Mini invasive skeletal muscle biopsy technique with a tri-axial end cut needle.

OBJECTIVE: Skeletal Muscle Biopsy is a minor surgical procedure for the diagnosis of different neuromuscular pathological conditions and has recently gained popularity also in the research field of age-related muscular modifications and sarcopenia. Few studies focused on the application of mini-invasive muscular biopsy in both normal and pathological conditions. The aim of our study was to describe a mini invasive ultrasound-guided skeletal muscular biopsy technique in complete spinal cord injured (SCI) patients and healthy controls with a tri-axial end-cut needle. PATIENTS AND METHODS: Skeletal muscle biopsies were collected from 6 chronic SCI patients and 3 healthy controls vastus lateralis muscle with a tri-axial end cut needle (Biopince© - Angiotech). Muscle samples were stained for ATPase to determine fibers composition, moreover, gene expression of cyclooxygenase-1 (COX-1) and prostaglandin E2 receptor has been analyzed by Real Time RT-PCR. RESULTS: All the procedures were perfomed easily without failures and complications. Control tissue was macroscopically thicker than SCI one. Control specimen displayed an equal distribution of type I and type II fibers, while SCI sample displayed a prevalence of type II fibers SCI specimen displayed a significant reduction in COX-1 gene expression. This mini-invasive approach was easy, accurate and with low complication rate in performing skeletal muscle biopsy in both SCI patients and controls. CONCLUSIONS: This technique could be useful in conditions in which the overall quantity of specimen required is small like for molecular biology analysis. For histological diagnostic purposes and/or conditions in which the original tissue is already pathologically modified, this technique should be integrated with more invasive techniques.

Serum activity of creatine kinase and aminotransferase aspartate enzymes of horses submitted to muscle biopsy and incremental jump test

The objective was to evaluate serum activity of the enzymes creatine kinase (CK) and aspartate aminotransferase (AST), which are leakage enzymes responsive to muscle injury, of athletic horses that underwent muscle biopsy and incremental jump test (IJT) involving incremental jumps. The animals were grouped as follows: the first group, horses with history of superior performance (SP); the second, with a history of inferior performance (IP); and lastly, a control group (CG). All groups underwent biopsy of the gluteus medius muscle, while groups SP and IP were also submitted to the incremental jump test (IJT) 24 hours after biopsy. The IJT consisted of three stages with 40 jumps each, where jump height increased progressively, from 40 to 60 and last, 80cm. Blood samples were drawn before biopsy, and 6 and 24 hours after the exercise as well. The levels of CK serum activity increased 6 hours after exercise and decreased 24 hours later in all groups, including CG. AST activity did not increase after biopsy and exercise. There was no increase of both enzyme activities that could be attributed to the exercise, possibly due to exercise short duration and/or low intensity. We conclude that the muscle biopsy was able to show that there was enough stimulus to cause CK enzyme leakage into the plasma, and consequent detection of increased serum activity, while the incremental jump test did not.

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.

MHC class I and II expression in juvenile dermatomyositis skeletal muscle

Objective To assess MHC I and II expressions in muscle fibres of juvenile dermatomyositis (JDM) and compare with the expression in polymyositis (PM), dermatomyositis (DM) and dystrophy. Patients and methods Forty-eight JDM patients and 17 controls (8 PM, 5 DM and 4 dystrophy) were studied. The mean age at disease onset was 7.1 +/- 3.0 years and the mean duration of weakness before biopsy was 9.4 +/- 12.9 months. Routine histochemistry and immunohistochemistry (StreptABComplex/HRP) for MHC I and II (Dakopatts) were performed on serial frozen muscle sections in all patients. Mann-Whitney, Kruskal Wallis, chi-square and Fisher`s exact statistical methods were used. Results MHC I expression was positive in 47 (97.9%) JDM cases. This expression was observed independent of time of disease corticotherapy previous to muscle biopsy and to the grading of inflammation observed in clinical, laboratorial and histological parameters. The expression of MHC I was similar on JDM, PM and DM, and lower in dystrophy. On the other hand, MHC II expression was positive in just 28.2% of JDM cases was correlated to histological features as inflammatory infiltrate, increased connective tissue and VAS for global degree of abnormality (p < 0.05). MCH II expression was similar in DM/PM and lower in JDM and dystrophy, and it was based on the frequency of positive staining rather than to the degree of the MCH II expression. Conclusions MHC I expression in muscle fibres is a premature and late marker of JDM patient independent to corticotherapy, and MHC II expression was lower in JDM than in PM and DM.

Myoelectric manifestations of sternocleidomastoid and anterior scalene muscle fatigue in chronic neck pain patients

Objective: This study compares myoelectric manifestations of fatigue of the sternocleidomastoid (SCM) and anterior scalene (AS) muscles between 10 chronic neck pain subjects and 10 normal matched controls. Methods: Surface electromyography (sEMG) signals were recorded from the sternal bead of SCM and AS muscles bilaterally during submaximal isometric cervical flexion contractions at 25 and 50% of the maximum voluntary contraction (MVC). The mean frequency, average rectified value and conduction velocity of the sEMG signal were calculated to quantify myoelectric manifestations of muscle fatigue. Results: For both the SCM and AS muscles, the Mann-Whitney U test indicated that the initial value and slope of the mean frequency in neck pain patients were greater than in healthy subjects (P < 0.05). This was significant both at 25 and 50% of MVC. Conclusions: These results suggest: (a) a predominance of type-II fibres in the neck pain patients and/or (b) greater fatigability of the superficial cervical flexors in neck pain patients. These results are in agreement with previous muscle biopsy studies in subjects with neck pain, which identified transformation of slow-twitch type-I fibres to fast-twitch type-IIB fibres, as well as the clinical observation of reduced endurance in the cervical flexors in neck pain patients. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Dominant and Recessive RYR1 Mutations in Adults with Core Lesions and Mild Muscle Symptoms

INTRODUCTION: Ryanodine receptor gene (RYR1) mutations have been associated with central core disease (CCD), multiminicore/minicore/multicore disease (MmD), and susceptibility to malignant hyperthermia (MH). METHODS: Patients with muscle symptoms in adulthood, who had features compatible with CCD/MmD, underwent clinical, histological, and genetic (RYR1 and SEPN1 genes) evaluations. Published cases of CCD and MmD with adult onset were also reviewed. RESULTS: Eight patients fulfilled the criteria for further analysis. Five RYR1 mutations, 4 of them unreported, were detected in 3 patients. Compound heterozygosity was proven in 1 case. CONCLUSIONS: To our knowledge, this is the only report of adult onset associated with recessive RYR1 mutations and central core/multiminicores on muscle biopsy. Although adult patients with CCD, MmD, and minimally symptomatic MH with abnormal muscle biopsy findings usually have a mild clinical course, differential diagnosis and carrier screening is crucial for prevention of potentially life-threatening reactions to general anesthesia.

The relationship between monocarboxylate transporters 1 and 4 expression in skeletal muscle and endurance performance in athletes

The purpose of this study was to examine the relationship between skeletal muscle monocarboxylate transporters 1 and 4 (MCT1 and MCT4) expression, skeletal muscle oxidative capacity and endurance performance in trained cyclists. Ten well-trained cyclists (mean +/- SD; age 24.4 +/- 2.8 years, body mass 73.2 +/- 8.3 kg, VO(2max) 58 +/- 7 ml kg(-1) min(-1)) completed three endurance performance tasks [incremental exercise test to exhaustion, 2 and 10 min time trial (TT)]. In addition, a muscle biopsy sample from the vastus lateralis muscle was analysed for MCT1 and MCT4 expression levels together with the activity of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD). There was a tendency for VO(2max) and peak power output obtained in the incremental exercise test to be correlated with MCT1 (r = -0.71 to -0.74; P < 0.06), but not MCT4. The average power output (P (average)) in the 2 min TT was significantly correlated with MCT4 (r = -0.74; P < 0.05) and HAD (r = -0.92; P < 0.01). The P (average) in the 10 min TT was only correlated with CS activity (r = 0.68; P < 0.05). These results indicate the relationship between MCT1 and MCT4 as well as cycle TT performance may be influenced by the length and intensity of the task.

Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase) used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50) and alkaline incubation (pH=10.50), at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue), type IIA (oxidative-glycolytic, intermediate blue) and type IIX (glycolytic, dark blue). There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding) and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

Preliminary study on age- and sex-dependent alterations in the composition of skeletal muscle fibers of Brasileiro de Hipismo horses

The aim of this work was to characterize the distribution of myofibers in the gluteus medius muscle of inactive horses of the Brasileiro de Hipismo (BH) breed at different ages by means of histochemical analyses, according to sex and depth of the biopsy. A total of 78 inactive horses (9 castrated males, 35 stallions, and 34 females) of the BH breed, aged 1 to 4 years, were used. A percutaneous muscle biopsy was obtained with a 6.0-mm Bergstrom-type needle, which allowed the removal of muscle fragments at depths of 20 and 60 mm. Myofiber types were determined based on myofibrillar adenosine triphosphatase (mATPase) and nicotinamide dinucleotide tetrazolium reductase (NADH-TR) techniques. Morphometry of the fibers was determined based on cross-sectional area (CSA), mean frequency (F), and relative cross-sectional area (RCSA). The current study demonstrated that BH horses 3 and 4 years of age show a greater percentage of, and area occupied by, type IIA fibers and lower percentage of type IIX fibers in the gluteus medius muscle compared with horses 1 and 2 years of age. No difference was found between sexes in the frequency of and area occupied by the different fiber types at any of the depths and ages studied. In this study, females showed a greater CSA for all fibers in comparison with males, at 1 year of age. The results of the current study indicate that the gluteus medius muscle of inactive BH horses shows modifications in its structural and biochemical composition during the growth of the animals, leading to a better oxidative capacity.

Aerobic training, but not creatine supplementation, alters the gluteus medius muscle

The aim of the present study was to investigate the effect of oral supplementation of creatine on the muscular responses to aerobic training. Twelve purebred Arabian horses were submitted to aerobic training for 90 d, with and without creatine supplementation, and evaluated with respect to BW and BCS and to the area and frequency of the different types of muscle fibers in the gluteus medius. Supplementation consisted of the daily administration of 75 g of creatine monohydrate mixed into the ration for the 90 d of training. Physical conditioning was conducted on a high-performance treadmill, and training intensity was stipulated by calculating the velocity at which blood lactate reaches 4 mmol/L, determined monthly for each animal. The individual intensity of physical force at 80% of aerobic threshold was established. Morphometry of glutens medius muscle fibers was performed on frozen sections processed for histochemical analysis of myosin adenosine triphosphatase and immunohistochemistry of slow-contracting myosin. The results demonstrated that the animals maintained a moderate BCS without alteration of BW during the course of training, providing evidence of equilibrium between food intake and caloric expenditure during the study period. The present study demonstrated that aerobic training for 90 d caused hypertrophy of fiber types I (P = 0.04), IIA (P = 0.04), and IIX (P = 0.01), as well as an increase in the relative area occupied by type I fibers (P = 0.02) at the expense of type IIX fibers (P = 0.03), resulting in modifications of the contractile and metabolic characteristics of the gluteus medius muscle. It was not possible to show any beneficial effect from creatine on the skeletal muscle characteristics examined.

Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Skeletal muscle major histocompatibility complex class I and II expression differences in adult and juvenile dermatomyositis

OBJECTIVE: To analyze major histocompatibility complex expression in the muscle fibers of juvenile and adult dermatomyositis. METHOD: In total, 28 untreated adult dermatomyositis patients, 28 juvenile dermatomyositis patients (Bohan and Peter's criteria) and a control group consisting of four dystrophic and five Pompe's disease patients were analyzed. Routine histological and immunohistochemical (major histocompatibility complex I and II, StreptoABComplex/HRP, Dakopatts) analyses were performed on serial frozen muscle sections. Inflammatory cells, fiber damage, perifascicular atrophy and increased connective tissue were analyzed relative to the expression of major histocompatibility complexes I and II, which were assessed as negatively or positively stained fibers in 10 fields (200X). RESULTS: The mean ages at disease onset were 42.0 +/- 15.9 and 7.3 +/- 3.4 years in adult and juvenile dermatomyositis, respectively, and the symptom durations before muscle biopsy were similar in both groups. No significant differences were observed regarding gender, ethnicity and frequency of organ involvement, except for higher creatine kinase and lactate dehydrogenase levels in adult dermatomyositis (p<0.050). Moreover, a significantly higher frequency of major histocompatibility complex I (96.4% vs. 50.0%, p<0.001) compared with major histocompatibility complex II expression (14.3% vs. 53.6%, p = 0.004) was observed in juvenile dermatomyositis. Fiber damage (p = 0.006) and increased connective tissue (p<0.001) were significantly higher in adult dermatomyositis compared with the presence of perifascicular atrophy (p<0.001). The results of the histochemical and histological data did not correlate with the demographic data or with the clinical and laboratory features. CONCLUSION: The overexpression of major histocompatibility complex I was an important finding for the diagnosis of both groups, particularly for juvenile dermatomyositis, whereas there was lower levels of expression of major histocompatibility complex II than major histocompatibility complex I. This finding was particularly apparent in juvenile dermatomyositis.

Incidence of adverse events associated with percutaneous muscular biopsy among healthy and diseased subjects

The development of the percutaneous muscle biopsy technique is recognized as one of the most important scientific contributions in advancing our understanding of skeletal muscle physiology. However, a concern that this procedure may be associated with adverse events still exists. We reported the incidence of adverse outcomes associated with percutaneous muscle biopsy in healthy and diseased subjects. Medical records of 274 volunteers (496 muscle biopsies) were reviewed. This included 168 healthy subjects (330 muscle biopsies) as well as 106 chronically ill patients (166 muscle biopsies). This latter group encompassed patients with type II diabetes (n=28), osteoarthritis (n=39), inclusion body myositis (n=4), polymyositis (n=4), and chronic heart failure (n=31). The most common occurrences were pain (1.27%), erythema (1.27%), and ecchymosis (1.27%). Panic episode, bleeding, and edema were also reported (0.21%, 0.42%, and 0.84%, respectively), while infection, hematoma, inflammation, denervation, numbness, atrophy, and abnormal scarring were not verified. The percent of incidents did not differ between healthy and ill individuals. In conclusion, the incidence of complications associated with percutaneous muscle biopsy is scarce and of minor clinical relevance. Additionally, the rate of adverse events is comparable between healthy and chronically ill subjects.

Adipogenic and myogenic gene expression in rotator cuff muscle of the sheep after tendon tear

Chronic rotator cuff tendon tears lead to fatty infiltration and muscle atrophy with impaired physiological functions of the affected muscles. However, the cellular and molecular mechanisms of corresponding pathophysiological processes remain unknown. The purpose of this study was to characterize the expression pattern of adipogenic (PPARgamma, C/EBPbeta) and myogenic (myostatin, myogenin, Myf-5) transcription factors in infraspinatus muscle of sheep after tenotomy, implantation of a tension device, refixation of the tendon, and rehabilitation, reflecting a model of chronic rotator cuff tears. In contrast to human patients, the presented sheep model allows a temporal evaluation of the expression of a given marker in the same individual over time. Semiquantitative RT/PCR analysis of PPARgammaã, myostatin, myogenin, Myf-5, and C/EBPbeta transcript levels was carried out with sheep muscle biopsy-derived total RNA. We found a significantly increased expression of Myf-5 and PPARgamma after tenotomy and a significant change for Myf-5 and C/EBPbeta after continuous traction and refixation. This experimental sheep model allows the molecular analysis of pathomechanisms of muscular changes after rotator cuff tear. The results point to a crucial role of the transcription factors PPARgamma, C/EBPbeta, and Myf-5 in impairment and regeneration of rotator cuff muscles after tendon tears in sheep.