Histochemical observations on calcium absorption in the various regions of the intestine of Esomus danricus (Ham.), Clarias batrachus (Linn.) and Channa striatus (Bloch) are given in this paper. The fish were studied at different time intervals after experimentally feeding them on a calcium diet. Three hr after feeding on the calcium diet, no absorption was recorded in these fish except for moderate absorption in the anterior intestine of Esomus danricus. At an interval of six hr, absorption of calcium was recorded in the anterior intestine of all the three fish. However, in Esomus danricus, the middle intestine also showed calcium absorption at this time interval while in the other fish, no absorption was recorded in this region. Nine hr after feeding, maximum absorption was observed in the anterior and middle intestine of Clarias batrachus and Channa striatus and weak absorption was recorded in the middle intestine of Esomus danricus. However, maximum absorption was noted in the pyloric caeca of Channa striatus at this time interval. No absorption was noted in the posterior intestine of all the three fish.
Immunohistochemical localization and biochemical activity of glutathione peroxidase (GSH-PO) in rat adrenal glands were studied on 1) normal untreated, 2) hypophysectomized and 3) ACTH administered groups in order to clarify the role of GSH-PO in the adrenocortical lipid metabolism. In the normal untreated group, GSH-PO was immunohistochemically localized in the zonae fasciculata and reticularis of the adrenal cortex. The hypophysectomized group showed marked atrophy of these zones which contained accumulated cholesterol esters. Thin layer chromatography of this group showed the presence of sterol hydroperoxides in mitochondrial, cytosol and microsomal fractions. Immunohistochemically, GSH-PO was localized in these atrophic zones. On ACTH administration, the numbers of GSH-PO positive adrenocortical cells were increased and the staining was more intensified. The activity of GSH-PO was higher in this group than in the control group. These findings suggested that GSH-PO in the rat adrenocortical cells may be playing an important role in reduction of both lipid peroxides such as cholesterol ester hydroperoxides in the reparative process of cell injury (hypophysectomy) and steroid hydroperoxides during steroidogenic process (ACTH administration).
Immunohistochemical localization of dipeptidyl aminopeptidase (DAP) IV in the rat endocrine organs was studied using the unlabeled antibody method. Fresh frozen, chloroform-acetone treated sections of the endocrine organs (pineal, pituitary, thyroid, parathyroid, adrenal gland and pancreas) were investigated for the present study. A fairly intense immunoreaction of DAP IV was detected in the capillary endothelium of the thyroid gland and adrenal cortex. The capillary endothelium of the pineal, pituitary, parathyroid gland and pancreatic islets showed faint immunoreaction. No immunoreaction could be seen in the parenchymal cells of these endocrine organs except for the follicular epithelial cells of the thyroid gland. These results indicated that DAP IV in the endocrine organs was restricted to the capillary endothelium, and showed organ-dependent differences. Especially, it seemed that DAP IV in the capillary endothelium might be of importance in the thyroid and adrenal glands.
The localization of three catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in some ganglia of dogs was studied by immunohistocytochemical methods. The suprarenal and superior cervical ganglion cells showed TH-, DBH-positive and PNMT-negative or TH-positive, DBH- and PNMT-negative reactions by immunofluorescent staining. In the superior cervical ganglia, small numbers of TH-positive, small intensely fluorescent (SIF) cells formed clusters. By immunoelectron microscopy, TH-positive reaction products were observed in the cell matrix and granules of the superior cervical ganglion. In the nodose ganglion, small numbers of TH-positive, DBH-negative dopamine cells with varicose fibers were scattered among many TH-negative principal ganglion cells.
Density and total number of dopaminergic (DA-) cells and indoleamineaccumulating (IA-) cells were studied by means of a histofluorescence technique in relation to specimen size in the retina of carp (Cyprinus carpio), ranging from 9.2 to 37.5cm in body length (tip-to-tip). Both classes of cells were simultaneously visualized in retinal flat-mounts following intravitreal injection of a mixture of noradrenaline and 5, 6-dihydroxytryptamine. Central, intermediate, peripheral and marginal regions of the retina were examined. The densities of both classes of cells were found to be higher in the peripheral than in the central retina, and higher in all the regions in smaller fish as compared to larger ones. The density reduction with growth appeared to be more pronounced in the central than in the peripheral retina. The density ratio of DA-cells to IA-cells was approximately 1:1 in any region of differentsized specimens. The total numbers of both classes of cells per retina, on the other hand, increased with growth (approximately 260 cells each per cm of body length) as a result of cell proliferation at the retinal margin.
It is known that administration of galactosamine in large quantities to the rat induces liver injury which shows morphological and functional features similar to those of acute human viral hepatitis. However, the changes in the ultrastructure of the parenchymal cell and in the histochemical localization of enzymes in this liver injury have not been analyzed systematically. In this study, transient injury to the rat liver was induced by a single intraperitoneal injection of galactosamine. After the administration of the drug, most of the liver cells showed synchronous morphological alteration accompanied by changes in the histochemical localization of several enzymes in the liver cell. Namely, after the administration of galactosamine, the histochemical reaction of sorbitol dehydrogenase at the light microscopic level was frequently restricted to one side of the cytoplasm and the peripheral cytoplasm near the cell border. At the electron microscopic level, the reaction deposits of sorbitol dehydrogenase were recognized in the hyaloplasm and its localization shifted with the movement of cell organelles. Moreover, the localization of G-6-Pase changed with alteration of the shape of the endoplasmic reticulum and its distribution, while the localizations of 5′-nucleotidase and Mg++-ATPase changed with the morphological alteration of the plasma membrane.
The authors investigated the localization of progesterone (P) and estradiol (E2) in human chorionic villi by immunohistochemical techniques and obtained the results to be presented in this report. 1. The tissues were fixed in 1.25-2.5% glutaraldehyde (GA) including 10% glycerol at 4°C for 2-4hr, then washed in phosphate buffer including 10% glycerol, frozen and microtomed to produce adequate specimens for further processing and evaluation. 2. The immunostaining was done with either anti P or anti E2 antisera, which were adsorbed with either bovine serum albumin (BSA) or steroid extracted powdered placentas to eliminate non-specific reactions. 3. The cytoplasm of the syncytiotrophoblastic layer and Langhans cells of chorionic villi from the first and second trimester, and the cytoplasm of the syncytiotrophoblastic layer excluding the areas of the vasculosyncytial membranes of chorionic villi in late pregnancy revealed the presence of immunoreactive P and E2.
The time of origin of the neuroblasts in the neural tube of the chick embryo was determined by histochemical observation of acetylcholinesterase (AChE) activity. In the central nervous system, the neuroblasts appeared first in the caudal half of the rhombencephalon of the 1.5-day chick embryo (13-somite, stage 11). They were round medium-sized cells and located in both the ventral and the dorsal halves of the wall of the neural tube. In the cervical, thoracic and lumbosacral neural tubes, the neuroblasts first appeared at stage 12, 14 and 17 respectively. At these levels, they were large and located in the ventral half of the wall of the neural tube. Occasionally they provided an axon which extended to the ventrolateral edge of the neural tube. The time of origin of the neuroblasts in the neural tube of the chick embryo is more accurately determined by histochemical observation of AChE activity than by 3H-thymidine autoradiography. The time difference between the actual neuroblastic differentiation and the appearance of AChE moderately positive cells is much smaller than that between the actual neuroblastic differentiation and the differentiation determined by 3H-thymidine autoradiography.
Effects of 5, 7-dihydroxytryptamine (5, 7-DHT) on indoleamine-accumulating (IA-) cells were investigated on the retinas (cryo-sectioned or flat-mounted) of adult carp (Cyprinus carpio), by means of a histofluorescence technique. The eyes, pretreated with a single intravitreal injection of 5, 7-DHT (15μg), were removed at various testing periods (4hr to 50d) with or without an intravitreal injection of 5, 6-dihydroxytryptamine (5, 6-DHT; one or 5μg) alone, or with the same injection of noradrenaline (NA; 5μg) plus 5, 6-DHT (5μg) 2hr prior to enucleation. In cryo-sections of the retinas loaded with 5, 6-DHT (5μg), the indoleamine was found to be taken up not only by IA-cells but also by other cells located in the amacrine cell layer. The latter class of cells was more weakly fluorescent and smaller in size than IA-cells. Two days after pretreatment with 5, 7-DHT, IA-cells already became invisible, while the accumulative ability of smaller cells for 5, 6-DHT remained. Subsequently, IA-cells were not seen again with 5, 6-DHT in flat-mounts up to 50d (the longest testing period in the present experiment) after pretreatment with 5, 7-DHT. The results obtained reveal that intravitreally given 5, 7-DHT (15μg) destroyed quasipermanently the somata of retinal IA-cells in the adult carp.
Actin filaments of hepatocytes in three types of experimental rat cholestasis were examined by a previously reported method (J. Histochem. Cytochem. 28; 335, 1980). Liver treated with cytochalasin B in vivo showed bile canalicular dilation and the obscure polygonal pattern of hepatocytes depended upon partial loss and granular aggregation in peripheral fluorescence. Liver treated with phalloidin showed an accumulation of fluorescence mainly around bile canaliculi in addition to an accumulation in the cytoplasma and nucleus in hepatocytes. Liver ligated with common bile duct showed increased fluorescence in the peripheral cytoplasma of hepatocytes, particularly around bile canaliculi, but this alteration varied according to the duration of ligation.
The acetylcholinesterase (AChE) activity at the myotendinous junction (MTJ) in several skeletal muscles of limbs and trunks of adult rats and in M. soleus and M. gastrocnemius of newborn rats with or without denervation of sciatic nerves was studied under a light microscope and an electron microscope. Under an electron microscope the AChE activity at the MTJ was detected in the lamina rara. In M, soleus and M. rectus abdominis of adult rats nearly all fibers exhibited AChE activity and in other muscles of adult rats a few fibers exhibited the activity. In M. gastrocnemius of newborn rats the percentage of AChE-positive MTJ was higher than that of adult rats. As they grew the activity gradually decreased. There were AChE-positive and AChE-negative MTJs in muscle. The Z lines of the muscle fiber, which had AChE-positive MTJ, were wider than those of the fiber, which had AChE-negative MTJ. In the denervated muscles, the AChE activity at the MTJ was detected three weeks after the denervation in adult rats and one month after the denervation in newborn rats. In the atrophic muscles the AChE activity could not be detected under a light microscope, but was detected slightly under an electron microscope. The present results suggest that AChE activity is detected at the MTJ in the red muscle fiber and the enzyme seemed to be myogenic.
To assess the appropriateness of the lead nitrate method using dimethyl sulfoxide (DMSO) for the cytochemical demonstration of adenylate cyclase (ACLase) and guanylate cyclase (GCLase), we have carried out a biochemical assay of ACLase and GCLase in parallel with the cytochemical study. Tissues from rats were fixed in a mixture of 2% paraformaldehyde, 0.25% glutaraldehyde and 5% DMSO in 0.1M cacodylate buffer, pH 7.4, for 30min, and then washed with 5% DMSO in 0.1M cacodylate buffer. Vibratome or Microslicer sections were incubated in the following medium for 15-60min at 37°C. Medium for ACLase: 80mM Tris-maleate buffer, pH 7.4; 4mM MgSO4; 10mM NaF; 2mM theophilline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM AMP-PNP. Medium for GCLase: 80mM Tris maleate buffer, pH 7.4; 3mM MnCl2 or MgSO4; 2mM theophilline; 2mM lead nitrate; 0.25M sucrose; 5% DMSO; 2.5mM levamisole; 0.5mM GMP-PNP. Some reaction medium for GCLase contained NaN3. ACLase and GCLase activities were biochemically and cytochemically enhanced by adding DMSO to the incubation medium as well as to fixatives. Cyclases were localized in the plasma membranes, gap junctions, endoplasmic reticulum and Golgi apparatus.
A new one-step lead citrate method is introduced for the histochemical and cytochemical demonstration of Ca2+-ATPase activity. The standard incubation medium contains 3mM ATP as the substrate, 10mM CaCl2 as the activator, 2mM lead citrate as the capture reagent, and 250mM glycine-NaOH or-KOH buffer, pH 9.0. The incubation medium was clear and stable. Cytochemical reactions in this incubation medium were examined in various tissues; liver, kidney, heart, small intestine, trachea, bone marrow, and femoral muscle. The reaction products were found in plasma membrane, mitochondria, myofilaments, axoneme of cilia, sarcoplasmic reticulum, transverse tubules, gap junctions, lysosomes, and others. The cytochemical reaction showed Ca2+ and substrate dependency. Preheating sections in boiling water resulted in complete loss of the reaction, but pretreatment with 10mMEDTA or EGTA did not affect it. Inhibitors such as 10mM PCMB, 0.1mM quercetin, and 0.1mM oligomycin, partly reduced the cytochemical reaction in some tissues, but 0.5mM bromotetramisole left it unchanged. Substrate specificity was proved by the substitution of various substrates. Reaction products were found to be composed of lead phosphate with X-ray microanalysis. From these results, it was concluded that the cytochemical demonstration of Ca2+-ATPase activity was successful by this method at both light and electron microscopic levels.