and index finger, while the other thumb and index finger stabilized and lightly tractioned the paw. The wrist was mobilized 5 cycles posterior to anterior, and 5 cycles in axial rotation, while maintaining the traction of the paw.

5. While the rat was stabilized at the shoulder, the treating thumb and fingers grasped the proximal upper limb and gently tractioned (a stroking traction) while sliding across the fur, rolling back and forth when reaching the forearm and continuing this to the rat’s fingers until they slid out of the treating hand.

Treatment was performed on both arms, and took less than 10 minutes per session depending on the cooperation of the individual rat.

Spontaneous changes in behaviors occurring during or after treatment

Although the rats seemed very comfortable with the treatments, we looked for and recorded on incidence any behaviors indicative of discomfort. These potential behaviors included: momentary freezing, prolonged freezing, strong escape behaviors, limb withdrawal during touching or mobilization, increased defecation or urination, increased biting or acts of aggression, and increased vocalization. Rats were then returned to their home cage, and for 1 hour after each treatment session, home-cage behaviors that could be related to pain or discomfort were recorded on incidence, including: excessive movement around the home cage, increased grooming, limb guarding, paw licking, or limping. Similar home-cage behaviors were also observed for in untreated FRC and HRHF-CON rats, for 1 hour per day, 5 days/week for 12 weeks.

Spontaneous changes in behaviors occurring during task performance

Trained observers tracked changes in spontaneous behaviors occurring during each period of HRHF task performance. Bilateral pulling of the lever bar was recorded upon occurrence, as was a switch in forearm used to pull the lever bar from the typically used “preferred” reach limb. On occasion, a rat would twist its forelimb into a supinated position when pulling on the lever bar rather than using a typical pronated position; this was recorded as a “supinated pull.” Fumbling, guarding, or pulling with one digit rather than the typical four-digit grasp were recorded upon occurrence, as was refusal to participate (typically seen as sitting in the corner of the chamber, and thus termed “sits in corner”). Data from the last day of each task week are reported for HRHF-CON and HRHF-MMT groups (FRC rats did not perform the task).

Force lever data were also recorded continuously during each task session for later calculation of the percentage of successful reaches via an automated script (MatLab; Mathworks, Natick, MA). A reach was defined as a force deflection that exceeded 2.5% of the baseline (or zero) force until the next sample in which the force fell below 2.5% of baseline. The number of successful reaches/week is reported for the HRHF-CON and HRHF-MMT groups (FRC rats did not perform the task).

Reflexive Grip Strength

Reflexive grip strength of the forelimbs was measured in 15 animals (FRC, n=10; HRHF-CON, n=10; HRHF-MMT, n=5) bilaterally, at baseline (the naive time point), after training (the 0 week time point of the HRHF task), and every 2 weeks thereafter, using a rat grip strength-recording unit (Stoelting, Wood Dale, IL), as described previously (Kietrys, Barr et al. 2011). These assays were performed by an examiner naive to group assignment. The test was repeated 5 times/limb/trial, and the maximum grip strength per trial reported in centinewtons (cN).

Serum biomarker assays

Serum was collected from 15 rats in the study for assay of TGF-β1 levels (FRC, n=10; HRHF-CON, n=10; HRHF-MMT, n=5). At 90 minutes after the last task session, rats were anesthetized with 5% isoflurane in oxygen and then euthanized by exsanguination. Blood was withdrawn from the heart via cardiac puncture and centrifuged immediately at 1000 x g for 20 min at 4°C. The supernatant (serum) was collected and stored at −80°C until analyzed using a commercially available ELISA kit for TGF-β1 (KAC1688, Invitrogen Corporation, Camarillo, CA). Results from the serum ELISA were normalized to ml of serum (pg TGF-β1/ml serum).

Histological and Immunohistochemical Analyses

Forelimb tissues were collected for histological analysis from FRC (n=7), HRHF-CON (n=6), and HRHF-MMT (n=5) rats were collected. Following euthanasia and after serum collection, animals were perfused transcardially with 4% buffered paraformaldehyde (pH 7.4). Tissues were collected and postfixeden bloc by immersion for two days. Forepaws were then separated from the forelimbs at the level of the wrist.

Forepaws were removed at the level of the wrist using a small Dremel bone saw. The forepaw tissues were processeden bloc, with skin, lumbricals and bone intact, and embedded in paraffin, sectioned into 5 μm thick cross-sections, mounted onto charged slides (Super Frost Plus, Thermo Fisher Scientific, Pittsburgh, PA), and allowed to dry overnight before storage at room temperature. After deparaffinization in xylene and rehydration, sections on slides were stained with Masson’s Trichrome, dehydrated, coverslipped with DPX mounting medium for bright field microscopy, and examined for fibrous collagen (stained blue). Adjacent sections were treated with 3% H₂O₂ in methanol to block for endogenous peroxidase for 30 min, washed in phosphate buffered saline (PBS), then permeabilized with 0.05% pepsin in 0.01N HCL, washed again in PBS, prior to blocking with 4% dried milk (Carnation) in PBS for 20 min at room temperature. Sections were then immunostained in batched sets by the same individual for TGF-β1 using a mouse monoclonal anti-TGF-β1 at a 1:500 dilution in PBS with 4% milk (MAB240, R&D Systems, Minneapolis, MN). Slides were incubated overnight at room temperature in a humid chamber. On the 2^nd day, after washing, sections were incubated with goat anti-mouse secondary IgG antibody conjugated to HRP (Jackson Immunoresearch Laboratories, West Grove, PA; diluted 1:100 in PBS, incubated 2 hours at room temperature before washing in PBS) and visualized using DAB (Fast DAB, Sigma, St. Louis, MO). These sections were dehydrated, and then coverslipped with DPX mounting medium for brightfield microscopy. A third set of adjacent sections was stained with hematoxylin and eosin.

After removal of the forepaw at the level of the wrist, the fixed forearm flexor digitorum muscle and tendon mass were removed from the forearm bones using a scalpel. A proximal region near the elbow was removed with a scalpel for cross-sectioning, while the remaining flexor digitorum muscles were separateden bloc as a flexor mass for longitudinal sectioning. These tissues were cryoprotected in 30% sucrose in PBS before cryosectioning into 15 μm cross sectional or longitudinal slices. Sections were then placed onto charged slides (Super Frost Plus, Thermo Fisher Scientific) and allowed to dry overnight before storage at −80°C. These cryosectioned tissues were immunostained in batched sets by the same individual for collagen type I and TGF-β1 using sequential double-labeling methods [mouse monoclonal anti-Collagen type I at 1:500 dilution (C2456, Sigma) and mouse monoclonal anti-TGF-β1 at 1:500 dilution (MAB240, R&D Systems)]. Briefly, after an antigen retrieval step of 0.5% pepsin for 15 minutes at room temperature, sections were washed in phosphate buffered saline (PBS), incubated for 30 minutes in 10% goat serum in PBS, before incubating with anti-Collagen type I at the listed dilution for overnight at room temperature. Sections were washed in PBS before incubation in a goat anti-mouse secondary IgG antibody, that had been preabsorbed to reduce non-specific cross-reactivity with rat antigens, and conjugated to a red fluorescent cyanine dye (AffiniPure F(ab′)₂ fragment, CY3, 550 nm excitation; Jackson ImmunoResearch Laboratories) at a dilution of 1:100 for 2 hours at room temperature in the dark. Sections were washed in PBS and reblocked in 10% goat serum in PBS for 20 min, before incubating overnight at room temperature with anti-TGF-β1 at the listed dilution. Sections were washed in PBS before incubation in a goat anti-mouse secondary IgG antibody conjugated to a green fluorescent cyanine dye (DYLIGHT 488; Jackson ImmunoResearch Laboratories) at a dilution of 1:100 for 2 hours at room temperature in the dark. The fluorescent tag-labeled sections were washed with PBS, incubated with DAPI (1:1000 dilution in PBS) for 15 minutes at room temperature, washed again, then coverslipped with 80% glycerol in PBS for epifluorescence microscopy. Adjacent sections were stained with hematoxylin and eosin.

Preabsorption control staining was also performed to demonstrate if the antibodies bound specifically to the antigen of interest using TGF-β1 human recombinant protein (GF111, Millipore, Billerica, Massachusetts) or Collagen type 1 purified rat protein (C7661, Sigma, St. Louis, MO). No labeling was observed in the tissues for any pre-absorbed antibody, as previously published and depicted (Fisher, Zhao et al. 2015). We also performed no-primary antibody controls in which serum was substituted for the primary antibody, followed by secondary antibodies; no labeling was observed, as previously published and depicted (Fisher, Zhao et al. 2015).

Quantification of staining and immunostaining was performed in batched sets by one person (MFB), blinded to group assignment, using a microscope (Nikon E600) interfaced with a digital camera (Retiga 4000R QImaging Firewire Camera, Surry, BC Canada) and an image analysis system (BioQuant Osteo II, Bioquant Image Analysis Corporation, Nashville, TN). First, in the forepaw and median nerve at the level of the wrist, the proportion of each field or region of median nerve, respectively, displaying the blue stain after Masson’s Trichrome staining (indicative of fibrous collagen) was quantified. The microscope’s light intensity (bright field), camera gain and exposure were standardized and remained constant. The blue color was designated as a separate threshold in the RGB component of the image BioQuant software. This threshold was saved in a subprogram, and then applied to all images for consistent auto-measurement of the blue stain. The Videocount Area Array option of the software was also utilized (defined as the number of pixels in a field that met a user-defined color threshold of staining). The number of thresholded blue pixels in each field were then counted and analyzed as a percentage of the total number of pixels in that field, using similar methods as previously described for liver fibrosis (del Pilar Alatorre-Carranza, Miranda-Diaz et al. 2009). For the glabrous region of dermis of the mid forepaw, 3 to 4 fields were counted per animal (in cross-sectional tissue slices) using a 20X objective, and the % area of blue staining (fibrous collagen) was averaged for each animal, before comparing results across groups. For the median nerve at the level of the wrist, 2–3 fields were counted per animal (in longitudinal sectioned tissue slices) using a 10X objective, and the % area with blue staining averaged for each animal, before comparing results across groups.

Next, the percent area of immunoreactivity for TGF-β1 in the paraffin embedded forepaws was performed in batched sets in the glabrous dermis and adjacent underlying loose connective tissue, and around the lumbricals, in 5 animals/group. The % area of immunoreactivity for TGF-β1 and collagen 1 in the cryosections were also quantified in batched sets in the proximal flexor digitorum muscle region that had been cross-sectioned, and in longitudinal sections of flexor digitorum tendons (near the wrist). The microscope’s epifluorescence light intensity, camera gain and exposure were standardized, and remained constant for each batch of acquired images. Again, a thresholded color was chosen (green or red), saved into a subprogram for consistent auto-measurement of green or red pixels in the field, versus total number of pixels in each field. Three to 5 fields were counted per region, per animal, using a 20X objective, and the % area with staining was averaged for each animal, before comparing results across groups.

Tissue Biochemical Assays

Following euthanasia and after serum collection, flexor digitorum muscles from 5 FRC rats and 5 HRHF-CON rats were collected from the distal forearm region of the preferred reach limb. Muscles were homogenized, as previously described (Barbe, Gallagher et al. 2013), and assayed by customized multiplex bead-based analysis kits (LXSARM, R&D Systems) for IL-1α, IL-6, IL-1β, and TNFα. ELISA assay data (pg of cytokine protein) were normalized to μg total protein, determined using a bicinchoninic acid protein assay kit.

Statistics

Behavior data across weeks was assayed using SAS (SAS Institute Inc, Cary, NC). Specifically, a Poisson regression with generalized estimating equation was used to estimate incidence rate, odds ratios, and 95% confidence intervals (CI) for spontaneous behaviors occurring in reach versus nonreach limbs of HRHF-MMT rats during MMT. A linear mixed-effects model was used to estimate and compare the incidence rate of altered movement strategies across weeks in HRHF-CON versus HRHF-MMT rats. Logistic regression with generalized estimating equation was used to compare differences in individual altered movement strategies in HRHF-CON versus HRHF-MMT rats. A linear mixed-effects model was also used to estimate and compare the change over time (slopes) in the number of successful reaches, and the maximum grip strength, between groups (FRC, HRHF-CON and HRHF-MMT). One-way ANOVAs were used to compare results of histochemical and immunohistochemical quantification, except for quantification of fibrosis in the median nerve, where a two-tailed t-test was used, using GraphPad PRISM v.6.02, GraphPad Software, Inc., La Jolla, CA. Bonferroni post-hoc corrections were used when multiple pair-wise tests were performed on the same data set to reduce the chances of obtaining false-positive results (Type I errors). In accordance with this method, an adjustment was made top values by the analysis program used by dividing the critical p value (α = 0.05) by the number of comparisons made, thus increasing the stringency of the analysis. Adjusted p values of < 0.05 were considered significant for all comparisons. Group means plus standard error of the mean (SEM) or standard error (SE) are reported.

Daily MMT led to a small increase in withdrawal of the preferred reach limb during MMT, and no observation of spontaneous behaviors suggestive of discomfort

For the 5 HRHF-MMT rats, Poisson regression analysis showed a small increase in spontaneous behaviors suggestive of discomfort was observed during MMT of the reach limbs, compared to the non-reach limbs [incidence rate ± SE for the reach limb = 0.65 ± 0.26; nonreach limb = 0.92 ± 0.23; incidence rate ratio (95% CI) = 0.7 (0.5, 0.99); p=0.047]. When these behaviors were examined individually, the HRHF-MMT rats showed no increased urination, defecation, or vocalization behaviors during MMT of either limb (Fig. 1), and no biting or acts of aggression (data not shown). There was a very small but equal incidence of strong escape behaviors during MMT of either limb in weeks 7 and 9 (Fig. 1), although these increases did not reach significance in post hoc tests.

Our monitoring of spontaneous signs of discomfort continued after return of rats to the home cage (excessive movement around the home cage, increased grooming, limb guarding, paw licking or limping). None were observed. These observations indicate that the treatment was well tolerated.

Spontaneous behaviors suggestive of discomfort occurring during HRHF task were less with MMT

Since we have previously observed increased spontaneous behaviors suggestive of discomfort during performance of this HRHF grasping and pulling task (Fisher, Zhao et al. 2015), these same behaviors were monitored here. A linear mixed-effects model was used to show that the incidence rate of altered movement strategies that may indicate discomfort or reduced function were lower across weeks of task performance in HRHF-MMT rats, compared to HRHF-CON rats (Fig. 2; HRHF-MMT overall incidence rate mean ± SEM = 1.38 ± 0.22; HRHF-CON = 1.97 ± 0.17; difference = −0.59 ± 0.26; p=0.027). Logistic regression analyses of individual subgroups of altered movement behaviors showed a decreased incidence rate of limb switching in HRHF-MMT rats, compared to HRHF-CON rats [Odds ratio (95% CI) = 0.026 (0.0066, 0.0999); p<0.001]. Also, guarding/avoidance behaviors did not occur in HRHF-MMT rats after Week 3, and pulling with only 1–2 fingers (rather than all 4 fingers) was not observed in HRHF-MMT rats after Week 7 (Fig. 2); low incidence of each were observed in HRHF-CON rats. Fumbling was observed only in 1 HRHF-CON rat in Week 4. These observations suggest that the treatment was beneficial to task performance.

Modeled manual therapy led to enhanced performance and reduced motor declines typically induced by the HRHF task

While the number of successful reaches per week increased slightly over time in the HRHF-CON rats, performance of the rats receiving MMT was significantly enhanced (Fig. 3A). A linear mixed-effects model was used to show that the slope of change in successful reaches across weeks of task performance in HRHF-MMT rats was 10.6 ± 2.95 (slope ± SE); in contrast, the slope in HRHF-CON rats was 1.08 ± 2.95 (difference = 9.5 ± 4.18; p=0.025). Post-hoc tests comparing the number of successful reaches per week showed that these effects were driven by the higher incidence of successful reaches by HRHF-MMT rats at testing weeks 4–12 (Fig. 3A). These data support a substantial increase in voluntary task performance due to the MMT treatments.

Consistent with our previous studies, grip strength in the preferred reach limbs of each HRHF group decreased during training and continued to decline with time in HRHF-CON rats, compared to FRC rats (Fig. 3B). A linear mixed-effects model was used to show that the slope of change in grip strength in HRHF-CON rats was −11 ± 1.66 cN/week (slope ± SE), while the slope in FRC rats −2.5 ± 1.57 cN/week (difference = −8.5 ± 2.29, p=0.0003). During this same task period, the grip strength of HRHF-MMT rats improved towards levels seen in FRC rats (Fig. 3B; HRHF-MMT slopes ± SE = −3.5 ± 2.24 cN/week, difference between HRHF-MMT and FRC rats = −1.02 ± 2.74, p=0.71) and away from levels seen in HRHF-CON (Fig. 1B; difference between HRHF-MMT and HRHF-CON slopes ± SEM = 7.5 ± 2.79 cN/week, p=0.008). Post-hoc tests showed that these effects were driven by the decreased grip strength of HRHF-CON rats at all points after training compared to age-matched FRC rats, but decreases in HRHF-MMT rats, compared to FRC rats, only post training (week 0) and through task week 2 (Fig. 3B). These data support an increase in reflexive forearm/forepaw motor strength due to the MMT treatments.

Maximum pulling force did not induce inflammation in HRHF rats by 12 weeks of task performance

H&E staining did not show increases in inflammatory cells in any tissues examined in the HRHF-CON and HRHF-MMT rats at the time of tissue collection (12 weeks after onset of HRHF task performance), compared qualitatively to FRC rats (n=5/group; data not shown). To further confirm a low inflammatory response in tissues, additional FRC and HRHF-CON animals (n=5/group) were examined using ELISA and then one-way ANOVAs for the presence of pro-inflammatory cytokines in flexor digitorum muscles (IL-1α, IL-6, IL-1β, and TNFα). No significant increases were observed in HRHF-CON rats, compared to FRC rats (data not shown).

Modeled manual therapy prevented HRHF-induced increased forepaw collagen deposition around the median nerves and lumbrical muscles

Consistent with our previous results showing increased collagen deposition in forepaw and forelimb tissues of HRHF rats (Clark, Al Shatti et al. 2004,Fedorczyk, Barr et al. 2010,Abdelmagid, Barr et al. 2012,Gao, Fisher et al. 2013,Jain, Barr-Gillespie et al. 2014,Fisher, Zhao et al. 2015), HRHF-CON rats had increased fibrous collagen deposition in the forepaw connective tissues surrounding branches of the median nerves, lumbrical muscles and their individual myofibers in HRHF-CON rats (Fig. 4A,B), compared to HRHF-MMT rats (Fig. 4C,D). As shown inFigures 4F and G, there was also increased collagen deposition around and within the median nerve at the level of the wrist (i.e., intraneurally between individual axons). These differences were confirmed by quantification of the blue staining in this latter region using a two tailed t-test (p=0.007;Fig 4E). Similar increases in fibrous collagen deposition were observed in their glabrous forepaw dermal extracellular matrix of HRHF-CON rats, compared to both FRC and HRHF-MMT rats (Fig. 5A,C-F). These differences were confirmed by quantification of the blue staining in this region (n=5/group; ANOVA, F_{2, 12} = 25.33,p < 0.001;Fig. 5B).

Modeled manual therapy attenuated HRHF task-induced increases in tissue TGF-β1 and collagen type 1

We observed increased TGF-β1 immunoreactivity in the dermis and underlying loose connective tissue of the glabrous dermal region of the forepaw in HRHF-CON rats, compared to FRC and HRHF-MMT rats (Fig. 6A,C, E). These differences were confirmed by quantification of the percent area with TGF-β1 immunostaining in this region (n=5/group; ANOVA, F_{2, 12} = 12.45,p = 0.001 (p<0.05 each posthoc test,Fig. 6G). The TGF-β1 immunoreactivity was localized within and around enlarged dermal fibroblast-like cells in HRHF-CON rats (Fig. 6C; representative cells are indicated by arrows). In the nearby lumbrical muscles, we observed low level increases TGF-β1 immunoreactivity in small fibroblast-like cells located on the perimeter of myofibers in HRHF-CON rats, compared to FRC and HRHF-MMT rats (Fig. 6B, D, F; representative cells are indicated by arrows). These differences were confirmed by quantification of the % area with TGF-β1 immunostaining in the lumbrical muscles (n=5/group; ANOVA, F_{2, 12} = 19.59,p = 0.0002 (p<0.05 each post hoc test;Fig. 6H). The latter increases were over 3 fold less than that seen in the connective tissue of the dermis.

Using cross-sections and longitudinal sections of mid-belly flexor digitorum muscles for immunohistochemistry, we examined TGF-β1 and collagen type I immunoreactivity. We found low levels in each of the FRC rats (Fig. 7A–C), higher levels in HRHF-CON rats (Fig. 7D–F), yet lower levels in HRHF-MMT rats, qualitatively, compared to HRHF-CON rats (Fig. 7G–I). The collagen type I immunostaining was localized to fibroblast-like cells and extracellular matrix immediately surrounding the myofibers, particularly in HRHF-CON rat tissues (Fig. 7D,F), while TGF-β1 immunostaining was localized just to the fibroblast-like cells in the perimeter of myofibers. These findings were quantified in cross-section slices of mid-belly flexor digitorum muscles and showed that the % area with TGF-β1 immunoreactivity in the mid-flexor digitorum muscle region was increased in HRHF-CON rats (n=6), compared to FRC (n=7) and HRHF-MMT rats (n=5; ANOVA, F_{2, 15} = 9.05,p = 0.002;Fig. 8A). The percent area with collagen type 1 immunoreactivity in this same region of mid-flexor digitorum muscle was also quantified and shown an increase in HRHF-CON rats, compared to FRC and HRHF-MMT rats (ANOVA, F_{2, 15} = 10.92,p = 0.001;Fig. 8B).

We also assayed serum for TGF-β1 levels using ELISA and observed increased serum TGF-β1 levels in HRHF-CON rats (n=5), compared to HRHF-MMT rats and compared to FRC rats (n=10 each; ANOVA, F_{2, 22} = 10.22,p = 0.0007;Fig. 8C). Note though, that this difference was driven mainly by an increase in serum TGF-β1 in two HRHF-CON rats.

Data fromFigures 4–8 support that the MMT treatments prevented increases in collagen deposition and TGF-β1 production in forearm and forelimb tissues.

Highlights.

1. Provision of modeled manual therapy for 5 days/week for the duration of a 12-week task paradigm reduced discomfort-related behaviors, improved grip strength, and enhanced task performance in rats performing a high repetition high force task.

2. The modeled manual therapy, performed 5 day/wk for 12 weeks, attenuated task-induced increases in collagen and TGF-β1 deposition in nerve and connective tissues of the forearm.

3. These observations support the investigation of manual therapy as a preventative for hand and wrist work-related musculoskeletal disorders, including median nerve fibrosis.

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