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PubMed1994

Kinetic microphotometric evaluation of in situ hybridization for mRNA of slow myosin heavy chain in type I and C fibres of rabbit muscle.

The present study was undertaken in order to test the possibility of microphotometric evaluation of in situ hybridizations. The histochemical detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a model. Scanning densitometry of Northern blot hybridizations showed that the detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. These findings were the basis for elaborating a comparative microphotometric evaluation of in situ hybridization in tissue sections by measuring the reaction rate of the alkaline phosphatase-catalysed formazan production. Relative amounts of HCI mRNA were thus determined by comparing reaction rates instead of by single point microphotometry. This method was applied to studies on the distribution of HCI mRNA in different fibre types of normal rabbit muscles and and muscles undergoing fast-to-slow fibre transformation in response to low-frequency stimulation. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjacent to the sections processed for in situ hybridization. On the average, type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded for C fibres. According to the pronounced scattering of the values measured in single C fibres, these fibres represented a heterogeneous population in the transforming muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Glucose-6-phosphate dehydrogenase is enriched in oligodendrocytes of the rat spinal cord. Enzyme histochemical and immunocytochemical studies.

Glucose-6-phosphate dehydrogenase (G6PD) was localized in rat spinal cord by catalytic enzyme histochemistry and immunocytochemistry. G6PD detected by either method was shown to be strongly enriched in cell bodies and processes of oligodendrocytes, whereas in the compact myelin G6PD was not detected. The enzyme histochemical procedure for the demonstration of G6PD was also adapted for microphotometric measurements of G6PD activity in the spinal cord white matter. There was a linear relationship between G6PD activity and section thickness up to 14 microns and between G6PD activity and reaction time up to 5-6 min as demonstrated by kinetic and end-point measurements. Significantly lower activities were measured in end-point measurements than in kinetic measurements because of formazan loss during rinsing. Methoxyphenazine methosulphate as an exogenous electron carrier and sodium azide as a blocker of the respiratory chain significantly increased the demonstrable G6PD activity. The Km was 0.62 mM and the Vmax 3 mumol glucose-6-phosphate/cm3 wet tissue and per min at 25 degrees C. It is concluded that G6PD in oligodendrocytes may be important for the generation of NADPH required for lipid biosynthesis related to myelogenesis, and reduction of glutathione required for protection of membrane sulphydryl groups.

Animals↗
PubMed1993

Distribution of NADPH-diaphorase activity in rat paravertebral, prevertebral and pelvic sympathetic ganglia.

Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive 'baskets' of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.

Animals↗
PubMed1995

Reperfusion injury to endothelial cells after cold storage of rat livers: protection by mildly acidic pH and lack of protection by antioxidants.

Lethal reperfusion injury to sinusoidal endothelial cells occurs after cold ischemic storage of livers and may be responsible for liver graft failure from storage injury. Here, we evaluated potential mechanisms underlying this reperfusion injury. In rat livers stored in Euro-Collins solution for 24 h and reperfused with Krebs-Henseleit bicarbonate buffer, nonparenchymal cell killing showed periportal predominance as assessed by nuclear staining with trypan blue. In livers reperfused in the retrograde direction, the lobular distribution of cell killing was reversed, indicating that cell killing was more rapid in oxygen-rich upstream regions. However, antioxidants, including allopurinol, desferrioxamine, catalase, superoxide dismutase, superoxide dismutase plus catalase, and U74006F, did not reduce cell killing. Similarly, reperfusion with anoxic buffer did not prevent lethal injury. Antioxidants and anoxic reperfusion also did not improve cell viability in livers stored in UW solution. Nevertheless, superoxide generation, as identified by formazan formation from nitroblue tetrazolium, was increased in Kupffer cells after lives storage and reperfusion as compared to unstored livers. Acidification of the reperfusion buffer from pH 7.4 to pH 7.15 reduced overall nonparenchymal cell killing from about 40% to 10%. Moreover, a pH gradient developed across the liver lobule during reperfusion with the effluent 0.2-0.4 pH units more acidic than the influent. This intralobular pH gradient appears to account for the relative sparing of cells in more acidic downstream regions of the lobule. Lower temperatures of reperfusion also reduced lethal injury. In conclusion, Kupffer cells generated superoxide after perfusion of stored rat livers, but formation of oxygen free radicals did not appear to contribute to lethal reperfusion injury to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

2,3,5-Triphenyl tetrazolium chloride (TTC) reduction as exponential growth phase marker for mammalian cells in culture and for myeloma hybridization experiments.

Triphenyl tetrazolium chloride in vitro reduction by cells produces a red formazan pellet which can be extracted and measured. We have shown that such reduction is associated with animal cell growth, and particularly with the specific growth rate, so the measurement of Triphenyl tetrazolium chloride reduction is proposed as a physiological marker of the exponential growth of cultured cells. Further application of this technique is shown using this Redox reaction for estimating plasmacytoma fusion potential for hybridoma cell line production.

Animals↗
PubMed1975

Rat liver alcohol dehydrogenase isozymes: influence of infection with Trypanosoma.

A study has been made of changes in alcohol dehydrogenase (ADH) isozymes in livers of rats infected with the following trypanosomes: Trypanosoma cruzi, T. rhodesiense, T. congolense, and six geographical isolates of T. lewisi. A control group of the same age was injected with saline, and another left uninjected. Liver homogenates were electrophoresed in noble agar gels followed by formazan staining according to the method of Ursprung and Leone (1965). Of a total of 25 replicates, 15 showed that the level of rat liver ADH isozymes was similar in the uninjected and saline injected animals; 6 replicates showed a slightly higher level of ADH isozymes in uninjected than saline injected control rat livers, and 4 replicates had a higher level in saline injected than in the uninjected. With either 1-octanol or ethanol as substrate, a marked elevation of ADH activity was observed beginning with day 7 post inoculation in rat livers infected with T. cruzi, T. lewisi, and T. congolense, or as early as day 4 post inoculation with T. rhodesiense. The elevation persisted 59 days or more in rat livers infected with T. lewisi, but rats infected with the pathogenic species usually died 5 to 14 days after infection. The elevation of ADH isozymes has also been demonstrated spectrophotometrically for the first 5 minutes of reaction time, using acetaldehyde as substrate. Also observed in livers infected with pathogenic species and certain strains of T. lewisi were: (1) ADH isozyme pattern changes involving an elevated level of certain isozymes only, and (2) abnormal isozyme mobilities.

Alcohol Oxidoreductases↗
PubMed1977

Light and electron-microscopic studies on guinea pig hearts after perfusion with nitro-blue tetrazolium (NBT).

Guinea pig hearts isolated by the Langendorff technique were perfused with a nitro-blue tetrazolium salt solution. In spite of a homogeneous dark-blue staining at the macroscopic examination, light-microscopic studies showed a spotty staining of the myocardial fibers. Since no microscopic alterations were found which could explain the unstained areas, ultramicroscopic studies were undertaken. At ultrastructural level, striking alterations of myocardial cells were observed. They consisted of mitochondrial lesions, alterations of the transversal tubuli, and intercalated discs. Arteriolar narrowing and capillary edema were found, suggesting that pathological changes in the microcirculation could be a possible reason for the spotty staining of the myocardial fibers. Some speculations on the nature and localization of the formazan granules are also reported.

Animals↗
PubMed1978

Endotoxin stimulated nitroblue-tetrazolium (NBT)-test in patients with hypoparathyroidism, pseudohypoparathyroidism and other forms of hypocalcemia.

Endotoxin-stimulated NBT-tests were carried out in 15 patients with hypocalcemia of varying etiology and in 14 normocalcemic children free of infection. In the control group the formazan cell percentage (FCP) was 73.8 +/- 1.6% (range 63% to 83%). In 5 patients with hypoparathyroidism or pseudohypoparathyroidism the FCP before treatment was lower than normal. Vitamin D3 therapy produced a rapid increase of serum calcium but normalisation of NBT-test was only achieved after a latent period of one or more months. Patients with hypocalcemic rickets and children with an acute relapse of the nephrotic syndrome also showed abnormal results. The clinical significance of the NBT-test in hypocalcemic conditions is discussed.

Adolescent↗
PubMed1977

[Light- and electronmicroscopic investigations on ultrastructure and enzyme distribution in the external sulcus of the cochlea (author's transl)].

The ultrastructure of the external sulcus of the cochlea of young healthy guinea pigs and hamsters was investigated as well as the distribution of lactate dehydrogenase (LDH) in this region. The demonstration of LDH activity was carried out as follows. The unfixed and undecalcified cochlea was cut in 80 micron thick frozen sections, which were incubated immediately in the reaction mixture for LDH and subsequently fixed with paraformaldehyde. For electron microscopy the usual procedure was applied. The epithelial cells of the external sulcus show deep reaching extensions into the vessel-rich connective tissue of the basal part of the spiral ligament. These roots are bound together into root bundles surrounded by the basement membrane and fibrills. The root cells differ in their ultrastructure. The light cells, which have been described previously, contain a number of stretched mitochondria and only few other cell organels. Beside these, dark cells can be found (Fig. 2). Their cytoplasm shows high contrast and contains many mitochondria, ribosomes, rough endoplasmatic reticulum and Golgi apparatus. In the dark cells big vasuoles can be seen. All these structures indicate a high rate of synthesis in the dark cells. The root cells are linked together by membranous foldings, which diverge in some parts, forming intercellular spaces in which small cell protrusions are found. Other intercellular gaps can also be detected building a system of small channels. It is possible that these structures serve for extracellular transport. The LDH activity, which is demonstrated by formazan deposits can be exclusively found in the root cells. This could be demonstrated in light microscopic semithin sections (Fig. 3) and with electron microscopy (Fig 4). The possible role of LDH in the root cells is discussed.

Animals↗
PubMed1975

The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide.

The indigogenic method for aminopeptidase of Pearson et al. (1963) was critically evaluated. The localization obtained with it is not correct due to diffusion artifacts. Ferricyanide cannot be used as an oxidation agent. Based on experiments with other oxidation agents (phenazonium methosulfate, nitro BT, tetranitro BT) a new method was devised. The recommended incubation medium contains 0.9 mM L-N-(5-bromoindol-3-yl) leucinamide hydrobromide (chloride), 0.73 mM tetranitro BT, 0.27 mM phenazonium methosulfate and 0.67 M phosphate buffer pH 7.4. The enzyme activity is indicated by the deposition of tetranitro BT formazan. Results with this method in rat kidney, jejunum, liver, lung, and submaxillary gland, in monkey kidney and jejunum, and in human jejunal biosies are almost identical with those obtained with L-leucyl-4-methoxy-beta-naphthylamide applied in a simultaneous azocoupling procedure. The given principle of the demonstration of aminopeptidase activity with an indolylamine substrate deserves a further exploration in the demonstration of peptidases "in situ" both on optical as well as electronmicroscopical levels.

Aminopeptidases↗
PubMed1978

[Azoindoxyl methods for the investigation of hydrolases. III. Histochemical studies of beta-D-N-acetylglucosaminidase (author's transl)].

The suitability of various azoindoxyl procedures for the light microscopical demonstration of beta-N-acetylglucosaminidase is described. The incubation media tried consist of 0.5 mg N-Acetyl-(5-bromindol-3-yl)-beta-D-glucosaminide (5-Br-3-indolyl-beta-D-N-acetylglucosaminide; 1 mg dissolved in 0.05 ml N,N-dimethylformamide) in 1 ml 0.1 M citric acid phosphate buffer, pH 4.5 or 5. 0.02 ml hexazotized p-rosaniline or new fuchsine/ml or tetrazotized BAXD or 0.5 mg Fast Blue B or Garnet GBC/ml were employed as a coupling reagent. Hexazotized new fuchsine yields the best results independent on the pretreatment of the tissue and the organ investigated followed by hexazonium-p-rosaniline. Compared with the azo dye method using naphthol AS-BI beta-D-N-acetyl-glucosaminide as a substrate and hexazotized p-rosaniline or new fuchsine or tetrazotized BAXD for simultaneous coupling especially the azoindoxyl technique with the new fuchsine is equvialent or superior. When the indolyl glucosaminide is used in the indigogenic, tetrazolium or metal precipitation method the results are mostly inferior with the exception of the tetrazolium reaction using BSPT. However, the main advantage of the azoindoxyl procedure is that at least the azoindoxyl dye deriving from hexazotized p-rosaniline can be osmificated and withstands treatment with organic solvents and resins. Therefore, the reaction product seems to be suitable for the electron microscopic demonstration of glucosaminidase. Among the other reaction principles this can reliably be achieved only with BSPT as a tetrazolium salt followed by osmification of its formazan. After fixation of blocks of tissue in form- or glutaraldehyde beta-D-N-acetylglucosaminidase can be localized with 5-Br-3-indoxyl-beta-D-N-acetylglucosaminide as a substrate and hexazotized new fuchsine for simultaneous coupling in the lysosomes of many rat organs.

Acetylglucosaminidase↗
PubMed1979

Quantitative succinate-dehydrogenase histochemistry. III. Variations in histochemical succinatedehydrogenase activity in different cross-sections of the same muscle fibre.

The variation in histochemical SDH-activity at different levels in the same muscle fibre was determined in muscle fibre cross-sections both by visual classification and quantitative determination of the formazan-deposits. This work resulted in a confirmation of the earlier micro-biochemical studies of Spamer AND Pette (1977, 1979) and Lowrey et al. (1978) that the activity of enzymes of the citric acid cycle is not homogeneously distributed in a muscle fibre over its entire length. In addition it is shown that the observed variations in histochemical SDH-activity strongly interfere with the visual muscle fibre typing. Some of the possible causes for these variations in histochemical SDH-activity (section-thickness, presence of the motor-endplate) and the implications of these findings for the relation between histochemical characteristics and functional properties of the muscle fibres are briefly discussed.

Animals↗
PubMed1978

[Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)].

Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.

Acetylglucosaminidase↗
PubMed1982

A new method for the enzyme cytochemical staining of individual cells with the use of a polyacrylamide carrier.

A new method for enzyme cytochemical studies on individual cells is developed. Cells are incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in a cytochemical medium. Five different kinds of individual cells, i.e. isolated rat hepatocytes, isolated mouse oocytes, cultivated human fibroblasts, rat thymocytes and human blood cells are used for testing the applicability of this method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT. The incorporation technique solves at least some of the problems occurring with enzyme cytochemistry on single cells. The morphology of the cell is very well preserved, the formazan precipitation due to enzyme activity occurs entirely within the cell cytoplasm, the nothing dehydrogenase activity can be kept very low and the loss of cells is completely prevented with all cell types used.

Acrylic Resins↗
PubMed1981

Visualization of isozymes which generate inorganic phosphate.

A sensitive, new enzymatic method for the detection of isozymes which liberate inorganic phosphate (or pyrophosphate) is described. The new method for the detection of phosphate differs from the established method for nucleoside phosphorylase by the substitution of one reagent. The new enzymatic method, compared to existing methods for the detection of phosphate on electrophoretic gels, is advantageous due to its sensitivity and generation of a nondiffusible formazan chromogenic product.

Alkaline Phosphatase↗
PubMed1978

Studies on the optimalisation and standardisation of the light microscopical succinate dehydrogenase histochemistry.

The present investigation was undertaken in order to establish an optimal tissue pretreatment and an optimal incubation medium for the histochemical demonstration of succinate dehydrogenase (E.C. 1.3.99.1). The investigations were performed on steroid producing (testicle, adrenal gland) and steroid dependent (Fallopian tube) tissues. We studied the influences fo formalin fixation, acetone, magnesium ions, cyanides, electron carries (phenazine methosulfate, menadione coenzyme Q10), osmolarity, substrate concentration and inhibitors (oxalacetate, oxalate, malonate, 4-chloromercuribenzoic acid). The following procedure yields blameless morphological integrity and enzyme localization as well as optimal SDH-activity: Freezing of tissue cubes (diameter less than 5 mm) in propane cooled with liquid nitrogen or in melting freon. Incubation of 5 micrometer cryostat sections in narrow jars in the following medium (38.5 ml):--10 ml of 0.2 M sodium phosphate buffer pH 7.6 (52 mM).--18 mg tetranitro-BT in 0.5 ml dimethylformamide and aqua bidest. ad 10 ml (0.5 mM).--2.6 mg KCN in 16 ml aqua bidest. (1 mM).--540 mg succinate (disodium salt, hexahydrate) in 2 ml aqua bidest. (52 mM).--3 mg PMS (phenazine methosulfate) in 0.5 ml aqua bidest. (0.25 mM). The incubation medium has an osmolarity of 440 mosm. The incubation is carried out for 10 min at 37 degree C in darkness. To avoid non specific formazan deposits in lipid containing tissues a preincubation of the cryostat sections in 100% acetone at--22 degree C or--40 degree C for 7--10 min and an incubation time of 20--30 min is recommended. Control incubations adduced proof at the specificity of the SDH demonstration. Parallel incubation without PMS in order to determine indirectly the content of endogenous CoQ10 is further recommended.

Adrenal Glands↗
PubMed1978

Methodological aspects of the histochemical localization and activity of some cerebellar dehydrogenases.

In a detailed study focused on the methodological problems in dehydrogenase histochemistry [e.g., fixation, diffusion of enzymes and of reduced inermediates, conversion of NADPH and NADP to NADH and NAD, respectively, penetration of tetrazolium salt and formazan substantivity, 'nothing dehydrogenase' reaction, use of exogenous CoQ10 and of flavoprotein substitute (PMS)], the distribution and activity of succinate dehydrogenase, NAD(P)H-tetrazolium reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase (H and M types), and of L-glutamate dehydrogenase (E.C.1.4.1.2 and E.C.1.4.1.3) have been investigated in the rat cerebellum. It was evident from the study that reliable results could only be obtained if all the aforementioned factors had been considered. The image of actual concentration of SDH in the neuropil of the molecular layer could only be recorded by adding CoQ10, while other structures exhibited greater balance between SDH and endogenous mitochondrial CoQ. Contrary to previous studies, a reversed localization of the activity of G-6-PDH and LDH was noticed. The elements of molecular and Purkinje layers were rich in G-6-PDH, while the granular layer was nearly depleted. The actual level of LDH could only be recorded if NADH-tetrazolium reductase was bypassed with PMS. The H and M types of LDH coexisted in the three cortical layers, the H type being prevalent and the M type attaining its highest level in synaptic glomeruli followed by the structures of the molecular layer and the Purkinje cells. High activity of GDH was noticed in Bergmann glia followed by synaptic glomeruli, while most other structures showed weak to moderate activity. The two GDH types coexisted in all structures showing activity, except for Bergmann cells, which only showed presence of the E.C. 1.4.1.3 type. Furthermore, Bergmann glia was exceptional by showing no activity of SDH and LDH, but strong activity of G-6-PDH and NADPH-tetrazolium reductase. The granular cells were exceptional by showing weak or no activity of all enzymes in question.

Animals↗
PubMed1988

Histochemical technique for the demonstration of pyruvate kinase activity.

We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP and D-glucose to yield D-glucose-6-phosphate and ADP. The D-glucose-6-phosphate is oxidized by exogenous and endogenous D-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP+, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Sodium azide and amytal are included to block electron transfer to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal muscle. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.

Adenosine Diphosphate↗