The skeletal muscle microbiopsy method in exercise and sports science research: A narrative and methodological review.

BACKGROUND: The skeletal muscle microbiopsy protocol was introduced to the Exercise and Sports Science (ESS) research field in 1999 and has been used as a protocol to directly examine muscular structural and biochemical changes. There is much variation in the reporting of the microbiopsy protocol and its related pre- and post-procedure for participant care and sample collection. The purpose of this narrative and methodological review is to compare the microbiopsy to the traditional Bergström protocol used in the ESS field, identify and summarize all related microbiopsy protocols used in previous ESS studies and determine the most frequently used microbiopsy protocols aspects and associated pre- and post-biopsy procedures. METHODS: A review of literature up to January 2022 was used following the PRISMA and Cochrane Methodological Review Guide to determine frequently used methods that may facilitate optimal and potential recommendations for muscle microbiopsy needle gauge (G), concentration or dose (% or ml) and administration of local anesthetic, co-axial/cannula introducer gauge (G), muscle depth (cm), muscle sample size collected (mg), passes to collect samples, time points of muscle sampling, and promotion of participant compliance and minimization of adverse events. RESULTS: Eighty-five articles were selected based on the inclusionary requirements related to the ESS field or methodological considerations. The most frequently reported aspects in previous research to suggest the location of the vastus lateralis is the midpoint between the patella and the greater trochanter of the femur or 1/3 or 2/3 the distance from the patella to anterior superior iliac spine, 14 G biopsy needle, subcutaneous injected lidocaine administration (2 ml, 1%), 13 G co-axial/cannula, 1-2 cm muscle depth, 10-20 mg of muscle sample, ~3-time points, and 2-3 passes. DISCUSSION: There is much variation in the reporting of the microbiopsy protocol and its related pre- and post-biopsy procedures. Standardization in reporting may promote recommendations to optimize data integrity, participant safety, participant adherence to the study design, and increase reproducibility. Recommendations are made for the microbiopsy procedure based on frequently reported characteristics.

Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease.

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.

Development of a multiplex assay to determine the expression of mitochondrial genes in human skeletal muscle.

NEW FINDINGS: What is the central question of this study? Can a custom-designed multiplex gene expression assay be used to quantify expression levels of a targeted group of mitochondrial genes in human skeletal muscle? What is the main finding and its importance? A custom-designed GeXP multiplex assay was developed, and the ability to accurately quantify expression of a targeted set of mitochondrial genes in human skeletal muscle was demonstrated. It holds distinct methodological and practical advantages over other commonly used quantification methods. ABSTRACT: Skeletal muscle is an important endocrine tissue demonstrating plasticity in response to external stimuli, including exercise and nutrition. Mitochondrial biogenesis is a common hallmark of adaptations to aerobic exercise training. Furthermore, altered expression of several genes implicated in the regulation of mitochondrial biogenesis, substrate oxidation and nicotinamide adenine dinucleotide (NAD+ ) biosynthesis following acute exercise underpins longer-term muscle metabolic adaptations. Gene expression is typically measured using real-time quantitative PCR platforms. However, interest has developed in the design of multiplex gene expression assays (GeXP) using the GenomeLab GeXP™ genetic analysis system, which can simultaneously quantify gene expression of multiple targets, holding distinct advantages in terms of throughput, limiting technical error, cost effectiveness, and quantifying gene co-expression. This study describes the development of a custom-designed GeXP assay incorporating the measurement of proposed regulators of mitochondrial biogenesis, substrate oxidation, and NAD+ biosynthetic capacity in human skeletal muscle and characterises the resting gene expression (overnight fasted and non-exercised) signature within a group of young, healthy, recreationally active males. The design of GeXP-based assays provides the capacity to more accurately characterise the regulation of a targeted group of genes with specific regulatory functions, a potentially advantageous development for future investigations of the regulation of muscle metabolism by exercise and/or nutrition.

Immunohistochemical and ultrastructural analysis of sporadic inclusion body myositis: a case series.

Sporadic inclusion body myositis (s-IBM) is a progressive, skeletal muscle disease with poor prognosis. However, establishing the final diagnosis is difficult because of the lack of clear biomarkers in the blood serum and very slow development of clinical symptoms. Moreover, most other organs function normally without any disturbance. Here, in patients with this untreatable disease, we have underlined the importance of immunohistochemical and ultrastructural assessment of skeletal muscle in patients diagnosed with s-IBM. The goal of this study was to identify the distribution of specific antigens and to determine morphological features in order to localize pathological protein aggregates, rimmed vacuoles, and loss of myofibrils, which are key elements in the diagnosis of s-IBM. All studied patients were between 48 and 83 years of age and were hospitalized in the Department of Rheumatology and Internal Medicine between 2011 and 2016. Anamneses revealed an accelerated progression of muscle atrophy, weakness of limb muscles, and difficulties with climbing stairs. Based on histopathology and transmission electron microscopy examination, inflammatory infiltrations consisting of mononuclear cells, severe atrophy and focal necrosis of myofibers, splitting of myofilaments, myelinoid bodies and rimmed vacuoles were observed. Primary antibodies directed against CD3, CD8, CD68, cN1A, beta-amyloid, Tau protein and apolipoprotein B made it possible to identify types of cells within infiltrations as well as the protein deposits within myofibers. Using a combination of immunohistochemistry and electron microscopy methods, we were able to establish the correct final diagnosis and to implement a specific treatment to inhibit disease progression.

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

Acid α-glucosidase (GAA) activity and glycogen content in muscle biopsy specimens of patients with Pompe disease: A systematic review.

Pompe disease is a rare genetic disorder characterized by a deficiency of acid α-glucosidase (GAA), leading to the accumulation of glycogen in various tissues, especially in skeletal muscles. The disease manifests as a large spectrum of phenotypes from infantile-onset Pompe disease (IOPD) to late-onset Pompe disease (LOPD), depending on the age of symptoms onset. Quantifying GAA activity and glycogen content in skeletal muscle provides important information about the disease severity. However, the distribution of GAA and glycogen levels in skeletal muscles from healthy individuals and those impacted by Pompe disease remains poorly understood, and there is currently no universally accepted standard assay for GAA activity measurement. This systematic literature review aims to provide an overview of the available information on GAA activity and glycogen content levels in skeletal muscle biopsies from patients with Pompe disease. A structured review of PubMed and Google Scholar literature (with the latter used to check that no additional publications were identified) was conducted to identify peer-reviewed publications on glycogen storage disease type II [MeSH term] + GAA, protein human (supplementary concept), Pompe, muscle; and muscle, acid alpha-glucosidase. A limit of English language was applied. Results were grouped by methodologies used to quantify GAA activity and glycogen content in skeletal muscle. The search and selection strategy were devised and carried out in line with Preferred Reporting of Items in Systematic Reviews and Meta-Analysis guidelines and documented using a flowchart. Bibliographies of papers included in the analysis were reviewed and applicable publications not already identified in the search were included. Of the 158 articles retrieved, 24 (comprising >100 muscle biopsies from >100 patients) were included in the analysis, with four different assays. Analysis revealed that patients with IOPD exhibited markedly lower GAA activity in skeletal muscles than those with LOPD, regardless of the measurement method employed. Additionally, patients with IOPD had notably higher glycogen content levels in skeletal muscles than those with LOPD. In general, however, it was difficult to fully characterize GAA activity because of the different methods used. The findings underscore the challenges in the interpretation and comparison of the results across studies because of the substantial methodological variations. There is a need to establish standardized reference ranges of GAA activity and glycogen content in healthy individuals and in Pompe disease patients based on globally standardized methods to improve comparability and reliability in assessing this rare disease.

Insulin-like growth factor-I biocompartmentalization across blood, interstitial fluid and muscle, before and after 3 months of chronic resistance exercise.

This investigation examined the influence of 12-week ballistic resistance training programs on the IGF-I system in circulation, interstitial fluid, and skeletal muscle, at rest and in response to acute exercise. Seventeen college-aged subjects (11 women/6 men; 21.7 ± 3.7 yr) completed an acute ballistic exercise bout before and after the training program. Blood samples were collected pre-, mid-, and postexercise and analyzed for serum total IGF-I, free IGF-I, and IGF binding proteins (IGFBPs) 1-4. Dialysate and interstitial free IGF-I were analyzed in vastus lateralis (VL) interstitial fluid collected pre- and postexercise via microdialysis. Pre- and postexercise VL muscle biopsies were analyzed for IGF-I protein expression, IGF-I receptor phosphorylation (p-IGF-IR), and AKT phosphorylation (p-AKT). Following training, basal serum IGF-I, free IGF-I, IGFBP-2, and IGFBP-3 decreased whereas IGFBP-1 and IGFBP-4 increased. Training reduced basal dialysate and interstitial free IGF-I but had no effect on basal skeletal muscle IGF-I, p-IGF-IR, or p-AKT. Acute exercise elicited transient changes in IGF-I system concentrations and downstream anabolic signaling both pre- and posttraining; training did not affect this acute exercise response. Posttraining, acute exercise-induced changes in dialysate/interstitial free IGF-I were strongly correlated with the changes in intramuscular IGF-I expression, p-IGF-IR, and p-AKT. The divergent influence of resistance training on circulating/interstitial and skeletal muscle IGF-I demonstrates the importance of concurrent, multiple biocompartment analysis when examining the IGF-I system. As training elicited muscle hypertrophy, these findings indicate that IGF-I's anabolic effects on skeletal muscle are mediated by local, rather than systemic mechanisms.NEW & NOTEWORTHY In the first investigation to assess resistance training's effects on the IGF-I system in serum, interstitial fluid, and skeletal muscle, training decreased basal circulating and interstitial IGF-I but did not alter basal intramuscular IGF-I protein activity. Posttraining, acute exercise-induced interstitial IGF-I increases were strongly correlated with intramuscular IGF-I expression and signaling. These findings highlight the importance of multibiocompartment measurement when analyzing IGF-I and suggest that IGF-I's role in hypertrophic adaptations is locally mediated.

Proximity ligation assay to detect DUX4 protein in FSHD1 muscle: a pilot study.

OBJECTIVE: Aberrant expression in skeletal muscle of DUX4, a double homeobox transcription factor, underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Although previous studies of FSHD muscle biopsies detected mRNAs encoding DUX4 and its target genes, no studies had reported detection of DUX4 protein. Our objective was to develop a proximity ligation assay (PLA) for DUX4 and to determine if this assay could detect DUX4 protein in FSHD muscle sections. RESULTS: We developed a PLA protocol using two DUX4 antibodies previously reported by Stephen Tapscott's group: P2G4, a mouse mAb specific for an epitope in the N-terminal region, and E5-5, a rabbit mAb specific for an epitope in the C-terminal region, in combination with commercial PLA secondary reagents. We validated the DUX4 PLA using cultured human myogenic cells in which DUX4 was ectopically expressed in a small fraction of nuclei. Using this two primary mAb PLA on an FSHD1 biceps biopsy, we observed nuclei with apparent DUX4 PLA signals associated with a small subset of myofibers (~ 0.05-0.1%). Though a limited pilot study, these results suggest that the two primary mAb PLA protocol could be useful for detecting DUX4 protein in FSHD muscle biopsies.

Intramuscular hematoma of the vastus lateralis following percutaneous skeletal muscle microbiopsy: a case report.

Recently, percutaneous microbiopsy needles have been used as a less invasive alternative to the Bergstrom needle for obtaining human skeletal muscle biopsy to assess changes in protein content, gene expression, and enzymatic activities. Unlike the Bergstrom muscle biopsy procedure, potential complications associated with microbiopsies of human skeletal muscle have not been documented. Therefore, the present case report follows a young male's recovery from a muscle biopsy-induced hemorrhage/hematoma of the right vastus lateralis with the specific aims of (1) informing future participants, researchers, and clinicians on expected time course of recovery and (2) informing methods to minimize future participant adverse event risk during and after the percutaneous microbiopsy procedure. The present case report demonstrates that the inadvertent hemorrhaging of a neighboring vessel by percutaneous microbiopsy procedure can be debilitating. To minimize the risk of muscle biopsy-induced hemorrhage/hematoma, we advise post-biopsy compression for up to 15 min and post-biopsy follow-up should be completed for up to 72 h. When there is indication of hematoma development, compression should be applied, and the participant should avoid exercise and physical activity.

Muscle2View, a CellProfiler pipeline for detection of the capillary-to-muscle fiber interface and high-content quantification of fiber type-specific histology.

Because manual immunohistochemical analysis of features such as skeletal muscle fiber typing, capillaries, myonuclei, and fiber size-related parameters is time consuming and prone to user subjectivity, automatic computational methods could allow for faster and more objective evaluation. Here, we developed Muscle2View, a free CellProfiler-based pipeline that integrates all key fiber-morphological variables, including the novel quantification of the capillary-to-fiber interface, in one single tool. Provided that the images are of sufficient quality and the settings are configured for the specific study, the pipeline allows for automatic and unsupervised analysis of fiber borders, myonuclei, capillaries, and morphometric parameters in a fiber type-specific manner from large batches of images in <10 min/tissue sample. The novel identification of the capillary-to-fiber interface allowed for the calculation of microvascular factors such as capillary contacts (CC), individual capillary-to-fiber ratio (C/Fi), and capillary-to-fiber perimeter exchange (CFPE) index. When comparing the Muscle2View pipeline to manual or semiautomatic analysis, overall the results revealed strong correlations. For several variables, however, there were differences (5-15%) between values computed by manual counting and Muscle2View, suggesting that the methods should not necessarily be used interchangeably. Collectively, we demonstrate that the Muscle2View pipeline can provide unbiased and high-content analysis of muscle cross-sectional immunohistochemistry images. In addition to the classical morphological measurements, the Muscle2View can identify the complex capillary-to-fiber network and myonuclear density in a fiber type-specific manner. This robust analysis is done in one single run within a user-friendly and flexible environment based on the free and widely used image software CellProfiler.NEW & NOTEWORTHY Here, we developed a freely available CellProfiler-based pipeline termed Muscle2View, which provides unbiased, high-content analysis of muscle cross-sectional immunohistochemistry images. In addition to fiber typing, myonuclei counting, and the quantification of fiber type-specific morphological measurements, the Muscle2View pipeline can identify the complex capillary-to-fiber network from a batch of images within minutes. Thus, the Muscle2View is a viable tool for researchers aiming to quantify immunohistochemical variables from skeletal muscle biopsies.

Establishment of primary myoblast cell cultures from cryopreserved skeletal muscle biopsies to serve as a tool in related research & development studies.

BACKGROUND: Primary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes. METHODS AND RESULTS: This protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at -80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens. CONCLUSIONS: This protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.

Muscle irisin response to aerobic vs HIIT in overweight female adolescents.

BACKGROUND: Exercise stimulates the production of fibronectin type III domain-containing protein 5 (FNDC5), which is cleaved to release a protein called irisin. This protein induces browning of white adipose tissue resulting in increased thermogenesis. Different studies have measured circulating irisin at baseline and in response to exercise among a wide variety of individuals; yet, regarding the effect of different exercise intensities in obese adolescent girls, limited insight is available. This study compares the effect of acute aerobic exercise of moderate intensity and high-intensity interval training (HIIT) on irisin levels in skeletal muscle and plasma of sedentary overweight or obese female adolescents. METHODS: The aerobic group (n = 15) and HIIT group (n = 15) underwent anthropometric and metabolic measurements, electrocardiogram, peak oxygen uptake (VO2peak), and two vastus lateralis muscle biopsies before and after session of workout. The session of aerobic exercise included cycling at 65% of their peak heart rate (HRpeak) for 40 min. In the HIIT group, exercise included six bouts of 1 min at 85-95% HRpeak separated by 1 min of recovery. Irisin levels were evaluated in samples of skeletal muscle (western blot) and plasma (ELISA). RESULTS: The levels of expression of irisin in skeletal muscle increased significantly after a session of HIIT (p < 0.05), while aerobic exercise no affect irisin levels. No significant differences between the groups in plasma irisin levels were found. CONCLUSIONS: The increase in muscle irisin levels was observed only following HIIT session. No increases in plasma irisin concentration were observed.

Investigating human skeletal muscle physiology with unilateral exercise models: when one limb is more powerful than two.

Direct sampling of human skeletal muscle using the needle biopsy technique can facilitate insight into the biochemical and histological responses resulting from changes in exercise or feeding. However, the muscle biopsy procedure is invasive, and analyses are often expensive, which places pragmatic restraints on sample sizes. The unilateral exercise model can serve to increase statistical power and reduce the time and cost of a study. With this approach, 2 limbs of a participant are randomized to 1 of 2 treatments that can be applied almost concurrently or sequentially depending on the nature of the intervention. Similar to a typical repeated measures design, comparisons are made within participants, which increases statistical power by reducing the amount of between-person variability. A washout period is often unnecessary, reducing the time needed to complete the experiment and the influence of potential confounding variables such as habitual diet, activity, and sleep. Variations of the unilateral exercise model have been employed to investigate the influence of exercise, diet, and the interaction between the 2, on a wide range of variables including mitochondrial content, capillary density, and skeletal muscle hypertrophy. Like any model, unilateral exercise has some limitations: it cannot be used to study variables that potentially transfer across limbs, and it is generally limited to exercises that can be performed in pairs of treatments. Where appropriate, however, the unilateral exercise model can yield robust, well-controlled investigations of skeletal muscle responses to a wide range of interventions and conditions including exercise, dietary manipulation, and disuse or immobilization.

Isolation and Expansion of Muscle Precursor Cells from Human Skeletal Muscle Biopsies.

One of the major issues concerning human skeletal muscle progenitor cells is represented by the efficient isolation and in vitro expansion of cells retaining the ability to proliferate, migrate and differentiate once transplanted. Here we describe a method (1) effective in obtaining human muscle precursor cells both from fresh and frozen biopsies coming from different muscles, (2) selective to yield cells uniformly positive for CD56 and negative for CD34 without FACS sorting, (3) reliable in maintaining proliferative and in vitro differentiative capacity up to passage 10.

Pathological characteristics of glycogen storage disease III in skeletal muscle.

We report a 25-year-old man with glycogenosis III who presented with a progressive 2 year history of fatigue, hand stiffness and cramping. The glycogenoses are a group of rare metabolic disorders which develop as a result of deficiencies in various enzymes involved in the metabolism of glycogen. Some, but not all, glycogenoses, may result in skeletal muscle pathology. Among those that result in vacuolar myopathic changes, glycogen storage disease III or debrancher enzyme deficiency, an autosomal recessive condition, is less commonly encountered than acid maltase (Type II) and myophosphorylase (Type V) deficiencies. Many patients with debrancher enzyme deficiency also have liver involvement. The neurological examination of our patient showed mild proximal limb weakness and decreased reflexes. He had elevated creatine kinase and aldolase levels. He also demonstrated some elevations in his liver function tests, suggesting possible liver involvement. A skeletal muscle biopsy demonstrated vacuolar myopathic changes (acid phosphatase negative) accompanied by focal endomysial fibrosis and chronic inflammation. An ultrastructural examination showed that his vacuoles were filled with glycogen material. An enzyme assay of skeletal muscle tissue showed a significant decrease in debrancher enzyme activity (11% of normal). We review the typical clinical presentation of patients with glycogenosis III and discuss the differential diagnoses of glycogenosis III versus the other glycogenoses resulting in vacuolar myopathy.

Avaliações funcionais e morfométrica de modelo de lesão muscular por agulha de biópsia em gastrocnêmio de ratos / Functional and morphometric evaluations of model of muscle lesion with biopsy needle in gastrocnemius of rats

Introdução: Dentre os modelos experimentais, o uso de agulha de biópsia mostrou ser eficaz para reproduzir as lesões em atletas. Objetivo: Avaliar a força de preensão, a nocicepção e as alterações morfológicas do músculo gastrocnêmio de animais submetidos à lesão muscular. Métodos: Foram utilizados seis ratos Wistar. A lesão foi realizada na junção miotendínea do músculo gastrocnêmio direito, utilizando agulha de biópsia. Para avaliação funcional utilizou-se o teste de força de preensão e avaliação nociceptiva em três momentos: 24 horas antes da lesão (AV1); no segundo dia pós-lesão (AV2) e no sexto dia pós-lesão (AV3). Ao final do experimento o músculo foi retirado para análise morfológica. Resultados: Houve redução da força de preensão entre AV1 e AV2 (p=0,0031). Para a nocicepção os valores de AV2 (p<0,001) e AV3 (p=0,0109) foram maiores que AV1. Na região das fibras musculares observou-se desarranjo do padrão fascicular, aumento do calibre de vasos sanguíneos, da quantidade de tecido conjuntivo, de fibroblastos e de células inflamatórias. Conclusão: A lesão muscular por agulha de biópsia provocou redução da força de preensão e da nocicepção do membro lesado, como também evidenciou as etapas características do processo de degeneração muscular e início do processo de regeneração.

Introduction: Among the experimental models, the use of a biopsy needle was shown to be effective to reproduce lesions that occur with athletes. Objective: To evaluate the grip strength, nociception and morphological alterations of the gastrocnemius muscle of animals submitted to muscle injury. Methods: Six Wistar rats were used. The lesion was performed at the myotendinous junction of the right gastrocnemius muscle using a biopsy needle. For functional evaluation, the grip strength test and nociceptive evaluation were used at three moments: 24 hours before the injury (AV1); on the second postoperative day (AV2) and on the sixth postoperative day (AV3). At the end of the experiment the muscle was removed for morphological analysis. Results: There was a reduction in grip strength between AV1 and AV2 (p = 0.0031). For the nociception the values of AV2 (p < 0.001) and AV3 (p = 0.0109) were higher than AV1. In the muscle fibers region, there was disarrangement of the fascicular pattern, increased blood vessel size, the amount of connective tissue, fibroblasts and inflammatory cells. Conclusion: Muscle lesion by biopsy needle caused reduction of grip strength and nociception of the damaged limb, as well as the characteristic stages of the muscular degeneration process and the beginning of the regeneration process.