Summary
本研究建立了一个方案,重点是对用于急性肾损伤研究的双侧肾缺血再灌注小鼠模型进行技术改进。
Abstract
心脏骤停会给公共卫生带来沉重的负担。急性肾损伤 (AKI) 是心肺复苏成功后自主循环 (ROSC) 恢复后心脏骤停幸存者的不良标志物。相反,AKI 肾功能恢复是有利神经系统结局和出院的预测指标。然而,缺乏有效的干预措施来预防ROSC后心脏骤停引起的肾脏损伤,这表明需要额外的治疗策略。肾灌注不足和再灌注是心脏骤停后引起AKI的两种病理生理机制。在临床环境中,双肾缺血再灌注诱导的 AKI (IR-AKI) 动物模型与 ROSC 后 AKI 患者相当。然而,双侧肾脏的IR-AKI在技术上难以分析,因为该模型与高死亡率和肾脏损伤的广泛差异相关,这可能会影响分析。选择轻量级小鼠,置于异氟醚全身麻醉下,采用背外侧入路进行手术,并在手术过程中保持体温,从而减少组织损伤并建立可重复的急性肾IR-AKI研究方案。
Introduction
在美国,心脏骤停每年发生超过 80,000 次 1,2。心脏骤停的死亡率极高 3,4,5,6。AKI 是 ROSC7、8、9、10、11、12、13 后心脏骤停患者高死亡率和不良神经系统结局的主要危险因素。AKI的恢复是有利的神经系统结局和出院的良好预测指标14,15,16。然而,IR-AKI的有效疗法仍然缺乏15,16,17,18,19。需要额外的治疗策略来进一步改善该疾病的临床结果。
IR-AKI与双侧肾缺血入路是用于AKI研究的动物模型之一20,21,22,23,24,25,26。肾脏 IR-AKI 动物模型在研究 ROSC6、27、28、29、30 后心脏骤停患者的 AKI 方面不如全身 IR 损伤模型复杂。这意味着肾脏IR-AKI动物模型的一致结果更容易实现,因为实验中存在的混杂因素较少。此外,肾脏 IR-AKI 方案通常涉及单侧或双侧肾蒂闭塞。双侧肾IR-AKI实验条件与成功心肺复苏后心脏骤停患者ROSC后AKI的临床条件相当。尽管两种模型中肾脏的病理特征都反映了人类肾IR损伤的病理特征31,32,33,但在人类病理条件下,如心力衰竭、血管收缩和感染性休克35,双侧肾缺血方法与AKI更相关。双侧肾脏 IR-AKI 动物模型适用于专注于 ROSC 后心脏骤停的肾 IR 损伤的研究。
双侧肾脏IR-AKI模型与技术难度、实验复杂性和手术时间长有关23,26,32,33,35,36。为了克服这些技术难题,本研究通过对小鼠进行一些技术修改,建立了可靠的双侧IR-AKI研究方案。所提出的方案减少了手术并发症,减少了组织损伤,并降低了手术期间的死亡可能性。因此,可用于研究ROSC后AKI的病理生理过程,以开发针对肾灌注不足和再灌注损伤的新治疗策略37,38,39。
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Protocol
所有动物实验均按照美国国立卫生研究院出版的 《实验动物护理和使用指南》(NIH 出版物编号 85-23,1996 年修订)进行。该研究方案已获得辅仁大学机构动物护理和使用委员会的批准并符合其指南。有关本协议中使用的所有材料和仪器的详细信息,请参阅 材料表 。
1. 准备小鼠
- 选择体重为21-23g的8周龄C57BL / 6雄性小鼠。
- 在受控温度(21±2°C)下在12小时光照和黑暗循环下饲养和维持小鼠,自由获取食物,标准小鼠食物颗粒和自来水。
2. 麻醉
- 戴上外科口罩和无菌手套。
- 将小鼠置于麻醉下,在诱导室中以1L / min的速度将2%异氟醚与氧气混合。
- 通过踏板反射评估麻醉水平。
注意:踏板反射是后爪因脚趾捏紧而缩回。当踏板反射消失时,麻醉完成。 - 麻醉完成后,将每只小鼠移动并俯卧在带有电热毯的手术平台上,以保持体温。手术前稳定体温,并用直肠温度探头监测。将眼药膏涂抹在双眼上,以防止干燥。
- 将小鼠的爪子粘在板上。
- 将面罩贴在小鼠的脸上,以提供1%异氟烷和1L / min氧气的持续供应
- 定期通过踏板反射评估麻醉水平,并在手术过程中相应地调整麻醉剂的输送。
3. 双侧肾IR-AKI手术
- 触摸背部并手动找到小鼠的腰椎。沿着脊柱向头移动,寻找位于小鼠最后一根肋骨两侧下方的肋脊角。
- 将脱毛化妆水涂抹在肋脊角区域的两侧约30秒,然后用生理盐水去除皮毛。
- 用棉球用三轮倍他丁溶液和75%酒精对剃光的皮肤进行消毒。
注意:在整个手术过程中保持手术的无菌区域是关键。使用手术单并使用无菌器械。 - 使用细尖镊子轻轻提起左肋脊角以下的皮肤,然后用剪刀从左侧腰椎中线沿皮肤张力线做一个 1 厘米的斜背外侧切口。用剪刀横切左侧肌肉壁,以可视化左肾。
- 重复上述外科手术以可视化右肾。用无菌棉签清除手术过程中产生的少量血液。
- 用镊子小心地将左肾与周围组织推开并分离。识别左肾暴露后的肾蒂。
注意:注意不要伤害肾上腺和周围的血管。 - 用微血管夹夹住左肾蒂25分钟。通过肾脏颜色从粉红色到深红色的明显变化来确认缺血。
- 用无菌盐水湿棉球覆盖钳夹的肾脏,以避免左肾椎弓根钳夹期间干燥。
- 重复上述外科手术,用微血管夹夹住右肾蒂25分钟。
- 用无菌盐水湿棉球覆盖钳夹的肾脏,以避免在右肾椎弓根钳夹期间干燥。
- 定期监测无菌盐水湿棉球的麻醉深度和湿度。
- 打开左微血管夹,开始左肾再灌注。通过左肾从深红色到粉红色的可见颜色变化来确认再灌注。
- 打开右微血管夹,开始右肾再灌注。
- 确认肾脏变色后,将肾脏放回腹腔。
- 用 6-0 可吸收缝合材料封闭腹腔和皮肤。
- 用甜菜碱溶液和75%酒精用棉球擦洗伤口。
- 仔细观察动物,直到它开始自由移动和进食。
注意:密切注意动物,直到它们恢复足够的意识以保持胸骨卧位。 - 给予卡洛芬(5 mg/kg,0.2 mL,皮下注射)2-3天,以防止术后疼痛。
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Representative Results
在进一步的显微镜或分子分析之前,应评估双侧肾脏 IR-AKI 手术的质量。在手术过程中,应通过观察肾脏是否在用微血管夹夹住肾蒂后不久将肾脏颜色从粉红色变为深红色来确认肾缺血(图1)。手术后,IR-AKI手术引起的肾脏损伤可以通过下颌下采血进行生化分析,用几微升血清进一步验证,结果表明血尿素氮和肌酐水平较基线增加(图2)。
图1:肾椎弓根钳夹后肾缺血。 肾脏颜色从粉红色变为深红色,表明肾脏灌注不足。 请点击这里查看此图的较大版本.
图 2:双侧 IR-AKI 手术后的肾功能不全。 肾再灌注后 2 天血清尿素、氮和肌酐水平升高。缩写:IR-AKI=缺血再灌注诱导的急性肾损伤;BUN = 血尿素氮;I/R = 缺血再灌注(n = 4,*p < 0.05 与对照组相比)。 请点击这里查看此图的较大版本.
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Discussion
拟议的双侧 IR-AKI 方案适用于研究双肾灌注不足和再灌注损伤的机制。该方案表明,轻量级小鼠、异氟醚全身麻醉、手术背外侧入路以及手术期间的体温维持减轻了相关的技术困难,缩短了手术持续时间,并增加了急性双侧肾脏 IR-AKI 研究的一致性。
技术难点影响双侧肾IR-AKI手术中肾损伤的严重程度33.除了小鼠品系、性别、年龄和加热系统36、40、41、42、43、44 之外,正确放置血管钳对于一致的结果至关重要。研究建议仔细解剖周围的脂肪组织,以释放肾脏和肾脏蒂或动脉23,26,32,35,36。与文献23,32,35,36中研究的8-20周龄小鼠通常体重25-28g相比,本研究使用相对年轻和轻便的小鼠(8周龄,体重21-23g)来减少肾周脂肪组织的数量,无需外周组织解剖和血管钳的正确放置即可轻松暴露肾脏和肾蒂。这将减少与手术相关的创伤和技术复杂性,缩短麻醉和手术时间,加快不熟悉研究程序的人的学习曲线,并提高研究的可重复性。
全身麻醉会影响 IR-AKI 研究的结果。长时间麻醉会增加手术期间的动物损失33.在文献中,苯巴比妥钠是一种抑制中枢神经系统的长效巴比妥类药物,已皮下注射用于 IR-AKI 手术 26,33,35。苯巴比妥在 5 分钟后起效,并有助于在至少 15 分钟内实现手术麻醉45.因此,苯巴比妥只能由熟练的外科医生给药,以避免延长麻醉时间(>60 mg/kg)和手术期间动物损失33。相比之下,本研究使用异氟醚(一种不易燃的吸入麻醉剂)诱导快速起效,在 7-10 分钟内达到手术麻醉,并在停止吸入后 15 分钟内停止作用46。异氟醚与氧气的输送易于操作者在手术过程中立即启动、维持和停止,建议用于肾脏 IR-AKI 手术。
最后,接近肾蒂的方法可能会影响IR-AKI手术的质量。一些 IR-AKI 研究使用中线剖腹手术检查了肾蒂,其中打开腹腔,将腹膜和肠推到一边以进入肾脏。然而,这样做可能会增加液体和热量损失、手术相关创伤和手术持续时间32,35。因此,该方案建议采用背外侧方法进行 IR-AKI 研究,以从侧面和腹膜后暴露肾脏,以保持体温并最大限度地减少与手术相关的损伤,从而改善手术条件和研究一致性。
该模型在旨在识别和表征 ROSC 后心脏骤停引起的双侧肾损伤标志物的研究中具有潜在应用。然而,在手术过程中由于手术损伤而释放的细胞因子可能会影响研究结果,使其与临床情况无关,并限制研究结果从实验室到床边的翻译。
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Disclosures
作者声明,本文的发表不存在利益冲突。
Acknowledgments
该模型是在台湾科学技术部(MOST 109-2320-B-030-006-MY3)的资助下开发的。本文由Wallace Academic Editing编辑。
Materials
Name | Company | Catalog Number | Comments |
Absorbable Suture, 6-0 | Ethicon | J510G-BX | |
Betadine solution | Shineteh Istrument | ||
Carprofen | Sigma | PHR1452 | |
Cotton balls | Shineteh Istrument | ||
Graefe Forceps | Fine Science Tools | 11051-10 | |
Heating pad | Shineteh Istrument | ||
Isoflurane | Piramal Critical Care Inc. | 26675-46-7 | |
Moria Vessel Clamp | Fine Science Tools | 18320-11 | |
Olsen-Hegar needle holder | Fine Science Tools | 12002 - 12 | |
Saline | Shineteh Istrument | ||
Scalpel blades | Shinva | s2646 | |
Small Animal Anesthesia Machine | Sheng-Cing Instruments Co. | STEP AS-01 | |
Tissue scissors | Fine Science Tools | 14072 - 10 |
References
- Holmberg, M. J., et al. Annual incidence of adult and pediatric in-hospital cardiac arrest in the United States. Circulation: Cardiovascular Quality and Outcomes. 12 (7), 005580 (2019).
- Benjamin, E. J., et al. Heart disease and stroke statistics-2018 update: a report from the American Heart Association. Circulation. 137 (12), 67 (2018).
- Lascarrou, J. B., et al. Targeted temperature management for cardiac arrest with nonshockable rhythm. The New England Journal of Medicine. 381 (24), 2327-2337 (2019).
- Chang, H. C., et al. Factors affecting outcomes in patients with cardiac arrest who receive target temperature management: The multi-center TIMECARD registry. Journal of the Formosan Medical Association. 121 (1), 294-303 (2022).
- Yu, G., et al. Comparison of the survival and neurological outcomes in OHCA based on smoking status: investigation of the existence of the smoker's paradox. Signa Vitae. 18 (2), 121-129 (2022).
- Chen, Y. C., et al. Major interventions are associated with survival of out of hospital cardiac arrest patients - a population based survey. Signa Vitae. 13 (2), 108-115 (2017).
- Sandroni, C., et al. Acute kidney injury after cardiac arrest: a systematic review and meta-analysis of clinical studies. Minerva Anestesiologica. 82 (9), 989-999 (2016).
- Patyna, S., et al. Acute kidney injury after in-hospital cardiac arrest in a predominant internal medicine and cardiology patient population: incidence, risk factors, and impact on survival. Renal Failure. 43 (1), 1163-1169 (2021).
- Storm, C., et al. Impact of acute kidney injury on neurological outcome and long-term survival after cardiac arrest - A 10 year observational follow up. Journal of Critical Care. 47, 254-259 (2018).
- Geri, G., et al. Acute kidney injury after out-of-hospital cardiac arrest: risk factors and prognosis in a large cohort. Intensive Care Medicine. 41 (7), 1273-1280 (2015).
- Guo, Q. Y., Xu, J., Shi, Q. D. Gasping as a predictor of short- and long-term outcomes in patients with cardiac arrest: a systematic review and meta-analysis. Signa Vitae. 17 (2), 208-213 (2021).
- Chen, P. C., et al. Prognostic factors for adults with cardiac arrest in the emergency department: a retrospective cohort study. Signa Vitae. 18 (3), 56-64 (2022).
- Lee, M. J., et al. Predictors of survival and good neurological outcomes after in-hospital cardiac arrest. Signa Vitae. 17 (2), 67-76 (2021).
- Deakin, C. D., et al. European Resuscitation Council guidelines for resuscitation 2010 section 4. adult advanced life support. Resuscitation. 81 (10), 1305-1352 (2010).
- Cha, K. C., et al. Recovery from acute kidney injury is an independent predictor of survival at 30 days only after out-of-hospital cardiac arrest who were treated by targeted temperature management. Signa Vitae. 17 (2), 119-126 (2021).
- Park, Y. S., et al. Recovery from acute kidney injury as a potent predictor of survival and good neurological outcome at discharge after out-of-hospital cardiac arrest. Critical Care. 23 (1), 256 (2019).
- Mah, K. E., et al. Acute kidney injury after in-hospital cardiac arrest. Resuscitation. 160, 49-58 (2021).
- Pelkey, T. J., et al. Minimal physiologic temperature variations during renal ischemia alter functional and morphologic outcome. Journal of Vascular Surgery. 15 (4), 619-625 (1992).
- Kim, H., et al. Effect of different combinations of initial body temperature and target temperature on neurological outcomes in out-of-hospital cardiac arrest patients treated with targeted temperature management. Signa Vitae. , 1-7 (2022).
- Wyss, J. C., et al. Differential effects of the mitochondria-active tetrapeptide SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) and its peptidase-targeted prodrugs in experimental acute kidney injury. Frontiers in Pharmacology. 10, 1209 (2019).
- Wang, Y., Wang, B., Qi, X., Zhang, X., Ren, K. Resveratrol protects against post-contrast acute kidney injury in rabbits with diabetic nephropathy. Frontiers in Pharmacology. 10, 833 (2019).
- Li, S., Yu, L., He, A., Liu, Q. Klotho inhibits unilateral ureteral obstruction-induced endothelial-to-mesenchymal transition via TGF-beta1/Smad2/Snail1 signaling in mice. Frontiers in Pharmacology. 10, 348 (2019).
- Godoy, J. R., Watson, G., Raspante, C., Illanes, O. An effective mouse model of unilateral renal ischemia-reperfusion injury. Journal of Visualized Experiments. (173), e62749 (2021).
- Chen, Q., et al. SIRT1 mediates effects of FGF21 to ameliorate cisplatin-induced acute kidney injury. Frontiers in Pharmacology. 11, 241 (2020).
- Li, H. D., et al. Application of herbal traditional Chinese medicine in the treatment of acute kidney injury. Frontiers in Pharmacology. 10, 376 (2019).
- Grenz, A., et al. Use of a hanging-weight system for isolated renal artery occlusion during ischemic preconditioning in mice. American Journal of Physiology-Renal Physiology. 292, 475-485 (2007).
- Gao, Q., et al. Accumulated epinephrine dose is associated with acute kidney injury following resuscitation in adult cardiac arrest patients. Frontiers in Pharmacology. 13, 806592 (2022).
- Oh, Y. T., et al. Vasoactive-inotropic score as a predictor of in-hospital mortality in out-of-hospital cardiac arrest. Signa Vitae. 15 (2), 40-44 (2019).
- Burne-Taney, M. J., et al. Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury. American Journal of Physiology-Renal Physiology. 285 (1), 87-94 (2003).
- Adams, J. A., et al. Periodic acceleration (pGz) prior to whole body ischemia reperfusion injury provides early cardioprotective preconditioning. Life Sciences. 86 (19-20), 707-715 (2010).
- Gaut, J. P., Liapis, H. Acute kidney injury pathology and pathophysiology: a retrospective review. Clinical Kidney Journal. 14 (2), 526-536 (2021).
- Hesketh, E. E., et al. Renal ischaemia reperfusion injury: a mouse model of injury and regeneration. Journal of Visualized Experiments. (88), e51816 (2014).
- Wei, Q., Dong, Z. Mouse model of ischemic acute kidney injury: technical notes and tricks. American Journal of Physiology-Renal Physiology. 303 (11), 1487-1494 (2012).
- Wei, Q., Dong, Z. Regulation and pathological role of bid in ischemic acute kidney injury. Renal Failure. 29 (8), 935-940 (2007).
- Grenz, A., et al. Use of a hanging-weight system for isolated renal artery occlusion. Journal of Visualized Experiments. (53), e2549 (2011).
- Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice. Journal of Visualized Experiments. (78), e50495 (2013).
- Han, S. J., Lee, H. T. Mechanisms and therapeutic targets of ischemic acute kidney injury. Kidney Research and Clinical Practice. 38 (4), 427-440 (2019).
- Huang, C. W., et al. A novel caffeic acid derivative prevents renal remodeling after ischemia/reperfusion injury. Biomedicine & Pharmacotherapy. 142, 112028 (2021).
- Spoelstra-de Man, A. M. E., Oudemans-van Straaten, H. M. Acute kidney injury after cardiac arrest: the role of coronary angiography and temperature management. Critical Care. 23 (1), 193 (2019).
- Burne, M. J., Haq, M., Matsuse, H., Mohapatra, S., Rabb, H. Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model. Transplantation. 69 (5), 1023-1025 (2000).
- Muller, V., et al. Sexual dimorphism in renal ischemia-reperfusion injury in rats: possible role of endothelin. Kidney International. 62 (4), 1364-1371 (2002).
- Schmitt, R., Marlier, A., Cantley, L. G. Zag expression during aging suppresses proliferation after kidney injury. Journal of the American Society of Nephrology. 19 (12), 2375-2383 (2008).
- Oxburgh, L., de Caestecker, M. P.
Ischemia-reperfusion injury of the mouse kidney. Methods in Molecular Biology. 886, 363-379 (2012). - Delbridge, M. S., Shrestha, B. M., Raftery, A. T., El Nahas, A. M., Haylor, J. L. The effect of body temperature in a rat model of renal ischemia-reperfusion injury. Transplantation Proceedings. 39 (10), 2983-2985 (2007).
- IBM Micromedx, I. Phenobarbital sodium. IBM Corporation. , Available from: https://www-micromedexsolutions-com.autorpa.mmh.org.tw/micromedex2/librarian/CS/53C834/ND_PR/evidencexpert/ND_P/evidencexpert/DUPLICATIONSHIELDSYNC/51EFF0/ND_PG/evidencexpert/ND_B/evidencexpert/ND_AppProduct/evidencexpert/ND_T/evidencexpert/PFActionId/evidencexpert.DoIntegratedSearch?SearchTerm=Phenobarbital+Sodium&fromInterSaltBase=true&UserMdxSearchTerm=%24userMdxSearchTerm&false=null&=null# (2022).
- IBM Micromedx, Isoflurane. IBM Corporation. , Available from: https://www-micromedexsolutions-com.autorpa.mmh.org.tw/micromedex2/librarian/PFDefaultActionId/evidencexpert.DoIntegratedSearch?navitem=headerLogout# (2022).