Harmonic analysis of perfusion pumps.

The controversy over the use of nonpulsatile versus pulsatile pumps for maintenance of normal organ function during ex vivo perfusion has continued for many years, but resolution has been limited by lack of a congruent mathematical definition of pulsatility. We hypothesized that the waveform frequency and amplitude, as well as the underlying mean distending pressure are all key parameters controlling vascular function. Using discrete Fourier Analysis, our data demonstrate the complexity of the pulmonary arterial pressure waveform in vivo and the failure of commonly available perfusion pumps to mimic in vivo dynamics. In addition, our data show that the key harmonic signatures are intrinsic to the perfusion pumps, are similar for flow and pressure waveforms, and are unchanged by characteristics of the downstream perfusion circuit or perfusate viscosity.

Life sciences flight hardware development for the International Space Station.

During the construction phase of the International Space Station (ISS), early flight opportunities have been identified (including designated Utilization Flights, UF) on which early science experiments may be performed. The focus of NASA's and other agencies' biological studies on the early flight opportunities is cell and molecular biology; with UF-1 scheduled to fly in fall 2001, followed by flights 8A and UF-3. Specific hardware is being developed to verify design concepts, e.g., the Avian Development Facility for incubation of small eggs and the Biomass Production System for plant cultivation. Other hardware concepts will utilize those early research opportunities onboard the ISS, e.g., an Incubator for sample cultivation, the European Modular Cultivation System for research with small plant systems, an Insect Habitat for support of insect species. Following the first Utilization Flights, additional equipment will be transported to the ISS to expand research opportunities and capabilities, e.g., a Cell Culture Unit, the Advanced Animal Habitat for rodents, an Aquatic Facility to support small fish and aquatic specimens, a Plant Research Unit for plant cultivation, and a specialized Egg Incubator for developmental biology studies. Host systems (Figure 1A, B: see text), e.g., a 2.5 m Centrifuge Rotor (g-levels from 0.01-g to 2-g) for direct comparisons between g and selectable g levels, the Life Sciences Glovebox for contained manipulations, and Habitat Holding Racks (Figure 1B: see text) will provide electrical power, communication links, and cooling to the habitats. Habitats will provide food, water, light, air and waste management as well as humidity and temperature control for a variety of research organisms. Operators on Earth and the crew on the ISS will be able to send commands to the laboratory equipment to monitor and control the environmental and experimental parameters inside specific habitats. Common laboratory equipment such as microscopes, cryo freezers, radiation dosimeters, and mass measurement devices are also currently in design stages by NASA and the ISS international partners.

Enhanced cardiac thermal dilution measurement of cardiac output, volumes, regurgitation, valve effective diameters, ventricular power and efficiency -- Feasibility analysis using digital simulation.

Cardiac output is measured by the thermal dilution method which uses a quadruple lumen catheter, with a thermistor on the tip, through the right atrium, right ventricle and into the pulmonary artery. Cold saline is injected into the right atrium and the resulting pulmonary artery temperature profile is integrated. The same procedure performed with three thermistors and three pressure sensors located on the catheter to measure temperature and pressure in the atrium, ventricle and artery respectively will produce a set of temperature and pressure curves with shapes determined by injectate temperature, injectate volume, heart rate, systolic time interval, body temperature, cardiac output, volumes, flow rates and valve openings. A digital computer program has been developed to optimize the fit of a lumped parameter model to the thermodilution curves in order to determine heart rate, systolic time as a fraction of cardiac cycle, right atrial systolic and diastolic volumes, ventricular systolic and diastolic volumes, cardiac output, inflow valve forward and reverse flow rates and effective diameters, outflow valve forward and reverse flow rates and effective diameters, ventricular power and efficiency. The program has been tested over a range of operating conditions including noise in the temperature and pressure signals, randomly varying heart rate and cardiac cycle. All of the data for the tests were produced by a digital computer simulation of a pulsatile artificial heart. The results of these tests indicate that the enhanced thermal dilution analysis method is feasible.

Enhanced cardiac thermal dilution analysis for cardiac output, volumes, stroke volumes, and regurgitation rates--sensitivity analysis using digital simulation.

Cardiac output is measured by placing a double lumen catheter with a thermistor on the tip through the right ventricle into the pulmonary artery, injecting cold saline into the right atrium, and integrating the resulting pulmonary artery temperature profile. If a similar procedure is performed with thermistors located in the right atrium, right ventricle, and pulmonary artery, the resulting temperature curves are determined by the known or easily measured quantities: injectate temperature, injectate volume, heart rate, systolic time interval, body temperature, and time in the cardiac cycle at which injection begins and the unknown quantities: right atrial, right ventricular, pulmonary artery mean and stroke volumes, inflow and outflow valve regurgitation rates, and cardiac output. A digital computer program using a lumped parameter model has been developed to use these quantities to produce thermal dilution curves and optimize the fit of the model curves to the temperature curves from the thermal dilution measurement to determine the unknown quantities. The program is used to investigate the effects of measuring system time constant, heat transfer, and noise on the accuracy of these measurements. The results indicate that the method is practical.

Signaling pathways involved in thrombin-induced cell protection.

This study examined the signal transduction pathways involved in thrombin-induced neuroprotection and compares these results with those of a similar study of thrombin-induced neuronal death. In thrombin-induced protection of astrocytes from hypoglycemia, pretreatment of astrocytes with tyrosine or serine/threonine kinase inhibitors, cytochalasin D, or exoenzyme C3, a potent inhibitor of the small GTPase RhoA, attenuated thrombin-induced protection. These same inhibitors were previously shown to block thrombin-induced cell death, implying a similarity in the cell death and cell-protective pathways. Biochemical assays determined that thrombin increased available RhoA activity, although more slowly and to a lesser extent than occurs in thrombin-induced cell death. A clear difference in these pathways was revealed when a time course study of thrombin-induced cell death indicated that unlike thrombin-induced protection, cells must be exposed to thrombin for >16 h to irreversibly enter the cell death pathway. Addition of lower doses of thrombin every 24 h also induced cell death. These studies indicate that exposure of cells to micromolar concentrations of thrombin alone does not induce cell death, but the continued exposure to thrombin is required. Thus the cell death and protective pathways may share initial signaling proteins, but differences in the amplitude as well as the duration of the signal may result in different final pathways.

Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities.

Thrombin activity is a factor in acute CNS trauma and may contribute to such chronic neurodegenerative diseases as Alzheimer's disease. Thrombin is a multifunctional serine protease that catalyses the final steps in blood coagulation. However, increasing evidence indicates that thrombin also elicits a variety of cellular and inflammatory responses, including responses from neural cells. Most recently, high concentrations of thrombin were shown to cause cell death in both astrocyte and hippocampal neuron cultures. The purpose of this study was to determine the mechanisms underlying thrombin-induced cell death. Our data show that thrombin appears to cause apoptosis as evidenced by cleavage of DNA into oligonucleosomal-sized fragments, fragmentation of nuclei, and prevention of death by inhibition of protein synthesis. Synthetic peptides that directly activate the thrombin receptor also induced apoptosis, indicating that thrombin-induced cell death occurred via activation of the thrombin receptor. The signal transduction cascade involves tyrosine and serine/threonine kinases and an intact actin cytoskeleton. Additional study revealed the involvement of the small GTP-binding protein RhoA. Thrombin induced RhoA activity in both astrocytes and hippocampal neurons, and inhibition of RhoA activity with exoenzyme C3 attenuated cell death, indicating that thrombin activation of RhoA was necessary for thrombin-induced cell death. Tyrosine kinase inhibitors blocked thrombin induction of RhoA, indicating that tyrosine kinase activity was required upstream of RhoA. These data suggest a sequential linkage of cellular events from which we propose a model for the second messenger cascade induced by thrombin in neural cells that can lead to apoptosis.

Thrombin receptor activation induces secretion and nonamyloidogenic processing of amyloid beta-protein precursor.

The amyloid beta-protein (A beta) and protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) are major constituents of senile plaques and cerebrovascular deposits in individuals with Alzheimer's disease and related disorders. It has been suggested that the coagulation protease thrombin may process A beta PP in a manner leading to the formation of A beta. Here we investigated the effects of thrombin on the secretion and processing of PN-2/A beta PP and the production of A beta in a cellular system. Incubation of glioblastoma cells with thrombin (1-5 nM) resulted in the accumulation of abnormally processed, carboxyl-terminal-truncated forms of secreted PN-2/A beta PP (approximately 85 kDa) in the culture medium. Higher concentrations of thrombin (> 10 nM) also increased the levels of secreted PN-2/A beta PP in cultured untransfected glioblastoma cells and glioblastoma cells that were stably transfected to overproduce the 695 isoform of A beta PP. Increased secretion of PN-2/A beta PP required the proteolytic activity of thrombin, was induced by activation of the thrombin receptor by agonist peptides, and required activation of protein kinase C. Incubation of the untransfected and transfected glioblastoma cells with thrombin led to decreased levels of soluble A beta in the culture medium consistent with previously suggested mechanisms regarding the secretion of PN-2/A beta PP. Although the present studies suggest that thrombin does not directly contribute to A beta formation, its proteolysis of secreted PN-2/A beta PP may disrupt regions near the carboxyl terminus of the secreted proteins that account for their neuroprotective and cell adhesive properties.

Regulation of protease nexin-1 target protease specificity by collagen type IV.

Recent studies have shown that serine protease inhibitors can be regulated in their activity, specificity, and location by glycoprotein or extracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a member of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly to and is regulated by the ECM. This interaction accelerates the inhibition of thrombin by PN-1 and blocks urokinase and plasmin inhibition by PN-1. Previous work showed that heparan sulfate proteoglycan is largely responsible for the acceleration of thrombin inhibition by PN-1. Our current studies were directed at identifying ECM component(s) that decreased the ability of PN-1 to inhibit urokinase and plasmin. These studies showed that collagen type IV decreased the formation of SDS-stable complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order rate constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate constant for inhibition of thrombin by PN-1 was unaffected by addition of collagen type IV. Other ECM components (collagen type I, vitronectin, fibronectin, and heat-denatured collagen type IV) did not affect complex formation or the rate of inhibition of proteases by PN-1, indicating that these effects were specific to collagen type IV. Binding of PN-1 to immobilized collagen type IV was demonstrated using an enzyme-linked immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was approximately 15 nM. Collagen type IV was also copurified with PN-1 from fibroblast-conditioned medium. These results demonstrate a novel regulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the inhibition of its target protease.

One-dimensional computer analysis of oscillatory flow in rigid tubes.

The dynamic characteristics of catheter-transducer systems using rigid tubes with compliance lumped in the transducer and oscillatory flow of fluid in rigid tubes were analyzed. A digital computer model based on one dimensional laminar oscillatory flow was developed and verified by exact solution of the Navier-Stokes Equation. Experimental results indicated that the damping ratio and resistance is much higher at higher frequencies of oscillation than predicted by the one dimensional model. An empirical correction factor was developed and incorporated into the computer model to correct the model to the experimental data. Amplitude of oscillation was found to have no effect on damping ratio so it was concluded that the increased damping ratio and resistance at higher frequencies was not due to turbulence but to two dimensional flow effects. Graphs and equations were developed to calculate damping ratio and undamped natural frequency of a catheter-transducer system from system parameters. Graphs and equations were also developed to calculate resistance and inertance for oscillatory flow in rigid tubes from system parameters and frequency of oscillation.

Design of a hydraulic analog of the circulatory system for evaluating artificial hearts.

A major problem in improving artificial heart designs is the absence of methods for accurate in vitro testing of artificial heart systems. A mock circulatory system has been constructed which hydraulically simulates the systemic and pulmonary circulations of the normal human. The device is constructed of 1/2 in. acrylic sheet and has overall dimensions of 24 in. wide, 16 in. tall, and 8 in. deep. The artificial heart to be tested is attached to the front of the device, and pumps fluid from the systemic venous chamber into the pulmonary arterial chamber and from the pulmonary venous chamber into the systemic arterial chamber. Each of the four chambers is hermetically sealed. The compliance of each chamber is determined by the volume of air trapped above the fluid in that chamber. The pulmonary and systemic resistances are set automatically by bellows-operated valves to simulate the barroreceptor response in the systemic arteries and the passive pulmonary resistance response in the pulmonary arteries. Cardiac output is measured by a turbine flowmeter in the systemic circulation. Results using the Kwan-Gett artificial heart show a good comparison between the mock circulatory system response and the calf response.