RESUMEN
BACKGROUND: We describe a new surgical technique of in situ intestinal ischemia-reperfusion injury in the pig, which includes transection of the small bowel, extrinsic autonomic denervation, lymphatic disruption, and finally in-situ cold ischemia of the graft by flushing through the first jejunal artery. MATERIAL AND METHODS: Ten female pigs were used for the study. All neural and lymphatic connections to the jejunoileum were transected. The stripped superior mesenteric vessels remained as the only connections. The skeletonized mesenteric vessels were clamped and the superior mesenteric artery was cannulated through the first jejunal artery. The isolated jejunoileum was flushed with cold IGL-1 solution. A small incision on the superior mesenteric vein was made to allow outflow of the effluent. After the flushing process was complete, the small incision in the superior mesenteric vein was closed and the vascular clamps were removed. The proximal 70% of the graft was resected. RESULTS: The mean preoperative weight of the animals was 25.8 +/-7.6 kg. The mean duration of the operation was 242.0 +/- 28.6 min. The mean cold ischemia time was 47.6 +/- 3.9 min. All animals survived the procedure and were sacrificed at day 8. At sacrifice, there were no adhesions. The small bowel appeared normal. On intestinal histology, there were no significant changes between specimens obtained from the animal immediately at the end of cold flushing (T0), 2 h after reperfusion (T1), and at sacrifice (T2). CONCLUSIONS: This novel technique for intestinal ischemia-reperfusion injury in the pig provides an extremely useful model for experimental studies of immunological and cold ischemia-reperfusion injury of transplanted small bowels.
Asunto(s)
Íleon/irrigación sanguínea , Yeyuno/irrigación sanguínea , Daño por Reperfusión , Animales , Desnervación Autonómica , Isquemia Fría/métodos , Modelos Animales de Enfermedad , Femenino , Íleon/inmunología , Intestino Delgado/trasplante , Yeyuno/inmunología , Arteria Mesentérica Superior/cirugía , Porcinos , Trasplante AutólogoRESUMEN
AIM: To compare, in a pig liver transplantation model, the protective effect of UW with that of IGL-1, a high-sodium preservation solution containing polyethylene glycol (PEG) as an oncotic supply. METHODS: All livers were harvested and grafted orthotopically according to standard techniques. The livers were washed out and preserved for 7 h in IGL-1 (n = 6) or in UW solution (n = 7) at 4 degree centigrade. In a sham group (n = 4), the livers underwent a 60-min warm ischemia at 37 degree centigrade. The hepatocellular injury was assessed in organ preservation solution washed out from the graft at the end of ischemic storage (before revascularization), and in serum 2 h after reperfusion and daily for up to 6 d. RESULTS: Livers preserved in IGL-1 solution released markedly less AST than that preserved in the UW solution before and after revascularization (P < 0.05). Besides, the activity of creatine kinase-BB, a marker of sinusoidal lining cells injury, was higher in the UW group than in the IGL-1 group (P < 0.05). Histological results showed less necrotic regions in livers preserved in IGL-1 solution; however, no difference was observed for inflammation. CONCLUSION: IGL-1 liquid effectively protects parenchymal and non-parenchymal cells against preservation-reperfusion injuries.
Asunto(s)
Trasplante de Hígado/métodos , Soluciones Preservantes de Órganos/farmacología , Animales , Creatina Quinasa/metabolismo , Estudios de Evaluación como Asunto , Femenino , Isquemia , Polietilenglicoles/química , Daño por Reperfusión/prevención & control , Porcinos , Factores de TiempoRESUMEN
A new Institut Georges Lopez (IGL-1) solution was used to preserve steatotic livers. Steatotic (obese [Ob]) and nonsteatotic (lean [Ln]) livers from Zücker rats (n = 16, 8 Ln and 8 Ob) were preserved for 24 hours at 4 degrees C in University of Wisconsin (UW) or IGL-1 solution, respectively, and then perfused ex vivo for 2 hours at 37 degrees C. Additionally, Ob and Ln livers (n = 16, 8 Ln and 8 Ob) were preserved in IGL-1 plus Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME). Hepatic injury and function (aminotransferases, bile production, bromosulfophthalein clearance), and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion injury, such as oxidative stress, mitochondrial damage, and vascular resistance, were studied. Nitric oxide (NO) production and constitutive and inducible NO synthase were also measured. Steatotic and nonsteatotic livers preserved in IGL-1 solution showed lower transaminases, malondialdehyde, glutamate dehydrogenase levels, and higher bile production than UW-solution-preserved livers. IGL-1 solution protected against oxidative stress, mitochondrial damage and the alterations in vascular resistance associated with cold ischemia-reperfusion. Thus, at the end of reperfusion period, aspartate aminotransferase levels in steatotic livers were 281 +/- 6 U/L in UW vs. 202 +/- 10 U/L in IGL-1 solution. Glutamate dehydrogenase was 463 +/- 75 U/L in UW vs. 111 +/- 4 U/L in IGL-1 solution, and oxidative stress was 3.0 +/- 0.1 nmol/mg prot in UW vs. 2.0 +/- 0.1 nmol/mg prot in IGL-1 solution. These beneficial effects of IGL-1 solution were abolished by the addition of L-NAME, which implicates NO in the benefits of IGL-1. In conclusion, IGL-1 solution provided steatotic livers with better protection against the deleterious effects of cold ischemia-reperfusion injury than did UW solution.
Asunto(s)
Hígado Graso/metabolismo , Hígado/metabolismo , Soluciones Preservantes de Órganos/metabolismo , Preservación de Órganos/métodos , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Bilis/metabolismo , Hígado Graso/patología , Glutamato Deshidrogenasa/metabolismo , Hígado/patología , Pruebas de Función Hepática , Malondialdehído/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Obesidad/metabolismo , Soluciones Preservantes de Órganos/química , Ratas , Ratas Zucker , Reperfusión , Temperatura , Resistencia VascularRESUMEN
INTRODUCTION: Renal ischaemia and reperfusion lesions partly determine short-term and long-term graft survival. Organ preservation conditions appear to play a decisive role. This article presents the preclinical experimental results obtained in renal transplantation with an extracellular organ preservation solution, in which polyethylene glycol (PEG) is used as colloid. METHODS AND RESULTS: The effects of inversion of Na+ and K+ gradients in the IGL-1 preservation solution compared to UW and replacement of hydroxylethyl starch (HES) by PEG were evaluated in an ex vivo isolated, perfused rat kidney model and then in a pig renal autotransplantation model. In these experimental models, after 24 hours of static storage, the sodium reabsorption fraction correlated with the quality of tubular function of the kidney and the glomerular filtration rate were constantly better in the IGL-1 group than in the UW group. In vivo, in the pig, resumption of renal function was significantly better in the IGL-1 group and histological examination demonstrated a significant reduction of expression of Major Histocompatibility Complex (MHC) type II, an indirect marker of inflammation, but also a reduction of markers of apoptosis and fibrosis for kidneys preserved in IGL-1. CONCLUSION: In animal renal transplantation, IGL-1 ensures better resumption of renal function than UW, which currently remains the "gold standard"preservation solution. Further studies must be conducted to determine whether this new generation solution can replace UW as the reference solution.
Asunto(s)
Trasplante de Riñón , Riñón , Soluciones Preservantes de Órganos/farmacología , Animales , Biomarcadores/metabolismo , Evaluación Preclínica de Medicamentos , Tasa de Filtración Glomerular , Antígenos de Histocompatibilidad Clase II/metabolismo , Masculino , Modelos Animales , Polietilenglicoles , Ratas , Ratas Sprague-Dawley , Trasplante AutólogoRESUMEN
Ischemia-reperfusion injury conditions short-term and long-term graft function. The effects of the inversion of K+ and Na+ concentrations and substitution with polyethylene glycol for hydroxyethyl starch in University of Wisconsin (K-UW) solution were evaluated in isolated perfused rat kidneys and in autotransplanted pig kidneys. In the rat model kidneys were cold-stored for 24 h in K-UW or Na-UW or Na-PEG UW solutions (IGL-1 solution). Fractional sodium reabsorption and glomerular filtration rate was better in kidneys preserved in Na-UW and IGL-1 solution than those preserved in K-UW solution. In the pig model the left kidney was harvested and preserved in K-UW or IGL-1 solution for 24 h and then transplanted. In the autotransplanted pig model, kidneys preserved in IGL-1 solution showed a better function and a significant reduction of MHC class II expression, cellular apoptosis and interstitial fibrosis. In conclusion, kidneys preserved in IGL-1 solution tolerated ischemia/reperfusion injury better than those preserved in K-UW solution.
Asunto(s)
Trasplante de Riñón , Soluciones Preservantes de Órganos/farmacología , Animales , Evaluación Preclínica de Medicamentos , Técnicas In Vitro , Masculino , Modelos Animales , Soluciones Preservantes de Órganos/química , Perfusión , Ratas , Ratas Sprague-Dawley , Porcinos , Trasplante AutólogoRESUMEN
BACKGROUND: Ischemia-reperfusion injury has been associated with both early and late effects on allografts in the form of delayed graft function and decreased graft survival. Recent studies demonstrated that functional parameters were influenced by cold storage conditions and particularly the ratio of Na+:K+ of the preservation solution. METHODS: We have extended this study to examine whether the high-Na+ low-K+ formulation of Belzer's solution (HEH) was efficient in an autotransplanted pig kidney model when compared with the classical low-Na+ high-K+ University of Wisconsin solution and the new high-Na+ low-K+ Celsior solution. Kidneys were harvested, cold flushed, and preserved for 24, 48, or 72 hr with HEH, Celsior solution, or University of Wisconsin solution and autotransplanted. Renal function was determined on days 1, 3, 7, and 14, and at 4 to 16 weeks after autotransplantation. Histologic changes and cell infiltration were assessed on kidney biopsy specimens taken after reperfusion (30-40 min), at days 5 and 14, and at 4 to 5 and 10 to 12 weeks after surgery. Peripheral benzodiazepine receptor (PBR), a structural mitochondrial protein, was also studied. RESULTS: Cold storage in HEH resulted in reduction of delayed graft function and renal damage, with a decrease in interstitial inflammation. HEH reduced interstitial fibrosis, tubular atrophy, and improved PBR expression. CONCLUSION: This study suggests that cold preservation in HEH has a beneficial action in in vivo renal preservation and reduces tubular necrosis, interstitial inflammation, and fibrosis in these groups. In addition, PBR detection was correlated to the level of preservation integrity.
Asunto(s)
Adenosina , Alopurinol , Glutatión , Supervivencia de Injerto/fisiología , Insulina , Trasplante de Riñón/fisiología , Riñón , Soluciones Preservantes de Órganos , Potasio/análisis , Rafinosa , Daño por Reperfusión/prevención & control , Sodio/análisis , Animales , Riñón/patología , Trasplante de Riñón/patología , Masculino , Modelos Animales , Preservación de Órganos/métodos , Porcinos , Factores de Tiempo , Trasplante AutólogoRESUMEN
The addition of polyethylene glycol (PEG) to hepatocyte storage medium is known to decrease lipid peroxidation and swelling and to protect the cell cytoskeleton from cold. We therefore decided to investigate the effect of substituting PEG for hydroxyethyl starch (HES) in an extracellular-like UW solution, with and without Ca++, on rat liver preservation. Isolated perfused rat livers were used to assess graft injury after 24h of cold storage. Four groups of preserved livers ( n=6 for each group) were compared to controls (non preserved livers, n=11). For this purpose, Belzer solution (K+-UW, group 1) was stepwise modified. Group 2 (Na+-UW) was treated with the same liquid, however with inverted concentrations of Na+ and K+. Group 3 was preserved in the first experimental solution (EPS-1) with Ca++ (0.5mM) added to the Na+-UW solution. In the EPS-2 (group 4), PEG-35 (0.03mM) was substituted for HES. The last group, EPS-3 (group 5) was treated with the same compounds as EPS-2, but without Ca++. After 24h of cold storage and 120min normothermic reperfusion, there was no statistical difference in transaminases (ALT and AST) release between the control and the Na+-UW groups. Furthermore, rat livers preserved in Na+-UW solution released less ( P<0.05) ALT and AST and excreted more ( P<0.05) indocyanine green (ICG) than livers preserved in K+-UW solution. The addition of 0.5mM Ca++ to Na+-UW solution (EPS-1) dramatically increased ( P<0.05) parenchymal (ALT, AST) and non parenchymal (creatine kinase-BB) cellular injury. The substitution of PEG (0.03mM) for HES (EPS-2) reduced ( P<0.05) membrane injuries due to Ca++ while bile flow was statistically increased ( P<0.05). Finally, the omission of Ca++ from EPS-2, that is EPS-3, has no statistically significant effect on the studied parameters. PEG effectively protected the rat liver grafts from the onset of hypothermic ischemia-reperfusion and Ca++ damages and thus may be a valuable additive to preservation solutions.