Down-regulation of membrane glycoprotein in amoeboid microglia transforming into ramified microglia in postnatal rat brain

Summary The present study describes the ultrastructural localization and labelling pattern of lectin in different microglial cell phenotypes in the postnatal rat brain using the isolectin, GSA I-B4. The nascent round and amoeboid microglial cells (round cells and cells displaying short processes) were labelled at their cytoplasmic membrane and the membrane of the subplasmalemmal vacuoles. In the course of their transformation into ramified forms with age, dense lectin labelling was observed successively at different sites in the differentiating cells. The most striking feature was the staining of the Golgi saccules on the trans face, the trans tubular network and associated vesicles and vacuoles in the intermediate ramified microglia (ramified cells bearing thick and long processes and those with thin and long processes). The vacuoles with accumulated reaction products were closely associated with many microtubules extending into the cytoplasmic processes. At the surface, the lectin-labelled vacuoles and vesicles appeared to fuse with the membrane and their contents communicated with the exterior. In the advanced or most differentiated ramified microglial cells (cells bearing attenuated processes), the lectin staining at all the above mentioned sites became diminished. In conclusion, in the transformation of the round microglia into their ramified derivatives, the glycoconjugates at the cytoplasmic membrane are progressively reduced. It is postulated from this study that the down-regulation of the glycoconjugates of the microglial plasma membrane is due primarily to their internalization during endocytosis. This process would trigger ade novo galactosyl protein synthesis and/or modification at the trans Golgi saccules and trans tubular network probably in an attempt to degrade the internalized membrane glycoproteins or to replenish the consumption of the membrane glycoconjugates.     

Expression of induced nitric oxide synthase in amoeboid microglia in postnatal rats following an exposure to hypoxia

The present study showed the expression of induced nitric oxide synthase (iNOS) immunoreactivity in amoeboid microglia following an exposure to transient hypoxia in postnatal rats. iNOS immunoreactivity was expressed mainly in the amoeboid microglia in corpus callosum and subependymal regions of the ventricles within 3 h after hypoxia. The expression declined after 5 h, and became undetectable after 15 h and in longer surviving rats. The immunoreactivity of these cells with OX-42, which is a marker for microglia cells and detects complement type three receptors (CR3), was comparable in the rats exposed to hypoxia and the control rats. Immunoglobulin G (IgG) immunoreactivity was observed in the amoeboid microglia up to 3 h after hypoxia but it was undetectable in longer surviving rats and in the control rats. The iNOS expression in the amoeboid mircoglial cells may be related to the host defense and maintenance of structural integrity of the highly vulnerable periventricular white matter after hypoxia. The immunostaining of amoeboid microglial cells with IgG following hypoxia indicates leakage of plasma immunoglobulin from the blood vessels and its removal by the amoeboid microglial cells.     

Immunocytochemical localization of CR3 complement receptors with OX-42 in amoeboid microglia in postnatal rats

Summary The present study described the labelling of amoeboid microglial cells in the postnatal rat brain with OX-42, an antibody that recognizes type 3 complement receptors CR3 in mononuclear phagocytes. Of the diverse morphological forms of amoeboid microglia present in the corpus callosum in early postnatal (2–5 days) rats, cells with a round regular outline, or showing short stout processes, were the most intensely stained. When traced from the main cell colony into the borderline zone with the cortex, the immunoreactivity of amoeboid microglia that assumed a ramified form was drastically reduced. Examination of materials from the late postnatal (8–12 days) age group showed that the majority of the OX-42 positive cells in the corpus callosum became oval, elongated and ramified. Immunoelectron microscopy confirmed the above observations, and also showed that the immunoreactivity in the round amoeboid microglia was localized in their plasma membrane, surface projections and invaginations, as well as in some of the subsurface vacuoles. The immunoreactivity was reduced in the oval cells, and diminished in the elongated or ramified form. It is proposed that the presence of CR3 membrane receptors in amoeboid microglial cells is related to their active role in endocytosis. These, however, diminish with the growth of the brain.     

Fabry disease: ultrastructural lectin histochemical analyses of lysosomal deposits

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from a deficiency of lysosomal α-galactosidase activity. Globotriaosylceramide accumulates predominantly in lysosomes of various tissues. Former studies have clarified the nature of this disease, and the accumulated materials in the lysosomes have been analyzed using biochemical techniques. In the present study, transmission electron microscopy was used to reveal the fine structure of these lysosomal deposits, and sugar residues in the lysosomal deposits in Fabry disease were examined by lectin histochemistry combined with enzyme digestion. This is the first report to describe the lysosomal sugar residues in Fabry disease analyzed using lectin histochemistry at the ultrastructural level. With these techniques, we were able to detect α-galactosyl, β-galactosyl and glucosyl sugar residues in the lysosomal deposits. The experimental procedures used in this study have considerable potential for use in investigations of glycolipid and glycoprotein storage diseases without the need for complex methodology and expensive materials. Received: 17 March 1999 / Accepted: 2 June 1999     

Cystic partially differentiated nephroblastoma in an adult: an immunohistochemical, lectin histochemical and ultrastructural study

AIMS: Cystic partially differentiated nephroblastoma (CPDN) is an uncommon renal multicystic tumour, usually affecting early infants. To our knowledge, this report describes the first case of CPDN occurring in an adult. METHODS AND RESULTS: A 45-year-old man was found incidentally to have a left renal cystic tumour, measuring 20 mm in diameter, at the lower pole far from the pelvis. The tumour was composed of multilocular cystic spaces of variable size and intervening septa without solid nodular areas. The cysts were lined by a single layer of flattened, hobnail, or columnar epithelium. The septa were made of mesenchymal cells, which were admixed with small numbers of loosely aggregated blastemal cells, occasional tubular structures in various stages of development, and a few glomeruloid structures. The tumour cells had no anaplasia, and mitoses were rare. Immunohistochemical and lectin histochemical studies revealed that the cyst lining epithelium and the tubular structures in the septa expressed predominantly the markers for distal tubules and collecting ducts. Ultrastructurally, the cyst lining cells closely resembled collecting duct cells while some tubular structures showed an immature nephrogenic morphology. The patient was alive and well without evidence of recurrence 11 months after surgery. CONCLUSIONS: CPDN does occur in adults, as experienced in Wilms' tumour, though its incidence is extremely low. This study suggests that CPDN may show maturation intermediate between cystic nephroma and Wilms' tumour, even in adult cases.     

Monocyte involvement in glomerular crescents: a histochemical and ultrastructural study

The biopsies from 14 patients with glomerulonephritis with crescents were studied to determine the extent of monocyte involvement in crescent formation. Antiglomerular basement membrane (GBM) disease was diagnosed in six and immune complex (IC) glomerulonephritis in eight. Alpha-Naphthyl acetate staining of frozen sections for nonspecific esterase was done in 11 cases (five anti-GBM and six IC), and the peroxidase-antiperoxidase method for lysozyme was done in two (both IC). Electron microscopy of crescents was carried out in 12 cases. The mean number of nonspecific esterase (or lysozyme-excluding neutrophils) positive cells was 6.0 (+/-5.8) in the anti-GBM cases and 1.1 (+/- 1.4) in the IC ones. The difference was significant (P less than 0.05). By electron microscopy the cellular crescents in cases of anti-GBM disease contained variable numbers of monocytes (macrophages) and epithelial cells. Those from cases of IC disease showed very few monocytes but numerous epithelial cells. The results show that monocytes participate in a numerically significant way in crescent formation in anti-GBM disease but appear to be of minor importance in crescents in IC disease.     

Pseudopyogenic granuloma: Enzyme histochemical and ultrastructural study

In an attempt to elucidate the nature of the abnormal vessels in pseudopyogenic granuloma, we performed enzyme histochemical analysis of 10 lesions and correlated them with electron microscopic findings in four lesions. The dermal vessels of pseudopyogenic granuloma possess voluminous endothelial cells demonstrating marked activity of various hydrolytic and respiratory enzymes. The alkaline phosphatase activity, however, is characteristically faint or absent. Electron microscopy reveals that mitochondria and the other common cytoplasmic organelles are abundant in the hypertrophic endothelial cells, probably reflecting increased metabolic cellular activity, whereas specific endothelial granules (Weibel-Palade bodies) are generally sparse in these abnormal cells. Other notable features occurring in several endothelial cells are cytoplasmic vacuolation and the acquisition in the subnuclear zone of dense bodies associated with fine actin-like filaments, 55 to 75 A thick. The latter change is interpreted as evidence of endothelial contraction. Classification of the abnormal dermal vessels using available criteria has proved difficult, but it is suggested that they arise predominantly from the venous side of the microcirculation and are mainly postcapillary venules, although involvement of both larger and smaller caliber vessels also occurs.     

Pulmonary blastoma: an ultrastructural and histochemical study

We present a unique case of pulmonary blastoma and describe its histology and ultrastructure. The stromal component exhibited bizarre multinucleated giant cells containing previously undescribed granules. These membrane-limited granules were eosinophilic, periodic acid-Schiff positive, diastase resistant, acid phospha-tase and nonspecific esterase negative, immunohisto-logically negative for lysozyme, albumin, and gamma globulin, and often homogeneously electron dense. A similar electron-dense material accumulated in the rough endoplasmic reticulum of these stromal giant cells, suggesting that the material in the granules is synthetic. The material appeared to be a glycoprotein but was not alpha_I -antitrypsin, alpha-fetoprotein, or human chorionic gonadotropin by immunohistologic techniques. These stromal cell granules differ from granules described in epithelial cells of other pulmonary blastomas but resemble granules found in some sarcomas.     

The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7–9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert’s resorcin–fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.     

Biological aspects of the tongue morphology of wild-captive WWCPS rats: a histological,histochemical and ultrastructural study

The aim of this study was to characterise the tongue in wild-type rats using several microscopic techniques. Warsaw Wild Captive Pisula Stryjek (WWCPS) rats belong to a lineage of wild-caught rats. The study was carried out on tongues of 15 male and 15 female WWCPS rats. Histological, histochemical and ultrastructural studies were carried out. There were no significant differences between the male and female WWCPS rat tongues. There was a median groove approximately 1 cm long in the apex of the tongue that faded caudally. The intermolar prominence was clearly marked in the distal part of the lingual body. Lingual mechanical papillae located on the surface of the tongue formed four subtypes based on their shape: small filiform papillae, giant filiform papillae, thin elongated filiform papillae and wide filiform papillae. Gustatory papillae formed the second group of papillae and were divided into bud-shaped fungiform papillae, a single vallate papilla surrounded by an incomplete papillary groove and foliate papillae, which were a well-formed and composed of several pairs of folds divided by longitudinal grooves. In the posterior lingual glands (mucoserous and serous), acidic sulphated mucin-secreting cells gave a strong AB pH 2.5 positive reaction, and a positive reaction with the AB pH 1.0 stain for acidic carboxylated mucin. Double AB/PAS staining showed the presence of the majority of mucous cells with predominant of acidic mucins. Positive PAS staining showed the presence of neutral mucin. HDI staining demonstrated a weak positive reaction within Weber’s glands of the WWCPS rat tongue.     

Askin tumor with metastasis to the scalp: a histochemical, immunohistochemical and ultrastructural study

A 29-year-old woman presented with facial edema, and imaging disclosed a tumor extending from the anterior chest wall to the anterosuperior aspect of the mediastinum. Transbronchial cytology of the primary tumor and biopsy of the metastatic scalp lesion were performed. Histologically, the tumor consisted of closely packed small round cells. The neoplastic cells generally had round nuclei, finely dispersed chromatin, and small to prominent nucleoli. Histochemically, the cytoplasm of the neoplastic cells contained abundant glycogen and stained with Grimelius silver. Immunohistochemically, the neoplastic cell membranes reacted with CD99 (MIC2) and the neoplastic nuclei reacted with Fli-1, but various other markers, including lymphocyte and skeletal muscle markers, were not detected. No neoplastic cells were also reactive for chromogranin A, synaptophysin, and neurofilament. Ultrastructurally, some neoplastic cells had delicate cytoplasmic processes and contained membrane-bound dense core granules in the cytoplasm. Even if results are immunohistochemically negative for neuroendocrine markers, the combination of immunohistochemistry of CD99 (MIC2) and Fil-1 may be useful in diagnosing Askin tumor or its metastatic lesion.     

Elastofibroma: a histochemical, immunohistochemical, and ultrastructural study of two patients

Elastofibroma is a rare neoplasm. In this article, we report our study of the pathogenesis of fibrosis in elastofibroma. Three tumors obtained from two patients were selected. One patient was a 57-year-old Japanese woman who had a bilateral tumor, and the other patient was a 83-year-old Japanese man. All tumors occurred in the infrascapular region. Macroscopically, the cut surface of all tumors showed a poorly defined and whitish mass with yellowish foci. Microscopically, the tumor consisted of collagen fiber bundles, abnormal elastic fibers, and spindle cells suggestive of fibroblasts. Elastica-van Gieson and Masson-trichrome stain identified abnormal elastic fibers and abundant collagen fibers, respectively, in elastofibroma. Immunohistochemically, fibroblasts were positive for CD34 but negative for alpha-smooth muscle actin and h-caldesmon. Additionally, the cytoplasm of many fibroblasts was positive for TGF-beta in all tumors. Ultrastructurally, some fibroblasts with abundant organelles in one tumor were observed in the adjacent area to amorphous elastic mass and bundles of collagen fibers. However, no myofibroblasts were ultrastructurally identified in the tumor. Finally, our study supplies further evidence that elastofibroma may show the proliferation of CD34-positive fibroblasts and contain no myofibroblasts, and that fibroblasts may produce both abnormal elastic fibers and collagen fibers through the secretion of TGF-beta.     

Glycoconjugate expression in human renal oncocytomas. A lectin histochemical study

A panel of lectins chosen for its specificity for segments of the nephron was used in an avidin-biotin-peroxidase reaction to stain 13 human renal oncocytomas and a specimen of oncocytomatosis. The lectins were derived from Lotus tetragonolobus, Glycine max, Dolichos biflorus, and Arachis hypogaea. Two patterns predominated: one of staining with the lectins of D biflorus and G max, suggesting distal tubular or collecting-duct differentiation, and one of staining with the lectin of L tetragonolobus, suggesting proximal tubular differentiation.     

The prenatal human submandibular gland: a histological, histochemical and ultrastructural study

The submandibular glands of 6 human fetuses, 13.5-16 weeks old, were studied using light and electron microscopic techniques. The developing gland at this stage consisted of a bush-like network of terminal buds (primitive acini) and primary ducts surrounded by a loose mesenchyme. Both components had a lumen which was surrounded by 1 or 2 layers of epithelial cells. Those cells adjacent to the lumen were attached by desmosomes but lacked well developed terminal bars. The cells were separated by an intercellular space, into which projected numerous microvilli. The cytoplasm of the epithelial cells contained the usual organelles with some cells containing large accumulations of glycogen granules. Serous granules and the luminal contents were both strongly PAS and AB positive. The function of this secretory material, at this stage of human development, is unknown. No mucus-like granules were observed. The terminal buds and primary ducts were surrounded by a well developed basal lamina and contained a few elongated cells which appeared morphologically as developing myoepithelial cells. Morphologically the development of the human submandibular gland, at 13.5-16 weeks of age, is roughly equivalent ot the developmental stage of the gland seen in the newborn rat or mouse. By birth, the human submandibular gland would likely reach a mature state, because there would be ample time remaining, in a normal gestation, for the maturation process to be completed.     

Intranephronic calculosis in rats: an ultrastructural study.

Female Sprague-Dawley rats weighing 45-55 g fed a purified diet for 18 days developed hydroxyapatite intratubular lithiasis, the earliest calcific lesions being detectable by light microscopy on Day 12. The kidneys from these rats revealed ultrastructural changes in proximal tubular cells prior to intraluminal microlith formation. These changes included evidence for increased intracellular calcium, accumulation of electron-dense cytoplasmic granules, and vesiculation and shedding of brush border microvilli within Segment I of the proximal tubule. It was concluded, on the basis of ultrastructural observation, that microvesicles were formed by the shedding of vesiculated microvilli and microvesicles initiated the formation of an intraluminal microurolith in Segment I of the proximal tubule. The initially formed microurolith grew, as it traveled down the nephron, to a size large enough to be visualized by light microscopy. When it reached Segment III (straight segment) of the proximal tubule, the microurolith reached a size so large that it became difficult for it to pass the loop of Henle.     

Increased expression of transferrin receptors and iron in amoeboid microglial cells in postnatal rats following an exposure to hypoxia

This study was aimed to ascertain the effects of hypoxia on regulation of iron in the brain of newborn rats. At 3 h and 1 day after hypoxic exposure transferrin receptor expression as detected immunohistochemically with the antibody OX-26, and the iron content as shown by Perls' staining of amoeboid microglial cells was markedly increased. The induced changes, however, were not evident at 10 min and in longer surviving rats killed at 3 and 7 days. It is suggested that the upregulation of transferrin receptor expression coupled with iron uptake by amoeboid microglial cells in the periventricular regions is a protective mechanism to facilitate the sequestration of excess iron that may have been released either from the iron-rich oligodendrocytes, or accumulated due to a disruption of its normal transportation following the hypoxic insult. This would help protect the brain from harmful effects of iron.     

The effect of vitamin A on CCl4-induced hepatic injuries in rats: a histochemical, immunohistochemical and ultrastructural study

In this study, we aimed to investigate the effect of vitamin A on the transformation of the Ito cells to fibrogenic form and suppression of the development of fibrosis. Carbon tetrachloride intoxication was performed on rats for 2, 8, 12 or 20 weeks and 5x10(4) IU vitamin A (as retinol palmitate) was injected subcutaneously once every 4 weeks. Ito cells were detected by gold chloride impregnation, as well as desmin and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry. Additionally, all groups were examined ultrastructurally. The number of Ito cells that were labelled positively with gold impregnation decreased in the fibrotic groups; however, alpha-SMA and desmin immunopositive Ito cells increased. The samples from animals that were treated with vitamin A showed an increase in labelling with gold impregnation but a decrease in alpha-SMA immunopositivity. The data showed that vitamin A can prevent hepatic injury, by suppressing the transformation of Ito cells to fibrogenic form. We conclude that vitamin A has potential for the treatment of hepatic fibrotic diseases. Alpha-SMA immunohistochemistry was found to be more informative than desmin immunohistochemistry for monitoring liver fibrosis.