Closed Perfusion System Research Articles

Normothermic <i>ex vivo</i> lung perfusion using a developed solution followed by orthotopic left lung transplantation (experimental study)

The continued unavailability of adequate organs for transplantation to meet the existing demand has resulted in a major challenge in transplantology. This is especially felt in lung transplantation (LTx). LTx is the only effective method of treatment for patients with end-stage lung diseases. Normothermic ex vivo lung perfusion (EVLP) has been proposed to increase the number of donor organs suitable for transplant – EVLP has proven itself in a number of clinical trials. The ability to restore suboptimal donor lungs, previously considered unsuitable for transplantation, can improve organ functionality, and thus increase the number of lung transplants. However, widespread implementation of ex vivo perfusion is associated with high financial costs for consumables and perfusate.Objective: to test the developed solution on an ex vivo lung perfusion model, followed by orthotopic LT under experimental conditions.Materials and methods. The experiment included lung explantation stages, static hypothermic storage, EVLP and orthotopic left LTx. Perfusion was performed in a closed perfusion system. We used our own made human albumin-based perfusion solution as perfusate. Perfusion lasted for 2 hours, and evaluation was carried out every 30 minutes. In all cases, static hypothermic storage after perfusion lasted for 4 hours. The orthotopic single-lung transplantation procedure was performed using assisted circulation, supplemented by membrane oxygenation. Postoperative follow-up was 2 hours, after which the experimental animal was euthanized.Results. Respiratory index before lung explantation was 310 ± 40 mmHg. The PaO2/FiO2 ratio had positive growth dynamics throughout the entire EVLP procedure. Oxygenation index was 437 ± 25 mm Hg after 120 minutes of perfusion. Throughout the entire EVLP procedure, there was a steady decrease in pulmonary vascular resistance (PVR). Initial PVR was 300 ± 100 dyn×s/cm5; throughout the EVLP, PVR tended to fall, reaching 38,5 ± 12 dyn×s/cm5 at the end of perfusion.Conclusion. A safe and effective EVLP using our perfusate is possible. The developed orthotopic left lung transplantation protocol under circulatory support conditions, supplemented by membrane oxygenation, showed it is efficient and reliable.

Placental transport of Erythromycin and its effect on placental inflammatory factors.

Erythromycin is used for prevention and control of infectious perinatal morbidity. It has been hypothesised that erythromycin crosses the placenta and has an effect on the production of placental inflammatory factors. We evaluated the transport of erythromycin in an ex-vivo closed perfusion system of the placenta and determined its effect on the production of placenta inflammatory markers. In 2013, a prospective basic science study was conducted at the placental laboratory of College of Medicine and Health Sciences, United Arab Emirates. Six term placentas from uncomplicated pregnancies were studied using the ex-vivo dual closed-loop human placental cotyledon perfusion technique. Erythromycin was added to the perfusate in the maternal compartment. Samples were obtained from the maternal and fetal up to 240 minutes. The reference antipyrine was detected in the fetal circulation in the first 15 minutes after addition of the drug. At this point the mean antipyrine was 49.90±2.10μg/ml in the maternal perfusate and 7.1±1.56μg/ml in fetal perfusate. The fetal and maternal concentration became similar at 120 minutes. The transfer of antipyrine from maternal to fetal compartment was 98.66%. The differences between perfusion groups were non-significant that indicates the perfusion of placentas was comparable. After media exchange in both sides, erythromycin was added to the maternal perfusate. The experimental period of four hours was continued with medium circulation on both maternal and fetal circulation. The concentration of erythromycin decreased in the maternal circuit by 36.4% and increased in the fetal circuit by 65%. The concentration of IL-6 in the maternal circuit was normal. Erythromycin crossed the placenta and did not inhibit the production of IL-6. Future studies are needed concerning neonatal adverse effects and the development of antibiotic resistance.

Proof of concept study for a closed ex vivo limb perfusion system for 24-hour subnormothermic preservation using acellular perfusate.

The two approaches to vascularized tissue machine perfusion use either the open (nonpressurized) or closed (pressurized) perfusion system. Most studies describing isolated limb perfusion preservation rely on open perfusion systems and report tissue edema exceeding 40% after 12 to 14 hours of preservation. A variant of machine perfusion places the limb and perfusate into a reservoir closed to atmosphere. It is hypothesized that the reservoir pressure, acting as a transmural pressure, has the advantage of reducing edema formation by counteracting the hydrostatic pressure gradient from the perfusion pressure. This proof-of-concept study aim was to demonstrate feasibility of the Universal Limb Stasis System for Extended Storage (ULiSSES) device (closed, vertical perfusion system) to preserve forelimbs of Sus scrofa swine for 24 hours of subnormothermic perfusion compared with an open, horizontal perfusion system. The ULiSSES is a compact, practical device that applies pulsatile, pressurized perfusion through the novel use of a diaphragm pump powered by compressed oxygen. Forelimbs from swine were preserved in ULiSSES device (closed perfusion system) (n = 9) and in an open perfusion system (n = 4) using subnormothermic modified Krebs-Henseleit solution. Physiological parameters were measured at the start and every 3 hours for 24 hours. Limbs were weighed before and after perfusion to compare weight gain. Edema and cellular integrity were evaluated using histopathology pre and post perfusion. Closed perfusion system showed superiority compared with the open perfusion system in terms of oxygen consumption, reduction in vascular resistance, and overall tissue integrity. The closed perfusion system demonstrated a 21% reduction in weight gain compared with the open perfusion system and significantly reduced intracellular edema. The ULiSSES closed, pressurized perfusion technology has translatable military applications with the potential to preserve porcine limbs for 24 hours with improved results compared with an open perfusion system.

The Global End-Diastolic Volume (GEDV) Could Be More Appropiate to Fluid Management Than Central Venous Pressure (CVP) During Closed Hyperthermic Intrabdominal Chemotherapy with CO2 Circulation

ABSTRACTBackground: Closed hyperthermic intraperitoneal chemotherapy (HIPEC) may increase abdominal pressure and effects of hemodynamic changes due to maintenance hyperthermia. Our aim was to analyze the safety and effectiveness of our closed technique with CO2 circulation in management fluid status and hemodynamic parameters by means of cardiac preload control measured by Global End Diastolic Values (GEDV) and a gas exchanger. Material and Methods: A Pilot Clinical Study that included 18 advanced ovarian cancer patients undergoing citoreductive surgery and HIPEC. We used a closed-perfusion system (PRS Combat®) that includes CO2 circulation and a gas exchanger. Transpulmonary thermodilutions and hemodynamic measurements (PiCCO2®) were performed after citoreductive surgery (Pre-HIPEC); At half time of the HIPEC (Intra-HIPEC); After HIPEC (Post-HIPEC). Results: No significant hemodynamic measurements changes in the three thermodilutions values of Cardiac Index (CI) (p = 0.227), Global End Diastolic Values (GEVD) (p = 0.966), Stroke Volume Variation (SVV) (p = 0,884) and Systemic Vascular Resistance Index (SVRI) (p = 0.082). No correlation between central venous pressure (CVP) and GEDV (Pre-HIPEC: r = 0.164, p = 0.211; Intra-HIPEC: r = 0.015, p = 0.900; Post-HIPEC: r = 0.018, p = 0.890). There was better correlation between GEDV and CI (Pre-HIPEC: r = 0.432, p = 0.071; Intra-HIPEC: r = 0.418, p = 0.074; Post-HIPEC: r = 0.411, p = 0.080). Conclusions: Closed intrabdominal chemotherapy with CO2 circulation model may be a safe model for HIPEC by means of a gas exchanger. GEDV and its changes significantly correlated to CI, and not observed for CVP. GEDV values may be more appropriate for monitoring cardiac preload, blood loss limitation and to predict changes in intravascular volume status during intraperitoneal chemotherapy.

Investigation of perfused platelet activation using a 3D tissue-engineered medial layer

Introduction: Haemostasis is a highly regulated physiological process of which platelets play a critical role in forming a blood clot, to prevent haemorrhage. The prothrombotic properties of the vasculature facilitate platelet activation upon damage whereas its antithrombotic properties prevent platelet activation, adhesion and aggregation. Objectives: To fabricate a 3-dimensional (3D) medial layer by seeding rat dermal fibroblast cells (RDFCs) in collagen hydrogel to mimic smooth muscle cells in collagen hydrogel and a blood flow chamber to enable investigation of the effect of culture time period and shear stress on platelet activation. Materials and methods: RDFCs were cultured within collagen type hydrogel, to mimic a component of the blood vessel construct, the tunica media. A blood flow chamber was constructed using polydimethylsiloxane (PDMS) mould to fit with the construct (dimensions 7.3A~3A~1.5cm), forming a rhombic-shaped gasket. This chamber provided physiologically based flow regimes and enabled examination of platelet activation under flow conditions. Washed human platelets were perfused into the closed perfusion system containing the medial layer cultured for 2 and 7 days. Results: RDFCs seeded in collagen hydrogel favourably survived within the 3D layer construct. Platelet aggregation is culture time dependent since perfused platelets aggregate quicker upon exposure to 7 days cultured RDFCs in collagen than 2 days culture. Conclusions: Medial construct containing RDFCs were shown to activate platelets highlighting the release of neocollagen. This in vitro perfusion chamber is a potential tool for haemostasis and platelet activation studies. Acknowledgements: Ying Yang and Faiza Musa for supervising and support. Dr Waleed Alshaqha for supervising the project trip.

Reply to Bauer et al.

We appreciate the comments from Bauer et al. regarding our previously published article in this journal [1, 2] and would like to take the opportunity to clarify some important points of our study. The experimental set-up of our study included a sample closed perfusion system without a reservoir and without an air removal device on the venous side, as the main goal was to analyse pressure dynamics during cardiopulmonary bypass (CPB) and their effects on the formation of gaseous microemboli (GME) using a minimized closed perfusion circuit (MPC). We did not focus on the minimal extracorporeal circulation (MECC) system with a venous bubble trap (VBT) and with an oxygenator with an integrated arterial filter. To the best of our knowledge, this is the first study providing continuous recordings of venous line pressures every 250 ms during CPB. Independent of the further development of CPB techniques, in our opinion, it remains very important to document these pressure changes. The observed pressure differences reflected mostly volume requirements and underlined the need for an appropriate sensitive sensor to detect them. As Bauer et al. comment, this is the case in most current systems. However, venous pressure monitoring is not always sensitive enough to detect these differences [3]. In addition, the current automatic pump flow regulation involves a delay and under certain circumstances, is not fast enough to compensate negative pressure peaks sufficiently, leading to flow reduction. Even experienced perfusionists and anaesthesiologists, carefully handling the volume management, cannot provide 100% safety. To address this problem, our group developed a new device for automatic pressure and volume compensation during MPC, which was also evaluated in this experimental model, and these data are currently in submission. A large number of references within the comment of Bauer et al. state that MPC may be more vulnerable to accidental air introduction—the main trigger that develops de-airing devices [1]. In the in vitro study of Kutschka et al. [4], clinical aspects such as volume depletion were not considered. The better neurocognitive outcome after MPC when compared with CPB by Anastasiadis et al. [5] may be related to differences in cerebral perfusion that are influenced by various factors. However, the used MPC system had no VBT and the presence of GME was not investigated. All components of our experimental perfusion system are nowadays in routine clinical use. We used the same CPB equipment in order to make a realistic comparison between MPC and CPB. We agree with Bauer et al., that modern MECC systems with de-airing devices are safe and show a clinical benefit in daily routine use. Nevertheless, some aspects regarding these specific devices such as the use of more priming volume, more foreign surface contacts and additional costs should be studied further. Our newly developed compensation device may ameliorate or avoid these effects. Finally, we would like to mention that, following our experimental results, we plan to clinically compare MPC with clampless off-pump strategies, as the latter are well known for excellent postoperative neurological outcomes [6].

The Effect of Anti-Rosetting Agents against Malaria Parasites under Physiological Flow Conditions

Rosetting remains the dominant malaria parasite adhesion phenotype associated with severe disease and pathogenicity in Africa. The formation of rosettes, whereby a Plasmodium falciparum infected erythrocyte (IE) adheres to two or more non-IEs, is thought to facilitate the occlusion of microvascular blood vessels by adhering to host endothelial cells and other bound IEs. Current methods of determining the rosette-disrupting capabilities of antibodies/drugs have focused on static assays. As IEs in vivo are exposed to shear stresses within the microvasculature, the effect of flow conditions on rosetting requires further examination. This study establishes a new rosetting flow assay using a closed perfusion system together with inverted fluorescence microscopy and image analysis, and confirms previous reports that rosettes exist under shear stresses equivalent to those present in the microvasculature (0.5–1.0 dyn/cm2). Furthermore, we tested the effectiveness of rosette-disrupting PfEMP1 antibodies, heparin and fucoidan over a range of concentrations on two P. falciparum strains, and found no statistically significant differences between the results of static and flow assays. The new flow assay is a valuable addition to the tools available to study rosetting. However, the static assay has good predictive value and remains useful as the standard screening test for rosette-disrupting interventions.

Tissue Engineering for Ex Vivo Repair of Ischemically Damaged Tubule Epithelium

Introduction: The ability to repair warm ischemically damaged renal tubule epithelium prior to transplantation would provide the potential to expand organ donor criteria to include uncontrolled DCD (uDCD) patients. We have demonstrated that an ex vivo acellular, near-normothermic perfusion can resuscitate oxidative metabolism after warm ischemia sufficiently for there to be new synthesis that restores cytoskeletal integrity. However, during the period of ex vivo perfusion, the tubule epithelium is not regenerated because rather than repair, cell replacement is needed. We now describe the ability to deliver progenitor renal epithelial cells (REC) during ex vivo perfusion to the renal tubule epithelium with homing to the sites of damage. Methods: Human REC were fluorescently labeled with PKH26 red fluorescent cell linker (Sigma-Aldrich, St. Louis, MO). Porcine kidneys were damaged by 60-minutes of postmortem warm ischemia. The damaged kidneys were then flushed and placed on exsanguinous metabolic support perfusion at 32°C for 60-minutes to restore oxidative metabolism and normalize perfusion pressures and vascular flow rates. 5.0 × 107 labeled human REC were then infused into the renal artery at the rate of 0.5 × 106 per minute. The perfusion was then continued for an additional 8-hours. Results: During the administration of the REC there were no adverse vascular reactions in terms of there being no rise in perfusion pressures nor diminution in the vascular flow rate. Post-perfusion the labeled human REC were only detected within the renal tubule epithelium. By using an ex vivo closed perfusion system we were able to determine the number of human REC that were introduced intra-arterially, the number of REC remaining in the perfusate post-perfusion and the number of cells removed from the vascular space by copious flushing post-perfusion. More than 90% of the fluorescently labeled human REC were taken up by the kidney and could be detected predominantly in the tubules of the outer medulla (Figure 1a & 1b). The human REC were not found in the vascular compartment.Figure: [Tissue Engineering Tubule Epithelium]Conclusions: These results demonstrate the ability to deliver REC to the renal parenchyma during an ex vivo perfusion with homing to the site of damaged tubule epithelium and the corresponding ability to quantify the number of REC retained within the renal tissue. The ability to target progenitor cell delivery with corresponding determination of the number of cells deposited could provide opportunities for enhanced ex vivo repair of ischemically damaged kidney allografts.

Tissue Engineering for Ex Vivo Repair of Ischemically Damaged Tubule Epithelium

Introduction: The ability to repair warm ischemically damaged renal tubule epithelium prior to transplantation would provide the potential to expand organ donor criteria to include uncontrolled DCD (uDCD) patients. We have demonstrated that an ex vivo acellular, near-normothermic perfusion can resuscitate oxidative metabolism after warm ischemia sufficiently for there to be new synthesis that restores cytoskeletal integrity. However, during the period of ex vivo perfusion, the tubule epithelium is not regenerated because rather than repair, cell replacement is needed. We now describe the ability to deliver progenitor renal epithelial cells (REC) during ex vivo perfusion to the renal tubule epithelium with homing to the sites of damage. Methods: Human REC were fluorescently labeled with PKH26 red fluorescent cell linker (Sigma-Aldrich, St. Louis, MO). Porcine kidneys were damaged by 60-minutes of postmortem warm ischemia. The damaged kidneys were then flushed and placed on exsanguinous metabolic support perfusion at 32°C for 60-minutes to restore oxidative metabolism and normalize perfusion pressures and vascular flow rates. 5.0 X 107 labeled human REC were then infused into the renal artery at the rate of 0.5 X 106 per minute. The perfusion was then continued for an additional 8-hours. Results: During the administration of the REC there were no adverse vascular reactions in terms of there being no rise in perfusion pressures nor diminution in the vascular flow rate. Post-perfusion the labeled human REC were only detected within the renal tubule epithelium. By using an ex vivo closed perfusion system we were able to determine the number of human REC that were introduced intra-arterially, the number of REC remaining in the perfusate post-perfusion and the number of cells removed from the vascular space by copious flushing post-perfusion. More than 90% of the fluorescently labeled human REC were taken up by the kidney and could be detected predominantly in the tubules of the outer medulla (Figure 1a & 1b). The human REC were not found in the vascular compartment.Figure: [Tissue Engineering Tubule Epithelium]Conclusions: These results demonstrate the ability to deliver REC to the renal parenchyma during an ex vivo perfusion with homing to the site of damaged tubule epithelium and the corresponding ability to quantify the number of REC retained within the renal tissue. The ability to target progenitor cell delivery with corresponding determination of the number of cells deposited could provide opportunities for enhanced ex vivo repair of ischemically damaged kidney allografts.

Thromboresistant Cardiopulmonary Bypass Circuits: Room for Improvement?

Circulating blood contact with the surfaces of non-thromboresistant cardiopulmonary bypass (CPB) circuits induces several harmful inflammatory and hematological effects. Hussaini and colleagues in an effort to develop a more biocompatible CPB circuit that reduces inflammation, thrombogenesis and end-organ injury tested a thromboresistant CPB circuit using a heparin-bonded circuit (HBC) (1). The use of HBC is controversial as prior human studies using HBC have reported modest reductions at best in complement activation (2) as well as postoperative length of stay and end-organ injury (3). However, these studies have been criticized for a possible selection basis. Nonetheless, the use of these circuits is associated with reduced circulating complement (4, 5), interleukin-6 (5) and activated granulocytes (4, 6). The authors of this current study hypothesized that the outcomes using HBC may be improved by: 1) maintenance of a higher hematocrit (Hct) > 25%; 2) use of a closed perfusion system to eliminate the blood-gas interface (another site of foreign surface exposure); and 3) systemic normothermia. To study this, the authors compared HBC to conventional CPB (non-heparin-bonded circuits; NHB) in a porcine model (1). Anticoagulation as measured by ACT was achieved in both groups, although supratherapeutic levels were often achieved in the NHB group during CPB. Intraoperative hemodynamics were similar, and blood loss was higher in the NHB group as might be expected given the higher ACT (347.8 ± 79.3 ml vs. 214.1 ± 34.5 ml). In addition, thrombin anti-thrombin complex (TATIII) formation occurred more rapidly and remained elevated in the NHB group compared to the HBC group. Alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase and gamma-glutamyl transferase levels were also higher in the NHB group, and postoperative leukocyte and platelet counts were significantly lower. As might be expected, animals in the HBC group required 51% less heparin and 61% less protamine than in the NHB group. Microscopically, the arterial filters of the NHB circuits were clogged by activated leukocytes, red blood cells and platelets while the filters from HBC circuits were free of debris. Like all studies, there are several unanswered questions. First, the modified perfusion strategy changed several variables. This begs the question: which changes from conventional CPB are responsible for the observed effects—the increased hematocrit, normothermia, the HBC? The use of normothermia may be especially concerning to some surgeons since hypoperfusion may still occur while on CPB and hypothermia offers some end-organ protection. Finally, no cytokine levels were measured to further quantify the pro-inflammatory effects of these perfusion strategies. Nonetheless, these data suggest modification of conventional perfusion strategies may reduce CPB-associated inflammatory, immunologic and hematologic derangements. Further study is needed to identify the mechanisms the lead to authors’ observed results. Hopefully further mechanistic insight will lead to new perfusion strategies and circuits for patients.

Multifactorial Comparison of Modified and Conventional Perfusion Strategies in A Porcine Model of Cardiopulmonary Bypass

Utilization of thromboresistant circuits in cardiopulmonary bypass (CPB) surgery has been controversial. However, due to the advantages associated with these types of circuits, we sought to evaluate the efficacy of use of low-dose heparin in conjunction with thromboresistant surfaces, closed perfusion system, elimination of blood-gas interface, maintenance of hematocrit to >25%, and systemic normothermia, with respect to the conventional strategy of non-thromboresistant open circuits with high-dose heparin, during 3 h of CPB in an animal model. Using an open-chest swine model, animals were placed on CPB for 3 h with additional monitoring for 1 h post-CPB. Pigs were randomized into either a heparin-bonded circuit (HBC) group (n = 10) or a non-HBC (NHB) group (n = 10). Hemodynamic, hematologic, and biochemical parameters and multiphoton microscopy were used to compare the two groups. Pigs in the HBC group showed a 38.4% reduction in post-CPB blood loss in comparison with the NHB group (P = 0.0007). Additionally, compared with the HBC group, the NHB group exhibited a 32.7% post-CPB reduction in platelets (P < 0.001) and significant increases in alkaline phosphatase, aspartate aminotransferase, and creatine phosphokinase enzymes (P < 0.0202, P = 0.0015, P < 0.0001; respectively). Multiphoton imaging of the arterial filters revealed no entrapment of RBC, WBC, and platelets in the HBC group, while the filters in the NHB group were clogged by these cells. Utilization of modified perfusion strategy employing low-dose heparin and closed thromboresistant circuits is successful in ameliorating the potential adverse hematologic and pro-inflammatory elements induced with open perfusion system of non-thromboresistant circuits most commonly used in cardiac surgery.

A Closed Perfusion System With Heparin Coating and Centrifugal Pump Improves Cardiopulmonary Bypass Biocompatibility in Elderly Patients

Cardiopulmonary bypass induces a systemic inflammatory and hemostatic activation, which may contribute to postoperative complications. Our aim was to compare the inflammatory response, coagulation, and fibrinolytic activation between two different perfusion systems: one theoretically more biocompatible with a closed-circuit, complete heparin coating, and a centrifugal pump, and one conventional system with uncoated circuit, roller pump, and a hard-shell venous reservoir. Forty-one elderly patients (mean age, 73 +/- 1 years, 66% men) undergoing coronary artery bypass grafting or aortic valve replacement were included in a prospective, randomized study. Plasma concentrations of complement factors (C3a, C4d, Bb, and sC5b-9), proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-6, and interleukin-8), granulocyte degradation products (polymorphonuclear elastase), and markers of coagulation (thrombin-antithrombin) and fibrinolysis (D-dimer, tissue plasminogen activator antigen and tissue plasminogen activator-plasminogen activator inhibitor-1 complex) were measured preoperatively, at bypass during rewarming (35 degrees C), 60 minutes after bypass, and on day 1 after surgery. The mean concentrations of C3a (-39%; p = 0.008), Bb (-38%; p < 0.001), sC5b-9 (-70%; p < 0.001), interleukin-8 (-60%; p = 0.009), polymorphonuclear-elastase (-55%; p < 0.003), and tissue plasminogen activator antigen (-51%; p = 0.012) were all significantly lower in the biocompatible group during rewarming. Sixty minutes after bypass, the mean concentrations of sC5b-9 (-39%; p = 0.006) and polymorphonuclear-elastase (-55%; p < 0.001) were lower in the biocompatible group. The results suggest that a closed perfusion system with a heparin-coated circuit and a centrifugal pump may improve cardiopulmonary bypass biocompatibility in elderly cardiac surgery patients in comparison with a conventional system.

Ex vivo human placental transfer of human immunodeficiency virus-1 p24 antigen

OBJECTIVE: Our purpose was to determine whether human immunodeficiency virus-1 p24 antigen crosses the human placenta and, if so, to determine its clearance index relative to antipyrine. STUDY DESIGN: Eight term human placentas from uncomplicated vaginal or cesarean section deliveries were studied by ex vivo placental perfusion to determine the incidence and concentration required to obtain passage of p24 antigen into the fetal circulation. The concentration of p24 antigen was determined by antigen-capture enzyme immunoassay. RESULTS: The passage of p24 antigen into the fetal circulation was observed in three of five placentas studied when the p24 antigen concentration in the maternal circulation was 2942.8 ± 401 pg/ml. When the p24 concentration in the maternal circulation was raised approximately fourfold to 14506 ± 4124 pg/ml, p24 antigen passed to the fetal circulation in two of three placentas and in three of three placentas in the closed perfusion system. CONCLUSIONS: p24 antigen crossed the human placenta to the fetal circulation in what appears to be a concentration-dependent manner.

Placental transfer of theophylline in an in vitro closed perfusion system of human placenta isolated lobule.

Theophylline (TH) is a methylated xanthine widely used in the treatment of asthmatic pregnant women. Because of the scant available information on the transplacental profile, the time course of TH transfer was studied by an in vitro human placental perfusion. 6 placentas were perfused with Earle's enriched bicarbonate buffer for 180 min using recirculating maternal and fetal circuits. The physiological and biochemical properties of the tissue were well maintained. TH data were compared to those of antipyrine (AP), an usual marker in placental perfusions. The disappearance of TH from the maternal circuit was studied after administration of 15 mg/l in maternal perfusate. TH appeared in the fetal circuit within 5 min. Equilibrium was achieved in both circuits. TH fetomaternal mass ratio became constant (FMM = 0.45 +/- 0.01) after 80 min of perfusion and maternal to fetal clearance was 2.59 +/- 0.24 ml/min. About 16% of TH maternal dose was recovered in the tissue, while 18% appeared in fetal circulation. TH recovery was 89 +/- 9%. On the basis of our results, similar concentrations could be predicted in mother and fetus after maternal TH intake. The TH transfer profile is consistent with in vivo values reported in humans and animals at delivery.

Effects of nucleoside transport inhibition on long-term ex vivo preservation of canine hearts

The effect of nucleoside transport inhibition on 24-hour preservation of canine hearts was studied in 36 hearts arrested either with a cold hyperkalemic cardioplegic solution without (group I) or with supplementation of a specific nucleoside transport inhibitor (R75231, 1 mg/L) (groups II and III). The hearts were excised and stored for 24 hours at 0.5 degrees C. Then they were reperfused for 3 hours with use of a closed perfusion system primed with normal blood (groups I and II) or with blood supplemented with the same nucleoside transport inhibitor (0.32 mg/L) (group III). Serial biopsy specimens for determination of myocardial purines were taken. Creatine kinase and heat-stable lactate dehydrogenase release from the myocardium were examined during reperfusion. Recovery of function was studied during reperfusion by measurement of isometric contraction in a fluid-filled intraventricular balloon. After 24 hours of preservation, without the use of the drug, myocardial inosine and hypoxanthine accumulated to, respectively, 4.05 +/- 1.18 and 0.28 +/- 0.08 mumol/gm dry weight. In the drug-treated groups (II and III pooled), significantly less inosine and hypoxanthine accumulated (1.68 +/- 0.33 and 0.05 +/- 0.02 mumol/gm dry weight, respectively) (p < 0.05 versus group I). Upon reperfusion, intramyocardial adenosine was lost in the control hearts and maintained in the drug-treated hearts. Hypoxanthine accumulated significantly (p < 0.05) during reperfusion in group I (1.08 +/- 0.43 versus 0.16 +/- 0.13 in group II and 0.03 +/- 0.03 mumol/gm dry weight in group III). The rate of creatine kinase and heat-stable lactate dehydrogenase release was significantly lower (p < 0.05) in group III (that is, pretreatment and posttreatment with the drug) than in the control group. Functional recovery of hearts in group III was superior to that in group II (p < 0.05), while hearts in group I showed no recovery at all. We conclude that nucleoside transport inhibition improves long-term preservation of the heart and that the mechanism of this protection may be related to an increase in endogenous adenosine and reduction of myocardial hypoxanthine content.

Mechanistic studies in the transcuticular delivery of antiparasitic drugs II: ex vivo/in vitro correlation of solute transport by Ascaris suum

Using live, intact Ascaris suum and a closed perfusion system, the absorption kinetics and tissue distribution of selected radiolabeled permeants were measured to determine the importance of the transcuticular pathway for drug absorption. The data support the conclusions established by previous in vitro transport studies which utilized excised cuticle-hypocuticle tissue preparations. The external surface of A. suum can be breached by drugs and the rate-determining barrier is the lipoidal hypocuticle tissue, provided the permeant is sufficiently small to traverse the aqueous-filled, negatively charged collagen matrix of the cuticle. The ex vivo permeability coefficients of the model permeants for the cuticle-hypocuticle barrier were in good quantitative agreement with the in vitro permeability coefficients. The lipophilic permeants hydrocortisone and p-nitrophenol were preferentially distributed in the gut tissue, whereas the hydrophilic permeant urea was distributed evenly throughout the organism and was extensively metabolized. Ligated and nonligated A. suum showed no significant differences in either uptake kinetics or tissue distribution of the permeants. This indicates that the transcuticular pathway is the major route of drug absorption as compared to oral ingestion.

Progesterone secretion by corpora lutea of the isolated perfused rabbit ovary during pseudopregnancy.

An ovarian in vitro perfusion method was adapted to examine rabbit corpus luteum (CL) function during pseudopregnancy. Ovaries were perfused in vitro with tissue culture Medium 199 with or without 3% bovine serum albumin (BSA). Samples were obtained from both arterial and venous cannulae, allowing rates of progesterone secretion to be determined. Two perfusion methods were compared: a closed system in which perfusion medium continuously recirculated through the tissue, and an open system in which the ovarian vein cannula was left outside the perfusion chamber. The addition of 3% BSA was found to prevent edema and distortion of the interstitial space, and to result in increased progesterone secretion. With the closed perfusion system, the progesterone secretion rate measured over the 6-h perfusion was significantly higher on Day 11 than on Day 1 of pseudopregnancy and had declined significantly on Day 18. There was no difference in secretion rates whether the perfusion system was open or closed, and the results obtained with both systems closely approximated in vivo progesterone secretion rates. Measurements of CL tissue progesterone content before and after in vitro perfusion indicated that the changes in progesterone secretion seen during pseudopregnancy resulted from differences in the synthesis and secretion of progesterone and not from leakage of progesterone already present in the CL prior to perfusion. Taken together, these results indicate that the modified in vitro perfused rabbit ovary preparation described herein is an appropriate model to examine progesterone secretion by ovaries bearing CL.

Intimal response of saphenous vein to intraluminal trauma by simulated angioscopic insertion

Endothelial damage after simulated angioscopic insertion was evaluated in 22 dogs by radioimmunoassay of endogenous 6-keto-prostaglandin F1α (stable product of prostacyclin produced by the endothelium) by means of a closed perfusion system and scanning electron microscopy. In 20 dogs, a 1.7 mm diameter polyethylene tube similar in size and physical characteristics to a commercially available angioscope was passed once into the distal venotomy site and out of the proximal venotomy site for a total distance of 15 cm in the left saphenous vein (group A) and 10 times in both directions in the right saphenous vein, destroying all valves (group B). The vein segments were examined immediately after simulated angioscopic insertion and at intervals of 2, 3, and 4 weeks. The remaining two dogs were used as control animals to establish baseline prostacyclin production. All veins were patent when harvested. Scanning electron microscopy revealed globular and contracted endothelial cells in some areas of group A veins, whereas partial longitudinal absence of endothelial cells was noted in group B veins. Intimal coverage was complete by 2 weeks in group A veins and between 3 and 4 weeks in group B veins. Prostacyclin synthesis of group B veins was significantly less than that of group A at day 1 and at 2 weeks after operation (p < 0.05), reaching that of control vein synthesis by 4 weeks. This study suggests that simulated angioscopic trauma of the luminal surface of the canine saphenous vein had no negative effect on early patency.

Rates of H 2O 2 generation from peroxisomal β-oxidation are sufficient to account for fatty acid-stimulated ethanol metabolism in perfused rat liver

Fatty acids generate H 2O 2 via peroxisomal β-oxidation and increase ethanol metabolism markedly in a system that involves catalase-H 2O 2. The present studies were conducted to understand why fatty acid-stimulated ethanol metabolism occurs much faster than rates of H 2O 2 generation reported previously in perfused rat liver. A new method was developed to measure rates of H 2O 2 generation based on the fact that methanol is oxidized only by catalase in rat liver. Rates of H 2O 2 generation were estimated from the time necessary for the steady-state level of catalase-H 2O 2 measured spectrophotometrically (660-640 nm) through a lobe of the liver to return to basal values following the addition of a known quantity of methanol in a closed perfusion system containing 4% bovine serum albumin. Under these conditions, basal rates of H 2O 2 production and rates of 4-methylpyrazole-insensitive ethanol oxidation were in a similar range (10 to 20 μmol/g/hr). Rates of H 2O 2 generation were increased up to 80 μmol/g/hr by addition of laurate, palmitate or oleate (1 mM); half-maximal increases in rates were observed with 0.6 mM oleate. Hexanoate, a short-chain fatty acid, did not stimulate H 2O 2 production or ethanol uptake. In these studies, rates of H 2O 2 generation compared well with rates of fatty acid-stimulated ethanol uptake measured in the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, with all fatty acids studied. It is concluded, therefore, that rates of H 2O 2 generation are sufficient to account for rates of fatty acid-stimulated ethanol metabolism via catalase-H 2O 2. In addition, these data indicate that catalase may contribute significantly to ethanol oxidation under physiological conditions in the presence of fatty acids.

Effects of dissolved carbon monoxide on the respiratory activity of perfused neuronal and muscle cell cultures.

In order to address the question of whether small amounts of dissolved CO may inhibit cellular respiration, cultured mouse neuroblastoma cells and primary cultures of chick neurons, rat astrocytes and chick skeletal muscle and heart cells were exposed to CO containing buffer solutions in a closed perfusion system. Oxygen uptake was measured simultaneously with two polarographic oxygen electrodes as the difference in partial pressure of oxygen between the inlet and outlet of the perfusion chamber. After registration of the basal respiratory activity, perfusion solutions containing 5 ul 02/ml were bubbled with CO or N2 at a rate of 200 ml/min for 120 sec. By this procedure the partial pressure of 02 was decreased to reach a value of about 50% of the initial 02 content for both gases. Perfusion was then continued for 30 min at a rate of 0.5 ml/min. The respiratory activity of all the perfused cell cultures, except chick neurons, was found to be inhibited (13-29%) by perfusion solutions bubbled for 120 sec with CO as compared to N2 controls. Of the cells from the nervous system, astrocytes were more sensitive than neurons. Apparently, small amounts of dissolved CO can inhibit cellular respiration in the presence of a physiologically adequate amount of oxygen.