RESUMEN
INTRODUCTION: Hypothermic machine perfusion, an organ preservation modality, involves flow of chilled preservation fluid through an allograft's vasculature. This study describes a simple, reproducible, human model that allows for interrogation of flow effects during ex vivo organ perfusion. MATERIALS AND METHODS: Gonadal veins from deceased human renal allografts were subjected to either static cold storage or hypothermic machine perfusion for up to 24 hours. Caspase-3, Krüppel-like factor 2 expression and electron microscopic analysis were compared between 'flow' and 'no-flow' conditions, with living donor gonadal vein sections serving as negative controls. RESULTS: The increase in caspase-3 expression was less pronounced for hypothermic machine-perfused veins compared with static cold storage (median-fold increase 1.2 vs 2.3; P < 0.05). Transmission electron microscopy provided ultrastructural corroboration of endothelial cell apoptosis in static cold storage conditions. For static cold storage preserved veins, Krüppel-like factor 2 expression diminished in a time-dependent manner between baseline and 12 hours (P < 0.05) but was abrogated and reversed by hypothermic machine perfusion (P < 0.05). CONCLUSIONS: Our methodology is a simple, reproducible and successful model of ex vivo perfusion in the context of human organ preservation. To demonstrate the model's utility, we establish that two widely used markers of endothelial health (caspase-3 and Krüppel-like factor 2) differ between the flow and no-flow conditions of the two predominant kidney preservation modalities. These findings suggest that ex vivo perfusion may mediate the induction of a biochemically favourable endothelial niche which may contribute tohypothermic machine perfusion's association with improved renal transplantation outcomes.
Asunto(s)
Trasplante de Riñón/métodos , Riñón/irrigación sanguínea , Modelos Biológicos , Soluciones Preservantes de Órganos/farmacocinética , Preservación de Órganos/métodos , Apoptosis , Biomarcadores/metabolismo , Cadáver , Caspasa 3/metabolismo , Frío , Endotelio Vascular/metabolismo , Humanos , Riñón/metabolismo , Riñón/ultraestructura , Factores de Transcripción de Tipo Kruppel/metabolismo , Microscopía Electrónica , Perfusión/métodos , Venas/metabolismo , Venas/ultraestructuraRESUMEN
BACKGROUND: Proximal tubule cells have specialized apical membranes with microvilli that provide an extensive surface area for unidirectional transport of solute from lumen to blood. The major structural solute component is F-actin, which interacts with transmembrane proteins, including ion transport molecules related to normal absorptive and secretory functions. Our study was to evaluate F-actin and fluid absorption (Jv) in proximal tubules after exposure to preservation solutions. METHODS: In vitro microperfusion technique and immunohistochemistry analysis. RESULTS: 1. Absorptions were similar in 1- and 24-hour-preserved tubules, as well as in fresh tubules. The exception was tubules for 24 hours in Euro-Collins solution, which did not show absorption, suggesting that it was affected. 2. Fluorescence intensity of actin tubules preserved for 1 hour in both solutions showed similar values to each other and to the control group; tubules preserved for 24 hours in both solutions were similar to each other, although statistically different than the control group and those preserved for 1 hour in Belzer (UW) solution. CONCLUSION: There were differences among groups in the distribution of F-actin; Jv values were different for 24-hour preservation in each solution, whereas fluorescence intensity was similar in both 24-hour solutions. Thus, actin cytoskeleton was not responsible for it, because 24-hour preservation in UW showed Jv results comparable to the control group.
Asunto(s)
Actinas/fisiología , Citoesqueleto/fisiología , Túbulos Renales Proximales/fisiología , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Absorción , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Actinas/ultraestructura , Adenosina/farmacología , Alopurinol/farmacología , Animales , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Glutatión/farmacología , Insulina/farmacología , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/fisiología , Túbulos Renales Colectores/ultraestructura , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/ultraestructura , Masculino , Microscopía Confocal/métodos , Microvellosidades/efectos de los fármacos , Microvellosidades/ultraestructura , Modelos Animales , Soluciones Preservantes de Órganos/farmacocinética , Perfusión/métodos , Conejos , Rafinosa/farmacologíaRESUMEN
OBJECTIVES: This study aims to investigate the importance of ISO11405 recommended storage regime for extracted teeth in surface disinfectant chloramine T (chlT) prior to use in biofilm or in vitro caries studies involving microorganisms. ChlT may be absorbed into dentin and undergoes breakdown with organic material. METHODS: Extracted roots were stored in chlT (2 days), rinsed and transferred to distilled deionised water. HPLC at regular intervals determined chlT elution. At 4 weeks roots were boiled in water and eluent assessed with HPLC. ChlT breakdown (+/-organic material) over time was monitored with HPLC. ChlT minimum inhibitory/bactericidal concentration (MIC/MBC) against Lactobacillus acidophilus was evaluated using L. acidophilus broth and chlT serial dilutions. RESULTS: No significant increase in chlT elution was detected between 2h and 4 weeks (ANOVA, Tukeys, p>0.05), although levels tended to increase with time. ChlT detected in water was 0.005%, corresponding to 0.05% in dentin. After boiling (4 weeks) chlT breakdown products in water corresponded to 0.015% in dentin. MIC/MBC of chlT against L. acidophilus was 0.031%. SIGNIFICANCE: ChlT breakdown is accelerated by organic material. L. acidophilus is highly sensitive to chlT. ChlT readily leaches from dentin but rinsing does not reduce chlT concentration below MIC/MBC. Low levels of chlT may remain but will probably be in a less active form. Teeth disinfected in chlT for use in research involving bacteria must be stored in distilled water for at least 2h to reduce chlT concentration below MBC, although longer will give greater elution and breakdown.
Asunto(s)
Cloraminas , Descontaminación/métodos , Desinfectantes Dentales , Compuestos de Tosilo , Cloraminas/metabolismo , Cloraminas/farmacología , Cromatografía Líquida de Alta Presión , Recuento de Colonia Microbiana , Desinfectantes Dentales/metabolismo , Desinfectantes Dentales/farmacología , Dentina , Permeabilidad de la Dentina , Humanos , Lactobacillus/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Soluciones Preservantes de Órganos/metabolismo , Soluciones Preservantes de Órganos/farmacocinética , Sulfonamidas/metabolismo , Tolueno/análogos & derivados , Tolueno/metabolismo , Compuestos de Tosilo/metabolismo , Compuestos de Tosilo/farmacologíaRESUMEN
Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.
Asunto(s)
Antimetabolitos/farmacología , Monóxido de Carbono/farmacología , Intestino Delgado/irrigación sanguínea , Intestino Delgado/trasplante , Soluciones Preservantes de Órganos/farmacología , Trasplante de Órganos/efectos adversos , Daño por Reperfusión/prevención & control , Adenosina/química , Adenosina/farmacocinética , Adenosina/farmacología , Alopurinol/química , Alopurinol/farmacocinética , Alopurinol/farmacología , Animales , Antimetabolitos/análisis , Antimetabolitos/farmacocinética , Monóxido de Carbono/análisis , Monóxido de Carbono/farmacocinética , Modelos Animales de Enfermedad , Glutatión/química , Glutatión/farmacocinética , Glutatión/farmacología , Supervivencia de Injerto/efectos de los fármacos , Insulina/química , Insulina/farmacocinética , Insulina/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , Intestino Delgado/metabolismo , Masculino , Microscopía Electrónica de Transmisión , Preservación de Órganos , Soluciones Preservantes de Órganos/química , Soluciones Preservantes de Órganos/farmacocinética , Rafinosa/química , Rafinosa/farmacocinética , Rafinosa/farmacología , Ratas , Ratas Endogámicas Lew , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: To determine the behavior of isolated primordial follicles that were exposed to different concentrations of dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH), and glycerol (GLY). DESIGN: Isolated primordial follicles were exposed to the cryoprotectant (CPA) solution and photographed to calculate their volume at different periods of exposure. SETTING: Laboratorio Renzo Giuliani, University of Florence, Italy. ANIMAL(S): Lambs, 30-40 days old. INTERVENTION(S): Isolation of primordial follicles and subsequent exposure to CPA. MAIN OUTCOME MEASURE(S): Follicular volume. RESULT(S): At 2 minutes of CPA exposure, all follicles appeared to be shrunken. At approximately 5 minutes, shrinkage ceased, and follicles started to swell, absorbing the CPA and water to maintain osmotic equilibrium. When DMSO was tested, follicular dehydration in all concentrations did not exceed 17%; with PROH and EG, it reached 33% and 27%, respectively. The highest degree of dehydration (48%) was seen with GLY. In almost all tested concentrations, follicular shrinkage occurred up to 5 minutes. CONCLUSION(S): Volume changes in isolated primordial follicles can fluctuate according to the CPA used and its concentration.
Asunto(s)
Permeabilidad de la Membrana Celular , Crioprotectores/farmacocinética , Soluciones Preservantes de Órganos/farmacocinética , Folículo Ovárico/fisiología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Crioprotectores/administración & dosificación , Citoprotección/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Soluciones Preservantes de Órganos/administración & dosificación , Permeabilidad/efectos de los fármacos , OvinosRESUMEN
La isquemia fría es el mejor método actual para preservar el riñón para trasplante. Sin embargo condiciona, en primer lugar, un proceso de necrosis celular directo. En segundo lugar, durante el período de hipotermia se desarrolla un proceso de lesión mitocondrial que hace que la célula entre en un estado de apoptosis latente. Esta apoptosis se completa definitivamente en esta población celular en el momento de la revascularización. Las soluciones de preservación actualmente disponibles no son perfectas, y no pueden evitar este proceso. La adición de ciertas sustancias en la solución de UW ha demostrado experimentalmente (Desferrioxamina) una reducción de las lesiones mitocondriales producidas durante la fase de hipotermia. La utilización de perfusión de riñón aislado podría, comparativamente con la hipotermia simple, reducir la aparición de disfunción inicial del injerto hasta en un 20%, lo que tendría consecuencias importantes tanto funcionales como económicas (AU)
Cold ischemia is the best known method to preserve kidneys for transplant. However, it produces several detrimental effects. First, cellular necrosis. Secondarily, during the hypothermic period a mithocondrial injury process develops which makes the cell entering a pre-apoptotic state. This apoptosis occurs definitively in the reperfusion. Preservation solutions currently available are not perfect and are not able to avoid cold-related cell injuries. The adition of certain substances to UW solution (desferrioxamine) has shown experimentally a reduction in mitochondrial cold-related lesions. Isolated hypothermic kidney perfusion reduces initial graft dysfunction about 20% in comparison to hypothermic storage. This fact relates to important either economical as functional consequences (AU)
Asunto(s)
Humanos , Trasplante de Riñón/métodos , Preservación de Órganos/métodos , Perfusión/métodos , Criopreservación , Soluciones Preservantes de Órganos/farmacocinética , Isquemia/prevención & controlRESUMEN
BACKGROUND: novel donor heart preservation solution was formulated to produce hyperpolarized arrest with the potassium channel opener, pinacidil. The superior cardioprotective efficacy of this solution has been demonstrated previously when compared to University of Wisconsin solution following 4 hours of hypothermic ischemia. This study tested the hypothesis that pinacidil solution may extend preservation time and provide superior cardioprotective efficacy following 12 hours of ischemia. METHODS: Sixteen rabbit hearts were assigned to receive either pinacidil solution or University of Wisconsin solution in a crystalloid-perfused Langendorff model. Thirty minutes of initial perfusion preceded baseline data acquisition. Left ventricle pressure-volume curves were generated by inflating an intra-ventricular latex balloon. Following cardioplegic administration, hearts underwent 12 hours of hypothermic storage. After 60 minutes of reperfusion, post-ischemic data were acquired. RESULTS: Pinacidil solution demonstrated significantly better myocardial preservation compared to University of Wisconsin solution, with better recovery of developed pressure (53.0 +/- 11.1% vs 20.7 +/- 4.3%, p = 0.017, respectively), post-ischemic coronary flow (55.3 +/- 12.6% vs 23.9 +/- 4.3%, p = 0.034), maximum systolic dP/dT (46.4 +/- 8.3% vs 20.2 +/- 5.1%, p = 0.018) and minimum diastolic -dP/dT (65.3 +/- 10.8% vs 20.2 +/- 5.1%, p = 0.002). Diastolic compliance, expressed as baseline/post-ischemic diastolic slope ratios, was also better preserved by pinacidil solution (0.55 +/- 0.09) vs University of Wisconsin solution (0.40 +/- 0.03) (p = 0.135). CONCLUSIONS: A novel pinacidil solution resulted in improved donor heart preservation during 12 hours of hypothermic ischemia compared to the "gold standard," University of Wisconsin solution. Adopting alternative strategies of hyperpolarized arrest may allow extension of preservation time beyond the limits of traditional depolarizing solutions.
Asunto(s)
Trasplante de Corazón , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Pinacidilo/farmacología , Vasodilatadores/farmacología , Animales , Hemodinámica/efectos de los fármacos , Isquemia Miocárdica/fisiopatología , Soluciones Preservantes de Órganos/farmacocinética , Pinacidilo/farmacocinética , Conejos , Factores de Tiempo , Vasodilatadores/farmacocinéticaRESUMEN
OBJECTIVE: The present study was performed to investigate the influence of different routes of perfusion on the distribution of the preservation solutions in the lung parenchyma and upper airways. METHODS: Pigs were divided into four groups: control (n = 6), pulmonary artery (PA) (n = 6), simultaneous PA + bronchial artery (BA) (n = 8), and retrograde delivery (n = 6). After preparation and cannulation, cardioplegia solution and Euro-Collins solution (ECS) for lung preservation were given simultaneously. After removal of the heart, the double lung bloc was harvested. Following parameters were assessed: total and regional perfusion (dye-labeled microspheres), tissue water content, PA, aorta, left atrial and left ventricular pressures, cardiac output and lung temperature. RESULTS: Our data show that flow of the ECS in lung parenchyma did not reach control values (9.4+/-1.0 ml/min per g lung wet weight) regardless of the route of delivery (PA 6.3+/-1.5, PA + BA 4.8+/-0.9, retrograde 2.7+/-0.9 ml/min per g lung wet weight). However, flow in the proximal and distal trachea were significantly increased by PA + BA delivery (0.970+/-0.4, respectively, 0.380+/-0.2 ml/min per g) in comparison with PA (0.023+/-0.007, respectively, 0.024+/-0.070 ml/min per g), retrograde (0.009+/-0.003, respectively, 0.021+/-0.006 ml/min per g) and control experiments (0.125+/-0.0018, respectively, 0.105+/-0.012 ml/g per min). Similarly the highest flow rates in the right main bronchus were achieved by PA + BA delivery (1.04+/-0.4 ml/min per g) in comparison with 0.11+/-0.03 in control, 0.033+/-0.008 in PA, and 0.019+/-0.005 ml/min per g in retrograde group. Flows in the left main bronchus were 0.09+/-0.02 ml/min per g in control, 0.045+/-0.012 ml/min per g in PA, and 0.027+/-0.006 ml/min per g in retrograde group. The flow rates were significantly (P = 0.001) increased by PA + BA delivery of the storage solution (0.97+/-0.3 ml/min per g). CONCLUSIONS: Our data show that the distribution of ECS for lung preservation is significantly improved in airway tissues (trachea and bronchi) if a simultaneous PA + BA delivery is used.
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Pulmón , Soluciones Preservantes de Órganos/farmacocinética , Sistema Respiratorio/metabolismo , Animales , Bronquios/irrigación sanguínea , Bronquios/metabolismo , Soluciones Hipertónicas/administración & dosificación , Soluciones Hipertónicas/farmacocinética , Pulmón/metabolismo , Masculino , Soluciones Preservantes de Órganos/administración & dosificación , Flujo Sanguíneo Regional , Sistema Respiratorio/irrigación sanguínea , Porcinos , Distribución Tisular , Tráquea/irrigación sanguínea , Tráquea/metabolismoRESUMEN
In rat liver and kidney preservation, hepatic and renal uptake of 3H-adenosine, 3H-glutathione, and 3H-raffinose from University of Wisconsin solution and diffusion to the interstitial space were measured. At 4 degrees C only 0.38+/-0.47% and 2+/-0.92% of the total 3H-adenosine remained in the kidney and in the liver, respectively, but at 37 degrees C the amount remaining was 1+/-1% and 12+/-3% (P<0.001). Hepatic and renal uptake of the impermeant 3H-raffinose was unaffected by temperature. During flush out, interstitial accumulation of adenosine was significantly higher in livers than in kidneys and decreased during 24-h cold storage. Glutathione accumulation in the interstitial space was two orders of magnitude lower than 3H-adenosine accumulation and comparable to the impermeant raffinose. In summary, the bioavailability of components of preservation solutions at 4 degrees C is lower than at physiological temperatures, so that the application of cytoprotectants at 37 degrees C to organ donors, rather than simple addition to the cold storage solution, might improve cold storage preservation of livers and kidneys.
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Riñón , Hígado , Soluciones Preservantes de Órganos/farmacocinética , Preservación de Órganos/métodos , Adenosina/farmacocinética , Alopurinol/farmacocinética , Animales , Glutatión/farmacocinética , Insulina/farmacocinética , Masculino , Microdiálisis , Perfusión , Rafinosa/farmacocinética , Ratas , Ratas Wistar , Temperatura , TritioRESUMEN
O transplante pulmonar tem sido realizado com sucesso no tratamento de pacientes selecionados com doenças pulmonares no seu estágio final, apesar de ainda muito limitado quanto ao seu uso, pela escassez de doadoes adequados.A aquisiçäo à distância de órgäos adequados, poderá aumentar consideravelmente o número de transplantes pulmonares e esforços se concentram na possibilidade de proteçäo dos pulmöes por tempos maiores de isquemia e de reduçäo das lesöes nas fases iniciais de reperfusäo. As técnicas de preservaçäo atualmente utilizadas para a aquisiçäo à distância de pulmöes, incluem a auto-perfusäo isolada, o bypass cardio-pulmonar com hipotermia profunda e a preservaçäo estática com várias formas de perfusäo rápida via artéria pulmonar.As complicaçöes nas anastomoses brônquicas, continuam sendo importante fator de morbidade e de mortalidade em transplantes. Entre outros fatores, a qualidade de proteçäo das vias aéreas parece contribuir para melhor cicatrizaçäo brônquica.Na maioria dos centros de transplante, a técnica de preservaçäo pulmonar mais utilizada é a perfusäo rápida via artéria pulmonar, seguida por armazenamento hipotérmico dos pulmöes. Com esta técnica, a soluçäo de preservaçäo parece atingir a circulaçäo tráqueo-brônquica por meio de anastomoses, talvez nem sempre suficientes, ocasionando complicaçöes isquêmicas nas anastomoses das vias aéreas superiores. Vias de perfusäo rápida alternativas säo representadas pela retrógrada, via átrio esquerdo e veias pulmonares e pela anterógrada simultânea, via artéria pulmonar e segmento de aorta contendo as artérias brônquicas. No presente trabalho, foi feito um estudo comparativo sobre a distribuiçäo da soluçäo Euro-Collins em pulmöes de porcos através de diferentes vias de perfusäo rápida e foi utilizada a técnica de micro-esferas coloridas, näo-radioativas, para a mensuraçäo de fluxo ao parênquima pulmonar e às vias aéreas. Os animais foram divididos em 4 grupos: grupo controle (n=6); grupo AP (artéria pulmonar (n=6); grupo AP + AB (artéria pulmonar e artérias brônquicas contidas em seguimento de aorta) (n=8) e grupo VP (veias pulmonares) (n=6). Após preparaçäo cirúrgica, canulaçöes e cardioplegia, iniciava-se infusäo rápida de Euro-Collins através de uma das diferentes vias descritas, conforme o grupo a que pertencia o animal. Removia-se entäo o coraçäo e, após, o bloco contendo os dois pulmöes...