Preparation of positional renal slices for study of cell-specific toxicity.

To reduce structural complexity, rabbit kidneys were sliced perpendicular to their cortical-papillary axis to isolate four distinct cell groupings. This positional orientation allows identification of each renal cell type based on its location within the slice. A mechanical slicer was used to make several precision-cut slices rapidly from an oriented cylindrical core of renal tissue, with minimal tissue trauma. Slices were then submerged under a gently circulating oxygenated media in a fritted glass support system that maintains viability (intracellular K+/DNA ratio) and structural integrity (histology) for at least 30 h. A high dose of mercuric chloride (10(-3) M) was used to demonstrate the structural and biochemical changes of intoxicated slices. This method provides a controlled subchronic in vitro system for the study of the individual cell types involved in cell-specific renal toxicities and may also be a useful tool for addressing other pharmacological and physiological research questions.

An ultrastructural cytochemical stain specific for neuroendocrine neoplasms.

Previous work from our laboratory has indicated that the uranaffin reaction, when run under specific conditions, will stain neurosecretory granules. In this ultrastructural cytochemical study, we analyzed the granule-staining properties of 13 normal, 10 abnormal (non-neoplastic), and 138 neoplastic tissues in an attempt to evaluate the specificity of the uranaffin reaction for diagnostic purposes when compared with routinely processed specimens. For the uranaffin reaction, previously fixed tissue stored in buffer was rinsed with 0.9% NaCl and reacted with a 4% aqueous solution of uranyl acetate (pH 3.9) for 48 hours. After three NaCl rinses, the tissue was dehydrated and processed for electron microscopy. The granules of normal or non-neoplastic neuroendocrine cells that stained positively with the uranaffin reaction included pancreatic islet cells, thyroid C cells, adrenal medullary cells, parathyroid chief cells, and the neuroendocrine cells of the intestine. All 42 neuroendocrine neoplasms studied possessed abundant uranaffin-positive granules and included carcinoids, oat cell carcinomas, islet cell neoplasms, medullary carcinomas of the thyroid, pheochromocytomas, carotid body paragangliomas, a pituitary adenoma, Merkel cell carcinomas, parathyroid adenomas, and a neuroblastoma. All 96 control neoplasms that were not classified as neuroendocrine in nature were negative for neurosecretory granules when studied with the uranaffin reaction and included 13 neoplasms derived from endocrine glands, 57 neoplasms from secretory epithelium, 10 of hematopoietic origin, and 16 miscellaneous neoplasms. It was determined that the uranaffin reaction is a useful ultrastructural cytochemical marker for neuroendocrine granules and helped distinguish these cytoplasmic organelles from ultrastructurally similar granules derived from non-neuroendocrine cells.

An ultrastructural evaluation of the cell organelle specificity of the uranaffin reaction in two human endocrine neoplasms.

The organelle specificity of the uranaffin reaction was determined by subjecting two human neoplasms (a pheochromocytoma and islet cell carcinoma) to four different experimental conditions. In Uranaffin Procedure (UP) I, fixed tissue was immersed in 0.9% sodium chloride (NaCl) before reacting with 4% uranyl acetate (pH 3.9) for 24 hours. In UP II, the tissue was prepared as in UP I with the exception that the tissue was immersed in uranyl acetate for 48 hours. In UP III, fixed tissue was prepared as in UP I with the exception that tissue was immersed in 0.1M cacodylate buffer (pH 7.2) instead of 0.9% NaCl. In UP IV, fixed tissue was prepared as in UP III with the exception that the tissue was immersed in uranyl acetate for 48 hours instead of 24 hours. When UPs I and II were utilized, only three cell organelles showed electron-dense reactivity: the nucleus, ribosomes, and cytoplasmic neurosecretory-like granules. In the nucleus, the nuclear chromatin, nucleolus, interchromatinic granules and perichromatinic granules were intensely stained. The reaction product in all of the uranaffin-positive organelles had a finely granular appearance. When fixed tissue was immersed in cacodylate buffer instead of isotonic saline, a non-specific reactivity was observed. The reaction product in some areas had a distinct crystalline appearance and filled some areas of the cytosol, the cisternae of the endoplasmic reticulum, the Golgi apparatus, the perinuclear cisternae, the nucleus, nucleolus, mitochondria, neurosecretory-like granules and larger lysosome-like bodies. There was a statistically significant (p less than 0.05) increase in the number of uranaffin-positive granules/mu2 when both endocrine neoplasms were reacted with uranyl acetate for 48 hours instead of 24 hours. The increase in uranaffin-positive granules using UP II did not result in an increase in non-specificity of the staining reaction.