R. L. Cabrini

Percutaneous absorption of uranium compounds

Percutaneous absorption of soluble and insoluble uranium compounds has been induced in order to obtain information on penetration routes and the tissue injury produced by uranium salts. The high electron density of uranium provided a reliable way to visualize, by electron microscopy, the precise localization of the heavy compounds within the tissues. Few minutes after topical application of uranyl nitrate, dense deposits of uranium were observed at the epidermal barrier level. A few hours later, dense deposits were seen filling the intercellular spaces and were also scattered in the cytoplasm and nucleus. Mortality and body weight measurements indicated the high toxicity of uranyl nitrate and ammonium uranyl tricarbonate; uranyl acetate and ammonium diuranate were less toxic. As no penetration was achieved after uranium dioxide, no variations were detected on these parameters.

Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.

Effects of acute intoxication with uranyl nitrate on bone formation.

The alteration of bone formation by an acute intoxication with uranyl nitrate is demonstrated by histologic and histometric methods. When compared with the controls, intoxicated animals showed a markedly lower density in healing sockets, while bone formation was reduced in healing sockets as well as in metaphyseal bone.

A quantitative microspectrophotometric study of the lead precipitation reaction for the histochemical demonstration of acid phosphatase

SynopsisThe possibility of quantitating microspectrophotometrically the lead precipitation reaction for the histochemical demonstration of acid phosphatase was studied using the following models: sections of rat kidney and human prostate gland, and polyacrylamide films with incorporated prostate gland homogenate. The activity of the polyacrylamide models was previously evaluated by a biochemical method and the specificity of the reaction was controlled with specific inhibitors. Linear relations between cytophotometric readings and enzymatic activity, and between optical densities and different incubation periods, were demonstrated. The results obtained indicate the validity of the microspectrophotometric quantitation for this reaction with direct readings over a wide range of the visible light spectrum.

Microspectrophotometric Study of Oxidative Enzymes in Irradiated Epidermis

SummaryEnzyme variations produced by local x-irradiation on epidermis have been studied by a microspectrophotometric technique.Analysis of enzyme response in function of the post-irradiation time showed clear changes after 2 days using 16 000 rads. A consistent total enzyme content (TEC) increase was found in all irradiation conditions, for the four oxidative enzymes under study (SDH, LDH, G-6-phDH and NADP diaph). The mean enzyme concentration (MEC = TEC/epithelial thickness) increased for NADP diaph and G-6-phDH, while decreasing for LDH and SDH activities. These findings suggest a metabolic adjustment to other oxidative pathways.

Microspectrophotometric study of histoenzymic reactions in rat epidermis subjected to 0-5 Mrads of deuteron radiation.

SummaryThe epidermis of new-born rats was irradiated with a deuteron beam of 26·5 MeV. 500 krads were delivered to 4 mm diameter circular areas in the infraorbicular left side of the heads.Quantitative histochemical determinations of NADP diaphorase, glucose-6-phosphate dehydrogenase, lactic and succinic dehydrogenase were performed, using a microspectrophotometric scanning technique, from 2 to 48 hours after irradiation.There was no clear change with LDh and SDh. A clear increment was detected in both total content and mean concentration of NADP Diaph. and Gl-6-phDh. No evidence of a real enzyme decrease shortly after irradiation could be detected by our techniques. This could imply that no significant direct radiation effects occur to the enzyme molecules in vivo.

Increased osteogenesis in alveolar wound healing elicited by demineralized bone powder

The purpose of this study was to quantitatively assess the role of DBP in postextraction alveolar wound healing. It was sought to establish whether DBP implanted in a postextraction socket 1) activated the process of osteogenesis involved in wound healing, 2) was rejected, or 3) led to infection

Practical applications of the microphotometric quantification of histoenzyme reactions

SummaryThere are many histochemical reactions which can be used for the accurate topographic detection of enzymes in tissues. There are also many reports of applications of these techniques in which the results have been evaluated subjectively. However, only a few of these methods have been the subject of quantification, using different methodologies, most of them based on microphotometry. A critical analysis of these procedures has generally shown that they are valid either relatively or at an absolute level. Nevertheless, the routine application of these methods is not as widespread as one might expect.In this paper, the methodological developments that have taken place in the histochemical assay of some enzymes by microspectrophotometry are reviewed briefly. The enzymes include acid phosphatase, non-specific esterase, succinate dehydrogenase and cytochrome oxidase. These methods have given consistent and useful results when applied to our experimental models of irradiated skin, and thus seem useful for various other applications.The reasons why there have been so few applications of quantitative histochemistry are also discussed together with possible ways of making its use more general.

Ultrastructural Stereology of X-Irradiated Epidermis

Variations in the amount of several epidermal cell components during the process of radiation-induced acute cell death were studied following single local exposures of the tails of rats to a high dose of x radiation. The results were obtained by applying methods of ultrastructural stereology and expressed in relative and absolute volumes, surfaces, and numbers of the most important keratinocyte constituents. The increment of the nuclear volumes, the nuclear membrane surfaces the number of mitochondria, as well as an increase of the volume of filament bundles during the first 48 hr after radiation exposure, reveal an increased metabolic activity. Seventy-two hours after irradiation, this phase of cell hyperactivity is followed by a period in which the degradative phenomena become evident, i.e., an increasing mitochondrial swelling represented by an extreme increment in the volume of the average mitochondrion, the appearance of a significant amount of lipid droplets, and the cessation of protein accumulation in the form of cytoplasmic filaments. These statistically significant changes indicate that during the early stage of acute radiation reaction in the epidermis, the keratinocytes exhibit an increased activity which after 48 hr is surpassed by degenerative phenomena leading to ultimate cell death and skin ulceration.

Quantitative histochemistry of esterases

Microspectrophotometry of enzyme reactions is a practical, fast and relatively easy method for studying enzyme activity in situ. The conditions of its applicability should be carefully studied for each of the quantitative techniques to be used. So far, they have only been established for a limited number of histo-enzymatic reactions. Using a method similar to one employed for studying the validity of acid phosphatase quantitation (Cabrini et al., 1975), we have investigated a technique for demonstrating esterases in a histochemical model system. Non-haemolized bovine serum, kept at -20~ was used as the source of esterase. Its esterase activity (3650 IU) was determined with the method of Burlina & Galzigna (1972), which measures the activities of aryl esterase and aliesterase. A 0.25 M ~-naphthyl acetate solution in a total volume of 2.5 ml, was used as substrate. The histochemical models consisted of a solution of different serum concentrations incorporated in an acrylamide preparation in 0.1 N phosphatase buffer, pH 7.2. The films were prepared according to Van Duijn et aI. (1967) and Van Duijn & Van der Ploeg (1972) by mixing acrylamide and bis-acrylamide with a solution of ammonium persulphate and TEMED, except that the acrylamide concentration was reduced to 7.5% in order to reduce the unspecific background staining usually produced when the films are introduced into the incubation solution. Using this concentration it is also probable that a higher substrate turnover is achieved in the models. The decrease in the polyacrylamide concentration did not, in our experience, produce an appreciable loss of incorporated enzyme (se e below). The polymerization was performed at 4~ between two glass plates with a 240 -+ 5 ~m width spacer. Gomoris (1952) technique for demonstrating unspecific esterases was carried out on the polymerized films using ~-naphthyl acetate as substrate and Fast Blue B as coupling agent. All the polyacrylamide films containing different concentrations of serum were processed simultaneously in order to avoid handling errors. Microspectrophotometric measurements were performed in a Zeiss Cytoscan. The total magnification was x 14.3 and the wavelength 540 nm. The microspectrophotometer can be used for measuring areas as small as 0.78/am 2 , which would amount to an assayed volume of 6.24/am 3 if, for example, 8/~m-thick sections are used. With polyacrylamide models, the homogeneity of the reaction permitted the more comfortable measurement of larger circular areas (314/~m 2). The homogeneity of the reactions in the films was demonstrated by carrying out several measurements at different areas of each model. Using this procedure, the same optical density values were obtained. Fig. 1 shows the optical density of the final reaction product as a function of the incubation time for each serum concentration investigated. These results indicate the validity of the microspectrophotometric quantitation of the esterase reaction. The