Increase In Perfusion Rate Research Articles

Bioreactor operation for transgenic Nicotiana tabacum cell cultures and continuous production of recombinant human granulocyte-macrophage colony-stimulating factor by perfusion culture

The production of hGM-CSF was investigated in both a flask and a 5-l bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g l −1 and 6.5 μg l −1, respectively, those in the bioreactor were 15.6 g l −1 and 7.6 μg l −1. No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day −1). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 μg accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day −1, respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g l −1 of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day −1, respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes.

A monoclonal antibody to the fibrinogen γ-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles

We previously reported that hamster monoclonal antibody 7E9, which reacts with the C-terminus of the gamma-chain of mouse fibrinogen, inhibits factor (F)XIIIa-mediated cross-linking, platelet adhesion to fibrinogen, and platelet-mediated clot retraction; in addition, it facilitates thrombolysis. To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab')2, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab')2, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25-fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan delta; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab')2, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa-mediated cross-linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.

Norepinephrine Inhibits the Pelvic Pressure Increase in Response to Flow Perfusion

We evaluated the effects of norepinephrine on transport pressures in the normal upper urinary tract of the pig during increasing perfusion rates. Anesthetized Danish landrace Yorkshire pigs weighing 38 to 40 kg were studied. Transparenchymally 2, 6Fr catheters were introduced into the left renal pelvis for pressure measurements and perfusion, respectively. An ultrasonic flow probe was inserted around the left renal artery to record blood flow. A 10Fr catheter was placed transurethrally for bladder drainage and the bladder was maintained empty during the entire study. In the 5 group 1 pigs the pelvic pressure increase was examined at increasing perfusion rates of the renal pelvis (2, 4, 6, 8, 10 and 15 ml per minute) in response to endoluminal administration of increasing concentrations of norepinephrine (0, 5, 50 and 100 microg/ml) in saline. In the 5 group 2 pigs the pressure flow study was also done 4 times per animal using isotonic saline. Endoluminal norepinephrine had a dose dependent effect on the pressure flow relationship. Perfusion with 5 and 50 microg/ml norepinephrine caused a delayed increase and a decrease in pelvic pressure in response to increasing flow rates, whereas perfusion with 100 microg/ml norepinephrine significantly inhibited and almost eliminated the pressure increase at all perfusion rates compared with saline perfusion. Importantly there were no changes in blood pressure, the heart rate or renal arterial blood flow. In group 2 perfusion with isotonic saline resulted in the same pressure response to increasing flow rates each time. Endoluminal administration of norepinephrine caused a dose dependent inhibition on the pressure phases of the pressure flow relationship of the upper urinary tract in pigs. No systemic changes were observed. These observations may provide a useful adjuvant treatment strategy for upper urinary tract stone treatment and endoscopy.

Endothelin receptor blockade as a therapeutic strategy in ameliorating postischemic damage to the liver microcirculation.

Ischemia leads to profound endothelin- (ET) related constriction of hepatic microvessels, causing disturbances in blood and oxygen supply. The aim of the study was to modulate hepatic microvascular diameters by blocking ET receptors to find the optimal therapeutic vessel width for reduction of ischemia-reperfusion injury. In an in vivo model (84 female Wistar rats, 250-300 g) with portal decompression by means of a splenocaval shunt, normothermic hepatic ischemia was induced for 30 min by crossclamping of the hepatoduodenal ligament. The ET receptor antagonist (ERA) bosentan was administered before induction of ischemia in different dosages [0.1, 1.0 and 10.0 mg/kg body weight (BW) i.v. and 10 mg/kg BW intraportally (i.p.)]. The effect on microcirculation was assessed by in vivo microscopy and influence on hepatocellular function by measurement of aspartate aminotransferase (AST) levels. Sinusoidal diameters were reduced as a result of ischemia to 76.3 +/- 7.4% compared with values received from sham-operated animals. After application of 0.1 mg/kg of bosentan, sinusoids remained constricted (89.7 +/- 9.9%, AST 255.0 +/- 12.8 U/l). Blocking ET receptors with 1 mg/kg bosentan avoided sinusoidal constriction (99.0 +/- 8.8%, p < 0.05) and led to the most effective reduction of AST level peak after 6 h of reperfusion (244.0 +/- 34.2 U/l vs 422.9 +/- 163.3 U/l in untreated ischemia). Bosentan (10 mg/kg i.v.) caused an increase in sinusoidal diameter to 109.1 +/- 6.4% (AST 311.7 +/- 33.6 U/l) and 10 mg/kg i.p. to 136.8 +/- 19.3% and even increase AST levels (618.90 +/- 209.32 U/l). After intravenous application of 1 and 10 mg/kg BW bosentan the perfusion rate was significantly increased and sticking of leukocytes in sinusoids and venules reduced (p < 0.05). In our model, diameters of sinusoids and postsinusoidal venules could be regulated gradually. We conclude that the avoidance of constriction, but not an excessive vasodilation leads to increased perfusion rate and hence improved hepatocellular function.

Flow dependence and time constant of the change in nitric oxide concentration measured in the vascular media

It has been considered that the concentration of endothelium-derived nitric oxide (NO) in the arterial vascular wall changes in response to flow-induced shear stress. In the present study, using an NO-sensitive electrode, the aim was to directly evaluate the relationship between perfusion rate and NO concentration in the arterial vascular wall. The NO microelectrode (diameter: 100 microns) was inserted into the vascular media of isolated canine femoral arteries, and the vessel was perfused with a Krebs-Henseleit buffer solution. A flow-related change in NO concentration in the vascular media was then evaluated by changing perfusion rate. NO concentration attained a peak value with a first-order time delay by a stepwise increase in perfusion rate, and the peak-level NO concentration was linearly correlated with perfusion rate in each vessel (10-154 pA at 2.1-72.3 ml min-1; n = 7, r2 = 0.89-0.99, p < 0.03). The average time constant for an increase in NO current with a stepwise increase in perfusion rate was 24 +/- 3 s (n = 5). NO production was increased by perfusing a solution containing 1 mmol l-1 L-arginine and was attenuated by 100 mumol l-1 NG-nitro-L-arginine, indicating the intactness of the endothelium, proper insertion of the NO electrode and selective detection of NO by the electrode. It is concluded that the NO microelectrode is applicable to NO measurement in the vascular media where NO controls vascular tone and that the concentration of NO in the arterial vascular media changes with perfusion rate in a rate-dependent manner as well as with a time constant of about 24 s for a stepwise increase in flow.

ENERGY METABOLISM OF RAT SKELETAL MUSCLE MODULATED BY THE RATE OF PERFUSION FLOW

In order to advance our understanding of the phenomenon of flow-induced increases in the metabolism of the relaxed muscle, the metabolic rate of the isolated rat gracilis muscle was investigated at 28¡C in vitro. The muscle was perfused with cell-free Krebs-Henseleit bicarbonate buffer containing 5 % bovine serum albumin and 5 mM glucose, saturated with a gas mixture of 95 % O2 and 5 % CO2 and simultaneously superfused with a medium saturated with a low O2 gas mixture (1 % O2, 5 % CO2 and 94 % N2). Two different perfusion flow rates (0á054 and 0á100 ml min-1) have been used. Their influence on oxygen consumption and lactate production has been measured. After a 100 min perfusion period, the muscle was freeze-clamped and analysed for ATP, phosphocreatine, creatine, lactate, pyruvate, inorganic phosphate and glycogen content. The energy state of the cell and the proportions of glycolytic and mitochondrial fluxes of ATP synthesis were evaluated. During perfusion at the low flow rate of 0á054 ml min-1, the oxygen uptake was 45 ± 9 nmol min-1 (g wet wt)-1, accompanied by a dominance of anaerobic glycolytic synthesis of ATP over mitochondrial ATP synthesis, even though the total delivery of oxygen to muscle was three times higher than oxygen consumption. Increasing the perfusion flow rate to 0á100 ml min-1 increased the oxygen uptake to 120 ± 6 nmol min-1 (g wet wt)-1, thus leading to a prevalence of mitochondrial ATP synthesis over glycolytic ATP synthesis. The inner stores of glycogen served as the main substrate of energy metabolism and the role of exogenous substrates in the flow-stimulated increase of oxygen uptake was negligible. The increase in perfusion rate also enhanced the energy state of the muscle fibres, which was expressed either as the creatine charge or as the value of the change of Gibbs free energy of ATP hydrolysis. Data indicate that the change of perfusion flow rate per se, apart from oxygen and exogenous substrate supply, elicits changes in the regulation of energy metabolism within non-contracting skeletal muscle under open microcirculation.

Energy Metabolism of Rat Skeletal Muscle Modulated by the Rate of Perfusion Flow

In order to advance our understanding of the phenomenon of flow-induced increases in the metabolism of the relaxed muscle, the metabolic rate of the isolated rat gracilis muscle was investigated at 28 degrees C in vitro. The muscle was perfused with cell-free Krebs-Henseleit bicarbonate buffer containing 5% bovine serum albumin and 5 mM glucose, saturated with a gas mixture of 95% O2 and 5% CO2 and simultaneously superfused with a medium saturated with with a low O2 gas mixture (1% O2, 5% CO2 and 94% N2). Two different perfusion flow rates (0.054 and 0.100 ml min-1) have been used. Their influence on oxygen consumption and lactate production has been measured. After a 100 min perfusion period, the muscle was freeze-clamped and analysed for ATP, phosphocreatine, creatine, lactate, pyruvate, inorganic phosphate and glycogen content. The energy state of the cell and the proportions of glycolytic and mitochondrial fluxes of ATP synthesis were evaluated. During perfusion at the low flow rate of 0.054 ml min-1, the oxygen uptake was 45 +/- 9 nmol min-1 (g wet wt)-1, accompanied by a dominance of anaerobic glycolytic synthesis of ATP over mitochondrial ATP synthesis, even though the total delivery of oxygen to muscle was three times higher than oxygen consumption. Increasing the perfusion flow rate to 0.100 ml min-1 increased the oxygen uptake to 120 +/- 6 nmol min-1 (g wet wt)-1, thus leading to a prevalence of mitochondrial ATP synthesis over glycolytic ATP synthesis. The inner stores of glycogen served as the main substrate of energy metabolism and the role of exogenous substrates in the flow-stimulated increase of oxygen uptake was negligible. The increase in perfusion rate also enhanced the energy state of the muscle fibres, which was expressed either as the creatine charge or as the value of the change of Gibbs free energy of ATP hydrolysis. Data indicate that the change of perfusion flow rate per se, apart from oxygen and exogenous substrate supply, elicits changes in the regulation of energy metabolism within non-contracting skeletal muscle under open microcirculation.

Muscle ATP synthesis and utilisation, balanced during flow-induced increase of respiration.

To investigate the control of cell energetic metabolism, creatine charge, ATP/ADP ratio and oxygen consumption (as indicators of an energetic status, the balance between ATP synthesis and degradation and the aerobic ATP turnover, respectively) were evaluated in the rat gracilis muscle, perfused-superfused in vitro. During the perfusion rate of 70 microl/min the ATP/ADP ratio, as well as the creatine charge are kept at the in vivo level. With the decrease of the rate toward 54 microl/min (of an abundant oxygen delivery), the values of both parameters are lower than levels in vivo. With the increase of the rate up to 100 microl/min, both parameters are kept at the in vivo level, when respiration increases by 125%. The data demonstrate the 'unmatched' control of ATP utilisation and synthesis steady rates during the low perfusion rate; during the increasing steady ATP turnover following the increased perfusion rate, the two fluxes are strikingly 'matched', i.e. precisely balanced.

A model to explain the rapid pressure decrease after air-inflation of diseased middle ears.

Air-inflation in humans and monkeys with significant negative middle ear pressure or with middle ear inflammation was shown to cause greater than ambient middle ear pressure initially, followed by a rapid rate of pressure decrease to approach the preinflation value. A mathematical model of middle ear pressure regulation is presented and used to simulate air-inflation of the normal and diseased middle ear. The model represents the total volume of the middle ear as consisting of three subcompartments representing the airspace, effusion, and mucosa/blood. Gas exchange among those compartments was assumed to be diffusion limited, and the gas exchange between the mucosa/blood compartment and systemic blood was assumed to be perfusion limited. Disease was modeled as an increase in mucosal blood flow or, alternatively, as an increase in the volumes of the effusion and mucosa/blood compartments. The predictions of the model agree better with the experimental data when the increased rate of pressure change after middle ear inflation in diseased ears is driven by an increased volume of the effusion compartment as opposed to an increased perfusion rate. The responsible mechanism is a rapid redistribution among subcompartments of the gas volume introduced into the air compartment. These results suggest that middle ear inflation with inert gas can be used to diagnose the presence and relative amount of middle ear effusion, and that current protocols for treating otitis media with effusion using inflation need to be modified to optimize their intended effect.

EXPERIMENTAL PARTIAL UNILATERAL URETER OBSTRUCTION. I. PRESSURE FLOW RELATIONSHIP IN A RAT MODEL WITH MILD AND SEVERE ACUTE URETER OBSTRUCTION

Purpose To create an animal model with mild and severe partial unilateral ureter obstruction in young rats using a modified Ulm and Miller technique and to characterize the model by acute renal pelvic pressure measurements. Material and Methods During anesthesia the upper fourth (n = 15) or the upper two-thirds of the left ureter (n = 15) was embedded into the psoas muscle causing either a mild or severe partial obstruction. Sham-operated control rats were prepared in paralllel (n = 20). The baseline pelvic pressure, the perfusion pelvic pressure (perfusion rates: 0.2 to 1.0 ml. per minute) and peristaltic waves were recorded after a resting period. Results Mean baseline pelvic pressure and perfusion pelvic pressure were significantly higher in obstructed kidneys than in non-obstructed kidneys, and significantly higher in severely obstructed kidneys than in mildly obstructed kidneys (p <0.05). A fair linear relationship existed between the increase in pelvic pressure and the increase in perfusion rates in all groups. In the severely obstructed kidneys, baseline pelvic pressure was 16.9 ± 2.3 cm. H 2 O and the perfusion pelvic pressure increased significantly from 41.7 ± 3.3 cm. H 2 O to 68.6 ± 6.3 cm. H 2 O. The pelvic peristaltic amplitude increased significantly following the increase in perfusion rate and there was a significant difference in the amplitude between severely obstructed and non-obstructed kidneys when the perfusion rate exceeded 0.4 ml. per minute (p <0.05). Conclusion Embedding either the upper fourth or the upper two-thirds of the ureter into the psoas muscle produced a mild or a severe partial obstruction. In the latter model a significant increase in pelvic pressure and amplitude was observed, indicating the existence of severe obstruction.

Reduced pulmonary metabolism of endothelin-1 in canine tachycardia-induced heart failure.

Plasma endothelin-1 (ET-1) increases in congestive heart failure (CHF). The pulmonary vascular bed could contribute to this increase through a reduced clearance. We evaluated the effect of tachycardia-induced CHF on pulmonary ET-1 kinetics. To discern between changes due to variations in pulmonary hemodynamics from true alterations of endothelial cell functions, we quantified ET-1 kinetics in isolated rat lungs under variable pressure and flow-rate conditions. Indicator-dilution studies were performed in anesthetized dogs (n = 14) before and 3 weeks after rapid ventricular pacing and in isolated lungs from healthy rats (n = 4). In isolated lungs, graded increases in perfusion rate from 5-25 ml/min caused gradual reductions in ET-1 extraction from 60 +/- 1.5% to 17 +/- 4.9% (mean +/- S.D.). The capacity to clear ET-1 from the circulation, as computed from the permeability-surface area product (PS), however did not vary over this range of flows. CHF increased plasma ET-1 (11.2 +/- 11.4 vs. 5.2 +/- 1.6 fmol/ml, p < 0.01), did not affect pulmonary ET-1 extraction (29.4 +/- 12.5% vs. 29.9 +/- 12.9%), but decreased the PS (8.3 +/- 5.4 cm3/s vs. 14.4 +/- 9.9 cm3/s, p = 0.038). Contrary to the invariability of the PS in normal isolated rat lungs, CHF was associated with a positive relationship between the PS and pulmonary plasma flow (r = 0.65, p < 0.01). ET-1 binding studies in lung tissues showed no significant variations in ETA and ETB receptors densities but revealed a threefold decrease in binding affinity (p < 0.01) that may explain the reduced clearance. CHF causes a reduction of pulmonary ET-1 clearance that likely contributes to the increased circulating ET-1 levels and reflects pulmonary metabolic dysfunction associated with this condition.

Assessment of the recovery of three lipophilic psoralens by microdialysis: an in vitro study

The microdialysis method is extensively developed in neuropharmacological experiments. It is a non-invasive technique with promising applications in biological research. The aim of this work was to establish and to optimize a process to microdialyse chemicals. Three lipophilic molecules used in dermatology were tested: 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and trimethylpsoralen (TMP). These psoralens have the same basic chemical structure (furocoumarin). The different radical substitutions are responsible for their different lipophilicities. In this work, different microdialysis parameters were studied: the relative recovery and the equilibrium time in relation to the perfusion rate and to the drug lipophilicity. The equipment was composed of a CMA/100® microinjection pump, CMA/20® microdialysis probes (membrane length of 10 mm, cut off 20 and 100 kDa) and a CMA/140® microcollector. The perfusate was a phosphate buffer 0.06 M (pH 7.4). The psoralen dialysates were assessed by high performance liquid chromatography. The relative recovery of psoralens increased with the decreasing perfusion rates and with the drug lipophilicity. The equilibrium time decreased with the increasing perfusion rates. TMP was not detected under these experimental conditions. This work defines the optimal parameters for in vivo studies and shows the limit of this technique for the investigation of lipophilic drugs.

Gills of antarctic fish.

We review the literature on the way the structure of icefish gills relates the physiology of these haemoglobin-less fishes. Vascular casting confirmed earlier reports that the only special feature of the gills is the large size of the blood vessels, especially the prominent and continuous marginal channels Isolated perfused gill arches were used to study the effects of changes in afferent and efferent pressure on gill resistance and tritiated water influx in Chionobathyscus dewitti. Increasing perfusion rate did not change gill resistance, but there were moderate proportional increases in water influx. Reducing efferent pressure increased gill resistance but did not affect water influx. In both C. dewitti and Cryodraco antarcticus gills perfused at constant flow rate, noradrenaline produced concentration-dependent decreases in gill resistance and, with high concentrations, increases in water influx. Fixation while perfusion continued was used to compare blood space dimensions in control, noradrenaline-treated and unperfused gills. Noradrenaline caused large increases in the thickness of the lamellar blood space and increased lamellar height, despite a greatly reduced afferent pressure. This suggests that modulation of pillar cell active tension might be involved in control of lamellar perfusion. The possible relationship between gill water fluxes and lamellar recruitment is discussed.

In vitro flow characteristics of the Ahmed and self-constructed anterior chamber shunts

SUMMARY Objective To compare in vitro opening pressures (OP) and closing pressures (CP) of the Ahmed VS-1 and VS-2 glaucoma valves with those of several selfconstructed valve ‘prototypes,’ and to assess their ability to maintain perfusion pressures between 6 and 21 mm of Hg. Sample Population Ahmed VS-1 (n = 6), 2 groups of Ahmed VS-2 (group 1: n = 12; group 2: n = 14), and self-constructed valves with linear incisions in the long axis of the tube wall (n = 6) or X-shaped incisions in the tube walls (n = 2). Procedure Valves were perfused with deionized water, lactated Ringer's solution (LRS), Dulbecco's modified Eagle's medium (DMEM), DMEM plus 50% equine serum (ES), and 100% ES. Flow rates of 2.85, 4.2, 6.0, 9.0, and 12.0 μl/min were used for each perfusate. Valves were tested 3 times for reproducibility, and OP/CP were compared for each system. Results OP/CP of the VS-1, VS-2 (group 1), VS-2 (group 2), and linear 1.0-cm incisional valves with thick tubing consistently increased with increasing perfusion rate. Linear 0.5-cm (thick tubing) and 1.0-cm (thin tubing) incisional valves had increasing OP/CP with increasing perfusion rate in all but a few instances. Mean OP/CP decreased with increasing perfusate osmolarity for all perfusates except LRS, using the VS-1 and V-2 (group 2) valves. Mean OP/CP were consistently lower for VS-1 than VS-2 (group 1) valves at any given flow rate and for any given perfusate. Mean OP/CP were consistently lower for VS-2 (group 1) than VS-2 (group 2) valves at any given flow rate and for any given perfusate. The linear 0.5-cm incisional valves with thick and thin tubing induced the highest mean OP/CP, maximizing at &gt; 30 mm of Hg. Conclusions Only the VS-2 (group 2) valves consistently had mean OP/CP between 6 and 21 mm of Hg for all perfusates and at all flow rates. Clinical Relevance Anterior chamber shunts, although imperfect, appear to offer a physiologically sound alternative for glaucoma management. (Am J Vet Res 1997;58:1332–1337)

The Fab-fragment of a PAI-1 Inhibiting Antibody Reduces Thrombus Size and Restores Blood Flow in a Rat Model of Arterial Thrombosis

The effect of PRAP-1, a Fab-fragment of a PAI-1-inhibiting polyclonal antibody, on thrombus size and arterial blood flow was studied in a rat model of arterial thrombosis. It was shown that exposure of the carotid artery to FeCl3 led to the rapid formation of an occlusive thrombus with a morphology similar to that of arterial thrombi found in humans. Tranexamic acid (50 mg/kg), an inhibitor of fibrinolysis, increased thrombus size (p = 0.014) when given intravenously (i.v.) prior to the FeCl3-exposure. Heparin (1000 U), when given i.v. after FeCl3, did not affect the thrombus size per se, but caused a reduction in the interindividual variation of the size of the thrombus (p < 0.05). Thus, heparin was included in all the subsequent experiments. An i.v. infusion of t-PA (1 mg/kg/h), starting before thrombus formation, induced a 3.3 fold increase in the perfusion rate (p = 0.006) and a 67% reduction in the thrombus size (P < 0.001). PRAP-1, an inhibitor of rat PAI-1 activity, was given i.v. as a bolus followed by an infusion. Two doses of PRAP-1 were studied (7.5 and 15 mg/kg/h), and the administration of the PAI-1 inhibitor was started 10 min before FeCl3. The lower PRAP-1 dose caused a 3.8 fold increase in perfusion rate (p = 0.036), a 1.44 fold increase in the time to occlusion (p = 0.034), and the thrombus size was decreased by 18% (p = 0.104). The corresponding effects of the high PRAP-1 dose were a 6.5 fold increase in perfusion rate (p < 0.001), a 1.6 fold increase in time to occlusion (p = 0.038) and a 32% reduction in thrombus size (p = 0.016). It is concluded that an inhibitor of PAI-1 activity, PRAP-1, caused a moderate decrease in thrombus size and partly restores blood flow in a rat model of arterial thrombus. This finding suggests a potential role for an inhibitor of PAI-1 in the treatment of arterial thrombosis.

Prevention of Mural Thrombus in Porous Inner Tube of Double-Layered Tube by Saline Perfusion

An in vitro experiment under laminar nonpulsatile blood flow and an acute canine ex vivo femoral A-V series shunt experiment were undertaken to investigate the effectiveness of saline perfusion through pores of porous tubes to prevent formation of mural thrombus. PS/SBR porous tubes were used for the in vitro experiment. Commercially obtained ePTFE porous tubes were etched by sodium naphthalenide, and the etched tubes were used for the ex vivo experiment. According to the results of the in vitro experiment, mural thrombus on the surface of the porous tube could be prevented by the saline perfusion. Adhered blood cells decreased semi-logarithmically with increased perfusion rate (up to 0.022 ml/min-cm2) of isotonic saline solution. According to results of the ex vivo experiment, mural thrombus decreased with increased perfusion rate (up to 0.060 ml/min-cm2).

Selective recycle of viable animal cells by coupling of airlift reactor and cell settler

A new system for the perfusion culture of animal cells in suspension is described. It consists of an airlift loop reactor and a settling tank for cell retention. Insufficient nutrient and oxygen supply of the cells in the settling tank was prevented by cooling the cell suspension before entering the settler. As a result, the catabolic activity of the cells in the settler was reversibly reduced. Furthermore, the density gradient induced by cooling caused a liquid motion through the settler. Thus, it was not necessary to pump medium containing shear, sensitive cells. With this simple system, it was possible to prduce 2 to 5 g of antibodies in a 5.4-L reactor in continuous runs of 400 to 600 h. The productivity was increased by a factor of 17 and the cell density was 4 times higher in comparison with the corresponding batch system. The cell retention system was found to have the property of separating viable and nonviable cells. With the increasing perfusion rate, dead cells and debris were preferably washed out. For perfusion rates up to 1.3 d(-1), the retention efficiency of the settler was nearly 100% for viable cells; hence, this system may show advantages at the industrial scale.

Experience in scale-up of homogeneous perfusion culture for hybridomas

A perfusion system for production of monoclonal antibodies was developed using an externally-mounted, hollow-fibre cartridge. The experimental apparatus was operated for 420 h and demonstrated increased steady-state viable cell concentration with increase in perfusion rate. Antibody titres were up to three times those measured for batch cultures and specific antibody productivity was doubled. The procedure was successfully scaled to a 10 dm3 system which produced antibody under conditions of Good Manufacturing Practice (GMP). A calculation of productivity between the scaled perfusion system and 260 dm3 batch cultures resulted in comparable antibody production, whereas the perfusion allowed a halving in medium utilisation. Reactivity assays conducted on the purified antibody from both batch and perfusion cultures showed no evidence of proteolysis or altered antibody activity in the final perfusion product. This study provides additional support for the use of homogeneous perfusion cultures in production of monoclonal antibodies under GMP conditions.

Effect of luminal perfusion rate on duodenal alkalinization in the rat in vivo and in vitro.

This study investigates the effects of luminal perfusion rate on the observed rate of alkalinization of an unbuffered saline by anesthetized rat distal duodenum. Increases in perfusion rate (range 0.3-22 ml/min) increased both alkalinization (3-15 mumol/cm hr) and intraluminal pressure (6-22 mm Hg). Increases in pressure (6-30 mm Hg) at constant perfusion rate (3 ml/min) did not change alkalinization. The effect of perfusion rate on alkalinization was rapid, reversible, and could be repeated several times. It was unaffected by indomethacin (10 mg/kg subcutaneously) or tetrodotoxin (1 mg/ml in perfusate). A limited in vitro study at two flow rates (0.3 and 22 ml/min) showed a similar enhancement of alkalinization. It is concluded that the rate of alkalinization is increased by raising the perfusion rate of the lumen both in vivo and in vitro. The mechanism remains to be established but may involve the reduction of a diffusion barrier between the mucosa and luminal bulk fluid. These results may have implications for the movement of other substances across intestinal segments.

Replacement of Excised Segments of Proximal Ureters by Tubularised Bladder Grafts Vascularised by Previously Established Omental Pedicles

This article reports the final study of experiments in which autologous bladder grafts have been used to replace excised segments of ureters in dogs. The technique described involves the use of bladder grafts on previously established omental pedicles as replacements for proximal ureters. Grafts were tubularised over silastic stents, then completely covered with secondary wrappings of omentum. Operated upper tracts were compared to control sides up to 12 months, then, at one year, intravenous urography was repeated at which time inspection was made of operated upper tracts (and contralateral controls) using an image intensifier prior to recording renal pelvic pressure responses to increasing perfusion rates and, finally, pathological examination. Only two of 14 upper tracts with pedicle grafts were failures radiologically and no upper tract was obstructed urodynamically. This approach is recommended for consideration as a treatment option in managing longitudinal defects in ureters in humans.