Frog Mesentery Research Articles

Pathological and anatomical basis of inflammation (Zeitschr. F. Pathologie, 1920, Bd. 24)

With his classical experiments on the frog's mesentery, Conheim established, as is known, that inflammation is based on the emigration of leukocytes per diapedesin through the vascular wall, and Conheim relates to this emigration the marks that appear in the tissues undergoing inflammation. Subsequently, Marchand, Herzog and others made a significant amendment to the teachings of Conheim'a - that, in addition to emigrated leukocytes, the cells of the inflammatory infiltrate located around the vessels also have a histogenic origin, being the results of the proliferation of especially adventitiae elements, and those found in infiltration of polynuclear cells, not to mention lymphocytes, mast and plasma cells.

MOSAICING OF DYNAMIC MESENTERY VIDEO WITH GRADIENT BLENDING.

In biomedical imaging using video microscopy, understanding large tissue structures at cellular and finer resolution poses many image acquisition challenges including limited field-of-view and tissue dynamics during imaging. Automated mosaicing or stitching of live tissue video microscopy enables the visualization and analysis of subtle morphological structures and large scale vessel network architecture in tissues like the mesentery. But mosacing can be challenging if there are deformable, motion-blurred, textureless, feature-poor frames. Feature-based methods perform poorly in such cases for the lack of distinctive keypoints. Standard single block correlation matching strategies might not provide robust registration due to deformable content. In addition, the panorama suffers if there is motion blur present in a sequence. To handle these challenges, we propose a novel algorithm, Deformable Normalized Cross Correlation (DNCC) image matching with RANSAC to establish robust registration. Besides, to produce seamless panorama from motion-blurred frames we present gradient blending method based on image edge information. The DNCC algorithm is applied on Frog Mesentery sequences. Our result is compared with PSS/AutoStitch [1, 2] to establish the efficiency and robustness of the proposed DNCC method.

Metacercariae of Strigeidae Parasitizing Amphibians of the Chaco Region in Argentina.

The goal of the present study was to analyze the metacercariae found in adult amphibians collected in the Argentinean Chaco region. A total of 194 frogs, Leptodactylus chaquensis, Leptodactylus latinasus, and Lepidobatrachus laevis was examined for digeneans. Three different larval trematodes of the genus Strigea (Digenea, Strigeidae) were found infecting the liver, mesentery, body cavity, and muscle of frogs. These metacercariae are described and illustrated for the first time and provide new information on the composition of metacercariae in amphibians, thus contributing to the knowledge of larval trematodes biodiversity in the Neotropical region. We also present new information on the morphology and morphometry of strigeid metacercariae.

Similar Endothelial Glycocalyx Structures in Microvessels from a Range of Mammalian Tissues: Evidence for a Common Filtering Mechanism?

The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at ∼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.

Transient regulation of transport by pericytes in venular microvessels via trapped microdomains

A phenomenon that has defied explanation for two decades is the time scale for transient reabsorption in the classic experiments of Michel and Phillips on individually perfused frog mesentery microvessels. One finds that transient reabsorption lasts <2 min before a new steady state of low filtration is established when the lumen pressure is abruptly dropped from a high to a low value. Our experiments in frog and rat venular microvessels under a variety of conditions revealed the same time trend for new steady states to be established as in Michel and Phillips' experiments. In contrast, one theoretically predicts herein that the time required for the tissue albumin concentration to increase to values for a new steady state to be achieved through reabsorption is in the order of several hours. In this paper we propose a new hypothesis and theoretical model for this rapid regulation, namely that pericytes covering the interendothelial cleft exits create small trapped microdomains outside the cleft exits which regulate this transient behavior. Our electron microscopy studies on rat mesenteric venular microvessels reveal an average pericyte coverage of approximately 85%. The theoretical model based on this ultrastructural study predicts an equilibration time on the order of 1 min when the lumen pressure is abruptly lowered. The basic concept of a trapped microdomain can also be extended to microdomains in the interstitial space surrounding skeletal muscle capillaries.

The anti‐angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo

Vascular endothelial growth factor (VEGF) is the principal agent that increases microvascular permeability during physiological and pathological angiogenesis. VEGF is differentially spliced to form two families of isoforms: VEGF(xxx), and VEGF(xxx)b. Whereas VEGF(165) stimulates angiogenesis, VEGF(165)b is anti-angiogenic. To determine the effect of VEGF(165)b on permeability, hydraulic conductivity (L(p)) was measured in individually perfused microvessels in the mesentery of frogs and rats. As with VEGF(165), VEGF(165)b increased L(p) in amphibian (2.4 +/- 0.3-fold) and mammalian (1.9 +/- 0.2-fold) mesenteric microvessels. A dose-response relationship showed that VEGF(165)b (EC(50), 0.65 pm) was approximately 25 times more potent than VEGF(165) (EC(50), 16 pm) in amphibian microvessels. VEGF(165) has been shown to increase permeability through VEGF receptor 2 (VEGF-R2) signalling. However, VEGF(165)b increased L(p) of frog vessels to the same extent in the presence of the VEGF-R2 inhibitor ZM323881, indicating that it does not increase permeability via VEGF-R2 signalling, and was inhibited by the VEGF receptor inhibitor SU5416 at doses that are specific for VEGF receptor 1 (VEGF-R1). VEGF(165)b, in contrast to VEGF(165), did not result in a sustained chronic increase in L(p). These results show that although VEGF(165)b is anti-angiogenic in the mesentery, it does signal in endothelial cells in vivo resulting in a transient, but not sustained, increase in microvascular L(p), probably through VEGF-R1.

Eugene M. Landis and the physiology of the microcirculation.

This essay looks at the historical significance of two APS classic papers that are freely available online: Landis EM. The capillary pressure in frog mesentery as determined by micro-injection methods. Am J Physiol 75: 548–570, 1926 ( http://ajplegacy.physiology.org/cgi/reprint/75/3/548 ). Landis EM. Micro-injection studies of capillary permeability. II. The relation between capillary pressure and the rate at which fluid passes through the walls of single capillaries. Am J Physiol 82: 217–238, 1927 ( http://ajplegacy.physiology.org/cgi/reprint/82/2/217 ).

An Electrodiffusion-Filtration Model for Effects of Endothelial Surface Glycocalyx on Microvessel Permeability to Macromolecules

Endothelial surface glycocalyx plays an important role in the regulation of microvessel permeability by possibly changing its charge and configuration. To investigate the mechanisms by which surface properties of the endothelial cells control the changes in microvessel permeability, we extended the electrodiffusion model developed by Fu et al. [Am. J. Physiol. 284, H1240-1250 (2003)], which is for the interendothelial cleft with a negatively charged surface glycocalyx layer, to include the filtration due to hydrostatic and oncotic pressures across the microvessel wall as well as the electrical potential across the glycocalyx layer On the basis of the hypotheses proposed by Curry [Microcirculation 1(1): 11-26 (1994)], the predictions from this electrodiffusion-filtration model provide a good agreement with experimental data for permeability of negatively charged a-lactalbumin summarized in Curry [Microcirculation 1(1), 11-26 (1994)] under various conditions. In addition, we applied this new model to describe the transport of negatively charged macromolecules, bovine serum albumin (BSA), across venular microvessels in frog mesentery. According to the model, the convective component of the albumin transport is greatly diminished by the presence of a negatively charged glycocalyx under both normal and increased permeability conditions.

Determination of Microvessel Permeability and Tissue Diffusion Coefficient of Solutes by Laser Scanning Confocal Microscopy

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.

Interactive effect of chondroitin sulphate C and hyaluronan on fluid movement across rabbit synovium.

The polysaccharide hyaluronan (HA) conserves synovial fluid by keeping outflow low and almost constant over a wide pressure range ('buffering'), but only at concentrations associated with polymer domain overlap. We therefore tested whether polymer interactions can cause buffering, using HA-chondroitin sulphate C (CSC) mixtures. Also, since it has been found that capillary filtration is insensitive to the Starling force interstitial osmotic pressure in frog mesenteries, this was assessed in synovium. Hyaluronan at non-buffering concentrations (0.50-0.75 mg ml(-1)) and/or 25 mg ml(-1) CSC (osmotic pressure 68 cmH(2)O) was infused into knees of anaesthetised rabbits in vivo. Viscometry and chromatography confirmed that HA interacts with CSC. Pressure (P(j)) versus trans-synovial flow (;Q(s)) relations were measured.;Q(s) was outwards for HA alone (1.2 +/- 0.9 microl min(-1) at 3 cmH(2)O, mean +/- S.E.M.; n = 6). CSC diffused into synovium and changed;Q(s) to filtration at low P(j) (-4.1 microl min(-1), 3 cmH(2)O, n = 5, P < 0.02, t test). Filtration ceased upon circulatory arrest (n = 3). At higher P(j), 0.75 mg ml(-1) HA plus CSC buffered;Q(s) to approximately 3 microl min(-1) over a wide range of P(j), with an outflow increase of only 0.04 +/- 0.02 microl min(-1) cmH(2)O(-1) (n = 4). With HA or CSC alone, buffering was absent (slopes 0.57 +/- 0.04 microl min(-1) cmH(2)O(-1) (n = 4) and 0.86 +/- 0.05 microl min(-1) cmH(2)O(-1) (n = 5), respectively). Therefore, polymer interactions can cause outflow buffering in joints. Also, interstitial osmotic pressure promoted filtration in fenestrated synovial capillaries, so the results for frog mesentery capillaries cannot be generalised. The difference is attributed to differences in pore ultrastructure.

Leukocyte adhesion and microvessel permeability.

To investigate the direct effect of leukocyte adherence to microvessel walls on microvessel permeability, we developed a method to measure changes in hydraulic conductivity (L(p)) before and after leukocyte adhesion in individually perfused venular microvessels in frog mesentery. In 19 microvessels that were initially free of leukocyte sticking or rolling along the vessel wall, control L(p) was measured first with Ringer-albumin perfusate. Blood flow was then restored in each vessel with a reduced flow rate in the range of 30-116 microm/s to facilitate leukocyte adhesion. Each vessel was recannulated in 45 min. The mean number of leukocytes adhering to the vessel wall was 237 +/- 22 leukocytes/mm(2). At the same time, L(p) increased to 4.7 +/- 0.5 times the control value. Superfusion of isoproterenol (10 microM) after leukocyte adhesion brought the increased L(p) back to 1.1 +/- 0.2 times the control in 5-10 min (n = 9). Superfusing isoproterenol before leukocyte adhesion prevented the increase in L(p) (n = 6). However, the number of leukocytes adhering to the vessel wall was not significantly affected. These results demonstrated that leukocyte adhesion caused an increase in microvessel permeability that could be prevented or restored by increasing cAMP levels in endothelial cells using isoproterenol. Thus cAMP-dependent mechanisms that regulate inflammatory agent-induced increases in permeability also modulate leukocyte adhesion-induced increases in permeability but act independently of mechanisms that regulate leukocyte adhesion to the microvessel wall. Application of ketotifen, a mast cell stabilizer, and desferrioxamine mesylate, an iron-chelating reagent, attenuated the increase in L(p) induced by leukocyte adhesion, suggesting the involvement of oxidants and the activation of mast cells in leukocyte adhesion-induced permeability increase. Furthermore, with the use of an in vivo silver stain technique, the locations of the adherent leukocytes on the microvessel wall were identified quantitatively in intact microvessels.

A New View of Starling's Hypothesis at the Microstructural Level

In this paper we quantitatively investigate the hypothesis proposed by Michel (Exp. Physiol. 82, 1–30, 1997) and Weinbaum (Ann. Biomed. Eng. 26, 1–17, 1998) that the Starling forces are determined by the local difference in the hydrostatic and colloid osmotic pressure across the endothelial surface glycocalyx, which we propose is the primary molecular sieve for plasma proteins, rather than the global difference in the hydrostatic and oncotic pressure across the capillary wall between the plasma and tissue, as has been universally assumed until now. A spatially heterogeneous microstructural model is developed to explain at the cellular level why there is oncotic absorption at low capillary pressures in the short-lived transient experiments of Michel and Phillips (J. Physiol. 388, 421–435, 1987) on frog mesentery capillary, but a small positive filtration once a steady state is achieved. The new model also predicts that the local protein concentration behind the surface glycocalyx can differ greatly from the tissue protein concentration, since the convective flux of proteins through the orifice-like pores in the junction strand will greatly impede the back diffusion of the proteins into the lumen side of the cleft when the local Peclet number at the orifice is >1. The net result is that the filtration in the capillaries is far less than heretofore realized and there may be no need for venous reabsorption.

Network Assessment of Capillary Hydraulic Conductivity after Abrupt Changes in Fluid Shear Stress

Intact capillaries are composed of endothelial cells that experience continuous fluid shear stress as blood flows. Endothelial cells grown in culture demonstrate changes in barrier function in response to changes in shear stress. Therefore, it was hypothesized that intact capillaries would alter filtration in response to changes in fluid shear stress. Capillaries (n= 25) located in frog mesentery were classified according to flow direction as arteriolar, true, or venular and cannulated at 30 cm H2O to induce an abrupt change in shear stress. Frog and human red blood cells acted as velocity markers before and after cannulation, respectively, for calculating fluid shear stress. Hydraulic conductivity (Lp) was measured at 30 cm H2O following a change in shear stress and ranged from 0.7 to 96.8 × 10−7cm s−1cm H2O−1. For true and venular capillaries,Lpwas related to the magnitude of the shear stress stimulus and accounted for the wide range in absolute values ofLp. Arteriolar capillaries did not respond to the mechanical stimulus. These data indicate that blood flow may modulate filtration in homogeneous subpopulations of capillaries in the capillary bed.

Microvascular permeability and number of tight junctions are modulated by cAMP.

We tested the hypothesis that increased endothelial cell adenosine 3',5'-cyclic monophosphate (cAMP) decreases microvascular permeability in vivo. The effects of cAMP-specific phosphodiesterase type IV inhibition and adenylate cyclase activation on microvascular hydraulic conductivity (Lp) were investigated in intact individual capillaries and postcapillary venules in mesentery of pithed frogs (Rana pipiens). Treatment with rolipram (10 microM) and forskolin (5 microM) for 25 min decreased Lp to 37% of control. Rolipram alone also significantly decreased Lp. Isoproterenol (10 microM) decreased Lp to 27% of control within 20 min. A subgroup of eight vessels treated with rolipram and forskolin, in which mean Lp fell to 25% of control, was examined with transmission electron microscopy. The mean number of tight junctions in the treated vessels was 2.2 per cleft (303 clefts), significantly higher than in a matched control group (192 clefts), which was 1.7 per cleft. The results indicate that microvascular Lp can be modulated by intracellular cAMP and that one of the structural end points of stimulated cAMP levels is an increase in the mean number of tight-junction strands between endothelial cells.

Effect of cell shrinkage on permeability of cultured bovine aortic endothelia and frog mesenteric capillaries.

1. We have tested the hypothesis that a reduction in endothelial cell volume increases microvessel permeability and that the degree of endothelial cell attachment to their basement membranes determines the magnitude of permeability changes caused by a reduction in endothelial cell volume. 2. A decrease in endothelial cell volume was imposed on both intact microvessels and cultured endothelial monolayers by raising osmolarity by 100 mosmol l-1. 3. We found that hypertonic solutions did not increase the hydraulic permeability (Lp) of individually perfused venular microvessels in frog mesentery when the perfusate contained albumin. Hypertonic solutions did increase Lp, however, after we perfused the microvessels with the peptide Gly-Arg-Gly Asp-Thr-Pro (GRGDTP; 0.3 mmol l-1), to disrupt integrin-dependent endothelial cell (EC) attachment to the extracellular matrix (ECM). 4. After albumin was removed from the perfusate, hypertonic solutions increased Lp of microvessels and the permeability of endothelial monolayers to alpha-lactalbumin. 5. Our findings indicate that endothelial cell integrin-ECM binding plays a role in transducing changes in cell volume and/or shape into changes in permeability. We hypothesize that removal of albumin from the vascular perfusate may compromise EC-ECM interactions via an integrin-dependent mechanism.

Vascular endothelial growth factor increases hydraulic conductivity of isolated perfused microvessels.

These experiments report the first direct measurement of microvessel permeability coefficients after exposure to vascular endothelial growth factor (VEGF). The Landis technique was extended to enable measurement of the resistance of the microvessel wall to water flow, hydraulic conductivity (Lp), on the same microvessel in the frog mesentery during the initial exposure to VEGF (acute) and 24 and 72 h after initial exposure (chronic). Control measurements of Lp showed no change either acutely or chronically. Exposure to 1 nM VEGF rapidly and transiently increased microvessel Lp within 30 s (to 7.8-fold greater than baseline values) and returned to control within 2 min. The baseline Lp was fivefold greater after 24 h than the initial baseline as a result of VEGF perfusion and returned to its original value after 72 h. These experiments confirm the hypothesis that VEGF acts both acutely (over a period of a few minutes) and chronically (over a few hours) to increase microvascular permeability.

Ca2+ entry through conductive pathway modulates receptor-mediated increase in microvessel permeability.

We investigated the relationship between receptor-mediated increases in cytoplasmic Ca2+ concentration ([Ca2+]i) and increased microvessel permeability. In individually perfused venular microvessels of frog mesentery exposed to 10 microM ATP, [Ca2+]i increased from 59 +/- 7 to 172 +/- 21 nM within 1 min and then fell back toward control values. The corresponding peak increase in the hydraulic conductivity (Lp) of the microvessel wall was 5.7 +/- 0.5-fold relative to control. After removal of extracellular Ca2+, there was no significant increase in Lp, and the initial increase in [Ca2+]i was attenuated but not abolished. Depolarization of the endothelial cell membrane with high-K+ Ringer solution reduced the peak increase in [Ca2+]i to 106 +/- 7 nM and attenuated the increase in Lp 1.8 +/- 0.4-fold. The results conform to the hypothesis that Ca2+ entry into endothelial cells is required for acute increase in venular microvessel permeability by inflammatory agents and that the pathway for Ca2+ entry has the properties of a passive conductance pathway. Similar conclusions were reached in previous experiments in frog microvessels exposed to Ca2+ ionophores and perfusates with no plasma proteins. In venular microvessels of hamster mesentery exposed to ATP and bradykinin, a similar pathway for Ca2+ entry was demonstrated in the present experiments. We did not measure permeability changes in hamster microvessels in this study, but these microvessels respond to histamine and ionophores with a transient increase in permeability to macromolecules similar to that measured in frog microvessels [Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1982-H1991, 1995].

Protein Tyrosine Phosphorylation Modulates Microvessel Permeability in Frog Mesentery

Protein Tyrosine Phosphorylation Modulates Microvessel Permeability in Frog Mesentery

A shear stress component to the modulation of capillary hydraulic conductivity (Lp).

Two major factors that determine transcapillary exchange are Starling forces and hydraulic conductivity (Lp) of capillary walls. Although chemical stimuli alter Lp, the influence of mechanical stimuli has been neglected. Therefore, we tested the hypothesis that the Lp of intact capillaries would increase with the magnitude of changes in shear stress presented to each capillary. Capillaries (n = 26) in the mesentery of pithed frogs (n = 23) were cannulated at 30 cm H2O (average in situ pressure = 9.2) to alter shear stress. For the calculation of shear stress, velocities of both frog red blood cells (RBC) and human RBC in the pipette solution were measured in each capillary before and after cannulation, respectively. Lp was assessed 2 min after cannulation. Venular capillaries only responded to the changes in shear stress. The magnitude of the changes in shear stress correlated positively with the values for capillary Lp (Kendall's tau = 0.56; p = 0.02). While Starling forces influence the movement of fluid by acting perpendicular to the long axis of capillaries, shear stress acts in a plane tangential to the capillary walls and appears to alter capillary Lp. In addition to chemical stimuli, the mechanical stimulation of capillaries can alter the barrier itself and consequently alter filtration rate.

A diffusion wake model for tracer ultrastructure-permeability studies in microvessels.

We developed a time-dependent diffusion model for analyzing the concentration profiles of low-molecular-weight tracers in the interendothelial clefts of the capillary wall that takes into account the three-dimensional time-dependent filling of the surrounding tissue space. The model provides a connecting link between two methods to investigate transvascular exchange: electron-microscopic experiments to study the time-dependent wake formed by low-molecular-weight tracers (such as lanthanum nitrate) on the tissue side of the junction strand discontinuities in the interendothelial cleft of frog mesentery capillaries (R. H. Adamson and C. C. Michel. J. Physiol. Lond. 466: 303-327, 1993) and confocal-microscopic experiments to measure the spread of low-molecular-weight fluorescent tracers in the tissue space surrounding these microvessels (R. H. Adamson, J. F. Lenz, and F. E. Curry, Microcirculation 1: 251-265, 1994). We show that the interpretation of the presence of tracer as an all-or-none indication of a pathway across the junctional strand is likely to be incorrect for small solutes. Large-pore pathways, in which the local tracer flux densities are high, reach a threshold concentration for detection and are likely to be detected after relatively short perfusion times, whereas distributed small-pore pathways may not be detected until the tissue concentrations surrounding the entire vessel approach threshold concentrations. The analysis using this approach supports the hypothesis advanced by Fu et al. (J. Biomech. Eng. 116: 502-513, 1994) that the principal pathways for water and solutes of < 1.0 nm diameter across the interendothelial cleft may be different and suggests new experiments to test this hypothesis.