Plasticity of innervation of the medulla of axillary lymph nodes in the rat after antigenic stimulation

The purpose of this investigation was to test the hypothesis that activation of the immune system in rats will lead to changes in the density of innervation in lymph nodes. In order to reduce the variability between animals, the rats were reared under sterile conditions and immunostimulation was effected by subcutaneous application of bovine albumin in a region draining to the axillary lymph nodes of both sides. Control animals received an equivalent application of sterile physiological saline. The animals were sacrificed 10 days and 27 days and 4 months after immunostimulation. The nerves in the axillary lymph nodes were quantified by light microscopy in silver impregnated sections and at the ultrastructural level on ultrathin sections. The survival times were chosen so that the first group was in the ascending phase of antibody production, the second group at the peak, and the third group in the declining phase. Both at the light and ultrastructural levels, there were statistically significant differences in the density of innervation of medulla between the groups, with a particularly pronounced increase in the group 4 months after immunostimulation. At the ultrastructural level, there was also an increase in the density of incompletely ensheathed axonal profiles in the parenchyma of the medulla, while the nerves associated with blood vessels were not increased. We conclude that immunostimulation leads to morphological changes in the innervation of the medulla of axillary lymph nodes, that are consistent with the concept of functional activation of the autonomic nervous system through the immune system. © 1994 Wiley-Liss, Inc.     

Morphology and endocrine production of cells in the islets of Langerhans of the Chacma baboon

Biopsies of the pancreas head, tail, and uncinate regions of 6 Chacma baboons (Papio ursinus) were processed for ultrastructural and immunocytochemical (ICC) studies using avidin-biotin peroxidase label for light microscopy (LM) and immunogold for electron microscopy (EM). Survey 0.5 micron sections of Spurrs resin embedded tissue revealed areas of suitable islets. Thin 100-nm sections were then cut and stained from the osmicated blocks for ultrastructural studies. For ICC investigations, 1 micron sections were immunolabeled for LM before areas were selected for thin sectioning for ultrastructural immunolabeling. The baboon endocrine pancreas ultrastructure was found to be similar to that of other mammals with minor differences in islet and secretory granule size and shape and in electron opacity of the secretory granule cores. Insulin glucagon, somatostatin and pancreatic polypeptide (PP) producing cells were described. A small number of cells were seen to contain both glucagon and PP and some D cells were observed to contain a few granules with both the appearance and immunoreactivity of A cell secretory granules. Statistical analysis of 100 secretory granule diameters of each of the 4 cell types in 6 baboons revealed significant differences (p less than 0.001) in size between all but those of the A and D cells. The insulin precursor subunit, C-peptide, and the glucagon precursor, glicentin, were each found together with the final hormone product in their respective secretory granules. The precursors were often located toward the periphery of the secretory granule, suggesting that the conversion of precursor to active hormone may be membrane associated. A nonrandom topographical association was observed between A and D cells, suggesting a strong functional implication.     

Occurrence of cell death (apoptosis) during the involution of liver hyperplasia

A single intravenous injection of lead nitrate at a dose of 10 mumoles/100 gm of body weight caused liver enlargement associated with hepatic cell proliferation. In the present study the involution of liver hyperplasia which follows the withdrawal of lead was studied in male Wistar rats. Histologic examination of liver sections from rats killed during the regression of the liver did not show any sign of massive lytic cell necrosis; no variation in the levels of serum glutamate pyruvate transaminase could be observed during the same time period; however, light microscopic observation of sections from the involuting liver showed the presence of several apoptotic bodies; the occurrence of apoptotic bodies was also confirmed by ultrastructural examination. Their incidence was found to be markedly increased at 5 days after treatment, a time period when the liver is already regressing; very few apoptotic bodies were observed in control animals or in treated rats 2 days after lead injection, a time point when mitotic index reached its maximum, or at 15 days, when the liver had returned to control values. These findings suggest that removal of excess liver which follows the initial hyperplasia caused by lead is due to a controlled mode of cell death, namely, apoptosis.     

Orientation of the wall of the small intestine for electron microscope studies]

A simple and reliable technique for orientation of small pieces of the intestine is suggested; the technique makes it possible to investigate semi-thin sections under light microscope, and ultrastructural topography of the intestine wall--in electron microscope.     

Fine-needle aspiration biopsy diagnosis of malignant rhabdoid tumor of the kidney

A series of six fine-needle aspiration biopsies from three patients with malignant rhabdoid tumor of the kidney is reviewed. The smears revealed round, polygonal, and irregularly shaped cells with large nuclei and prominent nucleoli. Some of the tumor cells contained light pink to purple cytoplasmic inclusions that correlated with the eosinophilic inclusions seen in histopathologic sections and filamentous cytoplasmic aggregates noted on ultrastructural examination. Diagnosis of malignant rhabdoid tumor of the kidney may be suggested from aspiration biopsy smears; however, further confirmation of the diagnosis by histologic or ultrastructural examination is desirable.     

A procedure for sampling specific portions of identified neurons for ultrastructural examination

A procedure is described which facilitates the ultrastructural examination of selected regions of a neuron identified because it is Golgi-impregnated or HRP-filled. The procedure involves assigning a two-dimensional coordinate to every point on a drawing of the neuron by superimposing a grid over the drawing. A complete series of thick sections through the block containing the identified cells can then be related to each point on the drawn cell. A thick section which contains a region of interest is remounted on a plastic blank. From this new block one obtains thin sections for ultrastructural examination.     

Characterization of a new neurosecretory cell type containing gastrin-cholecystokinin-like peptide in the locust pars intercerebralis: ultrastructural and immunocytochemical studies, in situ and in vitro

A new neurosecretory cell type of the locust pars intercerebralis, immunolabelled with an antiserum against a vertebrate peptide related to gastrin-cholecystokinin (CCK-8(s)), was characterized both in situ and in primary cell cultures. Semithin sections of pars intercerebralis were first immunostained in order to identify neurosecretory cells containing CCK-like material and then examined by electron microscopy. The neurosecretory cells containing CCK-like material were paraldehyde fuchsin negative and were unequivocally identified in ultrathin sections adjacent to immunostained semithin sections. They exhibited neurosecretory vesicles of variable electron density, ranging in diameter from 150 to 250 nm. Immunogold labelled ultrathin sections adjacent to unlabelled ultrathin sections allowed for the unambiguous localization of CCK-like immunoreactive material over the neurosecretory vesicles of the cells containing CCK-like material. Immunoreactivity towards CCK-8(s)-like peptide could also be detected in pars intercerebralis neurosecretory neurons grown in vitro. The CCK-like positive neurons showed a multipolar morphology with fine processes radiating from the cell body. The positive cells had the same ultrastructural characteristics as the in situ CCK-like neurons. The pattern of neurite outgrowth on reactive CCK-like neurosecretory cells in vitro and the neuroanatomical pathway of the CCK-like immunoreactive neurosecretory cells in situ could be correlated. On the basis of their number, size and localization in the locust pars intercerebralis, it is possible that the CCK-like neurosecretory cells correspond to neurosecretory cell type C, which has not, to date, been identified at the ultrastructural level.     

Quantitation of the nuclear DNA content in tissue prepared for electron microscopy

The possibility of determining the cellular DNA content in tissue prepared for electron microscopy (EM) was investigated. Comparative cell population DNA analyses of deparaffinized and Epon-embedded sections of equal thickness (2 micron) from the same tumor (osteosarcoma) showed almost identical DNA distribution curves. The methodologic prerequisites for both EM and DNA analyses of identical cells was then investigated. Using a special technique involving consecutive sections of 500-600 A and 2-micron thickness for EM morphology and Feulgen staining, respectively, nuclear parts of identical cells could be identified in both types of sections. Thus, cells selected in ultrathin sections could be identified in corresponding Feulgen-stained Epon-embedded sections for cytophotometric DNA analysis. All normal cells (lymphocytes, fibroblasts, macrophages), as defined and selected at the ultrastructural level, proved to be diploid (with normal DNA content) as determined in the corresponding Feulgen-stained Epon-embedded sections, whereas all analyzed osteosarcoma cells, except one, were aneuploid. The results indicate that 2-micron thick Feulgen-stained, Epon-embedded specimens from osteosarcoma, previously fixed in glutaraldehyde and OsO4, may be used for cytophotometric DNA measurements. Since aneuploidy is almost exclusively found in malignant cells, this criterion may serve as a marker for such cells. Consequently, whenever it is questionable whether in a mixed population a given cell selected for ultrastructural study is malignant or not, DNA cytophotometry may give decisive information, provided aneuploidy can be demonstrated.     

The corticotroph of the rat adenohypophysis: A comparative study

The immunological and cytological specificity of antiserum to adrenocorticotrophic hormone was established and the corticotroph defined at the light microscopic level by immunocytochemistry. Our observations substantiated recent reports by several investigators. By comparing thin methacrylate sections with adjacent 1 μ-methacrylate sections immunochemically stained with anti-ACTH, the ACTH cell was identified at the ultrastructural level and its morphology studied. Using the criteria established at light and electron microscopic levels, the corticotroph was readily found in tissue which had been fixed in paraformaldehyde-osmium mixture and embedded in epoxy resin. Ultrastructural changes in this cell in response to experimentally designed alterations in the pituitary ACTH levels confirmed this identification. The ACTH cell was likewise identified in glutaraldehyde-fixed material. Thyrotrophs were tentatively defined. Several ultrastructural studies responsible for conflicting reports in the literature were investigated. It was concluded that the corticotroph is a unique cell type and that granule size and morphology remain important criteria for adenohypophyseal cell identification regardless of the fixation used. The need for a definitive immunocytochemical study of thyrotrophs at the ultrastructural level was recognized.     

A morphometric analysis of the somata and organelles of regenerating hypoglossal motoneurons from the rat

Summary A detailed morphometric evaluation of the somata and organelles of regenerating hypoglossal motoneurons from the rat was conducted. The volume of the hypoglossal nucleus and various parameters used to appraise neuronal size were estimated from 50 m sections. The subcellular composition of randomly selected neurons was quantified from 1 m and ultrathin sections. The volume of neuronal nuclei, nucleoli, mitochondria and lysosomes as well as the surface area of intracellular membranes were determined. Seven to 30 days following axotomy the volume of the hypoglossal nucleus was significantly diminished, undoubtedly reflecting dendritic retraction (P < 0.05). Concomitantly, all estimates of neuronal size indicated significant neuronal enlargement (P < 0.05). Ultrastructural alterations were most prominent 7 days following nerve transection: nucleolar volume was significantly increased, rough endoplasmic reticulum surface area was reduced, and non-Golgi smooth membrane surface area increased (P < 0.05). In general, other organelles resisted the influence of axotomy and all ultrastructural parameters returned to control levels 21 to 30 days following the nerve transection. Functional recovery was detected in all animals 21 and 30 days following axotomy. The measured responses of axotomized hypoglossal motoneurons are similar to those reported for retinal ganglion cells of the goldfish (Whitnall & Grafstein, 1982, 1983), suggesting common metabolic events among these distinct neuronal populations following axonal transection.     

Ultrastructural plasticity in the hippocampus organotypic tissue culture

Hippocampal cross sections of 7-9 day old rats were explanted onto semipermeable microporous membranes for 14-16 days followed by electron microscopic investigation. Besides the structures typical for adult hippocampus in vivo, substantial ultrastructural signs of metabolism and synaptic connections in explants were revealed: redistribution of polysomes and cisterns of granular endoplasmic reticulum, numerous pinocytotic invaginations, growth cones, somatic filopods, dotted (perforated) contacts, new formation of synapses and vacant postsynaptic sites. The data obtained indicate the active compensatory processes of plasticity, which ensure the maintenance of structure and function of hippocampal tissue in vitro.     

Electron microscopy of the pathology and chemotherapy of Legionella pneumophila infection in the chick embryo

The ultrastructural pathology induced in organs of chick embryos previously infected with 100 times the yolk sac LD50 of Legionella pneumophila was examined by electron microscopy. Subtle pathological changes were observed three days post-inoculation while more extensive cellular and sub-cellular necrosis occurred at four days post-inoculation. Selected antimicrobial agents were administered three days after infection and evaluated for their ability to reduce or reverse ultrastructural lesions. In this study, ciprofloxacin was the only antibiotic that reversed tissue damage induced in response to infection with L. pneumophila while doxycycline, erythromycin, and rifampicin were also effective, but to a lesser degree. These results demonstrated the superior antimicrobial activity of ciprofloxacin and further illustrated the efficacy of the fertile hen's egg as a useful in vivo assay system for the evaluation at the ultrastructural level of putative chemotherapeutic agents for the treatment of Legionnaires' disease.     

Comparative study of two immunocytochemical techniques in the electron microscopical detection of alpha-1-antitrypsin in routinely processed liver biopsies

Two indirect immunocytochemical techniques using different markers, namely peroxidase and gold, were applied to ultrathin sections of liver biopsies to detect alpha-1-antitrypsin. The blocks used were taken from the routine electron microscopy files and had been processed optimally for maximum ultrastructural preservation. The immunogold technique provided the best method for localizing alpha-1-antitrypsin and was associated with ultrastructural preservation equivalent to that seen in routinely processed liver biopsies. The procedure may be a useful adjunct to understanding the pathogenesis of alpha-1-antitrypsin deficiency and in recognizing further variants of this disorder.     

Immunochemical studies of the endocrine cells of the gastrointestinal tract. II An immunoperoxide technique for the localization of secretin containing cells in human duodenum.

Endocrine cells containing secretin have been identified in the epithelium lining human duodenum by direct and indirect immunoperoxidase techniques using immune sera raised against pur natural secretin. The techniques were applied to sections of carbodiimide-fixed tissue embedded in polyethylene glycol. Some sections, stained by a modified indirect technique, were processed for electron microscopy; secretin-containing granules were present by ultrastructural preservation was too poor to be of value. The potential advantages of a peroxidase technique over fluorescein-coniugated antisera are discussed.     

Comparative Study of Two Immunocytochemical Techniques in the Electron Microscopical Detection of Alpha-1-Antitrypsin in Routinely Processed Liver Biopsies

Two indirect immunocytochemical techniques using different markers, namely peroxidase and gold, were applied to ultrathin sections of liver biopsies to detect alpha-1-antitrypsin. The blocks used were taken from the routine electron microscopy files and had been processed optimally for maximum ultrastructural preservation. The immunogold technique provided the best method for localizing alpha-1 -antitrypsin and was associated with ultrastructural preservation equivalent to that seen in routinely processed liver biopsies. The procedure may be a useful adjunct to understanding the pathogenesis of alpha-1-antitrypsin deficiency and in recognizing further variants of this disorder.     

Experimental injury of the optic nerve with optic disc swelling.

An ultrastructural study utilizing horseradish peroxidase was performed to determine the mechanism and consequences of leakage of vascular protein following injury of the optic nerve. Unilateral optic nerve injuries were produced in four rhesus monkeys by making a cautery lesion on the retrobulbar portion of the optic nerve. Optic disc changes were followed with stereo fundus photography and fluorescein angiography. Three to 14 days after injury horseradish peroxidase was given intravenously and the tissue was prepared for electron microscopy, including serial sections of selected tissue blocks. Fundus photography and fluorescein angiography showed edema of the optic disc in two animals. There was leakage of horseradish peroxidase into the optic nerve head from the optic nerve lesion and the peripapillary choriocapillaris. Although the pathway of horseradish peroxidase leakage in the injured optic nerve was not entirely clear, serial sections indicated intraendothelial channels as one possible route. Alterations of the optic nerve head were confined to the axon segments anterior to the injury, and included aggregation of mitochondria, disruption of neurotubules, and swelling. These findings suggest that optic nerve injury produces damming of axoplasmic flow and that swelling of the optic nerve head is the result of axon enlargement.     

Development of macrophages in the lungs of fetal rabbits,rats, and hamsters

Fetal rabbits (days 13–32), rats (days 14–22), and hamsters (days 11–15) and selected postnatal animals were examined for pulmonary macrophages or their precursors in 2-m?m sections stained by PAS-lead hematoxylin (all species), electron micrographs (rabbit and rat), and cytochemical incubations for acid phosphatase (rabbit and rat), aliesterase, and N-acetyl glucosaminidase (rabbits). All methods revealed macrophages in perinatal specimens. The appearance and distribution of these cells were compared in the different preparations to establish the reliability of PAS-lead hematoxylin for identifying them in less developed fetal lungs, where they are less active for lysosomal enzymes the earlier the stage examined. In the sections, macrophages are seen to possess a round or indented nucleus, an irregular contour, and a deep purplish-gray cytoplasm containing a variety of pink PAS-stained granules, equated with heterolysosomes by ultrastructural cytochemistry. In less developed lungs, macrophages occur along with putative precursors having a more rounded outline and fewer PAS-stained granules. In pseudoglandular lungs these precursors predominate over rather vacuolated macrophages resembling Hofbauer cells. In all three species both cell types first appear in the stroma during the bronchial bud stage and are frequently seen to divide from that time on. The earliest precursors have a relatively sparse cytoplasm which later increases in daughter cells. Hofbauer-like cells disappear during the canalicular stage of development, replaced by macrophages and transitional forms from the more rounded precursors. In day 21 rabbit lungs, scattered stromal cells are reactive for aliesterase, and, some days later, for acid phosphatase and glucosaminidase. Free mononuclear cells are rare in airways of pseudoglandular lungs but become common later. A day or two before birth in rats, free cells range between rather undifferentiated leukocytes to typical macrophages, but cells with the macrophage's complete repertory of inclusions are seen only after birth. In the fetus, typical monocytes were not identified in either the pulmonary stroma or the airways. A replicating population of macrophage-like cells therefore resides in fetal lungs. It is established before bone marrow is formed and, in rats, before monocytes have appeared in the circulation.     

Extraskeletal myxoid chondrosarcoma of the pleura. Report of a case clinically simulating mesothelioma.

A primary extraskeletal myxoid chondrosarcoma of the pleura that clinically mimicked a malignant mesothelioma in a 66-year-old man with a history of asbestos exposure is described. Although exceedingly rare in this location, the characteristic histologic features, immunohistochemical reactivities, and ultrastructural features support the diagnosis of extraskeletal myxoid chondrosarcoma. Many ferruginous (asbestos) bodies consistent with the exposure history were found in the lung tissue sections and confirmed by energy-dispersive spectrometry. This case demonstrates an unusual pleural primary neoplasm associated with asbestos.     

Ultrastructural immunocytochemistry of somatotrophs and mammotrophs in embryos of the dwarf mutant mouse

The embryonic development of somatotrophs and mammotrophs in the adenohypophysis in dwarf (dw) mutant mice was studied by means of ultrastructural immunocytochemistry. Embryos at 16-18 days of gestation were removed from litters consisting entirely of dwarf homozygotes obtained from homozygous matings of adult dwarfs in which reproductivity was induced by means of hormone supplementation and renal capsule implants of normal pituitaries. Litters from normal (+/+) matings served as controls. Pituitaries from adult normal and dwarf mice also were removed and processed with the embryonic material. Thin sections were exposed to anti-growth-hormone serum (anti-GH) or to antiprolactin serum (anti-PRL) and processed immunocytochemically with the colloidal gold procedure. In the normal pituitaries, a strong positive reaction to anti-GH occurred in the adult and in embryos as early as 16 days, whereas the reaction to anti-PRL was strong in the adults but relatively weak in the embryos. In dwarf embryonic and adult pituitaries, ambiguous cells showing combinations of features characteristic of somatotrophs and mammotrophs were present. However, neither these cells nor other granulated cells reacted to anti-GH or to anti-PRL, except for a questionable reaction to the latter at 18 days and in the adult. Thus, the dwarf pituitary shows functional as well as morphological abnormalities as early as 16 days of gestation.     

Clear cell variant of medullary carcinoma of the thyroid

A previously unreported clear cell variant of medullary thyroid carcinoma is described. This histologic appearance was observed in sections from both the primary thyroid tumor and a vertebral metastasis. This variant merits recognition because it may be confused with a variety of other tumors occurring in the thyroid that contain cells with optically clear cytoplasm. Immunohistochemical and ultrastructural studies can be helpful in establishing the diagnosis of these tumors.