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At least 19 recordsLinked to original sources
PubMed1992

Modified cerium-based and Gomori-based cerium methods for light microscopic phosphatase histochemistry: the cerium-perhydroxide-diaminobenzidine-nickel (Ce-H2O2-DAB-Ni and Ce/Ce-H2O2-DAB-Ni) two-step procedures.

Some modified cerium-based and Gomori-based cerium methods for the demonstration of phosphatase activity in cryostat sections were described. Dextrane as stabilizing agent was added to the incubation media for ATPase, 5'-Nase, and TPPase. The oxidation of the CeIII-phosphate primary reaction product in a separate step by H2O2 before the DAB incubation yielded an increase of the intensity of the DAB-based visualization reaction (Ce-H2O2-DAB-Ni two step method). The sensitivity of the histochemical enzyme reaction was remarkably increased if CeIII-ions were employed as amplifying agent (Ce/Ce-H2O2-DAB-Ni two-step method). A new suitable DAB medium consisting of 0.015% DAB, 2.0% Ni-sulphate, 15% methanol, and 0.005% H2O2 in 0.1 mol/l acetate buffer, pH = 5.2, was used. The disadvantage of diffuse background staining has been overcome by addition of 15% methanol to the DAB solution. Electrovalently bound CeIII (cerophilia) was removed by treatment of the incubated sections with CeIII-citrate (CeIII-complexation). In addition, a novel membrane floating incubation for sections is proposed. At present, the modified procedures are some of the most sensitive modes for the demonstration of phosphatases and improve the earlier described cerium-DAB one-step technique (Halbhuber et al. 1988b).

Animals↗
PubMed1991

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections.

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.

3,3'-Diaminobenzidine↗
PubMed1985

Light microscopical localization of enzymes by means of cerium-based methods. III. Visualization techniques for cerium phosphate.

Cerium-based methods are more and more used for the electron microscopic localization of phosphohydrolases. By means of the earlier described Ce-Pb-technique, it is possible to localize these enzymes on the light microscopical level. The final product of this reaction is lead sulfide. In addition to this technique, other visualization methods for the light microscopically not visible cerium phosphate are proposed. 3 successful techniques are described in the report: The cerium perhydroxide reaction. By means of H2O2 cerium phosphate is converted into cerium perhydroxide which has an orange-yellowish colour. The manganese dioxide reaction with the conversion of cerium phosphate into cerium oxalate, which is able to reduce permanganate into the hardly soluble brown coloured manganese dioxide. A silver technique (Ce-Pb-AgS-method), which is characterized by the conversion of cerium phosphate into lead phosphate and in a second step to lead sulfide. At the active sites of the lead sulfide, the reduction of silver ions takes place. The reduced silver is converted in a final step into silver sulfide. The enzyme activity is represented by a brown or black coloured staining.

Acid Phosphatase↗
PubMed1985

Light microscopical localization of enzymes by means of cerium-based methods. I. Detection of acid phosphatase by a new cerium-lead-technique (Ce-Pb-method).

Cerium-III-ions are more and more used as capturing reagent and opaque marker for the electron microscopic localization of a number of H2O2-generating enzymes as well as phosphohydrolases. Contrary to its advantages over common lead methods in the histochemical detection of enzyme activities at the electron microscopic level, cerium-based methods proved to be a failure for light microscopic investigations. Therefore, our cerium-based method for the ultrahistochemical detection of acid phosphatase was developed for further observations at the light microscopic level. The principle of that new Ce-Pb-method is the conversion of light microscopic not visible cerium phosphate into lead phosphate by the secondary capture reagent alkaline lead citrate. Finally, the lead phosphate can be visualized as lead sulfide in the section. The Ce-Pb-method in its finally proposed manner was compared with a common lead method and showed a range of advantages. Because of that fact, the new Ce-Pb-method is recommended for a broader use in histochemistry, e.g. for the light microscopic enzyme investigation parallel to ultrahistochemical preparations.

Acid Phosphatase↗
PubMed1987

Light microscopical localization of enzymes by means of cerium-based methods. V. Optimization of the cerium-lead (Ce-Pb)-technique for alkaline phosphatase.

A modification of the earlier published cerium-based technique for histochemical detection of alkaline phosphatase activity at light microscopical level (Halbhuber and Zimmermann 1985) is described. The reduction of the s-collidine concentration from 200 mmol to 50 mmol, increase of cerium ion concentration rom 1 mmol to 5 or 10 mmol, and sucrose concentration from 7.5% to 15% at increased from pH = 9.0 to 9.5 less than or equal to 9.9 in the incubation medium led to a high intensification of the histochemical reaction. The brush borders of the rat kidney (especially of the epithelial cells of the primary convoluted tubules) and of the enterocytes demonstrate black-brown tinged and precisely localized final reaction products. Moreover, a simplification of the histochemical procedure by employment of postfixed cryostat sections (small intestine) instead of the time consuming perfusion fixed material (kidney) is presented. Several fixatives were also tested. Nakane's periodate-lysine-paraformaldehyde (PLP) or the periodate-lysine-glutaraldehyde (GLP) fixations are superior to the classical glutaraldehyde/paraformaldehyde double fixation. The proposed optimized cerium-based techniques are recommended for a broad use.

Alkaline Phosphatase↗
PubMed1991

A new visualization principle of cerium phosphate reaction product of phosphatases in cryotome sections for light microscopy--the cerium-oxalate-osmium-silver (Ce-Ox-Os-Ag) method.

A new visualization principle for the detection of cerium phosphate reaction product of phosphatases in light microscopy is described. The new mode is based on the conversion of cerium phosphate into cerium oxalate. The latter is able to react with OsO4. In this way osmium black is formed, staining enzymatic activity sites grey or greyish-black. Utilizing the argyrophilia of osmium black, the staining contrast could be remarkably intensified by posttreatment with Ag-proteinate (Ce-Ox-Os-Ag procedure). This procedure is of similar sensitiveness in comparison to the DAB-Ni method, proposed earlier. The advantages are a very pale background staining and a complete suppression of the cerophilia. Moreover, it substitutes to DAB, a probable potent carcinogen. The method is time-saving when the processing of the reactions was stimulated in a microwave oven.

Animals↗
PubMedDate not supplied

Cerium tissue/organ distribution and alterations in open field and exploratory behavior following acute exposure of the mouse to cerium (citrate).

Cerium chloride (1:3 complex with sodium citrate) was administered to male Swiss mice (6 to 8 weeks old) either intragastrically or subcutaneously at the 7 day LD5 or LD25 level. Open field behavior (ambulations, rearings) was quantified and tissue/organ Ce levels determined at 4 hr., 1, 3 or 7 days post administration. Via the i.g. route, Ce was poorly absorbed resulting in no observable behavioral alterations and no correlations between behavior and tissue levels. Via the s.c. route, Ce significantly (p less than 0.05) depressed ambulations and rearings, mainly at short times following administration of the LD25 dose. Analogous findings were obtained in a separate study of exploratory behavior. There was a significant (p less than 0.05) correlation between open field behavior and tissue Ce levels: thus, rearings were inversely correlated with lung, stomach, cerebrum, cerebellum and brain stem Ce levels and ambulations were inversely correlated with liver, kidney, lung, blood, stomach, intestine, muscle, cerebrum, cerebellum and brain stem levels. Spleen Ce levels and ambulations were directly correlated and it is speculated that the spleen may serve a protective function in the case of Ce intoxication.

Administration, Oral↗
PubMed1976

Cerium nitrate: a new topical antiseptic for extensive burns.

The wounds of 60 burned patients were treated topically with cerium nitrate, which was applied either as a cream or in aqueous solution. Cerium nitrate has a potent antiseptic effect in human burn wounds, especially against gram negative bacteria and fungi. Pseudomonas aeruginosa was recovered from the wounds infrequently and never predominated. Fungi were practically never found. No patient treated with cerium developed a necrotizing wound infection. Analysis of the detailed bacteriological data indicated that, in contrast to previous results with use of the nitrate or sulfadiazine salts of silver, when gram negative species predominated, the flora tended to be predominantly gram positive when cerium was used. Therefore, some patients were treated simultaneously with cerium nitrate and silver sulfadiazine; this resulted in an even more efficient suppression of the wound flora than was observed previously with either cerium alone or silver salts alone; results with the simultaneous topical therapy in patients with injuries that previously were uniformly lethal were excellent. No toxicity attributable to the use of cerium was observed, although one instance of methemoglobinemia due to nitrate was documented. The adsorption of topically applied cerium essentially is nil. The use of cerium nitrate was associated with a nearly 50 percent reduction in the anticipated death rate. Cerium nitrate is a promising new topical antiseptic agent for the treatment of burns, particularly when it is used in combination with silver sulfadiazine.

Adolescent↗
PubMed1992

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.

Alkaline Phosphatase↗
PubMed1976

Differences in liver weight, mortality in cerium-treated mice and 144Ce levels in blood, liver, urine and faeces at various intervals after treatment with nafenopin and pregnenolone 16-alpha-carbonitrile (PCN).

Pretreatment with nafenopin and pregnenolone-16-alpha-carbonitrile (PCN) decreased the acute lethal toxicity in mice induced by cerium chloride (12 mg/kg resp. 15 mg/kg). Treatment with nafenopin and PCN increased the relative liver weight. More cerium accumulated in the liver of nafenopin- and PCN-treated animals than in controls during the first hours after injection. After 14 days the amount of cerium in the livers of nafenopin-treated animals was significantly higher. The concentration of cerium as a percentage of the injected dose calculated per mg liver was lower during the first day in the nafenopin group and higher in the PCN group than in the control group. After 6 days cerium concentration decreased significantly less in the livers of the nafenopin group. Cerium concentration in the blood was significantly lower in the nafenopin group during the first day. Only small amounts of the injected cerium were excreted in the urine, and no difference between treated and untreated animals was found. The amount of cerium excreted in faeces was somewhat smaller in the nafenopin-treated animals during the whole experiment.

Animals↗
PubMed1986

Light microscopical localization of enzymes by means of cerium-based methods. I.V. Optimization procedures for acid phosphatase.

The earlier described cerium based histochemical reaction for acid phosphatase [Ce-Pb-reaction, Zimmermann and Halbhuber (1985)] was optimized. The target tissues (kidney, intestine) were fixed by perfusion with glutaraldehyde in cacodylate or piperazine buffer in anesthetized animals. Postfixation of prefixed sections is not advantageous because of the detectable repressing of the enzyme activity. Moreover, the employment of unfixed cryostat sections, which were postfixed, was always connected with a complete abolition of the acid phosphatase activity. The optimal concentration of the primary capture cerium III chloride in the incubation medium is about 1 mmol. Lower concentrations lead to an incomplete histochemical detection of phosphatase activity in lysosomes. The treatment of cryostat sections of perfusion fixed tissue with borohydride (diminution of aldehyde induced cross links) or with dimethylsulfoxide (extraction of lysosomal materials or the well known vehicle property) brought about an improvement of the penetration capacity for cerium-III-cations into the target structures. After conversion of the cerium phosphate (primary specific reaction product) into cerium perhydroxide, oxalate or fluoride, the Ce-Pb-reaction was negative. Therefore, these blocking reactions represent specific inhibition controls, which indicates the formation and presence of cerium phosphate. On the basis of these reactions it is possible to check the specificity of the histochemical Ce-Pb-reaction for phosphatase activity in sections.

Acid Phosphatase↗
PubMed1989

Intracellular localization of cerium. A microanalytical study using an electron microprobe and ionic microanalysis.

Radioactive cerium is a nuclear toxicant. Metallic cerium is used in industry. Aspects of the intracellular metabolism of this element were studied following intraperitoneal injection and aerosol exposure in rat. Two microanalytic methods, an electron microprobe and ionic microanalysis, enabled the sites of incorporation and the process of intracellular concentration of cerium to be determined in the liver, lung, kidney, bone marrow and bone tissue. The very high sensitivity of ionic analysis enabled very low concentrations of cerium to be detected with a spatial resolution of 0.5 microns. Microanalysis by electron microprobe permitted: (i) the lysosomal localization of cerium to be determined; and (ii) the lysosomal coprecipitation of cerium with phosphorus to be demonstrated. Results are discussed in relation to aspects of radiological protection.

Administration, Inhalation↗
PubMed1992

Effect of cerium nitrate-silver sulphadiazine on deep dermal burns: a histological hypothesis.

Although there are excellent clinical results from using cerium nitrate plus silver sulphadiazine in the treatment of deep dermal burns in our burn centre, its mechanism of action remains unexplained. We set up a prospective study to establish a hypothesis which could explain the formation of the typical leathery crust. Burns treated with cerium nitrate plus silver sulphadiazine and silver sulphadiazine alone were compared histologically and metallurgically. Application of cerium nitrate plus silver sulphadiazine on deep dermal burns caused superficial connective tissue calcification with few deep dermal changes. Cerium penetration was low and silver was never detected intradermally. The precipitation of cerium and pyrophosphate destroys the calcification inhibitory function of the latter, creates multiple calcification nuclei and leads to superficial dermal calcification. No calcium was detectable in biopsies from silver sulphadiazine-treated wounds. No major toxicological side-effects were seen during this study. The clinical results were favourable, as have been reported previously.

Adolescent↗
PubMed1990

Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method.

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.

3,3'-Diaminobenzidine↗
PubMed1977

Topical chemotherapy for burns using cerium salts and silver sulfadiazine.

These experimental data indicate that cerium compounds exert a measurable antimicrobial action in vitro. In vivo the simultaneous use of cerium sulfadiazine and silver sulfadiazine was more effective than silver sulfadiazine alone or the combination of cerous nitrate and silver sulfadiazine. When cerous nitrate was used clically, gram-positive bacteria predominated. In contrast, wounds exposed to silver salts alone harbor a predominately gram-negative flora. The combination of cerous nitrate and silver sulfadiazine appears to provide a broad spectrum inhibitory to both types of organisms. It is apparent that a sampling problem exists in any attempt to monitor wounds that may exceed a square meter in extent. These bacteriologic data pertain only to the surface flora. The need to culture tissue samples of burn wounds has been emphasized. Our experience, however, is that invasion of deep tissue without dense surface colonization, greater than 10(5), is infrequent. Furthermore, fragments of eschar were submitted regularly for culture; those results confirmed the surface culture findins. The characteristic yellow-green color of cerium nitrate treated eschars may results from oxidation of trivalent cerium to yellow ceric ions. Free silver and sulfadiazine ions are available also in small amounts because of limited ionization of the highly insoluble silver sulfadiazine. This might promote the in vivo formation of cerous sulfadiazine and would provide a continuous source of ionic cerium for microbial inhibition. The modification of silver sulfadiazine cream by incorporating cerous nitrate into it strikingly enhances its topical antiseptic effect in burn wounds without increasing toxicity.

Administration, Topical↗
PubMed1988

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry.

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.

3,3'-Diaminobenzidine↗
PubMed1992

Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.

Animals↗