Cristina Escudero

Optical aggregometry versus the PFA–100™: experimental studies in pigs treated with propofol

An experimental study of platelet aggregation was performed in 22 male Landrace 2 Large-White crossbred pigs treated with propofol at different doses, to compare the results of optical aggregometry with those of the PFA-100™ (Dade Int., Miami, FL, USA), a new platelet function analyzer. Platelet aggregation was analyzed in basal blood samples by both methods, after which the pigs were divided into three groups: G1, anaesthetic induction with propofol (2 mg/kg intravenously (i.v.)); G2, anaesthetic induction with propofol (2 mg/kg i.v.), followed by a second dose of 1.5 mg/kg; and G3, anaesthetic induction with propofol (2 mg/kg i.v.), followed by 1 h of continuous i.v. infusion at 13 mg/kg/h. Four minutes after propofol injection, blood samples were again taken from each group and studied by both methods. In groups G2 and G3, both methods showed reduced platelet aggregation, while in group G1 neither evidenced an anti-aggregating effect of propofol. Under our experimental conditions: (1) the propofol effect on platelet aggregation depends on the plasma concentration; (2) the results obtained with the two methods are comparable; (3) PFA-100™ may provide an alternative to optical aggregometry for detecting the effects of anaesthetic agents ex vivo .

Mesothelial versus endothelial cell seeding: Evaluation of cell adherence to a fibroblastic matrix using 111In oxine

OBJECTIVES The aim of this study was to compare the behaviour of mesothelial cells (MC) to that of endothelial cells (EC) when seeded onto a PTFE, prosthesis coated with a fibroblastic matrix. DESIGN, MATERIAL AND METHODS Three study groups were examined: a control group (Control) of PTFE prostheses with a fibroblast matrix (n = 8); Group EC, PTFE prostheses seeded with EC on a fibroblastic matrix (n = 8); and Group MC, PTFE, prostheses seeded with MC on a fibroblastic matrix (n = 8). All cell types were labelled with 111In (100 microCi/ml) 24 h after seeding, when the cells had formed a monolayer on the prosthetic surface. Radioactive levels were measured at 2, 4, 6, and 24 h. RESULTS Both EC and MC showed optimal adherence. The MC had a better radioactive uptake and retention than the EC. The number of EC and MC cells that remained adherent to the matrix was large enough to ensure complete covering of the prosthetic surface. CONCLUSION The use of MC is therefore feasible as an optimal alternative for achieving a natural covering on vascular prostheses prepared with a fibroblastic matrix.

Ischemia-Reperfusion Injury During Experimental Heart Transplantation. Evaluation of Trimetazidine¿s Cytoprotective Effect

INTRODUCTION AND OBJECTIVES The objectives of this study were to analyze the ischemia-reperfusion injury due to free radicals that occurs during heart transplantation and to determine the potential cytoprotective effect of trimetazidine. MATERIAL AND METHOD A total of 21 orthotopic heart transplantations were performed in pigs. We divided the experimental animals into 2 groups: in group A (n=11),standard myocardial protection was used; in group B (n=10), trimetazidine was added to the cardioplegic solution used to protect the donor heart and to the solution administered to the recipient prior to release of the aortic clamp (trimetazidine, 10(-5) mol/L), and recipients were pretreated with trimetazidine, 2.5 mg/kg. Blood samples were taken from the recipients coronary sinus at three times: at baseline, during ischemia, and during reperfusion. We measured the levels of malondialdehyde, a marker of lipid peroxidation, and of several antioxidants: glutathione peroxidase, glutathione reductase, superoxide dismutase, alpha-tocopherol, and retinol. The total antioxidant status was also determined. RESULTS Malondialdehyde production and enzymatic antioxidant activity rose during ischemia and reperfusion, while the retinol level decreased. The increases in malondialdehyde level and glutathione peroxidase activity that occurred between baseline and reperfusion were significantly higher in group A. CONCLUSIONS. The degree of lipid peroxidation and the level of activity of intracellular antioxidant mechanisms increased progressively throughout transplantation. Trimetazidine had a cytoprotective effect. It ameliorated free radical-induced reperfusion injury and modified the response pattern of several defense mechanisms.

Daño por isquemia-reperfusión durante el trasplante cardíaco experimental. Evaluación del papel citoprotector de la trimetazidina

Introduccion y objetivos. El objetivo de este trabajo fueanalizar el dano por isquemia-reperfusion mediado por radicales libres que se produce durante el trasplante cardiaco y eva-luar el posible efecto citoprotector de la trimetazidina (TMZ). Material y metodo. Se realizaron 21 trasplantes cardiacos ortotopicos en cerdos. Dividimos los experimentos en 2 grupos: A (n = 11), en el que se realizo una proteccion miocardica estandar, y B (n = 10), en el que se administro TMZ en la cardioplejia empleada para parar el corazon donante (TMZ, 10?5 mol/l), como pretratamiento intravenosodel receptor (TMZ, 2,5 mg/kg) y como parte de la cardio-plejia infundida en el receptor antes de despinzar la aorta(TMZ, 10?5 mol/l). Se tomaron muestras de sangre del senocoronario del receptor en 3 momentos: basal, isquemia y reperfusion. Se determino la concentracion de malonildial-dehido como marcador de peroxidacion lipidica y de variosantioxidantes: glutation peroxidasa, glutation reductasa,superoxido dismutasa, a-tocoferol, retinol y estado de antioxidantes totales. Resultados. Durante la isquemia-reperfusion aumentola produccion de malonildialdehido y la actividad de losantioxidantes enzimaticos, mientras que el retinol disminuyo. El incremento de malonildialdehido y de la actividad de la glutation peroxidasa entre el momento basal y la reperfusion fue significativamente mayor en el grupo A. Conclusiones. Durante el trasplante se incremento progresivamente el nivel de peroxidacion lipidica y se activaronlos sistemas antioxidantes intracelulares. La TMZ ejercio un efecto citoprotector y limito el dano por isquemia-reperfusion generado por los radicales libres, ademas de modificar el patron de reaccion de parte de los sistemas de defensa.

Influence of hypothermia on right atrial cardiomyocyte apoptosis in patients undergoing aortic valve replacement

BackgroundThere is increasing evidence that programmed cell death can be triggered during cardiopulmonary bypass (CPB) and may be involved in postoperative complications. The purpose of this study was to investigate whether apoptosis occurs during aortic valve surgery and whether modifying temperature during CPB has any influence on cardiomyocyte apoptotic death rate.Methods20 patients undergoing elective aortic valve replacement for aortic stenosis were randomly assigned to either moderate hypothermic (ModHT group, n = 10, 28°C) or mild hypothermic (MiHT group, n = 10, 34°C) CPB. Myocardial samples were obtained from the right atrium before and after weaning from CPB. Specimens were examined for apoptosis by flow cytometry analysis of annexin V-propidium iodide (PI) and Fas death receptor staining.ResultsIn the ModHT group, non apoptotic non necrotic cells (annexin negative, PI negative) decreased after CPB, while early apoptotic (annexin positive, PI negative) and late apoptotic or necrotic (PI positive) cells increased. In contrast, no change in the different cell populations was observed over time in the MiHT group. Fas expression rose after reperfusion in the ModHT group but not in MiHT patients, in which there was even a trend for a lower Fas staining after CPB (p = 0.08). In ModHT patients, a prolonged ischemic time tended to induce a higher increase of Fas (p = 0.061).ConclusionOur data suggest that apoptosis signal cascade is activated at early stages during aortic valve replacement under ModHT CPB. This apoptosis induction can effectively be attenuated by a more normothermic procedure.

Hemodynamic and morphologic alterations after experimental administration of protamine sulfate

The hemodynamic, hematologic, and morphologic effects induced by protamine sulfate have been studied in 28 dogs divided into 6 groups. All of the groups were given heparin (3 mg/kg body weight) and Groups I, II, III, and IV were given protamine (5 mg/kg body weight). Group I (control group) was not subjected to extracorporeal circulation. The other groups had the following interventions: Group II, cardiopulmonary bypass without aortic clamp, hypothermia, or cardioplegia; Groups III and V, hypothermia of 25 degrees C, aortic clamping for 25 minutes, administration of cardioplegic solution, and slow rewarming; and Groups IV and VI, the same as Groups III and V, but with rapid rewarming. After injection of protamine sulfate, there was a decrease in mean arterial pressure due to peripheral vasodilation and an increase in the mean pulmonary pressure due to increased pulmonary vascular resistance; marked diminution of the number of circulating platelets aside from the extracorporeal circulation; a decrease in the contractility of both ventricles with augmented right ventricular work and decreased cardiac output; and right ventricular edema in Groups I, II, III, and IV. These alterations were most evident in Groups III and IV.

Survival of allogeneic hepatocytes transplanted into the thymus.

Background/Aim: Currently, when cell therapy is being considered instead of liver transplantation to treat terminal liver diseases, complete knowledge of the evolution and behavior of ectopically transplanted hepatocytes is a subject of utmost interest in the design of clinical trials. Hepatocytes survive in ectopic locations and have a therapeutic effect in different experimental models. Although it offers remarkable advantages over liver transplantation, hepatocyte transplantation presents several problems, among them the number of cells that can be injected at once and their rejection. Our main objective was to study the survival and functionality of hepatocytes transplanted into the thymus and, secondarily, to test whether the intrathymic transplant could induce any tolerogenic effect. Methods: Hepatocytes from F344 rats were transplanted into thymuses of Gunn rats, half of which received a unique dose of cyclosporine A. The recipients were sacrificed at different times. Light microscopy was performed and bilirubin levels were determined in serum and bile. Results/Conclusions: Transplanted hepatocytes survive for at least 6 weeks in the thymus of allogeneic animals without immunosuppressive therapy. The work provides interesting data about the behavior of hepatocytes injected into this unique ectopic site and shows that the thymus can be used as a recipient organ for cell therapy.

Prevention of arterial thrombosis by a monoclonal antibody against the 100 to 109 amino acid sequence stretch of the beta-subunit of the human platelet fibrinogen receptor: A comparative study with low dose aspirin

OBJECTIVES The aim of this study was to compare, in dogs, the antithrombotic activity of aspirin and the murine monoclonal antibody P37, which inhibits platelet aggregation and fibrinogen binding to activated platelets. BACKGROUND The antithrombotic activity of P37 has been somewhat predictable, given its in vitro platelet antiaggregating activity and localization at or very near the fibrinogen binding site in the platelet fibrinogen receptor, the glycoprotein IIb/IIIa or integrin alpha IIb-beta 3. METHODS The monoclonal antibody P37 of the immunogamma-globulin-1 isotype was prepared according to previously described immunization and fusion protocols and screening assays. To compare its antiaggregating capacity with that of aspirin, experimental thrombosis was induced in all dogs by means of direct current applied to the carotid artery. Autologous platelets had previously been labeled with indium-111 oxine. The dogs were assigned to three groups: group I (n = 18) was the control group; group II (n = 12) was treated orally with 5 mg of aspirin/kg body weight per day for 7 days before induction of thrombosis, and group III (n = 10) was treated intravenously with a single dose of P37 (0.8 mg/kg). RESULTS The indium-111 oxine activity deposited in the thrombi was 12.94 +/- 12.83% (mean +/- SD) in group I, 3.55 +/- 2.99% in group II and 0.03 +/- 0.03% in group III. The differences between groups were always statistically significant (p < 0.05). CONCLUSIONS We conclude that a single dose (0.8 mg/kg) of P37 in a canine model of arterial thrombosis is approximately 100 times more efficient than the administration of aspirin (5 mg/kg per day) in preventing platelet deposition during thrombus formation.

Electrophysiologic assessment of calcium channel blockers in transplanted hearts: An experimental study☆

The effects of calcium channel blockers on automaticity, conduction, and refractoriness were studied in a model of heterotopic heart transplantation in dogs, which combined an innervated heart (recipient) and a denervated one (donor). Following the surgical procedure, 0.2 mg/kg verapamil (n = 10), 0.15 mg/kg diltiazem (n = 10), or 5 microg/kg + 30 microg/kg/h nifedipine (n = 10) was administered intravenously. In basal situation and after drug administration, each heart was assessed for AV interval, cycle length, sinoatrial conduction time, atrioventricular node antegrade block point, and atrioventricular node and ventricular refractory periods; electrocardiographic PR and QT intervals and QRS complexes; systemic arterial, pulmonary artery, central venous, and pulmonary capillary wedge pressures; and cardiac output. The depressor effects of these calcium channel blockers on automaticity, refractoriness, and conduction were more intense in the transplanted hearts, very possibly because of the absence of adrenergic reflexes mediated by the autonomic nervous system; in particular, verapamil produced a great depression of sinus automaticity in a large number of cases.

In Utero Hepatocellular Transplantation in Rats

This work represents a step forward in the experimental design of an in utero hepatocellular transplantation model in rats. We focused on the enrichment optimization of isolated fetal hepatocytes suspension, arranging the surgery methodology of in utero transplantation, monitoring the biodistribution of the transplanted hepatocytes, and assessing the success of the transplants. Rat fetuses have been transplanted at the 17th embryonic day (ED17) with fetal hepatocytes isolated from rats at the end of pregnancy (ED21). We assessed possible differences between lymphocyte population, CD4 positive, CD8 positive, double-positive T-cells, and anti-inflammatory cytokines interleukins 4 and 10 (IL4 and IL10) as well. Cellular viability reached the rates of 90–95%. Transplanted groups had a limited success. Transplanted hepatocytes were not able to pass through the hematoplacental barrier. The hepatocytes injected were primarily located in the liver. There was an upward trend in the whole amount of T CD4 and T CD8 cells. There was an increased IL4 in the transplanted groups observed in the pregnant rats. The possibility to induce tolerance in fetuses with a hepatocyte transplant in utero could be a key point to avoid the immunosuppression treatments which must be undergone by transplanted patients.