Histological and Histochemical Changes

Atrophy and Hypertrophy

A common occurrence is atrophy of only some of the fibres in a biopsy, either singly or in small clusters. When this occurs, the small fibres are usually obvious in comparison to the remaining large fibres. This small group atrophy is characteristic, but not diagnostic, of denervation (see Fig. 4.2 ), and in some neurogenic disorders there is large group atrophy ( Fig. 4.3 ). This is accompanied by diffuse hypertrophy (see Fig. 4.2 ) or group hypertrophy (see Fig. 4.3 ). It should be noted that splitting of a larger fibre may produce an apparent group of small fibres and branching of fibres will also contribute to the impression of fibre size variability (see Fig. 4.27 ). For this reason caution should be exercised in interpreting small group atrophy in the presence of fibre splitting or other pathological changes suggestive of a myopathy. Serial sections may be necessary to track a splitting or branched fibre. Multiple branching of fibres is also seen at myotendinous junctions (see Fig. 3.8 ).

In some disorders, such as some congenital myopathies, very small fibres may be difficult to identify with routine stains as they may be similar in size to a nucleus or capillary. These very small fibres are scattered through out the biopsy and can be seen clearly with immunohistochemistry using antibodies to fetal myosin (see Ch. 6 , Fig. 6.25 ). It is important to remember that not all small fibres are atrophic, as some may be regenerating fibres or hypotrophic fibres that have not grown. In myopathic conditions the atrophic and hypertrophic fibres are randomly distributed through out the sample ( Fig. 4.4 ). Sometimes, as in congenital myopathies, two distinct populations of fibres of different sizes are apparent, but they are not grouped ( Fig. 4.5 ) as seen in denervating disorders (see Fig. 4.3 ).

In dermatomyositis, fibres at the periphery of the fascicles may be small. Some of these are atrophic and some are regenerating. This appearance is called perifascicular atrophy and is thought to reflect ischaemic changes secondary to disease of the blood vessels. It is only seen in dermatomyositis, but not universally ( Fig. 4.6 ), although a mild perifascicular atrophy can be seen in cases of eosinophilic fasciitis (Shulman syndrome). In antisynthetase syndromes perifascicular necrosis is seen.

Aspects of atrophy and hypertrophy relating to each fibre type are discussed in the next section.

Quantification

This section describes the main methods used to quantify the degree of change of fibre sizes and illustrates typical histograms that can be obtained, in relation to fibre types. Although it is now performed less often on a routine basis and computerized systems are available, we felt it would be helpful to retain the background to quantification.

The starting point is measurement of the ‘lesser diameter’, which combines simplicity and speed with reasonable accuracy. This is defined as the maximum diameter across the lesser aspect of the muscle fibre ( Fig. 4.11 ). This measurement is designed to overcome the distortion that occurs when a muscle fibre is cut obliquely, producing an oval appearance in the fibre. Unless the lesser diameter is measured, an erroneously large measurement will result, as Fig. 4.11 shows. Computer software such as ImageJ uses a similar tool, where it is called Feret’s diameter or calliper length.

Measurements are performed on adenosine triphosphatase (ATPase)-stained sections or ones immunolabelled with myosin isoforms so that involvement of each fibre type can be calculated. These can be performed with an eyepiece micrometer or by projecting the imaging on a suitable surface or computer screen. Several computerized systems are available (including ones available on the web at no cost), but full automation can rarely be achieved as most systems are unable to accurately define two closely adjacent fibres, and this has to be done manually. Each fibre can be defined by immunolabelling of a sarcolemmal protein such as laminin α ₂ , β-spectrin or dystrophin, but even then the sarcolemma of adjacent fibres can be difficult to decipher. Computerized systems, however, are useful for calculating cross-sectional area of each fibre type, if the section is perfectly orientated in the transverse plane, for plotting histograms and calculating mean values and standard deviations. A total of at least 100 fibres of each type is measured and a histogram of the diameters of each fibre type plotted. This number of fibres has been shown to be representative, although quantification of all fibres in a section is possible with computerized systems. A mean fibre diameter and standard deviation are calculated and compared with normal values. Ideally, each laboratory should establish its own normal values, but many workers rely on published data ( ). A limitation of this is that some biopsies used for establishing these normal data in old publications were taken for a clinical reason and, although the samples apparently showed no defects, this cannot be established beyond doubt.

In addition to mean fibre diameter, it is important to assess variability. A useful figure is the variability coefficient, which is calculated as follows:

In normal muscle, the variability coefficient is less than 250, and any sample with a variability coefficient greater than this is considered to demonstrate abnormal variability in the size of fibres. In children, the gradual increase in size with age has to be taken into account.

Atrophy and Hypertrophy Factors

In an effort to quantify the degree of change of fibre size in a biopsy, atrophy and hypertrophy factors were devised by . These factors are calculated from the histograms of the muscle fibres and are an expression of the number of abnormally small or large fibres in the biopsy. In normal adult muscle, most fibres in the histogram are between 40 and 80 μm in diameter in males and 30–70 μm in females. Considering first the abnormally small fibres, a few fibres in the 30–40 μm range in a histogram from a male biopsy would have less significance than the same number of fibres in the range of 10–20 μm or than a larger number of fibres in the same (30–40 μm) range. This is taken into account by multiplying the number of fibres in the histogram with a diameter between 30 and 40 μm by 1, the number of fibres with a diameter between 20 and 30 μm by 2, the number of those from 10 to 20 μm by 3 and the number in the group less than 10 μm by 4. These products are then added together and divided by the total number of fibres in the histogram to put the result on a proportional basis. The resulting number is then multiplied by 1000 and this is the ‘atrophy factor’ . The hypertrophy factor is similarly derived to express the proportion of fibres larger than 80 μm in the male. A diagrammatic calculation from a histogram is shown in Fig. 4.12 . In addition to making the calculations for the muscle biopsy as a whole, one can also consider each fibre type separately. Thus, for each histochemical fibre type there are two numbers: the atrophy and hypertrophy factors (abbreviated A or H factor). The histogram for a given biopsy may then be expressed as a series of four numbers for A1, H1, A2 and H2 (atrophy and hypertrophy of type 1 and type 2 fibres, respectively). If fibre subtypes are considered, there will be six numbers: A1, H1, A2A, H2A, A2B and H2B. In adult females the limits 30–70 μm and not 40–80 μm are used to calculate atrophy and hypertrophy factors in a similar way ( Table 4.1 ).

TABLE 4.1

Upper Limits for the Value of Atrophy and Hypertrophy Factors for Normal Adult Male and Female Muscles

	Type 1		Type 2A and Type 2B
	Atrophy	Hypertrophy	Atrophy	Hypertrophy
Biceps
Male	150	300	150	500
Female	100	200	150	150
Vastus
Male	150	150	150	400
Female	100	400	200	150

This statistical approach, although somewhat laborious, is useful in detecting the presence of atrophy or hypertrophy that may not be apparent on routine inspection of a muscle biopsy and for demonstrating the presence of selective atrophy of one fibre type in association with hypertrophy of another type. In practice it is often clear when variation in fibre is abnormal, and it can be graded as mild, moderate or severe.

Using the atrophy and hypertrophy factors, selective atrophy of fibre types can be readily confirmed. If, in a biopsy, only the atrophy factor for type 1 fibres is above the normal limits, the biopsy is said to show selective type 1 fibre atrophy. Similarly, selective hypertrophy may be seen in some biopsies. This type of analysis is a practical way of recognizing atrophy of one fibre type in the presence of hypertrophy of the other. It should be stressed that these atrophy and hypertrophy factors are used only in biopsies from adult muscle and are most useful only when the change is not obvious on inspection. Table 4.1 shows a summary of data obtained from normal muscle using this method. In children under the age of 14, the relative sizes of the type 1 and type 2 fibres are smaller, and this has to be taken into account ( Fig. 4.13 ). Although the data in Fig. 4.13 date from 1969, our own recent assessment of all fibres in over 80 muscle biopsies from paediatric cases aged a few months to 16 years of age using Definiens software is in agreement with . Similarly, regions of interest of 200 fibres showed a good correlation. Mean diameters of type 1 and type 2 fibres should not differ by more than 12% of the largest diameter of the largest fibre type. The variability coefficient is again less than 250. Fibre type disproportion, a characteristic of congenital myopathies, is said to occur if the type 1 fibres are at least 12% smaller than type 2 fibres, although Brooke revised this to 25%. Illustration of typical histograms from normal biopsies and classical pathological situations are shown in Figs. 4.14–4.18 .

Fibre Type Predominance

Type predominance is an excess of one fibre type ( Fig. 4.19 ). In the interpretation of fibre type predominance, it is important to make careful comparison with controls from the same muscle of similar age and sex. In addition, proportions can differ in different regions of a muscle. In the quadriceps the normal ratio of type 1 to 2 fibres is approximately 1:2. If the type 2 fibres are subdivided, then type 1, 2A and 2B comprise approximately one-third each. There is, however, some variation within the normal population around these figures. From our experience, type 1 predominance is said to occur when more than 55% of the fibres are type 1, and type 2 fibre predominance when more than 80% of the fibres are type 2. An appearance of type 2 predominance may occur in muscle biopsies from neonates and results from a high prevalence of fibres with fetal myosin.

Fibre type predominance may reflect type grouping if the biopsy has been taken from the centre of a very large group of a uniform fibre type. However, some disorders are associated with fibre type predominance in a sufficient number of biopsies to make this explanation unlikely. Type 1 fibre predominance is a common feature of myopathic conditions: for example, the muscular dystrophies and congenital myopathies (see Fig. 4.19 ; see also Fig. 4.7 ). Type 2 fibre predominance, on the other hand, is associated with motor neurone diseases.

Internal Nuclei

When more than 3% of the fibres in transverse section contain a nucleus that is in the substance of the muscle fibre and not at its periphery the biopsy is said to demonstrate internal nuclei. In our experience, however, this is probably an overestimate and even a few internal nuclei in paediatric muscle are probably significant. In normal adults they are more common, particularly in individuals involved in sporting activities. In some conditions the nuclei may be central within the fibre, and in longitudinal section they may form a chain down the centre of the fibre or be spaced. It is important to distinguish between internal and central nuclei and to accurately describe their position. Central nuclei are a characteristic feature of biopsies from patients with congenital myotonic dystrophy and some other congenital myopathies such as centronuclear myopathies and those associated with defects in the genes encoding the ryanodine receptor 1 and titin (see Chapter 14, Chapter 15 ). In other situations ( Fig. 4.20 ), they are scattered within the myofibrils and more than one per fibre may be seen ( Fig. 4.21 ). Internal nuclei are often seen along the fibrous septa in split fibres (see Fig. 4.28 ); some of these relate to nuclei of the capillary endothelial cells.

It is important when assessing the presence of internal nuclei to examine the transverse sections of muscle and not the longitudinal, since in a longitudinal section (which may be up to 10 μm in thickness) a peripherally placed nucleus may be seen through the overlying myofibrillar tissue and may give the false impression of being within the muscle fibre.

The significance of internal nuclei may be summarized by saying that a great profusion of internal nuclei would be suggestive of a myopathy. They are particularly abundant in myotonic dystrophies, but they can also occur in chronic neuropathies.