Reidar Myklebust

Lipid digestibility and ultrastructural changes in the enterocytes of Arctic char (Salvelinus alpinus L.) fed linseed oil and soybean lecithin

Arctic char (Salvelinus alpinus L.) were fed two isocaloric diets supplemented with 15% linseed oil or soybean lecithin, and subsequently analysed for ultrastructural changes in various intestinal segments (pyloric caeca region, midgut and hindgut) as well as apparent digestibility coefficients (ADC) of the diets. The ADC of dry matter, lipid and individual fatty acids were always highest in fish fed the soybean lecithin diet. In hindgut for example, ADC of lipid and dry matter were 94 and 96%, respectively, in fish fed the soybean lecithin diet compared to 85 and 94% in fish fed linseed oil.There were major ultrastructural differences in the enterocytes between fish fed the two experimental diets. In char fed the linseed oil diet there were substantial accumulations of lipid droplets in the enterocytes from the pyloric caeca and midgut but not in hindgut. These accumulations were not observed in fish fed the soybean lecithin diet. In some cases, deposition of fat in the enterocytes was so great that it resulted in epithelial damage with lipid droplets and cell debris appearing in the intestinal lumen. These changes are likely to be pathological and may lead to intestinal malfunction and represent a major infection route to pathogenic bacteria. On the basis of these results it is suggested that endogenous phospholipid synthesis is insufficient to maintain lipoprotein synthesis in Arctic char when fed diets containing high levels of polyunsaturated fatty acid of total lipid, and that phospholipids should be supplied with such diets.

Liquid Loss as Effected by Post mortem Ultrastructural Changes in Fish Muscle: Cod (Gadus morhua L and Salmon Salmo salar

This study was performed in order to assess the effect of early post mortem structural changes in the muscle upon the liquid-holding capacity of wild cod, net-pen-fed cod (fed cod) and farmed salmon. The liquid-holding capacity was measured by a low speed centrifugation test. Transmission electron microscopy was used to discover ultrastructural changes both in the connective tissue and in the myofibrils. Differential scanning calorimetric thermograms of the muscle proteins were recorded to elucidate whether fundamental differences did exist between the proteins of the raw material tested. Multivariate statistics were used to explicate the main tendencies of variations in the thermograms. The salmon muscle possessed much better liquid-holding properties than the cod muscle, and wild cod better than fed cod regardless of the storage time. Both fed cod and farmed salmon, underwent the most severe structural alterations, probably caused by the low muscle pH values. The higher liquid-holding capacity of the salmon muscle was related to species specific structural features and better stability of the muscle proteins. The myofibrils of the salmon muscle were denser and intra- and extracellular spaces were filled by fat and a granulated material. The differences in thermograms of muscle from wild and fed cod were largely explained by the variations in pH. The severe liquid loss of fed cod is due to a low pH induced denaturation and shrinkage of the myofibrils. Post mortem degradation of the endomysial layer and the sarcolemma may have further facilitated the release of liquid.

Histological changes in intestine of Atlantic salmon (Salmo salar L.) following in vitro exposure to pathogenic and probiotic bacterial strains

Furunculosis and vibriosis are diseases that cause severe economic losses in the fish-farming industry. The foregut of the Atlantic salmon (Salmo salar L.) was exposed in vitro to two fish pathogens, Aeromonas salmonicida (causative agent of furunculosis) and Vibrio anguillarum (causative agent of vibriosis), and to one probiotic strain, Carnobacterium divergens, at 6 × 104 or 6 × 106 viable bacteria per milliliter. Histological changes following bacterial exposure were assessed by light and electron microscopy. Control samples (foregut exposed to Ringer’s solution only) and samples exposed only to C. divergens had a similar appearance to intact intestinal mucosal epithelium, with no signs of damage. However, exposure of the foregut to the pathogenic bacteria resulted in damaged epithelial cells, cell debris in the lumen, and disorganization of the microvilli. Co-incubation of the foregut with a pathogen and C. divergens did not reverse the damaging effects caused by the pathogen, although these were alleviated when probiotic bacteria were used. Based on these results, we suggest that the probiotic bacterium, C. divergens, is able to prevent, to some extent, pathogen-induced damage in the Atlantic salmon foregut.

Damaging effect of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.

Effect of soybean oil and soybean lecithin on intestinal lipid composition and lipid droplet accumulation of rainbow trout, Oncorhynchus mykiss Walbaum

Rainbow trout (Oncorhynchus mykiss Walbaum) were fed purified diets containing fish oil for six weeks and then soybean lecithin or soybean oil for 25 days. The gastrointestinal tract segments, stomach, midgut and hindgut were then sampled for lipid and fatty acid composition and electron microscopy. Membrane lipid class composition was fairly similar in all three segments of trout fed fish oil. Hindgut contained slightly more phosphatidylserine than stomach and midgut, while midgut contained more phosphatidylcholine and less lysophospatidylcholine/sphingomyelin. Feeding soybean products appeared to marginally decrease free cholesterol. The fatty acid compositions of the main lipid classes showed significant regional differences. In control fish, stomach had higher levels of arachidonic acid (20:4n-6) and n-6 polyunsaturated fatty acids than midgut and hindgut, and lower content of docosahexaenoic acid (22:6n-3) and n-3 polyunsaturated fatty acids. Midgut phosphatidylethanolamine also had higher levels of saturated fatty acids and less n-3 polyunsaturated fatty acids than the other tissues. Feeding soybean products decreased the n-3/n-6 ratio mainly due to increases in linoleic (18:2n-6) and 20:4n-6 and decreases in 22:6n-3 and eicosapentaenoic acid (20:5n-3). Phosphatidylcholine and to a lesser extent phosphatidylethanolamine adapted more readily to dietary changes by major increases in 18:2n-6 and C20−22 n-6 polyunsaturated fatty acids. The composition of phosphatidyl-serine and -inositol appeared to be under more strict metabolic control. Linoleic acid hardly increased at all while the increase in other n-6 polyunsaturated fatty acids was less pronounced than for the other lipid classes. Regardless of lipid class, stomach resisted dietary changes more strongly than midgut and hindgut. Increases in n-6 polyunsaturated fatty acids were minor as were the loss of n-3 polyunsaturated fatty acids. The dead-end product 20:2n-6 accumulated to a higher degree in hindgut phosphatidyl-ethanolamine and -coline compared to midgut and stomach, suggesting that the activity of Δ6 desaturation is higher in the anterior part of the intestine where most of the lipid is absorbed. Feeding soybean oil caused massive accumulation of free lipid droplets in midgut enterocytes while little lipid droplets were observed in trout fed fish oil or soybean lecithin. Since both soybean products influenced intestinal composition to the same degree, altered fatty acid profiles in membranes is not responsible for the observed lipid accumulation. This supports previous observations in Arctic charr (Salvelinus alpinus L.), suggesting that fish may require exogenous phospholipids in order to sustain a sufficient rate of lipoprotein synthesis.

Epithelium‐associated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study

E. RINGØ, J.B. LØDEMEL, R. MYKLEBUST, T. KAINO, T.M. MAYHEW AND R.E. OLSEN. 2001.

Ultrastructural changes induced in the isolated rat heart by enzymatically generated oxygen radicals

This study describes the effect of oxygen radicals on the ultrastructure of the isolated Langendorff-perfused rat heart. Oxygen radicals were enzymatically generated by xanthine oxidase (0.025 U/ml) and hypoxanthine (0.96 mM). Hearts were perfusion-fixed for electron microscopy and stereological technique was performed to obtain estimates of volume fractions (Vv) of different tissue components. Perfusion with oxygen radicals resulted in areas with severely damaged myocardial cells. These changes included swelling and cristolysis of mitochondria, disruption of filaments, development of intracellular edema and focal disruption of the sarcolemma. Stereological examination revealed few alterations after 5 min perfusion with oxygen radicals. After 10 min perfusion with oxygen radicals, however, the Vv (myocyte/myocardium) increased from 0.542 +/- 0.042 (mean +/- S.D.) to 0.663 +/- 0.144, and this paralleled the development of Vv (cellular edema/myocyte) being 0.047 +/- 0.028. Vv (capillary wall/capillary) increased from 0.215 +/- 0.046 to 0.411 +/- 0.123 indicating endothelial swelling. Although the mitochondria appeared swollen, Vv (mitochondria/myocyte) remained constant. The effect of a 35 min recovery period on the ultrastructure was minor. The application of SOD and catalase together with xanthine oxidase and hypoxanthine reduced the observed changes significantly, thus proving the participation of oxygen radicals. This study confirms that oxygen radicals can induce major alterations in myocardial ultrastructure.

Functional impairment in isolated rat hearts induced by activated leukocytes: Protective effect of oxygen free radical scavengers

Ischemia-reperfusion activates polymorphonuclear leukocytes (PMN). Depletion of PMN has been shown to reduce the size of experimental myocardial infarction. We have studied whether PMN activated by phorbol myristate acetate (PMA) would depress function of the isolated rat heart, and if this effect was mediated by oxygen free radicals (OFR). Cells and/or drugs were added to the perfusate into the aortic cannula for 10 min, followed by a 30 min recovery period. Oxygen free radicals formation was verified by chemiluminescence (CL). PMA-activated PMN (n = 13) caused CL response of 27,493 +/- 5113 counts (mean +/- S.E.M.) and reduced left ventricular developed pressure (LVDP) to 30 +/- 9% and coronary flow (CF) to 49 +/- 7% of the baseline value at the end of the observation period. Addition of super-oxide dismutase (SOD) and catalase (CAT) (n = 11) reduced the CL response to 5623 +/- 806 counts, but did not influence either LVDP (36 +/- 15%) or CF (51 +/- 18%). Addition of thiourea (TU) to the activated cell suspension (n = 8) further reduced the CL response (3663 +/- 474 counts), and LVDP was 86 +/- 5% and CF was 87 +/- 3%. When TU + SOD + CAT was mixed with PMN + PMA (n = 11), the CL was almost abolished (117 +/- 21 counts) and LVDP was 73 +/- 8% and CF was 94 +/- 6%. When CF was reduced (n = 7) alike the CF reduction in the hearts receiving PMA + PMA, LVDP was not significantly changed at the end of the observation period (75 +/- 6%). Unactivated PMN (n = 8) caused minor response of LVDP and CF, similar to PMN + PMA + TU and PMN + PMA + SOD + CAT + TU. PMA alone (n = 8) was cardiotoxic and caused changes similar to PMN + PMA. This effect was not inhibited by scavengers (n = 6). The supernatant of the PMN + PMA suspension (n = 7) did not impair cardiac function, suggesting that no free PMA was available after mixing with PMN. We conclude that activated PMN in the coronary circulation depressed cardiac function and increased vascular resistance due to OFR production.

Bacterial penetration into tonsillar surface epithelium during infectious mononucleosis.

Bacterial penetration into epithelial cells, scraped from the palatine tonsils of 14 patients (10 males, four females; median age 16 years) with current infectious mononucleosis and concomitant membranous tonsillitis, was studied using the transmission electron microscopic (TEM) technique. Bacteria were seen to adhere to and penetrate the epithelial cells, some of which were completely filled with bacteria. This finding suggests intracellular proliferation of bacteria. Epstein-Barr virus, the causative agent of infectious mononucleosis, especially when associated with growth of beta-haemolytic streptococci on the palatine tonsils, induces bacterial penetration into tonsillar tissue, that in turn might be a causative mechanism in the development of peritonsillar abscess.

Granule containing cells and fibres in the sinus venosus of elasmobranchs.

SummaryThe concentrations of catecholamines in the heart chambers of elasmobranchs were measured by the fluorimetric method of Bertler et al. (1958). Noradrenaline (NA) can be detected in all the chambers, but the sinus venosus is by far the richest in NA. This can either be due to the presence of storage sites for this amine in the sinus wall, or to a transport of amine to the sinus venosus from the anterior chromaffin bodies. The sinus wall contains large numbers of “granule containing cells” and axon-like processes, both with numerous dense-core vesicles of about 1800 Å diameter. The dense-core vesicles contain a uranophilic matrix indicating the presence of protein, phospholipids and/or nucleic acid. The reactions failed to demonstrate amine, which may be due to a loss of amine by diffusion, to a relatively low intravesicular amine concentration, or, to the absence of amines in these granule-containing cells and processes. Heavy accumulations of granule-containing processes occur in the subendothelial area. The endothelium contains fenestrae and pores through which granule-containing fibres protrude into the venous cavity. Granule-containing cells are innervated by presumed cholinergic nerve endings. It is suggested that the granule-containing cells and fibres belong to a neurosecretory system with a cholinergic input, releasing the contents of the dense-core vesicles into the blood stream at the level of the venous cavity.