Fluid Efflux Research Articles

Abstract 061: Interstitial Matrix Flow-Regulation:Myocardial Fibroblasts Respond to Colloidosmotic Pressure

Ischemia-reperfusion injury induces large differences in hydration potential between ischemic and nonischemic areas of the myocardium. They influence the rate and volume of fluid transfer in myocardial explants; temperature-dependent hydration-potential differences of approximately 100 mmHg in at-risk areas, as opposed to contiguous areas that are not at risk, suggest prompt interstitial fluid-transfer control mechanisms (Circ. Res . 2012;11:A235) . Aim: We adapted osmotic-stress techniques to determine whether myocardial fibroblasts, which are known to respond to mechanical and flow signals, also respond to hydration-potential changes. Methods: Fibroblasts were isolated from midwall regions of the left ventricles of healthy pigs using standard procedures. They were incorporated into 3-dimensional collagen gels of 500 mm 3 volume at 150 cells/mm 3 and equilibrated overnight in cultures using the nontoxic, inert polymer polyethylene glycol 8000 (molecular radius ~26.5 Å; concentration range 0-10% w/w) to adjust colloidosmotic pressure from approximately 5 to 205 mmHg . After the gels were detached from the dish, fluid flux was derived from any time-dependent changes in their dimensions. Results: The volume of gels without fibroblasts did not change significantly at any coloidosmotic pressure. In those with fibroblasts, the volume decreased, more slowly in those subjected to higher-than-plasma pressure levels. Progression curves conformed well to a two-exponential term model (R 2 >0.9) suggesting that two parallel processes contribute to matrix efflux, one relatively fast, with decay constant 0.274 ± 0.014 (n = 3) , and another ~50-fold slower. Initial rates were calculated from the fitted curves, and linear regression analysis used to examine their dependence on colloidosmotic pressure. Initial efflux rates decreased with pressure, mean slope, - 0.29 ± 0.08 µl/h/mmHg (R 2 = 0.7; P-value = 0.006). Conclusion: In vitro , fibroblasts in collagen matrices regulate fluid efflux in response to colloidosmotic stresses within the range of hydration-potential differences measured in myocardial explants after ischemia-reperfusion injury.

Acetazolamide attenuates transvascular fluid flux in equine lungs during intense exercise

During intense exercise in horses the transvascular fluid flux in the pulmonary circulation (Jv-a) represents 4% of cardiac output (Q). This fluid flux has been attributed to an increase in pulmonary transmural hydrostatic forces, increases in perfused microvascular surface area, and reversible alterations in capillary permeability under conditions of high flow and pressure. Erythrocyte fluid efflux, however, accounts for a significant fraction of Jv-a. In the lung the Jacobs-Stewart cycle occurs with diffusion of CO2 into alveolar space with possible accompanying chloride (Cl-) and water movement from the erythrocyte directly into the pulmonary interstitium. We hypothesised that inhibition of carbonic anhydrase in erythrocytes inhibits the Jacobs-Stewart cycle and attenuates Jv-a. Five horses were exercised on a treadmill until fatigue without (control) and with acetazolamide treatment (30 mg kg(-1) 30 min before exercise). Erythrocyte fluid efflux, plasma fluid flux across the lung and Jv-a were calculated using haemoglobin, haematocrit, plasma protein and Q. Fluid fluxes were used to calculate erythrocyte, plasma and whole blood Cl- fluxes across the lung. Cardiac output was not different between control and acetazolamide treatment. During exercise erythrocyte fluid efflux and Jv-a increased in control (9.3±3.3 and 11.0±4.4 l min(-1), respectively) and was higher than after acetazolamide treatment (3.8±1.6 and 1.2±1.2 l min(-1), respectively) (P<0.05). Plasma fluid flux did not change from rest in control and decreased after acetazolamide treatment (-4.5±1.5 l min(-1)) (P<0.05). Erythrocyte Cl- flux increased during exercise in control and after acetazolamide treatment (P<0.05). During exercise plasma Cl- flux across the lung did not change in control; however, it increased with acetazolamide treatment (P=0.0001). During exercise whole blood Cl- flux increased across the lung in control (P<0.05) but not after acetazolamide treatment. The results indicate that Jv-a in the lung is dependent on the Jacobs-Stewart cycle and mostly independent of transmural hydrostatic forces. It also appears that Jv-a is mediated by Cl- and water egress from erythrocytes directly into the interstitium without transit through plasma.

Pathophysiology and Treatment Options in Trigeminal Meningoceles

Trigeminal meningoceles, lateral to the maxillary nerve (V2), have seldom been reported as underlying pathology for spontaneous rhinoliquorrhea. In contrast to sphenoid meningoceles arising from a persistent lateral craniopharyngeal canal (Sternberg–Cruveilhier, medial to V2), their occurrence seems to be generated by addition of erosive processes to the constitutively thin bony shell underneath the semilunar ganglion, lateral to the round foramen (and V2).The developmental and anatomical relationships of trigeminal meningoceles to the sphenoid bone are depicted, and in a review of the literature we present the different surgical approaches employed for sealing the dura leak. In view of these techniques we discuss an unusual case of therapy-resistant rhinoliquorrhea with left-sided trigeminal meningocele involving the Meckel cave at the lateral sphenoid and reaching the superior orbital fissure and the medial orbital space.In contrast to patients who have lateral sphenoidal meningoceles with a persistent lateral craniopharyngeal canal (Sternberg–Cruveilhier), who can be treated successfully using an endoscopic transsphenoidal approach (recurrence rate 13.7%), the recurrence rate of cerebrospinal fluid (CSF) efflux for trigeminal meningoceles lies much higher (endoscopically 66%, open craniotomy 33%). The surgical strategy thus has to be chosen individually, taking into account specific anatomical situations and eventually preceding operations.

Characteristics of the low density corneal endothelial monolayer

Corneal endothelial cells form a leaky barrier on the posterior surface of the cornea, allowing influx of nutrient-carrying aqueous humor through the paracellular space and efflux of excess fluid. Corneal edema arises when the density of these non-proliferative endothelial cells declines from endothelial disease or intraocular surgery. The cellular changes occurring at low densities are ill-defined. We therefore investigated the paracellular pathway of corneal endothelial cell monolayers of varying density to determine alterations occurring in paracellular permeability and monolayer morphology. Primary cultures of bovine corneal endothelial cells (BCECs) were passaged onto permeable supports under varying culture conditions to obtain confluent monolayers of <1000, 1000–1999 and >2000 cells/mm2. Culture growth was monitored by transendothelial electrical resistance measurements. Diffusional permeability to sodium fluorescein, FITC-dextran MW 4000 or FITC-dextran MW 20,000 was measured. Confluent cultures were also analyzed by immunofluorescence localization of the tight junction protein ZO-1 and by transmission electron microscopy. For comparison, we evaluated ZO-1 for low and high density human corneal endothelium. Our results showed that all BCEC cultures grew to the same final transendothelial electrical resistance regardless of final density. In the diffusional permeability assay, permeability increased significantly only for the smallest tracer molecule (sodium fluorescein) in the lowest density monolayers (<1000 cells/mm2). ZO-1 immunofluorescence distinctly localized to intercellular junctions in high density BCEC cultures but had more diffuse localization at lower densities. Transmission electron microscopy imaging revealed cells with thinner cross-sectional profiles and longer overlapping intercellular processes at low density relative to high density cultures. Low density human corneal endothelium lacked the diffuse ZO-1 distribution seen in BCECs. Our data supports the hypothesis that barrier integrity is the primary function disrupted in low density corneal endothelial monolayers and contradicts the idea of a linear decline in barrier function with decreasing cell density.

Mechanotransduction of Interstitial Fluid Stresses by Tumor Cells within 3D Collagen Scaffolds

Solid tumors are characterized by high interstitial fluid pressure, which serves as a barrier to drug delivery and an indicator for poor prognosis (Milosevic et al. Cancer Res. 2001). Elevated intratumoral fluid pressure drives fluid efflux from the tumor and high interstitial flow velocities at the tumor margin. Recently, we have demonstrated that interstitial flow (∼3 m/s) influences the direction of tumor cell migration through two competing mechanisms (Polacheck et al. PNAS 2011). At low cell density and active autocrine chemokine receptors, cells migrate in the direction of flow, while at high cell density or when autocrine chemokine receptors are blocked, cells migrate upstream, against the flow. Tumor cell migration in the direction of flow has been well characterized by the autologous chemotaxis model (Shields et al. Cancer Cell 2007), but the mechanism leading to upstream migration remains unclear. Here, we discuss the nature of fluid stresses imparted on cells embedded within a porous medium, and we introduce a microfluidic platform that we have developed to study the effect of these stresses on cell migration. We demonstrate that interstitial fluid stresses result in a transcellular gradient in matrix adhesion tension.We further show that these stresses result in a statistically significant accumulation of vinculin and actin on the upstream side of the cell, where matrix adhesion tension is maximized. Interstitial flow affects matrix remodeling and results in increased matrix degradation on the downstream side of the cell; however, we observe upstream vinculin and actin accumulation even when MMPs are blocked, further suggesting that the fluid stresses and not matrix remodeling are responsible for the observed vinculin and actin distribution.

Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of the human placental amnion

Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2′,7′-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation.

Perilymph Pharmacokinetics of Markers and Dexamethasone Applied and Sampled at the Lateral Semi-Circular Canal

Perilymph pharmacokinetics was investigated by a novel approach, in which solutions containing drug or marker were injected from a pipette sealed into the perilymphatic space of the lateral semi-circular canal (LSCC). The cochlear aqueduct provides the outlet for fluid flow so this procedure allows almost the entire perilymph to be exchanged. After wait times of up to 4 h the injection pipette was removed and multiple, sequential samples of perilymph were collected from the LSCC. Fluid efflux at this site results from cerebrospinal fluid (CSF) entry into the basal turn of scala tympani (ST) so the samples allow drug levels from different locations in the ear to be defined. This method allows the rate of elimination of substances from the inner ear to be determined more reliably than with other delivery methods in which drug may only be applied to part of the ear. Results were compared for the markers trimethylphenylammonium (TMPA) and fluorescein and for the drug dexamethasone (Dex). For each substance, the concentration in fluid samples showed a progressive decrease as the delay time between injection and sampling was increased. This is consistent with the elimination of substance from the ear with time. The decline with time was slowest for fluorescein, was fastest for Dex, with TMPA at an intermediate rate. Simulations of the experiments showed that elimination occurred more rapidly from scala tympani (ST) than from scala vestibuli (SV). Calculated elimination half-times from ST averaged 54.1, 24.5 and 22.5 min for fluorescein, TMPA and Dex respectively and from SV 1730, 229 and 111 min respectively. The elimination of Dex from ST occurred considerably faster than previously appreciated. These pharmacokinetic parameters provide an important foundation for understanding of drug treatments of the inner ear.

Investigation of the Preservation of the Fluid Seal Effect in the Repaired, Partially Resected, and Reconstructed Acetabular Labrum in a Cadaveric Hip Model

Background: Debate exists on whether hip labral tears should be surgically repaired, partially resected, or reconstructed. Furthermore, limited data exist regarding the fluid seal properties of hip labrum repair and/or reconstruction with iliotibial band autograft when compared with the labrum-intact condition. Hypothesis/Purpose: The purpose of this study was to investigate the fluid seal properties of hip labral repair and reconstruction techniques. We hypothesized that hip labral repair preserves the acetabular labral fluid seal greater than labral tear, partial-resection, and reconstruction conditions. Study Design: Controlled laboratory study. Methods: Six human cadaveric hemipelvises were dissected of all soft tissue, leaving the hip capsule intact. Fluid efflux was measured under 5 conditions using a custom fluid infusion device: (1) hip labrum intact, (2) labral tear, (3) labral repair with suture anchors, (4) partial labral resection, and (5) labral reconstruction using ipsilateral iliotibial band autograft. Joint fluid expression was measured as flow rate under 3 different pressure settings (2, 3, and 4 psi). Statistical differences between conditions were assessed using 2-way, repeated-measures analysis of variance. The Student-Newman-Keuls (SNK) multiple comparison test was used to determine differences between levels. Results: There was a significant increase in fluid efflux with a simulated labral tear (0.54 ± 0.3 mL/sec) when compared with the intact hip labrum condition (0.006 ± 0.008 mL/sec, P < .05). The labrum-repaired condition (0.21 ± 0.2 mL/sec) demonstrated significantly less fluid efflux when compared with the labral-tear condition. Hip labral repair significantly prevented greater fluid efflux when compared with partial labral resection (0.60 ± 0.4 mL/sec) and reconstruction with iliotibial band autograft (0.54 ± 0.3 mL/sec; P < .05). Labral repair did not preserve fluid efflux as effectively as in the labrum-intact condition (0.21 ± 0.2 > 0.006 ± 0.008 mL/sec; P < .05). There was no difference observed in fluid efflux between the labral reconstruction, tear, or resection conditions (P > .05). Conclusion: In this human cadaveric model, hip labral repair outperforms partial labral resection and reconstruction in preserving the joint fluid seal; however labral repair does not restore fluid seal characteristics as effectively as in the labrum-intact condition. Clinical Relevance: Further prospective studies are needed to determine whether hip labral repair outperforms partial labral resection and/or reconstruction in clinical practice.

Plasma kallikrein: Novel functions for an old protease

Editorial on Bird et al. Thromb Haemost 2012; 107: 1141-1150. Plasma kallikrein (PK) is a plasma protease that drives procoagulantand proinflammatory reactions via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. The structure and biochemistry of PK have been analysed in great detail; however, its in vivo functions are just beginning to emerge. In this issue of Thrombosis and Haemostasis, Bird et al. presents a mouse model with target ablation of the PK gene (1). Severe deficiency of PK abolishes experimental thrombosis in arterial and venous vascular beds. Despite the striking thromboprotective effects conferred by targeting the protease, PK deficiency has minor effect on the haemo static capacity of gene-deficient mice. This animal model provides a promising tool to study the pathogenetic role of the protease in models of thrombosis and inflammation with exciting therapeutic perspectives. In the original waterfall cascade model of plasma coagulation, fibrin formation may be initiated by two distinct pathways, triggered either by exposure of blood to tissue factor at an injured vessel wall or to blood-borne (intrinsic) factors. The intrinsic pathway of coagulation is initiated by factor (F)XII, in a reaction involving highmolecular-weight kininogen (HK) and PK, collectively referred to as the plasma contact system (2). Contact with negatively charged surfaces such as kaolin or polyphosphate activates FXII, and activated FXII (FXIIa) cleaves PK to generate active kallikrein, which in turn reciprocally activates FXII zymogen. FXIIa initiates fibrin formation through its principal substrate of the intrinsic coagulation pathway, FXI and also triggers liberation of the potent inflammatory mediator bradykinin (BK) by PK-mediated HK cleavage (3). Binding of BK to the kinin B2 receptor (B2R) causes an immediate nitric oxide-mediated vasodilation through an increase of cGMP in vascular smooth muscle cells, activates various pro-inflammatory signalling pathways that dilate vessels, induces chemotaxis of neutrophils and increases vascular permeability and fluid efflux (4). In a positive feedback loop, kallikrein also activates surfacebound FXII to FXIIa. Furthermore, PK has the capacity to convert pro-urokinase to urokinase, plasminogen to plasmin, and for triggering the renin-angiotensin system. Due to its BK-forming capacity, kallikrein has potent in vitro effects on leukocyte migration, stimulates monocytes, and induces neutrophil aggregation. Recent studies have suggested a role of PK for control of intracerebral haemorrhage in hyperglycaemia (5). In contrast, hereditary deficiencies in PK (Fletcher trait) and its substrate HK (Fitzgerald trait) were not associated with haemostatic abnormalities (6). The serpin C1 esterase inhibitor (C1INH) is the major plasma inhibitor of PK and controls enzymatic activity of the protease. Plasma prokallikrein (PPK) is the zymogen of the trypsin-like serine protease PK. PPK is expressed by hepatocytes in humans and mice and circulates in plasma at a concentration of 50 g/ml, predominantly in a noncovalent 1:1-complex with HK. PK is a paralog of FXI, and both genes have evolved from a common anchestor (7). The principal mechanism of PK is to cleave the high-molecular-weight precursor (HK) that liberates BK. Despite similar names, PK is not related to the large group of kininogenases called tissue-type (urinary or glandular) kallikreins. Analogous to FXI, PK is composed of four apple domains (Apple 1–4) that form the heavy chain of the molecule and a C-terminal protease domain (8). However, in contrast to human FXI, human PK is a monomer. PK migrates as a doublet with 86and 88-kDa on nonreducing SDS-polyacrylamide gels. The two bands represent differentially glycosylated proteins. Contact activation is the mechanism of contact-induced FXII zymogen activation. Contact activation provides the mechanistic basis for one of the most commonly used diagnostic clotting test the activated partial thromboplastin time (aPTT). The contact surface used in aPTT assays is typically a negatively charged non-physiological substance such as kaolin (aluminum silicate), celite (diatomaceous earth), or ellagic acid. The aPTT is widely used in clinical practice for preoperative screening and monitoring of anticoagulant therapy. Despite its important role in fibrin formation in the aPTT assay, PK-driven coagulation does not appear to have haemo static or other physiological functions in vivo. This notion is based on the observation that PK deficiency, a rare incidentally observed coagulation abnormality in humans, has not yet been associated with pathology, such as abnormal bleeding, despite causing a marked prolongation of the aPTT. Similar to PK-deficient individuals, humans lacking the other contact proteins, FXII or HK, do not have impaired haemo stasis. Recently, FXII-deficient (FXII-/-) mice were generated and phenotyped to study the function of coagulation FXII in vivo (9, 10). Similar to FXII-deficient humans, FXII-/mice have a normal haemostatic capacity as assessed by a tail-bleeding assay. Completely unexpected, intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in FXII-defi-

Abstract P350: Interstitial Volume Regulation in the Heart: Mechanistic Insights from ex Vivo Fluid Dynamics in Pig Myocardia

Developing and testing specific therapies for myocardial edema require understanding their mode and site of action. However, parsing determinants of interstitial volume/pressure relationships in vivo is difficult, particularly in the myocardium, where rhythmic contractions add to the confounding components of fluid-transfer driving pressures. Here, we describe a novel ex-vivo model system based on osmotic stress techniques and illustrate its application to analyses of local myocardial fluid dynamics that exclude systemic influences and systole/diastole compressive cycles. Freshly harvested ventricular explants were equilibrated in physiologic media at colloidosmotic pressures ranging from 3 to 219 mmHg, and fluid transfer in/out was measured gravimetrically as a function of time and pressure. The relationship between the change in explant volume and the imposed colloidosmotic pressure of the media was linear. The hydration potential , defined empirically as the pressure at which explant volume did not change, was calculated from the abscissa intercept at volume change = 0 , and the compliance from the slope of volume/pressure regression lines. Fluid-transfer rates and energies were derived from volume versus time trajectories measured at 4 and 37 °C. Hydration potential was 71.3 ± 10.6 and 23.0 ± 15.2 mmHg at 4 and 37°C (significant, P = 0.002), while compliance was 1.09 ± 0.3 and 1.22 ± 0.2 µl/g/ mmHg (not significant, P = 0.2, n = 5). Temperature-dependent differences between in/out flow rates were also significant, giving experimental activation energies of −5.9 ± 2.2 and 1.4 ± 0.7 kcal/mol for inflow and outflow, respectively. Results show that at physiologic temperatures, even without vascular hydrostatic pressure gradients and lymphatic drainage, the myocardial bias toward interstitial fluid efflux persists. These findings are consistent with local fluid-control mechanisms actuated by colloidosmotic and tensional forces rather than passive changes in flow resistance. This new approach allows quantitative evaluation of interstitial components of Starling’s forces and should help in mechanistic preclinical characterization of treatments to correct interstitial myocardial edema.

Generalization of experimental data on two-phase critical efflux through axisymmetric channels

The results of studying the characteristics of a two-phase flow being formed during adiabatic water efflux through axisymmetric cylindrical and Laval nozzle channels are presented. Experiments were carried out with saturated water in the range of initial pressures 0.6–4.0 MPa. The fluid efflux was performed into the air with atmospheric pressure. The generalizing relations for calculating thrust pulse and critical flowrate were obtained.

Oval capsulorhexis for phacoemulsification in posterior polar cataract with preexisting posterior capsule rupture

We describe use of an oval capsulorhexis rather than the conventional circular capsulorhexis for phacoemulsification in posterior polar cataract with preexisting posterior capsule rupture. An oval capsulorhexis minimizes the turbulence in the capsular bag by increasing the area available for efflux of fluid. It also enables end-to-end nuclear sculpting, removal of the nuclear fragment from the bag, intraocular lens (IOL) implantation, and vitrectomy without stretching the capsular bag. The smaller axis of the oval capsulorhexis facilitates optic capture of a sulcus-fixated IOL. The oval capsulorhexis can be used safely for phacoemulsification of all grades of nuclear sclerosis in posterior polar cataract with preexisting posterior capsule rupture.

Mast Cells Increase Vascular Permeability by Heparin-Initiated Bradykinin Formation In Vivo

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability invivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

Acute Subdural Haematoma in the Presence of an Intrathecal Catheter Placed for the Prevention of Post-Dural Puncture Headache

A 31-year-old term primigravid woman had an intrathecal catheter placed following a dural puncture during attempted epidural analgesia during labour. After 23 hours she developed sudden loss of consciousness and an urgent brain computed tomography scan demonstrated a large left hyper-acute subdural haematoma. Craniotomy revealed active bleeding from a ruptured left temporal bridging vein. She was extubated 12 hours after surgery and was discharged home 11 days later without neurological deficit. Although subdural haematoma is a recognised complication of dural puncture, it has not been reported in the presence of an intrathecal catheter. An intrathecal catheter may not always prevent cerebrospinal fluid efflux, so subdural haematoma remains a potential complication of inadvertent dural puncture.

Effect of acute postural variation on diabetic macular oedema

This study aimed to study the pathophysiology of diabetic macular oedema (DMO) by analysis of concomitant changes in macular volume (MV), mean arterial blood pressure (MABP), intraocular pressure (IOP), and retinal artery and vein diameters in response to acute postural changes in patients with DMO and healthy subjects. Thirteen patients with DMO (13 eyes) and five healthy subjects (five eyes) were examined after resting in a chair for 15 mins using optical coherence tomography to measure MV and fundus photography to assess retinal vessel diameters. The patients then lay down for 60 mins, during which they were examined repeatedly before they were reseated and examined again. Intraocular pressure was measured using pulse-air tonometry, arterial blood pressure by sphygomanometry and fluid columns using rulers and a spirit level. In healthy subjects, retinal artery (p = 0.02) and vein (p = 0.001) diameters decreased when subjects lay down, whereas MV remained stable. In patients with DMO, no orthostatic variation in retinal vessel diameters could be demonstrated, whereas MV had increased by 2.4 +/- 0.6% (mean +/- standard error of the mean; p = 0.006) 50 mins after assuming a recumbent position. In both healthy subjects and DMO patients, MABP decreased and IOP increased in a recumbent position, with no significant difference between the groups. The increase in MV that occurs in DMO when changing from a seated to a recumbent position is associated with a failure of retinal artery contraction, a response seen in healthy subjects that appears to counter-regulate the increase in ocular perfusion pressure caused by assuming a recumbent position.

Macromolecular Leak from Extrasplenic Lymphatics during Endotoxemia

The spleen has an important physiological role in maintaining blood volume; this study aimed to determine whether during pathophysiological circumstances, namely endotoxemia, the extrasplenic pathway is dysfunctional. We hypothesize that increased 'leakiness' of lymphatics in response to lipopolysaccharide (LPS) provides a route for loss of protein-rich fluid into third spaces and prevents the spleen from maintaining blood volume homeostasis. Male Wistar rats (200-280 g, n = 24) were anesthetized with thiopental (40-90 mg x kg(-1) x hr(-1), i.v.) to study the extrasplenic (vessels in mesentery adjoining the spleen) and ileal mesenteric microcirculation using fluorescently labeled albumin (66 KDa FITC-BSA, 0.02 g.100 g(-1), i.v.) with intravital microscopy. LPS (150 microg x kg(-1) x hr(-1) i.v.) induced constriction of rat extrasplenic venules (-14 +/- 2.4% from 40.4 +/- 7.8 microm, p < 0.05) and no change in arteriolar diameter (-4.6 +/- 4.7% from 32.6 +/- 4.3 microm). As the spleen is freely permeable to protein, a greater increase in venular versus arteriolar extrasplenic resistance increases intrasplenic capillary hydrostatic pressure, leading to fluid efflux into the lymphatics, draining the spleen. In agreement we report here increased extrasplenic venular resistance with LPS and lymphatic dilation to accommodate this fluid (13.5 +/- 6% from 18.5 +/- 4.8 microm, p < 0.05). However, the extrasplenic pathway then appeared to dysfunction, with macromolecular leak from extrasplenic venules (24.6 +/- 6.4%, p < 0.05) and lymphatics (12.1 +/- 3.4%, p < 0.05), indicated by increased interstitial FITC-BSA fluorescence. This was less than from ileal mesenteric venules (324 +/- 32%, p < 0.05). There was a concurrent decrease in mean arterial pressure (T(180): -15.1 +/- 6.9% from MAP of 130.3 +/- 8.8 mmHg at T(0), p < 0.05). Lymphatics are generally considered to demonstrate unidirectional and inward uptake of large molecules. However, during endotoxemia, we have demonstrated that extrasplenic lymphatics also allow the leakage of large protein molecules out into interstitial spaces. Fluid losses from extrasplenic lymphatics could therefore contribute to hypovolemia and hypotension associated with sepsis.

Use of PITA for Estimating Key Reservoir Parameters

Abstract Well testing is sometimes reduced to perforating the well in underbalanced conditions, capturing the pressure data and then analyzing it. The primary objective of a Perforation Inflow Test Analysis (PITA) is to estimate the initial reservoir pressure, permeability and skin for evaluating a future development strategy. However, special analytical procedures are required for analyzing the data. In this study, a systematic analytical procedure for estimating meaningful reservoir parameters from perforation inflow tests will be presented. Two major aspects of data interpretation will be discussed: diagnostic analysis and modelling. A straight-line approach is taken to analyze the early-time and late-time data. A special diagnostic technique is required for detecting and estimating positive or negative skins. A distinctive feature of PITA is that it does not require calculation of the inflow rates. Note that the same approach can be applied in overbalanced situations, when there is fluid efflux, rather than fluid influx. When employing straight-line analysis techniques, acceptable estimates of initial reservoir pressure, permeability and skin are only obtained if the test duration is sufficient to achieve radial flow. This is usually not a problem in high permeability reservoirs. However, in low permeability reservoirs, the test duration required to reach radial flow can be prohibitively long. In these cases, the test is often terminated during the transition from wellbore storage to radial flow. Consequently, acceptable estimates of initial reservoir pressure, permeability and skin can only be obtained by extending the analysis into modelling. A rigorous technique for estimating the radius of investigation during the test will be discussed. Introduction Well tests have been the primary and most reliable means of characterizing reservoirs for decades. However, there has been a growing trend over the last several years to search for alternatives that could yield the desired information in less time in a more environmentally-friendly manner, and at a cheaper cost than conventional well tests. The desired change has inevitably been towards tests of shorter duration(1–4). Although it is accepted that results from short tests with small radii of investigation may not be as reliable as those from conventional well tests, it is reasonable to accept that they could be of value in assisting with strategic decisions about field development when an increased margin of error can be tolerated. In offshore wells, in addition to the potentially exorbitant cost of testing (several millions of dollars), the drive towards green (shorter) tests is fuelled by environmental considerations, such as requirements for restricted flaring of hydrocarbons. In Alberta, and elsewhere in North America, the driving force towards inexpensive tests is the marginal economics of low deliverability wells. Either way, there is an increasing trend towards these green tests to replace conventional well tests. One such green test consists of simply allowing the well to flow into the closed wellbore after perforating when there is an underbalanced wellbore condition. As the fluid from the reservoir enters the wellbore (with a fixed volume), the wellbore pressure builds up.

Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.

A concept of a spacecraft with small initial mass placed in the near-earth orbit by a laser rocket engine

A method and results of calculating the laser radiation power values necessary to place an artificial earth satellite in orbit are presented; the values depend on the initial vehicle mass, velocity of the working fluid efflux from the laser rocket engine nozzle, velocity of vehicle motion, optimal values of thrust-to-weight ratio taking into account irreversible energy losses in the rocket engine jet. The possibility of creating a spacecraft with small initial mass is substantiated. A layout scheme of the propulsive system is proposed that makes it possible to divide total laser radiation power and to use atmospheric air as a working fluid.