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Laboratory study using a rat T9 contusion model of spinal cord injury.This study aims to examine whether a combinatory treatment of Pioglitazone (PGZ) and granulocyte colony-stimulating factor (GCSF) can support neural stem/progenitor cells (NSPCs) directly and provide a sustainable microenvironment through immunomodulatory mechanisms.Neuroinflammation plays a crucial role in the progression of spinal cord injury (SCI) and hinders NSPC-mediated repair and regeneration. Broad acting drugs that mitigate inflammation and support NSPC proliferation have not been tested together in SCI research models.Isolated NSPCs were treated with vehicle control, PGZ, GCSF, or both PGZ and GSCF for 24 hours and stained with proliferation marker Ki67. Adult female Sprague-Dawley rats sustained moderate-to-severe contusion-based SCI at T9 and were administered either vehicle control, PGZ, GCSF, or both PGZ and GCSF treatments.Immunocytochemistry revealed that cultured NSPCs treated with both drugs produced higher numbers of actively proliferating cells and total cell numbers. ELISA on spinal cord tissue lysates at 1, 3, and 7 days post-injury (DPI) demonstrated that animals treated with PGZ, GCSF, or combination therapy showed significantly higher doublecortin levels at 7 DPI compared to control animals (P 0.05). Immunohistochemistry of injured tissue at 3, 7, and 14 DPI revealed no difference of ependymal NSPC proliferation between groups, but showed a significant decrease in lesion size with combination therapy compared to controls. Functional recovery was assessed by the Basso, Beattie, Bresnahan locomotor rating scale. Animals treated with both drugs had significantly higher levels of function at 1 (P 0.001), 3 (P 0.001), 7 (P 0.05), and 14 (P 0.05) DPI compared to controls.These results indicate that PGZ and GCSF treatment synergistically enhance NSPCs numbers and improve functional recovery after SCI. Our findings support an immunomodulatory strategy to recruit native NSPCs as a potential acute care intervention for SCI.Level of Evidence: N/A.

Decellularized tendon xenografts offer a promising alternative for reconstruction by using ubiquitously available material. This study compares static and centrifugal seeding of avian tendon scaffolds with NIH 3T3 fibroblasts. Incorporation of viable cells was achievable with both techniques, represented by DNA content. Proliferation rate and viability assay showed neither damage by centrifugal force nor superiority of the technique. Cell proliferation after 10 days of culture demonstrated that the scaffold did not hinder 3-D culturing. Confocal laser microscopy revealed structural details as formation of focal adhesions, to provide deeper insight into the process of cell attachment and growth in xenografts.

Study design Laboratory study using a rat T9 contusion model of spinal cord injury (SCI). Objective The purpose of this study was to evaluate which method of delivery of soluble keratin biomaterials would best support functional restoration through the macrophage polarization paradigm. Summary of background data SCI is a devastating neurologic event with complex pathophysiological mechanisms that currently has no cure. After injury, macrophages and resident microglia are key regulators of inflammation and tissue repair exhibiting phenotypic and functional plasticity. Keratin biomaterials have been demonstrated to influence macrophage polarization and promote the M2 anti-inflammatory phenotype that attenuates inflammatory responses. Methods Anesthetized female Lewis rats were subjected to moderate T9 contusion SCI and randomly divided into: no therapy (control group), an intrathecally injected keratin group, and a keratin-soaked sponge group (n = 11 in all groups). Functional recovery assessments were obtained at 3- and 6-weeks post-injury (WPI) using gait analysis performed with the DigiGait Imaging System treadmill and at 1, 3, 7, 14, 21, 28, 35, and 42 days post-injury by the Basso, Beattie, Bresnahan (BBB) locomotor rating scale. Histology and immunohistochemistry of serial spinal cord sections were performed to assess injury severity and treatment efficacy. Results Compared to control rats, applying keratin materials after injury improved functional recovery in certain gait parameters and overall trended toward significance in BBB scores; however, no significant differences were observed with tissue analysis between groups at 6 WPI. Conclusion Results suggest that keratin biomaterials support some locomotor functional recovery and may alter the acute inflammatory response by inducing macrophage polarization following SCI. This therapy warrants further investigation into treatment of SCI.Level of Evidence: N/A.

Background: Schwann cells are integral to the regenerative capacity of the peripheral nervous system, which declines after adolescence. The mechanisms underlying this decline are poorly understood. This study sought to compare the protein expression of Notch, c-Jun, and Krox-20 after nerve crush injury in adolescent and young adult rats. We hypothesized that these Schwann cell myelinating regulatory factors are down-regulated after nerve injury in an age-dependent fashion. Methods: Adolescent (2 months old) and young adult (12 months old) rats (n = 48) underwent sciatic nerve crush injury. Protein expression of Notch, c-Jun, and Krox-20 was quantified by Western blot analysis at 1, 3, and 7 days post-injury. Functional recovery was assessed in a separate group of animals (n = 8) by gait analysis (sciatic functional index) and electromyography (compound motor action potential) over an 8-week post-injury period. Results: Young adult rats demonstrated a trend of delayed onset of the dedifferentiating regulatory factors, Notch and c-Jun, corresponding to the delayed functional recovery observed in young adult rats compared to adolescent rats. Compound motor action potential area was significantly greater in adolescent rats relative to young adult rats, while amplitude and velocity trended toward statistical significance. Conclusions: The process of Schwann cell dedifferentiation following peripheral nerve injury shows different trends with age. These trends of delayed onset of key regulatory factors responsible for Schwann cell myelination may be one of many possible factors mediating the significant differences in functional recovery between adolescent and young adult rats following peripheral nerve injury.

To evaluate the biological, immunological, and biomechanical properties of a scaffold derived by architectural modification of a fresh-frozen porcine patella tendon using a decellularization protocol that combines physical, chemical, and enzymatic modalities.Porcine patellar tendons were processed using a decellularization and oxidation protocol that combines physical, chemical, and enzymatic modalities. Scaffolds (n = 88) were compared with native tendons (n = 70) using histologic, structural (scanning electron microscopy, porosimetry, and tensile testing), biochemical (mass spectrometry, peracetic acid reduction, DNA quantification, alpha-galactosidase [α-gal] content), as well as in vitro immunologic (cytocompatibility, cytokine induction) and in vivo immunologic nonhuman primate analyses.A decrease in cellularity based on histology and a significant decrease in DNA content were observed in the scaffolds compared with the native tendon (P.001). Porosity and pore size were increased significantly (P.001). Scaffolds were cytocompatible in vitro. There was no difference between native tendons and scaffolds when comparing ultimate tensile load, stiffness, and elastic modulus. The α-gal xenoantigen level was significantly lower in the decellularized scaffold group compared with fresh-frozen, nondecellularized tissue (P.001). The in vivo immunological response to implanted scaffolds measured by tumor necrosis factor-α and interleukin-6 levels was significantly (P.001) reduced compared with untreated controls in vitro. These results were confirmed by an attenuated response to scaffolds in vivo after implantation in a nonhuman primate model.Porcine tendon was processed via a method of decellularization and oxidation to produce a scaffold that possessed significantly less inflammatory potential than a native tendon, was biocompatible in vitro, of increased porosity, and with significantly reduced amounts of α-gal epitope while retaining tensile properties.Porcine-derived scaffolds may provide a readily available source of material for musculoskeletal reconstruction and repair while eliminating concerns regarding disease transmission and the morbidity of autologous harvest.

The long-term adaptations to microgravity and other spaceflight challenges within the confines of a spacecraft, and readaptations to weight-bearing upon reaching a destination, are unclear. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected. Thus, the purpose of this study was to evaluate gait changes in response to spaceflight. A prospective collection of gait data was collected on 3 groups of mice: those who spent~35 days in orbit (FLIGHT) aboard the International Space Station (ISS); a ground-based control with the same habitat conditions as ISS (Ground Control; GC); and a vivarium control with typical rodent housing conditions (VIV). Pre-flight and post-flight gait measurements were conducted utilizing an optimized and portable gait analysis system (DigiGait, Mouse Specifics, Inc). The total data acquisition time for gait patterns of FLIGHT and control mice was 1.5–5 min/mouse, allowing all 20 mice per group to be assessed in less than an hour. Patterns of longitudinal gait changes were observed in the hind limbs and the forelimbs of the FLIGHT mice after ~35 days in orbit; few differences were observed in gait characteristics within the GC and VIV controls from the initial to the final gait assessment, and between groups. For FLIGHT mice, 12 out of 18 of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. This rapid DigiGait gait measurement tool and customized spaceflight protocol is useful for providing preliminary insight into how spaceflight could affect multiple systems in rodents in which deficits are reflected by altered gait characteristics.

Background Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use. Methods In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use. Results In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining. Conclusions The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application.

Traumatic spinal cord injury (SCI) is typically the result of direct mechanical impact to the spine, leading to fracture and/or dislocation of the vertebrae along with damage to the surrounding soft tissues. Injury to the spinal cord results in disruption of axonal transmission of signals. This primary trauma causes secondary injuries that produce immunological responses such as neuroinflammation, which perpetuates neurodegeneration and cytotoxicity within the injured spinal cord. To date there is no FDA-approved pharmacological agent to prevent the development of secondary SCI and induce regenerative processes aimed at healing the spinal cord and restoring neurological function. An alternative method to electrically activate spinal circuits is the application of a noninvasive electromagnetic field (EMF) over intact vertebrae. The EMF method of modulating molecular signaling of inflammatory cells emitted in the extra-low frequency range of

Study design A laboratory study using a rabbit annular puncture model of intervertebral disc degeneration (IDD). Objective The aims of this study were to assess whether an amniotic suspension allograft (ASA) containing particulated human amnion and amniotic fluid derived cells regains intervertebral disc height and morphology and improves histologic scoring in a rabbit model of IDD. Summary of background data In contrast to current surgical interventions for IDD, in which the primary goal is to relieve symptomatic pain, one novel strategy involves the direct injection of anabolic cytokines. Current therapies for IDD are limited by both the short half-life of therapeutic proteins and general decline in anabolic cell populations. Methods Intervertebral discs in New Zealand white rabbits were punctured using 18-gauge needle under fluoroscopic guidance. Four weeks post-puncture, two groups of rabbits were injected with either ASA or a vehicle/sham control, while a third group was untreated. Weekly radiographs were obtained for 12 weeks to assess disc height index (DHI). Magnetic resonance imaging (MRI) T2 relaxation time was evaluated at weeks 4 and 12 to assess morphological changes. Histologic sections were evaluated on a semi-quantitative grading scale. Results Before treatment at week 4, DHIs and normalized T2 relaxation times between the three groups were not significantly different. At week 12, ASA-treated rabbits exhibited significantly greater DHIs and MRI T2 relaxation times than vehicle and untreated control groups. The ASA group had higher mean histologic score than the vehicle group, which demonstrated extensive fiber disorganization and delamination with reduced proteoglycan staining on histology. Conclusion Minimally invasive intervention with intradiscal injection of ASA was successful in reducing IDD in a reproducible rabbit model, with significant improvement in disc height and morphology when compared with vehicle and untreated control groups on radiographic and MRI analyses. Level of evidence N/A.

Purpose: Various animal models have been proposed to mimic the pathophysiologic process of intervertebral disc degeneration, a leading cause of back pain. The purpose of this study is to describe a minimally invasive technique via percutaneous needle puncture of the annulus fibrosus in New Zealand white rabbits. Methods: Under fluoroscopic guidance, an 18-gauge spinal needle was inserted 2 cm lateral to the midline spinous process. The needle was slowly advanced at approximately 45° angle until it was adjacent to the L5/L6 disc space. Lateral and anteroposterior views were used to verify correct needle position before advancing into the nucleus pulposus. The rabbits underwent weekly X-rays for 4 weeks to assess disc height index. MRI T2 relaxation was evaluated at week four to assess morphological changes. Discs were histologically graded on a 12-point scale to assess degeneration and compared to discs obtained from uninjured rabbits. Results: There were no complications associated with the percutaneous needle puncture procedure. All animals survived the duration of the experiment. Four weeks after injury, the disc height had progressively narrowed to approximately 50% of baseline. MRI assessment at the 4-week time point demonstrated a mean T2 relaxation time at the L5/L6 level that was 20.9% of the T2 relaxation time at the uninjured L4/L5 disc level ( p < 0.001). Histological analysis demonstrated lamellar disorganization of the annulus and decreased cellularity and proteoglycan content within the injured nucleus compared to uninjured control discs. Conclusion: The present study demonstrated a reliable technique of inducing an annular tear via a percutaneous needle puncture. Compared to open surgical approaches, the percutaneous model produces similar progressive disc degeneration while minimizing harm to the animal subjects. Clinical Relevance: The present study establishes a technique for the introduction of novel therapeutic agents to treat disc degeneration that may translate to future clinical trials.

Background: Bone grafts are used in approximately one half of all musculoskeletal surgeries. Autograft bone is the historic gold standard but is limited in supply and its harvest imparts significant morbidity to the patient. Alternative sources of bone graft include allografts, synthetics and, less commonly, xenografts which are taken from animal species. Xenografts are available in unlimited supply from healthy animal donors with controlled biology, avoiding the risk of human disease transmission, and may satisfy current demand for bone graft products. Methods: In the current study, cancellous bone was harvested from porcine femurs and subjected to a novel decellularization protocol to derive a bone scaffold. Results: The scaffold was devoid of donor cellular material on histology and DNA sampling (p &lt, 0.01). Microarchitectural properties important for osteoconductive potential were preserved after decellularization as shown by high resolution imaging modalities. Proteomics data demonstrated similar profiles when comparing the porcine bone scaffold against commercially available human demineralized bone matrix approved for clinical use. Conclusion: We are unaware of any porcine-derived bone graft products currently used in orthopaedic surgery practice. Results from the current study suggest that porcine-derived bone scaffolds warrant further consideration to serve as a potential bone graft substitute.

Langerhans cell sarcoma of the skin is a rare tumor with aggressive behavior. There are reports of Langerhans cell sarcoma involving the skin in patients with underlying systemic Langerhans cell histiocytosis. However, to our knowledge, sarcomatous transformation of skin Langerhans cell histiocytosis, has not been previously described. We report a case of Langerhans cell sarcoma of the skin representing a probable transformation from Langerhans cell histiocytosis. The lesion had a clinical and pathological biphasic pattern, presenting as a plaque and nodule in the lower leg of a 66 year-old female. The plaque area showed a superficial epidermotropic proliferation of atypical Langerhans cells. The associated deep nodule had an overtly sarcomatous Langerhans cell proliferation. The immunohistochemistry profile was characteristic of Langerhans cells in both components. We consider this case a unique example of deep nodular Langerhans cell sarcoma of the skin, in which the associated superficial Langerhans cell proliferation is consistent with the precursor lesion.

Premature musculoskeletal joint failure is a major source of morbidity among childhood cancer survivors. Radiation effects on synovial joint tissues of the skeleton are poorly understood. Our goal was to assess long-term changes in the knee joint from skeletally mature rats that received total-body irradiation while skeletal growth was ongoing.14 week-old rats were irradiated with 1, 3 or 7 Gy total-body doses of 18 MV X-rays. At 53 weeks of age, structural and compositional changes in knee joint tissues (articular cartilage, subchondral bone, and trabecular bone) were characterized using 7T MRI, nanocomputed tomography (nanoCT), microcomputed tomography (microCT), and histology.T2 relaxation times of the articular cartilage were lower after exposure to all doses. Likewise, calcifications were observed in the articular cartilage. Trabecular bone microarchitecture was compromised in the tibial metaphysis at 7 Gy. Mild to moderate cartilage erosion was scored in the 3 and 7 Gy rats.Late degenerative changes in articular cartilage and bone were observed after total-body irradiation in adult rats exposed prior to skeletal maturity. 7T MRI, microCT, nanoCT, and histology identified potential prognostic indicators of late radiation-induced joint damage.

The influence of age on rotator cuff function and muscle structure remains poorly understood. We hypothesize that normal aging influences rotator cuff function, muscle structure, and regulatory protein expression in an established rat model of aging.Seventeen rats were obtained from the National Institute on Aging. The supraspinatus muscles in 11 middle-aged (12 months old) and 6 old (28 months old) rats were studied for age-related changes in rotator cuff neuromuscular function by in vivo muscle force testing and electromyography (EMG). Changes in muscle structure and molecular changes were assessed with quantitative immunohistochemistry for myogenic determination factor 1 (MyoD) and myogenic factor 5 (Myf5) expression.Old animals revealed significantly decreased peak tetanic muscle force at 0.5 N and 0.7 N preload tension (P.05). The age of the animal accounted for 20.9% of variance and significantly influenced muscle force (P = .026). Preload tension significantly influenced muscle force production (P.001) and accounted for 12.7% of total variance. There was regional heterogeneity in maximal compound motor action potential (CMAP) amplitude in the supraspinatus muscle; the proximal portion had a significantly higher CMAP than the middle and distal portions (P.05). The expression of muscle regulatory factors MyoD and Myf5 was significantly decreased in old animals compared with middle-aged animals (P.05).The normal aging process in this rat model significantly influenced contractile strength of the supraspinatus muscle and led to decreased expression of muscle regulatory factors. High preload tensions led to a significant decrease in force production in both middle-aged and old animals.

Peripheral nerve injuries requiring surgery can be repaired by autograft, the clinical "gold standard", allograft, or nerve conduits. Most published clinical studies show the effectiveness of nerve conduits in small size defects in sensory nerves. Many preclinical studies suggest that peripheral nerve regeneration through conduits can be enhanced and repair lengths increased with the use of a biomaterial filler in the conduit lumen. We have previously shown that a luminal hydrogel filler derived from human hair keratin (HHK) can improve electrophysiological and histological outcomes in mouse, rabbit, and non-human primate nerve injury models, but insight into potential mechanisms has been lacking. Based on the premise that a keratin biomaterial (KOS) hydrogel provides an instantaneous structural matrix within the lumen, the current study compares the cellular behavior elicited by KOS hydrogel to Matrigel (MAT) and saline (SAL) conduit fillers in a 1 cm rat sciatic nerve injury model at early stages of regeneration. While there was little difference in initial cellular influx, the KOS group showed earlier migration of dedifferentiated Schwann cells (SC) from the proximal nerve end compared to the other groups. The KOS group also showed faster SC dedifferentiation and myelin debris clearance, and decreased macrophage infiltration during Wallerian degeneration of the distal nerve tissue.

Nonhuman primates have similar shoulder anatomy and physiology compared to humans, and may represent a previously underutilized model for shoulder research. This study sought to identify naturally occurring bony and muscular degeneration in the shoulder of nonhuman primates and to assess relationships between structural and functional aspects of the shoulder and measures of physical function of the animals. We hypothesized that age-related degenerative changes in the shoulders of nonhuman primates would resemble those observed in aging humans.Middle-aged (n = 5; ages 9.4-11.8 years) and elderly (n = 6; ages 19.8-26.4 years) female vervet monkeys were studied for changes in mobility and shoulder function, and radiographic and histologic signs of age-related degeneration.Four out of 6 (4/6) elderly animals had degenerative changes of the glenoid compared to 0/5 of the middle-aged animals (P = .005). Elderly animals had glenoid retroversion, decreased joint space, walked slower, and spent less time climbing and hanging than middle-aged vervets (P .05). Physical mobility and shoulder function correlated with glenoid version angle (P .05). Supraspinatus muscles of elderly animals were less dense (P = .001), had decreased fiber cross-sectional area (P .001), but similar amounts of nuclear material (P = .085). Degenerative rotator cuff tears were not observed in any of the eleven animals.The vervet monkey naturally undergoes age-related functional, radiographic and histological changes of the shoulder, and may qualify as an animal model for selected translational research of shoulder osteoarthritis.

Predisposition to Achilles tendon (AT) ruptures in middle-aged individuals may be associated with age-related changes to inherent passive biomechanical properties of the gastrocnemius-Achilles (GC-AT) muscle-tendon unit, due to known muscle-tendon structural changes in normal aging. The goal of this study was to determine whether the passive biomechanical response of the GC-AT muscle-tendon unit was altered with age in 6 young (8 months) and 6 middle-aged (24 months) F344xBN hybrid rats from the National Institute on Aging colony. Fung's quasilinear viscoelastic (QLV) model was used to determine in vivo history and time-dependent load-relaxation response of the GC-AT. Effective stiffness and modulus were also estimated using linear regression analysis. Fung's QLV revealed a significantly decreased magnitude of the relaxation response (parameter C, p=0.026) in middle-aged animals compared to young animals (0.108±0.007 vs. 0.144±0.015), with similar time-dependent viscous GC-AT properties (τ(1), τ(2)). The product of elastic parameters (A*B), which represents the initial slope of the elastic response, was significantly increased by 50% in middle-aged rats (p=0.014). Estimated GC-AT stiffness increased 28% at peak tensions in middle-aged rats (2.7±0.2 N/mm) compared to young rats (1.9±0.2 N/mm; p=0.036). While the limitations of this animal model must be considered, the changes we describe could be associated with the observation that GC-AT pathology and injury is more common in middle-aged individuals. Further studies are necessary to characterize the load-to-failure behavior of AT in middle-aged compared to young animals.

Aim: To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N’,N’-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Materials & methods: TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. Results & conclusion: TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.

Background: Radiation-induced fibrosis (RIF) of musculoskeletal tissue is a common complication of radiation therapy for extremity soft-tissue sarcoma, with no standardized strategy for prevention and treatment. Angiotensin-(1-7) (Ang-[1-7]), a well-tolerated endogenous heptapeptide hormone with antitumor and antifibrotic properties, was tested as a radioprotectant for RIF and stiffening of irradiated muscles. Methods: Male CD-1 mice were randomized to one of three treatment groups: control, simulated sarcoma radiation therapy to the gastrocnemius and soleus muscles, or radiation therapy along with continuous Ang-(1-7) delivery initiated three days before radiation therapy. The biologically equivalent dose of radiation (∼100.3 Gy) absorbed by normal musculature during the course of radiation therapy for extremity sarcoma was delivered by means of four dose fractions of 7.3 Gy over two weeks. Fibrosis (n = 5 per group) and mechanical properties (n = 4 to 6 per group) of the muscles were measured at six weeks and four months after radiation therapy, and the intramuscular concentration of the profibrotic cytokines transforming growth factor-beta (TGF-β) and connective tissue growth factor (CTGF) (n = 8 to 10 per group) were measured at six weeks. Results: Interstitial (p < 0.01) and perivascular (p < 0.05) fibrosis increased significantly in the muscles treated with radiation therapy alone versus the nonirradiated controls at both six weeks (interstitial, +89%; perivascular, +112%) and four months (interstitial, +154%; perivascular, +88%). The muscles treated with radiation alone also exhibited increased tension (p < 0.01) versus nonirradiated controls at both six weeks (+779%) and four months (+1761%) when placed under 5% strain, and at four months (+1390%; p < 0.001) under 10% strain. At four months, muscle stiffness had increased in the mice treated with radiation therapy alone (+90%; p = 0.002) compared with nonirradiated controls. TGF-β production was also greater in this group at six weeks (+37%; p = 0.06) versus control. Ang-(1-7) administration prevented RIF and stiffening, with no differences observed for any other outcome between those receiving radiation therapy with Ang-(1-7) and the nonirradiated controls. Likewise, Ang-(1-7) mitigated the increase in TGF-β and CTGF concentration from radiation therapy. Conclusions: Ang-(1-7) attenuated RIF, stiffening, and production of profibrotic cytokines that were elevated in mouse skeletal muscles after simulated radiation therapy for extremity sarcoma. Clinical Relevance: Ang-(1-7) may serve as a potential therapy for the prevention of RIF in patients who require radiation therapy as adjuvant treatment for soft-tissue sarcoma.

Botulinum Neurotoxin A (BoNT-A) injections have been used for the treatment of muscle contractures and spasticity. This study assessed the influence of (BoNT-A) injections on passive biomechanical properties of the muscle-tendon unit. Mouse gastrocnemius muscle (GC) was injected with BoNT-A (n = 18) or normal saline (n = 18) and passive, non-destructive, in vivo load relaxation experimentation was performed to examine how the muscle-tendon unit behaves after chemical denervation with BoNT-A. Injection of BoNT-A impaired passive muscle recovery (15% vs. 35% recovery to pre-stretching baseline, p < 0.05) and decreased GC stiffness (0.531 ± 0.061 N/mm vs. 0.780 ± 0.037 N/mm, p < 0.05) compared to saline controls. The successful use of BoNT-A injections as an adjunct to physical therapy may be in part attributed to the disruption of the stretch reflex; thereby modulating in vivo passive muscle properties. However, it is also possible that BoNT-A injection may alter the structure of skeletal muscle; thus modulating the in vivo passive biomechanical properties of the muscle-tendon unit.

The purpose of this article is to review basic science studies using various animal models for rotator cuff research and to describe structural, biomechanical, and functional changes to muscle following rotator cuff tears. The use of computational simulations to translate the findings from animal models to human scale is further detailed. A comprehensive review was performed of the basic science literature describing the use of animal models and simulation analysis to examine muscle function following rotator cuff injury and repair in the ageing population. The findings from various studies of rotator cuff pathology emphasize the importance of preventing permanent muscular changes with detrimental results. In vivo muscle function, electromyography, and passive muscle–tendon unit properties were studied before and after supraspinatus tenotomy in a rodent rotator cuff injury model (acute vs chronic). Then, a series of simulation experiments were conducted using a validated computational human musculoskeletal shoulder model to assess both passive and active tension of rotator cuff repairs based on surgical positioning. Outcomes of rotator cuff repair may be improved by earlier surgical intervention, with lower surgical repair tensions and fewer electromyographic neuromuscular changes. An integrated approach of animal experiments, computer simulation analyses, and clinical studies may allow us to gain a fundamental understanding of the underlying pathology and interpret the results for clinical translation.

BACKGROUND Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone with anti-proliferative and anti-angiogenic properties. The primary objective of this study was to determine whether Ang-(1-7) effectively reduces prostate cancer metastasis in mice. METHODS Human PC3 prostate cancer cells were injected into the aortic arch via the carotid artery of SCID mice pre-treated with Ang-(1-7) or injected into the tibia of athymic mice, administered Ang-(1-7) for 5 weeks beginning 2 weeks post-injection. Tumor growth and volume were determined by bioluminescent and magnetic resonance imaging. The presence of tumors was confirmed by hematoxylin and eosin staining; TRAP histochemistry was used to identify osteolytic lesions. The effect of Ang-(1-7) on osteoclastogenesis was assessed in differentiated bone marrow cells. RESULTS Pre-treatment with Ang-(1-7) prevented metastatic tumor formation following intra-aortic injection of PC3 cells, while 83% of untreated mice developed tumors in metastatic sites. Circulating VEGF was significantly higher in control mice compared to mice administered Ang-(1-7). A 5-week regimen of the heptapeptide hormone attenuated intra-tibial tumor growth; Ang-(1-7) was significantly higher in the tibia of treated mice than in control animals. Osteoclastogenesis was reduced by 50% in bone marrow cells differentiated in the presence of Ang-(1-7), suggesting that the heptapeptide hormone prevents the formation of osteolytic lesions to reduce tumor survival in the bone microenvironment. CONCLUSIONS These findings suggest that Ang-(1-7) may serve as an anti-angiogenic and anti-metastatic agent for advanced prostate cancer. By extension, the heptapeptide hormone may provide effective therapy for bone metastasis produced from primary tumors of the lung and breast. Prostate 73: 71–82, 2013. © 2012 Wiley Periodicals, Inc.

12/15 lipoxygenase (12/15LO) oxidizes polyunsaturated fatty acids (PUFAs) to form bioactive lipid mediators. The role of 12/15LO in atherosclerosis development remains controversial. We evaluated atherosclerosis development and lipid metabolism in 12/15LO-LDL receptor (LDLr) double knockout (DK) vs. LDLr knockout (SK) mice fed a PUFA-enriched diet to enhance production of 12/15LO products. Compared with SK controls, DK mice fed a PUFA-enriched diet had decreased plasma and liver lipid levels, hepatic lipogenic gene expression, VLDL secretion, and aortic atherosclerosis and increased VLDL turnover. Bone marrow transplantation and Kupffer cell ablation studies suggested both circulating leukocytes and Kupffer cells contributed to the lipid phenotype in 12/15LO-deficient mice. Conditioned medium from in vitro incubation of DK vs. SK macrophages reduced triglyceride secretion in McArdle 7777 hepatoma cells. Our results suggest that, in the context of dietary PUFA enrichment, macrophage 12/15LO expression adversely affects plasma and hepatic lipid metabolism, resulting in exacerbated atherosclerosis.

BACKGROUND Surgical repair of large chronic rotator cuff tears can be technically demanding because it requires manipulation of a muscle-tendon unit that is scarred, retracted, and stiffer than normal, all of which contribute to increased tension at the repair site. The purpose of the present study was to characterize the in vivo rotator cuff muscle-tendon unit function after acute and chronic injury at surgically relevant preload tensions. METHODS Sixty-two Sprague-Dawley rats were divided into a healthy, uninjured (control) group (n = 22), an acute injury group (n = 20), and a chronic injury group (n = 20) and underwent in vivo muscle force testing and electromyographic testing of the supraspinatus muscle-tendon unit at various preload tensions. RESULTS Preload tension affected the maximum supraspinatus muscle contractile force in all groups (p < 0.05). At the peak tension required to repair an acute tear, there was a 28% to 30% reduction in maximum tetanic contraction amplitude in all groups (p < 0.05). At the peak tension required to repair a chronic tear, there was a 40% to 53% reduction in maximal tetanic contraction amplitude in all groups (p < 0.05). The uninjured (control) group showed increased muscle endurance (p < 0.05) in comparison with the acute injury and chronic injury groups at all preload tensions. The chronic injury group showed reduced compound motor action potential amplitude (p < 0.05). CONCLUSIONS Both the acute and chronic injury groups demonstrated functional impairment related to increasing preload tensions. Higher repair tensions, associated with the chronic injury setting, resulted in greater functional impairment. The present study also demonstrates an association between increased time from rotator cuff tendon injury and impaired in vivo rotator cuff muscle electromyographic findings.

Botulinum neurotoxin-A (BoNTA) is a potent neurotoxin used to alter muscle tone to manage spasticity and to provide tendon bioprotection; however, the appropriate dose and injection volume to administer is not defined. Male mice (n = 120) received BoNTA injections into one gastrocnemius with either a constant volume (10 µl) with a variable dose (1, 3, 6 U/kg) or a constant dose (3 U/kg) in a variable volume (2.5, 5, 10, 20, 30 µl). Electromyographic (EMG) examination, muscle force generation (MFG), and wet muscle mass were measured in the ipsilateral and contralateral limbs at 1, 2, 4, or 12 weeks post-injection. MFG and EMG responses decreased to approximately 40% of contralateral after a 1 U/kg injection and 0% of contralateral by 3 and 6 U/kg injection at 1 week after injection. Neuromuscular blockade was greatest with a 10 µl injection volume. MFG, EMG examination, and wet muscle mass reached contralateral values 12 weeks after injection for all injection doses and volumes tested. Effective injection doses and volumes were identified for producing full and partial neuromuscular blockade in the mouse gastrocnemius. These findings have important clinical implications in the intramuscular administration of BoNTA to manage muscle tone. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1764–1770, 2011

Ischemic heart disease remains one of the most common causes of mortality in developed countries. Recently, stem cell therapy is being considered for treating ischemic heart diseases. On the other hand, there has been evidence of chondro-osteogenic mass formation after stem cell injection in the heart. In a recent publication, Chiavegato et al. ( J Mol Cell Cardiol. 42 (2007) 746–759) has suggested that amniotic fluid-derived stem (AFS) cells cause chondro-osteogenic masses in the infarcted heart. The goal of the current study was to further examine the formation of such masses, specifically, the role of AFS cells in this process. Our results confirm the presence of similar bone-like masses in the left ventricular wall of infarcted rats; however, this phenomenon occurred independent of AFS cell injection into the myocardium. Moreover, AFS cell injection did not increase the presence of chondro-osteogenic masses. Echocardiographic analysis of large infarctions in rats frequently revealed the presence of echogenic structures in the left ventricular wall. We further demonstrated a significant relationship between the infarction size and chondro-osteogenic formation and subsequent decrease in cardiac function. Collectively, our study indicates that chondro-osteogenic differentiation can take place in infarcted rat heart independent of cell injection. These results have significant implications for future design and testing of stem cell therapy for treatment of cardiac muscle diseases.

The chemical denervation that results from botulinum neurotoxin A (BoNT-A) causes a temporary, reversible paresis that can result in easier surgical manipulation of the muscle-tendon unit in the context of tendon rupture and repair. The purpose of the study was to determine whether BoNT-A injections can be used to temporarily and reversibly modulate active and passive skeletal muscle properties.Male CD1 mice weighing 40-50 g were divided into a 1-week postinjection group (n = 13: n = 5 saline and n = 8 BoNT-A) and a 2-week postinjection group (n = 17: n = 7 saline and n = 10 BoNT-A). The animals had in vivo muscle force testing and in vivo biomechanical evaluation.There was a substantial decline in the maximal single twitch amplitude (p.05) and tetanic amplitude (p.05) at one week and at 2 weeks after BoNT-A injection, when compared to saline-injected controls. BoNT-A injection significantly reduced the peak passive properties of the muscle-tendon unit as a function of displacement at one week (p.05). Specifically, the stiffness of the BoNT-A injected muscle-tendon unit was 0.417 N/mm compared to the control saline injected group, which was 0.634 N/mm, a 35% reduction in stiffness (p.05).Presurgical treatment with BoNT-A might improve the surgical manipulation of the muscle-tendon unit, thus improving surgical outcomes. The results implicate neural tone as a substantial contributor to the passive repair tension of the muscle-tendon unit. The modulation of neural tone through temporary, reversible paresis is a novel approach that might improve intraoperative and postoperative passive muscle properties, allowing for progressive rehabilitation while protecting the surgical repair site.

Background: Despite the increased use of arthroscopy in pediatric orthopaedics, there is a paucity of data regarding the potential long-term effects of this procedure on the immature physis. The purpose of this study was to test the hypothesis that elevated intra-articular pressures used during arthroscopic surgery do not result in growth disturbances or morphologic alterations in the epiphyseal plate. Methods: Twenty-seven 6-week-old skeletally immature New Zealand white rabbits were divided into experimental (n = 21) and control groups (n = 6). In the experimental group, a hydraulic pump was used to pressurize 1 randomly assigned knee joint per rabbit to intra-articular pressures of 120mm Hg for 2 hours. In the control group, rabbits received a sham intervention. All rabbits were killed at 6 months of age (skeletal maturity), and their tissues were evaluated grossly, radiographically, and histologically. Data collection included gross measurements (femur and tibia lengths, evaluation of varus/valgus angulation, and knee joint range of motion) and histologic analyses to determine whether morphologic changes were present in the articular cartilage or physis. Confidence intervals were used to test for statistical equivalence. Results: The pressurized and control groups had statistically equivalent gross measurements. No significant articular cartilage or physeal lesions were identified in histologic sections or radiographic studies. Conclusion: This study provided no evidence that arthroscopic pressurization of the knee joint to 120 mm Hg for 2 hours significantly affected physeal growth in a skeletally immature rabbit model. Clinical Relevance: This study provides the first direct evidence that arthroscopic pressurization of immature joints has no clinically significant adverse long-term effects. Therefore, novel uses of arthroscopy in pediatric patients should be explored without undue concern with regard to premature physeal closure.

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (

Purpose The management of peripheral nerve injuries with segmental defects is a challenge to both patient and surgeon. Repairs under tension have a poor prognosis; sensory nerve allografts have donor site morbidity and suboptimal motor recovery, but remain the gold standard. The development of conduit-based repair strategies has evolved and these are promising for sensory nerves and short defects; however, no conduit filler is clinically available that improves motor recovery equivalent to sensory autografts. In this study, motor recovery using keratin-based hydrogel filler was compared with that for sensory nerve autografts and empty conduits. Methods Fifty-four mice were randomized into 3 treatment groups: empty conduit, sural nerve autograft, and keratin hydrogel-filled conduit. Animals were followed for 6 weeks, 3 months, and 6 months. Outcomes included compound motor action potential (CMAP), nerve area, myelinated axon number and density, and myelinated axon diameter. Results Neuromuscular recovery with keratin was greater than with empty conduits in most outcome measures. Nerves that regenerated through the keratin hydrogel had lower conduction delays, greater amplitudes, more myelinated axons, and larger axons than nerves that regenerated through empty conduits. Sensory nerve autografts and keratin hydrogel were statistically equivalent in CMAP measurements at 6 months. Moreover, keratin-filled conduits demonstrated greater axon density and larger average axon diameter than both empty conduits and autograft at 6 months. Conclusions In a mouse tibial nerve model, keratin hydrogels significantly improved electrophysiological recovery, compared with empty conduits and sensory nerve autografts, at an early time point of regeneration. Keratin hydrogels also produce long-term electrical and histological results superior to empty conduits and equivalent to sensory nerve autografts.

Evaluation of vascular disorders of the upper extremity requires an anatomic and functional approach. The combination of a good history and physical examination, laboratory testing, and specialized vascular laboratory studies will help detect any underlying collagen vascular disease or coagulopathy and provide physiologic-specific and patient-oriented management. This paper reviews the currently available noninvasive modalities for the evaluation of upper-extremity perfusion.

The management of trauma-associated nerve defects is difficult because of the absence of autologous donor motor or sensory nerves. Pre-clinical development and clinical experience has shown that damaged nerves can be surgically repaired using a tubular conduit interposed across the defect. Acceptable patient outcomes are achieved so long as the gap distance does not exceed a few centimeters. Although research in animals has demonstrated that nerve repair can be facilitated across slightly larger gaps by introducing a biomaterial filler into the conduit lumen, these biomaterials are not typically "neuroinductive" (i.e. capable of acting directly on regenerative cells to enhance nerve tissue formation beyond clinical limits). Moreover, their use does not often result in functional recovery equivalent to nerve autograft, the clinical gold standard. Here we show that a biomaterial gel made from the proteins found in human hair can mediate a robust nerve regeneration response, in part through activation of Schwann cells. In vitro, keratins extracted from human hair enhance the activity of Schwann cells by a chemotactic mechanism, increase their attachment and proliferation, and up-regulate expression of important genes. Moreover, these characteristics translate to improved functional nerve recovery in an animal model. These results suggest that a biomaterial derived from human hair keratins is neuroinductive and can facilitate an outcome comparable to autograft in a nerve injury model.

Neuromuscular junction destabilization following nerve injury contributes to irreversible functional impairment. Myogenic Regulatory Factors (MRF's) including myoblast determination factor (MyoD), MRF-4, Myogenin, and myogenic factors-5 (myf-5), and Growth-associated protein 43 KDa (GAP43) regulate gene expression of nicotinic acetylcholine receptor (nAChR) subunits (alpha, beta, delta, gamma, and epsilon). We hypothesized that nerve injury induces altered gene expression of MRF's, nAChRs, and GAP-43 in the skeletal muscle which destabilize neuromuscular junctions. The tibial nerve was transected in 42 juvenile male Sprague-Dawley rats. Denervated and contralateral control gastrocnemius m. mRNA for nAChR subunits, MRF's, and GAP-43 were determined by real time reverse transcription polymerase chain reaction (real time RT-PCR). After transection, muscle mass decreased for 1 year with a nadir of 75% at 3 months. Alpha, gamma, and epsilon subunit genes increased by 3 and peaked at 7 days before returning to control levels (P < 0.05). Beta subunits and GAP-43 tended to increase. Delta subunits peaked at 3 days returning to control levels by 30 days. By one month, most of the nAChR subunits had returned to control levels. Alpha, beta, gamma, and delta subunit expression remained significantly lower than control up to 1 year later (P < 0.05). MRF4, Myogenin, and MyoD expression paralleled that of alpha, gamma, and epsilon nAChR subunits (P < 0.05). Gene expression of nAChR alpha, gamma, delta and epsilon subunits was biphasic in the first month after nerve injury, similar to that of MRF's. nAChR subunits and MRF's may play a critical role in neuromuscular junction stability.

Background: Tendon-repair techniques have evolved to increase the construct strength of the repair site in order to permit early active range of motion without tendon gap or rupture. The present study evaluated the hypothesis that the injection of botulinum neurotoxin type-A (BoNT-A) into the gastrocnemius muscle will reduce the active force production of that muscle below the force required to rupture the associated, repaired Achilles tendon. Methods: Seventy-nine rat Achilles tendons were surgically bisected and were repaired with use of a two-strand core suture with a running epitenon repair. After the repair, the animals were treated with unilateral intramuscular (gastrocnemius) injections of either BoNT-A (6 U/kg body weight) (thirty-seven rats) or saline solution (forty-two rats). Operatively treated ankles were fixed in the neutral position with a percutaneous pin for the first two days after surgery. Unrestricted ankle motion and weight-bearing were allowed after the second postoperative day. An assessment of gap formation or rupture at the repair site, electrophysiologic measurements of force applied to the tendon, and an assessment of the strength of the repaired tendon were performed. Results: Intramuscular BoNT-A injections produced a significant, reversible reduction in active muscle force (p < 0.007). Twitch and tetanus contractions decreased to approximately 25% of the values for the control side within one week, remained at

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal deathin vivoto ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from α- to γ-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic geneBax. However, there was an increase in intrafusal muscle fibers (spindles) inBaxknock-out versus both wild-type andMyoGDNFmice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.

The effects of ethanol exposure on agonist-stimulated phosphorylation of p70S6 kinase and ribosomal S6 protein were determined in confluent astrocyte monolayers. Basal phosphorylation of p70S6 kinase and S6 protein was either unaffected or reduced, respectively, after exposure to 50 mM ethanol for 4 days. The abilities of norepinephrine, carbachol and epidermal growth factor to phosphorylate these proteins were significantly decreased after ethanol exposure. In contrast, ethanol exposure had no effect on the protein expression of either p70S6 kinase or S6 protein. Our data suggest that continuous ethanol exposure results in a generalized decrease in agonist-activation of the p70S6 pathway.

To study the effects of neuromuscular blockade using botulinum toxin A on juvenile muscles at a dosage of 6 units/kg body weight in a rat model.A total of 34 male Sprague-Dawley rats (1-mo old) were used. A small incision was made along the posterior aspect of the left hind leg with the exposure of the gastrocnemius. Botulinum toxin A was injected at a dosage of 6 units/kg body weight in the medial and lateral heads of the muscle. An equivalent volume of saline were injected into the right gastrocnemius (control). Motor evoked action potentials, muscle force generation, and muscle mass and neuromuscular junction morphometry were analyzed at different time intervals up to 1 yr after toxin injection.During the 1-2 wks after botulinum toxin A injection, muscle mass, electrophysiologic variables, and muscle force generation were significantly reduced but returned to nearly normal at 6 mos postinjection. In the study group, neuromuscular junction gutter depth became significantly shallower 2 mos after injection, then normalized at 1 yr. There was a nonsignificant trend toward larger neuromuscular junctions from the gastrocnemius injected with botulinum toxin A.Our findings provide scientific evidence to support the clinical situation in which the interinjection interval of 3-6 mos of botulinum toxin A at a similar dosage is used.

BACKGROUND Controlled joint extension followed by gradual distraction with use of an external fixator may facilitate primary repair of peripheral nerve defects by permitting end-to-end repair without tension. The hypothesis of the present study was that gradual lengthening of nerve repairs with use of incremental distraction would provide superior results compared with grafting or repair under tension. METHODS A median nerve segment measuring four times the diameter of the nerve was resected in thirty-six rabbits to create a 7-mm gap in the nerve. Neurorrhaphy was performed with use of one of three techniques. In Group 1 (cable graft), a tension-free medial antebrachial cutaneous graft was placed to allow full range of motion of the elbow postoperatively. In Group 2 (end-to-end repair without distraction), the elbow was externally fixed in hyperflexion and the nerve was repaired end-to-end. At fourteen days, the fixator was removed and unprotected elbow motion was permitted. In Group 3 (end-to-end repair with gradual distraction), the elbow was externally fixed in hyperflexion and primary neurorrhaphy was performed. At fourteen days, the elbow was extended 10 degrees every other day with use of the articulated external fixator until full extension was achieved. Median nerve amplitude, latency, and nerve-conduction velocity; flexor digitorum superficialis single-twitch force generation and maximum tetanic force generation; muscle mass; and elbow range of motion were measured at three or six months. In addition, histologic analysis of the median nerve distal to the repair site and the morphometry of the neuromuscular junction in the flexor digitorum superficialis were performed at six months. RESULTS All rabbits regained full active and passive range of motion. At three months, the nerve-conduction velocities in Groups 2 and 3 were significantly greater than that in Group 1. At six months, the nerve-conduction velocities and amplitudes in Group 3 were significantly greater than those in Groups 1 and 2. At six months, the tetanic force in Group 3 was significantly greater than those in Groups 1 and 2. There were no significant differences in muscle mass among the groups. There were no significant differences in histological findings among the three groups, although there was a trend toward larger fiber size in Group 3 as compared with the other two groups. The neuromuscular junctions in Group 3 had a significantly larger surface area than did those in Group 1 (p = 0.002) and Group 2 (p = 0.034). CONCLUSION The use of an articulated external fixator and controlled gradual distraction appears to facilitate the treatment of peripheral nerve defects.

We compared the in vivo metabolism of pre � HDL particles isolated by anti-human apolipoprotein A-I (apoA-I) immunoaffinity chromatography (LpA-I) in human apoA-I transgenic (hA-I Tg) mice with that of lipid-free apoA-I (LFA-I) and small LpA-I. After injection, preLpA-I were removed from plasma more rapidly than were LFA-I and small LpA-I. PreLpA-I and LFA-I were preferentially degraded by kidney compared with liver; small LpA-I were preferentially degraded by the liver. Five minutes after tracer injection, 99% of LFA-I in plasma was found to be as- sociated with medium-sized (8.6 nm) HDL, whereas only 37% of pretracer remodeled to medium-sized HDL. In- jection of preLpA-I doses into C57Bl/6 recipients re- sulted in a slower plasma decay compared with hA-I Tg re- cipients and a greater proportion ( � 60%) of the pre � radiolabel that was associated with medium-sized HDL. PreLpA-I contained one to four molecules of phosphati- dylcholine per molecule of apoA-I, whereas LFA-I contained less than one. We conclude that preLpA-I has two meta- bolic fates in vivo, rapid removal from plasma and catabo- lism by kidney or remodeling to medium-sized HDL, which we hypothesize is determined by the amount of lipid associ- ated with the preparticle and the particle's ability to bind to medium-sized HDL. —Lee, J-Y., L. Lanningham-Foster, E. Y. Boudyguina, T. L. Smith, E. R. Young, P. L. Colvin, M. J. Thomas, and J. S. Parks. Prehigh density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice. J. Lipid Res. 2004. 45: 716-728.

Staphylococcus aureus is the bacterial pathogen which is responsible for approximately 80% of all cases of human osteomyelitis. It can invade and remain within osteoblasts. The fate of intracellular Staph. aureus after the death of the osteoblast has not been documented. We exposed human osteoblasts to Staph. aureus. After infection, the osteoblasts were either lysed with Triton X-100 or trypsinised. The bacteria released from both the trypsinised and lysed osteoblasts were cultured and counted. Colonies of the recovered bacteria were then introduced to additional cultures of human osteoblasts. The number of intracellular Staph. aureus recovered from the two techniques was equivalent. Staph. aureus recovered from time zero and 24 hours after infection, followed by lysis/trypsinisation, were capable of invading a second culture of human osteoblasts. Our findings indicate that dead or dying osteoblasts are capable of releasing viable Staph. aureus and that Staph. aureus released from dying or dead osteoblasts is capable of reinfecting human osteoblasts in culture.

The effects of ethanol exposures on mitogen-activated protein kinase (MAPK) activity were determined in confluent astrocyte monolayers prepared from neonatal rat cerebral cortex. Acute 30 min exposure to 50 mM ethanol had no significant effect on MAPK activity. However, chronic exposure to ethanol for 4 days elicited a concentration-dependent increase in the basal level of this enzyme activity with no parallel increase in its protein expression. In addition, the magnitude of MAPK activation by epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor was significantly increased above corresponding control values in cells chronically exposed to ethanol. Immunolabeling experiments indicated that the protein expression of receptors for these growth factors was unaffected by ethanol treatment. Our results suggest that even after chronic ethanol treatment, MAPK phosphorylation and, hence, activation remains elevated.

Background Longitudinal studies of upper extremity aging in humans include logistical concerns that animal models can overcome. The vervet is a promising species with which to study aging-related processes. However, age-related changes in upper extremity muscle structure have not been quantified in this species. This study measured age-related changes to muscle structure, examined relationships between muscle structure and measures of physical performance, and evaluated the presence of rotator cuff tears. Methods Muscle structure (volume, optimal fiber length, and physiologic cross-sectional area (PCSA)) of 10 upper extremity muscles was quantified from the right upper limb of 5 middle-aged and 6 older adult female vervets. Results Total measured PCSA was smaller ( P = .001) in the older adult vervets than in the middle-aged vervets. Muscle volume reduction predominate the age-related reductions in PCSA. Total measured PCSA was not correlated to any measures of physical performance. No rotator cuff tears were observed. Supraspinatus volume was relatively larger and deltoid volume relatively smaller in the vervet compared with a human. Conclusions The vervet is an appropriate translational model for age-related upper extremity muscle volume loss. Functional measures were not correlated to PCSA, suggesting the vervets may have enough strength for normal function despite loss of muscle tissue. Reduced relative demand on the supraspinatus may be responsible for the lack of naturally occurring rotator cuff tears.

Major accomplishments this year include successful seeding of AFS into ANA. This accomplishment also documented that these cells remained viable up to 72 hours after seeding. The seeded constructs were used to repair critical-sized, large gap nerve injury in rats and their functional recovery was monitored longitudinally using digital video gait analysis as well as electrophysiologic and histologic outcomes. The data analysis is not complete, but preliminary data indicate that this AFS seeded ANA used for nerve repair results in an improved functional outcome for the rats compared to a nerve autograft, the current gold standard for tension-free repair of transected peripheral nerves. The coming year will utilize these techniques for repairing large-gap (6 cm) nerve injuries in non-human primates. These represent a more translational model of peripheral nerve injury and repair.

Background: Advanced patient age is associated with recurrent tearing and failure of rotator cuff repairs clinically; however, basic science studies have not evaluated the influence of aging on tendon-to-bone healing after rotator cuff repair in an animal model. Hypothesis/Purpose: This study examined the effect of aging on tendon-to-bone healing in an established rat model of rotator cuff repair using the aged animal colony from the National Institute on Aging of the National Institutes of Health. The authors hypothesized that normal aging decreases biomechanical strength and histologic organization at the tendon-to-bone junction after acute repair. Study Design: Controlled laboratory study. Methods: In 56 F344xBN rats, 28 old and 28 young (24 and 8 months of age, respectively), the supraspinatus tendon was transected and repaired. At 2 or 8 weeks after surgery, shoulder specimens underwent biomechanical testing to compare load-to-failure and load-relaxation response between age groups. Histologic sections of the tendon-to-bone interface were assessed with hematoxylin and eosin staining, and collagen fiber organization was assessed by semiquantitative analysis of picrosirius red birefringence under polarized light. Results: Peak failure load was similar between young and old animals at 2 weeks after repair (31% vs 26% of age-matched uninjured controls, respectively; P > .05) but significantly higher in young animals compared with old animals 8 weeks after repair (86% vs 65% of age-matched uninjured controls, respectively; P < .01). Eight weeks after repair, fibroblasts appeared more organized and uniformly aligned in young animals on hematoxylin and eosin slides compared with old animals. Collagen birefringence analysis of the tendon-to-bone junction demonstrated that young animals had increased collagen fiber organization and similar histologic structure compared with age-matched controls (53.7 ± 2.4 gray scales; P > .05). In contrast, old animals had decreased collagen fiber organization and altered structure compared with age-matched controls (49.8 ± 3.1 gray scales; P < .01). Discussion: In a rat model of aging, old animals demonstrated diminished tendon-to-bone healing after rotator cuff injury and repair. Old animals had significantly decreased failure strength and collagen fiber organization at the tendon-to-bone junction compared with young animals. This study implies that animal age may need to be considered in future studies of rotator cuff repair in animal models. Clinical Relevance: With increasing age and activity level of the population, the incidence of rotator cuff tears is predicted to rise. Despite advances in rotator cuff repair technique, the retear rate remains specifically high in elderly patients. The findings of this research suggest that aging negatively influences tendon-to-bone healing after rotator cuff repair in a validated animal model.

This study was designed to detail the history and symptoms of symptomatic radial artery vaso-occlusive disease and to evaluate the results of radial artery reconstruction. Thirteen patients with symptomatic vaso-occlusive disease of the radial artery unresponsive to management by nonoperative modalities were managed with arterial reconstruction. All cases were treated with reversed interpositional vein grafting from the radial artery in the forearm (end-to-side) to the deep arch distally (end-to-end). Patients completed preoperative and postoperative assessments of symptoms and function, cold sensitivity (cold intolerance), and digital microvascular perfusion (isolated cold stress test evaluation with laser Doppler fluxmetry). At the follow-up examination all vascular grafts were patent, as determined by Allen's testing and Doppler ultrasound. The patients reported symptoms and functional status that demonstrated significant improvement following reconstruction. Microvascular evaluations demonstrated a significant improvement in digital microvascular perfusion as assessed by laser Doppler fluxmetry and digital temperature recordings with a resultant resolution of ischemic pain, numbness, and ulceration.

This study assessed the accuracy of arteriography and laser Doppler perfusion imaging (LDPI) as predictors of digital ischemia and correlated upper extremity symptoms, function, and nutritional flow with arteriographic and laser Doppler assessments. Multiple-level occlusive disease was documented in 25 hands (23 patients) by arteriography. LDPI demonstrated one or more hypoperfused digits in 17 hands. Patient questionnaires were used to assess upper extremity symptoms, pain, cold sensitivity, and function. Spearman correlation coefficients indicated that arteriography is a poor indicator of nutritional perfusion as measured by LDPI. Neither arteriography or laser Doppler perfusion imaging served as an indicator of the severity of cold intolerance. However, upper extremity symptoms and functional status correlated with both laser Doppler measurements and the level of cold sensitivity. Laser Doppler perfusion imaging and arteriographic analysis provide complimentary data in the evaluation of upper extremity ischemia.

The role of nitric oxide (NO) in the control of arteriovenous anastomoses (AVAs) has not been studied in vivo in a thermoregulatory end organ. In this study. the effect of local inhibition of NO synthesis by N G -nitro-L-arginine methyl ester (L-NAME) on the microvasculature in the rabbit ear (n = 12) was observed in vivo through a chronically implanted ear microvascular chamber. Ear cutaneous blood perfusion (CBP), total auricular arterial flow (TAF), and ear temperature were monitored simultaneously with the direct microvascular observations. Results revealed that intrafacial artery infusion of L-NAME produced significant vasoconstriction of arterioles, AVAs, and venules (p < 0.05). A decrease of ear blood perfusion also was demonstrated by changes of CBP, TAF, and surface temperature. The data provide evidence that basal generation of NO influences the vascular resistance in the thermoregulatory end organ. Moreover, endogenous NO production may be more important in regulating the AVA flow than is flow in other parts of the rabbit ear microvasculature. The effects of NO inhibition on ear microvasculature were not abolished by superior cervical ganglionectomy, indicating that NO production in the rabbit ear is not a neurally mediated mechanism. Further study with a short-term rabbit ear preparation showed that inhibition of NO production with L-NAME enhanced microvascular constrictive responses to extraluminal application of norepinephrine. NO thus appears to play a role of basal vasodilator in opposition to the basal adrenergic vasoconstrictor tone in the rabbit ear.

The clinical effects of peripheral sympathectomy on patients with vaso-occlusive disease are often dramatic and include relief of pain, improved quality of life, and healing of ulcers. Peripheral periarterial sympathectomy is known to increase skin temperature and to maximize the nutritional component of peripheral blood flow, but the pathophysiology of vaso-occlusive disease and the physiologic mechanisms of this treatment are unknown. In this study, the acute effects of periarterial sympathectomy were directly observed in a rabbit ear model of digital microcirculation (arterioles, arteriovenous anastomoses, and venules). The effects of periarterial sympathectomy on cutaneous perfusion and total flow were also examined using laser Doppler perfusion imaging and digital temperature measurements. The central auricular artery became dilated (50-100%) immediately after sympathectomy; the arterioles, arteriovenous anastomoses, and venules dilated to 165, 156, and 223%, respectively, at 30 minutes and to 187, 174, and 204%, respectively, at 60 minutes, relative to their baseline diameters prior to sympathectomy. Laser Doppler perfusion imaging values and ear temperatures were noted to increase after sympathectomy (8.9%, 3 degrees C), although the core temperature of the rabbit did not change. Thus, acute periarterial sympathectomy can (a) effectively reduce the vascular tone of the distal microvasculature and (b) increase total microcirculatory perfusion-cutaneous and thermoregulatory-by both venular and arteriolar dilation. Periarterial sympathectomy has the clinical potential to increase nutritional blood flow, thereby ameliorating the signs and symptoms of ischemia associated with thermoregulatory abnormalities. Dilation of the arteriovenous anastomoses, with a subsequent reduction in vascular resistance, may contribute to the increased cutaneous temperature noted after sympathectomy.

Vasospastic disorders of the upper extremity are common and often difficult to treat. Using the proposed classification system (Table 2) allows management based upon pathologic condition, physiologic staging, and response to treatment. Identifying patients in this way also helps in determining which treatments are most appropriate. The basic approach to management includes environmental and behavioral modifications including cessation of tobacco use, protection of hands, and avoidance of situations that trigger the vasospastic response. Pharmacologic therapy may provide good results in a majority of patients. Surgical intervention is reserved for patients with vaso-occlusion, ischemia, and refractory symptoms in spite of attempts at medical management. Surgical options include vascular reconstruction, peripheral sympathectomy, or a combination of techniques. The goal of medical and surgical management is to increase total or nutritional blood flow in the digits.