，德博士Caestecker的实验室支持由NIH 1RO1 HL093057-01 1RC4DK090770的01。哈里斯博士的实验室DK38226，DK51265，DK62794和退伍军人管理局的资金支持。支持小鼠肾脏损伤手术，血清肌酐和纤维化检测也提供了的范德比尔特奥布莱恩肾损伤中心授予1P30 DK079341。

<ol><li>所有手术器械，手术前高压灭菌。请注意，如果一个人在不同的小鼠进行多次手术，冲洗工具使用后，然后消毒用热珠灭菌。这是不够的，在70％乙醇中浸泡。 </li><li> 0.5毫升S / C灭菌生理盐水术前和术后即刻，以弥补体内津液损耗，在手术过程中。 </li><li>权衡老鼠。 </li><li>麻醉鼠标使用I​​P甲苯噻嗪/氯胺酮的混合物。它的老鼠通常需要3-5分钟，以达到手术麻醉平面。 </li><li>剃须足够的边境地区，以保持手术部位的污染切口部位的头发。这需要执行，而不是在一个预备区域进行手术。 </li><li>应用的润滑眼用软膏的眼睛在手术过程中，以防止干燥。 </li><li>将鼠标易吸水板凳垫加热表面覆盖着。 ，胶带腿手术表面。 </li><li>如果使用水浴系统，设置加热38℃1小时，手术前。 </li><li>无菌制备使用聚维酮碘溶液拭棒的网页从中心朝周3次，每次其次与Nolvasan洗涤以除去聚维酮碘擦洗。应除去所有优碘外科领域切口前与Nolvasan冲洗。前启动无菌性程序手套应改为无菌。 </li><li>用于外科手术用无菌手术悬垂领域。这将防止杂散头发进入外科领域奠定无菌器械在手术过程中提供一个区域。 </li><li>通过皮肤触诊肾脏位置。 </li><li>切鼠标（约1.5厘米），用剪刀和镊子沿中线背部皮肤。 </li><li>用剪刀和镊子钝性剥离经此切口在左侧和右侧的背侧面的单独的皮肤和皮下组织层。 </li><li>做一个小切口，通过正确的FLANK肾脏上方的肌肉和筋膜，exteriorize右肾。 </li><li>小心解剖的肾脏不受周围组织的上部和下部磁极。需要注意的是从上极周围的脂肪组织中包含带有其自身的血液供应可推离肾脏，但显然不应该被删除（ 图1）从小鼠的肾上腺。解放后肾周围组织，配合4-0丝线缝合，周围肺门右肾使用双外科结。留下的一端缝合〜2托起肾脏切结远端，所以你可以想像被削减的网站。灌溉残端用无菌生理盐水。 </li><li>关闭的肌肉层，使用可吸收缝合线。 </li><li>做一个小切口，通过左翼肾脏上方的肌肉和筋膜，exteriorize左肾（ 图2A）。 </li><li>使用钝钳，同时释放肾肾谨慎持有蒂周围脂肪组织，使用镊子（ 图2B-2F）。使用钝钳对肾脏的压力太大可能会导致肾功能损伤，所以需要记住这个设计时，"假"控制研究。如果你发现你的假控件正在开发肾损伤（如血清肌酐上升可见一斑），一种替代方法，你可以考虑释放肾蒂周围脂肪组织是组织用盐水浸泡消毒棉轻轻摇动拭子。在我们的经验中，脂肪是相当紧紧地贴到肾蒂，并不能很容易地用棉签去除，所以这可能不是你想使用第一种方法。然而，这种模式的其他方面一样，这是后话，你将不得不建立自己的实践和经验。 </li><li>钳左肾蒂使用非创伤性血管钳持物钳（便于放置钳），使用定时器不同CLA根据熔点倍（ 见表1）的小鼠品系，损伤的严重程度。 </li><li>量左肾与皮肤（ 图2H）。 </li><li>切开皮肤，用生理盐水浸湿纱布覆盖。 </li><li>肾Exteriorize，确认昏暗的外观均匀，松开夹紧指定的时间后，确认昏暗的外观均匀逆转整个肾脏（ 图2I / G）。如果肾脏不快速而均匀地重新灌注（粉红色向上）鼠标应该从进一步的分析中排除。轻轻推回肾脏进入腹膜后间隙。 </li><li>关闭肌肉层，使用可吸收缝合线。 </li><li>关闭皮肤层采用尼龙单丝不吸收缝线。 </li><li>辖丁丙诺啡镇痛。 </li><li>远离外科领域转移小鼠到一个加热垫，直到他们醒来。 </li><li>返回小鼠动物室。 </li><li>密切监测小鼠，并给予额外的剂量丁丙诺啡如上所示，每8-12小时的疼痛或不适。镇痛药导致的临床症状包括抑郁症或其他行为上的改变，外观异常，或姿势如PILO勃起，弯腰驼背的姿势缺乏疏导和进食，或行动不便。 </li></ol>

<ol>
	<li>Singh, A. P., Junemann, A., Muthuraman, A., Jaggi, A. S., Singh, N., Grover, K., Dhawan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+renal+failure.">Animal models of acute renal failure.</a> Pharmacological reports: PR. 64, 31-44 (2012).</li><li>Heyman, S. N., Rosenberger, C., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+ischemia-reperfusion:+Biases+and+myths-the+proximal+vs.+Distal+hypoxic+tubular+injury+debate+revisited.">Experimental ischemia-reperfusion: Biases and myths-the proximal vs. Distal hypoxic tubular injury debate revisited.</a> Kidney Int. 77, 9-16 (2010).</li><li>Lieberthal, W., Nigam, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+renal+failure.+II.+Experimental+models+of+acute+renal+failure:+Imperfect+but+indispensable.">Acute renal failure. II. Experimental models of acute renal failure: Imperfect but indispensable.</a> Am. J. Physiol. Renal Physiol. 278, F1-F12 (2000).</li><li>Dunn, S. R., Qi, Z., Bottinger, E. P., Breyer, M. D., Sharma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+endogenous+creatinine+clearance+as+a+measure+of+renal+function+in+mice.">Utility of endogenous creatinine clearance as a measure of renal function in mice.</a> Kidney Int. 65, 1959-1967 (2004).</li><li>Yuen, P. S., Dunn, S. R., Miyaji, T., Yasuda, H., Sharma, K., Star, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+HPLC+determination+of+creatinine+in+mouse+serum.">A simplified method for HPLC determination of creatinine in mouse serum.</a> Am. J. Physiol. Renal Physiol. 286, F1116-F1119 (2004).</li><li>Hetu, P. O., Gingras, M. E., Vinet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+rapid+liquid+chromatography+isotope+dilution+tandem+mass+spectrometry+(LC-IDMS/MS)+method+for+serum+creatinine.">Development and validation of a rapid liquid chromatography isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for serum creatinine.</a> Clin. Biochem. 43, 1158-1162 (2010).</li><li>Keppler, A., Gretz, N., Schmidt, R., Kloetzer, H. M., Groene, H. J., Lelongt, B., Meyer, M., Sadick, M., Pill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+creatinine+determination+in+mice+and+rats:+An+enzymatic+method+compares+favorably+with+a+high-performance+liquid+chromatography+assay.">Plasma creatinine determination in mice and rats: An enzymatic method compares favorably with a high-performance liquid chromatography assay.</a> Kidney Int. 71, 74-78 (2007).</li><li>Yang, L., Besschetnova, T. Y., Brooks, C. R., Shah, J. V., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+cycle+arrest+in+G2/M+mediates+kidney+fibrosis+after+injury.">Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury.</a> Nat. Med. 16, 535-543 (2010).</li><li>Shanley, P. F., Rosen, M. D., Brezis, M., Silva, P., Epstein, F. H., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+focal+proximal+tubular+necrosis+after+ischemia+with+reflow+in+the+rat+kidney.">Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.</a> Am. J. Pathol. 122, 462-468 (1986).</li><li>Chawla, L. S., Amdur, R. L., Amodeo, S., Kimmel, P. L., Palant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+severity+of+acute+kidney+injury+predicts+progression+to+chronic+kidney+disease.">The severity of acute kidney injury predicts progression to chronic kidney disease.</a> Kidney Int. 79, 1361-1369 (2012).</li><li>Lo, L. J., Go, A. S., Chertow, G. M., McCulloch, C. E., Fan, D., Ordonez, J. D., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialysis-requiring+acute+renal+failure+increases+the+risk+of+progressive+chronic+kidney+disease.">Dialysis-requiring acute renal failure increases the risk of progressive chronic kidney disease.</a> Kidney Int. 76, 893-899 (2009).</li><li>Burne, M. J., Haq, M., Matsuse, H., Mohapatra, S., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+susceptibility+to+renal+ischemia+reperfusion+injury+revealed+in+a+murine+model.">Genetic susceptibility to renal ischemia reperfusion injury revealed in a murine model.</a> Transplantation. 69, 1023-1025 (2000).</li><li>Muller, V., Losonczy, G., Heemann, U., Vannay, A., Fekete, A., Reusz, G., Tulassay, T., Szabo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+in+renal+ischemia-reperfusion+injury+in+rats:+Possible+role+of+endothelin.">Sexual dimorphism in renal ischemia-reperfusion injury in rats: Possible role of endothelin.</a> Kidney Int. 62, 1364-1371 (2002).</li><li>Schmitt, R., Marlier, A., Cantley, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zag+expression+during+aging+suppresses+proliferation+after+kidney+injury.">Zag expression during aging suppresses proliferation after kidney injury.</a> J. Am. Soc. Nephrol. 19, 2375-2383 (2008).</li><li>Oxburgh, L., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+injury+of+the+mouse+kidney.">Ischemia-reperfusion injury of the mouse kidney.</a> Methods Mol. Biol. 886, 363-379 (2012).</li></ol>

作者有没有利益冲突的披露。

我们描述了两种型号的IR-AKI的中度和重度肾功能损伤的影响进行研究。这些模型使我们能够诱发一致的和可预见的伤害死亡率低。我们的协议列出了很多的困难和陷阱传统与此模型关联。此外，我们已经表明，根据对肾蒂夹紧长度，模型诱导很大程度上可逆的轻度和中度AKI，或更严重的AKI不完整的恢复和持续性肾脏纤维化。伤后纤维化严重的IR-AKI模型可能会导致老鼠能够生存比将有可能更加严重?...

各种实验模型急性肾损伤（AKI）已发展到人类生存条件的多样性和复杂性（见参考最近的，全面检讨1）匹配。这些模型中的每一个都有自己的长处和弱点，而每一个模仿人类的​​相应条件具有不同的效率，没有精确地模拟人类同行的病理生理机制。缺血再灌注剂AKI（IR）已经发展成为一个模型，在啮齿动物的急性缺血性肾损伤。虽然肾小管损伤的严重程度，在这个模型中看到很少观察到在患者的肾脏灌注不足，损伤2，尽管有其局限性，这主要是因为在此模型中的可重现的性质，提供了其广泛的用途，预期将继续AKI的共同的基本机制，修复，治疗3到许多重要的见解。 IR手术需要FAMiliarity与小鼠肾脏解剖，这是我们在图1中示出一个简化的形式。肾缺血再灌注（IR）损伤手术可以通过腹（开腹手术）或背（后腹膜）方法。我们用背的方法，因为它创伤小，允许更快的恢复时间，提高生存期（特别是当第一次学习的过程）。可以进行单侧或双侧肾缺血再灌注损伤。然而，有效的夹紧压力（这可能是由于插入肺门周围的脂肪之间的钳口）和/或不同响应肾缺血侧之间的差异导致更可变的结果。虽然这不是一个难以逾越的问题，它可以增加之间的变异实验，这是一个重大的问题，这个模型。可以进行单侧红外与对侧肾切除术。这是我们的方法的选择，因为它减少可变性椎弓根之间的夹紧，并且在同一时间，一个的llows一个评估肾功能，仅单方面IR不受影响。一直在讨论什么是最实用与优化的方法来评估肾功能的影响。血中尿素氮（BUN）提供了一个衡量肾功能是AKI的部分机型，包括缺血再灌注损伤的一个有用的"先看看"的标记。然而，可以BUN水平可能受影响的老鼠（体积）的状态的影响，特别是腹方法IR损伤，延迟恢复时降低若干天手术后的流体经口摄入。血肌酐水合状态的影响较小，但显然是受到肌肉质量。与血清肌酐测量的困难之一已经检测非肌酐色原体小鼠血清中，用苦味酸为基础的技术的问题。作为替代方案，一些中心已经开发了一个基于HPLC的方法来量化鼠标肌酐，不会受到这件神器4。但是，UNL艾克BUN和苦味酸，只需要5-10微升血清肌酐检测，高效液相色谱法检测需要25微升血清每次检测，如果重复进行，将需要约100微升全血每次检测。这可以限制对小鼠的研究。一些中心已经开发出更灵敏的高效液相色谱法和质谱为基础的方法，使分析更小的样本量，5，6。然而，这些技术没有得到广泛使用。另一种酶的级联法（只需要5-10微升血清）已在小鼠和大鼠的血清样本进行评估，并密切平行高效液相色谱法测量血清肌酐苦味酸检测总是高估肌酐值7。虽然这个实验没有被广泛应用于在AKI文学，检测市售，使用简单，我们发现这个模型小鼠IR剂AKI给出可靠的结果。

<table border="1">
	<tbody>
		<tr>
			<td>Drug or solution</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ketamine (anesthetic)</td>
			<td>Webster Veterinary</td>
			<td>07-881-9413 (100 mg/ml)</td>
			<td>Final concentration 30 mg/ml Ketamine with 3 mg/ml Xylazine administered at 3-4 ml/kg (100 mg/kg Ketamine and 10 mg/kg Xylazine)</td>
		</tr>
		<tr>
			<td>Xylazine (analgesic)</td>
			<td>Webster Veterinary</td>
			<td>07-808-1939 (100 mg/ml)</td>
			<td>As above</td>
		</tr>
		<tr>
			<td>Antisedan (anti-Xylazine)</td>
			<td>Webster Veterinary</td>
			<td>07-867-7097 (5 mg/ml)</td>
			<td>Optional: to reverse effects of Xylazine in order to increase the rate of recovery after surgery Use 0.5-0.7 mg/kg</td>
		</tr>
		<tr>
			<td>Buprenorphine (analgesic)</td>
			<td>Bedford Laboratories</td>
			<td>NDC 55390-100-10 (0.3 mg/ml)</td>
			<td>Dose 0.05-0.1 &mu;g/g every 8-12 hr. Make up a 1:5 dilution in sterile normal saline preoperatively and give 2.5-5 &mu;l/g of the diluted solution.</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>NDC 17033-211-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Betadine swab sticks</td>
			<td>Purdue Products L.P.</td>
			<td>NDC 67618-153-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nolvasan Surgical Scrub</td>
			<td>Pfizer Animal Care</td>
			<td>300253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Drugs and solutions for I/R surgery.</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Integra Miltex</td>
			<td>VWR 21909-404</td>
			<td>For skin and muscle dissection</td>
		</tr>
		<tr>
			<td>Curved iris forceps</td>
			<td>FST</td>
			<td>11065-07</td>
			<td>To hold skin and muscle, to remove peri-nephric fat</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>ROBOZ</td>
			<td>RS-5228</td>
			<td>To manipulate kidneys</td>
		</tr>
		<tr>
			<td>Vascular clamp (795 g pressure)</td>
			<td>ROBOZ</td>
			<td>RS-5459</td>
			<td>Good for C57BL/6 and BALB/c mice strains</td>
		</tr>
		<tr>
			<td>Schwartz clip applying forceps</td>
			<td>ROBOZ</td>
			<td>RS-5450</td>
			<td>For RS-5459 clamps</td>
		</tr>
		<tr>
			<td>Vascular clamp (75-85 g pressure)</td>
			<td>FST</td>
			<td>18055-02</td>
			<td>Optimal for CD1 mice strain, but also can be used for C57BL/6 and BALB/c</td>
		</tr>
		<tr>
			<td>Micro-Serrefine Clamp Applicator-14 cm with Lock</td>
			<td>FST</td>
			<td>18056-14</td>
			<td>For FST 18055-02 clamps</td>
		</tr>
		<tr>
			<td>Gayman T/Pump (water-bath heated platform)</td>
			<td>Braintree Scientific</td>
			<td>TP-650</td>
			<td>The water heater is set at 38 &deg;C 1 hr prior surgery</td>
		</tr>
		<tr>
			<td>15" W x 24" Heated platform</td>
			<td>Braintree Scientific</td>
			<td>HHP-2</td>
			<td>Connect to Gayman heated water pump</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree Scientific</td>
			<td>Model 39DP</td>
			<td>Uses microwave for heating. This is useful for warming during recovery</td>
		</tr>
		<tr>
			<td>Absorbable suture (Vicryl 5-0)</td>
			<td>ETHICON</td>
			<td>VCP834G</td>
			<td>For fascia and muscle</td>
		</tr>
		<tr>
			<td>Non-absorbable suture: monofilament nylon MONOMID 6/0 660B)</td>
			<td>CP Medical</td>
			<td>CP-B660B-03</td>
			<td>For skin</td>
		</tr>
		<tr>
			<td>Halsey needle holder: Tungsten Carbide/13 cm</td>
			<td>FST</td>
			<td>12501-13</td>
			<td>To hold suture needles</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>FST</td>
			<td>06-662-3</td>
			<td>To quantify ischemia time</td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>FST</td>
			<td>18000-45</td>
			<td>To sterilize instruments between mice if you are performing multiple surgeries (it is not sufficient to soak instruments in ethanol)</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Braintree Scientific</td>
			<td>CLP-22965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Durapore</td>
			<td>1538-0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>EXELint</td>
			<td>26044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicators</td>
			<td>Kendall</td>
			<td>8884541300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbent BenchPad</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical drapes</td>
			<td>VWR</td>
			<td>21902-985</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr>
			<td>2'' x 2'' Gauze Pads</td>
			<td>Medi-First</td>
			<td>60673</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Cardinal Health</td>
			<td>2D7251</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Equipment and instruments for I/R surgery.</td>
		</tr>
	</tbody>
</table>

ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice

缺血再灌注引起的急性肾损伤（IR-AKI）被广泛用于AKI的小鼠作为模型，但结果往往是相当高，经常未经报告的死亡率可能混淆分析变量。双侧肾蒂钳位常用的，诱导IR-AKI的，但有效的钳位压力和/或肾功能的响应之间的肾脏缺血之间的差异经常导致多个变量的结果。此外，被称为短钳次吸引更多变量的肾小管损伤，而小鼠进行双边损伤钳较长时间的发展更一致的肾小管损伤，他们往往在严重肾功能不全，由于伤后的第3天死亡。为了提高伤后生存和获得更加一致和可预测的结果，我们已经开发出两款车型的单方面缺血再灌注损伤对侧肾切除。这两种手术是用背的方法，减少手术应激产生的f罗腹的剖腹手术常用鼠标的IR-AKI的手术。对于中度伤害的BALB / c小鼠进行诱导单侧夹持肾蒂26分钟，也同时接受对侧肾切除术。 50-60％的小鼠使用这种方法，发展适度AKI 24小时后受伤，但90-100％的小鼠生存。要诱发更严重的AKI，BALB / c小鼠进行肾蒂阻断30分钟后对侧肾切除伤后第8天。这允许90-100％的生存伤后肾功能恢复的功能性评估。伤后早期肾小管损伤以及伤后纤维化是高度一致，采用这种模式。

中度IR剂AKI IR同时对侧肾切除单侧大大降低结果的变异，但与小鼠的生存之本程序所必需的钳时间较短，我们仍然发现，只有50-60％的小鼠开发了预期的肾功能不全24小时伤后（ 图3A）。在实际应用中，这造成了难以评估数据，除非该研究涉及的治疗方案，可以发起伤后至少24小时，让时间来评估肾功能和丢弃小鼠不发展为肾功能不全。 <...

Watch this Scientific Journal Video about Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice at JoVE.com

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

我们描述模型，中度和重度缺血再灌注引起的肾损伤的小鼠进行单侧肾蒂夹紧对侧肾切除术同期或延期，其次分别。这些车型一贯引起肾功能不全和损伤后纤维化，但损伤程度和生存都依赖于鼠标的背景，年龄和手术设备。

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

ischemia-reperfusion-model-acute-kidney-injury-post-injury-fibrosis

Research

JoVE Journal

Medicine

39.5K 次观看.  Vanderbilt University Medical Center. 我们描述模型，中度和重度缺血再灌注引起的肾损伤的小鼠进行单侧肾蒂夹紧对侧肾切除术同期或延期，其次分别。这些车型一贯引起肾功能不全和损伤后纤维化，但损伤程度和生存都依赖于鼠标的背景，年龄和手术设备。

视频: Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

常温心脏骤停和心肺复苏术:缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

大鼠心肌缺血再灌注后冠状动脉流动储备评价

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成人斑马鱼中西斯铂诱导的急性肾损伤模型

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

器官缺血-输液损伤模拟大鼠肝脏移植模型的血动力学变化

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

温缺血/再灌注损伤的大鼠肺移植模型:改善结局的优化

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

单侧肾缺血再灌注损伤的有效小鼠模型

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...