Inert gas clearance from tissue by co-currently and counter-currently arranged microvessels.

To elucidate the clearance of dissolved inert gas from tissues, we have developed numerical models of gas transport in a cylindrical block of tissue supplied by one or two capillaries. With two capillaries, attention is given to the effects of co-current and counter-current flow on tissue gas clearance. Clearance by counter-current flow is compared with clearance by a single capillary or by two co-currently arranged capillaries. Effects of the blood velocity, solubility, and diffusivity of the gas in the tissue are investigated using parameters with physiological values. It is found that under the conditions investigated, almost identical clearances are achieved by a single capillary as by a co-current pair when the total flow per tissue volume in each unit is the same (i.e., flow velocity in the single capillary is twice that in each co-current vessel). For both co-current and counter-current arrangements, approximate linear relations exist between the tissue gas clearance rate and tissue blood perfusion rate. However, the counter-current arrangement of capillaries results in less-efficient clearance of the inert gas from tissues. Furthermore, this difference in efficiency increases at higher blood flow rates. At a given blood flow, the simple conduction-capacitance model, which has been used to estimate tissue blood perfusion rate from inert gas clearance, underestimates gas clearance rates predicted by the numerical models for single vessel or for two vessels with co-current flow. This difference is accounted for in discussion, which also considers the choice of parameters and possible effects of microvascular architecture on the interpretation of tissue inert gas clearance.

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.

Role of villus microcirculation in intestinal absorption of glucose: coupling of epithelial with endothelial transport.

Capillaries in jejunal villi can absorb nutrients at rates several hundred times greater (per gram tissue) than capillaries in other tissues, including contracting skeletal muscle and brain. We here present an integrative hypothesis to account for these exceptionally large trans-endothelial fluxes and their relation to epithelial transport. Equations are developed for estimating concentration gradients of glucose across villus capillary walls, along paracellular channels and across subjunctional lateral membranes of absorptive cells. High concentrations of glucose discharged across lateral membranes to subjunctional intercellular spaces are delivered to abluminal surfaces of villus capillaries by convection-diffusion in intercellular channels without significant loss of concentration. Post-junctional paracellular transport thus provides the series link between epithelial and endothelial transport and makes possible the large trans-endothelial concentration gradients required for absorption to blood. Our analysis demonstrates that increases of villus capillary blood flow and permeability-surface area product (PS) are essential components of absorptive mechanisms: epithelial transport of normal digestive loads could not be sustained without concomitant increases in capillary blood flow and PS. The low rates of intestinal absorption found in anaesthetised animals may be attributed to inhibition of normal villus microvascular responses to epithelial transport.

Oxygen partial pressure in outer layers of skin of human finger nail folds.

To gain insight into oxygen transport by the cutaneous microcirculation, we have developed oxygen-sensitive microelectrodes (tip diameter approximately 5 micro m) to measure the distribution of PO2 in dermal papillae of the finger nail folds of healthy human subjects. Oxygen entry into the tissue was minimised by covering the skin with a layer of paraffin oil. The finger was held under a dissecting microscope and microelectrodes were guided into position. PO2 varied from 5-25 % of its atmospheric value, Pair (approximately 160 mmHg), depending on the location within the papilla. Along the axis of a papillary loop, PO2 decreased from 40.0 +/- 4.8 mmHg (mean +/- S.E.M., n = 6) at the base to 30.4 +/- 5.2 mmHg (n = 6) at the tip. The lowest values of PO2, in the range of 5 % of Pair, were measured in the epidermis where the metabolism of cells was highest and the steepest PO2 gradients were recorded in the vicinity of the epidermal-dermal boundary. When the local circulation was abruptly reduced or stopped, PO2 fell exponentially with time, with a time constant of 8.4 +/- 1.5 s (n = 7). When flow was reinstated, PO2 rose exponentially to a new value with a time constant of 4.8 +/- 0.8 s (n = 6). The steady state PO2 following reperfusion was approximately 23 % higher than the pre-occlusion value (P < 0.05, ANOVA and two-tailed Student's t test) indicating localised reactive hyperaemia.

Effects of blood flow on the in vivo recovery of a small diffusible molecule by microdialysis in human skin.

The aim of this study was to investigate the impact of changes in local blood flow on the recovery of a small, diffusible molecule (sodium fluorescein) from the extravascular tissue space of the skin, by microdialysis in vivo. Loss and recovery of fluorescein by linear microdialysis probes (5-kDa molecular mass cutoff, 0.2 mm diameter) inserted 1 mm apart in pairs, at three sites in the skin of the volar surface of the forearm of healthy volunteers, was measured under conditions of basal, reduced (noradrenaline, 0.005 mg/ml), and increased (glyceryl trinitrate, patch) blood flow. Whereas loss of tracer from the delivery probe appeared unaffected by changes in local blood flow, retrieval of fluorescein by the second probe was directly related to blood flux, measured using scanning laser Doppler imaging. Steady-state recovery at vasoconstricted sites was 4.0 +/- 0.7 microg. ml(-1) compared with 1.8 +/- 0.7 microg. ml(-1) at control sites (p < 0.001). Local vasodilatation reduced the retrieval of fluorescein by approximately 50% to give a steady-state concentration of fluorescein in the dialysate at 40 to 50 min after the start of perfusion of 0.9 +/- 0.3 microg. ml(-1) (p = 0.05). These studies in the skin are consistent with microdialysis theory. They suggest that clearance of solute by the blood will have a significant impact on microdialysis probe recovery and that, in the skin, the magnitude of this clearance is directly related to blood flow.

Interstitial fluid pressure surrounding rat mesenteric venules during changes in fluid filtration.

The interstitial fluid pressure (P(isf)) has been measured in the exposed superfused mesenteries of anaesthetised rats using the micropipette servo-null technique. When mesenteries were superfused with Ringer-Locke solutions, P(isf) was close to atmospheric pressure with mean +/- S.E.M. values of -0.46 +/- 0.14 cmH(2)O (n = 22). Superfusing with paraffin oil did not alter P(isf) significantly, but P(isf) could be lowered considerably by removing fluid from the upper surface of the mesentery. Measurements of P(isf) were also made in the tissues immediately outside mesenteric venules as the pressure inside these vessels and the filtration of fluid through their walls was varied. No significant changes in perivascular P(isf) could be detected even though the intravascular pressure varied from 20 to 70 cmH(2)O. Addition of histamine or the mast cell degranulating agent compound 48/80 to the superfusate had no significant effect on P(isf). The findings are relevant to experiments on the permeability of single perfused mesenteric microvessels. They strengthen the assumption, which is made in these studies, that P(isf) is close to atmospheric pressure and does not change significantly with changes in the filtration and reabsorption of fluid through the vessel walls. Experimental Physiology (2001) 86.1, 33-38.

Effects of temperature on the wall strength and compliance of frog mesenteric microvessels.

In single perfused mesenteric microvessels of pithed frogs, we assessed wall strength from the critical pressure, PB, which has to be applied within the vessel in order to induce openings in the walls through which fluid and cells can extravasate. PB was determined in capillaries and venules of tissues at 12-20 The P(B) (mean +/- S.E.M.) in 22 vessels between 12 and 20 degrees C, P(B) was 92.0 +/- 7.40 cm H2O which was significantly higher than at room temperatures (P<0.001). The compliance of the vessel wall was estimated using both the red cell method and the oil meniscus technique. There was no measurable effect of temperature on wall compliance. The compliance of vessels from which the cells had been removed by previous perfusion with detergent solutions was very similar to that of intact vessels between 12 and 20 degrees C and between 0 and 5 degrees C. The negligible effects of temperature upon compliance suggest that microvessel walls have to be distended to a greater extent in cold tissue before P(B) is reached. This, together with their rapid closure, is consistent with the hypothesis that pressure-induced openings in microvascular walls are dependent on an active response of the endothelium rather than being the result of stress failure of the basement membrane.

Modeling exchange of plasma proteins between microcirculation and interstitium of the renal medulla.

In the absence of evidence for lymphatics in the inner medulla of the kidney, it has been proposed that plasma proteins are cleared by convection out of the medullary interstitial fluid (ISF) directly into the ascending vasa recta (AVR). To clarify this hypothesis we have developed a mathematical model of the microvascular exchange of fluid, plasma proteins, and small solutes among the descending vasa recta (DVR), the AVR, and the ISF. The model represents the DVR and AVR as limbs of a countercurrent exchange loop separated and surrounded by the ISF. Steady-state exchange of fluid and solute are considered by using conservation and exchange equations. We have used values for parameters based on experimental measurements and investigated the effects of the properties of the vasa recta, the flow, and the gradient of small solutes on the distribution of plasma proteins. Results from the model agree reasonably well with experimental measurements, suggesting that convection may account for the clearance of plasma proteins from the renal medulla maintaining their concentration below that of the AVR.

Flow modulates the transport of K+ through the walls of single perfused mesenteric venules in anaesthetised rats.

1. We have investigated the effects of varying flow velocity (U) upon permeability to potassium ions (PK) of single perfused mesenteric venules in anaesthetised rats. PK was estimated using a development of the single bolus microperfusion technique at chosen flow velocities in the range of 300 to 6000 microm s-1. 2. In an initial study on 12 vessels, there was a strong positive correlation between PK and U. This was described by the relation: PK = 0.0053U + 8.86, where PK and U are both expressed in micrometres per second (microm s-1). 3. The addition of the nitric oxide (NO) synthase inhibitors (20 micromol l-1) N G-monomethyl-L-arginine (L-NMMA) and N G-nitro L-arginine (L-NNA) to the superfusate abolished the positive correlation between PK and U. The addition of D-NNA (20 micromol l-1) did not change the relation between PK and U where the median value for the slope of the relation was 57.7 (+/- 58.7 interquartile (IQR)) x 10-4 (n = 4). The addition of L-arginine (200 micromol l-1) restored the relation between PK and U where the slope of the relation was increased from 3.9 (+/- 16.3 IQR) x 10-4 to 69.2 (+/- 13.5 IQR) x 10-4 (n = 7). 4. The addition of the guanylate cyclase inhibitor LY83583 (10 micromol l-1) abolished the positive correlation between PK and U (n = 6). 5. Our data suggest that the flow modulates the potassium permeability through the walls of single perfused rat mesenteric venules via a NO-cGMP-dependent process.

Microvascular permeability.

This review addresses classical questions concerning microvascular permeabiltiy in the light of recent experimental work on intact microvascular beds, single perfused microvessels, and endothelial cell cultures. Analyses, based on ultrastructural data from serial sections of the clefts between the endothelial cells of microvessels with continuous walls, conform to the hypothesis that different permeabilities to water and small hydrophilic solutes in microvessels of different tissues can be accounted for by tortuous three-dimensional pathways that pass through breaks in the junctional strands. A fiber matrix ultrafilter at the luminal entrance to the clefts is essential if microvascular walls are to retain their low permeability to macromolecules. Quantitative estimates of exchange through the channels in the endothelial cell membranes suggest that these contribute little to the permeability of most but not all microvessels. The arguments against the convective transport of macromolecules through porous pathways and for the passage of macromolecules by transcytosis via mechanisms linked to the integrity of endothelial vesicles are evaluated. Finally, intracellular signaling mechanisms implicated in transient increases in venular microvessel permeability such as occur in acute inflammation are reviewed in relation to studies of the molecular mechanisms involved in signal transduction in cultured endothelial cells.

Openings through endothelial cells associated with increased microvascular permeability.

Rapid increases in microvascular permeability are associated with the appearance of openings in microvascular endothelium, which are believed to develop between the endothelial cells of venules. Reconstruction of these openings, from electron micrographs of serial sections of the endothelium reveal that many pass through the periphery of the endothelial cells close to intact intercellular junctions. Transcellular pathways are the principal type of opening induced in microvascular endothelium by the ionophore A23187, by VEGF, and by high transmural pressures. Some mediators induce the fusion of vacuoles with the luminal and abluminal surfaces of the endothelium, and it is suggested that the transcellular openings may develop from vacuolar channels.

Inhibition of effects of flow on potassium permeability in single perfused frog mesenteric capillaries.

1. We have investigated the effects of various potential inhibitors on flow-dependent K+ permeability (PK) of single perfused mesenteric microvessels in pithed frogs. 2. Neither superfusion with a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (10 or 100 micromol l-1), nor the addition of indomethacin (30 micromol l-1) to both perfusate and superfusate reduced the positive correlation between PK and flow velocity (U). 3. In the presence of agents known to raise intracellular levels of adenosine 3',5'-cyclic monophosphate (noradrenaline, 8-bromo-cAMP and a combination of forskolin and rolipram) the slope of the relation between PK and U was no longer significant, so that PK was no longer flow dependent. 4. These results confirm that the flow dependence of PK is a biological process and not an artefact of measurement and suggest a role for intracellular cAMP rather than nitric oxide or prostacyclin in the flow-dependent modulation of PK in frog mesenteric microvessels.

The effects of flow on the transport of potassium ions through the walls of single perfused frog mesenteric capillaries.

1. We have investigated the effects of varying flow velocity upon permeability to potassium ions (PK) of single perfused mesenteric microvessels in pithed frogs. 2. PK was estimated using a development of the single bolus microperfusion technique at chosen flow velocities (U) in the range of 150-7000 micron s-1. 3. In thirty-seven out of forty-three vessels, there was a strong positive correlation between PK and U. Average values (median) for PK (micron s-1) were related to U (micron s-1) by the expression: PK = 0.0043U + 4.05 (n = 43). 4. The correlation between PK and U was independent of microvascular pressure (and hence fluid filtration) over the range of 5-70 cmH2O. 5. The correlation between PK and U was independent of the potassium concentration in the bolus over the range of 2-40 mmol l-1 and of the direction of the potassium flux through the capillary walls.

Capillaries, caveolae, calcium and cyclic nucleotides: a new look at microvascular permeability.

Over the past 35 years much effort has been directed at identifying the pathways through microvascular endothelium and unravelling the interactions between the convective and diffusive forces which drive fluid and solutes through them. While increases in permeability induced by inflammatory mediators are known to result from the formation of gaps in venular endothelium, it is only recent advances in cell biology that have allowed the mechanisms regulating permeability to be investigated from a sound base. Results from the general biology of vesicular transport have been applied in studies on the caveolae of microvascular endothelium. Work on single perfused microvessels and on endothelial cell cultures have revealed the importance of intracellular Ca2+ and both cAMP and cGMP in regulating permeability. Even the belief that permeability is increased by gaps developing between the cells has been challenged. Although the mechanisms regulating permeability remain far from clear, sensible hypotheses can now be proposed and tested.

Effects of anastomoses on solute transcapillary exchange in countercurrent systems.

OBJECTIVE: To investigate effects of anastomoses between the descending vasa recta (DVR) and the ascending vasa recta (AVR) on the distribution of small solutes in the interstitial fluid of the renal medulla. METHODS: Countercurrent capillary loops, surrounded by a secretory epithelium, were used to model microvessels in the renal medulla. Anastomoses between the DVR and AVR were modeled as a decrease in cross-sectional area of the vessels and a decrease in flow velocity from the base to the tip of the capillary loop. When experimental data were used to evaluate parameters of the model, it was seen that diffusive transport of solute in the axial direction of the capillary was negligible, and the equations could be greatly simplified. RESULTS: General formulae of the solute concentration distribution were derived for different degrees of shunting between the two limbs of the capillary loop. Analytical solutions for the steady-state solute distribution were obtained when the sizes of capillaries and flows in them were assumed to decrease linearly with the distance from the base to the tip of the capillary loop. When the effects of reduction in the size of the limbs were compared with the effects of reduction of flow velocities on solute distribution, it was found that, in the presence of anastomoses, change in flow velocity increases the axial gradient of the solute concentration more than change in the cross-sectional area. The combined effects of a decrease in flow velocity and cross-sectional area can easily double the axial gradient of the solute concentration for a modest degree of anastomotic shunting. CONCLUSIONS: In this study, we separated effects of anastomotic flow between the DVR and AVR from other factors affecting the complicated countercurrent solute exchanges in the renal medulla. Results from the model show that anastomoses increase the solute concentration in the medullary interstitium and also the axial gradient of the solute concentration there.

Differing effects of histamine and serotonin on microvascular permeability in anaesthetized rats.

1. We have investigated simultaneous changes in the hydraulic permeability (Lp) and the retention of perfusate macromolecules in single mesenteric venules of anaesthetized rats during perfusion with either histamine or serotonin. 2. The venules were microperfused in situ. Retention of macromolecules was assessed from the effective oncotic pressure (omega delta pi) exerted by the perfusate across the vessel walls. Lp and omega delta pi were estimated by the red cell microperfusion technique. 3. Perfusion with histamine (at concentrations between 16 microM and 3.26 mM) and serotonin (at concentrations between 26 microM and 1.3 mM) transiently increased Lp and reduced omega delta pi. Maximal changes were seen at 6-9 min with histamine and at 3 min with serotonin. 4. Maximal increases in Lp were greater with histamine (approximately 3-fold) than with serotonin (1.5- to 2-fold). Serotonin, however, decreased omega delta pi from a baseline of 14-15 cmH2O to one of 6-7 cmH2O whereas the fall of omega delta pi with histamine was only from 14-15 cmH2O to 10-11 cmH2O. 5. The data are consistent with the hypothesis that serotonin increases permeability by inducing openings in the venular endothelium which do not retain macromolecules. If histamine also increases permeability by gap formation, these gaps are able to retain macromolecules to a significant extent.

Transcellular openings through frog microvascular endothelium.

Reconstructions from serial ultrathin sections of microvascular endothelium suggest that gaps, which are induced by a range of stimuli, may pass through endothelial cells as well as between them. To address the possibility that the transcellular gaps are not artefacts of aldehyde fixation, we have reconstructed fourteen gaps, induced by the ionophore A23187, in frog mesenteric microvessels where the primary fixative was osmium tetroxide. All fourteen gaps were transcellular. The different actions of osmium tetroxide and glutaraldehyde lead us to consider that it is highly unlikely that transcellular gaps are fixation artefacts.