Localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity along the rabbit, guinea pig, rat and mouse nephron was studied both in aldehyde-fixed and unfixed fresh frozen kidney sections under the K-phosphatase cytochemical method by Mayahara et al. (25), and the results were compared with those of reports on biochemical Na-K-ATPase activity. The distribution pattern of K-NPPase activity along the nephron was identical in the four species in unfixed sections; with the highest activity in thick ascending limbs of Henle's loop (TALH) and high in distal convoluted tubules (DCT), intermediate in cortical collecting tubules (CCT), low in proximal convoluted tubules (PCT), little or none in proximal straight tubules (PST), collecting ducts, and thin limbs. Fixation did not affect the distribution pattern, however, it inverted the relative intensity of staining between DCT and medullary TALH. In all species, K-NPPase activity was higher in medullary thick ascending limbs (MTALH) than in cortical ones (CTALH). This was due to the difference in epithelial thickness between these two segments, and especially apparent in the rabbit and least pronounced in the rat. As this alteration occurred in the boundary between the outer stripe and the inner stripe of the outer medulla, it was suggested that MTALH should be divided into these two segments, namely inner stripe and outer stripe ones. The cytochemical K-NPPase activity along the nephron is in good agreement with biochemical reports of Na-K-ATPase activity, and some discordance in details were explained reasonably. These observations validated the usefulness of K-phosphatase cytochemistry for detection of Na-K-ATPase.
The alterations in noradrenaline (NA) nerve terminals fixed by KMnO4 have been studied electron microscopically during regeneration after an injection of 6-OH dopamine (6-OHDA), a specific neurotoxin of aminergic fibers, in the periventricular region of the rat hypothalamus. In non-treated rats, only 7% of the total number of NA terminals had obvious synaptic structures with neighboring dendrites. In rats 30 days after 6-OHDA treatment, NA terminals, though rarely found, showed synaptic contacts with dendrites. In rats 60 days after the neurotoxin injection, NA terminals increased remarkably in number, and half of the total number of NA terminals observed had a membrane specialization between NA terminals and dendrites. Thereafter, NA terminals with synaptic structure decreased in incidence. In rats 90 days after 6-OHDA treatment, NA terminals were found in a cluster which sometimes contained huge axonal bulgings in peri- and paraventricular areas of the hypothalamus. Results obtained by fluorescence histochemistry indicated that giant fluorescent varicosities corresponded to the clusters of NA terminals.
Endogenous catalase activity in the ovary of two sea urchin species, Hemicentrotus pulcherrimus and Anthocidalis crassispina, was investigated by 3, 3′-diaminobenzidine (DAB) method. Osmicated DAB products were located at the lamellar (in H. pulherrimus) or stellate (in A. crassispina) structure of the cortical granule (Cg) in the mature egg and that of the presumptive cortical granule (pCg) in the oocyte. As for the A-type giant granule (A-g.) in the nurse cell, the same product was also found at the cortical granule-like structure (Cgls), but not at the other part of A-g.. In the present study of sea urchin ovary it is clear that the A-g. is “Heterophagosome”, and that the heterophagic substance may be regenerated as a nutrient for the growing oocyte after autolysis of nurse cell.
The retinas of the river lamprey (Lampetra japonica) were processed for fluorescence microscopic study with flat-mounts and cryosections. The eyes were enucleated 2hr after an intravitreous injection of a mixture of noradrenaline (2.5μg) and 5, 6-dihydroxytryptamine (2.5μg). The density (number/mm2) of dopaminergic (DA) cells was calculated for four arbitrary concentric regions of retinal flat-mounts; the central, intermediate, peripheral and marginal regions. The density was found to be higher (mean±S.D.= 131±29 cells/mm2; n=12) in the peripheral region than in the rest and extremely low (15±7cells/mm2) in the marginal region, and no DA cells existed in the circumferential edge immediately neighboring the ora terminalis. Oval or fusiform DA cells extended in various directions 2 to 3 main processes, which irregularly ramified and formed dense fiber networks in the amacrine cell and inner plexiform layers. Since no fluorescent fiber was seen in the horizontal cell layer, DA cells appeared to be a subset of amacrine cells in the lamprey retina. Differences in morphology and spatial distribution were discussed between DA cells of the lamprey and teleost fish retinas.
A histofluorescence study was carried out with flat-mounts and cryosections of the carp (Cyprinus carpio) retina in order to explore morphological changes of dopaminergic (DA) cells following intravitreous injection of 6-hydroxydopamine (6-OHDA). The eyes were injected with a mixture of 6-OHDA, pargyline and ascorbic acid (10μg each to small-sized fish or 20μg each to large-sized fish) on two successive days, and enucleated at various survival intervals (2hr to 7 days) after the second injection. DA cells, taking up 6-OHDA and emitting a white-greenish fluorescence, were apparently normal in shape in 2 to 24hr after the injection, while they became yellowish and began to exhibit deformed cell bodies (swollen or shrunken) with tattered processes within 2 to 3 days after the injection. These cells lost uptake capability for noradrenaline given intravitreously. Such changes became severer in 4 to 7 days; faint yellowish cell bodies with no processes were collapsed or broken down into pieces and finally disappeared. These successive changes are reasonably assumed to represent the cytolytic destruction of DA cells induced by intravitreous 6-OHDA.
The sequence of cytomorphological and cytochemical changes during 20-methylcholanthrene (20-MC)-induced malignant transformation of the bronchial epithelium was studied in six beagle dogs. The data show that submucosal injection of 20-MC in the bronchus results in the occurrence of metaplastic cells without atypia, metaplastic cells with mild, moderate, and severe atypia, and finally in cancer cells. In two control dogs treated with submucosal water injections squamous metaplastic cells without atypia, with mild, and occasionally with moderate atypia could be demonstrated. In individual morphologically classified cells quantitative cytochemical determinations of DNA and nuclear proteins were performed. In both groups of dogs squamous metaplastic cells without atypia, or with mild atypia, showed cytochemical changes similar to those found in growth-activated normal cells. In contrast, cells from 20-MC treated dogs morphologically classified as squamous metaplastic cells with severe atypia or cancer cells all showed highly increased and scattered amounts of nuclear proteins and DNA significantly deviating from those in normal proliferating cells.
Various techniques of fluorescence histochemistry have some methodological difficulties mainly due to the instability of amine fluorophores. The present study aims to develop a stable and relatively simple histofluorescence method which can be applied to the routine examination for diagnostic histopathologists. Dissected tissue pieces were fixed by immersing in a FAGLUPAGAS* solution overnight. Cryostat sections of fixed materials were washed in a FAGLUGAS solution, mounted on glass slides, dried by warm air and finally cover-slipped in Entellan. Amine fluorescences in thusly fixed tissues were stable for several months when the specimens, either tissue blocks or cryostat sections, were kept in the FAGLUPAGAS.
The behaviour of the multiple forms of soluble and Triton-solubilized acetylcholinesterase was investigated using microgel gradient electrophoresis at selected days of postnatal development. Three to four anodically migrating forms designated with A (fast migrating) to D (slow migrating) could be separated in the adult rat brain. The comparison of both enzyme fractions revealed differences in regard to the most active form at early stages of development and the maturation of the pattern of multiple forms of acetylcholinesterase.
A new one-step method for light and electron microscopic localization of Ca2+-ATPase activity was applied to the rat trachea. Under light microscopic examination, Ca2+-ATPase reaction was found on the luminal side of the tracheal epithelium and on the nerve fibers of the submucosa. On electron microscopic examination, the most intense Ca2+-ATPase reaction was observed on the ciliated cells in the tracheal epithelium. Cytochemical reaction products were observed in the axoneme, probably corresponding to the bridge-like structures between the axoneme and ciliary membrane, the outer and inner arms of doublets, the radial spoke heads, the central sheath, the central core bridge, and the outer surface of the ciliary membrane. Enzyme activities were also found on the peripheral structures of triplets, which may correspond to connectors, and fibrous elements of the rootlet bundle. The reaction completely disappeared when Ca2+ or ATP was removed from the incubation medium. Substrate specificity was observed by substituting NPP or GP for ATP in the incubation medium. Lead and phosphate were identified in the reaction product by X-ray microanalysis with an analytical electron microscope, suggesting that the reaction products were lead phosphate. Ca2+-ATPase reaction was also observed on the microvilli, lateral plasma membrane, and cytoplasmic projections in both ciliated and non-ciliated cells such as brush cells, secretory cells, intermediate cells, and basal cells. From these results, it was concluded that Ca2+-ATPase activities, considered to be related to ciliary movement and Ca2+-transport, have been clearly localized in the rat trachea.
Brains of the golden hamster were examined by immunofluorescent and peroxidase-antiperoxidase (PAP) methods using antibody to tyrosine hydroxylase (TH) or serotonin (5-HT). The localization of monoaminergic nerve cell bodies in the brain of the golden hamster was found to be similar to that of the rat by histofluorescent and immunofluorescent methods. Monoaminergic nerve fibers and terminals were widely distributed throughout the brain. Especially on the supraependymal part of the ventricle, 5-HT-positive varicose fibers were visualized, but a few TH-positive nerve processes in the ventromedial periventricular hypothalamus and in the caudal portion of the locus coeruleus were observed to penetrate into the ventricular lumen. By immunocytochemistry, TH- or 5-HT-positive reaction products were found in the cytoplasm except for cisternae of Golgi apparatus and mitochondria of neurons of Al cell group or raphe dorsalis. TH- or 5-HT-immunoreactive granules were occasionally found in each neuron, and their diameters were 100-140nm for TH or 110-120nm for 5-HT, respectively. Granules contained in nerve fibers were slightly smaller than those contained in nerve cell bodies.