Oxidative Enzymes

The most useful oxidative enzymes studied in muscle are reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH) and cytochrome c oxidase (COX).

The principle of the histochemical technique for NADH-TR and SDH is to employ a colourless, soluble tetrazolium salt as an electron acceptor which is reduced to a deeply coloured, insoluble formazan product at the site of the enzyme activity. The commonly used tetrazolium salt is nitroblue tetrazolium (NBT) [2,2′-di- p -nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-biphenylene) ditetrazolium chloride], which gives a bluish final end product. Thus the intensity of the formazan reaction product is a reflection of the number of mitochondria within a fibre and reveals the characteristic checkerboard pattern of fibre types. Some caution in interpretation, however, is needed with regard to specificity with the techniques for NADH-TR and SDH because tetrazolium salts have a strong affinity for phospholipids and with the reaction for NADH-TR the sarcoplasmic reticulum is also revealed. This can, however, be advantageous as the technique for NADH-TR is useful for showing disruption and distortion of myofibrils and the internal structure of whorled fibres (see Ch. 4 ). The technique for SDH, in contrast, is specific for mitochondria, as is the technique for COX.

COX is very sensitive to fixation and is inhibited by cyanide and azide. Even brief fixation in formaldehyde, glutaraldehyde or alcohol can produce negative results in the histochemical reaction, emphasizing the need for frozen sections. It is an integral component of the mitochondrial membrane and is encoded by mitochondrial DNA. Succinate dehydrogenase, in contrast, is encoded by nuclear DNA. The method commonly used to demonstrate COX activity uses diaminobenzidine as an electron donor and produces a brown end product that can be enhanced by osmium. The reaction for COX reveals differences in mitochondrial number and their distribution in different fibre types (see Ch. 3 ). It is also an important method for demonstrating fibres devoid of activity caused by certain mutations in mitochondrial DNA. A combination of the technique for COX and SDH provides a clear method for identifying fibres that are deficient in COX but retain SDH activity, as they appear blue in contrast to the brownish-blue/grey of normal fibres.

Transferases

Hydrolases

Additional Enzyme Studies

Additional methods that may be useful in association with certain clinical features are included in the list of methods. Although several of these formed part of a routine set of procedures in the early years of muscle pathology, they only add additional diagnostic information in certain situations. With increasing awareness of subtle changes associated with some genetic defects, however, some are of value.

Acid phosphatase is localized mainly in lysosomes and may thus be used to indicate foci of degeneration and necrosis within muscle fibres. Very little is apparent in normal muscle fibres, except in perinuclear regions where it is seen as focal deposits associated with lipofuscin. Lipofuscin is more abundant in muscle from adults than from children and there may therefore appear to be more perinuclear acid phosphatase activity in adults. In type II glycogenosis and lysosomal disorders, acid phosphatase is useful as it demonstrates subtle increases and the activity associated with the vacuoles. It also highlights the presence of macrophages. Acid phosphatase activity is also abundant in vitamin E deficiency and Batten disease, and the deposits are autofluorescent. The colour of the autofluorescence can be used to distinguish the two types of deposit as in vitamin E deficiency they are orange–yellow but yellow in Batten disease.

Alkaline phosphatase is found primarily in cell membranes where active transport processes occur, such as the endothelium of arterioles and the arterial part of capillaries, and also in endoplasmic reticulum, Golgi apparatus and pinocytotic vesicles. The reaction is usually negative in muscle fibres but may be positive in focal necrotic fibres in various disease situations, and in some regenerating or non-innervated fibres. Its major use is in the assessment of inflammatory myopathies when perimysial areas may be intensely stained.

Phosphofructokinase may be useful to study if a glycogenosis is suspected, but only a result of total absence can be relied on. A deficiency is difficult to access histochemically and requires biochemical analysis.

Menadione-linked α -glycerophosphate dehydrogenase reveals a fibre type pattern with type 2 fibres more intensely stained than type 1, but is of particular diagnostic value in distinguishing reducing bodies. These, and the abnormal accumulation of myofibrillar material in some myofibrillar myopathies (see Ch. 16 ), as well as some unusual granular structures observed in acid maltase deficiency ( ), are the only abnormal structures to stain with this technique, even without substrate. Tubular aggregates can also show a slight degree of staining but are more easily identified by other stains. As the occurrence of these structures is very rare, this technique is often not included in a routine panel. In this technique the menadione is reduced by sulphydryl-containing structures.

Staining for myoadenylate deaminase is favoured by some, as a deficiency may be the only feature of note in some patients. Interpretation of the significance of a deficiency is hampered, however, by the presence of a common polymorphism in the normal population that obliterates the enzyme. A secondary reduction in enzyme activity may also occur for unknown reasons. Abundant tubular aggregates are also revealed by the reaction, even without substrate.

Acetylcholinesterase highlights areas with high cholinesterase activity, such as neuromuscular junctions. Myotendinous junctions also stain, but the reason is not known. It is also useful for studies of the vacuoles in the X-linked myopathy with excess autophagy vacuoles (XMEA).

Non-specific esterase also stains neuromuscular and myotendinous junctions and, similar to acid phosphatase, highlights phagocytic areas. It also stains small denervated fibres. A two-fibre pattern may also sometimes be seen.

Glycogen

The periodic acid-Schiff (PAS) stain, which has a very long history in histochemistry, is frequently used to demonstrate glycogen in muscle. It is worth bearing in mind, however, that not only glycogen but also other polysaccharides, as well as neutral mucopolysaccharides, muco- and glycoproteins, glycolipids and some unsaturated lipids and phospholipids, are stained with this reaction. The glycogen is demonstrated with Schiff reagent (fuchsin-sulphurous acid), which produces a reddish-purple stain and shows a fibre type pattern. The specificity of the PAS reaction for glycogen may be checked by using α-amylase digestion, and the use of celloidin helps to retain the glycogen. Although glycogen storage may be rare, the PAS technique is also useful in revealing damaged and some denervated fibres in several disorders as these may be devoid of glycogen and appear white.

Neutral Lipid

Neutral lipid can be demonstrated in normal muscle and takes the form of small droplets with a distribution similar to that of mitochondria. It can be demonstrated with the Sudan black or oil red O technique. Nile red is also favoured by some workers and is fluorescent in the presence of high lipid ( ). The concentration and size of the droplets vary with the fibre type and this must be taken into consideration in interpretation. Membranous areas high in phospholipids, such as those with high numbers of mitochondria, are also highlighted by Sudan black and Nile red but are not apparent with oil red O. In disorders affecting lipid metabolism, the excessive accumulation of lipid shows up as larger and more extensive droplets. Assessing the number and size of lipid droplets is also useful in mitochondrial disorders. The routine inclusion of a stain for lipid is a matter of choice which can be driven by clinical information and the patient population referred.

Proliferation of adipose tissue is a common feature of muscular dystrophies but also occurs in spinal muscular atrophies and other disorders. The unstained content of fat cells is readily apparent on routine histological stains but it can also be strikingly demonstrated with lipid stains. Stains for lipid in the presence of adipose tissue may, however, lead to diffuse spread of reaction product over large areas of the section.

Amyloid

It has been found useful to look for the deposition of amyloid in inclusion body myositis ( ). In addition, amyloid can be pathologically deposited in muscle. In sporadic forms of inclusion body myositis and some hereditary myopathies with vacuoles many of the characteristic rimmed vacuolated fibres contain amyloid. These myopathies are often referred to as hereditary inclusion body myopathies and have several pathological features in common with sporadic inclusion body myositis but rarely show lymphocytic inflammation. Amlyoid is composed of protein in a β-pleated sheet conformation. Ultrastructurally, it appears as tangled masses of unbranched double filaments of variable length. Each filament is 2.5–3.5 nm in diameter and separated by a 2.5 nm space, giving a total diameter of 8–10 nm. The most common method for demonstrating amyloid uses Congo red, and one at high alkaline pH was recommended by Mendell ( ). Amyloid stained with Congo red is visible as a red deposit with normal bright field optics but also shows ‘apple-green’ birefringence with polarized light and is most easily seen using fluorescence with an excitation filter suitable for fluorochromes such as Texas red ( ). More recently, the luminescent conjugated oligothiophenes (LCOs), in particular pentamer formyl thiophene acetic acid (p-FTAA), have successfully been shown to bind protein inclusions in muscle biopsies from patients with inclusion body myositis ( ).