Continuous measurement of oxygen consumption in small laboratory animals]

A technique for measuring the oxygen consumption of small laboratory animals suitable for pharmacological and toxicological studies is described. The oxygen consumption of the animal in the respiratory chamber is compensated by means of a photoelectrically operating servo mechanism from an oxygen reservoir. The oxygen supplied is directly recorded via a transducer by an X-Y recorder. The carbon dioxide exhaled by the animal is entirely absorbed. Composition and pressure of air in the respiratory chamber correspond to the physiological medium. The usefulness of this technique is demonstrated by investigations on the effects of epinephrine and morphine on the oxygen consumption of rats.     

An improved method of bronchoalveolar lavage of lungs of small laboratory animals: short report

This report describes a semi-automatic method for standardized bronchoalveolar lavage of small laboratory animals. In essence the method is constructed from a dispenser and a pressure chamber.     

Exchange of oxygen across the epicardial surface distorts estimates of myocardial oxygen consumption

The rate of oxygen consumption of isolated, Langendorff-circulated, saline-perfused hearts of guinea pigs, rats, and rabbits was measured using the classical Fick Principle method. The heart was suspended in a glass chamber the oxygen partial pressure, PO2, of which could be varied. The measured rate of oxygen consumption was found to vary inversely with the ambient (heart chamber) PO2. This result prevailed whether the chamber was filled with air, saline, or oil, and whether the pericardium was present or the heart was wrapped in Saran. The effect varied inversely with heart size both within and across species. It is concluded that the epicardial surface is permeable to oxygen which will diffuse either into or out of the heart as the PO2 gradient dictates. In either case the classically measured rate of oxygen consumption will be in error. The error can be large in studies of cardiac basal metabolism. A simple model is developed to describe the observed rate of oxygen consumption as classically measured. The measured rate is partitioned into two components: the true rate of oxygen consumption of the heart, and the rate of loss of oxygen by diffusive exchange across the epicardial surface. The latter component is proportional to the gradient of oxygen partial pressure from myocardium to environment and to the diffusive oxygen conductance of myocardial tissue. Application of the model allows the true rate of oxygen consumption of the heart to be recovered from measured values which may be considerably in error.     

Development of an automated microbial sensor system

An automated whole cell biosensor system was developed by integration of immobilized microbial cells in a flow-through system with screen-printed flow-through electrodes as detectors. The detectors used were thick-film Pt-electrodes in a 3-electrode configuration constructed as sandwich flow-through cells with a volume of about 36 microliters polarized at -900 mV. The measuring principle was the determination of oxygen consumption due to the microbial metabolism. Fructose was used as model analyte. The microorganisms were immobilized on cellulose-acetate membranes and integrated into a newly created reaction chamber (membrane reactor). The microbial cells used were Rhodococcus erythropolis and Issatchenkia orientalis known to be suitable for the determination of biological oxygen demand.     

Design, operation, and modeling of a membrane photobioreactor to study the growth of the Cyanobacterium Arthrospira platensis in space conditions

A membrane photobioreactor was designed, implemented and used to grow the cyanobacterium Arthrospira platensis PCC 8005 in batch mode. Growth was followed directly by monitoring optical density and indirectly by measuring pressure increase due to the oxygen produced and separated from the liquid phase by diffusion through a hydrophobic membrane, and pH increase due to carbon consumption. When the pressure attained an upper limit, valves opened automatically, and the oxygen in the gas chamber was flushed out with nitrogen. As expected, two growth phases were observed, a short exponential phase followed by a linear phase, indicating limitation by light transfer. Growth rate during the second phase was measured easily and accurately, and consistency of optical density, pressure and pH data values was checked using a model of the system. Pressure measurement was found best suited to monitoring and measuring growth rate in space in terms of accuracy, precision and reliability.     

Water fleas (Daphnia magna) provide a separate ventilatory mechanism for their brood

The planktonic filter feeder Daphnia magna depends on a steady oxygen supply by convection. In the ventral carapace chamber, this convection is established by the feeding current which is generated by the movement of the thoracic limbs. The present study revealed that this movement can cause an additional flow of medium which passes through the brood chamber of the animal. To visualise this current, ink or fluorescent microspheres were released by a microcapillary near the posterior opening of the brood chamber. The tracks of these probes were monitored by video microscopy. Digital motion analysis was used for the determination of flow velocity and flow rate. Ambient medium entered the brood chamber at the abdomen of the animal and moved then to the anterior end of the brood chamber before entering the ventral carapace chamber. Two horizontal lamellae, which are attached at both sides of the trunk and project laterally to contact the carapace walls, almost completely separate the dorsal brood chamber from the ventral carapace chamber. Water can only pass these barriers through small depressions in these lamellae at the level of the 3rd and 4th appendages. Female daphnids with embryos at late developmental stages showed more rapid water currents in the brood chamber than those with younger embryos. Moreover, animals showed higher flow rates when exposed to hypoxic conditions. As the oxygen uptake rate of older embryos is approximately three times higher than that of younger embryos, the enhanced brood chamber current could improve the oxygen availability for both the mother and its brood under conditions of reduced oxygen availability.List of symbols:a specific oxygen consumption rate (nmol mm-³ h-¹), K Krogh constant for oxygen diffusion (nmol mm-¹ h-¹ kPa-¹, P_c critical oxygen partial pressure (kPa), P o₂ oxygen partial pressure (kPa), r radius (mm), s distance (mm), t time (ms)     

Role of endothelial nitric oxide in microvascular oxygen delivery and consumption

Nitric oxide (NO) is an important signaling molecule modulating diverse processes such as vasodilation, neurotransmission, long-term potentiation, and immune responses. The endothelium contributes a significant fraction of NO from endothelial NO synthase (eNOS). The objective of this work was to analyze the role of eNOS in the modulation of oxygen supply to the tissues and in adaptation to maintain oxygenation uncompromised. Oxygen delivery and consumption were measured in the microcirculation of homozygous mutant endothelial nitric oxide synthase-deficient (eNOS(-/-)) and wild-type mice. Animals were implanted with a dorsal window chamber, allowing us to assess the intact microvascular system. Hemodynamics and oxygen tension were assessed in the microcirculation of conscious animals. The eNOS(-/-) mice had significantly higher blood pressure and lower heart rate (146 +/- 8 mm Hg, 401 +/- 17 bpm) than wild type (127 +/- 6 mm Hg, 428 +/- 20 bpm). Microvascular hemodynamic parameters were not significantly different between groups. The eNOS(-/-) animals delivered less oxygen to the microcirculation and released more oxygen to the tissue; both differences were statistically significant compared to wild type. The arteriolar vessel wall oxygen gradient, a measure of vascular smooth muscle cells and endothelial cell wall oxygen consumption, was significantly lower for eNOS(-/-) than for wild type, suggesting that the inhibition of eNOS is an antianoxia (oxygen sparing) mechanism. Finally, the findings of the study support the argument that NO availability limits oxygen consumption by the tissue.     

Effects of hyperbaric pressure on oxygen concentration in the swim bladder of Poecilia latipinna.

Poecilia latipinna was subjected to varying hydrostatic pressures in the laboratory which demonstrated the following points. The hyperbaric chamber is a suitable apparatus for testing gas gland secretion in this teleostean fish. Oxygen concentration within the swim bladder increased with increasing hydrostatic pressure. Pressures encountered by fishes at various levels of the water column and their effects on oxygen concentration in the swim bladder may be simulated in the laboratory.     

A high-precision automatic closed-circuit respirometer for small animals

An automatic apparatus for the continuous measurement of O2 consumption of small laboratory animals is described. By use of a high-sensitivity pressure transducer with associated circuitry together with a peristaltic O2 delivery system, the closed respirometer chamber is maintained at atmospheric pressure +/- 0.5 mmH2O. O2 delivery is measured to within 0.25 ml by recording rotations of the peristaltic pump, following calibration by the withdrawal of a preset volume of air from the chamber. Static trials (with the chamber empty) indicate a high degree of reproducibility of data with the chamber pressure remaining at atmospheric pressure +/- 0.5 mmH2O as a result of the proportional, as opposed to fixed-volume, delivery of O2. Trials with mice and rats have likewise produced data with a high degree of reproducibility.     

New technology for poison delivery

A long-life poison bait dispenser, consisting of a tree-mounted platform that dispenses a highly attractive liquid bait only when triggered by actions characteristic of a possum (Trichosurus vulpecula), was developed. The liquid bait formulation prevents deterioration due to the action of oxygen, moisture, bacteria and insects. The prototype is designed to dispense 100 lethal doses of poison, and is expected to last more than five years in the field without attention. The equipment is designed to avoid fouling by algae, debris or nesting insects. The selectivity for possums provides a low risk of exposing non-target mammals, birds and insects to poison. A reset delay reduces the likelihood of over- consumption of bait.     

Behavior and physiology of mice in a closely controlled environment

Activity and oxygen consumption studies on mice were performed in a very closely controlled environmental chamber. Some results are presented from these experiments in regard to the animals' possible dependence to age, sex, barometric pressure, light cycle and similar environmental parameters.     

Reduced oxygen diffusion across the shell of Gray gull (Larus modestus) eggs

Gray gulls, Larus modestus, nest 1500 m above sea level in northern Chile's Atacama Desert, one of the driest in the world. Their eggshell gas permeability, one third of that found in other Larus species, is an adaptation that reduces water loss, but at the expense of oxygen diffusion into the air cell with resultant hypoxia and reduced metabolic rate. This contrasts with characteristics found in birds nesting at very high altitudes where oxygen diffusion across the egg shell is maximized at the expense of water conservation. The oxygen consumption (MO2) of Larus modestus is 66% that of Larus argentatus; the oxygen conductance (GO2) is equivalent to 48% of that obtained in 5 other bird species. The oxygen partial pressure (PAO2) in the air chamber of Larus modestus (84 Torr) is lower than that of 10 other bird species whose average (PAO2) is 106 Torr. The CO2 partial pressure (PACO2) in the air chamber of Larus modestus is 68 Torr, a higher value than that found in 9 other bird species whose average (PACO2) is 39 Torr.     

Der Respirationsfermentor – ein Labormeßsystem zur Charakterisierung aerober mikrobiologischer Reaktionen

By use of a laboratory fermenter with a gastight circle aeration system all interesting values connected with the respiration of microorganisms can be determined. The advantage of the respiration fermenter is the simple control of the oxygen partial pressure with high accuracy. The aeration is carried out by inert gas-oxygen-mixtures. This system was used to avoid errors in oxygen-concentration measurement and regulation by oxygen sensitive sensors in fermentation fluids. For each selected oxygen partial pressure in the course of fermentation the following date in connection with all desired analytical values can be determined by manual or automatical means. Total oxygen consumption, momentary oxygen consumption rate, kinetics of oxygen consumption rate, kinetics of respiratory quotient. Several opportunities of measurement are presented and examples for application in various microbial systems are given.     

A lab-built respirometer for plant and animal cell culture

A very simple off-line respirometer was developed to measure oxygen consumption rates of low respiring and shear-sensitive cell suspensions. The respirometer is composed of a 10 mL glass syringe in which the plunger was substituted with a polarographic dissolved oxygen probe. Mechanical agitation is provided by means of a magnetic stirring bar inside the measuring chamber and a stir plate placed below the respirometer. Abiotic oxygen fluxes occurring in the measurement chamber such as oxygen diffusion and probe oxygen consumption were investigated. The apparent oxygen uptake rate was then corrected for abiotic oxygen fluxes, leading to accurate measurements of respiration rates ranging from 0.5 to 25.0 mM x h(-1). Additionally, the effect of the stirring bar shape and of the test length on the integrity of plant (Eschschzoltzia californica) and animal (NS0) cells was evaluated. Animal cells showed a higher resistance to mechanical stirring inside the respirometer compared to plant cells (0% of broken cells and 78.1% respectively for a polygonal stirring bar and a 15 min test). For plant cells, cell damage inside the measurement chamber was reduced by optimizing the stirring bar shape and reducing the test length to 5 min or less. This very simple design was shown to provide reliable and low-cost quantification of the oxygen uptake rate of plant and animal cells and can be use even for more demanding measurements such as oxygen affinity studies.     

Impact of clay particles on the cutaneous exchange of oxygen across the chorion of Atlantic salmon eggs

Rates of oxygen consumption for Atlantic salmon Salmo salar embryos approaching hatching were determined. Values were recorded using a 'closed system' experimental set‐up. A magnetic stirrer was used to ensure that zones of oxygen depletion did not develop in the micro‐environment surrounding the respiring eggs. Recorded values of oxygen consumption ranged from 0·0024 to 0·0038 mg O₂ egg⁻¹ h⁻¹, with a mean consumption rate of 0·0032 mg O₂ egg⁻¹ h⁻¹. The values of oxygen consumption were similar to those reported in other studies using a closed system experimental set‐up, however, they were lower than those reported in a study adopting a flow‐through system. The introduction of clay‐sized sediment to the incubation chamber created a thin film (<1 mm) of sediment on the egg surface, and resulted in reduced rates of oxygen consumption. The additional 0·3 g of clay sediment reduced oxygen consumption by an average of 41% and the addition of a further 0·2 g of clay sediment reduced consumption by an average of 98%. Two explanations for the recorded reduction in consumption were proposed: (i) the creation of a low permeability seal around the eggs restricted the availability of oxygen to the incubating embryos and (ii) the clay‐sized fine sediment physically blocked the micro‐pore canals in the egg membrane, thereby restricting oxygen uptake.     

Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.     

Dinitrocresol and phosphate stimulation of the oxygen consumption of a cell-free oxidative system obtained from sea urchin eggs

1. A cell-free system capable of using oxygen with oxalacetate as substrate has been prepared from both unfertilized and fertilized sea urchin eggs. The oxygen uptake by this system is about twice that of an equivalent quantity of intact unfertilized eggs and half that of an equivalent quantity of intact fertilized eggs. 2. The oxygen consumption of this cell-free oxidative system can be stimulated by addition of suitable concentrations of 4,6-dinitro-o-cresol or by inorganic phosphate. This confirms, with a cell-free system obtained from sea urchin eggs, the observations of Loomis and Lipmann regarding stimulation of oxygen consumption by a system obtained from rabbit kidney. 3. A preliminary but unsuccessful attempt has been made to determine the conditions under which cell-free, aerobic, phosphorylating systems may be obtained from either unfertilized or fertilized sea urchin eggs.     

Theory and design of disposable clinical blood viscometer

A disposable clinical whole blood viscometer which can produce viscosity measures over a wide range of shear rates in a single rapid determination has been developed and is currently under test. The design is based upon the time varying flow of blood through a capillary. The flow is driven by the pressure in a fixed volume air chamber and transmitted to the sample through a compliant membrane. The time varying pressure in the air chamber is measured by a suitable transducer. The instantaneous shear stress of the blood in the capillary is proportional to the air pressure, while the instantaneous shear rate is proportional to the pressure-time derivative. Proper design ensures that the system operates as a first order dynamic system with flow resistance entirely determined by the nonlinear sample viscosity. By constructing the air chamber in two parts coupled by a quick disconnect fitting the design can allow for the blood-containing part of the instrument to be discarded, eliminating handling and cleaning of blood contacted components. The entire determination is completed in less than a minute, so that anticoagulants are not necessary. Tests on a prototype show that the instrument gives results in excellent agreement with those obtained on a cone-plate rheogoniometer.     

Respiration,temperature regulation and energetics of thermogenic inflorescences of the dragon lily Dracunculus vulgaris (Araceae)

Dracunculus vulgaris is a protogynous arum lily with thermogenic inflorescences consisting of male and female florets on a spadix within a floral chamber. Above the chamber, an odour-producing appendix and a carrion-coloured spathe attract flying insects. The inflorescence shows a triphasic warming pattern. The floral chamber warms weakly on the first night as the spathe opens. Then the appendix produces a large amount of heat and a powerful scent during the first day. As the appendix cools on the second night, scent production ceases and the floral chamber rewarms. Warming ceases when the pollen is shed on the second day. The heating pattern is associated with attraction of pollinating insects by the appendix on the first day, entrapment in the warm chamber at night and release after pollen shedding. The temperature in the floral chamber is regulated at around 18 °C during the second night. The oxygen consumption rate of the florets is inversely related to the ambient temperature as in other thermoregulatory flowers. Conversely, the oxygen consumption rate of the appendix is directly related to the ambient temperature, indicating that it does not thermoregulate. Thus, temperature regulation is not associated with scent production, but with some activity inside the floral chamber.     

Aerobic metabolism of American alligators, Alligator mississippiensis, under standard conditions and during voluntary activity

We measured the rate of consumption of oxygen by alligators in a dry metabolic chamber and in a tank of water where they were free to dive and surface at will at 10-35 degrees C, a range spanning most of the body temperatures experienced by alligators in nature. Neither the standard metabolic rate nor the rate of oxygen consumption during one hour of sustained, voluntary activity varied with body mass, month of the year, duration of fasting before measurement, or experimental condition (terrestrial vs aquatic). Voluntary diving is not accompanied by any reduction in standard metabolic rate; these results and those of others suggest that the "diving reflex" of alligators is probably employed only in emergencies. Spontaneous activity for one hour is accompanied by a 1.9-4.4 fold rise in oxygen consumption; this factorial increase is less than that for other reptiles induced to maximal activity for brief intervals. Both standard and active oxygen consumption rise significantly with body temperature.