David E. Sims

Diversity Within Pericytes

1. Pericytes are cells of microvessels (arterioles, capillaries and venules) that wrap around endothelial cells. They are most abundant on venules and are common on capillaries.

Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout, coho and Atlantic salmon

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.

Uptake, disposition and depuration of domoic acid by blue mussels (Mytilus edulis)

Abstract Domoic acid (DOM), the toxin involved in amnesic shellfish poisoning, was presented to live mussels (at 5°C, 28 0 00 ) in dissolved form (125 nM) and as food encapsulated in liposomes. Less than 1% of dissolved DOM and up to 6% of food-borne DOM was incorporated into mussel tissues. DOM absorbed from solution was most concentrated in gills and kidney, whereas DOM ingested as food was most concentrated in digestive gland and kidney. Gonad, muscle, foot and connective tissues retained the lowest concentrations of toxin. Compared to their proportion of body weight; kidney, digestive gland and gill retained larger than expected proportions of total toxin burden. The concentration of toxin in mussel tissues did not decrease consistently over a depuration period of 48 h, nor did DOM appear to be translocated to any tissue for storage. Small amounts of DOM were eliminated in faeces and larger amounts in dissolved form. Over 80% of intracellular DOM was associated with the TCA (trichloroacetic acid) soluble fraction of digestive gland tissue. DOM in the TCA insoluble fraction was positively correlated with overall DOM concentration.

Preservation of tracheal mucus by nonaqueous fixative.

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.

Differentiation of light-dye effects in the microcirculation

Activation of photosensitive compounds has been used in the treatment of tumors and as a technique to study various microcirculatory phenomena. This technique may be accompanied by deleterious effects which may complicate interpretations of experimental results. However, the relevant physiological mechanisms that induce toxicity and the light doses needed to produce different toxic reactions have not been well defined. In the current study, the rat cremaster muscle preparation was used with in vivo fluorescent television microscopy and subsequently with electron and light microscopy to evaluate toxic reactions of light activation of fluorescein isothiocyanate. The most sensitive photoactive reactions were macromolecular leakage and platelet activation, occurring with 120 J/cm2 activation energy. Macromolecular leakage was at least partially restricted by perivenular and pericapillary pericytes and there was no morphological damage with this light dose. Since macromolecular leakage was significantly inhibited by pretreatment with diphenhydramine or Compound 48/80, it is in part due to the release of histamine from tissue mast cells. 720 J/cm2 reduced the red blood cell column in the venules by over 50% due to platelet thrombus formation, an effect that was accentuated by pretreatment with indomethacin. This suggests an inhibitory role of prostaglandins in platelet thrombus formation. In addition, 720 J/cm2 caused endothelial and smooth muscle cell swelling and ruptures, gap formation, and leukocyte and protein accumulation in the vessel walls.

Distribution of Crenosoma Vulpis and Eucoleus Aerophilus in the Lung of Free-Ranging Red Foxes (Vulpes Vulpes):

Crenosoma vulpis and Eucoleus aerophilus are nematode parasites that can cause verminous pneumonia in wild carnivores. There is a paucity of information regarding the distribution of parasites in the lungs and the relationship between histopathological and parasitological diagnoses in naturally infected foxes. The objectives of this study were: first, to study the lobar and airway distribution of C. vulpis and E. aerophilus in wild red foxes and second, to investigate the relationship between fecal and histopathological diagnoses. Samples from 6 sites of the lung and fecal contents were obtained from 51 wild foxes in Prince Edward Island. By fecal examination, 78.4% of wild foxes tested positive for C. vulpis and 68.6% for E. aerophilus. In contrast, 66.6% and 49% of foxes had histopathological evidence of C. vulpis and E. aerophilus in the lungs, respectively. Anatomically, C. vulpis was observed in the small bronchi and bronchioles of all pulmonary lobes whereas E. aerophilus was restricted to the large bronchi and the caudal lobes. Affected airways exhibited severe epithelial glandular hyperplasia and bronchiolar mucous metaplasia. It was concluded that C. vulpis is widely distributed in airways of all pulmonary lobes, whereas E. aerophilus is mainly restricted to the bronchi of caudal lobes. Also, this study showed that histological examination of lung underestimates the infection with E. aerophilus.

Morphometric assessment of epidermal and mucous-biofilm changes caused by exposure of trout to chloramine-T or formalin treatment

Juvenile rainbow trout were exposed to therapeutic concentrations of formalin or chloramine-T to assess the effects of these chemicals on the morphology of the piscine epidermis and its mucous coat. Repeated treatment, once weekly for 4 weeks, with either chemical did not affect the mucous coat of the epithelium or the degree of folding of the basal lamina. However, treated fish had increased numbers of highly dense vesicles within the apical portions of epithelial cells. The epidermal mucous cells of chloramine-T-treated fish were significantly smaller than in controls. This effect was not noted in formalin-treated fish. Treatment with either chemical resulted in a significantly thinned epidermis. It is concluded that although chloramine-T and formalin may continue to be useful in the aquaculture industry they cause potentially harmful alterations to fish skin.

In vitro study of domoic acid uptake by gland tissue of blue mussel (Mytilus L.)

Abstract Domoic acid is a neurotoxic amino acid responsible for an outbreak of human food poisoning in late 1987 following consumption of contaminated blue mussels ( Mytilus edulis L.) from eastern Prince Edward Island, Canada. In vitro techniques were used to examine the uptake of domoic acid by digestive gland tissue of the blue mussel. Uptake of domoic acid over different periods of time by digestive gland tissue was compared with that of structurally related amino acids, glutamic add and kainic acid. Domoic acid uptake was observed to be a function of time and concentration. Uptake of glutamic acid, which is a physiological amino acid, was greater than that of domoic acid. Kainic add, a non-protein, rare amino acid, was least absorbed. In a series of experiments to determine the inhibitory actions of chemically similar and dissimilar amino acids, kainic acid, glutamic acid and proline inhibited the uptake of domoic add by 42, 38 and 34%, respectively, indicating competition for the same carrier site. Glycine, which showed least inhibition is likely to have a separate pathway. A marginal enhancing effect of ATP and slight inhibitory effects of metabolic inhibitors (NaCN and maleic acid) suggested that uptake of domoic add by digestive gland tissue is not totally an energy-dependent process.

A comparative study of uptake and release of glutamic acid and kainic acid by blue mussel (Mytilus edulis L.)

Abstract Glutamic acid (OA) and kainic acid (KA) are structurally related to domoic acid (DOM), a neurotoxic amino acid that caused human food poisoning in 1987, following consumption of toxic mussels from Prince Edward Island, Canada. To study the difference in response of mussels to a physiological amino acid (GA) and a non-physiological amino acid (KA), GA and KA were presented to mussels in dissolved form and as a food encapsulated in liposomes. Kainic acid was absorbed from solution to a lesser degree (0.4%), while GA was taken up more readily (1.1%). Both amino acids were absorbed across the gastrointestinal epithelium. Kainic acid was concentrated in the kidney, whereas glutamic acid showed equal distribution in all visceral tissues. Glutamic acid was steadily released from mussel tissues, whereas KA resisted depuration. Over 80% of intracellular KA was associated with the TCA soluble fraction of digestive gland tissue, while about 42% of GA was associated with the TCA insoluble fraction. Although KA is structurally related to GA, it was handled differently by mussels, but in the same way as DOM, suggesting that non-protein amino acids are recognized as undesirable compounds.

ADAPTATION OF A FLUOROCARBON-BASED NON-AQUEOUS FIXATION REGIME FOR THE ULTRASTRUCTURAL STUDY OF THE TELEOST EPITHELIAL MUCOUS COAT

Studies on the microanatomy of the mucus-rich biofilm surface of normal or damaged teleost skin tissue have been limited because conventional fixation regimes do not effectively retain mucus during tissue preparation. A non-aqueous fixation method, based on a technique devised to retain airway mucous for ultrastructural study, and consisting of the use of an inert perfluorocarbon solvent with osmium teroxide 1%, was successfully used to prepare skin tissues of healthy juvenile rainbow trout. The skins mucous coat was examined by transmission electron microscopy and the results were compared with those obtained with tissues prepared by a conventional glutaraldehyde-based method. In samples fixed with glutaraldehyde, the cell-surface structures retained were limited to microridges and a poorly discernible glycocalyx layer. In contrast, those fixed by the non-aqueous method had a more clearly demonstrated glycocalyx layer, and a second fibrillar layer, resembling mucus, which was separated from the glycocalyx layer by an electron-lucent zone.