搜索

Two-event Transfusion-related Acute Lung Injury Mouse Model
通过双重免疫刺激建立输血相关急性肺损伤的小鼠模型   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Transfusion-related acute lung injury (TRALI) is defined as acute lung injury that occurs within 6 hours of a blood product transfusion. TRALI continues to be a leading cause of transfusion-related mortality and we have developed a mouse model of TRALI to better understand the mechanisms by which injury occurs and to test therapeutic approaches. Our model is a two-event model based on immune priming and the challenge of BALB/c wild-type mice with cognate MHC Class I monoclonal antibody (MHC I mAb). Immune priming with LPS mimics the primed state of recipients (first event) that is important for the development of TRALI. Donor HLA antibodies are frequently implicated in TRALI reactions, and cognate MHC Class I antibody (second event) produces acute lung injury in primed animals. Here, we describe a detailed protocol with high reproducibility within animals.

Keywords: Transfusions(输血), Acute lung injury(急性肺损伤), Two-event model(两事件模型)

Materials and Reagents

  1. BALB/c WT mice (male, age 8-12 weeks)
  2. MHC Class I mAb (H2Kd, IgG2a,κ; stock concentration 0.65 mg/ml) is purified from a hybridoma (ATCC, catalog number: 34-1-2S ) using standard protein A affinity chromatography.
  3. Isotype-matched mAb (IgG2a,κ, stock concentration 1 mg/ml) (BD, catalog number: 553453 )
  4. LPS (Sigma-Aldrich, catalog number: L2880 )
  5. Ketamine (Henry Schein, catalog number: 010177 ) and Xylazine (Henry Schein, catalog number: 033198 ) for mouse anesthesia
  6. Heparin Sodium Injection (Sagent Pharmaceuticals, NDC number: 25021 )
  7. Phosphate buffered saline (PBS) (see Recipes)

Equipment

  1. Surgical scissors
  2. Curved blunt-ended forceps
  3. 25 gauge and 28G sterile needles
  4. Insulin syringe (0.5 ml) and attached needle (sterile)
  5. 1 ml sterile syringes
  6. 30-gauge sterile needle for jugular vein cannulation
  7. Polyethylene tubing PE-10 (Intramedic, catalog number: 427401 )
  8. Cotton tipped applicator
  9. 6-0 silk suture
  10. Warming pad
  11. Tape

Procedure

  1. Preparation
    All protocols using live animals must be approved by an Institutional Animal Care and Use Committee and must follow officially approved procedures for the care and use of laboratory animals. Furthermore, experimental animals treated with biohazard materials should be handled and disposed using recommended animal biosafety procedures. When appropriate, the person(s) handling the animals must have official certification for performing procedures on animals and for submitting protocols for ethical approval.
    1. Weigh the animal on a balance (grams) in order to calculate the LPS dose required for each mouse.
    2. Prepare LPS stock solution by diluting LPS with sterile water to a concentration of 0.1 mg/ml. Aliquot (500 µl) and store at -80 °C.
    3. Desired LPS priming dose = 0.1 mg/kg. From the body weight of the mouse (measured in grams), convert this number to µl. Pipette this amount from the LPS stock solution to an Eppendorf tube and dilute to 150 μl final volume with sterile PBS. For example, for a 25 gram mouse, add 25 µl of LPS stock solution (0.1 mg/ml) to an Eppendorf tube and dilute to 150 µl final volume.
    4. Prepare MHC Class I mAb or isotype control mAb (0.5-4.5 mg/kg) and dilute to 100 μl final volume.
    5. Prepare a sterile heparin solution (4 units/ml) to flush the PE-10 tubing.
    6. Load insulin syringe with attached needle with sterile heparin solution (4 units/ml) and insert needle into PE-10 tubing (6 inches; 15 cm) on one end, and attach a sterile 30 gauge needle on the opposite end (Figure 1).


      Figure 1. Representative setup for jugular vein cannulation

  2. Transfusion-related acute lung injury model
    First event: immune priming with LPS
    Immune priming is necessary to produce robust lung injury when using mice housed in pathogen-free barrier rooms (Looney et al., 2009).
    1. Twenty-four hours prior to challenge with MHC I mAb mice, inject mice with LPS (0.1 mg/kg, i.p.).
    Second event: challenge with MHC I mAb
    All surgical procedures should be performed using standardized aseptic techniques and all surgical tools should be autoclaved following the surgery.
    1. Anesthetize animals with Ketamine (50-80 mg/kg) and Xylazine (8-12 mg/kg) injected intraperitoneal using insulin syringes. The anesthetics may be mixed in the same syringe.
    2. Place the mouse on a warming pad. Check the level of anesthesia using a paw pinch stimulus a few minutes after delivery of the anesthetics. Once adequate anesthesia is observed, immobilize the mouse in the supine position using adhesive tape.
    3. Make a vertical cut (~0.5 cm) to the right of the trachea and isolate the right jugular vein (Figure 2A).
    4. Carefully introduce the needle into the vessel. The needle, syringe, and PE-10 tubing should be filled with a heparin solution (4 units/ml). Aspirate blood from the jugular vein to verify intravascular placement of the needle (Figure 2B).
    5. Remove the syringe with heparin solution and replace with an insulin syringe (with attached needle) that has been preloaded with the MHC Class I mAb vs. isotype control mAb.


      Figure 2. Isolation of right jugular vein (A), cannulation and aspiration of blood (B), and removal of needle and placement of cotton-tip applicator (C)

    6. Flush the whole syringe content at a constant and uniform rate with particular care of not introducing any air bubbles.
    7. Carefully remove the needle and apply a cotton-tipped applicator with slight pressure to prevent bleeding (Figure 2C). Pressure should be applied for at least 1 min.
    8. Close the incision with 6-0 silk suture and apply post-operative care based on the institution’s animal care guidelines. Mice should be kept on a warming pad for the duration of the experiment and closely monitored for evidence of abnormal breathing.
    9. Mice are killed at 2 h after injection of the mAb or when they appear moribund.
    10. After carefully opening the thorax, collect blood from the right ventricle using a 25 gauge needle and a 1 ml syringe.
    11. Remove the lungs by cutting the trachea being careful to not let pulmonary edema fluid escape.

Representative data



Figure 3. Typical survival curve in mice challenged with MHC Class I mAb against H2Kd (1 mg/kg, i.v.) vs. vehicle control

Recipes

  1. Phosphate buffered saline (PBS)
    0.2g/L KH2PO4
    2.16 g/L Na2HPO4
    0.2 g/L KCl
    8.0 g/L NaCl
    Sterile filtered

Acknowledgments

This work was supported by the National Heart, Lung, and Blood Institute grant R01 HL107386 (M.R.L.).

References

  1. Looney, M. R., Nguyen, J. X., Hu, Y., Van Ziffle, J. A., Lowell, C. A. and Matthay, M. A. (2009). Platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury. J Clin Invest 119(11): 3450-3461.

简介

输血相关的急性肺损伤(TRALI)定义为在血液制品输血的6小时内发生的急性肺损伤。 TRALI仍然是输血相关死亡率的主要原因,我们已经开发了TRALI的小鼠模型,以更好地了解损伤发生的机制和测试治疗方法。 我们的模型是基于免疫引发的双事件模型和具有同源MHC I类单克隆抗体(MHC I mAb)的BALB/c野生型小鼠的攻击。 用LPS的免疫引发模拟对TRALI的发展重要的受体的引发状态(第一事件)。 供体HLA抗体经常涉及TRALI反应,并且同源MHC I类抗体(第二事件)在引发的动物中产生急性肺损伤。 在这里,我们描述一个详细的协议,在动物中具有高重现性。

关键字:输血, 急性肺损伤, 两事件模型

材料和试剂

  1. BALB/c WT小鼠(雄性,年龄8-12周)
  2. 使用标准蛋白A亲和层析从杂交瘤(ATCC,目录号:34-1-2S)纯化MHC I类mAb(H2Kd,IgG2a,κ;原料浓度0.65mg/ml)。
  3. 同种型匹配的mAb(IgG 2a,κ,原液浓度1mg/ml)(BD,目录号:553453)
  4. LPS(Sigma-Aldrich,目录号:L2880)
  5. 用于小鼠麻醉的Ketamine(Henry Schein,目录号:010177)和Xylazine(Henry Schein,目录号:033198)
  6. 肝素钠注射液(Sagent Pharmaceuticals,NDC编号:25021)
  7. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. 外科剪刀
  2. 弯曲的钝端钳
  3. 25号和28G无菌针
  4. 肝素钠注射液(Sagent Pharmaceuticals,NDC编号:25021)
  5. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. 外科剪刀
  2. 弯曲的钝端钳
  3. 25号和28G无菌针
    ...
  4. Warming pad
  5. Tape

Procedure

  1. Preparation
    All protocols using live animals must be approved by an Institutional Animal Care and Use Committee and must follow officially approved procedures for the care and use of laboratory animals. Furthermore, experimental animals treated with biohazard materials should be handled and disposed using recommended animal biosafety procedures. When appropriate, the person(s) handling the animals must have official certification for performing procedures on animals and for submitting protocols for ethical approval.
    1. Weigh the animal on a balance (grams) in order to calculate the LPS dose required for each mouse.
    2. Prepare LPS stock solution by diluting LPS with sterile water to a concentration of 0.1 mg/ml. Aliquot (500 µl) and store at -80 °C.
    3. Desired LPS priming dose = 0.1 mg/kg. From the body weight of the mouse (measured in grams), convert this number to µl. Pipette this amount from the LPS stock solution to an Eppendorf tube and dilute to 150 μl final volume with sterile PBS. For example, for a 25 gram mouse, add 25 µl of LPS stock solution (0.1 mg/ml) to an Eppendorf tube and dilute to 150 µl final volume.
    4. Prepare MHC Class I mAb or isotype control mAb (0.5-4.5 mg/kg) and dilute to 100 μl final volume.
    5. Prepare a sterile heparin solution (4 units/ml) to flush the PE-10 tubing.
    6. 加载带无菌肝素的胰岛素注射器 溶液(4单位/ml)并将针插入PE-10管(6英寸; cm),并在相对端附接无菌30号针   (图1)。


      图1.颈静脉插管的代表性设置

  2. 输血相关急性肺损伤模型
    第一个事件:用LPS免疫引发
    当使用安置在无病原体的屏障室中的小鼠时,免疫引发对于产生强烈的肺损伤是必要的(Looney等人,2009)。
    1. 在用MHC I mAb小鼠攻击前24小时,用LPS(0.1mg/kg,腹膜内)注射小鼠。
    第二个事件:用MHC I mAb挑战
    所有外科手术应使用标准化的无菌技术进行,所有手术工具应在手术后进行高压灭菌
    1. 麻醉动物用氯胺酮(50-80毫克/公斤)和赛拉嗪(8-12 mg/kg)使用胰岛素注射器腹膜内注射。 麻醉剂 可以在同一注射器中混合。
    2. 将鼠标放在升温 垫。 检查麻醉的水平使用爪捏伤刺激几个 分钟后麻醉剂交付。 一旦充分麻醉 观察,使用粘合剂将小鼠固定在仰卧位置 胶带。
    3. 在气管右侧垂直切割(〜0.5厘米),隔离右颈静脉(图2A)。
    4. 小心地将针头插入血管。 针,注射器,   和PE-10管应填充肝素溶液(4单位/ml)。   从颈静脉抽吸血液以验证血管内放置 的针(图2B)。
    5. 取出带有肝素的注射器 溶液并更换为胰岛素注射器(附带针头)   已经预装有MHC I类mAb对同种型对照mAb

      图2:右颈静脉(A),插管和 抽吸血液(B),取出针头和放置 棉签敷料器(C)

    6. 以恒定和均匀的速率冲洗整个注射器内容物,特别注意不要引入任何气泡
    7. 小心取出针头,并涂上棉签 具有轻微的压力以防止出血(图2C)。 压力应该   应用至少1分钟。
    8. 用6-0丝关闭切口 缝合并根据机构的动物应用术后护理 护理指南。 小鼠应保持在温暖垫上的持续时间 的实验,并密切监测异常的证据 呼吸。
    9. 在注射mAb后2小时或当它们出现濒死时,杀死小鼠
    10. 小心打开胸腔后,使用25号针头和1ml注射器从右心室收集血液
    11. 通过切除气管切除肺部,小心不要让肺水肿液漏出。

代表数据



图3.用针对H2Kd(1mg/kg,i.v.)的MHC I类mAb攻击的小鼠相对于媒介物对照攻击的小鼠中的典型存活曲线

食谱

  1. 磷酸盐缓冲盐水(PBS)
    0.2g/L KH 2 PO 4 sub/
    2.16g/L Na 2 HPO 4
    0.2 g/L KCl
    8.0g/L NaCl 无菌过滤

致谢

这项工作得到国家心肺血液研究所拨款R01 HL107386(M.R.L.)的支持。

参考文献

  1. Looney,M.R.,Nguyen,J.X.,Hu,Y.,Van Ziffle,J.A.,Lowell,C.A。和Matthay,M.A。(2009)。 血小板消耗和阿司匹林治疗在输血相关急性肺损伤的双事件模型中保护小鼠。 J Clin Invest 119(11):3450-3461。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ortiz-Muñoz, G. and Looney, M. R. (2015). Two-event Transfusion-related Acute Lung Injury Mouse Model. Bio-protocol 5(12): e1505. DOI: 10.21769/BioProtoc.1505.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。