Manual Therapy With Rest as a Treatment for Established Inflammation and Fibrosis in a Rat Model of Repetitive Strain Injury.

Background: Repetitive strain injuries caused by repetitive occupational work are difficult to prevent for multiple reasons. Therefore, we examined the effectiveness of manual therapy (MT) with rest to treat the inflammation and fibrosis that develops through the performance of a repetitive task. We hypothesized that this treatment would reduce task-induced sensorimotor declines and neuromuscular inflammation. Methods: Twenty-nine female Sprague-Dawley rats performed a reaching and lever-pulling task for 14weeks. All ceased performing the task at 14weeks. Ten were euthanized at this timepoint (TASK). Nine received manual therapy to their upper extremities while resting 7weeks (MTR); 10 were assigned to rest alone (REST). Ten additional food restricted rats were included that neither performed the task nor received manual therapy (FRC). Results: Confirming previous experiments, TASK rats showed behavioral changes (forepaw mechanical hypersensitivity, reduced grip strength, lowered forelimb/forepaw agility, and noxious cold temperature sensitivity), reduced median nerve conduction velocity (NCV), and pathological tissue changes (myelin degradation, increased median nerve and muscle inflammation, and collagen production). Manual therapy with rest (MTR) ameliorated cold sensitivity seen in REST rats, enhanced muscle interleukin 10 (IL-10) more than in REST rats, lead to improvement in most other measures, compared to TASK rats. REST rats showed improved grip strength, lowered nerve inflammation and degraded myelin, and lowered muscle tumor necrosis factor alpha (TNFα) and collagen I levels, compared to TASK rats, yet maintained lowered forelimb/forepaw agility and NCV, and increased neural fibrosis. Conclusion: In our model of repetitive motion disorder, manual therapy during rest had modest effects on behavioral, histological, and physiological measures, compared to rest alone. These findings stand in contrast to the robust preventive effects of manual therapy in this same model.

Key indicators of repetitive overuse-induced neuromuscular inflammation and fibrosis are prevented by manual therapy in a rat model.

BACKGROUND: We examined the effectiveness of a manual therapy consisting of forearm skin rolling, muscle mobilization, and upper extremity traction as a preventive treatment for rats performing an intensive lever-pulling task. We hypothesized that this treatment would reduce task-induced neuromuscular and tendon inflammation, fibrosis, and sensorimotor declines. METHODS: Sprague-Dawley rats performed a reaching and lever pulling task for a food reward, 2 h/day, 3 days/week, for 12 weeks, while simultaneously receiving the manual therapy treatment 3 times per week for 12 weeks to either the task-involved upper extremities (TASK-Tx), or the lower extremities as an active control group (TASK-Ac). Results were compared to similarly treated control rats (C-Tx and C-Ac). RESULTS: Median nerves and forearm flexor muscles and tendons of TASK-Ac rats showed higher numbers of inflammatory CD68+ and fibrogenic CD206+ macrophages, particularly in epineurium, endomysium and epitendons than TASK-Tx rats. CD68+ and CD206+ macrophages numbers in TASK-Tx rats were comparable to the non-task control groups. TASK-Ac rats had more extraneural fibrosis in median nerves, pro-collagen type I levels and immunoexpression in flexor digitorum muscles, and fibrogenic changes in flexor digitorum epitendons, than TASK-Tx rats (which showed comparable responses as control groups). TASK-Ac rats showed cold temperature, lower reflexive grip strength, and task avoidance, responses not seen in TASK-Tx rats (which showed comparable responses as the control groups). CONCLUSIONS: Manual therapy of forelimbs involved in performing the reaching and grasping task prevented the development of inflammatory and fibrogenic changes in forearm nerves, muscle, and tendons, and sensorimotor declines.

Nerve transfer for restoration of lower motor neuron-lesioned bladder function. Part 2: correlation between histological changes and nerve evoked contractions.

We determined the effect of pelvic organ decentralization and reinnervation 1 yr later on urinary bladder histology and function. Nineteen canines underwent decentralization by bilateral transection of all coccygeal and sacral (S) spinal roots, dorsal roots of lumbar (L)7, and hypogastric nerves. After exclusions, eight were reinnervated 12 mo postdecentralization with obturator-to-pelvic and sciatic-to-pudendal nerve transfers, then euthanized 8-12 mo later. Four served as long-term decentralized only animals. Before euthanasia, pelvic or transferred nerves and L1-S3 spinal roots were stimulated and maximum detrusor pressure (MDP) recorded. Bladder specimens were collected for histological and ex vivo smooth muscle contractility studies. Both reinnervated and decentralized animals showed less or denuded urothelium, fewer intramural ganglia, and more inflammation and collagen, than controls, although percent muscle was maintained. In reinnervated animals, pgp9.5+ axon density was higher compared with decentralized animals. Ex vivo smooth muscle contractions in response to KCl correlated positively with submucosal inflammation, detrusor muscle thickness, and pgp9.5+ axon density. In vivo, reinnervated animals showed higher MDP after stimulation of L1-L6 roots compared with their transected L7-S3 roots, and reinnervated and decentralized animals showed lower MDP than controls after stimulation of nerves (due likely to fibrotic nerve encapsulation). MDP correlated negatively with detrusor collagen and inflammation, and positively with pgp9.5+ axon density and intramural ganglia numbers. These results demonstrate that bladder function can be improved by transfer of obturator nerves to pelvic nerves at 1 yr after decentralization, although the fibrosis and inflammation that developed were associated with decreased contractile function.

Blocking CTGF/CCN2 reverses neural fibrosis and sensorimotor declines in a rat model of overuse-induced median mononeuropathy.

Encapsulation of median nerves is a hallmark of overuse-induced median mononeuropathy and contributes to functional declines. We tested if an antibody against CTGF/CCN2 (termed FG-3019 or Pamrevlumab) reduces established neural fibrosis and sensorimotor declines in a clinically relevant rodent model of overuse in which median mononeuropathy develops. Young adult female rats performed a high repetition high force (HRHF) lever-pulling task for 18 weeks. Rats were then euthanised at 18 weeks (HRHF untreated), or rested and systemically treated for 6 weeks with either an anti-CCN2 monoclonal antibody (HRHF-Rest/FG-3019) or IgG (HRHF-Rest/IgG), with results compared with nontask control rats. Neuropathology was evident in HRHF-untreated and HRHF-Rest/IgG rats as increased perineural collagen deposition and degraded myelin basic protein (dMBP) in median nerves, and increased substance P in lower cervical dorsal root ganglia (DRG), compared with controls. Both groups showed functional declines, specifically, decreased sensory conduction velocity in median nerves, noxious cold temperature hypersensitivity, and grip strength declines, compared with controls. There were also increases of ATF3-immunopositive nuclei in ventral horn neurons in HRHF-untreated rats, compared with controls (which showed none). FG-3019-treated rats showed no increase above control levels of perineural collagen or dMBP in median nerves, Substance P in lower cervical DRGs, or ATF3-immunopositive nuclei in ventral horns, and similar median nerve conduction velocities and thermal sensitivity, compared with controls. We hypothesize that neural fibrotic processes underpin the sensorimotor declines by compressing or impeding median nerves during movement, and that inhibiting fibrosis using an anti-CCN2 treatment reverses these effects.

Blocking CTGF/CCN2 reduces established skeletal muscle fibrosis in a rat model of overuse injury.

Tissue fibrosis is a hallmark of overuse musculoskeletal injuries and contributes to functional declines. We tested whether inhibition of CCN2 (cellular communication network factor 2, previously known as connective tissue growth factor, CTGF) using a specific antibody (termed FG-3019 or pamrevlumab) reduces established overuse-induced muscle fibrosis in a clinically relevant rodent model of upper extremity overuse injury. Young adult rats performed a high repetition high force (HRHF) reaching and lever-pulling task for 18 weeks, after first being shaped for 6 weeks to learn this operant task. Rats were then euthanized (HRHF-Untreated), or rested and treated for 6 weeks with FG-3019 (HRHF-Rest/FG-3019) or a human IgG as a vehicle control (HRHF-Rest/IgG). HRHF-Untreated and HRHF-Rest/IgG rats had higher muscle levels of several fibrosis-related proteins (TGFß1, CCN2, collagen types I and III, and FGF2), and higher muscle numbers of alpha SMA and pERK immunopositive cells, compared to control rats. Each of these fibrogenic changes was restored to control levels by the blocking of CCN2 signaling in HRHF-Rest/FG-3019 rats, as were HRHF task-induced increases in serum CCN2 and pro-collagen I intact N-terminal protein. Levels of cleaved CCN3, an antifibrotic protein, were lowered in HRHF-Untreated and HRHF-Rest/IgG rats, compared to control rats, yet elevated back to control levels in HRHF-Rest/FG-3019 rats. Significant grip strength declines observed in HRHF-Untreated and HRHF-Rest/IgG rats, were restored to control levels in HRHF-Rest/FG-3019 rats. These results are highly encouraging for use of FG-3019 for therapeutic treatment of persistent skeletal muscle fibrosis, such as those induced with chronic overuse.

Forced treadmill running reduces systemic inflammation yet worsens upper limb discomfort in a rat model of work-related musculoskeletal disorders.

BACKGROUND: Musculoskeletal disorders can result from prolonged repetitive and/or forceful movements. Performance of an upper extremity high repetition high force task increases serum pro-inflammatory cytokines and upper extremity sensorimotor declines in a rat model of work-related musculoskeletal disorders. Since one of the most efficacious treatments for musculoskeletal pain is exercise, this study investigated the effectiveness of treadmill running in preventing these responses. METHODS: Twenty-nine young adult female Sprague-Dawley rats were used. Nineteen were trained for 5 weeks to pull a lever bar at high force (15 min/day). Thirteen went on to perform a high repetition high force reaching and lever-pulling task for 10 weeks (10-wk HRHF; 2 h/day, 3 days/wk). From this group, five were randomly selected to undergo forced treadmill running exercise (TM) during the last 6 weeks of task performance (10-wk HRHF+TM, 1 h/day, 5 days/wk). Results were compared to 10 control rats and 6 rats that underwent 6 weeks of treadmill running following training only (TR-then-TM). Voluntary task and reflexive sensorimotor behavioral outcomes were assessed. Serum was assayed for inflammatory cytokines and corticosterone, reach limb median nerves for CD68+ macrophages and extraneural thickening, and reach limb flexor digitorum muscles and tendons for pathological changes. RESULTS: 10-wk HRHF rats had higher serum levels of IL-1α, IL-1ß and TNFα, than control rats. In the 10-wk HRHF+TM group, IL-1ß and TNFα were lower, whereas IL-10 and corticosterone were higher, compared to 10-wk HRHF only rats. Unexpectedly, several voluntary task performance outcomes (grasp force, reach success, and participation) worsened in rats that underwent treadmill running, compared to untreated 10-wk HRHF rats. Examination of forelimb tissues revealed lower cellularity within the flexor digitorum epitendon but higher numbers of CD68+ macrophages within and extraneural fibrosis around median nerves in 10-wk HRHF+TM than 10-wk HRHF rats. CONCLUSIONS: Treadmill running was associated with lower systemic inflammation and moderate tendinosis, yet higher median nerve inflammation/fibrosis and worse task performance and sensorimotor behaviors. Continued loading of the injured tissues in addition to stress-related factors associated with forced running/exercise likely contributed to our findings.

Bladder reinnervation by somatic nerve transfer to pelvic nerve vesical branches does not reinnervate the urethra.

AIMS: We sought to determine whether somatic lumbar nerve transfer to the pelvic nerve's anterior vesical branch after sacral decentralization for detrusor muscle reinnervation also leads to aberrant innervation of the bladder outlet. METHODS: Twenty-six female mongrel hound dogs underwent transection of sacral dorsal and ventral spinal roots (ie, sacral decentralization). Immediately afterward, 12 received genitofemoral nerve transfer and 9 received femoral nerve branch transfer. Five were left sacrally decentralized. Controls included 3 sham-operated and 6 unoperated. Eight months postsurgery, the bladder and urethra were injected with retrograde tracing dyes cystoscopically. After 3 weeks, detrusor and urethral pressures were assayed electrophysiologically immediately before euthanasia and characterization of neural reinnervation. RESULTS: Electrical stimulation of spinal cords or roots did not lead to increased urethral sphincter pressure in nerve transfer animals, compared with decentralized animals, confirming a lack of functional reinnervation of the bladder outlet. In contrast, mean detrusor pressure increased after lumbar cord/root stimulation. In sham/unoperated animals, urethral and bladder dye injections resulted in labeled neurons in sacral level neural structures (dorsal root ganglia [DRG], sympathetic trunk ganglia [STG], and spinal cord ventral horns); labeling absent in decentralized animals. Urethral dye injections did not result in labeling in lumbar or sacral level neural structures in either nerve transfer group while bladder dye injections lead to increased labeled neurons in lumbar level DRG, STG, and ventral horns, compared to sacrally decentralized animals. CONCLUSION: Pelvic nerve transfer for bladder reinnervation does not impact urethral sphincter innervation.

Comparing effects of rest with or without a NK1RA on fibrosis and sensorimotor declines induced by a voluntary moderate demand task.

OBJECTIVES: Fibrosis is one contributing factor in motor dysfunction and discomfort in patients with overuse musculoskeletal disorders. We pharmacologically targeted the primary receptor for Substance P, neurokinin-1, using a specific antagonist (NK1RA) in a rat model of overuse with the goal of improving tissue fibrosis and discomfort. METHODS: Female rats performed a low repetition, high force (LRHF) grasping task for 12 weeks, or performed the task for 12 weeks before being placed on a four week rest break, with or without simultaneous NK1RA treatment. Results were compared to control rats (untreated, or treated 4 weeks with NK1RA or vehicle). RESULTS: Rest improved LRHF-induced declines in grip strength, although rest plus NK1RA treatment (Rest/NK1RA) rescued it. Both treatments improved LRHF-induced increases in muscle TGFß1 and collagen type 1 levels, forepaw mechanical hypersensitivity (Rest/NK1RA more effectively), macrophage influx into median nerves, and enhanced collagen deposition in forepaw dermis. Only Rest/NK1RA reduced muscle hypercellularity. However, LRHF+4wk Rest /NK1RA rats showed hyposensitivity to noxious hot temperatures. CONCLUSIONS: While the NK1RA induced hot temperature hyposensitivity should be taken into consideration if this or related drug were used long-term, the NK1RA more effectively reduced muscle hypercellularity and improved grip strength and forepaw mechanical hypersensitivity.

Nerve transfer for restoration of lower motor neuron-lesioned bladder and urethra function: establishment of a canine model and interim pilot study results.

OBJECTIVE: Previous patient surveys have shown that patients with spinal cord or cauda equina injuries prioritize recovery of bladder function. The authors sought to determine if nerve transfer after long-term decentralization restores bladder and sphincter function in canines. METHODS: Twenty-four female canines were included in this study. Transection of sacral roots and hypogastric nerves (S Dec) was performed in 6 animals, and 7 animals underwent this procedure with additional transection of the L7 dorsal roots (L7d+S Dec). Twelve months later, 3 L7d+S Dec animals underwent obturator-to-pelvic nerve and sciatic-to-pudendal nerve transfers (L7d+S Dec+Reinn). Eleven animals served as controls. Squat-and-void behaviors were tracked before and after decentralization, after reinnervation, and following awake bladder-filling procedures. Bladders were cystoscopically injected with Fluoro-Gold 3 weeks before euthanasia. Immediately before euthanasia, transferred nerves were stimulated to evaluate motor function. Dorsal root ganglia were assessed for retrogradely labeled neurons. RESULTS: Transection of only sacral roots failed to reduce squat-and-void postures; L7 dorsal root transection was necessary for significant reduction. Three L7d+S Dec animals showing loss of squat-and-void postures post-decentralization were chosen for reinnervation and recovered these postures 4-6 months after reinnervation. Each showed obturator nerve stimulation-induced bladder contractions and sciatic nerve stimulation-induced anal sphincter contractions immediately prior to euthanasia. One showed sciatic nerve stimulation-induced external urethral sphincter contractions and voluntarily voided twice following nonanesthetized bladder filling. Reinnervation was confirmed by increased labeled cells in L2 and the L4-6 dorsal root ganglia (source of obturator nerve in canines) of L7d+S Dec+Reinn animals, compared with controls. CONCLUSIONS: New neuronal pathways created by nerve transfer can restore bladder sensation and motor function in lower motor neuron-lesioned canines even 12 months after decentralization.

The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform.

Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, ß-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS). Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. The best resolution of type 1 procollagens is achieved using 4%-12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed. Also, BME must not be added to the sample buffer and samples should not be boiled. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.

Blocking CCN2 Reduces Progression of Sensorimotor Declines and Fibrosis in a Rat Model of Chronic Repetitive Overuse.

Fibrosis may be a key factor in sensorimotor dysfunction in patients with chronic overuse-induced musculoskeletal disorders. Using a clinically relevant rodent model, in which performance of a high demand handle-pulling task induces tissue fibrosis and sensorimotor declines, we pharmacologically blocked cellular communication network factor 2 (CCN2; connective tissue growth factor) with the goal of reducing the progression of these changes. Young adult, female Sprague-Dawley rats were shaped to learn to pull at high force levels (10 min/day, 5 weeks), before performing a high repetition high force (HRHF) task for 3 weeks (2 h/day, 3 days/week). HRHF rats were untreated, or treated in task weeks 2 and 3 with a monoclonal antibody that blocks CCN2 (FG-3019), or a control immunoglobulin G (IgG). Control rats were untreated or received FG-3019, IgG, or vehicle (saline) injections. Mean task reach rate and grasp force were higher in 3-week HRHF + FG-3019 rats, compared with untreated HRHF rats. Grip strength declined while forepaw mechanical sensitivity increased in untreated HRHF rats, compared with controls; changes improved by FG-3019 treatment. The HRHF task increased collagen in multiple tissues (flexor digitorum muscles, nerves, and forepaw dermis), which was reduced with FG-3019 treatment. FG-3019 treatment also reduced HRHF-induced increases in CCN2 and transforming growth factor ß in muscles. In tendons, FG-3019 reduced HRHF-induced increases in CCN2, epitendon thickening, and cell proliferation. Our findings indicate that CCN2 is critical to the progression of chronic overuse-induced multi-tissue fibrosis and functional declines. FG-3019 treatment may be a novel therapeutic strategy for overuse-induced musculoskeletal disorders. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2004-2018, 2019.

Manual therapy prevents onset of nociceptor activity, sensorimotor dysfunction, and neural fibrosis induced by a volitional repetitive task.

Painful and disabling musculoskeletal disorders remain prevalent. In rats trained to perform repetitive tasks leading to signs and dysfunction similar to those in humans, we tested whether manual therapy would prevent the development of the pathologies and symptoms. We collected behavioral, electrophysiological, and histological data from control rats, rats that trained for 5 weeks before performing a high-repetition high-force (HRHF) task for 3 weeks untreated, and trained rats that performed the task for 3 weeks while being treated 3x/week using modeled manual therapy (MMT) to the forearm (HRHF + MMT). The MMT included bilateral mobilization, skin rolling, and long axis stretching of the entire upper limb. High-repetition high-force rats showed decreased performance of the operant HRHF task and increased discomfort-related behaviors, starting after training. HRHF + MMT rats showed improved task performance and decreased discomfort-related behaviors compared with untreated HRHF rats. Subsets of rats were assayed for presence or absence of ongoing activity in C neurons and slow Aδ neurons in their median nerves. Neurons from HRHF rats had a heightened proportion of ongoing activity and altered conduction velocities compared with control and MMT-treated rats. Median nerve branches in HRHF rats contained increased numbers of CD68 macrophages and degraded myelin basic protein, and showed increased extraneural collagen deposition, compared with the other groups. We conclude that the performance of the task for 3 weeks leads to increased ongoing activity in nociceptors, in parallel with behavioral and histological signs of neuritis and nerve injury, and that these pathophysiologies are largely prevented by MMT.

Prolonged high force high repetition pulling induces osteocyte apoptosis and trabecular bone loss in distal radius, while low force high repetition pulling induces bone anabolism.

We have an operant rat model of upper extremity reaching and grasping in which we examined the impact of performing a high force high repetition (High-ForceHR) versus a low force low repetition (Low-ForceHR) task for 18weeks on the radius and ulna, compared to age-matched controls. High-ForceHR rats performed at 4 reaches/min and 50% of their maximum voluntary pulling force for 2h/day, 3days/week. Low-ForceHR rats performed at 6% maximum voluntary pulling force. High-ForceHR rats showed decreased trabecular bone volume in the distal metaphyseal radius, decreased anabolic indices in this same bone region (e.g., decreased osteoblasts and bone formation rate), and increased catabolic indices (e.g., microcracks, increased osteocyte apoptosis, secreted sclerostin, RANKL, and osteoclast numbers), compared to controls. Distal metaphyseal trabeculae in the ulna of High-ForceHR rats showed a non-significant decrease in bone volume, some catabolic indices (e.g., decreased trabecular numbers) yet also some anabolic indices (e.g., increased osteoblasts and trabecular thickness). In contrast, the mid-diaphyseal region of High-ForceHR rats' radial and ulnar bones showed few to no microarchitecture differences and no changes in apoptosis, sclerostin or RANKL levels, compared to controls. In further contrast, Low-ForceHR rats showed increased trabecular bone volume in the radius in the distal metaphysis and increased cortical bone area its mid-diaphysis. These changes were accompanied by increased anabolic indices, no microcracks or osteocyte apoptosis, and decreased RANKL in each region, compared to controls. Ulnar bones of Low-ForceHR rats also showed increased anabolic indices, although fewer than in the adjacent radius. Thus, prolonged performance of an upper extremity reaching and grasping task is loading-, region-, and bone-dependent, with high force loads at high repetition rates inducing region-specific increases in bone degradative changes that were most prominent in distal radial trabeculae, while low force task loads at high repetition rates induced adaptive bone responses.