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.

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. 2. The pericyte population is highly variable between different tissues and organs, probably in a manner reflecting postarteriolar hydrostatic pressures. Pericytes are more abundant in the distal legs and feet, again suggesting a hydrostatic pressure-driven mechanical role for pericytes as protectors of microvessel wall integrity. 3. Pericyte alteration or degeneration is linked directly with microangiopathy in diabetes, scleroderma, hypertension, dementias and, possibly, inappropriate calcification of blood vessels. 4. Pericytes are functionally codependent on endothelial cells. Each cell type influences each others' mitotic rate and probably phenotypic expression. 5. Pericytes are not randomly located around microvessels. Instead, they are located adjacent to or over endothelial cell junctions of venules and especially over gaps between endothelial cells during inflammation. Pericytes are emerging as essential components of the microvessel wall, with metabolic, signalling and mechanical roles to support the endothelial cell.

Effect of emu oil on auricular inflammation induced with croton oil in mice.

OBJECTIVE: To determine the acute anti-inflammatory effects of topically applied emu oil. ANIMALS: 96 male CD-1 mice assigned randomly to 4 groups, each comprising 24 mice. PROCEDURE: To induce auricular inflammation, 50 microl of a solution comprising 10 microl of croton oil dissolved in 1 ml of acetone was applied to the inner surface of the left auricle (pinna). One hour later, 3 or 5 microl of emu oil (low- and high-dose groups, respectively) or 5 microl of porcine oil (oil-control) was applied to the left pinna. Control mice remained untreated. Six mice per group were euthanatized 3, 6, 12, and 24 hours after induction of inflammation. Specimens of auricular tissue (ear plugs) were obtained, using a 6-mm biopsy punch. Magnitude of swelling was calculated as the weight difference between left (inflamed) and right (noninflamed) ear plugs; degree of edema was determined as the difference between wet and dry weights of the left ear plug. RESULTS: Magnitude of swelling was significantly reduced at 6 and 12 hours in mice treated with emu or porcine oil, compared with controls. The greatest reduction in swelling was detected in the high-dose emu group at 6 hours. Compared with controls, degree of edema was significantly reduced at 6 hours only in the high-dose group, whereas by 12 hours, all groups treated with oils had significantly less edema than controls. At 24 hours, magnitude of swelling and degree of edema did not differ among groups. CONCLUSION: Topically applied emu oil significantly reduced severity of acute auricular inflammation induced by croton oil in mice.

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.

N omega-nitro-L-arginine methyl ester inhibits inflammatory liver injury induced by interleukin-2.

Administration of interleukin-2 (IL-2) for treatment of metastatic disease often results in inflammatory liver injury. Previous studies have implicated increased leukocyte and platelet adhesion and enhanced nitric oxide production as causative factors in the development of IL-2-induced hepatic injury. This study investigated the capacity of N omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthesis inhibitor, to limit IL-2-induced hepatic edema and hepatocellular damage in mice. Using hepatic intravital microscopy, we also examined the effects of L-NAME on IL-2-induced increases in leukocyte and platelet adhesion. Administration of IL-2 increased leukocyte and platelet adhesion in post-sinusoidal venules and decreased hepatic perfusion. Cotreatment with L-NAME had no effect on leukocyte adhesion but increased platelet-endothelial adhesion and microvascular thrombosis. Chronic IL-2 treatment induced hepatic edema and hepatocellular injury. However, coadministration of L-NAME attenuated IL-2-induced edema and completely inhibited hepatocellular damage. These findings suggest that nitric oxide may play a central role in IL-2-induced inflammatory liver injury.

Heterogeneity of the composition and thickness of tracheal mucus in rats.

Inability to preserve airway mucus in situ has limited our understanding of its structure and function. This light- and transmission electron-microscopic study of rat tracheal mucus used a nonaqueous fixative that retains mucus (epiphase) over a lucent layer (hypophase). The fixative is a 1% solution of osmium tetroxide dissolved in a perfluorocarbon. The mean thickness of rat tracheal epiphase was 5 microns, with significant variation (0.1-50 microns) around the tracheal circumference. Tracheal mucus was thickest at the trachealis muscle region and contained cells, cellular debris, and a variable amount of surfactant and lipid, estimated at 4-16% of the total epiphase in five rats, with a mean composition of 9%. Lipid was observed on the surface of the epiphase, embedded within mucus, and at the epiphase-hypophase interface. Refined study of developmental, physiological, and pathological alterations to the airway coat may benefit from this approach.

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 skin's 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.

Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H/HeN mice.

Immunotherapy with interleukin-2 (IL-2) is limited by severe side effects thought to be mediated by the activation of immune effector cells and the induction of secondary cytokines including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In C3H/HeN mice the primary IL-2 toxicity is the production of pleural effusion with subsequent respiratory compromise. IL-10 is a cytokine that has been shown to inhibit the generation of secondary cytokines in vitro and in vivo. In this study, in C3H/HeN mice, we tested the ability of IL-10 to inhibit IL-2-induced mononuclear cell and alveolar macrophage activation and IL-2-induced increases in serum TNF-alpha and IFN-gamma, all of which may contribute to the generation of toxicity. IL-10 was ineffective at reducing IL-2-induced pleural effusion. However, IL-10 did inhibit IL-2-induced increases in serum TNF-alpha, which was accompanied by a decrease in Golgi apparatus and rough endoplasmic reticulum in alveolar macrophages. In addition, IL-10 combined with IL-2 increased mononuclear cell activation, which may limit the ability of IL-10 to inhibit IL-2-induced IFN-gamma production and pulmonary injury.

Mechanisms of interleukin-2-induced hepatic toxicity.

Interleukin 2 (IL-2) mediates the regression of metastatic cancer, but its clinical use is limited by associated toxicities including hepatic dysfunction. To determine the mechanism for IL-2-induced hepatic dysfunction, we hypothesized that IL-2 activation of Kupffer cells causes leukocyte-endothelial adhesion and decreases hepatic sinusoidal blood flow. C57BL/6 mice were given injections of latex particles and prepared for intravital hepatic microscopy 2 h after i.p. IL-2 administration. Liver tissue was also prepared to quantitate hepatic tumor necrosis factor (TNF) mRNA and processed for light and electron microscopy. Phagocytosing Kupffer cells and leukocytes adherent to the endothelium were counted, and surface sinusoidal blood flow was quantitated. Kupffer cell activity was quantitated as the ratio of phagocytosing Kupffer cells to sinusoidal blood flow. IL-2 significantly increased Kupffer cell activity (0.56 +/- 0.05 for controls versus 0.84 +/- 0.05 for IL-2), significantly caused leukocyte-endothelial adhesion (26.7 +/- 7.9 for controls versus 87.0 +/- 27.6 for IL-2, WBC/mm2 endothelial surface), and significantly decreased the number of sinusoids containing blood flow per microscopic field (6.66 +/- 0.15 for controls versus 5.79 +/- 0.13 for IL-2) without causing changes in systemic hemodynamic parameters. In IL-2 treated livers, light and electron microscopy showed the constriction of sinusoids associated with swollen or ruptured mitochondria, which was consistent with hypoxic deterioration near central venules. Adherent platelets, neutrophils, and lymphocytes within sinusoids and central venules were also observed. PCR revealed that IL-2 significantly induced TNF mRNA expression in the liver. These data suggest that IL-2 activates Kupffer cells in association with the release of monokines including TNF, which causes activation of circulating leukocytes as well as hepatic sinusoidal endothelial cells. The resultant leukocyte and platelet adhesion to the endothelium may then physically impede the sinusoidal microcirculation, resulting in microscopic areas of hepatic ischemia and explaining the mechanism of IL-2-induced hepatic dysfunction.

Comparative morphology of the scolices and microtriches among five species of Tetrabothrius (Eucestoda: Tetrabothriidae).

The structure of the scolex, neck, and adjacent strobila of 5 species of Tetrabothrius was evaluated using low to high magnification scanning electron microscopy. Species-specific patterns and morphotypes (spiniform, papilliform, and filiform) of microtriches were found on the scolices and strobilar tegument of Tetrabothrius (Oriana) affinis, Tetrabothrius (Oriana) filiformis, and Tetrabothrius (Culmenamniculus) laccocephalus; microtriches were absent in Tetrabothrius (Culmenamniculus) cylindraceus and Tetrabothrius (Tetrabothrius) sp. The presence, distribution, and form of microtriches may not be correlated with the subgeneric divisions of Tetrabothrius or the host taxon. Unique patterns do not appear to be shared with other groups of cestodes, particularly other tetraphyllideans. We suggest that the spiniform, filiform, and papilliform types of microtriches may be symplesiomorphic for the Eucestoda.

The effect of soluble tumor necrosis factor receptor-II on endotoxin-mediated hemodynamic instability.

Tumor necrosis factor-alpha (TNF-alpha), a central mediator in the hemodynamic response to injury and infection, is a primary mediator of endotoxin-induced hemodynamic instability. Two types of naturally occurring soluble TNF receptors circulate in human experimental endotoxemia and the recombinant proteins of both have been hypothesized as potential therapeutic agents antagonizing TNF-mediated effects of endotoxemia. The administration of recombinant sTNFr-I has been previously shown to attenuate the hemodynamic collapse of lethal bacteremia. In the current study, we investigated the role of recombinant sTNFR-II at low (0.5 mg/kg) and high (2.5 mg/kg) doses as a potential therapeutic agent for the inhibition of endotoxin lipopolysaccharide (LPS)-mediated hemodynamic instability. Eighteen male Sprague-Dawley rats were anesthetized and cannulated for continuous blood pressure monitoring and cardiac output measurement by thermodilution. Groups of animals received saline, LPS (1 mg/kg), or sTNFr-II (at 0.5 or 2.5 mg/kg) 15 min prior to LPS (1 mg/kg). Hemodynamic variables (blood pressure, cardiac output, heart rate) were monitored every 15 min for 2 hr. LPS caused a 30% decrease in mean arterial pressure by 60 min, which began to recover by 120 min. sTNFr-II was unable to prevent LPS-induced hypotension at low or high dose. Serum levels of immunoreactive TNF-alpha, undetectable in control animals, were significantly increased by sTNFr-II compared to LPS alone. Serum from animals treated with high-dose sTNFr-II showed significantly less TNF cytotoxicity than those treated with low-dose sTNFr-II, indicating that high doses of sTNFr-II are required for the inhibition of the bioactivity of TNF.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 alters the positions of capillary and venule pericytes in rat cremaster muscle.

Interleukin-2 (IL-2) mediates regression of metastatic cancer, but its therapeutic efficacy has been limited by toxicities which occur secondarily to IL-2 induced macromolecular leakage from microvessels. Previous studies have indicated that pericytes function in an endothelial cell junction-dependent manner in lung capillaries and cremaster muscle venules, possibly to limit cellular and macromolecular leakage. The purpose of this investigation was to determine if pericyte positioning on skeletal muscle capillaries was junction-related, and if it was altered by IL-2. Anesthetized rats received intravenous injections of fluoroscein isothiocyanate conjugated to albumin. The cremaster muscle model of microcirculation was used in conjunction with intravital fluorescence microscopy to monitor changes in interstitial fluorescence which was used as an index of macromolecular leakage. IL-2 induced a progressive increase in interstitial albumin which started within 30 min of application and continued to increase until the end of the experiment at two hours. Cremaster muscle tissue was fixed and prepared for examination by transmission electron microscopy. A digitizing platter and morphometric software were used to quantify pericytes near vs away from endothelial cell junctions of capillaries and venules. In microvessels from animals in the control group, pericytes were randomly positioned on capillaries, and concentrated at endothelial cell junctions of venules. After treatment with IL-2, capillary pericytes were concentrated at junctions. Venular pericyte density also increased in endothelial junctional regions. IL-2 appears to alter the distribution of pericytes in the microcirculation, perhaps by induction of contraction.

Tumor necrosis factor causes microvascular protein leakage independently of neutrophils or mast cells.

Tumor necrosis factor-alpha (TNF-alpha) has been implicated as an important mediator in the development of multiple system organ failure after either severe injury or infection. Using the rat cremaster muscle, we previously showed that systemically administered TNF-alpha caused hypotension, tachypnea, and microvascular protein leakage in association with leukocyte-endothelial adherence. In the current study, we hypothesized that topical administration of TNF-alpha to the cremaster muscle would cause microvascular protein leakage independent of changes in hemodynamic parameters. In addition, histological methods were used to study the role of neutrophils and mast cells in the TNF-alpha-induced microvascular protein leakage. Topically applied low-dose (1 ng/ml) TNF-alpha caused microvascular leakage in the cremaster, without changes in central hemodynamic parameters, but high-dose TNF-alpha (10 ng/ml) did not cause protein leakage. Histological studies did not demonstrate evidence of either neutrophil adhesion or mast cell degranulation in topically applied TNF-alpha-treated cremasters compared to controls. These data suggest that TNF-alpha-induced macromolecular leakage is a dose-dependent phenomenon which can occur independently of neutrophils or mast cell degranulation.

Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in situ study.

A fixative composed of 1% w/v osmium tetroxide dissolved in a tluorocarbon (FC-72) was used to perfusion-fix rat blood vessels for this ultrastructural study. With non-aqueous fixation, the lumenal surfaces of endothelial cells in cardiac and skeletal muscles retained an extracellular coat which was morphologically consistent with glycocalyx. Venules displayed the greatest density and depth (up to 870 nm thick) of glycocalyx, but all vessel types had at least 70 nm of surface coat. The results indicate that non-aqueous fixatives will be useful in studies of the endothelial cell lumenal surface, while offering excellent preservation of general ultrastructure.

Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in situ study.

A fixative composed of 1% w/v osmium tetroxide dissolved in a fluorocarbon (FC-72) was used to perfusion-fix rat blood vessels for this ultrastructural study. With non-aqueous fixation, the lumenal surfaces of endothelial cells in cardiac and skeletal muscles retained an extracellular coat which was morphologically consistent with glycocalyx. Venules displayed the greatest density and depth (up to 870 nm thick) of glycocalyx, but all vessel types had at least 70 nm of surface coat. The results indicate that non-aqueous fixatives will be useful in studies of the endothelial cell lumenal surface, while offering excellent preservation of general ultrastructure.

Limitations of monastral blue as a vascular label: rapid rate of clearance is age-dependent, and interactions with anesthetics depress arterial blood pressure in rats.

Monastral blue (MB) has been described as an inexpensive, nontoxic vascular label. Discrepancies as to its rate of removal from circulation and physiological side effects prompted this study in which retention time of MB in the vascular system and effects of MB upon arterial blood pressure with different anesthetics (halothane, isoflurane, and pentobarbital) were measured in rats. Arterial pressure was monitored during intravenous infusion of MB with or without Evans blue, an albumin label. Localized areas of leakage were created by injecting 30 microL of 10(-4) M histamine into abdominal dermis at -2, 0, 5, 7, 10, and 15 minutes from infusion of MB. Mean arterial pressure decreased by 25-30% after MB infusion when halothane or isoflurane was used, but not with pentobarbital. Sites which leaked at 10 and 15 minutes did not usefully label with MB, although Evans blue-labelled albumin appeared in the interstitium. Younger, lighter rats (125-200 vs. 200-250 gm) retained MB longer in circulation, and had a shorter duration of MB-induced hypotension. Spectrophotometric analysis of rat serum showed rapid elimination of MB from the vascular system, with a half-life of 3.5 +/- 1.9 minutes. While MB remains a useful vascular label, its rapid removal from the circulation and its hypotensive effect must be recognized.

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.

Interleukin 2 acutely induces platelet and neutrophil-endothelial adherence and macromolecular leakage.

The acute effects of interleukin 2 (IL-2) were determined in the rat cremaster microcirculation model by intravital, light, and electron microscopy to better understand the pathophysiology of the IL-2-induced vascular leak syndrome. Four groups of rats were studied over a 2-h monitoring period. One group received 1 x 10(6) units of IL-2/kg i.v. (n = 10), while the remaining groups received IL-2 topically applied to the cremaster muscle in dosages of either 1 x 10(5) (n = 9), 1 x 10(6) (n = 5), or 3 x 10(6) (n = 5) units. Each group was compared with controls (n = 9). IL-2 administered i.v. acutely induced platelet and polymorphonuclear leukocyte-endothelial adherence and microvascular macromolecular leakage that occurred synchronous with the development of tachycardia, hypotension, tachypnea, and hypoxemia. Topically applied IL-2 induced similar microvascular alterations but without changes in hemodynamic and respiratory parameters, which suggests that microvascular alterations were not caused by IL-2-induced changes in hemodynamic parameters. Electron microscopy of cremaster muscle sections demonstrated platelet and neutrophil adherence to the endothelium and endothelial injury. We conclude that IL-2 (or a locally generated mediator) acutely induces platelet and neutrophil-endothelial adherence in the rat skeletal muscle microcirculation that is associated with the development of macromolecular leakage from the microcirculation.

Recent advances in pericyte biology--implications for health and disease.

This review highlights the contributions of recent pericyte research towards our understanding of normal and pathological functioning of microvessels. Pericytes are implicated in a variety of microvascular alterations, including wound healing, diabetes, inflammation, hypertension and neoplasia. They are capable of changing into other mesodermally derived cell types, including smooth muscle cells, osteoblasts and chondrocytes. The contractile properties of pericytes are being systematically examined in vitro; in addition to their tendency to contract spontaneously, pericytes can contract further in response to mediators of inflammation. In vivo studies indicate pericytes are concentrated near endothelial cell junctions along venules where they likely participate in inflammatory events. As agents are identified which modify pericyte responses to disease states, better therapeutic approaches will become possible.

Alteration of primate ovary surface epithelium by exposure to hexachlorobenzene: a quantitative study.

Hexachlorobenzene (HCB) is a fungicide and a pollutant of increasing concern in industrialized regions of the world. Reproductive failure is one of the effects of HCB upon mammals. Alteration of cell shape in the ovary surface epithelium (OSE) of Cynomolgus monkeys following oral administration of HCB was observed in this study. At the lowest dose used, 0.1 mg/kg body weight, a dosage that is associated with follicular degeneration, HCB caused quantifiable changes in length-to-width ratios of OSE. Measurement of cell shape by light microscopy offers a reliable indication of OSE changes induced by HCB.