Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.
Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.
成功的肾移植肾功能衰竭的治疗在1955年首次被描述同卵双胞胎1之间,从那时起,它已成为患者在世界各地的终末期肾功能衰竭的一种革命性的治疗,提供了长寿命2质量均提高。但是长期移植物的存活已经阻碍了许多导致慢性移植物损伤3病理过程。
在人类的移植肾排斥仍发病率的主要原因,尽管在immunosupporessive方案显著改进。发展肾移植小鼠模型的目的是密切复制人类肾移植4中发现的过程和病理。 Skoskiewicz 等人首先描述肾移植的小鼠模型,1973年5。虽然先进的显微外科技巧是必需的,它是一个有价值的吨OOL有以下几个原因:小鼠基因组中已经得到很好的特点,并有很大的不同的实验方法和可用于小鼠实验技术。
使用肾移植的小鼠模型中的许多团体已经使用的移植肾的生命支持器官,但在其他研究中,并在我们所描述的受体小鼠的天然肾脏的方法1留在原位进行实验4的持续时间。这样做的好处是,鼠标经过一个单一的麻醉和手术从而降低了发病率的鼠标和死亡的第二个步骤的风险。此外,该鼠标不从逐步肾功能衰竭的不利影响受害。
虽然在其他器官,如心脏和皮肤存在异体排斥反应的模型,这并不总是直接相关的肾移植。有证据表明,这些模型引出不同的模式和dy排斥的动力学性能,例如拒绝在心脏同种异体移植物和肾移植的时间过程中的某些菌株的组合6显著不同。我们在BALB / c小鼠的捐助者说明移植肾急性排斥反应的模式到非转基因FVB / NJ小鼠,这种模式表现出与T细胞和巨噬细胞7积聚的细胞介导的损伤。另外,我们也描述了慢性移植物损伤模型,展示间质纤维化和肾小管萎缩,这将导致从移植肾的C57BL / 6 BM12捐助者到C57BL / 6受,因为这些小鼠的特点是一个单一的MHC II类基因位点错-match 8。
移植的多个方面使用肾移植的小鼠模型中,包括急性排斥反应,细胞免疫和体液排斥,局部缺血再灌注损伤,以及试用新治疗剂进行了研究。 我们已修改的手术吨echnique,以减少操作时间,提高了易用性手术。特别是我们所描述的同时供体和受体的准备,并利用连续的主动脉修补吻合简化的血管吻合技术。此视频稿件和提供的关键点在建立这种技术的帮助。
最充分描述的方式来执行的动脉吻合术是使用供体的远端主动脉,与肾动脉中继续,在一个端部到另一侧的方式向收件人主动脉。我们描述了使用主动脉补丁,类似于人类肾脏移植,我们认为是比较方便执行的“Carrell补丁'镜像。虽然在供体和受体手术时间的文献报道是稀疏我们相信,利用端 – 侧供体主动脉的主动脉补丁到收件人主动脉而不是向收件人主动脉是优选的。使用补丁否定,要剖析?…
The authors have nothing to disclose.
从肾脏研究英国资助,爱丁堡和器官移植的欧洲社会皇家外科学院赞助这项研究。
Surgical Instruments | |||
Blunt Dissecting Scissors | Fine Science Tools | 14072-10 | For skin cutting |
Curved Castoviejo scissors | Fine Science Tools | 15017-10 | For tissue cutting |
Spring Scissors – straight | Fine Science Tools | 15000-08 | For suture cutting |
Toothed forceps 1×2 teeth | Fine Science Tools | 11021-12 | |
2 x Fine Tip forceps (Dumont No.5) | Fine Science Tools | 11251-20 | |
Angled Fine Tip forceps (Dumont No. 5/45) | Fine Science Tools | 11253-25 | For blunt dissecting |
Curved Fine Tip forcep (Dumont No.7) | Fine Science Tools | 11273-22 | Useful to pass around vessels |
Curved Crile Haemostat | Fine Science Tools | 1300-04 | |
Micro clip applicator with lock | Fine Science Tools | 18056-14 | |
2 x Micro serrefines spring width 2mm, jaw length 4mm | Fine Science Tools | 18055-04 | Microvascular clamps |
2 x Colibri 3cm wire retractor | Fine Science Tools | 17000-03 | |
Castroviejo needle holder with lock | Fine Science Tools | 120660-01 | |
Wound clip applicator | Fine Science Tools | 12031-07 | |
7mm wound clips | Fine Science Tools | 12032-07 | Remove 7 to 10 days after surgery |
Equipment | |||
OPMI pico microscope | Carl Zeiss | S100 | |
Thermal cautery unit with fine tip | Geiger | 150A | |
Heat electronic pad | Cozee Cumfort | n/a | |
Euroklav 23-S | Melag | n/a | Autoclave |
Disposable equipment | |||
7/O Silk braided suture | Pearsall | 30514 | |
10/O Dafilon (polyamide) suture | B-Braun | G1118099 | |
6/O Vicryl (plygalectin) | Ethicon | W9537 | |
Regular bevel needle, 1 inch, 21G | Bection, Dickinson and Company | 305175 | For ureteric anastamosis |
Regular bevel needle, 5/8 inch, 25G | Bection, Dickinson and Company | 305122 | |
Regular bevel needle, 1/2 inch, 30G | Bection, Dickinson and Company | 304000 | |
Insulin needle 1ml, 29G | Bection, Dickinson and Company | 324827 | |
Insulin needle 0.3ml, 30G | Bection, Dickinson and Company | 324826 | |
1 ml syringe slip tip | Bection, Dickinson and Company | 300184 | |
5 ml syringe slip tip | Bection, Dickinson and Company | 302187 | |
Wypall paper swabs | Kimberley-Clark | L40 | sterilised by autoclave |
Cotton wool buds | Johnson and Johnson | n/a | sterilised by autoclave |
Plain drapes | Guardian | CB03 | sterilised by autoclave |
Cell culture dish 60mm x 15mm | Corning Incorporated | 430166 | |
Dispensing Pin | B-Braun | DP3500L / 413501 | Used with NaCl 0.9% |
Re-agents and Drugs | |||
(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2% | Allergan Ltd | 21956GB10X | |
(Videne) Povidone-iodine 10% | Ecolab Ltd | PL 04509/0041 | |
(Vetalar V) Ketamine hydrochloride | Pfizer Animal Health | Vm 42058/4165 | 100mg/ml solution (dose 200mg/kg) |
(Domitor) Medetomidine hydrochloride | Orion Pharma | Vm 06043/4003 | 1mg/ml (dose 0.5mg/kg) |
(Vetergesic) Bupernorphine hydrochloride | Alsto Animal Health | Vm 00063/4002 | 0.3mg/ml (dose 0.05mg/kg) |
(Antisedan) Atipamezole hydrochoride | Orion Pharma | Vm 06043/4004 | 5mg/ml (dose 2mg/kg) |
University of Wisconsin Solution | Belzer Bridge to Life | n/a | dose approximately 500 microlitres/mouse |
NaCl 0.9% | Baxter | FKE1323 | |
Heparin Sulphate | non-proprietary | n/a | 5000units/ml (dose 5units/mouse) |