Venular Microvessels Research Articles

Effect of nitric oxide synthase inhibitors on basal microvessel permeability and endothelial cell [Ca2+]i.

We evaluated the role of basal nitric oxide (NO) release in the regulation of microvessel permeability under resting conditions. We measured changes in microvessel hydraulic conductivity (Lp) and endothelial cytoplasmic calcium concentration ([Ca2+]i) after application of NO synthase (NOS) inhibitors to the lumen of individually perfused frog mesenteric venular microvessels. NOS inhibitors caused a transient increase in Lp. The mean ratios of peak test Lp values relative to control values in the presence of N omega-nitro-L-arginine methyl ester (L-NAME) at concentrations of 1, 10, and 100 microM were 2.5 +/- 0.6, 2.9 +/- 0.7, and 4.8 +/- 0.4, respectively. N omega-monomethyl-L-arginine (L-NMMA) showed a similar effect and a biologically inactive isomer of L-NMMA, D-NMMA, showed no effect. These results demonstrate that basal levels of NO play a role in modulating microvessel permeability different from that due to NO produced in response to inflammatory agents. In the activated state NOS inhibitors attenuated the increased microvessel permeability in response to ionomycin and ATP [P. He, B. Liu, and F. E. Curry. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H176-H185, 1997]. The transient increase in basal permeability induced by NOS inhibitors was not accompanied by an increase in endothelial cell [Ca2+]i and did not require the presence of extracellular calcium. Application of ketotifen, a mast cell stabilizer, and an iron-chelating reagent, deferoxamine mesylate, attenuated the transient increase in Lp induced by L-NMMA, suggesting that basal NO may have an important antioxidant role in regulating normal permeability.

Effect of nitric oxide synthase inhibitors on endothelial [Ca2+]i and microvessel permeability.

To investigate the mechanism whereby nitric oxide (NO) signaling pathways regulate microvessel permeability in vivo, we measured changes in microvessel hydraulic conductivity (Lp) and endothelial cytoplasmic calcium concentration ([Ca2+]i) in response to calcium ionophore, ionomycin (5 microM), and ATP (10 microM) before and after the use of NO synthase (NOS) inhibitors in single perfused frog mesenteric venular microvessels. Ionomycin induced a transient increase in endothelial [Ca2+]i and an associated increase in Lp. The NOS inhibitors N omega-nitro-L-arginine methyl ester (10 and 300 microM) and N omega-monomethyl-L-arginine (L-NMMA; 10, 50, and 100 microM) significantly attenuated the peak increase in Lp induced by ionomycin. A similar inhibitory effect was also observed with the increase in Lp mediated by ATP. In contrast, D-NMMA, a biologically inactive isomer of L-NMMA, showed no effect on ionomycin-induced increase in Lp L-Arginine (3 mM) reversed the inhibitory effect of L-NMMA (10 microM) on Lp. However, the NOS inhibitors did not alter the magnitude and time course of the biphasic increase in endothelial [Ca2+]i induced by both ionomycin and ATP. These data suggest that 1) calcium-dependent NO release is a necessary step to increase microvessel permeability, and 2) the action of NOS inhibitors in attenuating the permeability increase in response to ionomycin and ATP occurs down-stream from calcium entry and does not involve modification of the initial increase in endothelial [Ca2+]i.

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].

Tracheal microvascular responses to inhibition of nitric oxide synthesis in anesthetized rats.

We previously demonstrated that rat tracheal arterioles and large venules constrict in response to alpha-adrenergic agonists and dilate in response to beta-adrenergic agonists. To further investigate the vasodilatory mechanisms in these vessels, we hypothesized that the vascular tone in the tracheal microcirculation is regulated in part by endogenously released nitric oxide (NO). To test this hypothesis, rat tracheal microvessels were visualized in vivo with a video microscope. The change in diameter af adventitial arteriolar (14.5 to 42.0 microm initial diameter, n = 41) and large venular (50.0 to 100.0 microm initial diameter, n = 41) microvessels following the suffusion of vasoactive drugs was measured with video calipers. Significant constriction was observed in arterioles (to 70.0% of initial diameter) and large venules (to 76.6% of initial diameter) after 20 min of suffusion with L-NAME, an inhibitor of NO synthesis. This constriction was in large part reversed by L-arginine, a biochemical precursor of NO, in arterioles (to 93.2% of initial diameter) and in venules (to 90.8% of initial diameter). The combination of prazosin, a selective alpha1-adrenergic antagonist, and yohimbine, a selective alpha2-adrenergic antagonist, also reduced L-NAME-induced constriction in arterioles (to 87.9% of initial diameter) and in venules (to 85.2% of initial diameter). L-arginine or the combination of prazosin and yohimbine alone did not affect the diameter of tracheal microvessels. These data suggest that NO exerts an important influence on tracheal microvascular tone in rats, and may attenuate alpha-adrenergic constriction in these vessels.

Visualization of endothelial clefts and nuclei in living microvessels with combined reflectance and fluorescence confocal microscopy.

The cellular basis for spatial heterogeneity along postcapillary venules in their response to inflammatory mediators is not understood. To study permeability regulatory processes on an individual cell basis in intact microvessels, we developed methods to delineate individual endothelial cells within living, single perfused microvessels. Individual postcapillary microvessels in the mesenteries of frogs and hamsters were perfused with chloride-free Ringer's solutions containing AgNO3 (0.1 g per 100 ml) for 5--10 s. Vessels were immediately flushed with Ringer's solution containing serum albumin and some vessels subsequently perfused with the fluorescent nucleic acid stain YO-PRO-1 (1 microM) for 10-15 min. Vessels were imaged in situ using laser scanning confocal microscopy. A reflectance image from silver precipitate in the endothelial clefts and a fluorescence image of the nuclei were simultaneously recorded. Stacks of confocal images were merged and used to reconstruct the three-dimensional orientation of endothelial cells. Hydraulic conductivity of some frog mesenteric venular vessels, measured before and after perfusion with AgNO3, was used to assess the integrity of the vessels. The endothelial cell clefts were delineated with a very fine, readily imaged precipitate of silver. Hydraulic conductivity measured after AgNO3 perfusion in frog mesenteric venular microvessels was not significantly different from control. Vessels showed a slight reduction in reactivity to inflammatory stimuli. The three-dimensional pattern of endothelial cells in the living vessels was imaged. Combining confocal microscopy with silver staining in single perfused microvessels was shown to be an effective means to delineate the three-dimensional pattern of endothelial cells forming microvessel walls. It enables further study of the vascular structure and function relationships at the individual cell level in intact microvessels.

Measurement of Membrane Potential of Endothelial Cells in Single Perfused Microvessels

The membrane potential is an important modulator of calcium ion flux into endothelial cells of venular microvessels. We developed a method to measure the membrane potential of endothelial cells forming the walls of individually perfused microvessels under the same experimental conditions as those used to measure cytoplasmic calcium concentration and microvessel permeability. The membrane potential-sensitive fluorescent dye, bis-oxonol (1 μ M), was added to the perfusate and the changes in bis-oxonol fluorescence intensity (FI) were calibrated in terms of changes in membrane potential using the cationic ionophore, gramicidin. FI changes an average of 0.625% per millivolt. The resting membrane potential of endothelial cells measured in single perfused microvessels, each calibrated individually, was 51.6 ± 4.9 mV ( n = 9). In the presence of high potassium Ringer's solutions (57.9 and 100 m M, [K +] o), the membrane depolarized 25 ± 3 and 40 ± 5 mV, respectively. Conversely, low potassium solutions (0.1 m M [K +] o) hyperpolarized the membrane by 23 ± 4 mV. The endothelial membrane was also depolarized when the Na-K-ATPase was inhibited with ouabain. This method provides new data to test current hypotheses describing the role of the endothelial cell membrane potential as a modulator of microvessel permeability.

Solvent drag of LDL across mammalian endothelial barriers with increased permeability.

We investigated the mechanisms of hamster low-density lipoprotein (LDL) transport across the endothelial barrier in individually perfused venular microvessels in hamster mesentery. These experiments are the first to use microperfusion techniques and quantitative fluorescence microscopy to investigate LDL transport across mammalian microvessel endothelium. The apparent permeability coefficient for hamster LDL, PsLDL, rose from 2.7 x 10(-7) cm/s at control to 23.2 x 10(-7) cm/s at the peak of the biphasic increase in microvessel permeability after exposure of the vessels to 100 microM histamine. Close to the peak, PsLDL rose 1.85 x 10(-7) cm/s for every centimeter of H2O increase in hydrostatic pressure. Thus, at a mean pressure of 11.3 cmH2O, 90% of the LDL flux was coupled to transendothelial water flow by a solvent drag mechanism. The corresponding solvent drag reflection coefficient for hamster LDL was estimated to be approximately 0.8. These results are consistent with sieving hamster LDL (effective radius 14.9 nm) through equivalent pores of approximately 22 nm radius. Similar results were found with human LDL (effective radius 13.2 nm) in hamster microvessels. The results provide a bridge between studies of LDL transport across cultured endothelial barriers, where high diffusive permeability coefficients to LDL may obscure the contributions of solvent drag, and studies in whole animals, where the consequences of sieving of LDL at the vessel wall, even in the high permeability state, have not received much attention.

Imaging of Ca2+ transients in endothelial cells of single perfused capillaries: correlation of peak [Ca2+]i with sites of macromolecule leakage.

To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca2+]i, does not increase uniformly within microvessel endothelial cells. We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca2+]i using a cooled CCD camera. Control [Ca2+]i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca2+]i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca2+]i (274 +/- 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca2+]i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increase in [Ca2+]i were largest. These data indicate that variation in [Ca2+]i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.

E-Selectin Mediates Leukocyte Rolling in Interleukin-1–Treated Rabbit Mesentery Venules

The selectins are lectin-like cell surface glycoproteins that have been implicated in playing a crucial role in the initiation of leukocyte adhesion to endothelial cells (ECs) during inflammation. Binding of selectins under conditions of flow mediates leukocyte rolling, which in vivo is almost exclusively observed in venular microvessels. We have shown in previous experiments that intraperitoneal treatment of rabbits with interleukin-1 beta (IL-1) increases leukocyte rolling in exteriorized mesenteries. In the present study, we used immunohistochemistry of mesenteries and found that IL-1 induced a marked E-selectin immunoreactivity, preferentially in venules. We therefore hypothesized that the increased rolling in response to IL-1 may be related to the induction of E-selectin on venular ECs. Intravital microscopy was used to investigate interactions between leukocytes and ECs after intraperitoneal application of IL-1. The rabbit E-selectin monoclonal antibody (MoAb) 9H9 significantly reduced rolling of leukocytes by approximately 40%. Vehicle alone, class-matched control MoAb or the nonblocking anti-E-selectin MoAb 14G2 had no effect on rolling. These results indicate that leukocytes roll on inflamed venular ECs partly through interactions with E-selectin. Furthermore, we propose that the restricted E-selectin immunoreactivity by venular ECs contributes to the remarkable difference seen between arterioles and venules in exhibiting leukocyte rolling in vivo.

E-selectin mediates leukocyte rolling in interleukin-1-treated rabbit mesentery venules

The selectins are lectin-like cell surface glycoproteins that have been implicated in playing a crucial role in the initiation of leukocyte adhesion to endothelial cells (ECs) during inflammation. Binding of selectins under conditions of flow mediates leukocyte rolling, which in vivo is almost exclusively observed in venular microvessels. We have shown in previous experiments that intraperitoneal treatment of rabbits with interleukin-1 beta (IL-1) increases leukocyte rolling in exteriorized mesenteries. In the present study, we used immunohistochemistry of mesenteries and found that IL-1 induced a marked E-selectin immunoreactivity, preferentially in venules. We therefore hypothesized that the increased rolling in response to IL-1 may be related to the induction of E-selectin on venular ECs. Intravital microscopy was used to investigate interactions between leukocytes and ECs after intraperitoneal application of IL-1. The rabbit E-selectin monoclonal antibody (MoAb) 9H9 significantly reduced rolling of leukocytes by approximately 40%. Vehicle alone, class- matched control MoAb or the nonblocking anti-E-selectin MoAb 14G2 had no effect on rolling. These results indicate that leukocytes roll on inflamed venular ECs partly through interactions with E-selectin. Furthermore, we propose that the restricted E-selectin immunoreactivity by venular ECs contributes to the remarkable difference seen between arterioles and venules in exhibiting leukocyte rolling in vivo.

Lipoprotein lipase (LpL) affects low density lipoprotein (LDL) flux through vascular tissue: evidence that LpL increases LDL accumulation in vascular tissue.

A cardinal feature of the atherosclerotic lesion is increased low density lipoprotein (LDL) content of the arterial wall. Such increases in vascular wall LDL could result from either increased flux of circulating LDL across the arterial endothelial barrier or decreased efflux of LDL that has entered the vascular tissue. A number of studies have focused on factors that alter permeability of endothelial cell monolayers and intact blood vessels causing increased LDL influx. In contrast, the current studies were designed to test the hypothesis that lipoprotein lipase (LpL), increases LDL accumulation and decreases LDL efflux from vascular tissue. Frog mesenteric venular microvessels were cannulated and the rates of fluorescently labeled LDL accumulation (N/t) and efflux (T1/2) were measured by quantitative fluorescence microscopy. When the vessels were perfused with a solution containing bovine milk LpL (10(-5) g/ml) and human LDL (protein = 0.68 mg/ml), N/t was > 15x greater than that of control vessels which were perfused with LDL alone. LpL addition did not change albumin permeability, suggesting that increased N/t was not related to changes in vessel permeability. Increased LDL accumulation within the vessel could have resulted from either an increase in LDL influx from the vessel lumen into the vascular tissue or a decrease in efflux of LDL. Therefore, LDL efflux from vascular tissue was determined by measuring the rate of decline in fluorescence intensity of control and LpL-treated vessels after washout of the vessel lumen with a clear, nonfluorescent solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial cell hyperpolarization increases [Ca2+]i and venular microvessel permeability

We tested the hypothesis that Ca2+ influx into endothelial cells forming the walls of intact venular microvessels was increased when the cell membranes were hyperpolarized. Cytoplasmic free Ca2+ concentration ([Ca2+]i) was measured after the endothelial cells forming the microvessel wall were loaded with fura 2, endothelial cell membrane potential was measured with the membrane potential dye bis-oxonol, and hydraulic conductivity (Lp) of the vessels was measured by the modified Landis technique to follow changes in microvessel permeability. When microvessels were exposed to low-K+ (0.1 mM) Ringer solution, the membrane of the endothelial cells was hyperpolarized approximately 27 mV and [Ca2+]i increased from 47 nM to a peak value of 151 +/- 28 nM. Under the same experimental conditions, Lp increased to a peak 6.3 times control. In the presence of ionomycin (5 microM), the initial peak [Ca2+]i measured with low-K+ Ringer solution was 347 +/- 58 nM compared with 252 +/- 58 nM with ionophore and normal Ringer solution. The corresponding initial increases in Lp were 28 times control and 10 times control, respectively. The results conform to the hypothesis that vasoactive substances that hyperpolarize the endothelial cell membrane may initiate and/or potentiate the inflammatory response in venular microvessels.

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium-dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium-dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium-derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.

Extravascular distribution of low-density lipoprotein and dextran in a high-permeability state

Previous studies from this laboratory indicated that in the early phase of a high-permeability state, low-density lipoprotein (LDL) crosses the microvascular barrier by porous pathways ( J. C. Rutledge, F. E. Curry, J. F. Lenz, and P. Davis, 1989, Circ. Res. 1990 ; 66, 486–495). To determine the extravascular distribution of LDL (3,500,000 MW) and a smaller reference macromolecule [20,000 MW dextran (D20)] image processing techniques were employed, and extravascular accumulation of solutes from different microvessels was compared to quantitative fluorescence microscopy. Frog mesenteric venular microvessels ( n = 8) were cannulated and perfused with fluorescent-labeled LDL and D20 and extravascular distribution of both solutes was imaged at control and after permeability was increased with the calcium ionophore ionomycin (5 μ M). At the peak increase in apparent permeability (2–4 min after ionophore), the processed images of the microvessels demonstrated that the extravascular distribution of fluorescent-labeled solute was not uniform and that D20 accumulated outside the microvessel wall in some areas where LDL did not accumulate. The patterns of extravascular accumulation of LDL and D20 in a high-permeability state imply a distribution of effective microvascular pore sizes and/or a distribution of resistances to solute flow in the pore or in the tissue surrounding the microvessel.

Graded modulation of frog microvessel permeability to albumin using ionophore A23187

We investigated the exchange of water and macromolecules across venular microvessels after permeability was increased. Quantitative fluorescence microscopy was used to measure albumin permeability coefficients in individually perfused microvessels of decerebrate frogs. Control permeability coefficient was 2.3 +/- 0.25 X 10(-7) cm/s. Solvent drag increased the apparent solute permeability coefficient (Ps) by 0.57 +/- 0.05 X 10(-7) cm/s for each cmH2O increase in microvessel pressure. The divalent cation ionophore A23187 (0.1-5 microM) produced a transient increase in Ps to a peak value (within 1-3 min), followed (after 4-8 min) by a sustained increase in permeability (16-34% of peak values). Peak values of Ps were 13 and 80 times control for 0.1 and 5 microM A23187, respectively. Both diffusion and solvent drag contributed to the sustained increase in Ps. The equivalent pore radius of the structures determining diffusion and solvent drag was less than or equal to 25 nm during the sustained increase in permeability, smaller than observed gaps between adjacent endothelial cells. The basement membrane and a fibrous matrix secreted by endothelial cells into the gaps may offer resistance to exchange in the high permeability state.

Effect of a helium-neon laser on pial microcirculation in rats

, laser action. Lasers, which have a photodynamic and biostimulatingac tion on tissues, are being increasingly used to study the morphological and functional changes in organs and tissues [2, 5, 8]. The use of lasers in microcirculatory research has made it possible to apply precise conditions of stimulation to different parts of tile microcirculatory bed [6] and also a means of assessing the microcirculation of the blood in vivo [7]. The use of low-energy lasers, inducing a biostimulating effect, is promising as a means of studying the effect of outside influences on vessels of the microcirculatory bed [i, 3]. Among these lasers, the heliumneon laser (H/~L) has achieved the most widespread popularity. The aim of this investigation was to study the effect of HNL on the microcirculatory bed of the pia mater and the microcirculation in rats in order to discover the sensitivity of arteriolar and venular microvessels to this agent at different ages of postnatal development.

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.

Localization and measurement of microvascular and microcirculatory responses to venous pressure elevation in the rat.

The effect of increase in regional draining venous pressure on the response of mesoappendix microvasculature and microcirculation was studied by direct microscopic methods <i>in vivo</i>. In 9 out of 21 experiments in pentobarbital anesthetized rats, elevation of ileo-colic vein pressure (13.0 ± 1.3 mm Hg; mean and SE) above resting levels (9.9 ± 2.2 mm Hg) resulted in persistent lumen expansion of small venular microvessels (18-68 <i>µ</i>m<i>, </i>i.d.) and evoked a vasoconstrictor response of consecutive segments of pre-capillary vessels and the sphincter. The vasoconstricor response was stimulus-dependent and reversible. The lumen reductions in percent for 9 arterioles (21.0 ± 4.37 <i>µ</i>m<i>, </i>i.d.) 9 metarterioles (9.3 ± 1.57 <i>µ</i>m<i>, </i>i.d.), and 5 precapillary sphincters (5.7 ± 1.23 <i>µ</i>m<i>, </i>i.d.) were: 17.9 ± 6.60, 43.6 ± 4.20, and 96.0 ± 3.81, respectively. In none of the experiments did the response propagate to larger arteriolar segments ( > 35–38 <i>µ</i>m, i.d.) in the tissue under observation. These microscopic studies confirmed the presence of a venous-pre-capillary and sphincter constrictor response in splanchnic vasculature in the rat.