Localization of neurons in Onufrowicz's (Onuf) Group-X in rabbits was studied by the retrograde tracer method using two kinds of fluorescent substances, Evans blue (EB) and Primuline (PL). EB was injected into the external sphincter muscle of the anus, and PL into the ischiourethral muscle which corresponds to the external sphincter of the urinary bladder in rabbit. One day after the simultaneous injections, the animals were sacrificed by perfusion with 10% neutral formalin. Samples taken from the sacral cord were serially sliced at 25 μm in frozen sections and were surveyed with a fluorescence microscope. Through G-excitation filter system, a cluster of neurons in Group-X was shown to be labeled with EB, illuminating in red. On the same specimen, after changing the filter system to U-excitation, another cluster of neurons was seen labeled with PL, illuminating in yellow. Group-X of Onuf was confirmed to be composed of two different motoneuron subgroups which separately innervate the external sphincter muscles of the anus and the urinary bladder.
Frog adrenal chromaffin cells have very long processes. These chromaffin cells are scattered in cortical lipid cells and sometimes in summer cells. By immunofluorescent method, we first confirmed tyrosine hydroxylase (TH) -positive, dopamine-β-hydroxylase (DBH) -positive, phenylethanolamine-N-methyltransferase (PNMT) -positive adrenaline cells, and TH-positive, DBH-positive, PNMT-negative noradrenaline cells in frog adrenal glands. The activities of TH, DBH and PNMT in the adrenals of the bullfrogs were 215, 22905 and 302 pmoles/min/mg protein, respectively. Acidophilic summer cells have neither TH, DBH nor PNMT. These results were reconfirmed by fluorescence microspectrophotometry.
Using glucose-6-phosphatase (G6Pase) as a marker enzyme, ultracytochemistry was performed on human degenerative hepatocytes in liver biopsy specimens from 7 cases of chronic aggressive hepatitis and 3 cases of chronic persistent hepatitis. Some modifications of prefixation, incubation time and postwashing time were made to apply the ultracytochemical method for this enzyme originally designed for animals to biopsied human liver. Normal G6Pase activity was found in the smooth endoplasmic reticulum (SER), the rough endoplasmic reticulum (RER) and the nuclear envelope (NE) of nearly normal hepatocytes in chronic persistent hepatitis. In degenerative hepatocytes of chronic aggressive hepatitis, lead phosphate deposition was reduced in the dilated cisternae and observed only on the inner surface of the cisternal membrane. SER was vesiculated and contained no G6Pase activity. Occasionally, in markedly dense and shrunken hepatocytes which were detached from neighbouring cells, G6Pase activity had mostly disappeared in SER and RER but it was well preserved in NE.
Using double immunostaining procedures, this study clarifies the localization of immunoreactive somatostatin- and immunoreactive hCG-containing cells simultaneously in the human placental villi and decidua. Immunoreactive somatostatin-containing cells were distributed in the cytotrophoblastic monolayer and decidua, while immunoreactive hCG-containing cells distributed exclusively in the syncytiotrophoblastic layer. The immunostainability of endocrine cells in the villi reduced progressively with gestation, however this change was not detected in the decidua. The results indicate that the endocrine placenta is discriminated by a syncytiotrophoblastic layer containing tropic hormone (hCG) and a cytotrophoblastic layer containing suppressive hormone (somatostatin), and that gestation modifies the immunocytological features of the endocrine placenta. However the role of somatostatin in the placental villi and decidua remains unknown.
A routine procedure for electron microscopic radioautography in combination with phenidon developer and the domestic emulsion, Sakura NR-H2, was developed. Good stable radioautograms have not been obtained by simply developing the emulsion coated grids with phenidon developer at any temperature, but the development of these grids at 18°C for 1 min after 30-45 sec gold latensification in freshly prepared gold thiocyanate gives satisfactory results. Some electron microscopic radioautograms developed by this method are presented. The addition of the anionic surfactant, dioctyl sodium sulphosuccinate, to the emulsion was found to be very effective not only for preventing the burst of emulsion but also in obtaining a uniform spread of the emulsion for the wire loop method. Radioautograms obtained with this method are suitable for quantitative electron microscopic radioautographic studies. Details of this method are introduced.
Histochemical changes in lactate dehydrogenase (LDH), α-glycerophosphate dehydrogenase (α-GPDH), myofibrillar adenosine triphosphatase (ATPase), phosphorylase and glycogen were investigated in pectoral major muscle of guinea pig and albino mouse after 240 R thoracic X-irradiation. The enzymic activities of LDH, α-GPDH and ATPase showed marked reduction at 24, 48 and 72 hrs post-irradiation. Phosphorylase and glycogen, on the other hand, showed an increase in activity, but the damage was severe at 48 and 72 hrs as type I fibers also showed the presence of phosphorylase and glycogen. The phosphorylase reaction emerged from this study as the most sensitive histochemical parameter of radiation injury. The significance of these alterations with reference to muscle glycolytic energy metabolism after thoracic X-irradiation is discussed.
Thiazolylazocresol (TAC) was found to demonstrate cadmium and zinc in different colors in tissue sections, enabling differential staining by coloration without a masking agent. The metals in stained sections were microscopically identified by the aid of a laser microprobe. The absorption spectra obtained from TAC-metal chelates in a test tube show three absorption maxima: around 650 nm (λ2), 550 nm (λ1) and 400 nm. The ratio of the absorption of λ1 and λ2 with cadmium differs considerably from that of zinc. The difference in the ratios (λ2/λ1) is presumed to be the cause of the differential staining by coloration.
Sulfhydryl groups in the rabbit erythrocytic membrane, intact and ghost, were ultracytochemically demonstrated using the Fast blue BBN (FBBBN) method, the Mercury orange (MO) method and the Mercuric chloride (MC) method. Reaction products were localized in the form of small particles or globules with a diameter of mostly 8.0-8.9nm in the ghost membrane and 7.0-7.9nm in the intact membrane by the FBBBN method, mostly 9.0-9.9nm in the ghost membrane and 8.0-8.9nm in the intact membrane by the MO method, and mostly 8.0-8.9nm in the ghost membrane and 7.0-7.9nm in the intact membrane by the MC method. Particles were aligned in one or several rows within the membrane. Among the three methods, the most reproducible results were obtained with the FBBBN or MC method. Specimens incubated in the MC method and subsequently freeze-fractured showed intramembranous particles (IMP) which appeared slightly transformed when compared with the IMP in non-incubated freeze-fractured specimens. These results suggest that IMP are shown by the ultracytochemical demonstration of sulfhydryl groups in the rabbit erythrocytic membrane.