The high correlation between counts and area fractions of lipofuscin granules, a biomarker of oxidative stress in muscular dystrophies.

Images of cryostat unstained sections of two skeletal muscles, diaphragm and extensor digitorum longus (EDL), from wild-type normal and dystrophic mdx mice were captured with a fluorescence microscope, binarised and analysed by an automated procedure using ImageJ free software. The numbers, Feret diameters and areas of autofluorescent lipofuscin (LF)-like granules in the sections were determined from the binary images. The mean numbers of counted LF granules per mm3 muscle tissue correlated highly (r ≥ 0.9) with the area fractions of the granules in sections of both normal and mdx muscles. The similar distribution patterns of granule sizes in sections of diaphragm and EDL muscles are consistent with the high correlations.

Quantitative evaluation of the beneficial effects in the mdx mouse of epigallocatechin gallate, an antioxidant polyphenol from green tea.

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (-)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients.

Subcutaneous injection, from birth, of epigallocatechin-3-gallate, a component of green tea, limits the onset of muscular dystrophy in mdx mice: a quantitative histological, immunohistochemical and electrophysiological study.

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a component of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5 mg/kg body weight in saline) was injected subcutaneously 4x a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8 weeks after birth. Saline was injected into normal and mdx controls. EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology, and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase were reduced to normal levels. Second, the numbers of fluorescent lipofuscin granules per unit volume of soleus and diaphragm muscles were significantly decreased by about 50% compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm and soleus muscles, the relative area occupied by histologically normal muscle fibres increased significantly 1.5- to 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were substantially reduced. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Fifth, the amount of utrophin in diaphragm muscles increased significantly by 17%, partially compensating for the lack of dystrophin expression.

Quantitative assessment of succinate dehydrogenase activity of rat lateral pterygoid muscle in undecalcified fresh-frozen section.

The quantitative assessment of enzyme activities in situ in single muscle fibres is essential for understanding the functions of skeketal muscles. The function of the lateral pterygoid muscle (LPM) is not fully understood because it is a deeply located masticatory muscle and cannot be dissected in an intact configuration. Here we report how to measure the activities of mitochondrial succinate dehydrogenase (SDH) in single muscle fibres in situ in the LPM in sections of rat heads. Unfixed head sections were incubated on gel films containing SDH substrate and nitroblue tetrazolium. During incubation, images of the section due to deposition of the final reaction products, formazans, were captured at intervals of 10 s using a real-time image analysis system for absorbance measurements. We found that the belly of the LPM consisted of four areas with different mean activities of SDH. The lateral and upper areas of the muscle showed similarly high SDH activities. Mean activity in the lower area was the lowest, about half of those of the lateral and upper areas. These results agree with the hypothesis that the superior head of the LPM participates in more continuous contraction and is more resistant to fatigue than the inferior head.

Aquaporin-5 water channel in lipid rafts of rat parotid glands.

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or alpha1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or alpha1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence.

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M3 mAChRs in interlobular ducts of rat parotid gland.

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M(3) muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M(3) mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca(2+) signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

A new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress. Application to dystrophin-deficient DMD skeletal muscles.

This is the first report on the development of an immunohistochemical technique, combined with quantitative image analysis, for the assessment of oxidative stress quantitatively in nuclear DNA in situ, and its application to measure DNA damage in Duchenne muscular dystrophic (DMD) muscles. Three sequential staining procedures for cell nuclei, a cell marker, and a product of oxidative DNA damage, 8-oxoguanine (8-oxoG), were performed. First, the nuclei in muscle sections were stained with Neutral Red followed by the capture of their images with an image analysis system used for absorbance measurements. Second, the same sections were then immunostained for laminin in basement membranes as the cell marker. Next, the sections were treated with 2 N HCl to remove the bound Neutral Red and to denature tissue DNA. Third, the sections were immunostained for 8-oxoG in DNA, using diaminobenzidine (DAB) to reveal the antibody complex. This was followed by capture of the images of the immunostained sections as previously. The absorbances at 451.2 nm of bound Neutral Red and DAB polymer oxides, the final product of 8-oxoG immunostaining, were measured in the same myonuclei in the sections. Analysis of these absorbances permitted indices of the 8-oxoG content, independent of the nuclear densities, to be determined in nuclear DNA in single myofibres and myosatellite cells surrounded by basement membranes. We found that the mean index for the myonuclei in biceps brachii muscles of 2- to 7-year-old patients was 14% higher than that in age-matched normal controls. This finding of the increased oxidative stress in the myonuclei in young DMD muscles agrees with the previous reports of increased oxidative stress in the cytoplasm in the DMD myofibres and myosatellite cells. The present technique for the quantitative assessment of oxidative stress in nuclear DNA in situ is applicable not only in biomedical research but also in the development of effective drugs for degenerative diseases related to oxidative stress.

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.

Molecular mechanisms and drug development in aquaporin water channel diseases: the translocation of aquaporin-5 from lipid rafts to the apical plasma membranes of parotid glands of normal rats and the impairment of it in diabetic or aged rats.

Salivary secretion from rat salivary glands occurs in response to stimulation by acetylcholine and norepinephrine released from nerve endings. Aquaporin-5 (AQP5) localizes in lipid rafts under control conditions and is induced to traffic to the apical plasma membrane in interlobular ducts of rat parotid glands by the activation of M3 muscarinic acetylcholine receptors or alpha1-adrenoceptors. This review will focus on the mechanisms of the translocation of AQP5 from lipid rafts to the apical plasma membrane in the interlobular duct cells of parotid glands of normal rats and the impairment of its translocation in diabetic or senescent rats.

Cell death of primary afferent nerve cells in neonatal mice treated with capsaicin.

Capsaicin (50 mg kg-1) or vehicle (control) was injected into 2-day-old mice, and apoptotic cell death of the lumbar dorsal root ganglion (DRG) cells was examined using in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method at 5, 24, 48 and 72 h after treatment. Apoptotic cells were counted per section under a fluorescent microscope. The mean number of apoptotic cells showed a significant increase in the capsaicin-treated DRG at 24 h, but the number was at most 1-2 per section. Thus, many previous findings of an approximate 70-80% loss of the small primary afferent neurons induced by capsaicin could not be explained by apoptosis alone. Moreover, according to the morphological changes, it appeared that capsaicin preferentially induced necrosis rather than apoptosis, at least in mice.