Quantitative succinate-dehydrogenase histochemistry. II. A comparison between visual and quantitative msucle fibre typing.

Most muscles exhibit a mosaic pattern of staining intensities of their muscle fibres after the histochemical reaction for succinate dehydrogenase (SDH). Visually these muscle fibres are usually classified into three groups: with low (A-fibres), intermediate (B-fibres), and high (C-fibres) enzymatic activity (staining intensity). Cytospectrophotometric methods were employed to investigate whether discrete groups of muscle fibres could be discerned, comparable to those found after the visual classification. The classifications were based on quantitative parameters of the total absorbance per cell and the distribution of the coloured endproduct over the fibre cross area.     

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

The histochemical profiles of fibre types in porcine skeletal muscle

Using a variety of histochemical methods -mATPase staining after alkaline and acid preincubations, NADH-TR and alpha-MGPDH- we have investigated the fibre types in porcine skeletal muscle. The results reveal that four major fibre types -I, IIA, IIB and II*- can be separated histochemically in Longissimus lumborum muscle of Landrace pigs. The histochemical properties of the muscle fibre type II* are very similar to that of type IIX described in other mammals. The existence of IIX fibres in pig muscle has been recently demonstrated by molecular biology techniques and our results validate the use of histochemistry (mATPase) as an easy methodology to differentiate the three fast myosins (type II fibres) in pig muscle.     

Studies on 5'-nucleotidase histochemistry. III. 5'-Nucleotidase activity in smooth muscle cells of the rat's gastrointestinal tube.

The distribution pattern of histochemically detectable 5'-nucleotidase (5'-Nase) activity is described in smooth muscle cells of the rat's gastrointestinal tube (esophagus, stomach, small intestine, large intestine). Both, light and electron microscopic methods are used. Faint positive 5'-Nase activity is observed on smooth muscle cells of the lamina muscularis mucosae in the thoracal esophagus whereas it is completely absent from smooth muscle cells of the abdominal esophagus and the stomach. In the small and large intestine strong positive 5'-Nase reaction is found on smooth muscle cells of the lamina muscularis mucosae and the innermost part of the lamina muscularis externa. In the circular and longitudinal layer of the lamina muscularis externa a slight increase in 5'-Nase activity is observed from the proximal to the distal segments. The reaction product is restricted to the outer cell surface of smooth muscle cells. In the small intestine the strong enzymatic activity in the innermost part of the muscularis externa is found to be localized at small and dense muscle cells (sd-cells). Common morphological and histochemical characteristics of sd-cells and smooth muscle cells of the lamina muscularis mucosae are emphasized. Hypothetical functions e.g. uptake of precursors of nucleosidephosphates, possible functional connection to a high glycogen content, correlation between 5'-Nase activity and proliferation capacity and local vasodilatory effect are discussed.     

Intrafusal fibre types in rat limb muscle spindles: morphological and histochemical characteristics.

Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali- and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length. Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

Quantitative histochemistry of creatine kinase in rat myocardium and skeletal muscle

Summary Creatine kinase (ec 2.7.3.2) activity was demonstrated in rat myocardium using a polyvinyl alcohol-containing incubation medium and auxiliary enzymes. The activity was quantified by microdensitometry using both endpoint measurements and kinetic measurements. Control reactions were performed in the absence of creatine phosphate and ADP.The linear regression lines of the absorbances of reduced Nitro BT at the isobestic wavelength (585 nm) on incubation time were highly significant for both endpoint and kinetic measurements. The activity obtained from endpoint measurements was about 40% lower. This was caused by loss of the formazan reaction product from the tissue sections when the incubation medium was removed at the end of the reaction. The relationship between creatine kinase activity (test minus control reaction) and section thickness was not linear for either myocardium or skeletal muscle; control reactions, however, showed linear relationships with section thickness for both tissues. Limited penetration of auxiliary enzymes into the sections may be responsible for this disporportionality. Therefore, care should be taken in the interpretation of quantitative data obtained with different tissues.In conclusion, multi-step enzyme reactions can be used for quantitative histochemical purposes provided it is taken into account that the reactivity is not proportional to section thickness.     

Quantitative histochemical determination of muscle enzymes: biochemical verification

We established quantitative histochemical assays for the enzymatic activity of succinate dehydrogenase and alpha-glycerol phosphate dehydrogenase for cat skeletal muscle. A computer-enhanced image analysis system was used to quantitate the histochemical enzyme-activity reaction products. We describe a series of experiments that verify the reliability and validity of the assays. Histochemically determined enzyme activities were linear with respect to tissue thickness and reaction time. Biochemically determined enzyme activities were also linear with respect to tissue thickness and incubation time. Consecutive tissue sections, assayed either histochemically or biochemically, were used to establish a linear regression equation that allowed quantitative histochemically determined reaction rates, measured in optical density per minute, to be calibrated as nanomoles per minute.     

Variations in fibre composition of the gastrocnemius muscle in rats subjected to speed training

Thirty-six adult Wistar rats were divided into three groups. One group was used as a control, and the other two underwent different training programmes in which greater relevance was attached to the intensity of exercise than to its duration. Samples of the red and mixed portions of m. gastrocnemius (caput lateralis) were stained with m-ATPase to determine the percentage of type I, IIA and IIB fibres, and with NADH-TR in order to quantify variations in the percentage of low staining intensity (FG) fibres. The most notable results obtained were: a) the ratio of type I type II fibres remained unchanged; b) the proportion of IIA fibres increased, while that of IIB fibres decreased correspondingly; c) FG fibres, which were virtually absent from the red portion, recorded a clear decrease which was more marked, and occurred more rapidly, than in IIB fibres. These differences were all statistically significant in the mixed portion of the muscle. Adaptive changes in fibre composition in the red portion were less marked.     

Variations in the fibre repeat between samples of cellulose I from different sources

A powder X-ray diffractometer was used to measure the fibre repeat in cellulose I with sufficient precision to detect variations between samples from different sources. The variations were correlated with the lateral dimensions of the crystallites and were attributed to different minimum-energy fibre repeats for chains in the interiors and on the surfaces of crystallites. Results were interpreted in terms of a model for internal mechanical stress in which the interior chains were under compression and the surface chains under tension to ensure identical fibre repeats for all chains. The model was used to extrapolate the fibre repeat to a value of 1.043 nm for a hypothetical, infinitely large crystal, and to 1.029 nm for a crystallite so narrow that all chains were exposed on surfaces.     

pH lability of myosin ATPase activity permits discrimination of different muscle fibre types in crustaceans

Summary Myofibrillar actomyosin ATPase activity has been studied histochemically in the closer muscle of the crab Eriphia spinifrons. Preincubation at pH 4.6 and 5.0 reveals differences in the lability of the ATPase. This permits the discrimination of four fibre types. Of these, three represent subgroups of rapidly contracting fibres. The histochemically defined fibre types correspond well with four groups defined according to electrophysiological criteria.     

pH lability of myosin ATPase activity permits discrimination of different muscle fibre types in crustaceans

Myofibrillar actomyosin ATPase activity has been studied histochemically in the closer muscle of the crab Eriphia spinifrons. Preincubation at pH 4.6 and 5.0 reveals differences in the lability of the ATPase. This permits the discrimination of four fibre types. Of these, three represent subgroups of rapidly contracting fibres. The histochemically defined fibre types correspond well with four groups defined according to electrophysiological criteria.     

Distribution of isoenzymes of the glycogenolytic cascade in different types of muscle fibre.

Distinct isoenzyme patterns of the glycogenolytic enzymes exist in different fibre types. Fast twitch glycolytic and slow twitch oxidative fibres differ in the proportion of the two isoenzymes of cyclic AMP dependent protein kinase and in the type of phosphorylase kinase that is present. Slow twitch oxidative fibres and cardiac fibres resemble one another in these two respects, but differ in that the type I phosphorylase of cardiac muscle is absent in slow twitch oxidative fibres. In all examples, the functional differences between the isoenzymes seem to be related to the regulatory rather than the catalytic behavior of the molecules. In the case of cyclic AMP dependent protein kinase and phosphorylase kinase, it is a regulatory subunit that appears to be affected [16,23], while in the case of phosphorylase, the type I isoenzyme is known to have a five to eight-fold Ka for the allosteric activator 5' AMP [6]. However, the precise physiological significance of these differences remains to be elucidated.     

Quantitative enzyme histochemistry in the brain

Two main groups of quantitative methods are used in the brain to relate enzymatic processes to cellular structures, i.e. the methods of microchemistry and microscopic histochemistry. Microchemistry tries to quantify enzyme activities in very small brain regions by miniaturizing biochemical methods, whereas microscopic histochemistry applies staining procedures to tissue sections, preserving the structural relationship that is present in situ and giving topological information on the distribution of enzymes which is indispensable in structural heterogeneous tissue as is the brain. The present review deals preferentially with microscopic methods and, in particular, with scanning microphotometry (image plane scanning). Using this technique two measuring procedures can be applied for the quantification of enzyme activities, i.e. end-point and kinetic (continuous monitoring) measurements which are described in detail. Methods for the microphotometric demonstration of certain important dehydrogenases (isocitrate dehydrogenases, succinate dehydrogenase, NAD-linked malate dehydrogenase, glutamate dehydrogenase and glycerol 3-phosphate dehydrogenase), of cytochrome c oxidase, hexokinase and acetylcholinesterase are presented. These methods were adapted for giving optimal demonstration of enzyme activities in the rat hippocampus. The examples are given to illustrate the aptitude and possibilities of this technique in the quantification of enzymes in the complex matrix of the brain.     

The value of enzyme histochemical techniques in classifying fibre types of human skeletal muscle

Summary Fibre-type classification of human skeletal muscle into type I and type II fibres is mostly based on their slight or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in muscle specimens, with no apparent disease of the neuromuscular system. Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and glyconeogenesis, respectively. A most important feature of the biochemical findings is the constancy of the activity ratios of the examined enzymes. From these results and from the histochemical results it can be concluded that in apparently normal adult human skeletal muscle the ATP-ase technique for type I and type II typing is reliable. For fibres with an intermediate intensity of staining with the myosin ATPase technique of typing it is also necessary to apply other enzyme histochemical techniques.