Kidney transplantation in mice with intraureteral cannula technique for urinary reconstruction.
Kidney transplantation in mice with intraureteral cannula technique for urinary reconstruction.
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DOI：
10.1097/tp.0000000000000420
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发表时间：
2014-11
期刊：
影响因子：
6.2
通讯作者：
Ying Zhang;Donghang Zheng;R. Praseedom;H. Zhou;Dong-sheng Huang;Hao Chen
中科院分区：
文献类型：
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作者：
Ying Zhang;Donghang Zheng;R. Praseedom;H. Zhou;Dong-sheng Huang;Hao Chen
作为基因修饰菌株的M尿肾移植可用于研究同种异体肾移植损伤的分子机制（1,2）。然而，由于技术的复杂性和高死亡率，世界上很少有实验室掌握这一程序。Skoskiewicz等人(3)于1973年报道了第一例小鼠肾移植，后来Zhang和Wang（4,5）对其进行了改进，最近Song Rong(6)对其进行了改进。根据Mannon和Coffman（7,8）的描述，即使是经验丰富的外科医生，肾移植的成功率也在40%到70%之间。手术过程中的技术故障，主要表现为膀胱坏死、尿吻合处漏尿或结石形成(9)，仍然是小鼠肾移植术后长期成功的主要障碍。在大鼠模型中，已经描述了输尿管皮肤引流，尽管这带有感染的固有风险（10）。另一方面，在小鼠肾移植中，由于输尿管直径很小，输尿管-输尿管吻合尚未见报道。在文献中，在小鼠肾移植中进行尿路重建的方法是将输尿管直接植入膀胱而不进行吻合，或者最常见的是使用大膀胱贴片进行膀胱与膀胱吻合（6,11）。输尿管末端和膀胱补片的血液供应与移植物血管是隔绝的，容易缺血。为了避免泌尿重建的并发症，本研究描述了一种新的小鼠肾移植泌尿重建方法，将微小的塑料管依次插入供体和受体肾盂作为重建输尿管的桥梁，称为输尿管内插管技术。采用新型肾内插管技术行等基因肾移植30例，常规大膀胱补片膀胱吻合25例，于10 ~ 12周龄雄性C57Bl/6小鼠进行冷缺血时间、尿重建时间、膀胱坏死、尿漏、肾积水、结石形成、肾脏病理改变及长期生存率的比较。采用新型静脉插管技术，将氯胺酮（100 mg/kg）与噻嗪（5 mg/kg）的混合物肌内注射麻醉小鼠。结扎供体小鼠右肾动静脉，腹腔内灌注500 kl生理盐水。将左侧输尿管从丰富的组织中剥离，并用9-0丝线在输尿管中部结扎（图1A， a′）。左肾通过结扎和分离肾上腺和睾丸血管分离。主动脉和下腔静脉（IVC）在其连接处解剖。依次用9-0丝线结扎肾下主动脉、肾动脉下腔静脉和肾上主动脉，然后用28.5号针插入肾下主动脉，原位灌注0.5 mL冷的、肝素化的（100单位/mL）乳酸林格液45秒，切开肾静脉与肾下腔静脉连接处。主动脉斜分，位于肾动脉下方约2mm处。然后通过烧灼主要血管周围的组织完全释放肾脏和相关血管，然后取出并在4℃盐水中保存15Y20分钟，直到移植时间（图1A， A '）。在传统的肾移植中，从膀胱到肾门的左输尿管被切开，不结扎输尿管，切除一小块包含左侧输尿管-膀胱交界处的椭圆形膀胱，用于受体的泌尿重建（12）。在受体中，将右肾门与肾周脂肪分离，露出前壁切开的输尿管肾盂连接，将11厘米长的弯曲微钳（Regine Tweezers，项目号504155；World Precision Instrument, Inc., Sarasota， FL）的一个腿尖插入内径0.28 mm的引流管末端，术前用75%乙醇浸泡1小时(聚乙烯管PE 10；Becton Dickinson and Company, Spark， MD)，将引流管（长度5mm）插入受体输尿管，用5-0丝环绑扎固定，然后取出肾脏（图1B， B′）。两个微血管钳（直0.75 4 mm颌，项目号501779-G； World Precision Instruments, Inc.）在近端和远端放置于肾下主动脉和下腔静脉，以促进体内平衡。将11-0尼龙缝线穿过主动脉的全层，然后缩回，单切口（约血管直径的五分之一）做椭圆动脉切开术。用肝素化盐水冲洗主动脉。在受术者动脉切开术的近端和远端放置两根固定缝合线。使用连续的11-0尼龙缝线在供主动脉和受体主动脉之间进行端侧吻合（VT04A00N07-13; Arosurgical Instruments Corporation, Newport Beach， CA）。首先通过腔内入路缝合后壁，然后缝合前壁。每侧只需要缝合三到四次。静脉与动脉的吻合方式相同。根据腔静脉贴片的大小，在每一侧缝合5到7次静脉。吻合后先松开远端钳，再松开近端钳。血液回流到肾脏（图1C， C′）。受者的输尿管与插入的导管被自由地切开至膀胱，而不剥离其脂肪，使导管容易。轻轻地暴露供体的输尿管肾盂连接处，在其下5毫米处的前壁上做一个小切口，并使用给编辑的信
Cannula Technique for Urinary Reconstruction M urine kidney transplantation as genetically modified strains can be used to study the molecular mechanisms of renal allograft injury (1, 2). However, because of the technical complexity and high mortality rates, few laboratories in the world have mastered this procedure. Skoskiewicz et al. (3) reported the first mouse kidney transplantation in 1973, which was later improved by Zhang and Wang (4, 5), and recently modified by Song Rong (6). As described by Mannon and Coffman (7, 8), the success rates of kidney transplantation even for experienced surgeons varied between 40% and 70%. Technical failure during surgery, one of importantly presenting as bladder necrosis and urine leakage at the sites of urinary anastomosis or calculi formation(9) are still major obstacles to the long-term success after mouse kidney transplantation. In the rat model, ureterocutaneous drainage has been described, although this carries inherent risk of infection (10). On the other hand, in kidney transplantation in mice, because of the tiny diameter of the ureter, ureterto-ureter anastomosis has not been described. In the literature, urinary reconstruction in mouse kidney transplantation has been performed by direct implantation of the ureter into the bladder without anastomosis or most commonly by bladder-to-bladder anastomosis using a large bladder patch (6, 11). The blood supply of the terminal ureter and bladder patch is exclusive from the graft vasculature and prone to ischemia. To avoid the complications from the urinary reconstruction, in the present study, we describe a new method of urinary reconstruction in mouse kidney transplantation where the tiny plastic tube was inserted into donor and recipient renal pelvis sequentially as a bridge to reconstruct the ureter, called as intraureteral cannula technique. Thirty isogenic kidney transplantations with the new intraureteral cannula technique and 25 mice treated by conventional bladder-to-bladder anastomosis using a large bladder patch in male C57Bl/6 mice from 10 to 12 weeks were performed to compare the cold ischemia time and urinary reconstruction time, the incidence of bladder necrosis, urine leakage, hydronephrosis, calculi formation, kidney pathologic change, and the long-term survival. With the new intraureteral cannula technique, the donor mouse was anesthetized with an intramuscular injection of mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg). After that, the right kidney artery and vein of the donor mouse were ligated, followed by infusion of a 500-KL saline into abdominal cavity. The left ureter is dissected from abounding tissue and ligated at the middle of ureter with a 9-0 silk suture (Fig. 1A, A’). The left kidney was isolated by ligating and dividing the adrenal and testicular vessels. The aorta and inferior vena cava (IVC) were dissected at their junction. The infrarenal aorta, IVC below the renal artery and the suprarenal aorta were ligated with a 9-0 silk suture in turn, then introduced a 28.5-gauge needle into the infrarenal aorta for perfusion in situ with 0.5 mL of cold, heparinized (100 unit/mL), lactated Ringer solution for 45 sec. The renal vein at its junction with the IVC was divided and cut. The aorta was divided obliquely, approximately 2 mm below the renal artery. The kidney and associated vessels were then completely freed by cauterizing the tissue surrounding the major vessels, then removed and stored in saline at 4-C for 15Y20 min until the time of transplantation (Fig. 1A, A’). In conventional kidney transplantation, the left ureter was dissected from the bladder to renal hilum without ligating the ureter, a small, elliptical patch of bladder containing the left ureterovesical junction was excised and used for urinary reconstruction in the recipient (12). In the recipient, the right renal hilumwas separated from the perinephric fat to expose the ureteropelvic junction which was incised on the anterior wall, and placed one leg tip of the 11-cm-long curve microforceps (Regine Tweezers, item no 504155; World Precision Instrument, Inc., Sarasota, FL) into the end of drainage catheter with an internal diameter of 0.28 mm, soaked into 75% ethanol for 1 hr before surgery (Polyethylene Tubing PE 10; Becton Dickinson and Company, Spark, MD) and inserted the drainage catheter (5 mm in length) into the ureter of the recipient, secured with a circumferential 5-0 silk ligature, and then removed the kidney (Fig. 1B, B’). Two microvascular clamps (Straight 0.75 4 mm jaw, item no 501779-G; World Precision Instruments, Inc.) were placed proximally and distally on infrarenal aorta and IVC to promote homeostasis. An 11-0 nylon suture was placed through the full thickness of aorta and retracted to make an elliptical arteriotomy by a single cut (about one fifth of the diameter of the vessel). The aorta was irrigated with heparinized saline. Two stay sutures were placed at the proximal and distal apices of the recipient’s arteriotomy. An end-to-side anastomosis was performed between the donor aorta and recipient aorta using continuous 11-0 nylon sutures (VT04A00N07-13; Arosurgical Instruments Corporation, Newport Beach, CA). The back wall was sewn first, through a transluminal approach, then the anterior wall. Only three or four sutures were required for each side. The vein was anastomosed in the same fashion as the artery. Five to seven times, the vein was stitched on each side, depending on the size of the cava patch. After anastomoses, the first distal clamp was loosened and then the proximal one. Blood flowed back into the kidney (Fig. 1C, C’). The recipient’s ureter with the inserted tube was dissected freely down to the bladder without stripping its fat to make the tubing easily. Gently exposed the donor’s ureteropelvic junction, 5 mm under which a small incision was made on the anterior wall, and using a LETTER TO THE EDITOR