צעדים לעצמיים ב<em> Ex vivo</emניסוי כבד וכליה> perfused בשר חזירים
Summary
Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.
Abstract
השימוש במודלים לשעבר perfused vivo יכול לחקות את התנאים הפיסיולוגיים של הכבד לתקופות קצרות, אבל כדי לשמור על הומאוסטזיס נורמלי לתקופה ממושכת זלוף הוא מאתגר. הוספנו את הכליה לvivo לשעבר perfused מודל הניסוי הקודם שלנו בכבד לשחזר מצב פיסיולוגי מדויק יותר לניסויים ממושכים ללא שימוש בבעלי חיים. חמישה כבדים וכליות ללא פגע נאספו לאחר המוות מחזירים הקריבו בימים שונים וperfused עבור מינימום של 6 שעות. גזים בדם העורקי לשעה התקבלו לנתח pH, חומצת החלב, גלוקוזה ופרמטרי כליות. נקודת הסיום הראשונית היה לבדוק את ההשפעה של הוספת כליה אחת למודל על רמות איזון חומצת בסיס, גלוקוז, ואלקטרוליטים. התוצאה של ניסוי כבד בכליות זה הושוותה לתוצאות של חמישה דגמי זלוף קודמים בכבד בלבד. לסיכום, עם התוספת של כליה אחת לvivo לשעבר מעגל כבד, hyperglycemIA וחמצת מטבולית השתפרו. בנוסף מודל זה משחזר את התגובות הפיזיולוגיות וחילוף חומרים של הכבד מספיק מדויק כדי לייתר את הצורך בשימוש בבעלי חיים בשידור חי. לשעבר מודל זלוף כבד כליות vivo יכול לשמש כשיטת חלופית במחקרים ספציפיים איברים. הוא מספק ניתוק מהשפעות מערכתיות רבות ומאפשר התאמות ספציפיות ומדויקות של עורקים ולחצים וזרימה ורידית.
Introduction
Studying a large animal organ physiology in an artificial environment is challenging. Furthermore, maintaining normal physiological and biochemical parameters for a prolonged period requires a closely monitored perfusion method and model. We have previously described our ex vivo autologous perfused porcine liver model for the study of hepatic physiology and significant progress has been achieved, since we began our liver reperfusion ex vivo model in 2005, in areas such as harvesting technique, understanding hepatic artery anatomical variations1, inflammatory response2,3, histological changes following liver electrolytic ablation (EA)4, and changes in acid base balance during EA5. Unfortunately hyperglycemia and metabolic acidosis require external supplements of insulin and bicarbonate to maintain an accurate physiological environment. Stable blood sugar levels and acid base balance are crucial when studying islet auto-transplantation, especially in the early implantation phase. We have developed a new model incorporating the kidney to clear excess metabolites and improve the environment for the islet transplantation6,7.
Extracorporeal perfusion experiments lasting longer than 24 hr can also be performed, but these require a larger team to perform the experiments8.
Animals used in this study received human care and study protocols were in accordance with the United Kingdom laws. They were euthanized with final exsanguinations during the blood-harvesting procedure according to United Kingdom regulations. Porcine liver and a left kidney were retrieved from animals in the food chain and that would otherwise be discarded. In this way important ethical implications can be overcome and the economic burden is lower than more common in vivo studies. The short duration of liver and kidney perfusion time in this model, lasting from 6-24 hr, has been a major limitation of our studies; this can be improved by minimizing the exposure of the organs to the ischemic injury (cold and warm ischemia).
Protocol
Representative Results
Discussion
In our group the experience and the results achieved with the ex vivo perfusion of the liver have been paralleled over the years by concomitant modifications in the retrieval technique adopted. In the last few years our group has achieved considerable experience using an ex vivo porcine model to study the hepatic physiology5,11, new liver ablation techniques2,5, and liver immunology3. Despite the technical challenges derived from an additional organ, corrections of …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Sarah Hosgood from the Department of Transplantation, University Hospitals of Leicester, for technical support with the porcine kidney in this model.
Materials
| Epoprostenol sodium (Flolan) | Glaxo Smith Kline | PL10949/0310 | |
| Sodium Bicarbonate 8.4% | Polyfusor, Fresenius Kabi Ltd | PL8828/0043 | |
| Cefuroxime sodium (Zynacef) | Flynn Pharma Ltd | PL13621/0018 | |
| Calcium Chloride 14.7% | Martindale Pharmaceuticals | PL1883/6174R | |
| Heparin | LEO Laboratories Limited | PL0043/0038R | |
| Sodium taurocholate | Sigma-Aldrich | T4009-25G | |
| Insulin (Actrapid) | Novo Nordisk | EU/1/02/230/003 | |
| Soltran (preservation solution) | Baxter Health Care | FKB4708G | preservation solution |
| Atraumatic centrifugal pump (Bio-console 560) | Medtronic Inc., Minneapolis, Minnesota- United States; custom pack, cardiac surgery division Europe |
References
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