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 5weeks with either saline or a daily average of 3 or 6mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5weeks 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 180mg/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 patient

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 (5mg/kg body weight in saline) was injected subcutaneously 4× a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8weeks 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 expressio

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 mm(3) 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

Studies in fluorescence microscopy

Histochemical techniques suitable for fluorescence microscopy have been developed for the detection of the principal chemical groupings and substances likely to be present in tissue sections. The mechanisms and specificities of the chosen reactions were confirmed wherever possible. The following methods were found to be the most satisfactory for the detection of: Amines An extremely intense green fluorescent product was produced in sites of proteins in tissue sections treated witha methanolic solution of pyridine containing a few drops of aqueous cyanogen bromide solution (the König-Sassi reaction). Treatment of sections with an alkaline solution of salicylaldehyde gave a thermolabile, green fluorescent conjugate. Tryptophane residues gave a characteristic purple fluorescence after reaction with a solution of dimethylaminobenzaldehyde in hydrochloric acid. The complex formed by mixing solutions of Solochrome dyes and alum fluoresced red after adsorption onto acidophilic primary and polymethyl amino groups. Chloraraine T oxidised the primary amine groups of α-amino-acid residues to aldehydes which were subsequently demonstrated as their blue fluorescent salicyloylhydrasones. The specificity of the preceding reactions were confirmed by the following deamination experiments. The diazotisation of amine groups with a cold solution of sodium nitrite and sulphuric acid, followed by diazotisation with warm ethanol was usually effective, out took some time to accomplish. Oxidative deamination with a dilute solution of sodium hypochlorite within the pH range 5.5 - 7.0 worked very efficiently and rapidly. Stable aldehydes were produced predominantly in nuclear sites provided the excess hypochlorite in the sections was destroyed by washing them in a neutral solution of ammonia containing a trace of copper sulphate. Cytophasmic proteins were probably converted to ketoacids. If sodium thiosulphate was used for destroying the excess hypochlorite, the induced aldehydes were destroyed. Instead strongly besophillc groupings, probably derivatives of sulphonic acids, were formed in both protoplasmic and nuclear proteins. Many of the reagents suggested by Danielli (1950) for blocking amine groups were found to be ineffective. Thiols Thiols condensed with N-ethyl maleimide (NEM) selectively to give a product containing an unconjugated ketone group. The zinc complex of the non-fluorescent hydrazone of the NEM-thiol conjugate derived from salicyloyl hydrazide fluoresced bluish-green. When all the basophilic substances and compounds containing hydroxyl groups were extracted from tissue sections by treating them with methanolic hydrochloric acid at 60°-90° for several hours (drastic methylation), it was found that the remaining thiol groups could be converted to basophilic thiosulphonic acids by exposing dry sections afterwards to sulphuryl chloride vapour. These acid groups exhibited an anomalous bright blue fluorescence after the treated section had been stained in dilute solutions of auramine O or acridine yellow. Disulphides Oxidation with peracids yielded basophilic sulphinic acids which were unique in resisting methylation. Subsequent staining with coriphosphine gave an intense red fluorescence in the oxidised sites. Previous blocking of other basophilic materials with methanolic thionyl chloride and of thiols with iodoacetate was essential. Phenols Indirect method. Acidified solutions of 1-nitroso-2-naphthol containing a trace of sodium nitrite coupled with tyrosine-rich sites to form an unstable green fluorescent conjugate. This test relied on the activating influence of the phenolic group on the ortho- and para- positions in the aromatic nucleus. The specificity of the reaction was confirmed by iodinating these positions. Direct method. Dinitrofluorobenzene reacted only with phenols and thiols (but not amines) at a pH below 5.5. By blocking the latter with iodoacetate, only the former reacted. The nitro groups of the conjugate were reduced to amines, but unfortunately attempts to demonstrate these groups by fluorescent methods (1) were unsuccessful. Carboxylic acids C-terminal carboxyl groups have been converted to methyl ketones by treating them with a mixture of acetic anhydride and pyridine at 60°. The ketones thus formed were demonstrated as the Blue fluorescent zinc complexes of their salicyloyl hydrazones. This, and other experimental evidence, vitiated the hypothesis put forward by Karnovsky and Fasman (1960) that the principal reaction here was the conversion of the side-chain carboxyl groups to mixed acid anhydrides. Sites containing side-chain carboxyl groups were detected by the changes in the colour of their fluorescence from blue to green in tissue sections stained with 0.01% solutions of coriphosphine at pH 2 - 3 and at PH 5. The interference of the C- terminal carboxyl groups was eliminated by previously converting then to their methyl ketones (5a). RNA was also extracted beforehand with hot perchloric acid. Nucleic acids Zirconium ions had en affinity for the phosphate groups of both types of nucleic acid which were subsequently demonstrated as their greenish-yellow fluorescent complex with morin. DNA yielded an aldehyde after brief (Feulgen) hydrolysis which was subsequently detectable as its blue fluorescent salicyloyl hydrazone. Sulphates No direct chemical methods have been found, but the presence of this group (in acid mucopolysaccharides) was inferred from the following tests: Their basophilia (towards coriphosphine) when counterstained with the acid dye, thiazol yellow. Acid mucopolysaccharides, containing only acid groups, exhibited the brown or red fluorescence of coriphosphine, while the nucleic acids and proteins fluoresced a light yellow or blue as a result of the very strong interaction between their basic amino groups and the acid dye. Their basophilia (towards coriphosphine) after selective extraction of nucleic acids. Methylation and reduction of tissue sections with lithium aluminium hydride in hot dioxane removed phosphate groups selectively. Uronic acid groups and some protein carboxyl groups were reduced to primary alcohols. Subsequent saponification and staining showed a reddish-brown fluorescence in sites containing acid mucopolysaccharides which contrasted with the weak green fluorescence of the remainder of the section. Hot perchloric acid has been used for the selective extraction of RNA. Other mineral acids and nucleases were found to be unsatisfactory for the selective extraction of nucleic fields because acid mucopolysaccharides were removed at the same time. Mild methods of methylation (using methanolic thionyl chlorine or diazomethane) have been successfully used for both the temporary and permanent blocking of sulphate and other basophilic groups. The normal technique for methylating tissues, using hot methanolic solutions of hydrochloric acid (Fisher and Lillie, 195U) were found to be unsatisfactory; much of the reactive material was extracted from tissue sections instead of being methylated. Sulphated acid mucopolysaccharides were desulphated to a limited extent by this reagent. Using published methods based upon iron mordants (Hicks and Mathaei, 1958; Hale, 1946), it was found that acid mucopolysaccharides did not always take up ferric ions whereas nucleic acids did. Experimental evidence has been collected to show that the results published previously were fortuitously successful and were not strictly specific. Uronic acids Sections were reduced with lithium aluminium hydride (7b) to show that the non-fluorescent dye alcian blue had a selective affinity for this group. When sections were stained with coriphosphine, they were first stained with alcian blue so as to quench the potential fluorescence of coriphosphine adsorbed onto acid mucopolysaccharides which otherwise would have been difficult to distinguish from that of nuclei. An intense blue fluorescence and a visible purple colour was observed in sites known to contain uronic acid groups after immersing sections in concentrated sulphuric acid at 60 - 75°. vic-Glycols 1,2-glycols were cleaved to "dialdehydes" by periodic acid. Those in neutral mucopolysaccharides and glycogen were oxidised completely within 10 minutes. Those in acid mucopolysaccharide required 24 hours or longer before they were oxidised significantly. The engendered dialdehydes have been conjugated with: solutions of homo- and heterocyclic amines (particularly aminoacridine dyes) containing dissolved sulphur dioxide (pseudo-Schiff reagents). Preliminary blocking with methanolic thionyl chloride of the basophilic substance initially present in tissue sections was essential for the observation of specific staining of the engendered dialdehydes. Protein thiol groups nevertheless still interfered with the specificity of the reaction for glycols. Evidence has been accumulated in favour of the following mechanism for this type of reaction which differs from that suggested by Kasten (1958); the engendered dialdehydes take up sulphur dioxide from the dye solution to form sulphenic acids which subsequently combine with the basic dye in a salt-like linkage. Apart from p-aminosalicylic acid, no potentially fluorescent amine has been found to condense with engendered aldehydes in situ, contrary to what Kasten (1958) conjectured and to the numerous reports of fluorescent anil formation of aldehydes in solution. Recent chemical work (Guthrie and Honeyman 1959) has indicated that hydrazines and hydrazides always react with engendered dialdehydes whereas Schiff-type reagents may not, particularly if the dialdehydes are converted to hemiacetal or hemialdol forms. This fact has been confirmed histochemically: salicyloyl hydrazide formed intensely blue fluorescent complexes reliably and more extensively with engendered dialdehydes than do pseudo-Schiff reagents. Moreover, salicyloyl hydrazides did not form any fluorescent artefacts with protein thiol groups after oxidation in periodic acid (Cf.9a). The phenylhydrazones of the dialdehydes formed in situ, unlike those in vitro, did not form formazans except with diazotised aniline in pyrnine. Hydroxyl groups The basophilic properties (towards azure A or coriphosphine) of this group after it had been sulphated were studied. The sulphating efficiencies of various Lewis base complexes of sulphur trioxide were investigated in both acid end basic media. The dioxan-sulphur trioxide dissolved in the weak base dimethylformamide (DMF) or sulphur trioxide dissolved in DMF were very efficient sulphating reagents, even for glycogen which could not be sulphated by acidic sulphating reagents. The amine groups of proteins were sulphated simultaneously by the DMF-sulphur trioxide complex. However, the N-sulphate groups, unlike O- sulphate groups, were easily hydrolysed off by methanolic hydrochloric acid at room temperature. If the amine groups were protected beforehand with suitable blocking reagents, the access of the sulphating complex to hydroxyl groups was completely hindered. Although glycogen in tissue sections can be sulphated normally by sulphuryl chloride vapour, the hydroxyl groups of neutral mucopolysaccharides were not sulphated in the same way: it is Relieved that in the latter, non-basophilic cyclic sulphate groups were formed. Primary hydroxyl groups The selective sulphation and subsequent basophilia (towards azure A or coriphosphine) of this group by the pyridine-sulphur trioxide complex in the DMF was investigated. Lipids Unsaturated lipids were oxidised by peracetic acid to aldehydes and subsequently condensed with salicyloyl hydrazide to form a blue fluorescent hydrazone. Lipids emitted a reddish-pink fluorescence when sections were stained with an acidified solution of protoporphyrin IX. Ketosteroids formed blue fluorescent salicyloyl hydrazones, most of whom, unlike aldehydo-salicyloylhydrazones, were stable towards alkalis. The interfering aldehydes originating from the autooxidation of lipids were blocked with sulphanilic acid. </ol

The kinetics of enzymes in situ, with special reference to lactate and succinate dehydrogenases

The kinetics of two enzyme systems in situ that have been studied with real-time image analysis systems are reviewed in detail. The enzymes are a structurally-bound mitochondrial enzyme, succinate dehydrogenase (SDH) and a soluble cytoplasmic enzyme, lactate dehydrogenase (LDH). The image analysis system is used to capture successive images of a tissue section at constant time intervals whilst it is being incubated on a substrate-containing gel film. The increasing absorbances of the final reaction products in each cell are measured in the successive images as a function of incubation time. The absorbances of the formazan reaction products formed by SDH, for example, in sections of liver determined by such means increase linearly during the first minute of incubation, but non-linearly afterwards. The initial velocities of SDH in single hepatocytes in sections incubated on gel substrate films are calculated from the activities during the first 20 s of incubation. In contrast, the activities of LDH measured in various cell types, including hepatocytes, with the gel film technique increase nonlinearly during the first minute of incubation, but linearly for incubation times between 1 and 3 min. The initial velocities (vi) of LDH in single cells can be, calculated, however from the activities during the first interval, 10 S, of the image capturing sequence. Unfortunately, the experimental errors of the initial velocities of LDH determined in this way are relatively high. To overcome this problem, we have found empirically that the equations vi = al% and vi = 1+aZ0A enable reliable initial velocities (vi) of the LDH reaction in single cells of various types to be calculated using the data of the linear activities for incubation times between 1 and 3 min. Dependence of the initial velocities of the SDH and LDH reactions on substrate concentrations gave the Michaelis constants (Km) and maximum velocities (V,,,). The Km values determined in situ for SDH in hepatocytes and for LDH in various cell types with the gel film technique are in the same order of magnitude as the corresponding values determined biochemically. The constants al, a and Km of LDH are characteristic for each cell type aniseem to be related to the intracellular localization of the enzyme and to its ligand-binding rather than to the different isozyme compositions in various cell types