Persistent ultrastructural changes in pancreatic acinar cells induced by 4-acetylaminofluorene

Male Leeds rats were fed a diet containing 0.05% of the non-carcinogen 4-acetylaminofluorene (4-AAF) for 8–10 months. They were then returned to a normal diet and their pancreatic tissues examined by electron microscopy at intervals between 2 and 12 months after the end of 4-AAF treatment. 4-AAF was found to induce a persistent alteration in the morphology of the granular endoplasmic reticulum, involving distortion and dilatation of the cisternae. In some respects this lesion resembles that which is induced by the carcinogenic isomer, 2-acetylaminofluorene (2-AAF).     

Biochemical changes induced by Coxsackie B4 virus in short-term culture of mouse pancreatic islets

Isolated mouse pancreatic islets were infected in vitro with two strains of Coxsackie B4 virus — a tissue culture - adapted strain and a mouse pancreas-adapted strain. Within 48 h of infection changes had occurred in the biochemical activities of islets infected with the mouse pancreas-adapted strain of virus. Basal insulin release was increased two-fold in these islets, while glucose-induced insulin secretion remained unchanged. Insulin biosynthesis was greatly reduced at a sti, mulatory concentration of glucose (20 raM), thus leading to a reduced insulin content in these islets. These effects are of importance because they demonstrate that certain strains of Coxsackie B4 virus, like encephalo-myocarditis virus, may selectively alter B-cell function in vitro.     

Lactation-induced changes in calcium handling by rat pancreatic islets

Glucose-stimulated insulin release occurred at a lower rate in pancreatic islets removed from lactating than non-lactating rats. This defect was corrected in the presence of either gliclazide or a calcium-agonist. With both agents present, insulin release from islets of lactating rats was greater. When islets were prelabelled with 45calcium, gliclazide stimulated to the same extent 45Ca outflow in islets from lactating and non-lactating rats, respectively. However, when the islets were prelabelled with 45Ca in the presence of gliclazide, the administration of Ba2+ increased effluent radioactivity more markedly in islets from non-lactating than lactating rats. This suggests that lactation favours, in gliclazide-stimulated islets, the sequestration of 45Ca in non-labile subcellular pools. When D-glucose was used instead of Ba2+, the greater lability of 45Ca in islets from non-lactating animals was apparently masked by a lesser efficiency in the metabolism and cationic effects of D-glucose in the non-lactating rats. The calcium-ionophoretic effect of islet extracts was higher in lactating than non-lactating rats. These results support the view that a depletion of endogenous calcium stores accounts, in part at least, for the decreased insulin secretory responsiveness to D-glucose in lactation, since the latter apparently favours the function of those systems involved in either the entry of calcium into or its sequestration within the islet cells.     

An excursion through the ultrastructural world of quick-frozen pancreatic islets

In this article we have presented a philosophical and historical perspective on quick freezing, freeze-drying, freeze-substitution, and immunocytochemical localization of pancreatic islet hormones. A compilation of our findings indicates that quick-freezing does not produce any gross distortion of islet tissue; the amount of usable islet tissue for ultrastructural analysis is approximately 13 micron deep from the frozen edge; three different cell types can be identified in quick-frozen tissue based on general morphological characteristics; freeze-substitution with tetrahydrofuran produces a unique ultrastructural appearance in which ribosomes are particularly striking; with the use of protein A-gold, insulin and glucagon can be localized immunocytochemically on silver-gray (50-nm-thick) sections treated with 1% ovalbumin at room temperature overnight; secretory granules of quick-frozen alpha and beta cells may exist in either a swollen or condensed state; swollen beta cell secretory granules contain a filamentous material that demonstrates immunogold labeling for insulin; insulin and glucagon can be localized within the cisternae of endoplasmic reticulum; our methods provide not only discrete immunocytochemical localization of hormone, but also well-preserved cellular compartments; energy electron loss spectroscopy (EELS) has shown that quantifiable nitrogen maps can be used as an index of hormone packaging in secretory granules; and the sectioning properties of secretory granules at the ultramicrotome change when islet tissue is unosmicated and sectioned on glycerol.     

Functional and ultrastructural changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Cinnamomum verum essential oil

Aims: To study cellular damage induced by Cinnamomum verum essential oil in Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Methods and Results: The effect of cinnamon bark essential oil on these two strains was evaluated by plate counts, potassium leakage, flow cytometry and transmission electron microscopy (TEM). Exposure to this oil induced alterations in the bacterial membrane of Ps. aeruginosa, which led to the collapse of membrane potential, as demonstrated by bis‐oxonol staining, and loss of membrane‐selective permeability, as indicated by efflux of K⁺ and propidium iodide accumulation. Thus, respiratory activity was inhibited, leading to cell death. In Staph. aureus, cells treated with the oil entered a viable but noncultivable (VNC) state. The oil initially caused a considerable decrease in the metabolic activity and in the replication capacity of these bacterial cells. The loss of membrane integrity appeared later, as indicated by bis‐oxonol and Propidium iodide (PI) staining. Data provided by TEM showed various structural effects in response to cinnamon essential oil. In Ps. aeruginosa cells, coagulated cytoplasmic material was observed, and intracellular material was seen in the surrounding environment, while oil‐treated Staph. aureus showed fibres extending from the cell surface. Conclusions: Cinnamon essential oil damages the cellular membrane of Ps. aeruginosa, which leads to cell death. There is evidence of VNC Staph. aureus after exposure to the oil. Significance and Impact of the Study: Cinnamon essential oil shows effective antimicrobial activity and health benefits and is therefore considered a potential food additive. To use this oil as a natural food preservative, especially in combination with other preservation methods, a thorough understanding of the mechanism through which this oil exerts its antibacterial action is required.     

Reversible ultrastructural changes in the rhabdom of the locust eye are induced by long term light deprivation

Summary Quantitative electron microscopic analysis of the compound eyes of locusts (Schistocerca gregaria) subjected to long term light deprivation showed that disorganization of rhabdomal microvillus structure and disruption of rhabdomere fusion were significantly more prevalent in deprived animals than in controls. These structural changes accompanied a reduction in sensitivity to photic stimulation in the deprived eyes. The disorganization of microvillus structure was reversible after 14 days recovery in light while the disruption of rhabdomere fusion was irreversible. Deprivation also resulted in an irreversible increase in number of ommatidia bearing fewer than normal retinula cells. These results are consistent with disruption by light deprivation of some aspect of normal photoreceptor membrane synthesis or degradation.We thank Ms. Irene Kwan for her excellent technical assistance, Dr. H. Silverman for useful discussions through all stages of this study, and Drs. H. Injeyan and S. Tobe for supplying the hatchling locusts. This work was supported by Ontario Graduate Fellowships to J.W.B. and a National Research Council of Canada operating grant to H.L.A.     

Isolated pancreatic islets of the rat: an ultrastructural study.

Pancreatic islets from adult Wistar rats were isolated by an improved collagenase digestion technique. Examination of the preparations showed that they contained B cells possessing secretory granules, each having an eccentric electron-dense core surrounded by an electron-lucent halo; the Golgi bodies with their characteristic features were located in a juxtanuclear position. Roughly surfaced endoplasmic reticulum and mitochondria were present and were mostly normal in appearance. The cell possessed all the ultrastructural attributes indicating that they were fully functional and structurally intact.     

Insulin signaling proteins in pancreatic islets of insulin-resistant rats induced by glucocorticoid

Chronic administration of glucocorticoids induces insulin resistance that is compensated by an increase in p-cell function and mass. Since insulin signaling is involved in the control of p-cell function and mass, we investigated the content of insulin pathway proteins in pancreatic islets. Rats were made insulin resistant by daily administration of dexamethasone (1mg/kg, b.w., i.p.) for 5 consecutive days (DEX), whilst control rats received saline (CTL). Circulating insulin and insulin released from isolated islets were measured by radioimmunoassay whereas the content of proteins was analyzed by Western blotting. DEX rats were hyperinsulinemic and exhibited augmented insulin secretion in response to glucose (P < 0.01). The IRa-subunit, IRS-1, Shc, AKT, p-p70S6K, ERK1/2, p-ERK1/2, and glucocorticoid receptor protein levels were similar between DEX and CTL islets. However, the IRp-subunit, p-IRp-subunit, IRS-2, PI3-K, p-AKT and p70S6K protein contents were increased in DEX islets (P < 0.05). We conclude that IRS-2 may have a major role, among the immediate substrates of the insulin receptor, to link activated receptors to downstream signaling components related to islet function and growth in this insulin-resistant rat model.     

Functional and morphological characteristics of isolated pancreatic islets in tissue culture

Islets were isolated from the pancreas of female rats by using the collagenase technique. After culturing for 4 days, the islets were taken for measurement of insulin release biosynthesis as well as glucose utilization in subsequent short-time incubations. A low glucose concentration was insufficient to maintain a glucose-stimulated insulin release in vitro. A high glucose concentration had a protecting and restoring effect on the insulin release: ultrastructurally, such islets showed signs of an active biosynthesis in the electron micrograph. The enhancement of Mg++ in the culture medium resulted in an improvement of insulin storage in the islets, accompanied by a well-preserved action of glucose in a subsequent incubation.     

Functional and morphological study of cultured pancreatic islets treated with cyclosporine

Cyclosporine A (CsA), a potent immunosuppressive drug, has been found to induce glucose intolerance through its toxic effect on the endocrine pancreas. It is not exactly known whether CsA has a direct effect on the endocrine pancreas or induces its effect indirectly. The present study was therefore undertaken to examine the function and morphology of isolated pancreatic islets when they are directly exposed in vitro to CsA. Pancreatic islets were isolated from adult male Lewis rats using collagenase ductal perfusion technique. The islets were separated with the discontinuous Ficoll gradient technique and further purified by hand picking of the non-islet tissue. The islets were cultured in RPMI-1640, pH 7.4 and maintained at 37 degrees C in a humid atmosphere of 5% (v/v) carbon dioxide in air. Cyclosporine was added to the culture medium to give a final concentration of 1 microg/ml (therapeutic dose), 5 microg/ml (toxic dose), or vehicle (control). Islets were harvested at 1, 4 and 10 days of culture and processed for functional or histological study. The functional study of the islets cultured with 1 microg/ml CsA showed insulin and C-peptide contents similar to those of the control islets. The islets cultured with 5 microg/ml CsA showed a marked decrease in insulin and C-peptide contents. Glucose-dependent insulin release was variable. C-peptide release was lower than that of the control following both the therapeutic and toxic doses of CsA. Phase contrast microscopy showed that the islets cultured with 1 microg/ml CsA were mostly normal looking with a well-defined regular periphery; a few islets had ill-defined or irregular peripheries. The islets cultured with 5 microg/ml CsA had ill-defined irregular peripheries at 1 day, and were dense and forming clumps at 4 and 10 days following culture. There was a decrease in the islet number following the therapeutic dose; the decrease was more following the toxic dose of CsA. The islet diameters increased after the therapeutic dose, but slightly decreased following the toxic dose of CsA. Islets showed a weakly positive immunoperoxidase reaction for insulin that was weaker following the toxic dose of CsA. It is concluded that CsA has a direct effect on B-cells that was proved by the functional and morphological changes seen in the pancreatic islets cultured in vitro.     

Glucose-induced changes in cytosolic ATP content in pancreatic islets

The cytosolic and mitochondrial contents in ATP, ADP and AMP were measured in islets incubated for 45 min at increasing concentrations of D-glucose and then exposed for 20 s to digitonin. The latter treatment failed to affect the total islet ATP/ADP ratio and adenylate charge. D-Glucose caused a much greater increase in cytosolic than mitochondrial ATP/ADP ratio. In the cytosol, a sigmoidal pattern characterized the changes in ATP/ADP ratio at increasing concentrations of D-glucose. These findings are compatible with the view that cytosolic ATP participates in the coupling of metabolic to ionic events in the process of nutrient-induced insulin release.     

The ultrastructural basis for the identification of cell types in the pancreatic islets

Summary The pancreatic islets of rabbit, dog and opossum have been studied by light and electron microscopy. Silver-positive cells in the rabbit are predominantly sandwiched between the peripheral A and central B cells, and by electron microscopy are identified as D cells. Pancreatic islets in the tail of the dog pancreas have A, B, and D (silver-positive) cells, but the islets in the uncinate process of the dog pancreas lack phosphotungstic acid hematoxylin-positive A cells. By electron microscopy the characteristic D cells are found in both tail and uncinate process, but A cells are confined to the tail islets, confirming the identification of cell types. A unique cell type termed the F cell is found in the dog uncinate islets and it is characterized by secretory granules of angular profiles. In the opossum, the A cells contain considerable amounts of glycogen demonstrable by both light and electron microscopy. A unique cell type is also present in the opossum islets termed an E cell (Thomas, 1937), which has large secretory granules (400–500 m). The physiological implications of a multiplicity of cell types in pancreatic islets is discussed.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases. The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donahue.     

Erythropoietin treatment alleviates ultrastructural myelin changes induced by murine cerebral malaria

ABSTRACT: BACKGROUND: Cerebral malaria (CM) is a severe complication of malaria with considerable mortality. In addition to acute encephalopathy, survivors frequently suffer from neurological sequelae. The pathogenesis is incompletely understood, hampering the development of an effective, adjunctive therapy, which is not available at present. Previously, erythropoietin (EPO) was reported to significantly improve the survival and outcome in a murine CM model. The study objectives were to assess myelin thickness and ultrastructural morphology in the corpus callosum in murine CM and to adress the effects of EPO treatment in this context. METHODS: The study consisted of two groups of Plasmodium berghei-infected mice and two groups of uninfected controls that were either treated with EPO or placebo (n = 4 mice/group). In the terminal phase of murine CM the brains were removed and processed for electron microscopy. Myelin sheaths in the corpus callosum were analysed with transmission electron microscopy and stereology. RESULTS: The infection caused clinical CM, which was counteracted by EPO. The total number of myelinated axons was identical in the four groups and mice with CM did not have reduced mean thickness of the myelin sheaths. Instead, CM mice had significantly increased numbers of abnormal myelin sheaths, whereas EPO-treated mice were indistinguishable from uninfected mice. Furthermore, mice with CM had frequent and severe axonal injury, pseudopodic endothelial cells, perivascular oedemas and intracerebral haemorrhages. CONCLUSIONS: EPO treatment reduced clinical signs of CM and reduced cerebral pathology. Murine CM does not reduce the general thickness of myelin sheaths in the corpus callosum.     

Quantitation of ultrastructural changes in mouse pancreatic acinar cells caused by foreign serum

Quantitative changes in the pancreatic acinar cell organelles were studied in BALB/c mice injected with 1.0 ml fresh rabbit serum intraperitoneally. Groups of 5 mice were killed at 0, 1, 3, 6 and 12 h after the serum injection. Pancreatic tissue was processed for electron microscopy by glutaraldehyde and osmium tetroxide fixation and Epon embedding. The proportions of acinar cell cytoplasm (volume fractions) occupied by zymogen granules, granular endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes (including autophagosomes) were determined by the point counting method from electron micrographs. The volume fraction of lysosomes increased during the first 3 h and remained markedly elevated up to 12 h. The volume fractions of zymogen granules increased from 12 to 28% in 12 h. It was concluded that the secretory mechanism of pancreatic acinar cells was injured by the foreign serum. The injury caused accumulation of zymogen granules and increased autophagic activity in the acinar cells.     

Acetylcholine stimulates glucose metabolism by pancreatic islets

Acetylcholine stimulates insulin secretion in the presence of physiological concentrations of glucose. Stimulation of insulin secretion by acetylcholine is accompanied by an increase in glucose usage by isolated rat islets. Acetylcholine increased glucose usage by 38%, 28%, and 12% at 3.5 mM, 5.5 mM, and 10 mM glucose, respectively, compared to glucose usage by isolated islets incubated with glucose alone. Data showing increased glucose usage in islets treated with acetylcholine converge with data from an earlier report (J. Biol. Chem. 254 3921-3929 [1979]) showing a crossover point for glycolytic metabolites at phosphofructokinase to indicate that activation of glycolysis by acetylcholine results from increased phosphofructokinase activity and coordinate activation of hexokinase in intact islets.     

Lipid and exocrine pancreatic ultrastructural changes due to experimental diabetes.

The aim of this study was to establish if the changes in the ultrastructure of the exocrine part of the pancreas are correlated with changes in serum glucose, cholesterol and lipoprotein fractions during the progression of diabetes in rabbits. Diabetes mellitus was induced in male New Zealand rabbits by a single injection of alloxan into the auricular vein. On the day 7th the glucose level in the whole blood was measured and this day was designated as the first day of diabetes. Rabbits were divided into 5 groups: untreated control, 21-day diabetes, 42-day diabetes, 90-day diabetes and 180-day diabetes. The cholesterol, HDL (high-density lipoprotein) and LDL (low-density lipoprotein) levels were examined in the serum. The total pancreatic lipase activity was measured spectrophotometrically in the pancreatic homogenate. Histological specimens were examined under an electron microscopy. The glucose level increased significantly in all of the alloxan exposed animals. The significant elevation of cholesterol level was observed on day 21 and 180. The HDL level was increased (P<0.05) only on the day 21st. The LDL level and the total activity of pancreatic lysosomal lipase increased significantly on day 21, 42 and 90. Further dilation of granular endoplasmic reticular ducts and decrease in the number of zymogen granules were observed amongst exocrine cells. Fragmented mitochondrial and translucent matrix were also seen. Intensification of the pancreatic fibrosis was found on day 90. Microvascular changes were reported in exocrine cells after 180 days. Their nuclei were smaller with large bulges on the nuclear membrane, and the number of heterogeneous electron granules of zymogen further declined. We concluded that the intensification of ultrastructural changes of the exocrine part of the pancreas correlated with the changes of the pancreatic lipase activity, and glucose and lipoprotein levels.     

Functional and histochemical analysis on pancreatic islets of APA hamsters with SZ-induced hyperglycemia and hyperlipidemia.

To clarify how Syrian hamsters of the APA strain (APA hamsters) keep a diabetic condition for a long period, the functional and histochemical changes in the pancreatic islets of diabetic APA hamsters were examined. By glucose tolerance test, no glucose-induced insulin secretion was seen in the diabetic APA hamsters. By immunohistochemistry, it was revealed that at 24 hr after SZ-injection, the number of islets had decreased and that remnant islets had become markedly smaller. The islets had hardly any insulin-immunoreactive cells and consisted of cells stained by anti-glucagon and somatostatin antibodies. One, three and six months after SZ-injection, a small number of cells with vacuolative changes, which were positive for PAS staining, were observed in most islets and the vacuolated cells were stained mainly by anti-insulin antibody. In addition, a number of PCNA-positive cells were observed, especially in the periphery of the vacuolated cells, while TUNEL-positive cells were not detected. This data suggests that beta-cells proliferating as a result of the replication of the resident beta-cells in islets had fallen into degeneration and necrosis by a stress, such as the glycogen deposition in hyperglycemia and hyperlipidemia. Consequently, secretion of insulin was maintained at low levels, which allowed the hamsters to live without insulin therapy in the diabetic condition for over 6 months.     

Dissociation of Langerhans islets in the rabbit after pancreatic duct ligation. Immunocytochemical and ultrastructural studies

In the rabbit, pancreatic duct ligation leads to serious disturbances of the pancreatic endocrine parenchyma. Immunocytochemical studies conducted over a short period (between 5 and 30 days post ligation) allow observation of a progressive dissociation of the Langerhans islets which initially affects the splenic part of the pancreas, a region where numerous large islets are found. This dissociation is followed by a dispersion of small heterologous endocrine cell clusters or isolated endocrine cells in a connective tissue which replace the exocrine parenchyma. On the 30th day after ligation ultrastructural studies show marked degranulation of the B cells demonstrating the great fragility of these cells. These observations of insular dissociation, scattering of the different endocrine cells and impairment of B cells are often reported in experimental and pathological studies of the pancreas.