Understanding the course of fibre differentiation during development and ageing is important for analysis of normal and diseased muscles. Often, a certain fibre type of the developing or ageing muscle is more affected than another by altered conditions or diseases. However, postnatal differentiation of muscle fibre types and subtypes is not completely investigated to this day.

A fibre type can be identified by the myosin heavy chain (MHC) isoforms it contains. At first, two distinct fibre types are generated: slow fibres containing predominantly slow MHC and fast fibres containing predominantly fast MHC. This slow–fast differentiation begins early in histogenesis. Differentiation into subtypes occurs later during development, but the exact time point of subdifferentiation is unclear up to now. Indeed, the exact time point at which fibre subtypes become detectable may be important for investigations of developmental changes in muscle properties, adaption processes to altered conditions or muscle diseases.

Several studies have shown that the age at which different fibre types can be distinguished depends on the method used, and on species and muscles. For example, it was found that in man and rhesus monkeys the histochemical differentiation of leg muscles takes place before birth, whereas fibres of hindlimbs of newborn rats are histochemically undifferentiated. In this respect, one has to consider that muscle fibres can be differentiated on the basis of different classification systems. The criteria which underlie different classification systems are obviously not interchangeable. It means, for example, that fibre types IIA and IIB identified after histochemical ATPase activity staining are not metabolically differentiated and therefore do not strictly correlate with FOG and FG fibres as identified by cytophotometry. Some studies have reported developmental changes in MHC expression, in fibre population, and in fibre type-specific enzyme activities of several rat hindlimb muscles. However, a comprehensive study including developmental changes in fibre type population, fibre type-specific metabolic profile and the time of detectability of fibre types with the different classification systems during normal development and ageing of rat hindlimb muscles is missing to this day. Therefore, the aim of the present study was firstly to find out at which time of muscle development and ageing, the fibre types of different classification systems are detectable in soleus (SOL), extensor digitorum longus (EDL) and gastrocnemius (GAST) muscles of rats. To type the fibres according to different criteria and to compare different classification systems, fibre typing was performed by immunohistochemical, enzyme histochemical and cytophotometrical methods. Secondly, age-dependent changes in the fibre type population and metabolic profile of each fibre type were investigated.

The experiments were approved by the Regierungspräsidium Leipzig. The rats were kept under standardized conditions in the animal breeding station of the University. Twenty-five Wistar rats of different ages were used: 21st embryonic day (ED) as determined by the presence of vaginal plugs, first postnatal day (PD), 8th PD (beginning of more or less co-ordinated leg movements), 21st PD (weaning), and 75th PD (young adult). Five rats per age category were used. Animals were anaesthetized with ether and then decapitated. In the case of embryos, and 1-day-old and 8-day-old rats, the entire lower parts of hindlimbs were prepared, and in the case of 21- and 75-day-old rats SOL, EDL and GAST muscles were removed separately, powdered with talcum and frozen in liquid nitrogen. Samples of each age category were mounted together on a cryostat chuck, and 10-?m-thick cross sections were cut with a cryostat 1800 (Reichert Jung, Vienna, Austria) and then used for immunohistochemical, enzyme histochemical and cytophotometrical analysis. In this way, variations caused by differences in section thickness and incubation conditions were avoided, at least for those sections on the same glass slide.

Myosin heavy chain (MHC) isoforms were demonstrated using commercially available monoclonal antibodies (MABs) from mouse against slow, fast and neonatal MHC isoforms following the guidelines recommended by the manufacturers (Novocastra, Newcastle, UK). In brief: serial sections were incubated with the primary MABs (WB-myosin heavy chains (MHCs), dilution 1:50; WB-MHCf, dilution 1:10; and WB-MHCn, dilution 1:10) for 60 min at 37°C followed by incubation with rabbit anti-mouse secondary ABs (Jackson Immuno Research, West Grove, PA, USA) for 60 min at 37°C and with mouse PAP-complex (Jackson Immuno Research) for 60 min at room temperature. Visualization was performed by incubation with 3,3-diaminobenzidine and H₂O₂ as peroxidase substrates. After dehydration, sections were mounted in Entellan (Merck, Darmstadt, Germany).

Control incubations were: (i) omission of primary MAB; and (ii) substitution of primary MAB by rabbit IgG (Dianova, Hamburg, Germany) at the same final dilution as the primary MABs.

Activities of succinate dehydrogenase (SDH, E.C. 1.3.5.1) and mitochondrial glycerol-3-phosphate dehydrogenase (GPDH, E.C. 1.1.99.5) were demonstrated as described previously. The activity of myofibrillic adenosine triphosphate (ATPase, E.C. 3.6.1.32) was demonstrated at pH 9.4 as well as after acid and alkaline preincubations at various pH values.

End-point measurements were made with a computer-controlled microscope photometer MPM 200 with a scanning table (Carl Zeiss, Oberkochen, Germany). The mean absorbance of the final reaction product of the respective enzyme reactions (myofibrillic ATPase, GPDH and SDH) was measured and taken as a measure of relative enzyme activity. The correlation of absorbance of the final reaction product with the respective enzyme activity is shown. The cytophotometrical method was established and described as a tool in metabolic fibre typing. In each muscle section, approx. 30 fibres, containing all fibre types, were measured. Three sections were analysed per muscle and enzyme reaction.

The methods of fibre typing have been described previously. In brief: fibres were typed into slow and fast according to their slow or fast MHC isoforms as detected immunohistochemically. On the basis of the differences in acid and alkaline lability of ATPase activity of the MHC isoforms, 3 fibre types I, IIA, IIB (including IIX) were differentiated. The physiological metabolic fibre typing into slow oxidative (SO), fast oxidative glycolytic (FOG) and fast glycolytic (FG) is based on cytophotometrically-quantified activities of myofibrillic ATPase (marker of contractile activity), SDH (marker of oxidative activity) and GPDH (marker of glycolytic activity) in serial cross sections of the same fibre. These enzyme activities characterize the metabolic profile of muscle fibres. FOG fibres were subdivided into FOG I fibres with moderate SDH and moderate GPDH activity and FOG II fibres with high SDH and moderate or high GPDH activity.

Counting of fibres was performed with the Imaging System KS 100 (Kontron, Eching, Germany). A measuring field was set over the entire muscle cross section. Serial sections with GPDH, SDH and alkaline ATPase activities were analysed. SO fibres are the most remarkable (lightest) fibres in the sections incubated for GPDH activity and they are identical with type I fibres found as light fibres after the alkaline ATPase reaction. FG fibres are the lightest fibres after the SDH reaction. Therefore, SO fibres were identified in sections incubated for GPDH activity, whereas FG fibres were analysed in sections incubated for SDH activity. The percentage of FOG fibres was determined indirectly as a difference: %FOG=100%–%SO–%FG. The percentage of type II fibres was calculated by addition of FOG and FG fibres as percentages.