Ultrasound Pressure-Dependent Cytokine and Immune Cell Response Lost in Aged Muscle.

OBJECTIVE: Therapeutic ultrasound remains a highly discussed topic in physical therapy due to uncertainty between treatment regimens and biological benefits. Its impact on aged populations, who are vulnerable to insufficient healing after muscle injury because of sarcopenia, is understudied. Despite the coupling between muscle inflammation and regeneration, research on the immune response after therapeutic ultrasound is limited. The objective of this study was to evaluate structure, inflammatory cytokine signaling and immune cell infiltration after therapeutic ultrasound in young and aging murine muscle. METHODS: Young (6-week-old) and Adult (52-week-old) male and female mouse non-injured gastrocnemii were treated with either low-intensity pulsed ultrasound at 2 W/cm2 (∼0.243 MPa) or high-intensity pulsed focused ultrasound at 554 W/cm2 (∼5.96 MPa). Cytokine expression was evaluated at 1, 8 and 24 hours, cell infiltration was measured via flow cytometry at 1 and 24 hours and immunofluorescence assessed muscle fiber area, fibrosis and satellite cells at 24 hours after sonication. RESULTS: Low-intensity pulsed ultrasound induced an early, transient inflammatory response where interleukin (IL)-15 and macrophages (M2 > M1) were increased 1 hour post-sonication. High-intensity pulsed focused ultrasound caused a late, extended immune response where monocyte chemoattractant protein 1 (MCP-1), neutrophils, monocytes and macrophages (M1 > M2) were increased 24 hours post-sonication. Notably, these changes manifested solely in Young gastrocnemius. The Adult gastrocnemius exhibited decreased cytokine expression (IL-1α, IL-6, IL-15, macrophage colony-stimulating factor [M-CSF]) and no alteration in immune cell recruitment post-sonication. There was no damage to muscle structure. CONCLUSION: Therapeutic ultrasound induced a pressure-dependent inflammatory response that can augment or mitigate intrinsic muscle cytokine signaling and cell recruitment in adolescent or aged muscle, respectively.

Pleiotropic Effects of Simvastatin and Losartan in Preclinical Models of Post-Traumatic Elbow Contracture.

Elbow trauma can lead to post-traumatic joint contracture (PTJC), which is characterized by loss of motion associated with capsule/ligament fibrosis and cartilage damage. Unfortunately, current therapies are often unsuccessful or cause complications. This study aimed to determine the effects of prophylactically administered simvastatin (SV) and losartan (LS) in two preclinical models of elbow PTJC: an in vivo elbow-specific rat injury model and an in vitro collagen gel contraction assay. The in vivo elbow rat (n = 3-10/group) injury model evaluated the effects of orally administered SV and LS at two dosing strategies [i.e., low dose/high frequency/short duration (D1) vs. high dose/low frequency/long duration (D2)] on post-mortem elbow range of motion (via biomechanical testing) as well as capsule fibrosis and cartilage damage (via histopathology). The in vitro gel contraction assay coupled with live/dead staining (n = 3-19/group) evaluated the effects of SV and LS at various concentrations (i.e., 1, 10, 100 µM) and durations (i.e., continuous, short, or delayed) on the contractibility and viability of fibroblasts/myofibroblasts [i.e., NIH3T3 fibroblasts with endogenous transforming growth factor-beta 1 (TGFß1)]. In vivo, no drug strategy prevented elbow contracture biomechanically. Histologically, only SV-D2 modestly reduced capsule fibrosis but maintained elevated cellularity and tissue hypertrophy, and both SV strategies lessened cartilage damage. SV modest benefits were localized to the anterior region, not the posterior, of the joint. Neither LS strategy had meaningful benefits in capsule nor cartilage. In vitro, irrespective of the presence of TGFß1, SV (≥10 µM) prevented gel contraction partly by decreasing cell viability (100 µM). In contrast, LS did not prevent gel contraction or affect cell viability. This study demonstrates that SV, but not LS, might be suitable prophylactic drug therapy in two preclinical models of elbow PTJC. Results provide initial insight to guide future preclinical studies aimed at preventing or mitigating elbow PTJC.

Increased volume and collagen crosslinks drive soft tissue contribution to post-traumatic elbow contracture in an animal model.

Post-traumatic joint contracture (PTJC) in the elbow is a biological problem with functional consequences. Restoring elbow motion after injury is a complex challenge because contracture is a multi-tissue pathology. We previously developed an animal model of elbow PTJC using Long-Evans rats and showed that the capsule and ligaments/cartilage were the primary soft tissues that caused persistent joint motion loss. The objective of this study was to evaluate tissue-specific changes within the anterior capsule and lateral collateral ligament (LCL) that led to their contribution to elbow contracture. In our rat model of elbow PTJC, a unilateral surgery replicated damage that commonly occurs due to elbow dislocation. Following surgery, the injured limb was immobilized for 42 days. The capsule and LCL were evaluated after 42 days of immobilization or 42 days of immobilization followed by 42 days of free mobilization. We evaluated extracellular matrix protein biochemistry, non-enzymatic collagen crosslink content, tissue volume with contrast-enhanced micro-computed tomography, and tissue mechanical properties. Increased collagen content, but not collagen density, was observed in both injured limb capsules and LCLs, which was consistent with the increased tissue volume. Injured limb LCLs exhibited decreased normalized maximum force, and both tissues had increased immature collagen cross-links compared to control. Overall, increased tissue volume and immature collagen crosslinks in the capsule and LCL drive their contribution to elbow contracture in our rat model.

The Role of Periarticular Soft Tissues in Persistent Motion Loss in a Rat Model of Posttraumatic Elbow Contracture.

BACKGROUND: Elbow injuries disrupt the surrounding periarticular soft tissues, which include the muscles, tendons, capsule, ligaments, and cartilage. Damage to these tissues as a result of elbow trauma causes clinically significant contracture in 50% of patients. However, it is unclear which of these tissues is primarily responsible for the decreased range of motion. We hypothesized that all tissues would substantially contribute to elbow contracture after immobilization, but only the capsule, ligaments, and cartilage would contribute after free mobilization, with the capsule as the primary contributor at all time points. METHODS: Utilizing a rat model of posttraumatic elbow contracture, a unilateral soft-tissue injury was surgically induced to replicate the damage that commonly occurs during elbow joint dislocation. After surgery, the injured limb was immobilized for 42 days. Animals were evaluated after either 42 days of immobilization (42 IM) or 42 days of immobilization with an additional 21 or 42 days of free mobilization (42/21 or 42/42 IM-FM). For each group of animals, elbow mechanical testing in flexion-extension was completed post-mortem with (1) all soft tissues intact, (2) muscles/tendons removed, and (3) muscle/tendons and anterior capsule removed. Total extension was assessed to determine the relative contributions of muscles/tendons, capsule, and the remaining intact tissues (i.e., ligaments and cartilage). RESULTS: After immobilization, the muscles/tendons and anterior capsule contributed 10% and 90% to elbow contracture, respectively. After each free mobilization period, the muscles/tendons did not significantly contribute to contracture. The capsule and ligaments/cartilage were responsible for 47% and 52% of the motion lost at 42/21 IM-FM, respectively, and 26% and 74% at 42/42 IM-FM, respectively. CONCLUSIONS: Overall, data demonstrated a time-dependent response of periarticular tissue contribution to elbow contracture, with the capsule, ligaments, and cartilage as the primary long-term contributors. CLINICAL RELEVANCE: The capsule, ligaments, and cartilage were primarily responsible for persistent motion loss and should be considered during development of tissue-targeted treatment strategies to inhibit elbow contracture following injury.

Muscle does not drive persistent posttraumatic elbow contracture in a rat model.

INTRODUCTION: Posttraumatic elbow contracture is clinically challenging because injury often disrupts multiple periarticular soft tissues. Tissue specific contribution to contracture, particularly muscle, remains poorly understood. METHODS: In this study we used a previously developed animal model of elbow contracture. After surgically inducing a unilateral soft tissue injury, injured limbs were immobilized for 3, 7, 21, and 42 days (IM) or for 42 IM with 42 days of free mobilization (42/42 IM-FM). Biceps brachii active/passive mechanics and morphology were evaluated at 42 IM and 42/42 IM-FM, whereas biceps brachii and brachialis gene expression was evaluated at all time points. RESULTS: Injured limb muscle exhibited significantly altered active/passive mechanics and decreased fiber area at 42 IM but returned to control levels by 42/42 IM-FM. Gene expression suggested muscle growth rather than a fibrotic response at 42/42 IM-FM. DISCUSSION: Muscle is a transient contributor to motion loss in our rat model of posttraumatic elbow contracture. Muscle Nerve 58:843-851, 2018.

Temporal Patterns of Motion in Flexion-extension and Pronation-supination in a Rat Model of Posttraumatic Elbow Contracture.

BACKGROUND: The elbow is highly susceptible to contracture, which affects up to 50% of patients who experience elbow trauma. Previously, we developed a rat model to study elbow contracture that exhibited features similar to the human condition, including persistently decreased ROM and increased capsule thickness/adhesions. However, elbow ROM was not quantitatively evaluated over time throughout contracture development and subsequent mobilization of the joint. QUESTIONS/PURPOSES: The purposes of this study were (1) to quantify the time-dependent mechanics of contracture, including comparison of contracture after immobilization and free mobilization; and (2) to determine what changes occur in capsule and joint surface morphology that may support the altered joint mechanics. METHODS: A total of 96 male Long-Evans rats were randomized into control and injury (unilateral soft tissue injury/immobilization) groups. Flexion-extension and pronation-supination joint mechanics (n = 8/group) were evaluated after 3, 7, 21, or 42 days of immobilization (IM) or after 42 days of IM with either 21 or 42 days of free mobilization (63 or 84 FM, respectively). After measuring joint mechanics, a subset of these limbs (n = 3/group) was prepared for histologic analysis and blinded sections were scored to evaluate capsule and joint surface morphology. Joint mechanics and capsule histology at 42 IM and 84 FM were reported previously but are included to demonstrate the full timeline of elbow contracture. RESULTS: In flexion-extension, injured limb ROM was decreased compared with control (103° ± 11°) by 21 IM (70° ± 13°) (p = 0.001). Despite an increase in injured limb ROM from 42 IM (55° ± 14°) to 63 FM (83° ± 10°) (p < 0.001), injured limb ROM was still decreased compared with control (103° ± 11°) (p = 0.002). Interestingly, ROM recovery plateaued because there was no difference between injured limbs at 63 (83° ± 10°) and 84 FM (73° ± 19°) (p > 0.999). In pronation-supination, increased injured limb ROM occurred until 7 IM (202° ± 32°) compared with control (155° ± 22°) (p = 0.001), representative of joint instability. However, injured limb ROM decreased from 21 (182° ± 25°) to 42 IM (123° ± 47°) (p = 0.001), but was not different compared with control (155° ± 22°) (p = 0.108). Histologic evaluation showed morphologic changes in the anterior capsule (increased adhesions, myofibroblasts, thickness) and nonopposing joint surfaces (surface irregularities with tissue overgrowth, reduced matrix), but these changes did not increase with time. CONCLUSIONS: Overall, flexion-extension and pronation-supination exhibited distinct time-dependent patterns during contracture development and joint mobilization. Histologic evaluation showed tissue changes, but did not fully explain the patterns in contracture mechanics. Future work will use this rat model to evaluate the periarticular soft tissues of the elbow to isolate tissue-specific contributions to contracture to ultimately develop strategies for tissue-targeted treatments. CLINICAL RELEVANCE: A rat model of posttraumatic elbow contracture quantitatively described contracture development/progression and reiterates the need for rehabilitation strategies that consider both flexion-extension and pronation-supination elbow motion.

Pronation-Supination Motion Is Altered in a Rat Model of Post-Traumatic Elbow Contracture.

The elbow joint is highly susceptible to joint contracture, and treating elbow contracture is a challenging clinical problem. Previously, we established an animal model to study elbow contracture that exhibited features similar to the human condition including persistent decreased range of motion (ROM) in flexion-extension and increased capsule thickness/adhesions. The objective of this study was to mechanically quantify pronation-supination in different injury models to determine if significant differences compared to control or contralateral persist long-term in our animal elbow contracture model. After surgically inducing soft tissue damage in the elbow, Injury I (anterior capsulotomy) and Injury II (anterior capsulotomy with lateral collateral ligament transection), limbs were immobilized for 6 weeks (immobilization (IM)). Animals were evaluated after the IM period or following an additional 6 weeks of free mobilization (FM). Total ROM for pronation-supination was significantly decreased compared to the uninjured contralateral limb for both IM and FM, although not different from control limbs. Specifically, for both IM and FM, total ROM for Injury I and Injury II was significantly decreased by ∼20% compared to contralateral. Correlations of measurements from flexion-extension and pronation-supination divulged that FM did not affect these motions in the same way, demonstrating that joint motions need to be studied/treated separately. Overall, injured limbs exhibited persistent motion loss in pronation-supination when comparing side-to-side differences, similar to human post-traumatic joint contracture. Future work will use this animal model to study how elbow periarticular soft tissues contribute to contracture.

Persistent motion loss after free joint mobilization in a rat model of post-traumatic elbow contracture.

BACKGROUND: Post-traumatic joint contracture (PTJC) in the elbow is a challenging clinical problem due to the anatomical and biomechanical complexity of the elbow joint. METHODS: We previously established an animal model to study elbow PTJC, wherein surgically induced soft tissue damage, followed by 6 weeks of unilateral immobilization in Long-Evans rats, led to stiffened and contracted joints that exhibited features similar to the human condition. In this study, after 6 weeks of immobilization, we remobilized the animal (ie, external bandage removed and free cage activity) for an additional 6 weeks, after which the limbs were evaluated mechanically and histologically. The objective of this study was to evaluate whether this decreased joint motion would persist after 6 weeks of free mobilization (FM). RESULTS: After FM, flexion-extension demonstrated decreased total range of motion (ROM) and neutral zone length, and increased ROM midpoint for injured limbs compared with control and contralateral limbs. Specifically, after FM total ROM demonstrated a significant decrease of approximately 22% and 26% compared with control and contralateral limbs for injury I (anterior capsulotomy) and injury II (anterior capsulotomy with lateral collateral ligament transection), respectively. Histologic evaluation showed increased adhesion, fibrosis, and thickness of the capsule tissue in the injured limbs after FM compared with control and contralateral limbs, which is consistent with patterns previously reported in human tissue. CONCLUSION: Even with FM, injured limbs in this model demonstrate persistent joint motion loss and histologic results similar to the human condition. Future work will use this animal model to investigate the mechanisms responsible for PTJC and responses to therapeutic intervention.

Development and use of an animal model to study post-traumatic stiffness and contracture of the elbow.

Post-traumatic joint stiffness (PTJS) of the elbow is a debilitating condition that poses unique treatment challenges. While previous research has implicated capsular tissue in PTJS, much regarding the development and progression of this condition remains unknown. The objective of this study was to develop an animal model of post-traumatic elbow contracture and evaluate its potential for studying the etiology of PTJS. The Long-Evans rat was identified as the most appropriate species/breed for development due to anatomical and functional similarities to the human elbow joint. Two surgical protocols of varying severity were utilized to replicate soft tissue damage seen in elbow subluxation/dislocation injuries, including anterior capsulotomy and lateral collateral ligament transection, followed by 6 weeks of unilateral joint immobilization. Following sacrifice, flexion-extension mechanical joint testing demonstrated decreased range-of-motion and increased stiffness for injured-immobilized limbs compared to control and sham animals, where functional impact correlated with severity of injury. Histological evaluation showed increased cellularity, adhesion, and thickness of capsule tissue in injured limbs, consistent with clinical evidence. To our knowledge, this is the first animal model capable of examining challenges unique to the anatomically and biomechanically complex elbow joint. Future studies will use this animal model to investigate mechanisms responsible for PTJS.