目前的研究由美国国家研究院​​卫生部授予R01 - HL092188，R01 - DK083385和R01HL098294，麻醉教育及研究基金会（FAER）港灯资助，FAER授予JYD和东风集团（德意志研究联合会）研究的支持奖学金（GR2121/1-1）公司。

总论所有操作应执行下一个堂堂正正的解剖显微镜（Leica），并通过使用手术凝固剂。应在实验组小鼠在年龄和体重相匹配，以确保结果的可比性。在实验过程中，应该是稳定的，体温，血压，麻醉和流体管理和监测。 1。麻醉和手术<ol><li>肾缺血实验中，可以同时申请两个肾缺血再灌注后，或单肾切除术相结合，与其余肾缺血治疗。由于双边的两个肾脏缺血治疗是增加技术上的困难，更复杂的实验设置和较长的手术，我们将执行一个正确的悬重系统利用剩余的左肾治疗缺血性肾切除术。我们也利用这个吊重系统在心脏冠状动脉阻塞。但是，该过程是不同的，因为肾动脉，可以从周围组织中分离，而在于心脏肌肉的冠状动脉内。 </li><li>小鼠（8-14周龄，雌雄不限）将被麻醉的剂量70mg/kg戊巴比妥。我们将保持约麻醉。 10毫克/公斤/小时戊巴比妥钠。过量可显着降低血压，从而可以改变的结果。在我们最初的研究中，我们也使用氯氨酮和甲苯噻嗪。然而，这些化合物诱导强大的心动过缓（150 /分）。的观察心动过缓旁边也有证据表明，这两种化合物是在缺血的情况下保护机制所涉及的。挥发性麻醉剂不能使用或是由于他们的能力，也诱发肾脏保护和低血压。因此，我们最终选择麻醉的是苯巴比妥。此外，氯胺酮和甲苯噻嗪主要由肾脏排出体外。肾切除术和缺血可能延长氯胺酮麻醉。 </li><li>一旦疼痛反射缺席（镊子挤压脚趾测试）将被置于小鼠在加热温度控制表（RT，埃芬博格，德国慕尼黑，体温保持在37.0 ° C）在左边侧卧位置。 </li><li>用胶带将较低和上肢表。 </li><li>对于非生存的手术，如视频演示中，用矿物油覆盖切口区，以防止切割皮肤吸入鼠毛，从而防止鼠毛过敏的风险。在求生存实验的情况下，适当的无菌技术，包括裁剪的头发，皮肤消毒用碘伏（如优碘），蒸压仪器的无菌处理和使用口罩一个外科医生的无菌手套是必需的。生存手术中不使用矿物油。为了生存手术使用无菌氯化钠</li><li>用剪刀和凝血电极（ERBE，ICC50，德国），以防止肌肉和皮肤血管出血，右侧切口将执行。在通过中线开腹访问肾脏，这种技术不需要进入腹腔。 </li><li>使用4 / 0丝线缝合，右肾剪刀将被削减，而不会干扰与肾上腺皮质血管，包括肾动脉和肾静脉，肾蒂结扎。 </li><li>手术伤口是使用肌壁和皮肤用4 / 0缝线连续缝合两针封闭。 </li><li>动物，然后放置在右侧卧的位置和左翼切口将被执行。 </li><li>左肾小心取出的结缔组织，避免肾上腺和船只。 </li><li>接下来，打开肾脏腹侧下降到一个有机玻璃杯。有机玻璃杯应消毒的生存手术。与周围组织的一个钳，肾极小心推入有机玻璃杯。此后，极是仔细有机玻璃杯推到一个镊子。肾脏将保持潮湿和温暖，用湿棉签用清水浸泡在37.0 ° C。 </li><li>肾者，暴露左肾和安置到一个有机玻璃杯克制腹侧肾血管紧张。一个足够的限制是很重要的一个肾动脉下成功就业的缝合。此后，肾脏的人是固定的，可以很容易地确定肾动脉，肾静脉上运行。 </li><li>肾动脉解剖从​​邻近组织，从腹主动脉用细镊子起飞。从最高层的一个弯钳的基本静脉动脉剥离。 </li><li>被放置在肾动脉，通过一个8 / 0丝线缝合（爱惜康，日本东京，德国）作为一个领先的指南钳。这种技术允许在不中断只有动脉血流肾肾静脉受压，较肾蒂夹紧。 </li><li>缝合是放置在一个小杆（可事先准备好的安全别针），重量约。 1G是连接到每个（Eppendorf管）。肾脏大小或添加到Eppendorf管中的水的重量，可适应。然而，动脉应稍微拉向上，使没有血液通过动脉，但没有对动脉的主要舒展运行。主要拉伸应力对动脉可引起动脉壁的增厚和减少再灌注血管壁损伤。 </li><li>虽然重量是不受支持的，立即肾动脉闭塞。相反，当的权重支持，立即恢复血流的器官。成功闭塞证实的颜色从红色变为白色。 </li><li>不同缺血时间可以进行实验的要求。一个完整的再灌注缺血时间的长短取决于将发生。缺血时间长达45分钟让可收回肾损伤。在我们的实验设置中，我们用20高达30分钟，缺血与一个中等的肾损伤，这让我们看到了改善，以及根据小鼠品系和治疗的肾脏损害减值。 </li><li>完成再灌注后肾脏可以更换的有机玻璃杯。因此肾脏的持有人是不固定的，对肾脏的紧张被释放。颅极轻轻的杯子中剔除出去，不愠不火的镊子和尾杆紧随。 </li><li>可以释放鼠标的固定（磁带）和鼠标就可以放置在仰卧姿势如FITC -菊粉标记间隙。 </li><li>在实验的子集，血压和心率，可以记录整个手术过程。血压测量得到不断通过颈内动脉导管。总之，颈内动脉通过钝性分离气管肌肉暴露。经过进一步的接触和小心避免组织创伤（尤其是迷走神经），导管插入使用两个缝线和一个小钳的船只。测量血压是一个statham元素（280 WK，王氏港建，Kaltbrunn，瑞士）。 </li><li>对于FITC标记的菊粉间隙导管将放在左侧颈内静脉滴注氯化钠和FITC标记的菊粉。此外，膀胱导管插入尿液收集在一个随时间变化的的方式将在下面详细解释。 </li></ol> 2。肾损伤的测量<ol><li>一般建议:下面的结果参数，以确定肾损伤，肾缺血: 执行FITC标记的菊粉的间隙，以评估肾小球滤过率（GFR）。作为替代方案确定通过代谢笼调查24小时，肾功能损伤后的肌酐清除率和血清肌酐。执行肾H＆E染色和一个过氧化物酶（MPO）ELISA法，以确定肾功能损害和炎症24小时，肾缺血。 </li><li>代谢笼调查: <ol><li>左肾缺血转身进入腹膜后的位置，如上所述，伤口闭合。 </li><li>小鼠在手术结束时，收到0.3毫升生理盐水IP和下加热灯2小时的恢复。 </li><li>此后，老鼠将被放入代谢笼（Tecniplast德国，Hohenpeissenberg，德国），肾功能参数测定24小时的2倍。 </li><li>每只小鼠的取水口已确定称重水空和填充瓶供应前鼠标比24小时后再次称重满瓶。鼠标的重量以及待定。 </li><li> 24小时后约75μl血从眼睛后方的静脉丛通过短isoflorane麻醉下的毛细管。 </li><li>通过测量肾使用市售的colorimtric方法，根据制造商的协议（LT - SYS的，劳动+技术公司，德国柏林）缺血后血浆和尿肌酐24小时的肾功能评估。血浆和尿钠 ，钾浓度+决心与火焰发射光度计（6361 ELEX的Eppendorf公司，汉堡，德国）。肾脏的排泄和血液动力学的价值观是使用 15-17标准的公式计算。 </li></ol></li><li>菊粉间隙:FITC -菊粉清除实验将执行以下肾缺血。 <ol><li>左侧颈内静脉插管连续的Infusion。解剖后从邻近组织的静脉用一备剪刀（杜蒙，WPI），放置两个缝线周围静脉（4 / 0）。放置之前，阻止血液流向proximale缝合静脉钳。此后，关闭远端结和重量放在远端缝合结束应变静脉紧张。静脉导管，这是非常重要的。使用微型剪刀切一个小口进入静脉。保持开放与罚款钳（杜蒙，WPI），并放入静脉导管。为了推进导管钳断。确保没有导管的张力，让你可以无需拿着它下来。随后关闭近端缝合静脉导管在第二。 </li><li>导管放置在定时收集尿液的膀胱。因此，你切一个小切口，在皮肤，膀胱。通过皮肤孔，小心地取出膀胱。用湿纸巾修复膀胱。放置在膀胱（4 / 0）准备缝合。 </li><li>剪下一小段导管（1.5厘米）结束的热量，这使得它更容易在膀胱修复导管周围的一环。 </li><li>削减其极顶一个孔进入膀胱。请削减之间的船只，以防止出血。插入导管进入膀胱的大底和关闭通过导管和膀胱的缝合。 </li><li>所有的老鼠收到0.85％氯化钠溶液中静脉推注，金额相等于体重的20％。连续输液维持在800ul/h/25g体重和FITC标记的菊粉是评价整个肾脏肾小球滤过率（GFR）的输液率。 20分钟的动物稳定后，20分钟定时尿液集合进行测定尿流率和FITC -菊粉（四次）。血液是在每测量FITC -菊粉期间中获得。 </li><li>当开始20分钟收集期间使用一个计时器。收集尿液，由一个小的Eppendorf管放入膀胱导管结束。测量前后收集尿液，使你能确定尿量管重量。 retroorbital血液收集的形式，通过在每个收集期中的毛细管静脉丛通过采取血液样本。 </li></ol></li><li>后进一步分析，如MPO ELISA法，细胞因子的EISA，组织学的每个程序，可采集血液和肾脏。 </li></ol> 3。代表性的成果: <img src="/files/ftp_upload/2549/2549fig1.jpg" alt="图1"/> 图1吊重系统为肾缺血模型:一个悬重系统已经建立，导致左肾肾缺血。因此8.0尼龙缝线划归肾动脉和一个小，重量轻（1G）附加在每个缝合结束。这个实验设计允许申请和释放的重量负荷可逆的肾动脉闭塞。

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综上所述，本研究介绍了一种在小鼠模型使用一个吊重系统为肾动脉闭塞的肾缺血的新技术。事实上，这项研究表明高度重复性的伤害，从而最大限度地减少钳夹肾蒂的变异。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>戊巴比妥钠</td><td> Vortech药业LS，有限公司</td><td> VPL 9372 </td><td> 4mg/mL在生理盐水</td></tr><tr><td>缝合，丝绸4.0 </td><td>哈佛器械</td><td> 517698 </td><td></td></tr><tr><td>缝合后，普理灵8.0 </td><td>美国爱惜康， </td><td> M8739 </td><td>可重复使用</td></tr><tr><td>解剖显微镜</td><td>徕卡</td><td> N / A </td><td>考虑慷慨的工作距离</td></tr><tr><td>暖气表</td><td> RT，Effenberger，德国</td><td> N / A </td><td>只和单一供应商</td></tr><tr><td>手术器械</td><td> WPI的，杜蒙</td><td></td><td>罚款微血管剪刀</td></tr></tbody></table>

use of a hanging-weight system for isolated renal artery occlusion

在住院的病人中，超过50％的急性肾损伤（AKI）的情况下引起的肾缺血1-3。一个住院的病人最近的一项研究显示，只有血清肌酐水平轻度增加（0.3〜0.4毫克/升）大于70％，在没有任何增加 1人死亡风险。沿着这些路线，手术过程中，需要交叉夹紧的主动脉和肾血管与肾功能衰竭率高达30％，4 。同样，超过10％的患者在正常情况下，心脏手术后AKI的发生与死亡率大幅增加有关。 AKI的是肝移植术后常见的并发症。至少有8-17％的患者最终需要肾脏替代治疗5。此外，由于肾小管细胞损伤在肾移植肾功能延迟是经常与缺血相关AKI的6。此外，AKI的发生在约20％的患者患败血症 6。AKI的发生是与急剧增加的发病率和死亡率 1关联。治疗方法是非常有限的，多数在AKI的介入试验，在人类的失败。因此，迫切需要额外的治疗方式，以防止缺血肾损伤3，7-9。 为了阐明肾损伤的机制因缺血和小鼠模型可能的治疗策略是集中需要7-13。小鼠模型提供利用不同的遗传模型，包括基因靶向小鼠和组织特异性的基因靶向小鼠（CRE - flox系统）的可能性。然而，小鼠肾缺血是在技术上具有挑战性和实验细节显着影响的结果。我们进行了系统评价的一种新型模式，为分离出小鼠肾动脉闭塞，特别是避免使用夹紧或缝合肾蒂 14 。这种模式需要一个肾切除右肾，因为缺血，可只有一个肾，由于实验设置执行。事实上，通过使用吊重系统，肾动脉只有仪表曾经在整个手术过程。此外，无静脉或尿道梗阻发生这种技术。通过测量血清肌酐，我们能证明与缺血时间剂量依赖性和高度重复性的肾损伤。此外，缺血预处理与传统的夹紧整个蒂，肾保护这种新的模式进行比较时，，是更深刻，更可靠。因此，他的新技术可能会在急性肾损伤领域工作的其他研究人员的有用。

Watch this Scientific Journal Video about Use of a Hanging-weight System for Isolated Renal Artery Occlusion at JoVE.com

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

因缺血而精确的小鼠模型为急性肾损伤（AKI）是一个重要的工具，调查急性肾损伤，并有可能找到治疗肾损伤的治疗手段。悬重系统提供了一个即时和可靠的肾动脉闭塞再灌注的工具，没有引起肾充血。

一个悬重系统使用隔离肾动脉闭塞

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

use-of-a-hanging-weight-system-for-isolated-renal-artery-occlusion

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

15.9K 次观看.  University of Colorado. 因缺血而精确的小鼠模型为急性肾损伤（AKI）是一个重要的工具，调查急性肾损伤，并有可能找到治疗肾损伤的治疗手段。悬重系统提供了一个即时和可靠的肾动脉闭塞再灌注的工具，没有引起肾充血。

视频: Use of a Hanging-weight System for Isolated Renal Artery Occlusion

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

悬重系统的使用在小鼠冠状动脉闭塞

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

科研

医学

Use of a Hanging-weight System for Liver Ischemia in Mice

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and can result in liver failure which has a high mortality rate1-2. Therefore murine studies of hepatic ischemia have become an important field of research by providing the opportunity to utilize pharmacological and genetic studies3-9. Specifically, conditional mice with tissue specific deletion of a gene (cre, flox system) provide insights into the role of proteins in particular tissues10-13 . Because of the technical difficulty associated with manually clamping the portal triad in mice, we performed a systematic evaluation using a hanging-weight system for portal triad occlusion which has been previously described3. By using a hanging-weight system we place a suture around the left branch of the portal triad without causing any damage to the hepatic lobes, since also the finest clamps available can cause hepatic tissue damage because of the close location of liver tissue to the vessels. Furthermore, the right branch of the hepatic triad is still perfused thus no intestinal congestion occurs with this technique as blood flow to the right hepatic lobes is preserved. Furthermore, the portal triad is only manipulated once throughout the entire surgical procedure. As a result, procedures like pre-conditioning, with short times of ischemia and reperfusion, can be easily performed. Systematic evaluation of this model by performing different ischemia and reperfusion times revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels3,9. Taken together, these studies confirm highly reproducible liver injury when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Thus, this technique might be useful for other investigators interested in liver ischemia studies in mice. Therefore the video clip provides a detailed step-by-step description of this technique.

We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.

使用吊重系统在小鼠肝缺血

Acute liver injury due to ischemia can occur during several clinical procedures e.g. liver transplantation, hepatic tumor resection or trauma repair and ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

大鼠原位肾移植: 一种新的简化的手术方法

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) is overactivated. In this context, there is a narrowing of the renal arteries that stimulate an increase in the expression and release of renin, the rate-limiting protease in RAAS. The resulting rise in renin expression is a known driver of renovascular hypertension, frequently associated with kidney injury and end organ damage. Thus, there is a great interest in developing novel treatments for this condition. The molecular and cellular mechanism of renin control in renal artery stenosis is not fully understood and warrants further investigation. To induce renal artery stenosis in mice, a modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed. The right kidney was stenosed in wild type mice and sham operated mice were used as control. After renal artery stenosis, we determined renin expression and kidney injury. Kidneys were harvested, and fresh cortices were used to determine protein and mRNA expression of renin. This animal model is reproducible and can be used to study pathophysiological responses, molecular and cellular pathways involved in renovascular hypertension and kidney injury.

A modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed using polyurethane tubing to initiate renal artery stenosis, inducing an increase in renin expression and kidney injury. Here, we describe a detailed procedure of preparing and placing the cuff onto the renal artery to generate a reproducible and consistent 2K1C mouse model.

肾动脉狭窄肾素调节的改良双肾单夹小鼠模型

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

通过肾脏动脉直接向肾脏输送药物

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

临时双性肾动脉闭塞的急性肾损伤大型动物模型

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

利用酶生物传感器的量化内源性ATP或H<sub&gt; 2</sub&gt; 0<sub&gt; 2</sub&gt;在肾脏

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

生物学

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 ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Technical Refinement of a Bilateral Renal Ischemia-Reperfusion Mouse Model for Acute Kidney Injury Research

Cardiac arrest poses a large public health burden. Acute kidney injury (AKI) is an adverse marker in survivors of cardiac arrest following the return of spontaneous circulation (ROSC) after successful cardiopulmonary resuscitation. Conversely, recovery of kidney function from AKI is a predictor of favorable neurological outcomes and hospital discharge. However, an effective intervention to prevent kidney damage caused by cardiac arrest after ROSC is lacking, suggesting that additional therapeutic strategies are required. Renal hypoperfusion and reperfusion are two pathophysiological mechanisms that cause AKI after cardiac arrest. Animal models of ischemia-reperfusion-induced AKI (IR-AKI) of both kidneys are comparable with patients with AKI following ROSC in a clinical setting. However, IR-AKI of both kidneys is technically challenging to analyze because the model is associated with high mortality and wide variation in kidney damage, which may affect the analysis. Lightweight mice were chosen, placed under general anesthesia with isoflurane, subjected to surgery with a dorsolateral approach, and their body temperature maintained during operation, thereby reducing tissue damage and establishing a reproducible acute renal IR-AKI research protocol.

This study established a protocol focusing on the technical refinement of a mouse model of bilateral renal ischemia-reperfusion for acute kidney injury research.

用于急性肾损伤研究的双侧肾缺血再灌注小鼠模型的技术改进

Cardiac arrest poses a large public health burden. Acute kidney injury (AKI) is an adverse marker in survivors of cardiac arrest following the return of ...