RESUMO
Background: In lung transplantation (LT), the length of ischemia time is controversial as it was arbitrarily stablished. We ought to explore the impact of extended cold-ischemia time (CIT) on ischemia-reperfusion injury in an experimental model. Methods: Experimental, randomized pilot trial of parallel groups and final blind analysis using a swine model of LT. Donor animals (n=8) were submitted to organ procurement. Lungs were subjected to 6h (n=4) or 12h (n=4) aerobic hypothermic preservation. The left lung was transplanted and re-perfused for 4h. Lung biopsies were obtained at (i) the beginning of CIT, (ii) the end of CIT, (iii) 30min after reperfusion, and (iv) 4h after reperfusion. Lung-grafts were histologically assessed by microscopic lung injury score and wet-to-dry ratio. Inflammatory response was measured by determination of inflammatory cytokines. Caspase-3 activity was determined as apoptosis marker. Results: We observed no differences on lung injury score or wet-to-dry ratio any given time between lungs subjected to 6h-CIT or 12h-CIT. IL-1β and IL6 showed an upward trend during reperfusion in both groups. TNF-α was peaked within 30min of reperfusion. IFN-γ was hardly detected. Caspase-3 immunoexpression was graded semiquantitatively by the percentage of stained cells. Twenty percent of apoptotic cells were observed 30min after reperfusion. (AU)
Antecedentes: En el trasplante de pulmón (TP), la duración del tiempo de isquemia es controvertida, ya que se estableció de forma arbitraria. Sería útil explorar el impacto del tiempo de isquemia fría (TIF) prolongado sobre la lesión de isquemia-reperfusión en un modelo experimental. Métodos: Ensayo piloto experimental aleatorizado de grupos paralelos y análisis ciego final utilizando un modelo de TP en cerdos. Se extrajeron los órganos de los animales donantes (n=8). Los pulmones se conservaron durante 6 horas (n=4) o 12 horas (n=4) en hipotermia aeróbica. El pulmón izquierdo se trasplantó y reperfundió durante 4 horas. Se obtuvieron biopsias de pulmón (i) al comienzo del TIF, (ii) al final del TIF, (iii) 30 minutos después de la reperfusión y (iv) 4 horas después de la reperfusión. Los injertos de pulmón se evaluaron histológicamente mediante la puntuación de daño histológico pulmonar y la relación de peso húmedo y peso seco. La respuesta inflamatoria se valoró mediante la determinación de citoquinas inflamatorias. Se determinó la actividad de caspasa-3 como marcador de apoptosis. Resultados: No observamos diferencias en la puntuación de daño histológico pulmonar o en la relación de peso húmedo y peso seco en un momento dado entre los pulmones sometidos a 6 h-TIF o 12 h-TIF. Las IL-1β e IL-6 mostraron una tendencia ascendente durante la reperfusión en ambos grupos. El TNF-α alcanzó su punto máximo dentro de los 30 minutos posteriores a la reperfusión. Apenas se detectó IFN-γ. La inmunoexpresión de caspasa-3 se clasificó semicuantitativamente por el porcentaje de células teñidas. Se observó un 20% de células apoptóticas 30 minutos después de la reperfusión. (AU)
Assuntos
Animais , Lesão Pulmonar , Transplante de Pulmão , Traumatismo por Reperfusão , Isquemia Fria , Preservação de Órgãos , SuínosRESUMO
BACKGROUND: In lung transplantation (LT), the length of ischemia time is controversial as it was arbitrarily stablished. We ought to explore the impact of extended cold-ischemia time (CIT) on ischemia-reperfusion injury in an experimental model. METHODS: Experimental, randomized pilot trial of parallel groups and final blind analysis using a swine model of LT. Donor animals (n=8) were submitted to organ procurement. Lungs were subjected to 6h (n=4) or 12h (n=4) aerobic hypothermic preservation. The left lung was transplanted and re-perfused for 4h. Lung biopsies were obtained at (i) the beginning of CIT, (ii) the end of CIT, (iii) 30min after reperfusion, and (iv) 4h after reperfusion. Lung-grafts were histologically assessed by microscopic lung injury score and wet-to-dry ratio. Inflammatory response was measured by determination of inflammatory cytokines. Caspase-3 activity was determined as apoptosis marker. RESULTS: We observed no differences on lung injury score or wet-to-dry ratio any given time between lungs subjected to 6h-CIT or 12h-CIT. IL-1ß and IL6 showed an upward trend during reperfusion in both groups. TNF-α was peaked within 30min of reperfusion. IFN-γ was hardly detected. Caspase-3 immunoexpression was graded semiquantitatively by the percentage of stained cells. Twenty percent of apoptotic cells were observed 30min after reperfusion. CONCLUSIONS: We observed that 6 and 12h of CIT were equivalent in terms of microscopic lung injury, inflammatory profile and apoptosis in a LT swine model. The extent of lung injury measured by microscopic lung injury score, proinflammatory cytokines and caspase-3 determination was mild.
RESUMO
BACKGROUND: In lung transplantation (LT), the length of ischemia time is controversial as it was arbitrarily stablished. We ought to explore the impact of extended cold-ischemia time (CIT) on ischemia-reperfusion injury in an experimental model. METHODS: Experimental, randomized pilot trial of parallel groups and final blind analysis using a swine model of LT. Donor animals (n=8) were submitted to organ procurement. Lungs were subjected to 6h (n=4) or 12h (n=4) aerobic hypothermic preservation. The left lung was transplanted and re-perfused for 4h. Lung biopsies were obtained at (i) the beginning of CIT, (ii) the end of CIT, (iii) 30min after reperfusion, and (iv) 4h after reperfusion. Lung-grafts were histologically assessed by microscopic lung injury score and wet-to-dry ratio. Inflammatory response was measured by determination of inflammatory cytokines. Caspase-3 activity was determined as apoptosis marker. RESULTS: We observed no differences on lung injury score or wet-to-dry ratio any given time between lungs subjected to 6h-CIT or 12h-CIT. IL-1ß and IL6 showed an upward trend during reperfusion in both groups. TNF-α was peaked within 30min of reperfusion. IFN-γ was hardly detected. Caspase-3 immunoexpression was graded semiquantitatively by the percentage of stained cells. Twenty percent of apoptotic cells were observed 30min after reperfusion. CONCLUSIONS: We observed that 6 and 12h of CIT were equivalent in terms of microscopic lung injury, inflammatory profile and apoptosis in a LT swine model. The extent of lung injury measured by microscopic lung injury score, proinflammatory cytokines and caspase-3 determination was mild.
Assuntos
Lesão Pulmonar , Transplante de Pulmão , Traumatismo por Reperfusão , Animais , Caspase 3 , Citocinas , Isquemia/patologia , Pulmão/patologia , Lesão Pulmonar/etiologia , Preservação de Órgãos , Projetos Piloto , Distribuição Aleatória , Traumatismo por Reperfusão/prevenção & controle , SuínosRESUMO
Background and objectives: The aim of this study was to evaluate the effects of remote ischemic preconditioning by brief ischemia of unilateral hind limb when combined with dexmedetomidine on renal ischemia-reperfusion injury by histopathology and active caspase-3 immunoreactivity in rats. Methods: 28 Wistar albino male rats were divided into 4 groups. Group I (Sham, n = 7): Laparotomy and renal pedicle dissection were performed at 65th minute of anesthesia and the rats were observed under anesthesia for 130min. Group II (ischemia-reperfusion, n = 7): At 65th minute of anesthesia bilateral renal pedicles were clamped. After 60 min ischemia 24 h of reperfusion was performed. Group III (ischemia-reperfusion + dexmedetomidine, n = 7): At the fifth minute of reperfusion (100 μg/kg intra-peritoneal) dexmedetomidine was administered with ischemia-reperfusion group. Reperfusion lasted 24 h. Group IV (ischemia-reperfusion + remote ischemic preconditioning + dexmedetomidine, n = 7): After laparotomy, three cycles of ischemic preconditioning (10 min ischemia and 10 min reperfusion) were applied to the left hind limb and after 5 min with group III. Results: Histopathological injury scores and active caspase-3 immunoreactivity were significantly lower in the Sham group compared to the other groups. Histopathological injury scores in groups III and IV were significantly lower than group II (p = 0.03 and p = 0.05). Active caspase-3 immunoreactivity was significantly lower in the group IV than group II (p = 0.01) and there was no significant difference between group II and group III (p = 0.06). Conclusions: Pharmacologic conditioning with dexmedetomidine and remote ischemic preconditioning when combined with dexmedetomidine significantly decreases renal ischemia- reperfusion injury histomorphologically. Combined use of two methods prevents apoptosis via active caspase-3. .
Justificativa e objetivos: Avaliar os efeitos do pré-condicionamento isquêmico remoto, mediante breve isquemia de membro posterior unilateral, em combinação com dexmedetomidina em lesão de isquemia-reperfusão renal por meio de histopatologia e imunorreatividade da caspase-3 ativa em ratos. Métodos: Foram divididos em quatro grupos 28 ratos machos albinos Wistar. Grupo I (Sham cirurgia controle], n = 7): laparotomia e dissecção do pedículo renal foram feitas em 65 minutos de anestesia e os ratos foram observados sob anestesia por 130 minutos. Grupo II (isquemia-reperfusão, n = 7): no 65° minuto de anestesia, os pedículos renais bilaterais foram pinçados; após 60 minutos de isquemia, foi feita reperfusão de 24 horas. Grupo III (isquemia-reperfusão + dexmedetomidina, n = 7): no quinto minuto de reperfusão, dexmedetomidina (100 mg/kg intraperitoneal) foi administrada ao grupo com isquemia-reperfusão. A reperfusão durou 24 horas. Grupo IV (isquemia-reperfusão + pré-condicionamento isquêmico remoto + dexmedetomidina, n = 7): após a laparotomia, três ciclos de pré-condicionamento isquêmico (10minutos de isquemia e 10minutos de reperfusão) foram aplicados no membro posterior esquerdo e depois de cincominutos ao grupo III. Resultados: Os escores de lesão histopatológica e imunorreatividade da caspase-3 ativa foram significativamente menores no grupo Sham em comparação com os outros. Os escores de lesão histopatológica dos grupos III e IV foram significativamente menores do que os do II (p = 0,03 e p = 0,05). A imunorreatividade da caspase-3 foi significativamente menor no grupo IV do que no II (p = 0,01) e não houve diferença significante entre os grupos II e III (p = 0,06). Conclusões: O condicionamento farmacológico com dexmedetomidina e o pré...
Introducción y objetivos: El objetivo de este estudio fue evaluar los efectos del precondicionamiento isquémico remoto mediante breve isquemia del miembro posterior unilateral en combinación con la dexmedetomidina en la lesión de isquemia-reperfusión renal por medio de histopatología e inmunoreactividad de la caspasa-3 activa en ratones. Métodos: 28 ratones machos albinos Wistar fueron divididos en 4 grupos. Grupo I (Sham cirugía control], n =7): se realizó laparotomia y disección del pediculo renal en 65 min de anestesia y los ratones fueron observados bajo anestesia durante 130min. Grupo II (isquemia-reperfusión, n = 7): en el sexagésimo quinto minuto de anestesia, los pídiculos renales bilaterales fueron pinzados; después de 60min de isquemia, se realizaron 24h de reperfusión. Grupo III (isquemia-reperfusión + dexmedetomidina, n = 7): al quinto minuto de reperfusión, la dexmedetomidina (100 μg/kg intraperitoneal) fue administrada en el grupo con isquemia-reperfusión; la reperfusión duró 24 h. Grupo IV (isquemia-reperfusión + precondicionamiento isquémico remoto + dexmedetomidina, n=7): después de la laparotomía, se aplicaron 3 ciclos de precondicionamiento isquémico (10 min de isquemia y 10 min de reperfusión) en el miembro posterior izquierdo y después de 5 min en el grupo in. Resultados: Las puntuaciones de lesión histopatológica e inmunoreactividad de la caspasa-3 activa fueron significativamente menores en el grupo Sham en comparación con los otros grupos. Las puntuaciones de lesión histopatológica de los grupos III y IV fueron significativamente menores que las del grupo II (p = 0,03 y p = 0,05). La inmunorreactividad de la caspasa-3 fue significativamente menor en el grupo IV que en el grupo II (p = 0,01) y no hubo diferencia significativa entre los grupos II ...