Ethchlorvynol-induced pulmonary edema in rats. An ultrastructural study.

Studies of ethchlorvynol (ECV)-induced pulmonary edema were undertaken for determination of the structural basis of increased microvascular permeability. Rats were administered an intravenous bolus dose of 15 mg/kg ECV and killed at time intervals between 5 minutes and 72 hours. Oyster glycogen and ferritin were used as permeability probes for identification of the sites of altered microvascular permeability. Edema fluid containing ferritin begins to accumulate in the alveolar interstitium 10 minutes after EVC . Thirty minutes after ECV, marked intersitial edema fluid is present containing both permeability probes. The absence of any appreciable transendothelial movement of either probe via vesicles and the presence of open endothelial junctions led the authors to propose the latter as the principal determinant of the increase in permeability. In addition to open endothelial junctions, prominent subendothelial blebs occur. These blebs develop in an otherwise intact endothelium and increase in frequency and size with time following their appearance at 10 minutes. Ferritin and glycogen progressively accumulate within the blebs. At 15 minutes the concentration of ferritin in blebs appears to equal that in plasma, whereas glycogen is absent or sparsely present in a few blebs. At 60 minutes both permeability probes have become concentrated in the blebs. The mechanism of formation of the blebs and concentration in them of the permeability probes cannot yet be specified. The lesion caused by ECV is completely reversible, so that by 72 hours after ECV there is complete resolution of interstitial edema, disappearance of the subendothelial blebs, and closure of endothelial junctions. A small amount of exudate remaining in the alveoli is cleared by 72 hours.     

Primary myocardial disease in the diabetic mouse. An ultrastructural study.

The hearts from C57BL/KsJ db+/db+ mice and controls were examined by light and electron microscopy at intervals during 5 to 28 weeks of age. C57BL/6J ob/ob mice and their lean littermates served as other controls. The percentage of increase in body and heart weights of the diabetic animals was 150% and 64% greater, respectively, than that of the controls. Over the period of observation there was progressive damage to the ventricular myocytes and intramural small arteries and arterioles of the diabetic animals. Initially, the cardiac muscle cells of both ventricles contained large numbers of lipid droplets. Subsequently, there was shrinkage and increased electron density of mitochondria that were enveloped by single limiting membranes that in turn gave rise to large residual bodies. This was followed by loss of myofilaments and atrophy of myocytes. Similar changes occurred in the smooth muscle cells of intramural arteries and arterioles but not in those of epicardial arteries. Reduplicated layers of basal laminae were seen around interstitial capillaries. Degenerative changes also occurred in perivascular nerve endings. These changes are discussed in relation to the altered metabolism of the diabetic state. It is concluded that the pathologic lesions in the cardiac muscle cells and intramural arterial vessels and capillaries constitute a primary myocardial disease in the genetically diabetic mouse.     

Peripheral neuropathy in the diabetic mutant mouse. An ultrastructural study.

Structural changes in peripheral nerves were studied sequentially in mutant diabetic mice. Axonal changes were found in both myelinated and unmyelinated fibers, the outstanding early change being the development of honeycombed Schwann cell-axon networks followed by axonal atrophy in both myelinated and unmyelinated fibers. In the late stages of the diabetic syndrome, this axonopathy was accompanied by secondary corrugated myelin breakdown. The changes, indicative of a primary axonal degeneration, are comparable to findings in human diabetic neuropathy for which this seems a suitable experimental model.     

An ultrastructural study of normal and injured hypoglossal nuclei after injection of horseradish peroxidase

Summary The left hypoglossal nuclei of normal rats, and rats which had received left hypoglossal nerve axotomies 7–21 days previously, were studied by electron microscopy after injection of horseradish peroxidase as a marker of extracellular space and pinocytosis.Quantitative analysis showed that the number of pinocytotically-derived structures in presynaptic boutons was significantly increased in rats at 7, 14, and 21 days after axotomy when compared with normal rats. It was suggested that presynaptic boutons which became detached from injured neurones retracted by a membrane cycling mechanism involving pinocytotic uptake of bouton plasmalemma, without compensatory membrane production elsewhere.It was confirmed that the channels in the microglial cells communicated with the extracellular space.     

The fate of intraportally transplanted islets in diabetic rats. A morphologic and immunohistochemical study.

Streptozotocin-induced diabetes in the rat can be reversed by the transplantation of isogenic islets of Langerhans from neonatal donors. We studied the morphology of intraportally transplanted islets with the aid of the immunoperoxidase staining technique to identify insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-containing cells at 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 39 weeks, and 65 weeks after transplant. Embolized pancreatic tissue, composed of approximately 80% acini and 20% islets, is initially distributed throughout the liver mainly to terminal branches of the portal system. Endothelialization and organization occur rapidly with the smaller fragments and within the first 4 weeks for larger thrombi. Exocrine pancreatic elements largely disappear as islet cells move into the hepatic lobules from the portal spaces. At 65 weeks after transplant, all islet cell types can be identified within large complex islet structures. The results of this study establish the survival and continued function of all known rat pancreatic islet cell types long after transplantation and support the theory that islet transplantation may represent the most physiologic replacement of hormonal deficiencies in the diabetic recipient.     

Effects of oxygen concentrations on human fibroblasts treated with Fe(3+)-NTA

Free radicals derived from the reaction of iron and oxygen are thought to be one of the causes of tissue injury. In order to identify whether oxygen concentrations are an important factor in iron-mediated damage to cells, cytotoxic effects of Fe(3+)-NTA on human fibroblasts (KMST-6 line) were studied under the conditions of 1% and 20% oxygen concentrations in an incubator. A comparison of the effects of Fe(3+)-NTA on cells cultured in 1% and 20% oxygen environments showed that the following features were more prominent under the usual culture concentrations of 20% oxygen: i) cytotoxicity, ii) increase in intracellular reactive oxygen species, iii) increase in H(2)O(2) production in the cells, and iv) formation of 8-hydroxydeoxyguanosine. To elucidate the roles of endogenous antioxidants, the levels of manganese superoxide dismutase (MnSOD) and catalase were measured by Western blotting. The increase in MnSOD in the presence of Fe(3+)-NTA was greater under the condition of 20% O(2) than under the condition of 1% O(2). The expression of catalase was significantly up-regulated at 20% O(2). However, when the cells were treated with Fe(3+)-NTA, the expression of catalase was markedly down-regulated under the condition of 20% O(2). Hydroxyl radical scavengers such as vitamin E, dimethyl-sulfoxide (DMSO) and mannitol reduced endogenous ROS generation and alleviated the cytotoxic effects of iron. On the other hand, superoxide dismutase (SOD), vitamin C and catalase did not show any protective effects against Fe(3+)-NTA. These findings suggest that enhanced cytotoxic effects of Fe(3+)-NTA at 20% O(2 )are due to endogenously produced hydroxyl radicals.     

Cystic cholangiomas after transplantation of pancreatic islets into the livers of diabetic rats

Islet transplantation is increasingly used as a therapy for human type 1 diabetes mellitus. In our study, we investigated the effect of the transplantation of a low number (n=350) of pancreatic islets into the right liver part on the neighboring portal bile ducts. Male streptozotocin- diabetic Lewis or autoimmune-diabetic BB/Pfd rats (n=1065) were subdivided into 11 experimental groups. A few days after low-number islet transplantation, cholangiocytes adjacent to the grafts showed an increase in proliferative activity. During the next 12–24 months, many peri-insular ductules progressed via tumor-like cystic lesions to large cystic cholangiomas, accompanied by a translocation of the insulin receptor into the cytoplasm and an increase in expression of insulin-related signaling proteins (Insulin-receptor-substrate-1, Raf-1, Mek-1). After 24 months, 53% of rats with low-number transplantation exhibited at least one cholangioma >10 mm, significantly outnumbering tumor development in the transplant-free left liver part and in any control group. No cholangiocarcinomas emerged. A graft cell origin of the tumors was excluded by Y chromosome in situ hybridization in cross-gender transplantations. Conclusively, low-number intrahepatic islet transplantation, most likely acting by permanent local hyperinsulinism, leads to prolonged cholangiocellular proliferation in streptozotocin- and in autoimmune-diabetic rats, resulting in the development of benign cystic cholangiomas.     

Pancreatic islets of malnourished rats: quantitative histologic and electron microscopic findings.

Young rats were maintained for three weeks on a low-protein diet. These animals developed many of the features of human protein-calorie deficiency, including dextrose intolerance and diminished insulin release. Quantitative histologic and ultrastructural studies showed that malnourished rats had (1) a reduced total pancreatic islet volume, and (2) a preponderance of pale granules in the B cells. It is suggested that pale B granules may contain increased amounts of insulin, which accumulate in the cells because of defective insulin release. The mechanism responsible for this has not been elucidated.     

Teratological study of stobadin after single and repeated administration in rats.

The toxic developmental potential of the anti-arrhythmic drug stobadin was assessed after single intravenous or repeated oral doses to pregnant rats. Stobadin was studied in the form of dihydrochloride (DH 1011) at doses of 2 and 6 mg/kg, given in single intravenous injections on days 3, 6, 9, or 12 of gestation. Immediately after injection of the 6-mg/kg dose of DH 1011 to pregnant rats, saccade abdominal respiration, tremor of hindlimbs, and sedative behaviour were observed on each day of medication. No deaths of females occurred in either the control or experimental groups. Slight foetal toxicity was manifested by significantly decreased foetal weight only after treatment on day 3 of gestation at 6 mg/kg and by significantly increased incidence of delayed ossification of the parietal and supraoccipital bone also at 6 mg/kg DH 1011 given on day 12 of gestation. The effect of repeated oral treatment in the form of dipalmitate salt (DP 1031) was studied in doses of 5, 15, and 45 mg/kg from days 2-15 of gestation. Oral exposure to 45 mg/kg DP 1031 resulted in significant reduction of maternal body weight gain and in embryofoetal toxicity, namely, increased preimplantation foetal loss, anomalies of sternebrae, and, after 15 and 45 mg/kg DP 1031, significantly decreased foetal weight and smaller litter size. The relevance of the two routes of stobadin administration for risk extrapolation is discussed.     

Lung lymphatics increase after hyperoxic injury. An ultrastructural study of casts.

The microscopic lymphatics of the lung can be cast and studied with scanning electron microscopy. This technique shows several different forms of lymphatics and the interstitial space that leads into lymphatics as no other method can. To study changes in lymphatic forms, rats were placed in 85% oxygen for 7 days to produce pulmonary edema. Methyl methacrylate resin was injected into the lung vasculature at various times after the animals were removed from hyperoxia. In the animals not exposed to hyperoxia, no artery, vein, or airway was surrounded by a lymphatic cast. However, in rats that were in the hyperoxic chamber, 22% of arteries, 30% of veins, and 51% of indeterminate blood vessels (which could be arteries or veins) were encompassed by saccular lymphatic casts. These lymphatics were still observed 7 days after recovery from hyperoxia. Fourteen days after hyperoxia, the lymphatics returned to control values. Only 9% of the pleural surface of the animals not exposed to hyperoxia had initial lymphatics. Fifty-two percent of the hyperoxia-exposed animals had initial lymphatics, measured 3 days after exposure. This decreased to 14% 14 days after exposure to hyperoxia (P < 0.01). Conduit lymphatics were found on the pleural surfaces of 33% of animals exposed to ambient air and 100% of animals exposed to the high-oxygen environment (P < 0.05). The median percentage of the pleural surface covered with lymphatics was 0 in the animals exposed to ambient air. It was 65% in animals exposed to hyperoxia, 3 days after returning to room air. It was again 0 in animals exposed to hyperoxia, 14 days after returning to room air (P < 0.001). The lymphatics around veins expanded more than around arteries (P < 0.0001). These results indicate that in the rat all compartments of the lung lymphatics expand after the injury and edema caused by oxygen and return to normal with the resolution of the edema.     

Acute murine adrenalitis induced by reovirus 3. An ultrastructural study.

Reovirus 3 infection of neonatal mice, although characterized primarily by encephalitis, hepatitis, and pancreatitis, also induces an adrenalitis. Histologically, the latter is characterized initially by foci of coagulative necrosis which later enlarge and become surrounded by leukocytic infiltration. Ultrastructurally, the virus was shown to replicate in the paranuclear region of mainly adrenocortical cells but also medullary and endothelial cells. Apoptosis is the most common form of necrosis exhibited and is quickly followed by an infiltration of mononuclear phagocytes which eventually ingest the virus and the cellular debris.     

Male breast carcinoma. An ultrastructural study.

A case of infiltrating ductal carcinoma of the male breast was studied by electron microscopy. The ultrastructure of the tumor is similar to that described in the female breast. Myofibroblasts are the preponderant cells in the stroma. Their significance and possible relation to hormonal stimuli are discussed.     

Leiomyomatosis peritonealis disseminata. An ultrastructural study

Electron microscopic features of the eighth documented case of leiomyomatosis peritonealis disseminata identify the main proliferative cell type as a mature leiomyocyte with all its associated ultrastructural features. The histogenesis of this neoplasm is considered to be a multicentric metaplastic change determined by an abnormal tissue response to the elevated hormonal levels occurring in pregnancy or ovarian functioning tumors. It is suggested that the cell of origin is the multipotent subcelomic mesenchymal cell, which retains the capacity to differentiate into several tissues, among them smooth muscle. To illustrate this possibility, myofibroblasts, cells with hybrid characters of fibroblasts and leiomyocytes, were observed; these may represent a transitional stage between the mesenchymal cell and the smooth muscle proliferating cell.     

Human lingual tuberculosis. An ultrastructural study

According to morphological criteria, the predominant cells from human oral tuberculosis granulomas are classified as monocytes, epithelioid cells, and multinucleated giant cells. The morphology of each cell type is related to its speculated function. It is theorized that macrophages and epithelioid cells represent an in vivo line of differentiation from undifferentiated monocytes and that giant cells form from a coalescence or syncytium of macrophages. The role of these phagocytic cells and other participating inflammatory cells in granulomatous inflammation is discussed.     

Histogenesis of dermatofibrosarcoma protuberans. An ultrastructural study

Ultrastructural study of five typical lesions of dermatofibrosarcoma protuberans revealed that the basic cell is fusiform and has a somewhat indented nucleus and an even distribution of cytoplasmic organelles. Fragments of basal lamina, intercellular junctions, abundant intercellular fibers, and a tendency to form cellular sheaths also were found. The tumor cells had a strong resemblance to pericytes and perineural cells, as well as marked ultrastructural similarities to neurofibroma. The authors conclude that dermatofibrosarcoma protuberans arises from a primitive fibroblastic cell with a "sheath forming cell" differentiation that has a closer relationship to the perineural cell than to the pericyte.     

Liposarcoma. An ultrastructural study of 15 cases

Fifteen liposarcomas from 13 patients were examined by electron microscopy. These included nine primary tumors, four recurrent tumors after primary surgery or irradiation, and two metastatic lesions. Twelve of the liposarcomas were located in the thigh, and 11 were of the myxoid variety. All neoplasms were composed of cells having the ultrastructural characteristics of some stage of lipoblastic differentiation, i.e., lipid droplets, micropinocytotic vesicles, glycogen, external lamina, intermediate filaments, Golgi apparatuses, rough and smooth endoplasmic reticulum, and mitochondria. The nuclear-cytoplasmic ratio and nuclear pleomorphism were related inversely to the size and number of lipid droplets. Lipoblasts were frequently in close association with capillaries and pericytes, and in four cases lipid droplets were found in pericytes. Multivacuolated, mitochondria-rich lipoblasts, resembling brown fat cells, also were seen. Most tumors contained lipid-free, poorly differentiated mesenchymal cells that showed a continuum of morphologic differentiation to cells that closely resembled early lipoblasts that contained nonmembrane-bound lipid vacuoles. Fibrolipoblasts, cells containing lipid droplets and abundant rough endoplasmic reticulum, were observed only in well-differentiated liposarcomas. Some soft tissue sarcomas contain vacuolated cells that simulate lipoblasts by light microscopy but prove to be reactive or malignant fibroblasts, histiocytes, or smooth muscle cells ultrastructurally. Therefore, use of electron microscopy may be necessary to establish the line of differentiation in these neoplasms.     

An ultrastructural study of subacute necrotizing lymphadenitis.

Fifteen cases of a unique lymphadenitis called subacute necrotizing lymphadenitis were studied electron-microscopically. The large lymphoreticular cells proliferating at cortical or paracortical areas of the lymph nodes mainly consisted of immunoblasts and histiocytoid cells, which were characterized by numerous intracytoplasmic myelinlike inclusions. Such histiocytoid cells seemed to be derived from the immunoblasts. Tubuloreticular structures, which had been often noticed within endothelial cells or lymphocytes of the patients with systemic lupus erythematosus (SLE) or SLE-related diseases, were also observed with high frequency in most cases examined. They were present within the cytoplasm of immunoblasts, endothelial cells, and histiocytoid cells. Immunoblasts in mitosis occasionally contained these structures. We offer the hypothesis that subacute necrotizing lymphadenitis with still unknown etiology may reflect a self-limited SLE-like autoimmune condition induced by virus-infected transformed lymphocytes.     

An ultrastructural study of a nasal haemangioendothelioma.

A malignant haemangioendothelioma of the nose has been examined with the electron microscope. The tumour is predominantly poorly differentiated with only little evidence of its vascular ancestry identified. The cells are polygonal with a wide spectrum of organelles, including microfilaments. A conspicuous feature was the presence of cytoplasmic fenestrae developing into intracytoplasmic vacuolated spaces. The satisfactory progress of this patient is noted, and the value of electron microscopy in enabling an early and accurate diagnosis of this tumour to be made is emphasised.