Vitalfärbung von Lysosomen und anderen Zellorganellen der Ratte mit Neutralrot Vital Staining of Lysosomes and Other Cell Organelles of the Rat with Neutral Red

Abstract

Microscopical studies on cells or tissues vitally stained with Neutral red (NR) were hitherto almost invariably confined to a few objects that could be investigated without tissue sectioning, for instance tissue culture. For NR, a cationic dye (molecular weight 289, PK 6,75) is easily soluble in water and organic solvents and diffuses during histological preparation for paraffin sectioning and even from cryostat sections. Thus comprehensive studies of vital staining of laboratory animals such as mouse and rat don't exist yet.This explains why it still remains doubtful, wether NR stains ‘preexisting’ or ‘newly formed’ granules, ‘paraplasmic’ cytoplasmic inclusion bodies or - complete or only partially - lysosomes and why it does so. We tried to solve these problems. Moreover we found that after intravital injection of the dye NR ‘stained’ the cells of the APUD-series (PEARSE) rather selectively as do catecholamines or catecholamine precursors. So it was to follow up wether NR could serve as a model for distribution studies of biogenic amines, too.The histological method that allows the localization of intravitally injected NR in tissue sections is freeze-drying. We applied freeze-drying to cryostat sections. The main advantages of this modiftcation are both the short time needed for the drying procedure and the large number of different tissues that can be cut and frozen-dried in the same time. In this way nearly all organs and tissues of the rat could be investigated.1 min after the intravenous injection of the dye NR has disappeared from the blood - at least in concentrations that are demonstrable in tissue sections. By using fluorescence microscopy the dye instead can be localized in all tissues being investigated mainly intracellularly. Apart from the diffuse staining of the cytoplasm in some tissues stained cell nuclei are observed. Intensely coloured nuclei together with a diffuse to scattered staining of the cytoplasm are signs of cell death.In some endocrine cells - mostly belonging to the APUD-series - a strong, often granular, reddening of the cytoplasm is seen; the nuclei are not stained. While the dosis and the form of application necessary to stain these endocrine cells, the intracellular localization and even the reactive groups (presumably carboxylic groups of the granule matrix) to which NR and catecholamines and their precursors are bound seem to be rather identic, some essential differences do exist: NR is easily and rapidly taken up and stored for a relatively short period, whereas biogenic amines accumulate in APUD - cells by active transport in a time - consuming process; their precursors are only stored following decarboxylation. - Secretory granules of some exocrine gland cells, too, may be vitally stained by NR.NR-staining of lysosomes is a well-known fact. We can add some details: a) There are-very characteristic for each of the tissues investigateddifferences in the time needed to stain lysosomes as well as in the duration of lysosomal staining: For instance lysosomes in the kidney proximal convolution are stained very rapidly directly after the injection of the dye and are destained in about 24 h, lysosomes in the thyroid epithelium are rapidly stained and destained, lysosomes in other organs need 45 to 60 min to get stained. c) There is no correlation between the vital staining of lysosomes and the characteristics lysosomes exhibit when stained by histologicalhistochemical methods in tissue sections. This may be due to the extraction from the tissue section of that particular component that, intravitally, binds the dye to lysosomes. - Differences in the composition of the lysosomal matrix in various organs are discussed as one major point of the heterogeneity of lysosomal vital staining with cationic dyes. d) NR vital staining of lysosomes of younger animals is less, that of older animals much more pronounced; lipopigment may, but must not neccessarily do so, exhibit a strong binding capacity for the cationic dye. e) In dehydration experiments the binding capacity oflysosomes is stronger, after premedication with reserpine less pronounced than normally. Desmethylimipramin has no effect at all.The so-called NR-crinom appears only in a few organs, resulting from autophagic as well as from heterophagic cell activity.Following albumin injection enlarged lysesomes are stained vitally in the renal proximal convolution. ‘Vacuoles’ induced by premedication with Macrodex® and glycerol remain unstained.NR is concentrated in the urine of the distal nephron and in the gastric lumen. Reabsorbtion occurs in the distal intestine.Essentially two factors are believed to be responsible for the pattern of NR vital staining: a) Its solubility that explains the distribution of the dye all over the organism, its diffusion through cell membranes and its elimination from the organism by ‘Non-ionic diffusion’. b) Its qualities as a light cationic dye that cause its - electrostatical - binding (‘storage’) to anionic sites of the tissue, for instance to carboxylic groups of the secretory granules of the APUDcells and carboxylic and/or phosphate groups of the lysosomal matrix.