Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.     

Cryopreservation of murine embryos with trehalose and glycerol

Several concentrations of trehalose (0.0, 0.04, 0.1, 0.25 M) in combination with three concentrations of glycerol (1.0, 1.5, 2.0 M) were evaluated for the cryopreservation of murine embryos. Embryos were transferred through increasing concentrations of glycerol in Dulbecco's phosphate-buffered saline with 10% fetal calf serum (PBS + FCS) to reach the final glycerol concentrations. They were then randomly assigned to one of the concentrations of trehalose. A total of 506 morulae were packaged individually in 0.25-ml plastic straws and cooled from ambient temperature at 1.0 degrees C/min in a programmable methanol freezer. Embryos were seeded at -7 degrees C and then cooled to -25 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. After thawing and a one-step dilution of glycerol, embryos were cultured for 48 hr and viability was determined by blastocoel formation. Highest viability (70.0%) after 48 hr in culture was obtained for embryos frozen in 1.5 M glycerol plus 0.10 M trehalose as compared to 31% viability for embryos frozen with glycerol alone. These observations suggest that trehalose can be used in combination with glycerol as a cryoprotectant and that a high rate of viability can be achieved after a one-step dilution of the cryoprotectants.     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Vitrification media: toxicity,permeability, and dielectric properties

The aim of this study was to select a cryoprotectant for use in attempts to preserve tissues and organs by vitrification. The first step was to select a cell line with which to compare the toxicity of a range of commonly used cryoprotectants. An immortal vascular endothelial cell (ECV304) was exposed to vitrifying concentrations of four cryoprotectants: dimethyl sulfoxide (Me(2)SO; 45% w/w); 2,3 butanediol (BD; 32%); 1,2-propanediol (PD; 45%); and ethanediol (ED; 45%). Three times of exposure (1, 3, and 9 min) and two temperatures (22 and 2-4 degrees C) were studied. After removal of the cryoprotectant, the ability of the cells to adhere and divide in culture over a 2-day period was measured and expressed as a Cell Survival Index (CSI). There was no measurable loss of cells after exposure to the four cryoprotectants but 3-min exposure to BD, PD, or Me(2)SO at room temperature completely destroyed the ability of the cells to adhere and divide in culture. In contrast, exposure to all four cryoprotectants at 2-4 degrees C for up to 9 min permitted the retention of significant cell function, the CSIs, as a proportion of control, being 76.3+/-7.0% for BD, 63.6+/-7.1% for PD, 37.0+/-4.1 for Me(2)SO, and 33.2+/-3.0 for ED. The permeability properties of the cells for these four cryoprotectants was also measured at each temperature. Permeability to water was high, L(p) approximately equal 10(-7) cm/s/atm at 2-4 degrees C with all the cryoprotectants, but there were substantial differences in solute permeability: BD and PD were the most permeable at 2-4 degrees C (P(s)=4.1 and 3.0 x 10(-6) cm/s, respectively). Equilibration of intracellular cryoprotectant concentration was rapid, due in part to high water permeability; the cells were approximately 80% of their physiological volume after 10 min. Treatment at 2-4 degrees C with BD was the least damaging, but PD was not significantly worse. Exposure to vitrifying concentrations of ED and Me(2)SO, even at 2-4 degrees C, was severely damaging. Segments of rabbit carotid artery were treated with vitrifying concentrations of each of the two most favorable cryoprotectants, BD and PD, for 9 min. It was shown that each cryoprotectant reduced smooth muscle maximum contractility to a similar extent and abolished the acetylcholine response. However, vital staining revealed that exposure to BD also caused substantial damage to the endothelial lining, whereas the endothelium was completely intact after PD exposure, raising the possibility that the effect of PD on NO release may be reversible. In later stages of this project it is planned to use dielectric heating to rewarm the tissues and thereby avoid devitrification. The effects of each cryoprotectant on this mode of heating was therefore studied. Gelatin spheres containing vitrifiable concentrations of each cryoprotectant were rewarmed from -60 degrees C in a radiofrequency applicator. Because the uniformity of heating is related to the dielectric properties of the material, these properties were also measured. PD was the most suitable. These physical measurements, combined with the measurements of toxicity and permeability, indicate that PD is the most favorable cryoprotectant of those tested for use in subsequent stages of this study.     

Cryomicroscopic observations of cattle embryos during freezing and thawing

A cryomicroscope was used to observe changes in the appearance of day 6 1 2 to 7 1 2 cattle embryos during cooling and warming in 1.4M glycerol/PBS. Embryos were cooled at various rates between 0.2 and 25 degrees C/min to temperatures between -25 and -60 degrees C and then cooled rapidly ( approximately 250 degrees C/min) to temperatures below -140 degrees C. The volume of the embryos calculated from the cross-sectional area during slow cooling decreased at -25 degrees C to about 50% of the isotonic volume. Fracture planes could be observed in the extracellular ice matrix surrounding the embryos after rapid cooling to approximately -140 degrees C. The fracture planes often touched the zona pellucida and sometimes caused cracks in the zona. Cracks in the zona pellucida were observed more often after rapid cooling from temperatures between -20 to -35 degrees C (9 13 ) than from temperatures between -36 to -60 degrees C (2 7 ). When embryos were warmed rapidly ( approximately 250 degrees C/min) from temperatures below -140 degrees C, no change was observed in the appearance of either the embryo or its surroundings except the melting of the extracellular ice. However, when embryos were warmed slowly (2 or 5 degrees C/min), a series of events was observed; first, at approximately -70 degrees C the cytoplasm and the extracellular space gradually darkened and reached maximum darkness at approximately -55 degrees C. Then, on continued slow warming, the dark material gradually disappeared and finally the large extracellular ice crystals melted.     

Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates?

Mouse spermatozoa in 18% raffinose and 3.8% Oxyrase in 0.25 x PBS exhibit high motilities when frozen to -70 degrees C at 20-130 degrees C/min and then rapidly warmed. However, survival is <10% when they are frozen at 260 or 530 degrees C/min, presumably because, at those high rates, intracellular water cannot leave rapidly enough to prevent extensive supercooling and this supercooling leads to nucleation and freezing in situ (intracellular ice formation [IIF]). The probability of IIF as a function of cooling rate can be computed by coupled differential equations that describe the extent of the loss of cell water during freezing and from knowledge of the temperature at which the supercooled protoplasm of the cell can nucleate. Calculation of the kinetics of dehydration requires values for the hydraulic conductivity (Lp) of the cell and for its activation energy (Ea). Using literature values for these parameters in mouse sperm, we calculated curves of water volume versus temperature for four cooling rates between 250 and 2000 degrees C/min. The intracellular nucleation temperature was inferred to be -20 degrees C or above based on the greatly reduced motilities of sperm that underwent rapid cooling to a minimum temperature of between -20 and -70 degrees C. Combining that information regarding nucleation temperature with the computed dehydration curves leads to the conclusion that intracellular freezing should occur only in cells that are cooled at 2000 degrees C/min and not in cells that are cooled at 250-1000 degrees C/min. The calculated rate of 2000 degrees C/min for IIF is approximately eightfold higher than the experimentally inferred value of 260 degrees C/min. Possible reasons for the discrepancy are discussed.     

Effects of cooling and warming rate to and from -70 degrees C, and effect of further cooling from -70 to -196 degrees C on the motility of mouse spermatozoa

We have previously reported high survival in mouse sperm frozen at 21 degrees C/min to -70 degrees C in a solution containing 18% raffinose in 0.25 x PBS (400 mOsm) and then warmed rapidly at approximately 2000 degrees C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to -70 degrees C at rates ranging from 0.3 to 530 degrees C/min. The survival curve was an inverted "U" shape, with the highest motility occurring between 27 and 130 degrees C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130 degrees C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was -70 degrees C. When sperm were frozen to -70 degrees C at optimum rates, lowering the temperature to -196 degrees C had no adverse effect.     

Ferulic acid inhibits endothelial cell proliferation through NO down-regulating ERK1/2 pathway

The aim of this study was to determine the antiproliferative mechanism of ferulic acid (FA) on serum induced ECV304 cell, a human umbilical vein endothelial line. The results suggest that FA significantly suppressed ECV304 cells proliferation and blocked the cell cycle in G0/G1 phase. Treatment of the cells with FA increased nitric oxide (NO) production and inactivated the extracellular signal-regulated kinase (EERK1/2), and the NO donor, sodium nitroprusside, inhibited both ECV304 cells proliferation and phosphorylation of ERK1/2. However, the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester, caused ECV304 cells proliferation. PD 98059, the inhibitor of ERK1/2, had no effect on the NO production. These results indicate that NO suppressed ECV304 cells proliferation through down-regulating ERK1/2 pathway. Moreover, the inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression and phosphorylation of retinoblastoma protein (pRb) by increment of p21 level. The findings not only present the first evidence that FA is a potent inhibitor on ECV304 cells proliferation, but also reveal the potential signaling molecules involved in its action.     

H-Ras is a negative regulator of alpha3beta1 integrin expression in ECV304 endothelial cells

We have examined the role of Ras in integrin expression in ECV304 endothelial cells. Among the integrins examined in stable ECV304 transfectants expressing dominant active H-Ras (DAR-ECV), expression of alpha3beta1 integrin showed a prominent reduction in all the DAR-ECV clones when compared to the parental ECV304 cells. This implies that H-Ras negatively regulates the expression of alpha3beta1 integrin in ECV304 cells. When treated with inhibitors of the Ras downstream pathway (LY294002, PD98059, SB203580), the expression of alpha3beta1 integrin was up-regulated most significantly by LY294002, suggesting that among the downstream pathways of Ras, phosphatidylinositol 3-kinase is a major determinant. With the application of blocking antibody to alpha3beta1 integrin (2 - 2 x 10(4) nM), migration of ECV304 cells was enhanced to maximal (18%) at 20 nM. These results suggest that migration of endothelial cells could be modulated by H-Ras via alteration of the expression levels of alpha3beta1 integrin.     

Influence of skeletal muscle glycogen on passive rewarming after hypothermia

To examine the influence of muscle glycogen on the thermal responses to passive rewarming subsequent to mild hypothermia, eight subjects completed two cold-water immersions (18 degrees C), followed by 75 min of passive rewarming (24 degrees C air, resting in blanket). The experiments followed several days of different exercise-diet regimens eliciting either low (LMG; 141.0 +/- 10.5 mmol.kg.dry wt-1) or normal (NMG; 526.2 +/- 44.2 mmol.kg.dry wt-1) prewarming muscle glycogen levels. Cold-water immersion was performed for 180 min or to a rectal temperature (Tre) of 35.5 degrees C. In four subjects (group A, body fat = 20 +/- 1%), postimmersion Tre was similar to preimmersion Tre for both trials (36.73 +/- 0.18 vs. 37.26 +/- 0.18 degrees C, respectively). Passive rewarming in group A resulted in an increase in Tre of only 0.13 +/- 0.08 degrees C. Conversely, initial rewarming Tre for the other four subjects (group B, body fat = 12 +/- 1%) averaged 35.50 +/- 0.05 degrees C for both trials. Rewarming increased Tre similarly in group B during both LMG (0.76 +/- 0.25 degrees C) and NMG (0.89 +/- 0.13 degrees C). Afterdrop responses, evident only in those individuals whose body core cooled during immersion (group B), were not different between LMG and NMG. These data support the contention that Tre responses during passive rewarming are related to body insulation. Furthermore these results indicate that low muscle glycogen levels do not impair rewarming time nor alter after-drop responses during passive rewarming after mild-to-moderate hypothermia.     

Direct observation of cold-shock effects in ram spermatozoa with the use of a programmable cryomicroscope

Cryoinjury in individual ram spermatozoa was investigated in cells cooled at 10 degrees C/min on a programmable cryomicroscope. In physiological buffer and cryoprotective media, there was a smooth decline in sperm swimming speed with decreasing temperature; cooling in buffer caused a marked decline in the proportion of cells displaying forward progression, especially once the temperature fell below 16 degrees C. Spermatozoa cooled in the presence of rhodamine 123, a mitochondrial-specific dye, showed that abolition of sperm motility by cold shock in buffer was not due to mitochondrial inactivation. Temperature decline through the region of 10 degrees C caused a number of spermatozoa in buffer to undergo a sudden asymmetric bending of the flagellum in the region of the midpiece. Ultrastructural studies suggest that this was caused by an unstable, asymmetric membrane lesion. Spermatozoa cooled in the presence of cryoprotective media showed better recovery of motility after rewarming and failed to exhibit the bending effect described above.     

Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling

The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to -70 degrees C or below. This approach, often called interrupted rapid cooling, is the subject of this paper. Mouse oocytes were suspended in 1.5M ethylene glycol (EG)/PBS, rapidly cooled (50 degrees C/min) to -25 degrees C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 degrees C/min) to -70 degrees C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 degrees C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at -25 degrees C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to -70 degrees C nor during warming.     

SDF—1α对单核细胞的黏附作用

目的探讨pcDNA3．1-基质细胞衍生因子1α重组质粒对单核细胞的黏附作用。方法将pcDNA3．1-2基质细胞衍生因子1α质粒用脂质体的方法导入人脐静脉内皮细胞株ECV304细胞中,G418筛选细胞克隆。通过逆转录聚合酶链反应法在转录水平检测基质细胞衍生因子1α在转基因ECV304细胞中的表达。在6孔板上种植转染基质细胞衍生因子1α基因的ECV304细胞,加THP-1细胞37℃孵育30min,磷酸缓冲液轻洗3次,去除未黏附细胞,倒置显微镜下计数上、下、左、右、中5个视野,取其平均值得到每个视野黏附单核细胞数。结果获得了稳定表达基质细胞衍生因子1α基因的ECV304细胞克隆,pcDNA3．1-基质细胞衍生因子1n重组质粒对单核细胞具有黏附作用。结论基质细胞衍生因子1α对单核细胞具有明显黏附作用。     

Is oxygen supply a limiting factor for survival during rewarming from profound hypothermia?

It has been postulated that unsuccessful resuscitation of victims of accidental hypothermia is caused by insufficient tissue oxygenation. The aim of this study was to test whether inadequate O2 supply and/or malfunctioning O2 extraction occur during rewarming from deep/profound hypothermia of different duration. Three groups of rats (n = 7 each) were used: group 1 served as normothermic control for 5 h; groups 2 and 3 were core cooled to 15 degrees C, kept at 15 degrees C for 1 and 5 h, respectively, and then rewarmed. In both hypothermic groups, cardiac output (CO) decreased spontaneously by > 50% in response to cooling. O2 consumption fell to less than one-third during cooling but recovered completely in both groups during rewarming. During hypothermia, circulating blood volume in both groups was reduced to approximately one-third of baseline, indicating that some vascular beds were critically perfused during hypothermia. CO recovered completely in animals rewarmed after 1 h (group 2) but recovered to only 60% in those rewarmed after 5 h (group 3), whereas blood volume increased to approximately three-fourths of baseline in both groups. Metabolic acidosis was observed only after 5 h of hypothermia (15 degrees C). A significant increase in myocardial tissue heat shock protein 70 after rewarming in group 3, but not in group 2, indicates an association with the duration of hypothermia. Thus mechanisms facilitating O2 extraction function well during deep/profound hypothermia, and, despite low CO, O2 supply was not a limiting factor for survival in the present experiments.     

Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation.

Attempts to cryopreserve bovine oocytes result in low survival because of their sensitivity to temperatures near 0 degrees C. This study evaluates the effects of chilling germinal vesicle-stage (GV) oocytes on their formation of microtubules and the meiotic spindle. In experiment 1, five groups of GV-stage oocytes, each consisting of approximately 90 oocytes, were held at 39 degrees C as controls, or at 31 degrees C, or cooled to 24, 4 or 0 degrees C for 10 min. After being treated, all oocytes were cultured at 39 degrees C for 24 hr. Compared to the controls, holding oocytes for 10 min at 31 or 24 degrees C did not significantly alter the formation of normal spindles, but chilling them to 4 or 0 degrees C did. After 24 hr of maturation, the respective percentages of oocytes containing normal meiotic spindles observed in the controls or those held at 31 or 24 degrees C were 69.8%, 71.9%, or 69.4% (P > 0.05). In contrast, the percentages of oocytes with normal spindles after they had been cooled to 4 or 0 degrees C were 44.0% or 29.1%, respectively. In experiment 2, approximately 90 oocytes/group were cooled to 4 degrees C for various times before being warmed and cultured. Regardless of the time of exposure, cooling oocytes to 4 degrees C reduced the formation of normal spindles. The percentages of oocytes cooled to 4 degrees C for 10, 20, 30, 45, or 60 min with normal spindles were 44.0%, 38.4%, 37.5%, 34.5% and 30.9%, respectively. In experiment 3, approximately 60 oocytes per group that had been held at 31 degrees C or cooled to 24, 4 or 0 degrees C for 10 min were allowed to mature for 24 hr before being subjected to in vitro fertilization. The cleavage rates of oocytes subjected to various chilling treatments exhibited the same pattern as that of oocytes with normal spindles. That is, there were no significant differences in cleavage rates among the control oocytes and those held at 31 or 24 degrees C (70.4%, 71.8%, and 72.4%; P > 0.05). However, only 37. 0% and 30.4% of oocytes chilled to 4 or 0 degrees C cleaved after fertilization. These results suggest that: (1) chilling bovine oocytes no lower than 24 degrees C does not reduce formation of normal meiotic spindles; (2) however, chilling oocytes to 4 degrees C or lower for as little as 10 min drastically reduces the formation of normal meiotic spindles and of fertilization; (3) the rates of fertilization and cleavage of resultant zygotes mimic that of formation of normal spindles.     

Mouse sperm desiccated and stored in trehalose medium without freezing

Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4 degrees C and 22 degrees C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22 degrees C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4 degrees C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22 degrees C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4 degrees C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.     

Effects of sympathetic stimulation during cooling on hypothermic as well as posthypothermic hemodynamic function

This experimental study was performed to explore hemodynamic effects of a moderate dose epinephrine (Epi) during hypothermia and to test the hypothesis whether sympathetic stimulation during cooling affects myocardial function following rewarming. Two groups of male Wistar rats (each, n=7) were cooled to 15 degrees C, maintained at this temperature for 1 h, and then rewarmed. Group 1 received 1 microg/min Epi, i.v., for 1 h during cooling to 28 degrees C, a dose known to elevate cardiac output (CO) by approximately 25% at 37 degrees C. Group 2 served a saline solution control. At 37 degrees C, Epi infusion elevated CO, left ventricular systolic pressure, maximum rate of left ventricle pressure rise, and mean arterial pressure. During cooling to 28 degrees C, these variables, with the exception of mean arterial pressure, decreased in parallel to those in the saline solution group. In contrast, in the Epi group, mean arterial pressure remained increased and total peripheral resistance was significantly elevated at 28 degrees C. Compared with corresponding prehypothermic values, most hemodynamic variables were lowered after 1 h at 15 degrees C in both groups (except for stroke volume). After rewarming, alterations in hemodynamic variables in the Epi-treated group were more prominent than in saline solution controls. Thus, before cooling, continuous Epi infusion predominantly stimulates myocardial mechanical function, materialized as elevation of CO, left ventricular systolic pressure, and maximum rate of left ventricle pressure rise. Cooling, on the other hand, apparently eradicates central hemodynamic effects of Epi and during stable hypothermia, elevation of peripheral vascular vasopressor effects seem to take over. In contrast to temperature-matched, non-Epi stimulated control rats, a significant depression of myocardial mechanical function occurs during rewarming following a moderate sympathetic stimulus during initial cooling.     

Phosphatidylserine-dependent adhesion of T cells to endothelial cells

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.     

Effect of warming rate on mouse embryos frozen and thawed in glycerol

Mouse embryos (8-cell) fully equilibrated in 1.5 M-glycerol were cooled slowly (0.5 degrees C/min) to temperatures between - 7.5 and - 80 degrees C before rapid cooling and storage in liquid nitrogen (-196 degrees C). Some embryos survived rapid warming (approximately 500 degrees C/min) irrespective of the temperature at which slow cooling was terminated. However, the highest levels of survival of rapidly warmed embryos were observed when slow cooling was terminated between -25 and -80 degrees C (74-86%). In contrast, high survival (75-86%) was obtained after slow warming (approximately 2 degrees C/min) only when slow cooling was continued to -55 degrees C or below before transfer into liquid N2. Injury to embryos cooled slowly to -30 degrees C and then rapidly to -196 degrees C occurred only when slow warming (approximately 2 degrees C/min) was continued to -60 degrees C or above. Parallel cryomicroscopical observations indicated that embryos became dehydrated during slow cooling to -30 degrees C and did not freeze intracellularly during subsequent rapid cooling (approximately 250 degrees C/min) to -150 degrees C. During slow warming (2 degrees C/min), however, intracellular ice appeared at a temperature between -70 and -65 degrees C and melted when warming was continued to -30 degrees C. Intracellular freezing was not observed during rapid warming (250 degrees C/min) or during slow warming when slow cooling had been continued to -65 degrees C. These results indicate that glycerol provides superior or equal protection when compared to dimethyl sulphoxide against the deleterious effects of freezing and thawing.     

Lipids in blood-brain barrier models in vitro I: Thin-layer chromatography and high-performance liquid chromatography for the analysis of lipid classes and long-chain polyunsaturated fatty acids

The objectives of this study were to optimize a sensitive high-performance liquid chromatography (HPLC) method for fatty acid (FA) analysis for the quantification of polyunsaturated FAs (PUFAs) in cell lipid extracts and to analyze the lipid and FA patterns of three cell lines used in blood-brain barrier (BBB) models: RBE4, ECV304, and C6. Thin-layer chromatographic analysis revealed differences in the phosphatidylcholine-phosphatidylethanolamine (PC:PE) ratios and the triglyceride (TG) content. The PC:PE ratio was <1 for RBE4 cells but >1 for ECV304 and C6 cells. ECV304 cells displayed up to 9% TG depending on culture time, whereas the other cell lines contained about 1% TG. The percentages of docosahexaenoic acid were 9.4 +/- 1.7% of the unsaturated FAs in RBE4 cells (n = 5; 4 d in culture; 9.9% after 10 d), 8.1 +/- 2.0% in ECV304 cells (n = 11; 10 to 14 d), and 6.7 +/- 0.6% in C6 cells (n = 6; 10 to 14 d) and were close to the published values for rat brain microvascular endothelium. The percentage of arachidonic acid (C20:4) was about half that in vivo. ECV304 cells contained the highest fraction of C20:4, 17.8 +/- 2.2%; RBE4 cells contained 11.6 +/- 2.4%; and C6 cells 15.8 +/- 1.9%. It is concluded that a sensitive HPLC method for FAs is now optimized for the analysis of long-chain PUFAs. The results provide a useful framework for studies on the effects of lipid modulation and give reference information for the development of further BBB models.