Micropipette System Research Articles

IN VITRO MODELS OF MICROCIRCULATION IN THE HUMAN FETUS, NEONATE AND ADULT

Intravital studies on microcirculation in humans have been limited to superficial structures such as the skin or eye. We have used the lubrication theory to describe blood flow in capillaries with diameters of 3 to 6 μm. According to this model red blood cell (RBC) surface area and volume, plasma viscosity and capillary diameter determine the necessary driving pressure for a given RBC flow velocity, the intracapillary hematocrit and blood viscosity, and the critical capillary diameter for RBC passage. Plasma viscosity determines the friction in the gap between RBC and capillary wall (“lubrication”). Plasma viscosity (tube viscometer), surface area and volume of RBCs and the validity of the model (micropipette system) were studied using blood from 10 adults and cord blood from 10 fetuses (18-22 wk), 20 preterm (24-36 wk) and 10 full-term neonates. Results. Plasma viscosity increased with increasing maturity due to rising plasma protein concentrations. Volume and surface area of RBCs increased with increasing maturity. The flow model lead to the following conclusions: If the cells are suspended in the same medium, fetal and neonatal RBCs require 27% (term neonates) to 100% (fetuses) higher driving pressures than adult RBCs to achieve the necessary elongation for passing through a 4.5-μm capillary. However, the different RBCs require similar driving pressures if the cells are suspended in the corresponding autologous plasma. This suggests that the disadvantage of the large size of neonatal RBCs is compensated for by the low plasma viscosity. Blood viscosity is 9% (term neonates) to 29% (fetuses) less than in adults. Below a critical vessel diameter (3.3 μm for adults, 3.6 μm for full-term neonates, 3.8 μm for preterm infants, and 4.1 μm for fetuses), the driving pressure and blood viscosity increase steeply as a result of rising friction between RBC and vessel wall. Direct microscopic observation of RBCs flowing in pipettes with diameters of 3.5 and 4 um showed the validity of the model.

Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells.

Lipid A is responsible for the activities of endotoxin and may cause circulatory failure and haemolysis. This study evaluated the effects of different lipid A concentrations on red blood cell (RBC) deformation (rheoscope), the aspiration pressure required to aspirate RBC into 3.3 microns pipettes, the membrane shear elastic modulus (i.e. membrane rigidity) and cellular geometry (micropipette system) after 15 min of incubation. Lipid A concentrations of 10 and 100 micrograms ml-1 of RBCs decreased RBC deformability by 26% and 39%, respectively. The aspiration pressure for RBCs into a 3.3 microns micropipette increased by 235% at a lipid A concentration of 10 micrograms ml-1 and by 586% at a concentration of 100 micrograms ml-1. The elastic shear modulus almost doubled at a lipid A concentration of 10 micrograms ml-1 and tripled at 100 micrograms ml-1. At a lipid A concentration of 100 micrograms ml-1, 37% of RBCs showed spicules. These echinocytes were less deformable than discocytes. Mean corpuscular volume, RBC volume and surface area were not affected by lipid A. We conclude that lipid A causes marked reduction of RBC deformability due to increasing membrane rigidity.

Small artery resistance increases during the development of renal hypertension.

Vascular pressures were measured in the principal (A1) arteriole and in upstream small arteries of the rat cremaster muscle to investigate vascular resistance changes associated with one-kidney, one clip Goldblatt hypertension. Pressure measurements were made at a proximal and distal site of each vessel using a servonull micropipette system. Vessel diameters were measured using video microscopy. A1 arteriole and external spermatic artery diameters were both decreased after 2 and 4 weeks of hypertension. Mean arterial pressure was elevated after 2 weeks of hypertension (106 +/- 4 mm Hg versus 140 +/- 5 mm Hg). Likewise, vascular pressures were elevated at every site: pudicepigastric artery (36%), external spermatic artery (47%), and A1 arteriole (38%). The pressure drop along the external spermatic artery was increased (87%) after 2 weeks of hypertension. Mean arterial pressure was further elevated from 2-4 weeks of hypertension (105 +/- 4 mm Hg versus 162 +/- 7 mm Hg) but only the proximal pudic-epigastric artery pressure was further elevated during this time from 2 to 4 weeks (131 +/- 5 mm Hg versus 147 +/- 7 mm Hg) of hypertension development. This was associated with an increased pressure drop (87%) along the artery compared with the situation at 2 weeks. These data indicate that small arteries upstream from the microcirculation contribute significantly to the increase in vascular resistance during hypertension. In addition, these data indicate that the increases in small artery resistance do not develop uniformly throughout all vessel branches.

Deformability and Volume of Neonatal and Adult Leukocytes

Volume and deformability of blood cells are important determinants of the microcirculation. Leukocytes are larger and considerably less deformable than erythrocytes. In our study, volume and deformability of polymorphonuclear neutrophils (PMN), lymphocytes, and monocytes in adults and full-term neonates were studied by means of a micropipette system. Neonatal immature granulocytes were also investigated. Membrane cytoplasm tongues were aspirated into 2.5-microns (diameter) micropipettes over a period of 1 min. Adult and neonatal PMN were totally aspirated into 5-microns micropipettes. Tongue growth and final tongue length of PMN were about twice those of monocytes and lymphocytes. At a pressure of -2 cm H2O, tongue growth of lymphocytes and monocytes was similar. At a pressure of -4 cm H2O, however, tongue growth of monocytes was faster and the final tongue was longer than those of lymphocytes (p less than 0.05). Cellular volume and deformation behavior of the different leukocyte subpopulations (PMN, monocytes, and lymphocytes) were similar in neonates and adults. Compared to mature neonatal PMN, immature neonatal neutrophilic granulocytes were significantly less deformable (final tongue length of 5.4 +/- 1.52 versus 9.3 +/- 1.48 microns at -2 cm H2O) and larger (421 +/- 68 versus 360 +/- 38 fL). The entry time of PMN into 5-microns micropipettes was similar in neonates and adults at aspiration pressures of -2, -3, and -4 cm H2O. We conclude that the deformability of neonatal and adult leukocytes is not different despite functional differences and that immature granulocytes may contribute to impaired microcirculation in neonates with severe septicemia or hypoxemia.

182 INCREASED PLASTIC DEFORMATION AND FRAGILITY OF RED CELL MEMBRANES IN TERM AND PRETERM NEONATES: A POSSIBLE CAUSE OF ACCELERATED RED CELL AGING

Elastic and plastic deformation (i.e. flexibility and fragility) of single red blood cell (RBC) membranes were studied microscopically in 5 term and 5 preterm neonates and 10 healthy male adults. Three methods were used: (1) A micropipette system was used to determine the membrane elastic shear modulus (μ) and the tension (T) required for local fragmentation of RBC membrane tongues aspirated into pipettes with internal diameters of 1 μm. (2) The micropipette system was also used to study the rate of plastic growth of RBC membrane tethers at given shear stress and the relaxation behavior of the tethers. (3) A flow channel system was applied to estimate μ and the tether growth rate at given shear stress. Salient results were: (1) The resistance to elastic nembrane deformation (i.e. μ) was approximately 1051 less in term and preterm neonates than in adults. (2) T was 3.8±0.8×10−3 dyn/cm in preterm infants, 6.2±0.7×10−3 dyn/cm in term neonates and 8.1±1.0×10−3 dyn/cm in adults. (3) At a shear stress of 2.5 dyn/cm2 membrane tether growth was 0 in adults, 0.27±0.9 μm/s in term neonates and 0.78±0.15 μm/s in preterm infants. (4) Tethers were partially reversible with recovery times of about 0.5 s in all three groups. The decreased resistance to plastic deformation and fragmentation may contribute to accelerated aging and shorter lifs span of neonatal RBC.

179 LIPID A BINDING AND DEFORMABILITY OF RED CELL MEMBRANE

Endotoxin may disrupt the microcirculation and cause hemolysis. Lipid A is considered to be primarily responsible for the toxicity of endotoxin. The present study was designed to evaluate the effect of lipid A on adult red blood cell (RBC) deformability. A rheoscope was used to study whole cellular deformability and a micropipette system was used for analysis of RBC membrane elasticity (shear elastic modulus) and RBC geometry (volume and surface area). RBC were suspended in buffer solution containing 1, 10 or 100 μg of lipid A per ml of RBC. Lipid A markedly diminished cellular and membrane deformability of RBC. After 15 min of incubation, 10 μg lipid A/ml RBC decreased cellular deformability by 25% and membrane elasticity by 45%. 100 μg lipid A/ml RBC caused a reduction in cellular deformability of 39% and in membrane elasticity of 65%. 1 μg lipid A/ml RBC did not affect RBC deformation. Volume and surface area of RBC were not altered by lipid A. The effect of lipid A on RBC deformation was time-dependent. The lowest RBC deformability was observed after 20 min of incubation. After 30 min RBC deformability improved and reached pre-incubation values after 60 min. We conclude that lipid A strongly diminishes RBC deformability. We speculate that RBC are able to detoxify lipid A.

26 FLOW BEHAVIOR OF NEONATAL AND ADULT ERYTHROCYTES IN VESSELS WITH DIAMETERS OF 3 TO 6 μm

This study was designed to analyse the flow behavior of red blood cells (RBC) by means of a mathematical model. According to this model, the flow resistance depends on the gap between RBC and vessel wall and on the plasma viscosity. Surface area (SA) and volume (V) of RBC from ten term neonates (N) and ten adults (A) were measured by means of a micropipette system and plasma viscosity (PV) was determined using a capillary viscometer. Neonatal RBC had larger V and SA than adult RBC (107 ± 6 vs 90±4 fl, and 154±7 vs 137±7 μm3). PV was lower in neonates (1.04±0.10 cP) than in adults (1.26±0.13 cP). The critical vessel diameter below that the gap becomes too small to allow sufficient lubrication is higher for neonatal RBC (3.3μm) than that for adult RBC (2.9μm). The driving pressure, hematocrit and viscosity of neonatal RBC is higher than that of adult RBC if both cell types have been suspended in the same medium. However, the driving pressure and viscosity of neonatal and adult RBC is similar for neonatal and adult RBC if the cells have been suspended in the corresponding neonatal and adult plasma. We conclude that the flow properties of neonatal and adult RBC in vessels with diameters of 3.5 to 6 μm are not different since the larger size of neonatal RBC is compensated by a lower PV.

Isolation and culture of heterokaryons following fusion of protoplasts from sexually compatible and sexually incompatible Medicago species

Medicago falcata L. and M. quasifalcata Sinsk. protoplasts were fused with those of M. sativa L. (alfalfa, lucerne) using polyethylene glycol (PEG). Heterokaryons were identified by their dual fluorescein diacetate-chlorophyll fluorescence and selected manually using a micro-pipette system. Fusion products mixed with albino M. sativa nurse protoplast cultures divided when cultured in the dark. Green colonies which appeared amongst the white albino nurse cells following transfer of cultures to the light produced callus tissues which were confirmed as being somatic hybrid in nature by isoenzyme and cytological analyses.

Hemodynamic characteristics of the intestinal microcirculation in renal hypertension.

This study investigated the microvascular changes that affect vascular resistance in the rat small intestine during two-kidney, one clip renal hypertension 4 weeks after renal artery stenosis. To study the intestinal microcirculation, a loop of the small intestine was exteriorized with intact circulation and innervation and a section of the bowel wall was prepared for observation with an intravital video microscopy system. Microvascular diameter, pressure, and flow velocity were measured for first, second, and third branch order arterioles and venules, using an image shearing monitor, servo-null micropipette system, and an optical Doppler velocimeter, respectively. The diameters of the first order arterioles and venules were significantly (p less than 0.05) reduced in hypertensive rats; however, diameters were unaltered in smaller second and third order arterioles and venules as compared with normotensive vessels. In hypertensive rats, mean arterial pressure was significantly (p less than 0.05) elevated (47%) and pressures also were elevated significantly (p less than 0.05) throughout the microcirculation, although by a proportionally smaller amount. Total network flow (i.e., first order arteriole flow) was significantly (p less than 0.05) reduced (40%) in hypertensive rats, but volume flows in individual second and third order arterioles were similar to flows measured in normotensive rats. Calculated total network resistance was increased (124%) in hypertensive rats. Thus, the intestinal microcirculation in rats with two-kidney, one clip renal hypertension is disturbed by elevated pressure and decreased total flow. The presence of normal flows in individual second and third order arterioles without any demonstrable difference in their diameters suggests that the predominant cause of elevated resistance across this segment of the intestinal microcirculation is a reduction in the number of perfused small arterioles.

Measurement of Endolymphatic and Perilymphatic Pressure in the Guinea Pig Cochlea

Hydrostatic pressure in the endolymph and perilymph was measured by a servo-controlled micropipette system in guinea pigs anesthetized with pentobarbital. During and after 3 minutes of anoxia, endolymphatic pressure showed changes similar to those observed in systemic arterial pressure and cochlear blood flow. Fine fluctuations with influences of heartbeat and respiration were observed in the endolymphatic pressure. These facts indicate that the system is applicable for the accurate measurement of inner ear pressure, especially of endolymphatic pressure. In guinea pigs with middle ear filled with a saturated sodium chloride solution, both endo- and perilymphatic pressure measurements showed a gradual decline for 15 minutes and a slow recovery during the next 25 minutes. These pressure changes indicate that water is transferred through the semipermeable membrane of the inner ear due to modified osmolarity. The absence of a pressure gradient between the two fluids during the period of observation suggests that Riessner's membrane is not stiff.

Tracheal volume deformation in a developmental rabbit model.

Alterations in tracheal volume were evaluated in a developmental in vivo rabbit model during and following the application of continuous positive pressure (CPP). Sequential volume changes were recorded during 60 min of CPP to a bypassed tracheal segment. The distal trachea was utilized for spontaneous ventilation. CPP of 10 cm H2O was applied to three developmental groups: group I, 5 term newborn pups; group II, 5, 7-day-old pups, and group III, 5 adult female rabbits of 18 +/- 6 months old. Changes in tracheal volume were measured by a micropipette system. Following 60 min of CPP, tracheal volumes increased (p less than 0.01) by 39.8% in group I; 36.1% in group II and 5.1% in group III. During a recovery period (60 min), return towards initial resting volume was observed in all groups, though maximal persistent volume deformation was observed in groups I and II. Thus, these data indicate tracheal barotrauma in the form of persistent dimensional deformation at early stages of development in an in vivo rabbit model.

Ascending and descending components of the medial forebrain bundle in the rat as demonstrated by the horseradish peroxidase-blue reaction. I. Forebrain and upper brain stem.

The ascending and descending components of the medial forebrain bundle (MFB) were investigated by means of horseradish peroxidase (HRP) with a sensitive substrate. The HRP was injected iontophoretically into the MFB at various levels from the anterior commissure to the posterior hypothalamus. In order to prevent the diffusion of HRP to other brain areas, a double micropipette system was used. The descending components of the MFB are derived from (1) the anterior cingulate area, infra- or prelimbic area, and sulcal cortex, (2) the lateral septal nucleus and diagonal band, (3) the bed nucleus of the stria terminalis, (4) the paraventricular nucleus (5) the substantia innominata, (6) the amygdaloid complex (AM), (7) the ventromedial (VM) and dorsomedial (DM) hypothalamic nuclei, (8) the entopeduncular nucleus and (9) nucleus periventricularis stellatocellularis. The ascending components of the MFB originate in: (1) the medial preoptic nucleus, (2) the nucleus periventricularis stellatocellularis and rotundocellularis, (3) the posterior hypothalamic nucleus, (4) the parafascicular nucleus, (5) the ventral premammillary nucleus, (6) the substantia grisea periventricularis, (7) the lateral habenular nucleus, (8) the VM and DM, (9) the paratenial nucleus, (10) the AM and (11) the arcuate nucleus.

A development of the Landis technique for measuring the filtration coefficient of individual capillaries in the frog mesentery.

Two methods are described for measuring the filtration coefficient of individually perfused frog mesenteric capillaries. Both methods involve the perfusion of capillaries via a micropipette with a solution in which a small number of human red cells are suspended. After a short period of perfusion, the capillary is occluded at a point some 500 µm or more downstream from the point of cannulation. Movements of the red cells in the isolated capillary micropipette system are interpreted to be the consequence of fluid movements across the capillary wall. The filtration coefficient of the capillary is determined either (method I) from a series of different filtration rates measured at different capillary hydrostatic pressures when intracapillary colloid osmotic pressure is constant, or (method II) from the changes in filtration rate as the fluid within the capillary concentrates at a constant pressure. Values for the filtration coefficient obtained by both methods have a skewed distribution. Determinations made by method I at 14–16°C have a peak value of 2 x 10–3 µm.sec–1.cm H2O–1 and determinations made by method II at 22–26°C have a peak value of 5 x 10–3 µm.sec–1.cm H2O–1. The assumptions underlying both methods are discussed and a mathematical model of the change in protein concentration in a closed off capillary at constant pressure is presented in the appendix.