Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis.

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.

Experimental study to test the potential tumor promotion effect of a tooth bleaching agent.

Carbamide Peroxide is routinely employed as a whitener for tooth enamel. Oral mucosa protection is recommended to avoid inflammatory reactions. Experimental work has demonstrated its irritative effect on gastric mucosa when swallowed. The activity of certain oxidizing agents as tumoral promoters has been demonstrated and associated to their capacity to induce hyperplasia. Within this context it seemed of interest to assess the possible action of carbamide peroxide as a tumoral promoter in oral mucosa with or without a precancerous condition. Its action was tested in 2 models which are highly sensitive to chemical cancerization: a) Dorsum skin or SENCAR mice treated with carbamide peroxide daily or twice a week with or without prior initiation with dimethylbenz(a)anthracene (DMBA). Control mice were submitted to the standard carcinogenesis protocol, i.e. initiation with DMBA and promotion with 12-O-tetradecanoyl phorbol acetate (TPA). b) Hamster cheek pouch submitted to topical application of carbamide peroxide 3 times a week with or without prior initiation with DMBA, hamster cheek pouch submitted to repeated topical application of DMBA as a complete carcinogen: application twice a week in the control group and identical treatment + 1 weekly application of carbamide peroxide to evaluate its capacity to enhance the process. The effects were assessed between 1 and 14 weeks of treatment at different intervals for the different experimental protocols. The control cases exhibited hyperplasia and tumor induction in keeping with the known sequence for both carcinogenesis models. None of the cases revealed a promoter or enhancer capacity of carbamide peroxide. These results indicate the lack of risk involved in the application of carbamide peroxide even in oral mucosa with a precancerous condition due to the action of initiation agents such as tobacco and alcohol.

Ultrastructural study of osteodentin formation induced by irradiation.

A single dose of 16,000 rads was delivered to a 1 X 1.5 cm area of the cheek of 2 day old Wistar rats. The lower first molar region was removed 1, 3, 5 and 8 days postirradiation and processed for light and electron microscopy observation. The most distinct structural feature at optical level was osteodentin production in the dental papilla, which began at the level of the initiation of Hertwig's epithelial sheath and continued until the pulpal area was completely filled by osteodentin. At the ultrastructural level, ameloblasts exhibited large lipidic vacuoles and irregular intracytoplasmic secretory granules. Odontoblasts underwent multiple regressive alterations with vacuolization and numerous myelin figures. Pulpal cells exhibited a capacity for odontoblastic differentiation with formation of one or more odontoblastic processes and dentin matrix secretion. Thus, irradiation resulted in stimulation of a great potential for dentin production in these cells.

Photographic microdensitometry for evaluation of acid phosphatase activity at the electron microscope level.

The dry mass of reaction products in ultrathin sections was determined using electron micrographs of polystyrene spheres of known weight deposited on Formvar membranes and evaluating the negatives photometrically. This method was applied to the quantification of the final reaction product of the acid phosphatase reaction in a model system in which enzyme was incorporated in gelatin. The enzyme activity was demonstrated by the lead precipitation method and quantified by direct microphotometry at the light microscope level. Models were then embedded and sectioned for electron microscopy. Microphotometric values afforded by the electron negatives were in linear correlation with incubation times and enzyme concentration. Section thickness and its possible variations due to deformation or contamination under the electron beam were also evaluated. Measurements of lysosomal acid phosphatase activity in rat kidney sections served to illustrate the application of the technique.

Experimental study to test the potential tumor promotion effect of a tooth bleaching agent

Carbamide Peroxide is routinely employed as a whitener for tooth enamel. Oral mucosa protection is recommended to avoid inflammatory reactions. Experimental work has demonstrated its irritative effect on gastric mucosa when swallowed. The activity of certain oxidizing agents as tumoral promoters has been demonstrated and associated to their capacity to induce hyperplasia. Within this context it seemed of interest to assess the possible action of carbamide peroxide as a tumoral promoter in oral mucosa with or without a precancerous condition. Its action was tested in 2 models which are highly sensitive to chemical cancerization: a) Dorsum skin or SENCAR mice treated with carbamide peroxide daily or twice a week with or without prior initiation with dimethylbenz(a)anthracene (DMBA). Control mice were submitted to the standard carcinogenesis protocol, i.e. initiation with DMBA and promotion with 12-O-tetradecanoyl phorbol acetate (TPA). b) Hamster cheek pouch submitted to topical application of carbamide peroxide 3 times a week with or without prior initiation with DMBA, hamster cheek pouch submitted to repeated topical application of DMBA as a complete carcinogen: application twice a week in the control group and identical treatment + 1 weekly application of carbamide peroxide to evaluate its capacity to enhance the process. The effects were assessed between 1 and 14 weeks of treatment at different intervals for the different experimental protocols. The control cases exhibited hyperplasia and tumor induction in keeping with the known sequence for both carcinogenesis models. None of the cases revealed a promoter or enhancer capacity of carbamide peroxide. These results indicate the lack of risk involved in the application of carbamide peroxide even in oral mucosa with a precancerous condition due to the action of initiation agents such as tobacco and alcohol.