A microdialysis method for the in situ investigation of the action of large peptide molecules in human skeletal muscle: detection of local metabolic effects of insulin

The possibility of using microdialysis catheters with a large pore size dialysis membrane (100 kDa) to investigate the action of macromolecules perfused into the interstitial space of peripheral tissues was explored. This was made possible by increasing the colloid osmotic pressure of the perfusate with 40 g/l of dextran-70 to prevent perfusate loss across the dialysis membranes. Microdialysis catheters were inserted into the quadriceps femoris muscle of 13 human subjects. With different perfusion flow rates (1. 33, 0.66, 0.33 and 0.16 microl/min) the recorded concentrations of glucose, lactate, and urea were in agreement with values previously obtained using a conventional membrane with a smaller pore size (20 kDa) [Rosdahl H, Hamrin K, Ungerstedt U, Henriksson. J Am J Physiol 1998;274:E936-45.]. When insulin was added to the perfusate, the concentration of glucose was significantly reduced, indicating that insulin diffuses across the dialysis membrane and has cellular effects that can be simultaneously recorded. The present findings are the first documentation on the use of microdialysis to study the local metabolic action of large peptide molecules in human tissues and may open new avenues for in-vivo metabolic research.     

Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production

Aquaporin (AQP) water channels are expressed in the eye at sites of aqueous fluid production and outflow: AQP1 and AQP4 in nonpigmented ciliary epithelium, and AQP1 in trabecular meshwork endothelium. Novel methods were developed to compare aqueous fluid dynamics in wild-type mice versus mice lacking AQP1 and/or AQP4. Aqueous fluid production was measured by in vivo confocal microscopy after transcorneal iontophoretic introduction of fluorescein. Intraocular pressure (IOP), outflow, and anterior chamber compliance were determined from pressure measurements in response to fluid infusions using micropipettes. Aqueous fluid volume and [Cl(-)] were assayed in samples withdrawn by micropipettes. In wild-type mice (CD1 genetic background, age 4-6 wk), IOP was 16.0 +/- 0.4 mmHg (SE), aqueous fluid volume 7.2 +/- 0.3 microl, fluid production 3.6 +/- 0.2 microl/h, fluid outflow 0.36 +/- 0.06 microl/h/mmHg, and compliance 0.036 +/- 0.006 microl/mmHg. IOP was significantly decreased by up to 1.8 mmHg (P < 0.002) and fluid production by up to 0.9 microl/h in age/litter-matched mice lacking AQP1 and/or AQP4 (outbred CD1 and inbred C57/bl6 genetic backgrounds). However, AQP deletion did not significantly affect outflow, [Cl(-)], volume, or compliance. These results provide evidence for the involvement of AQPs in intraocular pressure regulation by facilitating aqueous fluid secretion across the ciliary epithelium. AQP inhibition may thus provide a novel approach for the treatment of elevated IOP.     

Tracheal blood flow and luminal clearance of 99mTc-DTPA in sheep.

Tracheal blood flow and 99mTc-labeled diethylenetriamine pentaacetic acid (DTPA) clearance were measured in the sheep trachea in vivo. The tracheal arteries were isolated and perfused. An isolated segment of tracheal lumen was filled with Krebs-Henseleit solution containing 99mTc-DTPA, and radioactivity was measured in blood from a catheterized tracheal vein. Infusions at constant pressure of methacholine (n = 5), albuterol (n = 6), and histamine (n = 5) increased arterial inflow [+250 +/- 73.0, +74.2 +/- 22.9, +68.9 +/- 39.2% (SE), respectively] and venous outflow (+49.5 +/- 13.8, +11.6 +/- 4.5, +6.2 +/- 13.9%) but decreased 99mTc-DTPA output (-36.8 +/- 8.4, -20.4 +/- 6.2, -58.1 +/- 11.7%) and concentration (-53.9 +/- 10.1, -27.3 +/- 7.5, -49.3 +/- 14.4%). Phenylephrine (n = 9) decreased arterial inflow (-49.4 +/- 10.0%) and venous outflow (-4.1 +/- 5.9%) but increased 99mTc-DTPA output (+74.6 +/- 44.2%) and concentration (+94.4 +/- 56.6%). When the tracheal arteries were initially perfused at constant flow and the flow rate was then changed, 50% increases in flow (n = 5) increased perfusion pressure (+35.9 +/- 2.2%) and venous outflow (+10.5 +/- 3.8%) but decreased 99mTc-DTPA output (-24.4 +/- 7.8%) and concentration (-30.4 +/- 8.8%). Decreases in flow of 50% (n = 3) and 100% (n = 10) decreased perfusion pressure (-34.2 +/- 4.2, -80.1 +/- 3.5%, respectively) and venous outflow (-11.0 +/- 4.8, -29.7 +/- 7.2%) but increased 99mTc-DTPA output (+45.9 +/- 27.5, +167.4 +/- 70.4%) and concentration (+64.7 +/- 26.7, +305.7 +/- 110.2%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.     

High surface tension pulmonary edema induced by detergent aerosol

The effect of the detergent dioctyl sodium sulfosuccinate on pulmonary extravascular water volume (PEWV) was studied in adult anesthetized mongrel dogs. The detergent was dissolved as a 1% solution in a vehicle of equal volumes of 95% ethanol and normal saline and administered by ultrasonic nebulizer attached to the inspiratory tubing of a piston ventilator. Two hours following detergent aerosol PEWV measured gravimetrically was increased compared with either animals receiving no aerosol or those receiving an aerosol of vehicle alone. Loss of surfactant activity and increased alveolar surface tension were demonstrated by Wilhelmy balance studies of minced lung extracts, by a fall in static compliance, and by evidence of atelectasis and instability noted by gross observation and by in vivo microscopy. No significant changes in colloid oncotic pressure or pulmonary microvascular hydrostatic pressure were observed. These data suggest that pulmonary edema can be induced by increased alveolar surface tension and support the concept that one of the major roles of pulmonary surfactant is to prevent pulmonary edema.     

Reduction of myocardial infarction by postischemic administration of the calpain inhibitor A-705253 in comparison to the Na(+)/H(+) exchange inhibitor Cariporide in isolated perfused rabbit hearts

The calpain inhibitor A-705253 and the Na(+)/H(+)-exchange inhibitor Cariporide were studied in isolated perfused rabbit hearts subjected to 60 min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid solely or in combination at the beginning of reperfusion. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were performed. Myocardial infarct size and area at risk (transiently not perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 72.7+/-4.0% of the area at risk in untreated controls, but was significantly smaller in the presence of the inhibitors. The largest effect was seen with 10(-6) m A-705253, which reduced the infarcted area to 49.2+/-4.1% of the area at risk, corresponding to a reduction of 33.6%. Cariporide at 10(-6) m reduced the infarct size to the same extent. The combination of both inhibitors, however, did not further improve cardioprotection. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, heart rate, and in the release of lactate dehydrogenase and creatin kinase from heart muscle.     

A Physiological Approach to Perfusion-Flxation of Tissues with Formalin

A method of perfusion-fixation with formalin is here presented which is based upon well established physiological principles. It represents an attempt to preserve more accurately the structure and relationships of nervous tissue found in the living state. The essential points include the following: (1) Perfusion at an hydrostatic pressure equivalent to the mean arterial pressure of the animal to be fixed to maintain the normal patency of the vascular bed. For the guinea pig, it is approximately 70 mm. Hg; for the cat and monkey, it is approximately 120 mm. Hg. (2) The addition of a colloid to the perfusion fluid which will exert an osmotic pressure equal to the hydrostatic pressure in the capillaries and thus prevent edema. Gum acacia is used in this method. Its concentration depends upon the hydrostatic pressure to be used: for 70 mm. Hg, in the guinea pig it is 2.4%; for 122 mm. Hg, in the cat and the monkey, it is 5.6% gum acacia. (3) The addition of an electrolyte to the perfusion fluid to make it isotonic to the tissue fluid and thus prevent a disturbance in the distribution of water in the animal with resultant tissue distortion. NaCl (0.9%) is used in this method. (4) For further fixation or for preservation of the tissues, they are placed in a solution of 10% formalin which contains 0.9% NaCl to minimize swelling.     

Local effect of vanadate on interstitial glucose and lactate concentrations in human skeletal muscle

The aim of this study was to investigate the local effect of the insulin-mimetic agent vanadate on glucose metabolism in human skeletal muscle in vivo. Interstitial concentrations of glucose and lactate were determined by microdialysis at a low flow rate in the quadriceps femoris muscle of 18 men. In the same leg two microdialysis catheters were inserted. In one catheter, the perfusion medium was supplemented with sodium metavanadate (10-100 mM) after a basal period, the other catheter served as control. In the catheter perfused with metavanadate, the interstitial glucose concentration was decreased by 13-50% compared to the control catheter (p<0.05). The lactate concentration was higher in the 50 mM and 100 mM metavanadate catheters compared to control (39-89%, p<0.05). There was no difference between control and metavanadate catheters in urea concentrations. Five of the subjects were insulin-resistant and for them the results were similar, although the effect was somewhat smaller. The decreased interstitial glucose concentration, and the increased lactate concentration, in the vicinity of the microdialysis catheter most likely reflects an increased cellular glucose uptake. The present study thus indicates that vanadate mimics the effect of insulin in human skeletal muscle in vivo.     

Adenosine deaminase activity in rat intestine: assay with a microdialysis technique

Using microdialysis, we measured adenosine deaminase activity in rat intestine by detecting inosine, a breakdown product of adenosine. The dialysis probe consisted of a 3 x 0.22 mm dialysis fiber with a 50,000 mol wt cut off. When the probe was perfused at 1 microl/min in vitro, the average relative recovery rate of inosine was 22.1+/-0.9%). The dialysis probe was implanted in the intestinal mucosa and perfused with Tyrode solution containing adenosine at 1 microl/min. The dialysate samples were analyzed for inosine by high-performance liquid chromatography with ultraviolet (HPLC-UV) detection at 260 nm. When adenosine (100-1000 microM) was perfused, the level of inosine increased dose-dependently and was saturatable at about 1 mM adenosine. The ED50 of adenosine was 192.6 microM, with a maximum attainable inosine concentration of 59.7 microM. In the presence of aminoguanidine, a adenosine deaminase inhibitor (10 mM or 10 n mol/microl/min), the elevation of inosine was not observed. The dialysis technique makes it possible to measure adenosine deaminase activity in intestinal mucosa.     

Capacity and exchange functions of the kidney blood vessels at various leveles of venous outflow

In perfusion of the cat hemodynamically isolated kidney with a constant blood flow volume, responses of venous vessels to noradrenaline did not depend on the venous outflow pressure level and only involved a diminishing of the blood filling which distinguishes kidneys from other organs. The renal veins' capacity decreased in response to noradrenaline practically completely disappears in high values of the venous outflow pressure. The renal capillary filtration coefficient was shown to equal 0.21 +/- 0.11, whereas the effect of changes in renal vein's pressure on implementation of the microvessels' exchange function in determined by the shifts of capillary hydrostatic pressure.     

Measurement of Solute Transport Across the Blood–Brain Barrier in the Perfused Guinea Pig Brain: Method and Application to N-Methyl-α-Aminoisobutyric Acid

A technique for the vascular perfusion of the guinea pig head in vivo, suitable for measurements of blood-to-brain transport under controlled conditions of arterial inflow, has been developed. With a perfusion pressure ranging between 13 and 18 kPa and PCO2 in the arterial inflow of 5 and 5.5 kPa, cerebral blood flow, measured with [14C]butanol, was about 1 ml min-1 g-1 in the cerebral cortex, hippocampus, and caudate-putamen of the ipsilateral hemisphere; in the cerebellum and pontine white matter it was considerably less, and much higher perfusion pressures were required to establish equal blood flow throughout the whole brain. Regional water content, Na+/K+ ratio, ATP, energy charge potential, and lactate content of the ipsilateral side of perfused and nonperfused brain were not significantly different after 10 min perfusion. The D-[3H]mannitol space did not exceed 1% after 30 min of perfusion, indicating the integrity of the barrier. Over this period, EEG, ECG, and respiratory waveform remained normal. When [14C]N-methyl-alpha-aminoisobutyric acid (MeAIB), and D-[3H]mannitol were perfused together over periods extending to 30 min progressive uptakes of both solutes by the parietal cortex could be measured, and the unidirectional transfer constants estimated from multiple time-uptake data. The Kin for MeAIB (0.75 X 10(-3) ml min-1 g-1) was some three times that for mannitol. It is concluded that the technique provides a stable, well-controlled environment in the cerebral microvasculature of the ipsilateral perfused brain hemisphere suitable for examining the transport of slowly penetrating solutes into the brain.     

A flow-through amperometric sensor based on dialysis tubing and free enzyme reactors.

A generic flow-through amperometric microenzyme sensor is described, which is based on semi-permeable dialysis tubing carrying the sample to be analyzed. This tubing (300 microm OD) is led through a small cavity, containing the working and reference electrode. By filling this cavity with a few microl of an appropriate enzyme solution, an amperometric enzyme sensor results. As the dialysis tubing is impermeable for large molecular species such as enzymes, this approach does not require any immobilization chemistry, and as a consequence the enzyme is present in its natural free form. Based on this principle, amperometric sensors for lactate, glucose, and glutamate were formed by filling cavities, precision machined in Perspex, with buffered solutions containing respectively, lactate-, glucose-, and glutamate-oxidase. All sensors showed a large linear range (0-35 mM for glucose, 0-3 mM for lactate, and 0-5 mM for glutamate) covering the complete physiological range. The lower detection limit was in the order of 15-50 microM. Applicability in flow injection analysis systems is demonstrated.     

The application of fluidics technology for organ preservation

A prototype design of a portable, pulsatile, perfusion preservation device based on a novel application of fluidics technology was tested to evaluate its ability to oxygenate preservation solution and to examine the relationship between organ resistance, perfusion pressure, and perfusion flow characteristics. The effects of organ resistance on pulse rate, perfusion pressure, and perfusion flow were modeled. Interstitial PO2 in canine hearts stored at 4 degrees C for 12 hours in the fluidics device (n = 5) and in static hypothermic storage (n = 5) was also compared. Increasing outflow resistance did not have an effect on operating frequency of the fluidics actuator. Perfusion pressure rose as outflow resistance was increased, and the flow of preservation solution decreased proportionately. The PO2 of the preservation solution increased to 300 mm Hg in two hours and reached a plateau that exceeded 400 mm Hg within six hours. The aortic flow profile during pulsatile perfusion resembled a square wave function with a mean pulse duration of 0.30 +/- 0.05 seconds. Oxygen delivery by the fluidics perfusion device exceeded the oxygen requirements of the hypothermically preserved organs at all resistance levels. Initial interstitial PO2 in the hearts of both groups was greater than 150 mm Hg. In perfused hearts, PO2 declined 30% by the 12th hour, whereas complete depletion of oxygen was noted in the static storage group within six hours. The fluidics organ perfusion/transport apparatus weighs less than 18 kg, uses no electrical power, and can operate continuously for 10 to 12 hours expending 780 L of oxygen.     

Standing up to the challenge of standing: a siphon does not support cerebral blood flow in humans

Model studies have been advanced to suggest both that a siphon does and does not support cerebral blood flow in an upright position. If a siphon is established with the head raised, it would mean that internal jugular pressure reflects right atrium pressure minus the hydrostatic difference from the brain. This study measured spinal fluid pressure in the upright position, the pressure and the ultrasound-determined size of the internal jugular vein in the supine and sitting positions, and the internal jugular venous pressure during seated exercise. When the head was elevated approximately 25 cm above the level of the heart, internal jugular venous pressure decreased from 9.5 (SD 2.8) to 0.2 (SD 1.0) mmHg [n = 15; values are means (SD); P < 0.01]. Similarly, central venous pressure decreased from 6.2 (SD 1.8) to 0.6 (SD 2.6) mmHg (P < 0.05). No apparent lumen was detected in any of the 31 left or right internal veins imaged at 40 degrees head-up tilt, and submaximal (n = 7) and maximal exercise (n = 4) did not significantly affect internal jugular venous pressure. While seven subjects were sitting up, spinal fluid pressure at the lumbar level was 26 (SD 4) mmHg corresponding to 0.1 (SD 4.1) mmHg at the base of the brain. These results demonstrate that both for venous outflow from the brain and for spinal fluid, the prevailing pressure approaches zero at the base of the brain when humans are upright, which negates that a siphon supports cerebral blood flow.     

Venous waterfalls in coronary circulation

Several studies of flow through collapsible tubing deformed by external pressures have led to a concept known as the "vascular waterfall". One hallmark of this state is a positive zero-flow pressure intercept (Pe) in flow-pressure curves. This intercept is commonly observed in the coronary circulation, but in blood-perfused beating hearts a vascular waterfall is not the only putative cause. To restrict the possibilities, we have measured flow-pressure curves in excised non-beating rabbit hearts in which the coronary arteries were perfused in a non-pulsatile way with a newtonian fluid (Ringers solution) containing potent vasodilator drugs. Under these circumstances, vascular waterfalls are believed to be the only tenable explanation for Pe. In physical terms the waterfall is a region where the vessel is in a state of partial collapse with a stabilized intraluminal fluid pressure (Pw). It is argued that the most probable site of this collapse was the intramural veins just before they reached the epicardial surface. In accord with the waterfall hypothesis, Pe increased as the heart became more edematous, but flow-pressure curves also became flatter, implying multiple waterfalls with differing Pws, leading to complete collapse of some of the venous channels. The principal compressive force is believed to have been the interstitial fluid pressure as registered through a needle (Pn) implanted in the left ventricular wall, but a small additional force (Ps) was probably due to swelling of interstitial gels. A method is presented for estimating Ps and Pw. Unlike rubber tubing, blood vessels are both collapsible and porous. Apparently because of increased capillary filtration, Pn was found to increase linearly with the perfusion pressure. Thus, Pw was not the same at all points on the flow-pressure curve. This finding has interesting implications with respect to the concept of coronary resistance.     

Bioavailability of Ziconotide in brain: influx from blood, stability, and diffusion

Ziconotide is a selective peptide antagonist of the N-type calcium channel currently in clinical trials for analgesia. Ziconotide reached a maximal brain concentration of between 0.003 and 0.006% of the injected material per gram of tissue at 3-20 min after i.v. injection, and this decayed to below 0.001%/g after 2 h. The structurally distinct conopeptide SNX-185 (synthetic TVIA) was considerably more persistent in brain after i.v. administration, with 0.0035% of the injected material present at 2-4 h after i.v. injection, and 0.0015% present at 24 h. Similar results (i.e. greater persistence of SNX-185) were obtained when the peptides were perfused through in vivo dialysis probes implanted into the hippocampus. Image analysis and serial sectioning showed that diffusion of Ziconotide in the extracellular fluid around the dialysis probe was minimal, with the peptide located within 1 mm of the probe after 2 h. In vitro diffusion through cultured bovine brain microvessel endothelial cells (BBMEC) verified that a close structural analog of Ziconotide (SNX-194) passed through this blood-brain barrier (BBB) model as expected for peptides of similar physical properties (permeability coefficient of 6.5 x 10(-4) cm/g). Passage from blood to brain was also verified by in situ perfusion through the carotid artery. A statistically greater amount of radioactivity was found to cross the BBB after perfusion of radioiodinated Ziconotide compared to [14C]inulin. Capillary depletion experiments and HPLC analysis defined the brain location and stability.     

Reduction of myocardial infarction by calpain inhibitors A-705239 and A-705253 in isolated perfused rabbit hearts

Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9+/-2.3% of the area at risk in untreated controls ( n =12). The infarct size was significantly reduced in the presence of both calpain inhibitors. The best effect was achieved with 10 -8 M A-705253 ( n =8), which reduced ( p <0.001) the infarcted area to 49.3+/-3.9% of the area at risk, corresponding to an infarct reduction of 61.8%. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, and in the release of lactate dehydrogenase and creatine kinase from heart muscle.     

The effects of vitamin C and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart

The aim of this study was to assess the effects of vitamin C (ascorbic acid) on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by N(ω)-nitro-L-arginine monomethyl ester (L-NAME) in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40-120 cm H2O). Coronary flow, nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were determined. The experiments were performed during control conditions and in presence of vitamin C (100 μM) alone or vitamin C (100 μM) + L-NAME (30 μM). Administration of vitamin C induced only increase of nitrite levels, while vitamin C + L-NAME induced significant decrease of coronary flow above autoregulatory range, i.e. especially at higher coronary perfusion pressure (CPP) values, accompanied with similar dynamic in nitrite outflow. Vitamin C + L-NAME also induced significant decrease in TBARS production. The results of our study show no significant effects of vitamin C administration either on ROS levels or on coronary flow in isolated rat heart.     

Cardiac dynamics of the Langendorff perfused heart

The Langendorff perfused heart is studied in a closed system with (i) automatic regulations to maintain constancy of the perfusion column (Krebs-Henseleit + 0.5% albumin or 25-30% washed erythrocyte suspension), (ii) continuous recording of rate, coronary flow, and supravalvular aortic pressure. A microcomputer with software interface is used for storage treatment and on-line analysis of the recorded variables. In 38 preparations perfused with Krebs-Henseleit, minimal diastolic (61.2 +/- 2.8 mm Hg) is significantly below and peak systolic (98.7 +/- 3.6 mm Hg) significantly above perfusion pressure (80 mm Hg). Pressure difference between minimal diastolic and peak systolic (delta P) is 37.5 +/- 1.8 mm Hg. Increases in perfusion pressure will be associated with increases of coronary flow and delta P, which is also increased by isoprenaline administration. Oxygen consumption decreased by 76% when perfusion pressure was lowered from 80 to 60 mm Hg in hearts perfused with a 30% erythrocyte suspension. All of these experimental results were interpreted as indicating that delta P measured in this system resulted from an ejected volume (x acceleration) from the heart. The ejected volume corresponds to a valvular leak caused by the rigid nature of the system which is devoid of aortic compliance. delta P may be considered an index of left ventricular performance, an indication that the Langendorff preparation studied under the present conditions is a working heart. A 100-microliter volume constant infusion syringe for time administration of cardioactive drugs may be inserted at the base of the perfusion column to obtain dose-response effects.