Effect of acute pancreatitis on porcine intestine: a morphological study.

AIM: The ultrastructural changes in the intestine were studied during experimental acute edematous and necrotizing porcine pancreatitis. The immunohistochemical expression of E-cadherin and ß-catenin in the jejunum and colon was assessed to characterize changes in the adherens junctions. METHODS: Twenty-four pigs were randomized to controls (n = 8) or to develop mild edematous (n = 8, saline infusion to pancreatic duct) or severe necrotizing pancreatitis (n = 8, taurocholic acid infusion). The ultrastructure of the mesenteric artery and the vein and epithelium of the jejunum and colon was analyzed at baseline and after 540 min with electron microscopy. The expression of E-cadherin and ß-catenin was assessed with immunohistochemistry. RESULTS: In the colon the microvilli and their glycocalyx shortened and reduced in density the most in necrotizing pancreatitis. In necrotizing pancreatitis adherens and tight junctions were occasionally open in the colon but rarely in the jejunum. Mitochondria in the colon epithelial cells were degenerated in necrotizing pancreatitis, swollen in edematous pancreatitis, and remained intact in the control case. In necrotizing pancreatitis, capillaries of the colon showed a broken endothelial lining with narrow lumens. The expression of E-cadherin immunoreactivity showed a trend toward a decrease in the colon in both edematous and necrotizing pancreatitis. CONCLUSION: Ultrastructural abnormalities in acute pancreatitis appear early in the colon, where they seem to be more damaging than in jejunum. Epithelial cell damage seems to include mitochondrial injury and an opening of tight and adherens junctions, which are more pronounced in necrotizing pancreatitis. Damage is seen in the mucosal and mesenteric endothelial cells.

Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery.

Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.

Transhepatic intravascular ultrasound for evaluation of portal venous involvement in patients with cancer of the pancreatic head region.

The aim of this study was to evaluate the ability of intravascular ultrasound to diagnose tumor involvement of the portal and the superior mesenteric veins using the preoperative percutaneous, transhepatic approach, and to compare the findings with those made at concomitant direct portography, surgery, and histopathological examination. Ten patients with a preoperative diagnosis of a resectable tumor in the pancreatic head region were examined with percutaneous transhepatic portography (PTP) and intravascular ultrasound (IVUS). The surgeon's intraoperative evaluation and the histopathological examination in combination revealed tumor involvement of the portal or superior mesenteric veins in six of the ten patients. Percutaneous transhepatic portography suggested tumor involvement of the veins in six patients but two of the examinations were false positive and another two were false negative. Intravascular ultrasound showed signs of tumor involvement in eight patients. The examination was, however, false positive in two patients, but there were no false negatives. Complications of the percutaneous transhepatic procedure occurred in six patients including severe pain, bleeding, and related death. Percutaneous transhepatic IVUS of the portal vein may be a useful tool in the preoperative selection of the subgroup of patients with tumor of the pancreatic head region that could benefit from surgery. There is a need for technical improvement as well as studies with larger patient series to definitely decide the role of the technique.

Contractile properties and proteins of smooth muscles of a calponin knockout mouse.

The role of h1-calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic calponin gene. These calponin knockout (KO) mice displayed no detectable h1-calponin in their smooth muscles. The amplitudes of Ca2+ sensitization, force and Ca2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild-type (WT) mice, nor were the delays in onset and half-times of Ca2+ sensitization, initiated by flash photolysis of caged GTPgammaS, different. The unloaded shortening velocity (Vus) of thiophosphorylated fibres was significantly (P<0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous calponin to approximate WT levels; the concentration dependence of exogenous calponin slowing of Vus was proportional to its actomyosin binding in situ. Actin expression was reduced by 25-50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms. We conclude that the faster Vus of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by calponin. Our results show no detectable role of calponin in the signal transduction of the Ca2+-sensitization pathways in smooth muscle.

Animal model of vascular inflammation.

Inflammatory mechanisms play a central role in the pathology of a variety of conditions ranging from atherosclerosis, arthritis, cancer and Alzheimer's disease. Under normal conditions the inflammatory response initiates protective actions, but triggers tissue damage under pathological conditions. Acute or chronic inflammation is mediated by nascent expression of a host of proteins such as the cytokines interleukins (IL), tumor necrosis factor (TNF), and interferons. Currently available in vitro or in vivo methods do not offer the specificity to probe the complex inflammatory cascade. We developed an animal model in which a single injection of the proinflammatory cytokines TNF-alpha and IL-1 beta in live rodents initiates a rapid inflammatory reaction which can be monitored by video microscopy and electron microscopy. This model exhibits the characteristic feature of inflammatory reaction such as adhesion and transmigration of leukocytes, and activation and degranulation of platelets and mast cells. This model is applicable to inflammatory processes in the peripheral and cerebral vasculature including the blood-brain barrier disruption in Alzheimer's disease. The animal model of inflammation reported here may prove to be a valuable tool in investigating the pathophysiology of a number of inflammatory diseases and identifying potential targets as well as agents for therapy.

Changes of vascular architecture independent of blood pressure in experimental uremia.

Striking alterations of the structure of arterial vessels of different caliber are a well-known feature of renal failure, but it is currently unknown to what extent they are a reflection of hypertension or of uremia per se. To address this issue further we studied subtotally nephrectomized rats, sham-operated and pair-fed with matched controls. After uremia of 14 days' duration, stereologic measurements were carried out on perfusion-fixed tissue. To eliminate a potential influence of hypertension, subgroups of animals received furosemide and hydralazine in the drinking fluid to yield daily doses of 15 mg/kg and 20 mg/kg, respectively. At the end of the experiment, systolic blood pressure (tail plethysmography) was 110 +/- 13.3 (mean +/- SD) mm Hg and 99.4 +/- 8.1 mm Hg in untreated and treated controls, respectively, and 132 +/- 20.7 mm Hg and 103 +/- 13.0 mm Hg in untreated and treated uremic animals, respectively (n = 5 to 10 animals per group). The wall:lumen ratio of intramyocardial small arteries was 0.056 +/- 0.011 and 0.052 +/- 0.006 in untreated and treated controls, respectively. In untreated and treated uremic animals, the corresponding values were 0.077 +/- 0.011 and 0.066 +/- 0.007 (P < .01; control v uremia, ANOVA). A similar increase, unaffected by blood pressure treatment, was found for wall thickness of intramyocardial arteries. Analogous changes were also noted in mesenteric arterioles and veins. Finally, aorta media thickness was significantly (P < .005) higher in uremic animals than in controls (138 +/- 29 micrometers v 103 +/- micrometers).(ABSTRACT TRUNCATED AT 250 WORDS)

A microscopical assay using a densitometric application of image analysis to quantify neurotransmitter dynamics.

We have attempted to demonstrate the technical requirements and performance of a microscopical assay using a densitometric application of image analysis to measure immunohistochemical stain intensity. Not surprisingly, the techniques required were more demanding than those used for the quantification of field and object parameters in the nervous system. The following areas of methodology have been shown to be important: (1) use of buffers free of metallic ions for tissue processing, (2) selection and titration of first and second layer antibodies, (3) reduction and control of fading of fluorescence, (4) selection of microscopical and imaging equipment to give accurate, sensitive and uniform representations of low-light biological images, and (5) use of appropriate image analysis algorithms in order to generate binary images that match the spatial and intensity distributions of immunostaining. Incorporation of these techniques into our assay system gave sensitive measurements of the time-scale of uptake of 5-hydroxy-tryptamine (5-HT) into sympathetic nerve terminals. The microscopical assay appears to have advantages over alternative approaches used for studies of neurotransmitter dynamics, particularly in small, heterogeneous tissue samples.

Effects of long-term laxative treatment on neuropeptides in rat mesenteric vessels and caecum.

In order to investigate the toxic effects of long-term treatment with anthraquinone laxatives, rats were fed either chocolate alone, or chocolate adulterated with senna or danthron (1,8-dihydroxyanthraquinone) for 5 months. Mesenteric blood vessels and the outer muscle layers of the caecum, together with the myenteric plexus, were examined using ultrastructural, histochemical, immunohistochemical and immunoassay techniques. There was no ultrastructural evidence of degeneration in either the mesenteric vessels or the caecum. In the mesenteric vessels, levels of neuropeptide Y were significantly reduced in the danthron-fed rats, but levels of substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were unaffected by all treatments. In the caecum, VIP-, SP- and CGRP-immunoreactivity and catecholamine-fluorescence were unchanged by the laxative treatments.

Intraganglionic portal sinus located between small intensely fluorescent (SIF) cells and principal ganglionic neurons in the inferior mesenteric ganglion of the guinea pig.

The vascular system in the inferior mesenteric ganglion of the guinea pig was studied to clarify the transport pathway of transmitters released by the small intensely fluorescent (SIF) cells to the principal ganglionic neurons. Reconstruction of about 1500 1-micron-thick serial sections of the ganglion demonstrated its portal system. SIF cells were tightly packed and formed two or three clusters under the capsule of the ganglion. Branches from the inferior mesenteric artery ran directly toward these clusters and broke up into a number of coiled and looped sinusoid capillaries among the SIF cells. They then drained into a large sinus surrounding the clusters in the ganglion. Capillaries were derived from this sinus and ramified among the principal ganglionic neurons. After supplying the neurons, these vessels drained into veins surrounding the ganglion. Therefore, as we observed two distinct groups of capillaries, we call this sinus the "intraganglionic portal sinus". All the transmitters secreted from the SIF cells are collected into this intraganglionic portal sinus and are then conveyed through the capillaries to the principal ganglionic neurons.

Quantitative comparisons of hydraulic permeability and endothelial intercellular cleft dimensions in single frog capillaries.

1. We have investigated the ultrastructure of the intercellular clefts of the walls of single capillaries and venules of the frog mesentery in which the hydraulic permeability (Lp) and the reflection coefficient of the vessel walls to serum albumin (sigma BSA) had been measured using the micro-occlusion technique of Michel (1980). Our aim was to investigate whether the dimensions of the clefts were sufficient to accommodate the pathways through the vessel walls necessary to account for the measured permeability. 2. Lp was measured in seventeen individually perfused vessels. The walls of fourteen of these were relatively impermeable to macromolecules with a sigma to albumin greater than 0.66 (mean value 0.83, S.E.M. +/- 0.04). The Lp of these fourteen vessels ranged from 1.8 x 10(-7) to 12.5 x 10(-7) cm s-1 cmH2O-1 and had a mean value of 5.9 (S.E.M. +/- 0.85) x 10(-7) cm s-1 cmH2O-1. 3. Cleft dimensions estimated from electron micrographs of 642 transversely sectioned endothelial cell junctions from the same seventeen vessels gave a value for the mean cleft width (W) of 0.0220 micron (S.E.M. +/- 0.0064 micron). The mean depth of the clefts from luminal to abluminal surface of the endothelium (delta x) was 0.395 micron (S.E.M. +/- 0.091 micron) with a range of 0.104-1.70 micron. The cleft length per unit area of cell wall (L), calculated using the formulation of Bundgaard & Frøkjaer-Jensen (1982), was 2064 (S.E.M. +/- 112) cm cm-2. Measurements were also made of cleft dimensions from longitudinally sectioned junctions from five of the seventeen vessels. 4. The fraction of the surface area of capillary wall occupied by the clefts (Ap = LW) had a mean value of 0.0048 (+/- 0.00014) for all seventeen vessels with a range of 0.0030-0.0074 when estimated from transverse sections. There was no correlation between the variation of Lp between different vessels and the variations of Ap. 5. Data from the fourteen vessels when sigma BSA was greater than 0.66 revealed a correlation between values of Lp and the reciprocal of delta x (r = 0.6675, P less than 0.01). No correlation was found between Lp and the mean thickness of the endothelial cells in the vicinity of the clefts. This is strong evidence for the intercellular cleft being the principal pathway for fluid movements. Variation in cleft depth appears to be a factor determining variation in permeability between different capillaries.(ABSTRACT TRUNCATED AT 400 WORDS)

The ultrastructure of frog microvessels following perfusion with the ionophore A23187.

The ultrastructure of frog mesenteric capillaries and venules has been examined after the permeability of these vessels to fluid and macromolecules has been increased to a measured extent by perfusion with the ionophore A23187. An average 4.5-fold increase in hydraulic permeability in thirteen vessels was associated with the presence of gaps between the endothelial cells and marked attenuation of endothelial cytoplasm with the appearance of many fenestrations. The changes in ultrastructure suggested much larger increments in permeability than had been measured in these same vessels in vivo.

Leukocyte endothelium adhesion and microvascular hemodynamics.

In the present study we have attempted to provide quantitative details of hemodynamic determinants of leukocyte to endothelium adhesion in the microvasculature. To this end, several hypotheses have been advanced to suggest that the preferential adhesion of leukocytes in the larger venules (30-50 microns diameter) rests upon the inherent ability of the microvasculature to compensate for small perturbations in resistance and that WBC deformability may play a significant role in this process. Flow redistribution and attendant arteriolar vasomotor adjustments may forestall LEA in the larger collecting venules of the network, where venous obstruction may be countered by bringing the full weight of the arteriovenous pressure gradient to oppose WBC adhesion. Direct measurements of the force of adhesion suggest that with diminishing vessel diameter, as for example in the immediate post-capillary venules, WBC dispersal forces will be greatest due to dramatic increases in the proportionality between force and wall shear stress. This event would tend to preclude adhesion in the smallest venular microvessels. It has also been shown that there is a strong potential for WBC deformability to affect the adhesion process by modification of the shear stresses acting on the WBC surface, as evidenced by an inverse relationship between force and wall shear stress and direct observations of WBC shape changes with increasing shear.

Perfusion and superfusion fixation effects on rat mesentery microvascular beds. Intravital and electron microscope analyses.

The effects of glutaraldehyde on dimensions and ultrastructure of microvascular beds in rat mesentery were studied in two kinds of experiment, administering the fixative by intra-arterial perfusion at a pressure of 80 mm Hg and by superfusion of the exteriorized mesenteric membrane. The microvascular segments were observed by means of intravital microscopy and recorded on videotape before, during, and after glutaraldehyde reached the microvascular segment being observed. Vascular outer diameters were measured at exactly the same points before and after fixation; in Epon embedded whole-mounts; and in sections analyzed by light and transmission electron microscopy, confirming positively the various segments of the microvascular bed and yielding information concerning the preservation of cellular components. Both experiments confirmed that neither perfusion nor superfusion of glutaraldehyde changes the outer diameter of any segment of the microvascular bed compared to the dimensions 5-10 sec before the blood vessels are reached by the fixative. They remain unaltered also after embedding in epoxy resin. During superfusion, there is a 20-50 sec delay until the blood flow comes to a complete stop. This delay is assumed to give rise to the recorded small undulations of luminal endothelial cell membranes and slight buckling of the entire endothelial layer, probably due to a gradual fall in intravascular pressure. Occasionally, the ultrastructure of some endothelial cells is less well preserved after superfusion fixation. This study demonstrates that intraarterial perfusion of glutaraldehyde renders an instantaneous fixation of mesenteric microvessels, preserving the prefixation dimensions of the various segments and the ultrastructure of the cells. Superfusion of glutaraldehyde is slower in reaching the microvessels and may change slightly the appearance of the vascular wall, and cause some impairment of microvascular functions, such as increased postcapillary leukocyte margination and extravasation.

Thrombus formation by the application of thrombin to the outer surface of mouse mesenteric vein: comparison with the application of ADP.

Mesenteries of mice under anesthesia were stretched over an inverted microscope. A micropipette filled with solution containing various concentrations of ADP or thrombin was brought into contact with the outside of a mesenteric vein by micromanipulation, and then poured over the outer surface of the vein. Morphological characteristics of the thrombi and the time needed for thrombus formation were examined. Application of either thrombin or ADP to the adventitia of mesenteric veins caused thrombus formation. Although thrombi by application of ADP seemed to be anchored by direct adhesion of platelets to the exposed subendothelium, thrombi by application of thrombin seemed to be anchored by deposited fibrin.

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm.

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.

A morphological study of the effects of 5-hydroxytryptamine and indomethacin on rat mesenteric venules.

A method is described for preparing venules of the rat mesentery for electron microscopy after the application of 5-hydroxytryptamine (5-HT) and pretreatment with indomethacin. Local application of 5-HT caused the leakage of colloidal carbon and the emigration of leucocytes into the venule wall. 5-HT also caused endothelial cells to bulge and their nuclei to contort. It increased the number of protrusions on both the luminal and abluminal surfaces of the endothelium and increased the width of the subendothelial space, and the degree of vesiculation in the endothelial cells. Systemic treatment with indomethacin significantly decreased the amount of carbon passing through the endothelium after the local application of 5-HT, but enhanced some of the other effects of 5-HT. Thus it increased the bulging of endothelial cells and contortion of their nuclei, and further increased the number of surface protrusions and the subendothelial space. It had no effect on the emigration of leucocytes resulting from the application of 5-HT.

Observations of the microcirculatory bed in rat mesocecum using differential interference constrast microscopy in vivo and electron microscopy.

The microvascular bed of the rat mesocecum has been examined in vivo using differential interference (Nomarski) optics and subsequently by electron microscopy. The preferential channel, from terminal arteriole to collecting venule, has been examined. In the terminal arteriolar segment the endothelial layer is covered by a continuous layer of smooth muscle cells which, in turn, are surrounded by adventitia. In the metarteriolar segment the periendothelial cells still resemble smooth muscle cells but the tunica media is discontinuous. In the distal segment periendothelial cells are more scattered and have the appearance of pericytes. There appears to be a continuous transition of the periendothelial cell layer from terminal arteriole to distal segment. Nerve endings were seeen in both the terminal arteriolar and metarteriolar segments. During contraction smooth muscle cells, oriented circumferentially, shorten and thicken. Endothelial cells appear anchored by myoendothelial junctions. Endothelial cells have filaments which show increased banding during vasoconstriction, suggesting that such cells may contract. Capillary offshoots leave the preferential channel, usually at right angles. Smooth muscle cells are oriented to form a sphincter and there are many myoendothelial junctions at the branch point. Within a short distance the capillary branch loses its periendothelial coat.