Muscle Biopsy for Diagnosis of Neuromuscular and Metabolic Diseases

Histological Examination

Hematoxylin and eosin (HE) stain has been the most universal and traditional method for examination of formalin-fixed, paraffin-embedded tissue sections of all tissues for more than a century, and its application to cryostat sections of the muscle biopsy is no exception. The amount of diagnostic information yielded by histological examination alone is limited, but it can detect the presence of inflammation; foreign tissues including Trichinella and other parasites; neoplasia, both primary and metastatic; myofiber necrosis; perimysial and endothelial connective tissue proliferation; and patterns of distribution of atrophic myofibers. Subcellular structural abnormalities, such as ragged-red fibers, are subtler and harder to see than with histochemical stains and some, such as nemaline rods, are almost impossible to detect because of lack of color contrast.

Histochemical Examination

The purpose of histochemistry is to distinguish myofiber types and subtypes, certain metabolic products such as glycogen and neutral lipids, and many subcellular abnormalities not well seen with HE. Histochemical studies are traditionally performed on freshly frozen sections only, though some enzymatic activities and fiber typing using myosin and actin antibodies can also now be demonstrated in paraffin sections by immunocytochemistry. The most useful and frequently applied histochemical stains are:

• (1)

  Modified Gomori trichrome. The chromotrope-2 R component of this stain has a high affinity for phospholipids and is perceived as a red color; mitochondrial and endoplasmic reticulum membranes have high phospholipid content in contrast with most plasma membranes that have a higher ratio of glycolipids. In normal myofibers in transverse section, a thin subsarcolemmal rim of red staining may be seen because of mitochondrial concentration, particularly evident at motor endplates. The intermyofibrillar sarcoplasm appears as a red reticular network because of mitochondria and endoplasmic reticulum against the green staining of myofibrils from the fast green FCF component. The third component of this trichrome stain is hematoxylin, which stains nuclear chromatin (DNA) purple. This stain is ideal for demonstrating ragged-red fibers of some mitochondrial cytopathies. Chromotrope-2 R also stains tropomyosin, so nemaline rods arising from the Z-band also are well demonstrated. Myelin sheaths of normal intramuscular nerve twigs are also intensely red stained.

• (2)

  Myofibrillar calcium-mediated adenosine triphosphatase (ATPase) stains. These stains are highly reliable for distinguishing fiber types and subtypes, depending on the pH of preincubation. At alkaline pH (usually 9.4 to 10.2), type II fibers are dark and type I fibers are light. At acid pH (usually 4.6–4.3), the reverse is true, and subtypes of II can be distinguished. Types of intrafusal myofibers of muscle spindles differ from those of extrafusal fibers. These stains are very helpful for those myopathies showing type-specific atrophies. In chronic neurogenic atrophy with many cycles of denervation and reinnervation, a phenomenon of histochemical type-grouping is seen, in which the normal intermixed checkerboard distribution of types I and II myofibers is replaced by small and large groups of myofibers, all of the same type, adjacent to other groups of the opposite type. Some groups may be so large as to occupy an entire fascicle.

• (3)

  Respiratory chain complexes, or “oxidative mitochondrial enzymes.” The three most relevant stains include nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR; respiratory complex I), succinate dehydrogenase (SDH; complex II) and cytochrome c oxidase (COX; complex IV). These activities are greatly increased in ragged-red zones. In many mitochondrial diseases, scattered myofibers that are otherwise well preserved and not degenerating show loss of COX activity, even if complex IV is not selectively impaired. One caution is that the intensity of histochemical activity of COX is generally lower than either NADH-TR or SDH, particularly in type II myofibers, hence normally low activity should not be misinterpreted as pathologically absent. Modification of the SDH stain by the addition of phenazine methosulfate is useful because this compound suppresses activity in normal mitochondria but does not affect SDH expression in abnormal mitochondria. Because complex II has only four subunits, all encoded by nDNA, it remains reactive in all mitochondrial diseases regardless of any mtDNA point mutations. The other four complexes include a combination of mtDNA and nDNA encoded subunits. These mitochondrial enzymes also are markers of fiber types, showing more intense activity in type I fibers than those of type II, but caution must be exercised in interpretation because severely atrophic myofibers of either type show intense activity due to loss of myofibrils and concentration of sarcoplasmic mitochondria. NADH-TR marks not only mitochondria but sarcoplasmic (i.e. endoplasmic) reticulum as well, unlike SDH and COX, which are pure mitochondrial stains.

• (4)

  Acid phosphatase. This stain is useful for showing abnormal lysosomal activity in diseases such as infantile (Pompe) and adult acid maltase deficiencies. It appears as granular material within the myofiber. Nonspecific esterase can used as an alternative. Alkaline phosphatase is not a suitable alternative.

• (5)

  Oil Red O and Sudan Black B. These stains are used to demonstrate neutral lipids within myofibers and in the connective tissue, but complex phospho- and glycolipids, such as sphingomyelin and galactocerebroside, are not stained.

• (6)

  Periodic acid-Schiff (PAS) reaction. Increased intramuscular glycogen occurs in some glycogenoses, but may also represent a physiological condition of a patient who is receiving good nutrition, even parenterally for an extended time, and who has little physical activity to utilize the glycogen that accumulates in myofiber, as in children in intensive care units. PAS also is good for demonstrating peripheral nerve myelin in both cryostat and paraffin sections.

Immunocytochemical Reactivities

These techniques are usually applied to frozen cryostat sections, but also may be applied to paraffin-embedded sections, except for dystrophin. They can demonstrate features not shown by the routine histochemical battery of stains, including sarcolemmal-region proteins both of the sarcolemma itself and those that attach the sarcolemma to the basal lamina (basement membrane). They are highly specific for many muscular dystrophies, such as the dystrophinopathies, merosin deficiency, and dystroglycanopathies. Other immunoreactivities can substitute for histochemistry in distinguishing fiber types if only formalin-fixed, paraffin-embedded sections are available, for example heavy- and light-chain (“slow” and “fast”) myosin immunoreactivities that provide similar information as myofibrillar ATPase stains. Immunocytochemistry can also distinguish types of inflammatory cells, such as T-lymphocytes (CD-43), B-lymphocytes (CD-20), and macrophages (CD-68). Endothelial cells of capillaries and other small intramuscular blood vessels are well demonstrated by vimentin and nestin immunoreactivities, or by the more specific CD-31 and CD-34 antibodies.

Silver Impregnations

This group is useful for showing axons in peripheral nerve, including intramuscular nerves, and includes variations of technique such as those of Bielschowsky, Bodian, Sevier-Munger, and others. Reticulin stains demonstrate reticulin fibers around intramuscular capillaries.

Transmission Electron Microscopy (EM)

Since the 1950s, EM has provided ultrastructural insight into the subcellular anatomy of myofibers ( Figure 4.1 ) and of aberrations in many myopathies, to both confirm and verify the interpretation of histochemical stains and to offer additional detail at a resolution not possible by light microscopy. In recent years, it has been used more sparsely and selectively because of its labor intensity for both technologists and pathologists and because of cost, including the maintenance of the equipment. Special tissue preparation is required, with fixation in glutaraldehyde, post-fixation in osmium tetroxide, and staining with uranyl acetate and lead citrate. Ultrafine sections must be cut on special diamond knife blades. EM can, however, demonstrate many features of diagnostic importance if used for selective muscle biopsies. Nemaline rods and disarrays of myofilaments are well shown. One of its most important contemporary applications is to demonstrate ultrastructural changes in mitochondrial cytopathies. There are several mitochondrial alterations, including simplification of the cristae, stacked and whorled cristae, increased numbers of osmophilic spheroids within the mitochondrial matrix and paracrystalline structures. The latter are an array of geometrical crystal-like structures within the mitochondria and are a highly specific finding. Within myofibers, paracrystalline structures (sometimes called “inclusions,” though this is incorrect because they are not foreign particles) often assume a brick shape with sharp corners and elongation. The ultrastructural periodicity of these structures is constant and predictable; these structures are in the space between the cristae and are made of creatine kinase. Often, fragments of preserved cristae can also be seen within the same mitochondrion that has a paracrystalline structure, confirming their location as intramitochondrial. Endothelial cells of intramuscular capillaries in young infants with mitochondrial disease also contain paracrystalline-like structures suggestive of intramitochondrial location but without preserved cristae. Normal Weibel-Palade granules of endothelial cells that contain von Willebrand factor V consist of parallel microtubules that, on oblique or longitudinal sections, have a repetitive pattern reminiscent of a crystal.

Biochemical Assay of Respiratory Chain Complexes

The measurement of specific activities of each of the five respiratory chain complexes, both individually and in combination (e.g. complexes 1+III), may be performed from homogenates of frozen tissue and provides quantitative numbers. Citrate synthetase is usually examined as a control because if it is decreased, the other specific activities may be unreliable due to delayed freezing of the tissue. At times, one may find convincing histochemical and ultrastructural evidence of mitochondrial disease, associated with normal activities of the complexes, particularly in infants. This anomaly is due to the measurement of an average of the muscle tissue homogenate rather than an examination of individual myofibers. If only a few scattered myofibers are affected, they may be diluted by the normal activities of the majority of myofibers. Interpretation of these assays must be made cautiously, and clinicians must understand that normal quantitative assay results do not necessarily exclude mitochondrial disease. Similarly, decreased specific activity in one complex may be an artifactual result and, in the absence of supportive evidence in other aspects of the muscle biopsy, should not be taken as conclusive for mitochondrial cytopathy. Diminished activity in all or most complexes with normal citrate synthetase activity is a primary criterion of the “mitochondrial depletion syndrome” of infancy, a genetically heterogeneous group with many clinical phenotypical features in common.

Molecular Genetic Studies

Muscle tissue can be used for molecular genetic studies of many muscular dystrophies. It is also useful in showing both mitochondrial DNA (mtDNA) point mutations and nuclear DNA (nDNA) mutations or deletions in mitochondrial disorders. Ideally, the tissue should be freshly frozen for such studies, but DNA extraction can also be made from formalin-fixed tissue, though this is less reliable. Details of techniques and discussion of the many mutations that have been demonstrated in mitochondrial diseases are beyond the scope of this chapter.