Peter J. Stoward

Early Onset of Lipofuscin Accumulation in Dystrophin-Deficient Skeletal Muscles of DMD Patients and mdx Mice

Lipofuscin, the so-called ageing pigment, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals and redox-active metal ions. Usually it accumulates in post-mitotic, long-lived cells such as neurons and cardiac muscle cells. In contrast, it is rarely seen in either normal or diseased skeletal muscle fibres. In this paper, we report that lipofuscin accumulates at an early age in both human and murine dystrophic muscles. Autofluorescent lipofuscin granules were localized, using confocal laser scanning microscopy and electron microscopy, in dystrophin-deficient skeletal muscles of X chromosome-linked young Duchenne muscular dystrophy (DMD) patients and of mdx mice at various ages after birth. Age-matched normal controls were studied similarly. Autofluorescent lipofuscin granules were observed in dystrophic biceps brachii muscles of 2–7-year-old DMD patients where degeneration and regeneration of myofibres are active, but they were rarely seen in age-matched normal controls. In normal mice, lipofuscin first appears in diaphragm muscles nearly 20 weeks after birth but in mdx muscles it occurs much earlier, 4 weeks after birth, when the primary degeneration of dystrophin-deficient myofibres is at a peak. Lipofuscin accumulation increases with age in both mdx and normal controls and is always higher in dystrophic muscles than in age-matched normal controls. At the electron microscopical level, it was confirmed that the localisation of autofluorescent granules observed by light microscopy in dystrophin-deficient skeletal muscles coincided with lipofuscin granules in myofibres and myosatellite cells, and in macrophages accumulating around myofibres and in interstitial connective tissue. Our results agree with previous biochemical and histochemical data implying increased oxidative damages in DMD and mdx muscles. They indicate that dystrophin-deficient myofibres are either more susceptible to oxidative stress, or are subjected to higher intra- or extracellular oxidative stress than normal controls, or both.

The histochemical localization of xanthine oxidase

Certain cells initially convert much of the oxygen they censume to the superoxide anion (O-a), which in turn may initiate the peroxidation of the lipids of cell membranes if it is not immediately destroyed. There is some evidence that xanthine oxidase is the principal enzyme catalysing the formation of O~ in this situation and thus it may be a useful marker of lipid peroxidation and, as a consequence, cell ageing (Mead, 1976). Unfortunately, none of the three methods published previously for the histochemical demonstration of this enzyme in mammalian tissues (Sackler, 1966; Mclndoe et al., 1974; Auscher & Amory, 1976) is particularly satisfactory. However, we have found that a high xanthine oxidase activity can be localized discretely in the sarcolemmae of aerobic skeletal muscle fibres (Fig. 1) and in the cytoplasm of rodent enterocytes (Fig. 2), hepatocYtes and renal collecting tubule cells by incubating 10/ira cryostat sections of unfixed tissue for 1-2 h at 37~ in the following medium: 1 mM hypoxanthine, 1 mM NAD § and 1 mg/ml NitroBT dissolved in 0.2 g phosphate buffer, pH 7.4, containing 22% polyvinyl alcohol (grade BO5/140). After incubation, the sections are washed and fixed for 5 min at room temperature in 10% formalin containing 0.9% saline, followed by a brief rinse in water, dehydration through the ethanols, clearing in xylene, and mounting in DPX. The following inhibitor tests appear to substantiate the specificity of the technique. Preincubation of unfixed sections at room temperature for 30 min-2 h in 22% polyvinyl alcohol containing 2 M MnC12 (which presumably removes the FAD component of the enzyme; Komal et al., 1969), 1 mM 1,10-phenanthroline, 1 mM arsenite, 10 mM KCN or 0.05 M formaldehyde inhihits nearly all the enzyme activity. In contrast, when phenanthroline, arsenite or KCN are incorporated in the normal incubation medium, very little inhibition occurs. Allopurinol, which is generally assumed to be a specific inhibitor of the enzyme, behaves differently. Preincubation in a 1 mM solution of this inhibitor (in 22% polyvinyl alcohol) normally only partially represses the activity of the enzyme. In some tissues, such as liver, it has no effect. It also appears to have no effect (in any tissue) when included in the complete incubation medium. On the contrary, allopurinol can be substituted as a substrate in place of hypoxanthine in our technique. These observations are consistent with the conclusion that our technique localizes the NAD-stimulated D-form of xanthine oxidase as distinct from the NAD-nonrequiring, allopurinol-sensitive O-form of the enzyme (cf. Waud & Rajago, palan, 1976). About half the amount of reaction product is deposited in sites assumed to :contain NAD-stimulated xanthine oxidase when control sections are incubated in media from which either the substrate or NAD + has been omitted; the mean absorbance (as

Localisation and quantification of dehydrogenase activities in single muscle fibres of mdx gastrocnemius

Abstract The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate subsarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions.

Some quantitative histochemical studies of the periodic acid oxidation of glycogenin situ

SynposisThe periodic acid oxidation of the ‘glycogens’ in sections of ox liver fixed in Carnoys fluid has been studied quantitatively. It was found that in 0.02 M acetate buffer at pH 5.0 the oxidation was rapid at first but levelled off after I hr. Even after prolonged oxidation (12 days), not more than 23–24% of the total available glycogen was oxidized. However, in the presence of electrolytes (e.g. 0.2.m sodium chloride) the oxidation was much greater. After 2.5 hr, for example, 40% of the available glycogen was oxidized. There was little difference in the velocity of oxidation in sections mounted on glass-slides and free-floating ones, or in free-floating sections of different thicknesses.Mounted and unmounted sections consumed on average 11–17 times more periodate than could be accounted for by the oxidation of their glycogen content.The results are interpreted in terms of a complex formed between periodate ions and the cuter glucosyl residues of the glycogen aggregate. The negative charge on the complex, it is suggested, prevents free periodate ions approaching and oxidizing the inner glycosyl residues.

A histochemical study of the apparent deamination of proteins by sodium hypochlorite.

SummaryThe possible chemical mechanisms by which neutral solutions of sodium hypochlorite containing a high concentration of sodium chloride abolish the acidophilia of proteins in sections of fixed tissue are reviewed. The most probable one is the chlorination of the protein terminal amino groups, followed by the breakdown of the N-chloramine so formed into α-ketocarboxylic acid, nitrile or aldehyde groups. Hypochlorite solutions certainly do not deaminate tissue sections as was previously thought.Experimental evidence for the formation of relatively stable N-chloramine groups in situ and their limited conversion to aldehydes is reported. For example, the acidophilia of hypochlorite-treated sections was found to be restored after flooding them with hydriodic acid followed by the extraction of the liberated iodine with an alcohol. The significance of these experimental findings is discussed.

The histochemical basis of quantitative histology

The interaction between histochemistry and microscopy for quantifying the morphology and function of cells in sections of tissue is reviewed.

Endogenous peroxidase in mast cells localized with a semipermeable membrane technique

SynopsisHamster mast cells have been found to give strong peroxidatic reactions at pH 5, 7.5 and 10 when sections of skeletal muscle are incubated for 2.5 h in the dark at room temperature on semipermeable membranes covering a gelled incubation medium consisting of 0.01% hydrogen peroxide, 5.5 mM diaminobenzidine and 1.36% agar dissolved in Universal buffer. The techniques is very efficient: with it, all mast cells react in marked contrast to the negative reaction they usually give with conventional techniques.The peroxidatic reactions are abolished if tissues are perfused beforehand with either aminotriazole or KCN but not if these inhibitors are incorporated in the gelled incubation medium. This and other evidence suggests that the mast cell reactions are not due to either catalase or haemoglobin adsorbed onto mast cell granules from lysed red blood cells.Skeletal muscle fibres do not exhibit any visible-peroxidase activity with the membrane technique.

Some investigations of the mechanism of the so-called “methylation” reactions used in mucosubstance histochemistry

SummaryThe results reported in this paper substantiate Vilters (1968) hypothesis that the absence of basophilia of acid mucosubstances after a so-called histochemical methylation with acidified methanol may be due to, among other factors, the lactonisation of carboxyl groups rather than, as is generally held, their esterification. Sections from several fixed tissues of Syrian hamster and mouse were treated at 60 ° or at room temperature with dry methanol, ethanol, butan-l-ol, benzene or cyclohexane containing 0.35% hydrogen chloride or 1% thionyl chloride. Others were treated with methanolic methyl iodide or etheral diazomethane. With most of these solutions, the azurophilia attributable to mucosubstance carboxyl and sulphate half-ester groups was eventually abolished; subsequent saponification restored only the azurophilia due to carboxyl groups. However, some variation was found for each “methylating” agent, and also between different mucosubstances.

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle

SummaryThe reproducibility, specificity and validity of Meijers semipermeable membrane simultaneous coupling technique for the assay of acid phosphatase activity in sections of skeletal muscle have been investigated quantitatively, using naphthol AS-BI phosphate as the substrate and hexazotised Pararosanaline (HPRA) as the coupler. With this technique, unlike conventional techniques, presumed specific final reaction product (FRP) is evident in three different histological sites in normal skeletal muscle; first, as intensely coloured red granules within muscle fibres; second, as a diffuse reddish colouration throughout the sarcoplasm of all muscle fibres (the intrafibre areas); and third, in certain connective tissue elements between the muscle fibres (the interfibre areas). The mean absorbance of the FRP (at its absorption maximum, 530 nm) formed in each of these sites after a constant incubation time does not differ significantly in serial sections. 6 mM sodium molybdate, an acid phosphatase inhibitor, reduces the mean absorbance by 50% in the intrafibre areas, but in the interfibre connective tissue areas, 1 mM is sufficient. In contrast, 10 mM EDTA, an alkaline phosphatase inhibitor, has a negligible effect on the formation of specific FRP. Thus, Meijers technique appears to be reproducible and specific. The mean absorbance of the FRP formed in each of the three reactive histological areas increases linearly with incubation time and section thickness. The maximum amount of FRP is formed at pH 5 and when the substrate concentration is above about 4 mM. However, some of the FRP in the intrafibre areas is unspecific, and arises from the transformation of adsorbed HPRA to a purple-coloured product having an absorption maximum at 570 nm. Much of the non-specific FRP appears after the incubation has been terminated with formalin, and reaches a maximum several hours after the sections have been subsequently mounted. As a consequence, Meijers technique is not entirely valid.

The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell.

SummaryThe kinetics of lactate dehydrogenase in mouse cardiac muscle fibres, skeletal muscle fibres, gastric parietal cells, parotid gland ductal and acinar cells, oocytes and mouse and human hepatocytes were studied as a function of substrate concentration in sections of unfixed mouse and human tissues incubated at 37°C on lactate agarose gel films. The absorbances of the final reaction products deposited in single cells of various types were measured continuously as a function of incubation time using an image analysis system. The initial velocities (vi) of the dehydrogenase were calculated from two equations deduced previously by us, vi = a1∘A (equation 1) and vi = v + a2∘A (equation 2), where v and ∘A are, respectively, the gradient (steady-state velocity) and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min, and a1 and a2 are constants characteristic for each cell type.Hanes plots using vi, calculated from equation 2 gave more consistent estimates of the Michaelis constant (Km) and the maximum reaction velocity (Vmax) than those employing either steady-state velocity measurements or vi calculated from equation 1. The Km thus found for mouse skeletal muscle fibres (10.4–12.5 mM) and hepatocytes (14.3–16.7 mM) agreed well with values determined previously in biochemical assays. However, the Km for cardiac muscle fibres (13.4 mM) was higher. The Km of the enzyme in gastric parietal cells, parotid gland cells and oocytes was in the range 7.6–9.7 mM. The Vmax were more diverse, ranging from 29 μmoles hydrogen equivalents/cm3 cytoplasm/min units in mouse parotid gland acinar cells, 59–68 units in skeletal and cardiac muscle fibres, 62–65 units in gastric parietal cells and oocytes, and 102–110 units in hepatocytes. The diversity found for Km and Vmax in different cell types confirms the value of the quantitative histochemical approach in revealing the heterogeneity of cellular metabolism in situ.