Summary

大鼠异位心脏和心肌细胞移植的免疫模型

Published: May 08, 2020
doi:

Summary

我们描述了大鼠异位腹心移植的模型,这意味着对当前策略的修改,从而导致简化的手术方法。此外,我们还描述了一种新的排斥模型,通过注射重要的心肌细胞的耳朵内,允许进一步的移植免疫学分析在大鼠。

Abstract

50多年来,大鼠异位心脏移植一直是各种免疫学研究的常用模型。自1964年第一次描述以来,已经报告了若干修改。在大鼠进行异位心脏移植30年后,我们开发了一种简化的手术方法,无需进一步手术训练或背景,即可轻松教授和进行。

在解剖上升主动脉和肺动脉和上等类腔和肺静脉结扎后,供体心脏被收获,然后注入冰冷盐水溶液补充肝素。在夹紧和切开受体腹血管后,捐赠者提升主动脉和肺动脉分别被麻醉到受体腹部主动脉和劣质的vena卡瓦,使用连续的导联。

根据不同的供体-受体组合,此模型允许分析异体移植的急性或慢性排斥。该模型的免疫意义进一步增强,通过一种新的注射重要心肌细胞的耳内注射方法,以及随后对排空颈淋巴组织的分析, 进一步增强了该模型的免疫意义。

Introduction

异位心脏移植是一种常用的实验模型,用于不同研究移植耐受性、急性和慢性异体移植排斥、缺血再灌注损伤、机器灌注或心脏重塑等。除其他优点外,移植功能可以通过触觉进行非侵入性监测,移植失败不会导致接受者与其他器官(如肾脏或肝脏)相比的重要损伤。

1964年,Abbott等人最初描述了大鼠1的异位腹心移植。后来,在1966年,原子塔等人对阿纳斯托莫的端到端技术作了描述。1969,奥诺和林赛报告了目前使用的模型的基础。在过去的几十年中,已经发表了一些修改,以创建不同类型的卸载,部分加载或加载左心室心脏移植,包括联合异位心肺移植44,5,6。5,6对于免疫分析,最常见的是非体积加载心脏移植移植。在这种情况下,血流逆行进入供体上升主动脉,随后进入冠状动脉。静脉排水沿冠状窦进入右中庭和心室(图1A-B)。因此,除了来自Thebesian静脉的少量血液外,左心室被排除在血流之外。这也使它成为研究左心室辅助装置治疗7期间的病理生理机制的有用模型。

异位心脏移植已在各种物种中进行,包括小鼠、兔子、猪,甚至被用作,人类8、9、10、119的单体或双心形辅助8装置。10,11大鼠仍然是移植模型的流行实验动物,特别是因为不同大鼠菌株组合的移植存活时间在过去已经明确,大量的免疫试剂是可访问的12,13。12,与老鼠不同,大鼠做手术和获得淋巴组织进行免疫分析更可行12。此外,近年来在大鼠身上引入商业性克隆技术,很可能导致对实验鼠模型14的反复关注。

一般来说,异位心脏移植可以通过进行宫颈或腹部麻醉附着在受体血管上。然而,一些研究表明,由于更好的获得手动触觉或跨体波他心动图,因此可以更精确地检测移植失败15,16,因此,股骨性麻醉有助于改善监测。15,

结果表明,两种阿生虫技术在操作时间、并发症率、结果和移植生存时间上没有差异。显然,必须提及有足够的排空淋巴结,作为宫颈麻醉的好处;但是,需要较长的培训期。相反,腹部麻醉对于免疫学研究来说不那么复杂,同样有价值,特别是当结合一种新疗法的异源性心肌细胞和随后的宫颈淋巴切除术的结果时。两种模型的组合提供了广泛的干预后免疫分析。

以下协议是指对外科医生进行手术,以减少缺血时间。但是,所有实验都可以由一个人执行。图2A-B显示了心脏外植和植入仪器和材料的设置。

Protocol

所有动物经验均按照下萨克森州(德国奥尔登堡州拉维斯)消费者保护和食品安全区域当局当地道德动物审查委员会的准则进行,并经批准,12/0768 和 17/2472。 1. 心脏外植和灌注 注:作为移植捐赠者,使用7-22周年龄的雌性或雄性大鼠。 通过吸入的二氟拉纳对供体大鼠进行麻醉(感应5%,维持3%,O2流量为1升/分钟)。注射5毫克Carprofen下皮每…

Representative Results

过去,不同的免疫问题都根据该模型得到解决,在工作小组中,500多例移植进行了验证,存活率超过95%,13、18、19、20、21、22、23、24。23,2413,18,19,20,21,22,总手术时间(包?…

Discussion

先前描述的大鼠异位心脏移植方法主要基于1969年对小野和林赛的描述。自那时以来,对各种物种进行了若干修改,导致该模型的广泛多样性。结合其中几个修改,并介绍我们自己的经验,在实验室进行了超过30年的异位心脏移植,我们创造了一个可行的手术方法,不需要长时间的训练或手术背景。在下面,我们将讨论此模型的一般限制,并强调协议的关键步骤。此外,我们将强?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们要感谢布里塔·特劳特维格、科林娜·勒伯特和英格丽德·梅德的承诺。

Materials

Anesthesia device (including isoflurane vaporizer) Summit Anesthesia Solutions No Catalog Number available
Cannula (27 G) BD Microlance 302200
Carprofen Pfizer Rimadyl 50 mg/mL
Cellstar Tubes (15 mL) GreinerBioOne 188271
Cell strainer (40 µm) BD Falcon 2271680
Collagenase Type CLSII Biochrome C2-22
Compresses 5×5 cm Fuhrmann 31501
Compresses 7.5×7.5 cm Fuhrmann 31505
Cotton swabs Heinz Herenz Medizinalbedarf 1032128
Dexpathenol (5 %) Bayer "Bepanthen"
DPBS BioWhittaker Lonza 17-512F
Forceps B. Braun Aesculap BD557R
Forceps B. Braun Aesculap BD313R
Forceps B. Braun Aesculap BD35
Heating mat Gaymar Industries "T/Pump"
Hemostatic gauze Ethicon Tabotamp
Heparin-Natrium 25 000 I.E. Ratiopharm No Catalog Number available
Isofluran CP CP-Pharma No Catalog Number available
Large-pored sieve (stainless steel) Forschungswerkstätten Hannover Medical School No Catalog Number available
Lidocaine Astra Zeneca 2 % Xylocain
Metamizol-Natrium Ratiopharma Novaminsulfon 500 mg/mL
Micro forceps B. Braun Aesculap BD3361
Micro needle holder Codman, Johnson & Johnson Medical Codmann 80-2003
Micro needle holder B. Braun Aesculap BD336R
Micro needle holder B. Braun Aesculap FD241R
Micro scissors B. Braun Aesculap FD101R
Micro scissors B. Braun Aesculap FM471R
Needle holder B. Braun Aesculap BM221R
Penicillin/Streptomycin/Glutamine (100x) PAA P11-010
Peripheral venous catheter (18 G) B. Braun 4268334B
Peripheral venous catheter (22 G) B. Braun 4268091B
Probe pointed scissors B. Braun Aesculap BC030R
Retractors Forschungswerkstätten Hannover Medical School No Catalog Number available
RPMI culture medium Lonza BE12-702F
Saline solution (NaCl 0.9 %) Baxter No Catalog Number available
Scissors B. Braun Aesculap BC414
Surgical microscope Carl-Zeiss OPMI-MDM
Sutures (anastomoses) Catgut Mariderm 8-0 monofil
Sutures (ligature) Resorba Silk 5-0 polyfil
Sutures (skin, fascia) Ethicon Mersilene 3-0
Syringe (1 mL) B. Braun 9166017V
Syringe (10 mL) B. Braun 4606108V
Syringe (20 mL) B. Braun 4606205V
Vascular clamp B. Braun Aesculap FB708R

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Weigle, C. A., Lieke, T., Vondran, F. W. R., Timrott, K., Klempnauer, J., Beetz, O. An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats. J. Vis. Exp. (159), e60956, doi:10.3791/60956 (2020).

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