Transfer of magnesium across the perfused choroid plexus of sheep

Isolated perfused choroid plexus preparations from sheep were used to study the effects of low concentrations of magnesium in the perfusion fluid on the transfer of magnesium into choroid plexus fluid (CPF). A perfusion fluid of similar electrolyte composition to sheep blood resulted in CPF similar to ventricular cerebrospinal fluid at a rate of 2.2 microliter min-1 mg-1 dry choroidal tissue. Decreasing the concentration of magnesium in the perfusion fluid caused a fall in the concentration of magnesium in the CPF, although it remained higher than in the perfusion fluid. The rate of transfer of magnesium from the perfusion fluid to the CPF decreased in the presence of high levels of potassium in the perfusion fluid. But decreasing the concentration of calcium in the perfusion fluid had no effect on magnesium transfer rates. These results suggest that the ability of the choroid plexus to transport magnesium against a concentration gradient is an important control of the concentration of the cerebrospinal fluid. However, this ability is insufficient to maintain cerebrospinal fluid concentrations of magnesium at normal levels when the blood magnesium concentration is below about 0.5 mmol l-1.     

Recoil and stiffening by adherent leukocytes in response to fluid shear

Prolonged exposure to fluid shear stress alters leukocyte functions associated with the immune response. We examined the initial response of freshly isolated human leukocytes to fluid shear stress under high magnification. Adherent leukocytes exhibit a rapid biomechanical response to physiological levels of fluid shear stress. After passive displacement in the direction of a constant fluid shear stress, adherent leukocytes actively recoil back in the opposite direction of the fluid flow. Recoil is observed within seconds of the applied fluid shear stress. Simultaneously, fluid shear stress induces a stiffening of the cell. The immediate cell displacement in response to a step increase in fluid shear stress is greatly attenuated in subsequent steps compared to the initial fluid shear stress step. Recoil is not mediated by actin polymerization-dependent mechanisms, as cytochalasin D had no effect on this early response. However, stiffening was determined in part by an intact actin cytoskeleton. Inhibiting myosin force generation with ML-7 abolished the recoil and stiffening responses, implicating force generation by myosin as an important contributor to the early leukocyte response to fluid shear stress. This initial shear stress response may be particularly important in facilitating leukocyte attachment under sustained fluid shear stress by the flowing blood in the microcirculation.     

Two-Fluid Model of Biofilm Disinfection

We consider a dynamic model of biofilm disinfection in two dimensions. The biofilm is treated as a viscous fluid immersed in a fluid of less viscosity. The bulk fluid moves due to an imposed external parabolic flow. The motion of the fluid is coupled to the biofilm inducing motion of the biofilm. Both the biofilm and the bulk fluid are dominated by viscous forces, hence the Reynolds number is negligible and the appropriate equations are Stokes equations. The governing partial differential equations are recast as boundary integral equations using a version of the Lorenz reciprocal relationship. This allows for robust treatment of the simplified fluid/biofilm motion. The transport of nutrients and antimicrobials, which depends directly on the velocities of the fluid and biofilm, is also included. Disinfection of the bacteria is considered under the assumption that the biofilm growth is overwhelmed by disinfection. Supported by NSF award DMS-0612467.     

Applications of supercritical fluid extraction and chromatography in forensic science

Supercritical fluid technology is a rapidly expanding analytical technique. Here we give a brief insight into the background of supercritical fluid technology and how supercritical fluid extraction and supercritical fluid chromatography work in analysis. The applications of these two techniques in forensic science are known to be important. The main area of forensic use of supercritical fluid technology is in the sample preparation and separation of drugs of abuse particularly opiates, cannabinoids, cocaine and sedatives. Supercritical fluid technology can be used for both time-of-death-related drug analysis and for obtaining information relating to long term drug abuse. We also give a review of the use of supercritical fluids in two other major forensic areas, fingerprinting and the extraction and separation of explosives from both bombing events and gunshot residues. Overall we show that supercritical fluid technology is fast becoming a major part of forensic investigations and that it is an invaluable analysis technique.     

Swimming of spermatozoa in a linear viscoelastic fluid.

A modified resistive force theory is developed for a spermatozoon swimming in a general linear viscoelastic fluid. The theory is based on a Fourier decomposition of the flagellar velocity, which leads to solving the Stokes flow equations with a complex viscosity. We use a model spermatozoon with a spherical head which propagates small amplitude sinusoidal waves along its flagellum. Results are obtained for the velocity of propulsion and the rate of working for a free swimming spermatozoon and the thrust on a fixed spermatozoon. There is no change in propulsive velocity for a viscoelastic fluid compared to a Newtonian fluid. The rate of working does change however, decreasing with increasing elasticity of the fluid, for a Maxwell fluid. Thus the theory predicts that a spermatozoon can swim faster in a Maxwell fluid with the same expenditure of energy for a Newtonian fluid.     

Peristaltic transport of a couple stress fluid in a uniform and non-uniform channels

The problem of peristaltic transport of a couple stress fluid in uniform and non-uniform two-dimensional channels has been investigated under zero Reynolds number with long wavelength approximation. Blood is represented by a couple stress fluid (a fluid which its particles size are taken into account, a special case of a non-Newtonian fluid). It is found that the pressure rise decreases as the couple stress fluid parameter gamma increases (i.e., small size fluid particle). So the pressure rise for a couple stress fluid (as a blood model) is greater than that for a Newtonian fluid. Also the pressure rise increases as the amplitude ratio phi increases for different values of gamma. Further, the pressure rise in the case of non-uniform geometry is found to be much smaller than the corresponding value in the case of uniform geometry. Finally, the maximum pressure rise when the mean flow rate over one period of the wave, Q = 0, increases as phi increases and gamma decreases.     

Electro-osmosis and the reabsorption of fluid in renal proximal tubules

The lateral intercellular spaces (LIS) are believed to be the final common pathway for fluid reabsorption from the renal proximal tubule. We postulate that electrogenic sodium pumps in the lateral membranes produce an electrical potential within the LIS, that the lateral membranes bear a net negative charge, and that fluid moves parallel to these membranes because of Helmholtz-type electro-osmosis, the field- induced movement of fluid adjacent to a charged surface. Our theoretical analysis indicates that the sodium pumps produce a longitudinal electric field of the order of 1 V/cm in the LIS. Our experimental measurements demonstrate that the electrophoretic mobility of rat renal basolateral membrane vesicles is 1 micron/s per V/cm, which is also the electro-osmotic fluid velocity in the LIS produced by a unit electric field. Thus, the fluid velocity in the LIS due to electro-osmosis should be of the order of 1 micron/s, which is sufficient to account for the observed reabsorption of fluid from renal proximal tubules. Several experimentally testable predictions emerge from our model. First, the pressure in the LIS need not increase when fluid is transported. Thus, the LIS of mammalian proximal tubules need not swell during fluid transport, a prediction consistent with the observations of Burg and Grantham (1971, Membranes and Ion Transport, pp. 49-77). Second, the reabsorption of fluid is predicted to cease when the lumen is clamped to a negative voltage. Our analysis predicts that a voltage of -15 mV will cause fluid to be secreted into the Necturus proximal tubule, a prediction consistent with the observations of Spring and Paganelli (1972, J. Gen. Physiol., 60:181).     

A fatigue microcrack alters fluid velocities in a computational model of interstitial fluid flow in cortical bone

Targeted remodeling is activated by fatigue microcracks and plays an important role in maintaining bone integrity. It is widely believed that fluid flow-induced shear stress plays a major role in modulating the mechanotransduction process. Therefore, it is likely that fluid flow-induced shear stress plays a major role in the initiation of the repair of fatigue damage. Since no in vivo measurements of fluid flow within bone exist, computational and mathematical models must be employed to investigate the fluid flow field and the shear stress occurring within cortical bone. We developed a computational fluid dynamic model of cortical bone to examine the effect of a fatigue microcrack on the fluid flow field. Our results indicate that there are alterations in the fluid flow field as far as 150 microm away from the crack, and that at distances farther than this, the fluid flow field is similar to the fluid flow field of intact bone. Through the crack and immediately above and below it, the fluid velocity is higher, while at the lateral edges it is lower than that calculated for the intact model, with a maximum change of 29%. Our results suggest that the presence of a fatigue microcrack can alter the shear stress in regions near the crack. These alterations in shear stress have the potential to significantly alter mechanotransduction and may play a role in the initiation of the repair of fatigue microcracks.     

Magnetic particle translation as a surrogate measure for synovial fluid mechanics

The mechanics of synovial fluid vary with disease progression, but are difficult to quantify quickly in a clinical setting due to small sample volumes. In this study, a novel technique to measure synovial fluid mechanics using magnetic nanoparticles is introduced. Briefly, microspheres embedded with superparamagnetic iron oxide nanoparticles, termed magnetic particles, are distributed through a 100 μL synovial fluid sample. Then, a permanent magnet inside a protective sheath is inserted into the synovial fluid sample. Magnetic particles translate toward the permanent magnet and the percentage of magnetic particles collected by the magnet in a given time can be related to synovial fluid viscosity. To validate this relationship, magnetic particle translation was demonstrated in three phases. First, magnetic particle translation was assessed in glycerol solutions with known viscosities, demonstrating that as fluid viscosity increased, magnetic particle translation decreased. Next, the relationship between magnetic particle translation and synovial fluid viscosity was assessed using bovine synovial fluid that was progressively degenerated via ultrasonication. Here, particle collection in a given amount of time increased as fluid degenerated, demonstrating that the relationship between particle collection and fluid mechanics holds in non-Newtonian synovial fluid. Finally, magnetic particle translation was used to assess differences between healthy and OA affected joints in equine synovial fluid. Here, particle collection in a given time was higher in OA joints relative to healthy horses (p < 0.001). Combined, these data demonstrate potential viability of magnetic particle translation in a clinical setting to evaluate synovial fluid mechanics in limited volumes of synovial fluid sample.     

Nitric oxide production by bone cells is fluid shear stress rate dependent

Shear stress due to mechanical loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. Moreover, the rate (determined by frequency and magnitude) of mechanical loading determines the amount of bone formation. Whether the bone cells' response to fluid shear stress is rate dependent is unknown. Here we investigated whether bone cell activation by fluid shear stress is rate dependent. MC3T3-E1 osteoblastic cells were subjected for 15 min to fluid shear stress of varying frequencies and amplitudes, resulting in peak fluid shear stress rates ranging from 0 to 39.6 Pa-Hz. Nitric oxide production, a parameter for bone cell activation, was found to be linearly dependent on the fluid shear stress rate; the slope was steepest at 5 min (0.11 Pa-Hz(-1)) and decreased to 0.03 Pa-Hz(-1) at 15 min. We conclude that the fluid shear stress rate is an important parameter for bone cell activation.     

Effects of hypoxia on alveolar fluid transport capacity in rat lungs.

There is little information regarding the effect of hypoxia on alveolar fluid clearance capacity. We measured alveolar fluid clearance, lung water volume, plasma catecholamine concentrations, and serum osmolality in rats exposed to 10% oxygen for up to 120 h and explored the mechanisms responsible for the increase in alveolar fluid clearance. The principal results were 1) alveolar fluid clearance did not change for 48 h and then increased between 72 and 120 h of exposure to hypoxia; 2) although nutritional impairment during hypoxia decreased basal alveolar fluid clearance, endogenous norepinephrine increased net alveolar fluid clearance; 3) the changes of lung water volume and serum osmolality were not associated with those of alveolar fluid clearance; 4) an administration of beta-adrenergic agonists further increased alveolar fluid clearance; and 5) alveolar fluid clearance returned to normal within 24 h of reoxygenation after hypoxia. In conclusion, alveolar epithelial fluid transport capacity increases in rats exposed to hypoxia. It is likely that a combination of endogenous norepinephrine and nutritional impairment regulates alveolar fluid clearance under hypoxic conditions.     

Numerical study of the impact of non-Newtonian blood behavior on flow over a two-dimensional backward facing step

Endothelial cell (EC) responsiveness to shear stress is essential for vasoregulation and plays a role in atherogenesis. Although blood is a non-Newtonian fluid, EC flow studies in vitro are typically performed using Newtonian fluids. The goal of the present study was to determine the impact of non-Newtonian behavior on the flow field within a model flow chamber capable of producing flow disturbance and whose dimensions permit Reynolds and Womersley numbers comparable to those present in vivo. We performed two-dimensional computational fluid dynamic simulations of steady and pulsatile laminar flow of Newtonian and non-Newtonian fluids over a backward facing step. In the non-Newtonian simulations, the fluid was modeled as a shear-thinning Carreau fluid. Steady flow results demonstrate that for Re in the range 50-400, the flow recirculation zone downstream of the step is 22-63% larger for the Newtonian fluid than for the non-Newtonian fluid, while spatial gradients of shear stress are larger for the non-Newtonian fluid. In pulsatile flow, the temporal gradients of shear stress within the flow recirculation zone are significantly larger for the Newtonian fluid than for the non-Newtonian fluid. These findings raise the possibility that in regions of flow disturbance, EC mechanotransduction pathways stimulated by Newtonian and non-Newtonian fluids may be different.     

An Apparatus for Measuring Volumes of Small Objects

An apparatus for measuring volumes of small objects such as tissue blocks is described. The apparatus measures volumes by fluid displacement and consists of a micropipette adapted to fit the mouth of an Erleiuneyer flask, a Luer adaptor fused to the side of the flask, and a glass syringe. When assembled with fluid enclosed, the fluid rises to a low level in the micropipette. Withdrawal of fluid into the syringe lowers the fluid level below the mouth of the flask. The micropipette is raised, the object to be measured is placed in the flask, and the micropipette is joined to the flask again. Fluid returned to the flask from the syringe rises to a higher level in the micropipette. The difference between the two fluid levels equals the volume of the object measured.

This apparatus gives reproducible measurements and can be calibrated for absolute volume determination. It is inexpensive to construct and easy to use.     

Effect of membrane potential on the mechanical equilibrium of biological membranes

The mechanical equilibrium of a membranous sac, whose wall is sandwiched by two oppositely charged fluid layers, is investigated as a mathematical model of a living cell. In so doing, it is assumed that the space charge density in the inner and the outer charged fluid layer is constant. It is also assumed that the fluid inside and outside of the charged fluid layer is a perfect conductor. By solving Maxwell's equation, the electric field and the thickness of the inner and the outer charged fluid layer is determined as a function of the geometry of the sac. Then, the fluid pressure in the charged fluid layer is derived by considering the body force created by the electrostatic field. The condition of mechanical equilibrium of the sac membrane yields an equation which reveals the inter-relation between the geometry, the sac fluid pressure and the membrane potential. According to this equation, the change of membrane potential causes a deformation of the sac. If the wall of the membranous sac is permeable, increase (decrease) of the absolute value of the membrane potential results in swelling (shrinking) of the sac. On the other hand, the mechanical change of the sac volume results in the change of the membrane potential. This analysis provides also an explanation of how the red blood cell maintains the biconcave shape, when the red blood cell is assumed to be a fluid filled membranous sac with non-zero membrane potential.     

Suction blister fluid as potential body fluid for biomarker proteins

Early diagnosis is important for effective disease management. Measurement of biomarkers present at the local level of the skin could be advantageous in facilitating the diagnostic process. The analysis of the proteome of suction blister fluid, representative for the interstitial fluid of the skin, is therefore a desirable first step in the search for potential biomarkers involved in biological pathways of particular diseases. Here, we describe a global analysis of the suction blister fluid proteome as potential body fluid for biomarker proteins. The suction blister fluid proteome was compared with a serum proteome analyzed using identical protocols. By using stringent criteria allowing less than 1% false positive identifications, we were able to detect, using identical experimental conditions and amount of starting material, 401 proteins in suction blister fluid and 240 proteins in serum. As a major result of our analysis we construct a prejudiced list of 34 proteins, relatively highly and uniquely detected in suction blister fluid as compared to serum, with established and putative characteristics as biomarkers. We conclude that suction blister fluid might potentially serve as a good alternative biomarker body fluid for diseases that involve the skin.     

Functional recovery of fluid drainage precedes lymphangiogenesis in acute murine foreleg lymphedema

Secondary lymphedema in humans is a common consequence of axillary lymph node dissection (ALND) to treat breast cancer. It is commonly hypothesized that lymphatic growth is required to increase fluid drainage and ameliorate lymphedema. Although there is a pronounced alteration in the balance of interstitial forces regulating fluid transport that sustains the chronic form of lymphedema, it is presently unknown whether changes occur to the balance of interstitial forces during acute lymphedema that may play a role in the recovery of fluid drainage. Here, we compared the relative importance of lymphangiogenesis of lymphatic vessels and interstitial flows for restoring fluid drainage and resolving acute lymphedema in the mouse foreleg after ALND. We found that removal of the axillary lymph nodes reduced lymph drainage in the foreleg at days 0 and 5 postsurgery, with fluid tracer spreading interstitially through subcutaneous tissues. Interstitial fluid drainage returned to normal by day 10, whereas functional regrowth of lymphatic vessels was first detected by indocyanine green fluorescence lymphography at day 15, demonstrating that the recovery of interstitial fluid drainage preceded the regrowth of lymphatic vessels. This was confirmed by the administration of VEGF receptor-3-neutralizing antibodies, which completely blocks lymphatic regrowth. It was found that the recovery of interstitial fluid drainage and the natural resolution of acute lymphedema produced by ALND were not hindered by VEGF receptor-3 neutralization, demonstrating that interstitial fluid drainage recovery and the resolution of acute lymphedema are lymphangiogenesis independent. The data highlight the central role of the interstitial environment in adapting to lymphatic injury to increase fluid drainage.     

Oscillating fluid flow regulates cytosolic calcium concentration in bovine articular chondrocytes.

Mechanical loading is a well-known regulator of cartilage metabolism. This suggests that a loading-induced physical signal regulates chondrocyte behavior. Previous studies have focused on the effects of steady fluid flow on chondrocytes. In contrast to steady flow, loading induced fluid flow occurs in an oscillatory pattern and includes a reversal of flow direction with each loading event. In this study we examined the hypothesis that oscillating fluid flow increases cytosolic Ca2+ concentration ([Ca2+]i) in bovine articular chondrocytes (BAC) in a frequency-dependent manner and that the presence of serum affects this response. The aims of our study were to examine (1) whether BAC respond to physiologic oscillating fluid flow in vitro and compare these results to steady fluid flow, (2) the effect of fetal bovine serum on fluid flow responsiveness of BAC and (3) whether the response of BAC to fluid flow is flow rate and/or frequency dependent. [Ca2+]i was quantified using the fluorescent dye fura-2. BAC were exposed to steady, 0.5, 1, or 5 Hz sinusoidal oscillating fluid flow at five different flow rates in a parallel plate flow chamber. Our findings demonstrate that BAC respond to oscillating fluid flow with an increase in [Ca2+]i (p > 0.05), and furthermore, chondrocyte responsiveness to fluid flow increases with peak flow rate (p < 0.0001) and decreases with increasing frequencies (p < 0.0001). Finally, the presence of serum in the media potentiated the responsiveness of BAC to fluid flow (p < 0.0001). Our results suggest an important role for mechanical load-induced oscillating fluid flow in chondrocyte mechanotransduction.     

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid

Abstract. There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential [26]. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential [5, 8–10]. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No troph-ectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.     

Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera

Seminal fluid is a biochemically complex mixture of glandular secretions that is transferred to the females sexual tract as part of the ejaculate. Seminal fluid has received increasing scientific interest in the fields of evolutionary and reproductive biology, as it seems a major determinant of male fertility/infertility and reproductive success. Here we used the honeybee Apis mellifera, where seminal fluid can be collected as part of a male's ejaculate, and performed a series of experiments to investigate the effects of seminal fluid and its components on sperm viability. We show that honeybee seminal fluid is highly potent in keeping sperm alive and this positive effect is present over a 24 h time span, comparable to the timing of the sperm storage process in the queen. We furthermore show that the presence of proteins within the seminal fluid and their structural integrity are crucial for this effect. Finally, we activated sperm using fructose and provide evidence that the positive effect of seminal fluid proteins on sperm survival cannot be replicated using generic protein substitutes. Our data provide experimental insights into the complex molecular interplay between sperm and seminal fluid defining male fertility and reproductive success.     

Absorption of amniotic fluid by amniochorion in sheep

Swallowing of amniotic fluid and lung fluid inflow were eliminated in 10 chronically instrumented fetuses. The urachus was ligated, and fetal was urine drained to the outside. At the beginning and the end of 21 experiments of 66 +/- 5 (SE) h duration, all amniotic fluid was temporarily drained to the outside for volume measurement and sampling. Amniotic fluid osmolalities and oncotic pressures were experimentally controlled. Amniochorionic absorption of amniotic fluid depended strongly on the osmolality difference between amniotic fluid and fetal plasma (P < 0.001), but at zero osmolality difference there still was a mean absorption rate of 23.8 +/- 4.7 (SE) ml/h (P < 0.001). Absorption was unaffected by the protein concentration difference between amniotic fluid and fetal plasma, but infused bovine albumin in the amniotic fluid was absorbed at a rate of 1.8 8 +/- 0.4 g/h (P < 0.001), corresponding to a volume flow of fluid of 33.8 8 +/- 6.1 ml/h (P < 0.001). Fluid absorption in the amniochorion is driven in part by crystalloid osmotic pressure, but about 25 ml/h is absorbed by a path that is permeable to protein. That path has the physiological characteristics of lymphatic drainage, although no anatomic basis is known to exist for a lymphatic system in the amniochorion.