Immunocytochemical Investigations Research Articles

Muscarinic supersensitivity in the rat urinary bladder after capsaicin pretreatment

The effects of capsaicin on urinary bladder function have been investigated in adult rats. Ten days after capsaicin treatment immunocytochemical investigations showed a nearly complete disappearance of substance P (SP) and calcitonin gene-related peptide (CGRP) in all parts of the bladder. Recordings of micturition patterns and cytometrical investigations in conscious animals revealed no functional effects of capsaicin treatment. In-vitro experiments showed that the contractile response to substance P was similar before and after capsaicin treatment and CGRP exerted no contractile effects on the urinary bladder in either group of rats. The concentration-response curve to carbachol as well as the frequency-response curve to electrical stimulation were significantly shifted to the left in bladder muscle after capsaicin treatment. However, the maximal responses were similar in control and capsaicin-treated bladders. In the presence of scopolamine the maximal response to electrical stimulation was clearly lower in bladders subjected to capsaicin treatment than in controls. In conclusion, depletion of substance P and CGRP in the rat urinary bladder by capsaicin induced no supersensitivity to these peptides. However, the increased sensitivity to carbachol and to electrical stimulation seen after capsaicin treatment indicates the development of a supersensitivity to muscarinic receptor stimulation. Despite this supersensitivity in vitro no functional effects of capsaicin treatment were found in vivo.

Uterine Estrogen Receptors Are Increased by RU486 in Late Pregnant Rhesus Macaques but not after Spontaneous Labor*

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.

Effect of EGF Administration on EGF Receptor mRNA in hCG Producing Tumor.

In this study we examined the expression of EGF receptor mRNA after EGF administration in hCG producing tumor (choriocarcinoma). We transplanted the tissue of choriocarcinoma into female nude mice and investigated the effects of EGF on the growth of tumors, the binding activity of EGF receptor and the expression of EGF receptor mRNA in the tumor tissues. Two doses of EGF 5.0 micrograms, 50 micrograms and phosphate buffered saline as a control were injected subcutaneously every day for four weeks. Removed tumors were used for immunocytochemical studies and EGF receptor mRNA investigations. HCG and EGF receptors were detected immunocytochemically in the tumor. The low dose EGF employed stimulated the tumor growth while the high dose EGF inhibited the tumor growth compared with that of the control group. The binding activity of EGF receptor and the expression of EGF receptor mRNA also changed in accordance with the stimulation or inhibition of tumor growth. The growth of hCG producing tumor by EGF administration appeared to be dependent upon the binding activity of EGF receptor and the expression of EGF receptor mRNA.

Sarcoplasmic reticulum (SR) Ca2+-ATPase as a marker of muscle cell differentiation: Immunohistochemical investigations of rhabdomyosarcomas and enhancement of the immunostaining after sodium methoxide pretreatment

An immunocytochemical investigation of sarcoplasmic reticulum (SR) Ca2(+)-ATPase (SR-Ca-ATPase) was performed on formalin-fixed paraffin-embedded specimens of different types of rhabdomyosarcomas such as variants of embryonal and pleomorphic forms. Immunostaining frequency of tumours using SR-Ca-ATPase was compared with that of traditionally used muscle specific markers myoglobin, and desmin. Utilizing the possible cleaving of ester bounds sodium methoxide pretreatment was found to be very effective in enhancement of SR-Ca-ATPase immunostaining reaction. In 11 of 15 tissue specimens of 5 cases round shaped and elongated rhabdomyoblasts with definite cytoplasm exhibited positive immunoreactions with all of the polyclonal antibodies tested, using the streptavidin-biotinylated peroxidase complex (S-ABC-method). In formalin-fixed and paraffin-embedded material of 2 cases of undifferentiated rhabdomyosarcomas composed of small round tumour cells with scanty cytoplasm pretreatment with sodium methoxide induced the immunostaining of SR-Ca-ATPase. After that pretreatment a staining of the paranuclear cytoplasm occurred in many of these undifferentiated tumour cells. In these 2 cases, neither myoglobin nor desmin antibodies could react. However, when frozen sections of one of the poorly differentiated tumours were used monoclonal and polyclonal desmin antibodies reacted immunocytochemically in all of the small cells. Sodium methoxide induced or enhanced SR-Ca-ATPase immunocytochemical reaction can be a further addition to the diagnosis of rhabdomyosarcomas in formalin-fixed paraffin-embedded sections, even when desmin antibody fails to react.

Insulin-like immunoreactivity in the brain of two hagfishes, Eptatretus stouti and Myxine glutinosa

Immunocytochemical investigation of the brains of hagfish Myxine glutinosa and Eptatretus stouti with antisera raised against salmon insulin revealed the presence of groups of immunoreactive cells discretely localized in the mid- and hindbrain of both species. Subpopulations of these cells reacted weakly with antisera against Myxine islet insulin and equivocally with anti-bovine insulin serum. Extracts prepared from Myxine brain were subject to gel filtration and were found by radioimmunoassay to contain two forms of insulin-like material, one of large molecular weight (<66 kDa) and another smaller molecule (6 kDa). The relationship of these molecules to the insulin-related growth factor family of neurohormonal peptides is discussed and their potential function assessed in terms of a possible homology with Muller-type cells and involvement in axonal regeneration phenomena.

GABA, glycine, glutamate, aspartate and taurine in the perihypoglossal nuclei: an immunocytochemical investigation in the cat with particular reference to the issue of amino acid colocalization.

The differential distribution of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA) and taurine (Tau) was investigated in the cat's perihypoglossal nuclei. Serial semi-thin (0.5 micron) sections through the perihypoglossal nuclei were incubated with antisera raised against the mentioned amino acids with the aim of studying possible co-localization. In each experiment different measures were undertaken in order to screen for possible cross-reactivities, and all sections were processed together with test conjugates in order to ascertain the specificity of the antisera used. A very high proportion of the neurons in the perihypoglossal nuclei (about 90%) shows strong immunostaining for Asp and also displays distinct immunoreactivity for Glu in neighbouring sections. About 25% of the cells in the perihypoglossal nuclei are intensely immunostained for Gly, but very few cells show immunoreactivity for GABA. Only glial cells appear to be immunostained for Tau. Neurons that are Gly(+) also display Glu and Asp immunoreactivities. The neuropil of the perihypoglossal nuclei shows a high density of GABA(+), Gly(+) and Glu(+) puncta mainly representing stained axons and terminals. Fewer Asp(+) puncta and very few Tau(+) nerve terminal-like puncta are seen. Details of the regional distribution of immunopositive neurons and puncta within the perihypoglossal nuclei are described. The findings are discussed with particular reference to the possible role of the mentioned amino acids as transmitter substances in the known synaptic circuitry of the perihypoglossal nuclei.

Adenohypophysial Changes in Mice Transgenic for Human Growth Hormone-Releasing Factor: A Histological, Immunocytochemical, and Electron Microscopic Investigation*

The effect of protracted GH-releasing factor (GRF) stimulation on adenohypophysial morphology was investigated in six mice transgenic for human GRF (hGRF). All animals had significantly higher plasma levels of GH and GRF and greater body weights than controls. Eight-month-old mice were killed, and the markedly enlarged pituitaries were studied by histology, immunocytochemistry, electron microscopy, and immunogold method, using double labeling at ultrastructural level. In all pituitaries, a massive hyperplasia, chiefly of mammosomatotrophs, was found. These bihormonal cells, containing GH and PRL, were demonstrated by light microscopy and ultrastructural immunocytochemistry. Electron microscopy revealed the presence of cells with characteristics of GH cells in three pituitaries and cells resembling human adenomatous mammosomatotrophs in the other three glands. All of these cells, regardless of their ultrastructural features, contained secretory granules heavily labeled for GH by immunogold technique; PRL labeling varied from cell to cell, with the predominance of a weak immunostaining and was colocalized with GH in secretory granules. These results indicate that chronic exposure to GRF excess leads to mammosomatotroph hyperplasia. It is suggested that GH cells proliferate and transform to mammosomatotrophs in response to GRF stimulation. Focal PRL cell hyperplasia noted in three pituitaries could also be due to a GRF effect. Longer exposure to GRF is needed to clarify whether GRF can cause adenoma.

Review of Neuroendocrine Carcinomas of the Larynx

Neuroendocrine carcinomas of the larynx are uncommon tumors of considerable scientific interest and clinical importance. They include typical carcinoid tumors, atypical carcinoid tumors, and small cell neuroendocrine carcinomas. This paper considers these neoplasms from a personal experience and reviews the relevant medical literature. About 200 cases of neuroendocrine carcinomas of the larynx have been reported. The diagnosis is based on light microscopy and is confirmed by ultrastructural evidence of neurosecretory granules. Histochemical and immunocytochemical investigations may support it. Paraneoplastic syndromes associated with laryngeal neuroendocrine carcinomas have been reported occasionally. The histogenesis, treatment, and prognosis of these lesions also are discussed.

An Ultrastructural and Immunocytochemical Analysis of Human Endothelial Cell Adhesion on Coated Vascular Grafts

Human adult endothelial cells were enzymatically harvested from adipose tissue. Cell viability was established by Trypan blue exclusion and transmission and scanning electron microscopy. Endothelial cells were identified by immunocytochemical investigation at light microscopy, transmission electron microscopy, and scanning electron microscopy. Isolated cells were positive for actin and vimentin, negative for desmin. Factor VIII RA was mainly expressed at cell surface and occasionally disclosed in the cytoplasm. Reactivity for UEA I and J15 was weak or undetectable. Human endothelial cells were seeded and left to adhere for one hour onto different nonvascular substrates (glass, poly-l-lysine, formvar-carbon, fibronectin, Teflon). Scanning electron microscopy defined surface features, suggesting tenacious cell adhesion on the substrate. Different vascular substrates were tested (preclotted Dacron, albumin Dacron, Hemashield Dacron, Gelseal Dacron, ePTFE, fibronectin-ePTFE). Commercially available coated grafts showed qualitative and quantitative differences in cell adhesion. In particular, Gelseal Dacron provided the best quantitative results, even though a wide variability was observed. In contrast, fibronectin-coated ePTFE gave more reliable results and high spreading efficiency. In the short term, coated grafts do not seem to offer greater advantages than fibronectin-coated ePTFE. However, specific incubation times for each coated graft should be selected and the long-term approach (graft culture) should also be attempted.

Immunocytochemical investigations of two ‘new’ fetal antigens (FA-1 and FA-2) isolated from amniotic fluid

Immunocytochemical investigations of two ‘new’ fetal antigens (FA-1 and FA-2) isolated from amniotic fluid

FMRFamide-like Immunoreactivity in the Nervous System of the StarfishAsterias rubens

The nervous system of the starfish Asterias rubens was subjected to immunocytochemical investigation using antisera raised against the molluscan neuropeptide FMRFamide. Immunoreactivity was detected in the radial nerve cords and the circumoral nerve ring, as well as in the sub-epithelial nerve plexus of the tube foot system. The hyponeural part of the radial cords contained numerous immunoreactive cell bodies. In the ectoneural tissue, immunoreactive cells were present in the epithelium, with cell bodies especially abundant in the lateral parts of the nerve, close to the site of emergence of the innervation to the tube feet. The sub-epithelial nerve plexus of the tube feet contained immunoreactive fibers that were continuous with an extensive system of ectoneural immunoreactive fibers in the radial nerve cords. Immunoreactive fibers were particularly evident in the regenerating radial nerves of previously sectioned arms.

Thiamine-like molecules in the cerebellar cortex of the rat: light and electron microscopic immunocytochemical investigation.

Immunocytochemical methods were used for studying the location of thiamine-like molecules in the cerebellar cortex of the rat. A positive reaction was observed in several synapses and in several dendrites. This reaction was associated with tubular formations and synaptic vesicles. A positive reaction was also found in several mitochondria and in glial cells. The possible role of thiamine-like molecules in cerebellar cortex is discussed.

Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops.

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.

The human oral raphe system

Serial sections (15 microns, 120 microns, and 400 microns) of nine brain stems treated with a combined lipofuscin pigment-Nissl stain were examined in order to delineate the three-dimensional conformation and subdivisions as well as the neuronal types of the human oral raphe system. Characteristic lipofuscin deposits within the somata of various cell types facilitated the demarcation of the oral raphe nuclei from surrounding structures. The dorsal, central, and linear raphe nuclei, e.g. the major subdivisions of the oral raphe system, share common traits as far as neuronal composition and pigmentation is concerned. The interfascicular subnucleus, the dorsofascicular subnucleus, and the intercalate subnucleus are minor subdivisions of the dorsal raphe nucleus. The intercalate one cannot be differentiated from surrounding areas in preparations solely stained for Nissl-material, while it can facilely be identified in combined pigment-Nissl preparations by virtue of differences in the pigmentation pattern. Our architectonical concept of the oral raphe system is in good accordance with the one derived from immunocytochemical investigations of serotonin-containing neurons in the human brain stem. Furthermore, five main neuronal types are described which constitute the oral raphe nuclei. They have been differentiated according to their characteristics as seen in combined pigment-Nissl preparations. I) Large ovoid to polygonal neurons with densely packed and intensely stained pigment granules. II) Similarly featured cells displaying dust-fine and faintly stained pigment granules. III) Medium-sized, ovoid to polygonal neurons with loosely distributed, small pigment granules. IV) Small ovoid neurons devoid of pigment or with only few, intensely stained granules. V) Small spindle-shaped nerve cells with various amounts of intensely stained pigment granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical investigation of L-glutamic acid decarboxylase in the rat hippocampal formation: the influence of transient cerebral ischemia.

The hippocampus is especially vulnerable to ischemic damage. Neurons in the CA3c region and dentate hilus demonstrate fast progressive damage while CA1 pyramidal cells demonstrate delayed neuronal damage. The delayed CA1 pyramidal cell loss could be caused by postischemic neuronal hyperactivity if hippocampal interneurons are lost after ischemia. Therefore we have counted the L-glutamic acid decarboxylase (GAD)-immunoreactive neurons in the hippocampus from control rats and rats surviving 4 or 11 days after 20 minutes of cerebral ischemia. All rats were injected intraventricularly with colchicine before they were killed. The hippocampal cell counts showed an increase in GAD-immunoreactive somata visualized on the fourth postischemic day. Eleven days after ischemia, the number of GAD-immunoreactive neurons visualized in the hippocampus CA1 and CA3c region decreased. GAD-immunoreactive baskets were visualized in the pyramidal cell layer and the granule cell layer in controls and 4 days after ischemia, but not in the CA1 and CA3c pyramidal cell layer 11 days after ischemia. We suggest the number of GAD-immunoreactive neurons visualized on the fourth postischemic day increases because somatal GAD accumulation increases and, therefore, ischemia may enhance GAD production. Our previous counts of CA1 interneurons 21 days after ischemia in toluidine-stained semithin sections demonstrated no interneuron loss. Therefore we suggest that the decreased number of CA1 and CA3c GAD-immunoreactive neurons visualized 11 days after ischemia is related to a decreased GAD production. It is possible at this stage after ischemia that the interneurons have decreased their GAD production because they have lost their input and/or target cells. We conclude that our counts of GAD-immunoreactive neurons visualized after ischemia express changes in the content of somatal GAD rather than the actual number of GAD-immunoreactive somata. Finally, we conclude that the delayed loss of CA1 pyramidal cells seen 4 days after ischemia is not preceded by loss of hippocampal GAD-immunoreactive neurons.

Effects of cyclosporin a on the endocrine pancreas of the rat. Morphological and immunohistochemical studies

Treatment with the immunosuppressive agent cyclosporin A frequently gives rise to functional alteration of the islets of Langerhans which may result in diabetes. Light microscopy, immunocytochemical, and electron microscopy investigations demonstrate degranulation, vacuolization, and destruction of endocrine pancreatic cells in treated animals. Similar changes, but of a milder degree, are observed in the A-cells. The morphological alterations described are likely to be the result of inhibition of synthesis and secretion in B-cells.

Intraocular transplants of a human gastrinoma in immuno-suppressed rats: morphological, chromatographic and functional studies

Tissue pieces of a metastatic human gastrinoma (ultrastructural Type II) were successfully transplanted to the anterior eye-chamber of rats immunosuppressed with Cyclosporin A. Immunocytochemical investigation of the transplants showed evidence for preserved endocrine activity of tumour cells with immunoreactivity towards the C-terminal of the gastrin/cholecystokinin molecule. Studies of gastric acid secretion in tumour-bearing rats and sham-operated controls with chronic gastric fistulas showed that the basal acid output did not differ between the groups during 3 weeks of study. However, the stimulated gastric acid secretion decreased after 5 days in both groups to remain significantly depressed throughout the study, an effect probably due to Cyclosporin A treatment of the groups. The concentration of immunoreactive gastrin in plasma from rats with tumours in oculo was 5 times higher than in sham-operated rats. Gastrin-34 was the major immunoreactive component in both patient serum and rat plasma. An immunoreactive fraction corresponding to component I was found in the patient serum, but not in the rat plasma, although present in the chamber fluid. Components corresponding to gastrin-17 were found both in the patient serum and in the rat plasma. The chromatographic pattern of the tumour was similar to that in rat chamber fluid. The dominating component corresponded to gastrin-17, while gastrin-34 represented the quantitatively smaller component. Gastrin-34 was, however, relatively more abundant in the tumour extract than in the chamber fluid. The study also indicates that a gastrin-producing tumour transplanted in oculo in immunosuppressed rats may increase the rat plasma concentration of the same molecular forms of gastrin as seen in the clinical situation.

Ribosomal protein interactions in yeast

We have isolated three different monoclonal antibodies specific for mammalian type-I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type-I DNA topoisomerase.

Chromophobe cell renal carcinoma and its variants—a report on 32 cases

This paper reports on 32 chromophobe cell renal carcinomas observed in 697 renal cell cancers (RCC) of adults (peak in the sixth decade of life). The chromophobe cell-type differs from other types of RCC macroscopically, the cut-surface being predominantly of grey-beige colour. Histologically, there are two variants: one is the typical (light) variant (n = 22) and the other is eosinophilic (n = 10). Both variants have in common (a) reaction of the cytoplasm with Hale's acid iron colloid; (b) electron microscopic detection of cytoplasmic microvesicles (150-300 nm), frequently with 'inner vesicles', and (c) low glycogen content in comparison with the clear cell carcinoma. Immunocytochemical investigations on the intermediate filaments show a positive reaction for cytokeratins No. 18 (uniformly) and Nos. 7 and 19 (to varying extents) for both variants, whereas vimentin was not found in any of these carcinomas, in contrast to the clear-cell type. The cytomorphological grading revealed predominantly G II tumours. A lymph node metastasis was found in one patient. On the basis of the mortality curves determined, the prognosis for patients with chromophobe cell carcinomas is more favourable than that of the clear-cell type. In terms of differential diagnosis, on the one hand, the typical (light) variant of the chromophobe cell RCC must be delimited from the clear-cell RCC, and on the other hand, the eosinophilic variant must be distinguished from the chromophilic or 'granular' RCC. Microscopic, histological, histochemical, electron microscopic, and intermediate filament analysis results document that the chromophobe cell type of RCC is a distinct entity. The implications for the nomenclature of RCC, especially with regard to the 'granular' type, are discussed.

Dopaminergic terminals in the rat locus coeruleus: an immunocytochemical investigation.

Dopaminergic terminals in the rat locus coeruleus: an immunocytochemical investigation.