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Muscle histochemistry is a technique used to identify the chemical composition of muscle tissue. It involves the use of chemical reactions to produce colours that can be easily visualised under a microscope. This technique is particularly useful in diagnosing muscle diseases and distinguishing between different types of muscle fibres. For example, the myosin ATPase (mATPase) assay can be used to distinguish between fast- and slow-contracting muscle fibres. Muscle biopsies are often used in muscle histochemistry to obtain specimens for analysis. These biopsies can be open biopsy specimens or needle biopsy samples from a moderately affected site. The specimens are then processed using various techniques, such as enzyme histochemistry, immunohistochemistry, and histopathological methods, to identify muscle disorders or defects.
| Characteristics | Values |
|---|---|
| Definition | Histochemistry is a technique used to identify the nature of chemical components within muscle tissue by means of visually observable chemical reactions. |
| Use | Muscle biopsies are useful in diagnosing denervating diseases through myofiber type atrophy or groupings. |
| Types of Assays | Myosin ATPase (mATPase) assay, succinate dehydrogenase (SDH) assay, and a-glycerophosphate dehydrogenase (αGPD) assay. |
| Function of ATPase Assay | Distinguish between fast- and slow-contracting muscle fibers. |
| Function of SDH Assay | Distinguish between oxidative and nonoxidative fibers. |
| Function of αGPD Assay | Distinguish among fibers based on their relative glycolytic potential. |
| Quantitative Analysis | Planimetry can be used as a quantitative method for muscle fibre determination. |
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What You'll Learn
Muscle biopsies
The muscle biopsy procedure can be performed using either an open biopsy or a needle biopsy. The open biopsy method involves making an incision of 30 mm, while the needle biopsy requires only a 5 to 10 mm incision. In both methods, sharp dissection is performed down to the fascia, and a specimen is withdrawn. The needle biopsy technique is preferred in some institutions as it minimises trauma. To obtain sufficient tissue for analysis, the Tarnopolsky suction-modified Bergström technique can be employed, which provides larger tissue samples while maintaining safety.
Once the specimen is obtained, it should be handled and stored appropriately to ensure its quality and suitability for analysis. The muscle tissue should not be immersed in saline, fixative, or other liquids. Instead, it can be saved in saline-moistened gauze for several hours and kept cool. For long-distance transportation, the specimen can be frozen in isopentane pre-cooled to -160°C using liquid nitrogen and shipped overnight with adequate dry ice. The freezing process should be rapid to prevent artefacts, and the frozen muscle should be stored at -80°C.
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Histochemical assays
Histochemistry is a technique used to identify the nature of chemical components within tissues by observing the colour produced by chemical reactions. Histochemical assays are used to determine muscle fibre types.
Myosin ATPase (mATPase) assay
The myosin ATPase assay is one of the three histochemical assays used to determine muscle fibre types. It is used to distinguish between fast- and slow-contracting muscle fibres. The first step of this assay is pre-incubation with either an acid (pH ~4) or basic (pH ~10) solution. Acid pre-incubations inhibit the myosin ATPase activity in fast (mammalian type 2A, 2X, and 2B) fibre types, but not slow (mammalian type 1). Fast-contracting muscle fibres hydrolyse ATP faster than slow-contracting fibres. When given equivalent times, fast-contracting fibres appear dark histochemically, and slow-contracting fibres appear light.
Succinate Dehydrogenase (SDH) assay
The succinate dehydrogenase (SDH) assay is another one of the three histochemical assays used to determine muscle fibre types. It is used to distinguish between oxidative and non-oxidative fibres. The SDH enzyme is located in the inner membrane of the mitochondrion, bound to the cristae. SDH is responsible for oxidising succinate to fumarate in the citric acid cycle. As this reaction proceeds, succinate is oxidised, and the reduced form of NADH is produced. The electron acceptor is chemically reacted with nitro blue tetrazolium (NBT), a purple salt, to visualise the reaction. This results in a speckled pattern of the mitochondria, which is proportional to the number of mitochondria. Oxidative fibres have a relatively dense, purple speckled appearance, while non-oxidative fibres have only scattered purple speckles.
Α-Glycerophosphate Dehydrogenase (αGPD) assay
The α-glycerophosphate dehydrogenase (αGPD) assay is the third of the three histochemical assays used to determine muscle fibre types. The αGPD enzyme is used to distinguish between fibres based on their relative glycolytic potential. Recall that glycolysis is used to generate ATP in the absence of oxygen. The chemical reactions involved in glycolysis take place in the muscle cell cytoplasm (myoplasm). The role of αGPD in glycolysis is to shuttle the NADH that is produced into the mitochondria, where ATP can be produced. It is thus related to glycolytic activity, as the more NADH that can be shuttled into the mitochondrion, the more energy that can be produced.
Applications of Muscle Histochemistry
Muscle biopsies are useful in diagnosing denervating diseases through myofibre type atrophy or groupings. Certain dystrophic fibres are deficient in type 2B fibres, which is also detectable with enzyme histochemistry procedures. Histochemistry and cytochemistry are also important tools for analysing the toxic effects of xenobiotics, as changes in metabolic functions can be directly related to the morphology of a tissue or the type of cell involved.
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Muscle fibre types
Histochemistry is a technique used to identify the chemical composition of tissues by observing the colour changes resulting from chemical reactions. It is often used to study skeletal muscle fibres, which are the long multinucleated cells that make up skeletal muscle tissue. Skeletal muscles are one of the three types of vertebrate muscle tissue, alongside cardiac and smooth muscle.
Skeletal muscle fibres can be classified based on two criteria: the speed of muscle fibre contraction and how fibres regenerate adenosine triphosphate (ATP). Using these criteria, there are three main types of skeletal muscle fibres: slow oxidative (Type I), fast oxidative (Type IIa), and fast glycolytic (Type IIx). Slow oxidative fibres contract slowly and use aerobic respiration (oxygen and glucose) to produce ATP. Fast oxidative fibres contract quickly and also use aerobic respiration to generate ATP. Fast glycolytic fibres also contract quickly but rely on anaerobic glycolysis to produce ATP.
The different types of muscle fibres can be identified through histochemical assays, which produce colour changes that can be easily visualised. The myosin ATPase (mATPase) assay can distinguish between fast- and slow-contracting muscle fibres by inhibiting myosin ATPase activity in fast fibres with an acid preincubation. The succinate dehydrogenase (SDH) assay distinguishes between oxidative and non-oxidative fibres by producing a purple stain, with oxidative fibres having a dense speckled appearance and non-oxidative fibres having scattered speckles. The a-glycerophosphate dehydrogenase (αGPD) assay identifies fibres based on their relative glycolytic potential.
Muscle biopsies are also used to diagnose muscle diseases by examining muscle fibre atrophy or groupings. For example, certain dystrophic fibres deficient in type 2B fibres can be detected with enzyme histochemistry procedures. Resistance exercises can also affect muscle fibres by increasing the formation of actin and myosin, leading to more structured muscle fibres.
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Muscle diseases
Muscle histochemistry is a technique used to identify the nature of chemical components within muscle tissue by means of visually observable chemical reactions. The colour produced by these reactions can be easily visualised. Histochemical reactions can be used to identify muscle diseases.
Muscle biopsies are an essential component of the investigation of patients with neuromuscular disorders. The biopsy technique has been used for over 50 years and has been the chief technique in evaluating muscle biopsies since the 1960s. Specimens are prepared for cryostat sectioning to allow for histochemical investigations. Histochemical techniques on cryostat sections of muscle biopsies provide a better opportunity for defining fibre types and detecting any alterations in muscle fibres.
Muscle biopsies are useful in diagnosing denervating disease through myofibre type atrophy or groupings. Certain dystrophic fibres are deficient in type 2B fibres, which can be detected with enzyme histochemistry procedures. Histopathological techniques are also used in the investigation of muscle diseases.
There are three broad categories of muscle diseases: dystrophic, congenital/structural, and inflammatory myopathies. Histochemistry can be used to identify normal antigenic constituents in skeletal muscle and their loss, accumulation, or maldistribution in corresponding myopathies. For example, histochemistry can be used to identify microvasculature activation in muscle from adult patients with dermatomyositis.
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Muscle structure
Muscle histochemistry is a technique used to identify the nature of chemical components within muscle tissue by means of visually observable chemical reactions. Histochemical reactions produce colour and can, therefore, be easily visualised.
The human body has more than 600 muscles that help us do everything from moving our bodies to breathing and staying alive. The three main types of muscle tissue are skeletal, cardiac, and smooth muscle groups. Skeletal muscle is attached to the bone by tendons, and together they produce all body movements. Each skeletal muscle consists of thousands of muscle fibres wrapped together by connective tissue sheaths. The outermost connective tissue sheath surrounding the entire muscle is known as the epimysium. The connective tissue sheath covering each fasciculus (a bundle of muscle fibres) is known as the perimysium, and the innermost sheath surrounding the individual muscle fibre is known as the endomysium.
Skeletal muscle fibres are crossed with a regular pattern of fine red and white lines, giving the muscle a distinctive striated appearance. When bundled together, all the myofibrils are arranged in a unique striated pattern forming sarcomeres, which are the fundamental contractile units of a skeletal muscle. The two most significant myofilaments are actin and myosin filaments, which are arranged distinctively to form various bands on the skeletal muscle.
Histochemical assays are used to determine muscle fibre types and distinguish between fast- and slow-contracting muscle fibres. The three histochemical assays typically used are the myosin ATPase (mATPase) assay, the succinate dehydrogenase (SDH) assay, and the a-glycerophosphate dehydrogenase (αGPD) assay.
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Frequently asked questions
Muscle histochemistry is a technique used to identify the nature of chemical components within muscle tissue.
The chemical reactions used in muscle histochemistry produce colours that can be easily visualised.
There are three types of muscle histochemistry: myosin ATPase (mATPase) assay, succinate dehydrogenase (SDH) assay, and a-glycerophosphate dehydrogenase (αGPD) assay.
Muscle histochemistry has a variety of applications, including diagnosing denervating diseases, identifying different muscle fibre types, and distinguishing between fast- and slow-contracting muscle fibres.
Muscle histochemistry is not routinely performed in many laboratories. It is also limited in differentiating certain types of carcinomas and may require fresh frozen tissue samples for analysis.
