Enhanced Diaphragm Muscle Function upon Satellite Cell Transplantation in Dystrophic Mice.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

Restricted physical activity after volumetric muscle loss alters whole-body and local muscle metabolism.

Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle, resulting in chronic functional deficits and pathological comorbidities, including altered whole-body metabolic rate and respiratory exchange ratio (RER), despite no change in physical activity in animal models. In other injury models, treatment with ß2 receptor agonists (e.g. formoterol) improves metabolic and skeletal muscle function. We aimed first to examine if restricting physical activity following injury affects metabolic and skeletal muscle function, and second, to enhance the metabolic and contractile function of the muscle remaining following VML injury through treatment with formoterol. Adult male C57Bl/6J mice (n = 32) underwent VML injury to the posterior hindlimb compartment and were randomly assigned to unrestricted or restricted activity and formoterol treatment or no treatment; age-matched injury naïve mice (n = 4) were controls for biochemical analyses. Longitudinal 24 h evaluations of physical activity and whole-body metabolism were conducted following VML. In vivo muscle function was assessed terminally, and muscles were biochemically evaluated for protein expression, mitochondrial enzyme activity and untargeted metabolomics. Restricting activity chronically after VML had the greatest effect on physical activity and RER, reflected in reduced lipid oxidation, although changes were attenuated by formoterol treatment. Formoterol enhanced injured muscle mass, while mitigating functional deficits. These novel findings indicate physical activity restriction may recapitulate following VML clinically, and adjunctive oxidative treatment may create a metabolically beneficial intramuscular environment while enhancing the injured muscle's mass and force-producing capacity. Further investigation is needed to evaluate adjunctive oxidative treatment with rehabilitation, which may augment the muscle's regenerative and functional capacity following VML. KEY POINTS: The natural ability of skeletal muscle to regenerate and recover function is lost following complex traumatic musculoskeletal injury, such as volumetric muscle loss (VML), and physical inactivity following VML may incur additional deleterious consequences for muscle and metabolic health. Modelling VML injury-induced physical activity restriction altered whole-body metabolism, primarily by decreasing lipid oxidation, while preserving local skeletal muscle metabolic activity. The ß2 adrenergic receptor agonist formoterol has shown promise in other severe injury models to improve regeneration, recover function and enhance metabolism. Treatment with formoterol enhanced mass of the injured muscle and whole-body metabolism while mitigating functional deficits resulting from injury. Understanding of chronic effects of the clinically available and FDA-approved pharmaceutical formoterol could be a translational option to support muscle function after VML injury.

Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

Early initiation of electrical stimulation paired with range of motion after a volumetric muscle loss injury does not benefit muscle function.

NEW FINDINGS: What is the central question of this study? First, how does physical rehabilitation influence recovery from traumatic muscle injury? Second, how does physical activity impact the rehabilitation response for skeletal muscle function and whole-body metabolism? What is the main finding and its importance? The most salient findings were that rehabilitation impaired muscle function and range of motion, while restricting activity mitigated some negative effects but also impacted whole-body metabolism. These data suggest that first, work must continue to explore treatment parameters, including modality, time, type, duration and intensity, to find the best rehabilitation approaches for volumetric muscle loss injuries; and second, restricting activity acutely might enhance rehabilitation response, but whole-body co-morbidities should continue to be considered. ABSTRACT: Volumetric muscle loss (VML) injury occurs when a substantial volume of muscle is lost by surgical removal or trauma, resulting in an irrecoverable deficit in muscle function. Recently, it was suggested that VML impacts whole-body and muscle-specific metabolism, which might contribute to the inability of the muscle to respond to treatments such as physical rehabilitation. The aim of this work was to understand the complex relationship between physical activity and the response to rehabilitation after VML in an animal model, evaluating the rehabilitation response by measurement of muscle function and whole-body metabolism. Adult male mice (n = 24) underwent a multi-muscle, full-thickness VML injury to the gastrocnemius, soleus and plantaris muscles and were randomized into one of three groups: (1) untreated; (2) rehabilitation (i.e., combined electrical stimulation and range of motion, twice per week, beginning 72 h post-injury, for ∼8 weeks); or (3) rehabilitation and restriction of physical activity. There was a lack of positive adaption associated with electrical stimulation and range of motion intervention alone; however, maximal isometric torque of the posterior muscle group was greater in mice receiving treatment with activity restriction (P = 0.008). Physical activity and whole-body metabolism were measured ∼6 weeks post-injury; metabolic rate decreased (P = 0.001) and respiratory exchange ratio increased (P = 0.022) with activity restriction. Therefore, restricting physical activity might enhance an intervention delivered to the injured muscle group but impair whole-body metabolism. It is possible that restricting activity is important initially post-injury to protect the muscle from excess demand. A gradual increase in activity throughout the course of treatment might optimize muscle function and whole-body metabolism.

Resistance wheel running improves contractile strength, but not metabolic capacity, in a murine model of volumetric muscle loss injury.

The primary objective of this study was to determine if low- or high-resistance voluntary wheel running leads to functional improvements in muscle strength (i.e., isometric and isokinetic torque) and metabolic function (i.e., permeabilized fibre bundle mitochondrial respiration) after a volumetric muscle loss (VML) injury. C57BL/6J mice were randomized into one of four experimental groups at age 12 weeks: uninjured control, VML untreated (VML), low-resistance wheel running (VML-LR) and high-resistance wheel running (VML-HR). All mice, excluding the uninjured, were subject to a unilateral VML injury to the plantar flexor muscles and wheel running began 3 days post-VML. At 8 weeks post-VML, peak isometric torque was greater in uninjured compared to all VML-injured groups, but both VML-LR and VML-HR had greater (∼32%) peak isometric torque compared to VML. All VML-injured groups had less isokinetic torque compared to uninjured, and there was no statistical difference among VML, VML-LR and VML-HR. No differences in cumulative running distance were observed between VML-LR and VML-HR groups. Because adaptations in VML-HR peak isometric torque were attributed to greater gastrocnemius muscle mass, atrophy- and hypertrophy-related protein content and post-translational modifications were explored via immunoblot; however, results were inconclusive. Permeabilized fibre bundle mitochondrial oxygen consumption was 22% greater in uninjured compared to VML, but there was no statistical difference among VML, VML-LR and VML-HR. Furthermore, neither wheel running group demonstrated a change in the relative protein content of the mitochondrial biogenesis transcription factor, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). These results indicate that resistance wheel running alone only has modest benefits in the VML-injured muscle. NEW FINDINGS: What is the central question of the study? Does initiation of a resistance wheel running regimen following volumetric muscle loss (VML) improve the functional capacity of skeletal muscle? What is the main finding and its importance? Resistance wheel running led to greater muscle mass and strength in mice with a VML injury but did not result in a full recovery. Neither low- nor high-resistance wheel running was associated with a change in permeabilized muscle fibre respiration despite runners having greater whole-body treadmill endurance capacity, suggesting resilience to metabolic adaptations in VML-injured muscle. Resistance wheel running may be a suitable adjuvant rehabilitation strategy, but alone does not fully mitigate VML pathology.

Xenotransplantation and exotransplantation: Strategies to expand the number of donor organs.

Cardiovascular disease is common and has a high mortality. Due to the limited number of organs available for orthotopic heart transplantation, alternative therapies have received intense interest. In this commentary we contrast xenotransplantation and blastocyst complementation to produce pigs that will serve as donors for organ transplantation. These strategies hold tremendous promise and have the potential to provide an unlimited number of organs for chronic, terminal diseases.

Compromised Muscle Properties in a Severe Hypophosphatasia Murine Model.

Hypophosphatasia (HPP) is a rare metabolic bone disorder characterized by low levels of tissue non-specific alkaline phosphatase (TNAP) that causes under-mineralization of the bone, leading to bone deformity and fractures. In addition, patients often present with chronic muscle pain, reduced muscle strength, and an altered gait. In this work, we explored dynamic muscle function in a homozygous TNAP knockout mouse model of severe juvenile onset HPP. We found a reduction in skeletal muscle size and impairment in a range of isolated muscle contractile properties. Using histological methods, we found that the structure of HPP muscles was similar to healthy muscles in fiber size, actin and myosin structures, as well as the α-tubulin and mitochondria networks. However, HPP mice had significantly fewer embryonic and type I fibers than wild type mice, and fewer metabolically active NADH+ muscle fibers. We then used oxygen respirometry to evaluate mitochondrial function and found that complex I and complex II leak respiration were reduced in HPP mice, but that there was no disruption in efficiency of electron transport in complex I or complex II. In summary, the severe HPP mouse model recapitulates the muscle strength impairment phenotypes observed in human patients. Further exploration of the role of alkaline phosphatase in skeletal muscle could provide insight into mechanisms of muscle weakness in HPP.

Mechanisms and strategies to promote cardiac xenotransplantation.

End stage heart failure is a terminal disease, and the only curative therapy is orthotopic heart transplantation. Due to limited organ availability, alternative strategies have received intense interest for treatment of patients with advanced heart failure. Recent studies using gene-edited porcine organs suggest that cardiac xenotransplantation may provide a future source of organs. In this review, we highlight the historical milestones for cardiac xenotransplantation and the gene editing strategies designed to overcome immunological barriers, which have culminated in a recent cardiac pig-to-human xenotransplant. We also discuss recent results of studies on the engineering of human-porcine chimeric organs that may provide an alternative and complementary strategy to overcome some of the major immunological barriers to producing a new source of transplantable organs.

Temporal changes in the muscle extracellular matrix due to volumetric muscle loss injury.

PURPOSE/AIM: Volumetric muscle loss (VML) is a devastating orthopedic injury resulting in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. However, there is only limited mechanistic understanding of VML-induced fibrosis. Herein we examined the temporal changes in the fibrotic deposition at 3, 7, 14, 28, and 48 days post-VML injury. MATERIALS AND METHODS: Adult male Lewis rats (n = 39) underwent a full thickness ~20% (~85 mg) VML injury to the tibialis anterior (TA) muscle unilaterally, the contralateral TA muscle served as the control group. All TA muscles were harvested for biochemical and histologic evaluation. RESULTS: The ratio of collagen I/III was decreased at 3, 7, and 14 days post-VML, but significantly increased at 48 days. Decorin content followed an opposite trend, significantly increasing by day 3 before dropping to below control levels by 48 days. Histological evaluation of the defect area indicates a shift from loosely packed collagen at early time points post-VML, to a densely packed fibrotic scar by 48 days. CONCLUSIONS: The shift from early wound healing efforts to a fibrotic scar with densely packed collagen within the skeletal muscle occurs around 21 days after VML injury through dogmatic synchronous reduction of collagen III and increase in collagen I. Thus, there appears to be an early window for therapeutic intervention to prevent pathologic fibrous tissue formation, potentially by targeting CCN2/CTGF or using decorin as a therapeutic.

Lifelong Ulk1-Mediated Autophagy Deficiency in Muscle Induces Mitochondrial Dysfunction and Contractile Weakness.

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.

Musculoskeletal Regeneration, Rehabilitation, and Plasticity Following Traumatic Injury.

The musculoskeletal system has an integral role throughout life, including structural support to the body, protection, and allowing a range of fine to complex movements for daily living to elite sporting events. At various times, injuries to the musculoskeletal system occur resulting in varying levels of impact to the person both acutely and chronically. Specifically, there is a spectrum of complexity in orthopedic injuries, with some such as common muscle strains, that while burdensome will have no impact on life-long functional ability, and others that can result in long lasting disability. Focusing on extremity injuries, this review highlights: i)the current impact of orthopedic injuries in sport and daily life; ii) the foundation of bone and skeletal muscle repair and regeneration; and iii) the disruptions in regenerative healing due to traumatic orthopedic injuries. This review seeks to maximize the broad and collective research impact on sport and traumatic orthopedic injuries in search of promoting ongoing innovation for treatment and rehabilitation approaches aimed to improve musculoskeletal health throughout life.

A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated.

KEY POINTS: The female hormone oestrogen may protect muscle from injury by reducing inflammation but this is debatable. In this study, the inflammatory response of injured muscle from oestrogen-replete mice was comprehensively compared to that from oestrogen-deficient mice. We show that oestrogen markedly promotes movement of neutrophils, an inflammatory white blood cell type, into muscle over the first few days after injury but has only a minor effect on the movement of macrophages, another inflammatory cell type. Despite the enhancement of inflammation by oestrogen in injured muscle, we found strength in oestrogen-replete mice to recover faster and to a greater extent than it does in oestrogen-deficient mice. Our study and others indicate that lower doses of oestrogen, such as that used in our study, may affect muscle inflammation and injury differently from higher doses. ABSTRACT: Oestrogen has been shown to protect against skeletal muscle injury and a reduced inflammatory response has been suggested as a possible protective mechanism. There are, however, dissenting reports. Our objective was to conduct an unbiased, comprehensive study of the effect of oestradiol on the inflammatory response following muscle injury. Female C57BL6/J mice were ovariectomized and supplemented with and without oestradiol. Tibialis anterior muscles were freeze injured and studied primarily at 1-4 days post-injury. Oestradiol supplementation increased injured muscle gene expression of neutrophil chemoattractants (Cxcl1 and Cxcl5) and to a lesser extent that of monocyte/macrophage chemoattractants (Ccl2 and Spp1). Oestradiol markedly increased gene expression of the neutrophil cell surface marker (Ly6g) but had less consistent effects on the monocyte/macrophage cell surface markers (Cd68, Cd163 and Cd206). These results were confirmed at the protein level by immunoblot with oestradiol increasing LY6G/C content and having no significant effect on CD163 content. These findings were confirmed with fluorescence-activated cell sorting counts of neutrophils and macrophages in injured muscles; oestradiol increased the proportion of CD45+ cells that were neutrophils (LY6G+ ) but not the proportion that were macrophages (CD68+ or CD206+ ). Physiological impact of the oestradiol-enhanced neutrophil response was assessed by strength measurements. There was no significant difference in strength between oestradiol-supplemented and -unsupplemented mice until 2 weeks post-injury; strength was 13-24% greater in supplemented mice at 2-6 weeks post-injury. In conclusion, a moderate level of oestradiol supplementation enhances neutrophil infiltration in injured muscle and this is associated with a beneficial effect on strength recovery.

Impact of volumetric muscle loss injury on persistent motoneuron axotomy.

INTRODUCTION: Volumetric muscle loss (VML) occurs following significant traumatic injury or surgical removal of skeletal muscle, resulting in nonrecoverable loss of tissue and long-term dysfunction. Perhaps less recognized is that VML injuries inherently disrupt the neuromuscular unit, resulting in fiber denervation and presumptive motor unit rearrangement, expansion, and/or loss. To characterize neural dysfunction we quantified motoneuron axotomy, in efforts to understand how this relates to the temporal coordination of neuromuscular and morphological alterations due to injury. METHODS: In an established rat tibialis anterior (TA) VML injury model, we examined the motoneuron, skeletal muscle, and maximal isometric torque at 3, 7, 14, and 21 days postinjury. RESULTS: Significant axotomy of 57-79% of all TA muscle motoneurons was observed through 21 days postinjury, which was coupled with a 45-90% TA maximal torque deficit. DISCUSSION: A ∼20% partial ablation of the TA muscle causes disproportionate damage across the motor unit acutely postinjury. Muscle Nerve 57: 799-807, 2018.

Early rehabilitation for volumetric muscle loss injury augments endogenous regenerative aspects of muscle strength and oxidative capacity.

BACKGROUND: Volumetric muscle loss (VML) injuries occur due to orthopaedic trauma or the surgical removal of skeletal muscle and result in debilitating long-term functional deficits. Current treatment strategies do not promote significant restoration of function; additionally appropriate evidenced-based practice physical therapy paradigms have yet to be established. The objective of this study was to develop and evaluate early rehabilitation paradigms of passive range of motion and electrical stimulation in isolation or combination to understand the genetic and functional response in the tissue remaining after a multi-muscle VML injury. METHODS: Adult male mice underwent an ~ 20% multi-muscle VML injury to the posterior compartment (gastrocnemius, soleus, and plantaris muscle) unilaterally and were randomized to rehabilitation paradigm twice per week beginning 2 days post-injury or no treatment. RESULTS: The most salient findings of this work are: 1) that the remaining muscle tissue after VML injury was adaptable in terms of improved muscle strength and mitigation of stiffness; but 2) not adaptable to improvements in metabolic capacity. Furthermore, biochemical (i.e., collagen content) and gene (i.e., gene arrays) assays suggest that functional adaptations may reflect changes in the biomechanical properties of the remaining tissue due to the cellular deposition of non-contractile tissue in the void left by the VML injury and/or differentiation of gene expression with early rehabilitation. CONCLUSIONS: Collectively this work provides evidence of genetic and functional plasticity in the remaining skeletal muscle with early rehabilitation approaches, which may facilitate future evidenced-based practice of early rehabilitation at the clinical level.

Impairment of early fracture healing by skeletal muscle trauma is restored by FK506.

BACKGROUND: Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration. METHODS: We hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle). RESULTS: The salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque production CONCLUSION: Collectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.

Regenerative and Rehabilitative Medicine: A Necessary Synergy for Functional Recovery from Volumetric Muscle Loss Injury.

Volumetric muscle loss (VML) is a complex and heterogeneous problem due to significant traumatic or surgical loss of skeletal muscle tissue. The consequences of VML are substantial functional deficits in joint range of motion and skeletal muscle strength, resulting in life-long dysfunction and disability. Traditional physical medicine and rehabilitation paradigms do not address the magnitude of force loss due to VML and related musculoskeletal comorbidities. Recent advancements in regenerative medicine have set forth encouraging and emerging therapeutic options for VML injuries. There is significant potential that combined rehabilitative and regenerative therapies can restore limb and muscle function following VML injury in a synergistic manner. This review presents the current state of the VML field, spanning clinical and preclinical literature, with particular focus on rehabilitation and regenerative medicine in addition to their synergy. Moving forward, multidisciplinary collaboration between clinical and research fields is encouraged in order to continue to improve the treatment of VML injuries and specifically address the encompassing physiology, pathology, and specific needs of this patient population. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel.

Analysis of fluid movement in skeletal muscle using fluorescent microspheres.

INTRODUCTION: Regional variability in interstitial fluid pressure confounds use of intramuscular pressure measurement to assess muscle force. It is hypothesized that interstitial flow is dependent on intramuscular pressure. The goal of this study was to assess the feasibility of using fluorescent microspheres to evaluate movement of interstitial fluid in skeletal muscle. METHODS: Two diameters of fluorescent microspheres were injected into the rat tibialis anterior during both static (n = 6) and passively lengthened (10% strain) experimental conditions (n = 6). Microsphere dispersion was evaluated using confocal imaging of transverse muscle sections. RESULTS: Fluorescent microspheres tracked interstitial fluid while not penetrating the muscle fiber. When compared with the static condition, significantly greater dispersion (P = 0.003) was seen with passively lengthened conditions (17 ± 9% vs. 31 ± 7%, respectively). Dispersion did not differ for the 2 microsphere sizes (P = 0.811). CONCLUSIONS: Fluorescent microspheres track movement of interstitial fluid, and dispersion is dependent on passive lengthening. Muscle Nerve 54: 444-450, 2016.