Monolayer cultures of rat lymphatic endothelial cell were obtained from explants of rat thoracic ducts. The cells displayed a typical cobblestone morphology, expressed von Willebrand factor (factor VIII-associated antigen) and angiotensin converting enzyme, and took up acetylated-low density lipoprotein, being indistinguishable by these criteria from blood vessel endothelial cells. The availability of these cells will facilitate the use of rat experimental models for functional studies of lymphatic endothelium.
This study tested the hypothesis that the rarefaction of cutaneous capillaries with age is compensated by an increase in the response of remaining capillaries to heat stress. Experiments were performed on unanesthetized male Sprague Dawley rats of the following age groups: young (Y--age 4 mo.), middle aged (M--age 16 mo.) and old (O--age 27 mo.). The subepidermal vascular plexus of the tail was viewed by video microscopy from which measurements of blood cell velocity (BCV) in single plexus capillaries and the density of flowing capillaries (DFC) were made. The animal's body and tail were housed in separate chambers for respectively indirect and direct heat stress trials. At a neutral temperature of 25 degrees C there were no significant age differences in BCV, but DFC was decreased from the Y to the M to the O groups. During an indirect heat stress of 35 degrees C, there was a modest increase in DFC and the Y greater than M greater than O rank order persisted. By contrast, all groups experienced a marked increase in BCV during both indirect and direct heat stress, and the degree of the response was significantly greater for the O rats compared to the M or Y groups. These results support the hypothesis being tested.
In a group of 12 subjects with essential hypertension (EH), we evaluated the erythrocyte membrane fluidity and red cell membrane transverse fluidity gradient. We also evaluated the total red cell Ca content, the red cell cytosolic free calcium, the red cell membrane cholesterol/phospholipid ratio and the red cell membrane individual phospholipids.From the data obtained, it is evident that the erythrocyte membrane fluidity and red cell membrane transverse fluidity gradient discriminate normals from hypertensives. None of the red cell metabolic parameters is able, however, to differentiate normals from hypertensive subjects.Our data underline the abnormality of the red cell membrane dynamic properties in hypertension; this abnormality is not, however, related to the red cell metabolic parameters considered.
The role of leukocytes in the decreased perfusion following ischemia in skeletal muscle was examined in the microcirculation of the rat cremaster muscle. The isolated muscle was viewed with an intravital microscope. Diameters of A1 and A2 arterioles and collecting venules were determined hourly. The number of leukocytes rolling along the venular walls was determined from a videotape. Nonischemic (control) rats (n = 10) were observed for 6 hours. The ischemic group (n = 10) was observed for one hour, the iliac and femoral arteries and veins were then clamped for 4 hours, released, and the muscle was observed for another two hours. No change in arteriole or venule diameters occurred in the control group. The diameters of the arterioles in the ischemic group decreased significantly during reperfusion but, the venule diameters did not. There was a significant reduction during reperfusion but, perfused capillaries following ischemia compared to control. There was a small but not significant increase in the number of rolling leukocytes in the ischemic group. The extent of leukocyte rolling in postcapillary venules was found to not correlate with the decrease in capillary perfusion that occurs after ischemia and reperfusion. However, the decrease in capillary flow was associated with reduced arteriole diameters.
Endothelium-derived relaxing factor, which is believed to be nitric oxide (NO), mediates vasodilation of arteries perfused with hypoxic solutions. The purpose of the present study was to determine if NO mediates the response of arterioles in the hamster cheek pouch to changes in superfusion solution PO2. This was accomplished by comparison of constriction of fourth order arterioles to increases in superfusate PO2 before and during superfusion with NG-nitro-L-arginine (L-NAG), a stereospecific inhibitor of NO synthesis. The efficacy of L-NAG was assessed by comparison of dilations induced by topical application of methacholine (MCH), an endothelium-dependent vasodilator. We found that 10-15 min superfusion with 30 microM L-NAG significantly inhibited MCH-induced arteriolar dilation. However, this concentration of L-NAG had no significant effect on resting arteriolar diameters, O2-induced constrictions, constrictions induced by phenylephrine or dilations induced by sodium nitroprusside (SNP). Increasing the concentration of L-NAG to 100 microM similarly inhibited MCH-induced dilations, but did not affect SNP reactivity and may have increased vasoconstriction induced by O2. Thus, effective inhibition of NO synthesis in the hamster cheek pouch does not inhibit responses to elevated oxygen. Therefore NO does not mediate arteriolar O2 reactivity in this tissue. Furthermore, there is little evidence for tonic modulation of arteriolar reactivity by NO in the microvessels observed in this study.
In a group of subjects with essential obesity, in a group of obese subjects with non-insulin dependent diabetes mellitus (NIDDM), and in a group of obese subjects with impaired glucose tolerance (IGT), we evaluated whole-blood filtration, mean erythrocyte aggregation, erythrocyte membrane fluidity and red cell lipid pattern. From these data, it is evident that the macro- and microrheological determinants are able to discriminate normals from each group of obese subjects. Regarding the red cell lipids, few are the variations between each group of obese subjects and normal controls.
In a group of diabetics subdivided for type (12 of type 1 and 12 of type 2), we evaluated the total red cell Ca content, red cell cytosolic free calcium, erythrocyte membrane fluidity and erythrocyte membrane protein lateral mobility. From the results obtained, it is evident that the total red cell Ca content does not discriminate normals from type 1 and 2 diabetics, whereas the red cell cytosolic free calcium does differentiate between these diabetic types. Erythrocyte membrane fluidity and erythrocyte protein lateral mobility discriminate normals from type 1 and 2 diabetics. In normals and in diabetics of type 1 and 2, no relationship is evident between total red cell Ca content, membrane fluidity and membrane protein lateral mobility. A slight, but significant negative correlation between red cell cytosolic free calcium values and parameters reflecting the red cell dynamic properties is present in type 2 diabetics only.
The effect of topically applied papaverine (PAP) and norepinephrine (NE), on the microvascular pressure distribution was studied in the cremaster muscle of anesthetized rats. The cremaster muscle was exteriorized into a tissue bath containing Krebs bicarbonate buffer, and microvessel diameters and pressures were measured using a video caliper and the resistance servo-null method, respectively. Pressures were measured in the 1st through 4th branch order arterioles (1A to 4A) and 1st through 4th branch order venules (1V to 4V). Resistances were calculated across each segment from pressure gradients and 1A blood flow which was estimated from red cell velocities. PAP (10 microM) produced approximately a 15% decrease in arteriolar pressures with a concomitant increase in venular pressures of between 15 and 50%. In contrast, NE (0.1 microM) significantly increased arteriolar pressure by approximately 30% and decreased venular pressure by about 25%. Changes in systemic pressure during treatment with PAP and NE were small or insignificant and could not account for the observed changes in microvascular pressure. Significant dilation was observed in 3A, 4A, and 3V vessels after papaverine. In comparison, NE caused significant constriction in all vessel orders except 4V. PAP decreased resistance across all segments between 1A and 4V by 22-42% and increased venular resistance by almost 400%. NE increased resistance in all microvascular segments with the largest changes occurring in 2A to 3A (+276%) and 4A to 4V (+277%) segments. These data demonstrate that PAP and NE induce significant and opposite changes in arteriolar and venular pressures. Such network alterations in microvascular pressure should be considered when evaluating microvascular reactivity and exchange in the presence of vasoactive agents.
To estimate functional diffusion distances, the distribution of perfused capillaries was calculated in dog myocardium. A fluorescent dye was injected via a femoral vein in 6 anesthetized, open-chest dogs, and passed once through the coronary circulation (23 +/- 3 s). In 6 animals the dye circulated 4-20 min. In 6 animals the dye circulated for one pass following 2-3 min of asphyxia. The heart was then removed and frozen. Frozen sections from the left ventricule were cut, illuminated to excite the dye to fluoresce, and photographed. They were then stained by silver methenamine to mark all capillaries. The density of all capillaries was compared to that of capillaries containing fluorescent label. The distributions of capillaries were estimated by morphometry. In one pass of the normoxic coronary circulation, 66(+/- 4 SE)% of subepicardial and 60(+/- 4)% of subendocardial capillaries were detectably labeled. Their distribution approached a random pattern, and maximal distances to the nearest labeled capillary were lengthened by 50% compared to all capillaries. With multiple passes of the dye, or with asphyxia 76-79% of the capillaries were detectably labeled and their distribution approached the ordered pattern of the total capillary bed. We speculated that the unlabeled capillaries represented a spatially heterogeneous blood flow reserve.
The present article summarizes the author's perennial research on the flow of red blood cells in microvessels, the major determinant of rheological properties of blood in the microcirculation. Two main patterns of blood flow structure in microvessels, in the smallest arteries and veins and in the capillaries are described. The red cell concentration (hematocrit) in the blood flowing in microvessels undergoes regular alterations with changes of blood flow rate and vessel diameter in the microvascular beds. Further, the red blood cell concentration and flow velocity gradients are found in the cross-section of microvessels that should considerably affect the blood rheological properties in the microcirculation. In addition, radial displacements and blood velocity fluctuations of red cells in the flow are discovered in the larger microvessels during ischemic decrease of blood flow rate. The main factor disturbing the normal blood flow structure, and hence the normal rheological properties of blood, is the intravascular aggregation of red blood cells, which is to be diagnosed and eliminated in patients with blood rheological disturbances.
The small blood vessels were examined in 31 premenopausal and 31 postmenopausal women. In each group there were 14 non-smokers and 17 smokers of age groups 35-45 and 45-59. The women underwent full gynecological examination. Blood pressure was normal in each. The small blood vessels were examined by capillaroscopy of conjunctivae and nailfolds, by oscillometry of the radial artery, skin thermometry of the wrist and terminal phalanx, and by capillary fragility. When all abnormalities in non-smokers were compared with those in smokers in the menopausal groups, a significant difference was found, (Table 4), to the disadvantage of the smokers.
A normotensive (1.0 microgram per 100 g b.w.) or hypotensive (10.0 micrograms per 100 g b.w.) dose of serotonin (5-HT) was administered endoportally while changes in microcirculation at the inlet and outlet regions of hepatic lobules were measured on-line using quantitative in vivo microscopy. The number of sinusoids with decreased (cellular) flow also was counted to index intralobular perfusion in video recordings of microvasculature examined off-line. The normotensive and hypotensive doses of 5-HT elicited decreases in intralobular perfusion within periportal and centrivenous sinusoids. Hypoperfusion was accompanied by a transient decrease in volumetric flowrate (Q) at the outlet of centrivenous sinusoids in 40% of normotensive and in 100% of hypotensive rats. At the inlet of periportal sinusoids, Q was depressed in 75% of hypotensive and in 27% of normotensive rats. The remainder of these segments had either an increase or no change in Q at the inlet and outlet. These results suggested that during conditions of 5-HT induced (lobular) hypoperfusion: (a) Q at the inlet is maintained in 73% of normotensive rats by redistribution of intralobular blood flow, and decreased in all but 25% of hypotensive rats as a function of transient reductions in total hepatic (arterial) and/or portal (venous) blood flow(s), and (b) Q at the outlet is depressed in 40% of normotensive rats by apparent increases in flow redistribution and resistance to flow generated during sinusoidal constriction, whereas in all hypotensive rats this mechanism is aggravated by decreased total hepatic (arterial) and/or portal (venous) inflow(s). Therefore, although the initial time course for microvascular responses tended to be similar for normo- and hypo-tensive doses of 5-HT, quantitative differences in regional flow distribution and Q emphasize (a) the importance of intra- and extra-hepatic determinants in the regulation of blood flow within hepatic (unit) lobules, and (b) the presence of microvascular heterogeneity within these lobular units.
The aim of this study was to determine the gastric microcirculatory alterations occurring during reperfusion after a period of ischemia and the possible role of oxyradicals in the microcirculatory disturbance. An in vivo microscopy technique was used to observe the superficial mucosal blood flow during reperfusion. After reperfusion, mucosal blood flow resumed quickly and then slowed with eventual cessation of flow. Thirty minutes of ischemia followed by reperfusion resulted in cessation of flow in 50 +/- 4% and 81 +/- 8%, of the capillaries in the microscopic field at 15 and 30 min, respectively, after reperfusion. During this mucosal microcirculatory change, numerous white thrombi were observed flowing in the mucosal microvessels. In rats pretreated with allopurinol to inhibit oxyradical formation, blood flow was maintained to a significant and markedly greater extent. Study of the submucosal microvasculature after reperfusion revealed a marked delay in transit of a fluorescein-albumin bolus from terminal submucosal arterioles through the mucosal microvasculature and back to submucosal collecting venules. Submucosal vascular diameter change could not explain the altered mucosal blood flow. These findings indicate that there is marked slowing and cessation (in many microvessels) of gastric mucosal blood flow during reperfusion after a period of ischemia, and that the obstruction to flow occurs in the mucosal microvessels. The results of the study with allopurinol suggest that oxygen-derived free radicals generated by xanthine oxidase may play a major role in the genesis of this gastric mucosal microcirculatory disturbance.
The suffused noneverted cheek pouch of pentobarbital anesthetized hamsters was used to study the effects of localized, selective mast cell degranulation on vascular permeability. Fluorescein isothiocynate dextran (FITC-D, 70,000 Da) was utilized as a tracer, and intra-vital light microscopy was employed to monitor the formation of vascular leakage sites while direct measurement of plasma and suffusate tracer concentrations were used to monitor tracer clearance. Varying the time at which the FITC-D tracer was injected i.v. relative to the start of the Compound 48/80 suffusion permitted direct determination of the duration of any observed increase in vascular permeability. Selective, local mast cell degranulation was triggered by suffusing the cheek pouch with Compound 48/80 for 10 minutes which stimulated the formation of focal FITC-D leakage sites in the postcapillary venules resulting in increases in [FITC-D]S, [FITC-D]S / [FITC-D]P . 10(-6), and clearance. In contrast, suffusion of the cheek pouches with saline failed to trigger the formation of venular FITC-D leakage sites or to promote increases in [FITC-D]S, [FITC-D]S / [FITC-D]P . 10(-6), and FITC-D clearance. The increase in permeability produced by Compound 48/80 was marked but transient (duration less than 20 minutes), and subject to inhibition by treatment with either the H-1 receptor antagonist diphenhydramine or the endothelial cell stabilizer isoproterenol. There was no evidence for for a non-histamine mediated or delayed-onset increase in vascular permeability to macromolecules during the course of these experiments.
The suffused noneverted cheek pouch of pentobarbital anesthetized hamsters was used to study the effects of various inhibitors of receptor/cellular function on inflammatory mediator stimulated increases in vascular permeability. Fluorescein isothiocynate dextran (FITC-D, 70,000 Da) was utilized as a tracer, and intra-vital light microscopy was employed to monitor the formation of vascular leakage sites while direct measurement of plasma and suffusate tracer concentrations were used to monitor tracer clearance. Vascular permeability increases were triggered by suffusing the cheek pouch with histamine, bradykinin, or Compound 48/80 which stimulated the formation of focal FITC-D leakage sites in the postcapillary venules resulting in marked increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. Saline, calmidazolium, and papaverine lacked intrinsic permeability increasing activity, and failed to alter histamine, bradykinin, and compound 48/80 stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. In contrast, treatment with cytochalasin B, DDAVP, diphenhydramine, tubulazole C, or verapamil inhibited histamine and Compound 48/80 stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance. Bradykinin stimulated formation of venular FITC-D leakage sites and increases in [FITC-D]S, [FITC-D]S/[FITC-D]p. 10(-6), and FITC-D clearance were not affected by treatment with calmidazolium, cytochalasin B, DDAVP, diphenhydramine, tubulazole C, or verapamil. These findings demonstrate that inflammatory mediator stimulated increases in vascular permeability may be differentially affected by inhibitors of receptor/cellular function.
Cerebromicrovascular membrane fluidity was studied in two model systems: 1) hepatic encephalopathy and 2) cultured endothelium exposed to free arachidonic acid alone or with H2O2. The membrane fluidity was measured by fluorescence anisotropy using 1, 6-diphenyl-1,3,5-hexatriene as a fluorescent probe. In addition, the effect of arachidonic acid with or without H2O2 on cellular permeability to trypan blue-albumin was investigated in endothelial cultures. The findings indicate that the hepatic encephalopathy and the arachidonic acid treatment of endothelium causes an increase in membrane fluidity. This modulation of endothelial membrane fluidity is not associated with changes in cellular permeability to trypan blue-albumin complex. An increased cellular permeability to trypan blue-albumin complex was seen after endothelial exposure to arachidonic acid and H2O2.
The relationship between blood flow (xenon washout method), edema formation (percent total water content), and the number of polymorphonuclear leukocytes (PMNLs), as measured by the level of the enzyme myeloperoxidase, has been investigated in post-ischemic skeletal muscle of rats. A tourniquet model of temporary, complete ischemia of one hindlimb for 3 or 4 hours was used. Biopsies were taken after 0.5, 5 and 12 hours of reperfusion (6 experimental groups) and from a control group that had received only anesthesia. After 4 hours, but not 3 hours of ischemia there was a restricted blood flow during the early reperfusion phase, the "no-reflow" phenomenon, indicating severe ischemia. There was no significant accumulation of PMNLs in the skeletal muscle nor was there a correlation between the number of PMNLs in the post-ischemic muscle and the restricted bloodflow. With 4 hours of ischemia and 0.5 hours of reperfusion there was a statistically significant, positive correlation between the number of PMNLs and the amount of edema; no such correlation was evident in either of the other groups. These results suggest that PMNLs are not the major cause of reduced bloodflow or of edema in the early reperfusion phase after total ischemia.
We evaluated the total red cell Ca2+ content and the macro- and microrheological determinants in a group of subjects with vascular atherosclerotic disease (VAD). From the results obtained it is evident that, compared to controls, the VAD group presents an alteration of the macro- and microrheological parameters. No difference is evident for the red cell Ca2+ content in normals and in VAD subjects. No relationship is present between total red cell Ca2+ content, VBC (volume blood cells), MEA (mean erythrocyte aggregation) and red cell membrane fluidity expressed as Iex/Im ratio. In contrast to the latter, are the correlation between the red cell Ca2+ content and the degree of polarization obtained with fatty acid probes.
Intra-arterial infusion of a racemic mixture of the beta 2-agonist terbutaline blocks histamine-mediated increases in lymph flow and protein concentration in the canine forelimb. In the current study we have assessed the relative anti-inflammatory potencies of the purified stereoisomers of terbutaline. Infusion of histamine (4 micrograms base/min) increased lymph flow, protein concentration and protein transport. The intra-arterial infusion of 1-terbutaline (1 microgram/min) significantly decreased forelimb arterial pressures and prevented any changes in lymph parameters due to subsequent histamine infusion. Intra-arterial infusion of d-terbutaline (1 microgram/min) did not significantly affect forelimb vascular pressures but subsequent to histamine administration, lymph parameters increased similar to that seen with histamine alone. Infusion of a high dose of d-terbutaline (100 micrograms/min) slightly decreased forelimb arterial pressures but failed to inhibit histamine-mediated increases in lymph parameters. Infusion of 1-terbutaline alone (1 microgram/min) significantly decreased forelimb arterial pressures, lymph flow and protein transport and slightly but significantly increased lymph protein concentration. These data indicate that the beta 2-agonistic and anti-inflammatory properties of terbutaline are confined solely to the levorotatory enantiomer.
A detailed description is made of an acute closed cranial window method. The method is used for the study of cerebral pial microcirculation by intravital microscopy in the rat. Using these methods and techniques, the effects of systemic hypotension by SNP, i.v., on pial microvessel hemodynamics and on ICP were simultaneously measured and characterized under normophysiological conditions. The pH, PO2, PCO2 and temperature of the artificial cerebrospinal fluid (CSF) in the closed cranial window, intermittently measured, remained relatively constant, 30 to 60 min following the period of stabilization of the preparation. The infusion of SNP (6.2-35.0 micrograms/kg/min, 0.02% sol., i.v.) significantly decreased BP (52.1 +/- 13.4 mm Hg, mean +/- SD). From measurement of microvessels internal diameter (I. D.) and microhemodynamics, significant increases in pial arteriolar I.D. (from 35.4 +/- 10.1, microns, to 47.1 +/- 5.7, microns, mean and S.D., 33.0%) and estimated bulk flow (51.2%), occurred during the hypotension. The changes in hemodynamic parameter were predominantly in the arteriolar system. Only minimal changes in the venular diameter occurred during the SNP hypotension. The observed moderate (22.0%) increase in ICP during SNP hypotension in pentobarbital anesthetized rat correlates well with the microhemodynamic changes of the cerebral microcirculatory system.
