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PubMed1987

Ultrastructural visualization of anterogradely transported horseradish peroxidase in developing corticospinal tract of rat.

Until now a satisfactory method for electron microscopic (EM) detection of anterogradely transported horseradish peroxidase (HRP) in developing neural tissue, using sensitive chromogen tetramethylbenzidine (TMB), has not been described. Use of the stabilizing agent ammoniumheptamolybdate (AHM), in combination with a modified prolonged osmication [4 hr at pH 5.0 in 0.1 M phosphate buffer (PB)] made possible visualization of HRP-TMB-(AHM) reaction product at the ultrastructural level in outgrowing corticospinal tract (CST) fibers of young postnatal rat. This reaction product appeared to be very distinctive and clearly detectable, making ultrastructural identification of HRP-labeled outgrowing CST fibers in rat spinal cord rather easy. In addition, the procedure described in this report preserves the ultrastructural details of the developing neural tissue.

Animals↗
PubMed1989

Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques.

Several immunogold techniques were used to determine the ultrastructural localization of calcitonin gene-related peptide (CGRP), tachykinin, somatostatin, and gamma-amino-butyric acid (GABA) immunoreactivity in the dorsal horn of rat spinal cord. The immunocytochemical reactions were carried out directly on ultrathin sections from non-osmicated frozen tissue, non-osmicated low temperature-embedded (Lowicryl K4M) tissue, and osmicated epoxy-embedded material. Preservation of ultrastructural morphology and immuno-labeling efficiency were compared. Morphology of subcellular organelles was relatively good in ultra-thin frozen sections, which showed the highest immunoreactivity. However, only very small samples of tissue could be examined. Although there was relatively good immunolabeling in the Lowicryl K4M-embedded tissue, the ultrastructure of the neuropil, and particularly that of synapses, was poorly maintained. In contrast, the osmicated epoxy-embedded material offered optimal morphological preservation together with accurate subcellular localization of all antigens under study. The latter approach thus enabled clear visualization of CGRP, tachykinin, and somatostatin immunoreactivity restricted to large dense-cored vesicles (90-150 nm diameter) in many axonal and synaptic profiles in the superficial layers of the dorsal horn. CGRP- and tachykinin-positive profiles were also present in the tract of Lissauer. GABA immunoreactivity was present mainly in axons and terminals, and less frequently in somatic and dendritic profiles. In terminals, which often formed symmetrical synapses on immunonegative dendritic profiles, it was associated with small (30-60 nm diameter) clear vesicles and mitochondria. Double immunolabeling was possible on all preparations, but the osmicated, epoxy-embedded material clearly showed co-localization of peptides, especially of CGRP and tachykinins, within the same dense-cored vesicles in axonal fibers and/or terminals. On the other hand, peptide and GABA immunoreactivity were consistently seen in different nerve profiles. In a few cases, GABAnergic terminals were seen to synapse on tachykinin-positive fibers.

Animals↗
PubMed1991

Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.

Animals↗
PubMed1993

Diamine oxidase-gold labels histamine in human mast-cell granules: a new enzyme-affinity ultrastructural method.

We developed a post-embedding ultrastructural enzyme affinity-gold technique to label histamine. Diamine oxidase (DAO)-gold complex was prepared and tested for specificity with routinely processed, Epon-embedded, histamine-rich human mast-cell granules and histamine-agar test blocks, both of which were labeled. Specificity controls included removal of staining by DAO digestion of Epon sections containing mast cells or by filtering DAO-gold over histamine-agarose beads, and a wide variety of controls (for these specificity controls) that did not abrogate mast-cell granule or histamine-agar block labeling with DAO-gold. Internal negative controls in each sample included the failure of DAO-gold to stain Type II alveolar pneumocyte lamellar bodies or altered, swollen mast-cell granules from which histamine has been released. The new post-embedding enzyme affinity-gold technique is effective in optimally prepared ultrastructural samples that allow clear identification of key ultrastructural morphology.

Amine Oxidase (Copper-Containing)↗
PubMed2006

Oncocytic carcinoma of parotid gland: a case report with clinical, immunohistochemical and ultrastructural features.

BACKGROUND: Oncocytic carcinoma is an extremely rare neoplasm of the salivary glands. We report a case of oncocytic carcinoma arising in a parotid gland in a 66-year-old female. METHOD: An excisional biopsy of the parotid tumor was performed. The specimen was submitted for histology and after fixation in formalin solution and inclusion in paraffin, 3-5 microm sections were stained with hematoxylin and eosin for conventional evaluation and Periodic acid Schiff stain. Immunohistochemical studies were performed using antibodies against mitochondrial antigen, keratin, S-100, alpha-actin, vimentin, alpha-1-antichymotrypsin as well as an ultrastructural analysis was performed. RESULTS: Frozen sections revealed an infiltrative growth pattern and the diagnosis of a malignant epithelial lesion was made. Permanent sections stained with haematoxylin and eosin revealed a neoplasm that had replaced a wide area of the parotid gland and had invaded subcutaneous adipose tissue. Perineural invasion was evident, but vascular invasion was not found. Neoplastic elements were large, round or polyhedral cells and were arranged in solid sheets, islands and cords. The cytoplasm was abundant, eosinophilic and finely granular. The nuclei were large and located centrally or peripherally. The nucleoli were distinct and large. Periodic acid Schiff stain demonstrated a granular cytoplasm. Immunohistochemistry demonstrated mitochondrial antigen, keratin, and chymotrypsin immunoreactivity in the neoplastic cells. Ultrastructural analysis revealed numerous mitochondria packed into the cytoplasm of the neoplastic cells. Thus, the final diagnosis was that of oncocytic carcinoma of parotid gland. CONCLUSION: This neoplasm shows clinical, microscopical, histological and ultrastructural features of oncocytic carcinoma and this must be considered in the differential diagnosis of other proliferations in the parotid gland with abundant granular cytoplasm and metastatic oncocytic carcinomas.

Journal Article↗
PubMed2004

Effect of phospholipase A2 inhibitory peptide on inflammatory arthritis in a TNF transgenic mouse model: a time-course ultrastructural study.

We evaluated the therapeutic effect of secretory phospholipase A2 (sPLA2)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA2 inhibition.

Animals↗
PubMed1980

Fetal rat hepatic ultrastructural changes associated with organ culture and glucocorticoid treatment.

Fetal hepatic ultrastructure was examined from 18 days gestation to term. Such structure was compared to that of tissue obtained at 18 days gestation which had been maintained in organ culture for 24, 48, and 72 h. In addition, the effects of maternal glucocorticoid administration in vivo and in vitro organ culture glucocorticoid exposure upon fetal hepatic morphogenesis were examined. Enhanced fetal hepatocyte ultrastructural maturation, characterized by a reduction in hematopoietic percursors, an increased frequency of hepatocyte-hepatocyte contact, a complete compliment of intracellular organelles, and the development of bile canaliculi by either maternal in vivo or in vitro fetal organ culture glucocorticoid exposure, was demonstrated. These studies extend previous observations concerning bile salt synthesis and secretion by fetal hepatic tissue both in vivo and in vitro by providing an ultrastructural basis for the functional changes observed and reported to occur in response to steroid therapy

Animals↗
PubMed1979

Limb muscle endplates in ocular myasthenia gravis: quantitative ultrastructural study.

Motor endplate ultrastructure in the biceps brachii was quantitatively analyzed in five patients with ocular myasthenia gravis (56 endplates), six patients with generalized myasthenia gravis (83 endplates), and five controls (64 endplates). Ultrastructural changes of the motor endplates were observed in nonweak limb muscles of patients with ocular myasthenia gravis as well as in generalized myasthenia; these changes were mostly restricted to the postsynaptic region. Decrease of the mean presynaptic and postsynaptic membrane length, and postsynaptic membrane density, were more remarkable in generalized myasthenia than in ocular myasthenia. Widening of the primary and secondary synaptic clefts was also observed more frequently in generalized myasthenia. There was no correlation between ultrastructural alterations and the duration of symptoms or treatment, severity of disease, or titers of antiacetylcholine receptor antibody.

Adolescent↗
PubMed1982

Santavuori disease: diagnosis by leukocyte ultrastructure.

Since Santavuori's 1973 description of infantile neuronal ceroid lipofuscinosis, 46 of the 58 reported cases have been Finnish. We recognized the disorder in three children from two different American families by leukocyte ultrastructure and clinical picture. These patients had the cardinal features of early developmental deterioration, retinal blindness, microcephaly, and seizures. Ultrastructural study of buffy coats revealed compact, granular, osmiophilic membrane-bound cytoplasmic inclusions in approximately 15 to 21% of lymphocytes and larger mononuclear cells. Similar cytoplasmic inclusions were seen in neurons, astrocytes, macrophages, and endothelial cells of a frontal lobe biopsy from one patient. The use of leukocyte ultrastructure combined with an awareness of the characteristic clinical picture should lead to the increased recognition of this disorder in American children.

Biopsy↗
PubMed1982

Supratentorial subarachnoid epithelial cyst: case report and ultrastructural study.

Supratentorial epithelial cysts are rare entities that have evoked controversy regarding their pathogenesis. Recent ultrastructural studies have failed to resolve this controversy. We present a case report and an ultrastructural study of such a cyst. The cyst wall was lined by columnar epithelial cells with prominent cilia and microvilli at their apical surface. They were joined by well-developed junctional complexes and contained abundant intracytoplasmic, electron-dense, granular material, some of which was membrane-bound. These ultrastructural findings suggest that epithelial cysts are derived from elements of the embryological tela choroidea.

Adult↗
PubMed2005

Postischemic reperfusion: ultrastructural blood-brain barrier and hemodynamic correlative changes in an awake model of transient forebrain ischemia.

OBJECTIVE: In nonrecovery models of cerebral ischemia, blood-brain barrier (BBB) and cerebral blood flow (CBF) changes are known to occur during reperfusion. It is unknown, however, whether those CBF and BBB alterations occur after brief, transient ischemia with neurological recovery. The purpose of this study was to characterize the time course of CBF and BBB ultrastructural changes during reperfusion in an awake, recovery model of transient global forebrain ischemia (GFI). METHODS: Forty-five adult Sprague-Dawley rats were subjected, while awake, to 10 minutes of GFI by the nine-vessel occlusion method. Thirty-five age-matched animals composed a sham-operated group. Normal control (n = 5), sham-operated (n = 5), and nine-vessel occlusion/reperfusion (n = 15) rats were selected for ultrastructural analysis. Electroencephalography was performed, and CBF, mean arterial blood pressure, and intracranial pressure were measured during ischemia and reperfusion up to 24 hours. Quantitative morphological analysis of cortical BBB capillaries was performed by transmission electron microscopy at the same time points at which specific CBF changes occurred during reperfusion. RESULTS: CBF decreased to 6% of preocclusion values during GFI. This correlated with coma and decerebrate rigidity. During reperfusion, short-lived hyperemia (225 +/- 18%, P < 0.001) was characterized by increased intracranial pressure (28.3 +/- 2.6 mm Hg, P < 0.001) and isoelectric electroencephalogram. This was followed by hypoperfusion, which reached a nadir of 59.7% (59.7 +/- 8.8%, P < 0.01) from baseline by 90 minutes. At this time point, the electroencephalogram recovered, and intracranial pressure and mean arterial blood pressure showed no abnormalities. By 8.5 hours, CBF returned to normal, and this coincided with complete recovery of the animal. Ultrastructural BBB analysis revealed astrocyte end-foot process edema and patent capillaries during hyperemia. Severe interstitial BBB edema and capillary lumen collapse was observed during hypoperfusion. Detachment and migration of pericytes was observed during hypoperfusion and beyond. CONCLUSION: A biphasic CBF response is elicited during reperfusion after brief nonlethal GFI under awake conditions.

Animals↗
PubMed2006

Ultrastructure of perinidal capillaries in cerebral arteriovenous malformations.

OBJECTIVE: The ultrastructure of perinidal capillaries in cerebral arteriovenous malformations (AVMs) was examined to clarify their pathomorphological features. METHODS: Fifteen AVM specimens were dissected and divided into perinidal and intranidal groups and processed for ultrastructural study immediately after surgical removal. Eleven of the patients had presented with hemorrhage. Tissue from four normal controls was also studied. Electron microscopy was used to compare features of the blood-brain barrier and endothelial cells (ECs) of capillaries in perinidal, intranidal, and controls. RESULTS: Perinidal capillaries demonstrated abnormal ultrastructure of the blood-brain barrier with no basement membranes and astrocytic foot processes. ECs had fenestrated luminal surfaces. Large gaps were observed at endothelial intercellular junctions. ECs contained numerous filopodia, large numbers of cytoplasmic processes, numerous micropinocytotic vesicles, and the cytoplasm contained more filaments than those observed in controls. Pericytes were rich in pinocytotic vesicles, vacuoles, and filaments. Their processes were in close contact with ECs. Weibel-Palade bodies were present in perinidal ECs. CONCLUSION: The absence of blood-brain barrier components in perinidal capillaries may contribute to extravasation of red blood cells into the surrounding brain in the absence of major hemorrhage and explain the gliosis and hemosiderin occasionally seen around AVMs. Cellular differentiation and proliferation in perinidal capillaries should be included in a systematic study aimed at a better understanding of the mechanisms underlying the recurrence of surgically removed AVMs.

Adolescent↗
PubMed1987

The ultrastructure of normal fetal and neonatal pig testis germ cells and the influence of fetal decapitation on the germ cell development.

The development of germ cells in the male pig was investigated ultrastructurally in normal and decapitated fetuses. The age ranged respectively from 30 days p.c. till one month after birth and from 52 days p.c. until birth. The ultrastructural organization of the germ cells changes dramatically between 30 days p.c. and 52 days p.c. which coincides with the formation of 'true' sex cords. From 52 days p.c. onwards the morphology is rather stable: cells show a 'hydrated' appearance and typical cell bridges. There is no obvious difference in the ultrastructure of germ cells in decapitated animals, their normal littermates and control animals. Therefore germ cell development in the pig is likely to be insensitive to gonadotropins during the fetal period. The development of pig germ cells follows closely the pattern described for several species. Quantitatively there is an increase in the ratio of germ cell/Sertoli cell per cross sectional diameter in the decapitated animals.

Animals↗
PubMed2004

The ultrastructure of the Chlamydomonas reinhardtii basal apparatus: identification of an early marker of radial asymmetry inherent in the basal body.

The biflagellate unicellular green alga Chlamydomonas reinhardtii is a classic model organism for the analysis of flagella and their organizers, the basal bodies. In this cell, the two flagella-bearing basal bodies, along with two probasal bodies and an array of fibers and microtubules, form a complex organelle called the basal apparatus. The ultrastructure of the basal apparatus was analysed in detail by serial thin-section electron microscopy of isolated cytoskeletons and several newly discovered features are described, including a marker for the rotational asymmetry inherent in the basal bodies and probasal bodies. In addition, the complex three-dimensional basal apparatus ultrastructure is resolved and illustrated, including the attachment sites of all basal apparatus elements to specific microtubular triplets of the basal bodies and probasal bodies. These data will facilitate both the localization of novel basal apparatus proteins and the analysis of mutants and RNA interference cells with only subtle defects in basal apparatus ultrastructure. The early harbinger of radial asymmetry described here could play a crucial role during basal body maturation by orienting the asymmetric attachment of the various associated fibers and therefore might define the orientation of the basal bodies and, ultimately, the direction of flagellar beating.

Animals↗
PubMed1980

Ultrastructure of polytene chromosomes of Drosophila isolated by microdissection.

Drosophila polytene chromosomes prepared by a new micromanipulative procedure, which avoids acid squashing, have been examined at the ultrastructural level in the electron microscope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along with the chromocentre and various interbands. The ultrastructure of these chromosomes, which have never been exposed to acid protein denaturants, compares favourably with that of classical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized in linear arrays and evidence is adduced for looped transcription units. Particles with characteristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands. In interbands RNP particles and 'superbead'-like chromatin particles may be distinguished. Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for the localization of native chromosomal proteins at an ultrastructural level.

Animals↗
PubMed2000

The interplay among cardiac ultrastructure, metabolism and the expression of oxygen-binding proteins in Antarctic fishes.

We examined heart ventricle from three species of Antarctic fishes that vary in their expression of oxygen-binding proteins to investigate how some of these fishes maintain cardiac function despite the loss of hemoglobin (Hb) and/or myoglobin (Mb). We quantified ultrastructural features and enzymatic indices of metabolic capacity in cardiac muscle from Gobionotothen gibberifrons, which expresses both Hb and Mb, Chionodraco rastrospinosus, which lacks Hb but expresses Mb, and Chaenocephalus aceratus, which lacks both Hb and Mb. The most striking difference in cellular architecture of the heart among these species is the percentage of cell volume occupied by mitochondria, V(v)(mit,f), which is greatest in Chaenocephalus aceratus (36.53+/-2.07), intermediate in Chionodraco rastrospinosus (20.10+/-0.74) and lowest in G. gibberifrons (15.87+/-0.74). There are also differences in mitochondrial morphologies among the three species. The surface area of inner mitochondrial membrane per volume of mitochondria, S(v)(imm, mit), varies inversely with mitochondrial volume density so that S(v)(imm,mit) is greatest in G. gibberifrons (29.63+/-1.62 microm(-)(1)), lower in Chionodraco rastrospinosus (21.52+/-0.69 microm(-)(1)) and smallest in Chaenocephalus aceratus (20.04+/-0.79 microm(-)(1)). The surface area of mitochondrial cristae per gram of tissue, however, is greater in Chaenocephalus aceratus than in G. gibberifrons and Chionodraco rastrospinosus, whose surface areas are similar. Despite significant ultrastructural differences, oxidative capacities, estimated from measurements of maximal activities per gram of tissue of enzymes from aerobic metabolic pathways, are similar among the three species. The combination of ultrastructural and enzymatic data indicates that there are differences in the density of electron transport chain proteins within the inner mitochondrial membrane; proteins are less densely packed within the cristae of hearts from Chaenocephalus aceratus than in the other two species. High mitochondrial densities within hearts from species that lack oxygen-binding proteins may help maintain oxygen flux by decreasing the diffusion distance between the ventricular lumen and mitochondrial membrane. Also, high mitochondrial densities result in a high intracellular lipid content, which may enhance oxygen diffusion because of the higher solubility of oxygen in lipid compared with cytoplasm. These results indicate that features of cardiac myocyte architecture in species lacking oxygen-binding proteins may maintain oxygen flux, ensuring that aerobic metabolic capacity is not diminished and that cardiac function is maintained.

Animals↗
PubMed1987

Ultrastructural preservation of plasma membranes by non-lethal slow freezing to liquid nitrogen temperature.

Secondary hyphae of Lyophyllum ulmarium were shown to tolerate slow freezing, which allowed extracellular freezing, to -196 degrees C. A freeze-fracture study showed that under this non-lethal freezing condition, the plasma membrane of the secondary hyphae did not show any ultrastructural changes as compared with the control, except gross cellular shrinkage. Tertiary hyphae of Lyophyllum ulmarium, on the other hand, were completely injured by slow freezing to -196 degrees C, and the plasma membrane showed distinct intramembrane particle aggregation as a result of direct membrane contact caused by severe cellular deformation. It is suggested that the absence of freezing injury in the secondary hyphae was due to ultrastructural preservation of the plasma membrane, which resulted from avoidance of severe cellular deformation, while occurrence of freezing injury in the tertiary hyphae is considered to be due to ultrastructural changes in the plasma membrane caused by severe cellular deformation.

Cell Membrane↗
PubMed1985

Histochemical, ultrastructural and cytochemical study of reperfusion effect on ischemic myocardial injury.

We assessed the histochemical, ultrastructural and cytochemical effects of reperfusion on ischemic myocardial cells during the early and late reperfusion phases in two groups of dogs. Group A were 8 dogs undergoing 1 hour occlusion of LAD, and Group B were 14 dogs undergoing 1 hour occlusion of LAD followed by 2 hour reperfusion period. The results of the histochemical study (PAS stain) demonstrated that in Group A, a patchy distribution of glycogen occurred primarily in the subepicardial region. Three-dimensional analysis of this distribution revealed peninsulas of glycogen running parallel with a vessel. The cells in Group B, mainly subepicardium, showed a moderate glycogen content which was more extensive than those in Group A. The ultrastructural changes were assessed after a 60-minute ischemia and subsequent recovery (after 5 minutes and 120 minutes of reflow) using transmural biopsy specimens. Each myocardial cell was graded from 0-4 according to the degree of ischemic injury and recovery. The degree of ischemic damage varied in intensity from slight to severe, in both the subepicardium and the subendocardium. Ca++-ATPase activity was examined cytochemically in myocardial cells of Group B. After 60-minute occlusion, the moderately ischemic cells (especially in the subepicardium) that were without amorphous dense bodies or marked sarcolemmal lifting-off made significantly greater ultrastructural recovery (p less than 0.05) with restoration of Ca++-ATPase activity on sarcoplasmic reticulum and mitochondria after 120 minutes of reflow. This occurred even though after 5 minutes of reflow the cell showed temporary deterioration such as contraction bands, vacuoles and severe destruction of some mitochondria.

Animals↗