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Intra-amniotic Injection of Mouse Embryos
羊膜内注射小鼠胚胎   

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EBioMedicine
Jun 2017

Abstract

Recent outbreaks of infectious neuro-developmental diseases such as congenital Zika syndrome - have led to a demand for prognosis data from animal models. We developed an intra-amniotic injection mice model that allows Zika virus (ZIKV) infected mice to grow to puberty. In this system, ZIKV is injected into the amniotic fluid of pregnant mice and infected embryos thereafter. ZIKV-infected mice show several symptoms of clinical ‘congenital Zika syndrome’, including decreased brain volume and mis-laminated retina. We also evaluated several behavioral functions of these ZIKV-infected mice, for example, after the mice reach puberty, they have visual and motor defects. This technique can be used to screen and evaluate drug candidates and may help evaluate the prognosis of infectious neuro-developmental diseases.

Keywords: Intra-amniotic (羊膜内), Embryos (胚胎), Virus (病毒), Infection (感染)

Background

It has been more than sixty years since the first human case of ZIKV infection was reported but in 2007 only 14 human cases of ZIKV infection had been recorded. Prognosis data have lagged far behind the recent outbreak of ZIKV in 2015. There is some evidence that mouse models may be effective for prognosis studies because previous reports have proved neurovirulence of ZIKV in mice. Therefore, various methods of ZIKV infection including intravenous injection, intraperitoneal injection, foot-pad injection and brain injection have been used to study congenital Zika syndrome in individuals during the embryonic period or infancy. The intra-amniotic injection model presented here has two advantageous features. First, wild-type mice (C57 BL/6J) can be used and studies are not limited to mice with immunologic deficiencies. Second, ZIKV infected mice can grow into puberty, which is beneficial to studies trying to evaluate prognosis.

Materials and Reagents

  1. Glass pipettes (World Precision Instrument, catalog number: 4878 )
  2. Sterile gauze (Winner Medical Group, catalog number: 016935 )
  3. Cotton ball (Winner Medical Group, catalog number: 50401050 )
  4. 1 ml Syringe (KDL, catalog number: 60017031 )
  5. 50 ml centrifuge tube (Corning, catalog number: 430828 )
  6. Dropper (Shanghai Baiqian Biotechnology, catalog number: J00082 )
  7. Suture needle (Ningbo Medical Needle Co., LTD, 7/0)
  8. Pregnant mouse (E15) (Shanghai Yison Biotechnology Company, strain: C57 BL/6J)
  9. 75% alcohol (Sinopharm Chemical Reagent, catalog number: 80176960 )
  10. Oxygen (Shanghai Lvmin Gas company, purity > 99%)
  11. Cyanoacrylate (Pattex®, catalog number: PSK12CT-2 )
  12. Isoflurane (RWD Life Science, catalog number: R510-22 )
  13. Iodophor (Shanghai Likang Disinfectant Hi-Tech, catalog number: 310100 )
  14. Lidocaine (MP Biomedicals, catalog number: 190111 )
  15. Ampicillin (Inalco, catalog number: 1758-9314 )
  16. Dulbecco’s modified Eagle’s medium (DMEM) (Corning, catalog number: 10-013-CV )
  17. 10% fetal bovine serum (FBS) (Biological Industries, catalog number: 04-001-1ACS )
  18. Pen-Strep Solution (Penicillin: 10,000 U/ml, Streptomycin: 10 mg/ml) (Biological Industries, ISRAEL, catalog number: 03-031-1B )
  19. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  20. Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Sigma-Aldrich, Fluka, catalog number: 71645 )
  21. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  22. 0.1 M Phosphate buffer solution (PBS) (see Recipes)
  23. Ampicillin solution (see Recipes)
  24. Culture medium (see Recipes)
  25. 1% lidocaine solution (see Recipes)

Equipment

  1. Flaming/brown micropipette puller (Sutter Instrument, model: P-97 )
  2. Anesthesia machine (RWD Life Science, catalog number: R610 )
  3. Water bath (Jinghong Experimental Equipment, model: XMID-8222 )
  4. Tweezers (VETUS, catalog number: ST-11 )
  5. Scissors (RWD Life Science, catalog number: S12003-09 )
  6. Shaver (Codos, catalog number: KP-3000 )

Procedure

  1. Sterilization: sterilize all tools that will be used in this surgery by autoclaving at 121 °C, 0.12 MPa or with 75% alcohol.
  2. Fill two 50 ml centrifuge tubes with PBS and Ampicillin, respectively. Before use, warm these solutions in a 45 °C water bath.
  3. Pipette preparation: We couple a glass pipette with a 1 ml syringe for injecting a large amount of solution (Figure 1). The diameter of the pipette tip is ~50 µm. If the diameter of pipette tip is too large, amniotic fluid will overflow and this may affect the development of fetus. If the diameter of the pipette tip is too small, the pipette can be easily blocked, which increases the duration of time that the embryos are exposed (Figure 2).


    Figure 1. Glass pipette and syringe coupled together


    Figure 2. Glass pipette tip with different diameters. A. The tip diameter is too fine. B. The tip diameter is good. C. The tip diameter is too large.

  4. Anesthetize a pregnant mouse (E15) with 1.5% isoflurane in oxygen in an induction chamber at a respiratory rate of 60 times per minute.
    Note: If the age of embryos is older than E17 or younger than E14, the survival rate of mice will decrease.
  5. Remove the pregnant mouse from the induction chamber. Place the head of the mouse in a respiratory mask infused with 0.5-1% isoflurane in oxygen. Maintain a respiratory rate between 80 and 100 times per minute to ensure that the embryos do not achieve hypoxia status and that the pregnant mouse is fully anesthetized during surgery.
  6. Shave the mouse abdomen using a shaver that was sterilized with cotton ball with 75% alcohol and iodophor.
  7. Drape sterile gauze over the abdomen and make a 2 cm long slit to expose the abdominal incision.
  8. Cut a 2 cm long incision on the abdominal skin and peritoneum around the position of the womb being careful to cut from outside to inside. Be careful not to damage the capillary vessels on the peritoneum, because bleeding from damaged vessels can lead to wound infection.
  9. Hold the uterine wall with tweezers and pull out embryos that are in the uterus. Be careful not to damage vessels on the uterine wall especially those that are close to the placenta. Keep the surface of mouse embryos moist using sterilized PBS during the injection process (Figure 3).


    Figure 3. Embryos before injection

  10. With one hand, hold the tweezers and gently lift the uterine wall. With the other hand, hold the injector to inject 0.1 ml culture medium (see Recipes) (injection speed: ~10 µl/sec) into the amniotic at the position close to the tweezers (Video 1).

    Video 1. The posture of intra-amniotic injection

  11. Inject the other embryos with a similar volume and injection speed. The survival rate of embryos is typically about 85%.
  12. Insert the embryos back into the abdominal cavity with tweezers after the injection.
  13. Fill the abdominal cavity with 2 ml 0.1 mg/ml warm ampicillin in PBS to prevent infection. Remove the extra solution with gauze at the surface of the abdomen.
  14. Carefully suture the peritoneum and abdominal skin from inside to outside.
  15. To ease pain, apply 1% lidocaine solution (see Recipes) to the wound on the abdominal skin. The time it takes to complete the surgery (Step 6 to Step 15) should not exceed half an hour. The embryo survival rate decreases if the surgery exceeds 30 min.
    Note: The specific operation of Steps 6-15 is shown in Video 2.

    Video 2. Intra amniotic Injection of Mouse Embryos

  16. Move the pregnant mouse back to her home cage and kept her warm with a heating pad at 37 °C for 3-5 h until it recovers from anesthesia and can move freely.
  17. ZIKV infected mice show walking defects or paralysis. Videos of ZIKV mice exhibiting this behavior can be found here: http://www.ebiomedicine.com/article/S2352-3964(17)30184-6/addons.

Data analysis

Analysis of behavioral tests including open filed test, tail suspension test, rota-rod test, catwalk and elevated plus maze in the visual and motor deficits of ZIKV infected mice can be found online at http://www.ebiomedicine.com/article/S2352-3964(17)30184-6/fulltext#s0010.

Notes

  1. The diameter of the suture needle should be about 120 µm. The diameter of a stitch should be about 70 µm. The larger the diameter of the suture needle and stitch, the greater the wound injury will be, especially to the abdominal wound. A fine stitch used to suture will help healing.
  2. To ensure that the culture medium is injected into the amniotic fluid, you need to inject about 5 µl culture medium near to the sterile gauze before injecting into the amniotic fluid. If the tip is not blocked, you will see culture medium flowing. Similarly, check to ensure the tip is not blocked after you inject into the amniotic fluid.
  3. If the pregnant mouse keeps lying down on its abdomen, you need to apply 1% lidocaine solution on the wound again and inject 0.3 ml 0.9% sodium chloride solution in the neck to avoid hydropenia.

Recipes

  1. 0.1 M PBS solution (pH 7.4)
    0.02 M potassium phosphate monobasic (KH2PO4)
    0.04 M sodium phosphate dibasic (Na2HPO4)
    0.1567 M sodium chloride (NaCl)
  2. Ampicillin solution
    100 μg ampicillin in 1 ml 0.1 M PBS
  3. Culture medium
    DMEM containing:
    10% FBS
    100 units/ml of penicillin
    100 µg/ml of streptomycin
  4. 1% lidocaine solution
    1 g lidocaine in 100 ml 0.9% sodium chloride solution
    A drop of hydrochloric acid (~40 µl) was added into the solution to help dissolve

Acknowledgments

The authors declare no competing interests. This protocol was adapted from Cui et al. (2017). J.Z. thanks the following funding agencies for supporting this work: the NSF of China (31771195, 81790640), the Young 1000 Plan and Ministry of Science and Technology of the People’s Republic of China (2015AA020512). L. L. thanks the following funding agencies for supporting this work: Ministry of Science and Technology of the People’s Republic of China (2015AA020930 and 2016YFC1202901).

References

  1. Cui, L., Zou, P., Chen, E., Yao, H., Zheng, H., Wang, Q., Zhu, J. N., Jiang, S., Lu, L. and Zhang, J. (2017). Visual and motor deficits in grown-up mice with congenital Zika virus infection. EBioMedicine 20: 193-201.

简介

最近爆发的传染性神经发育疾病,例如先天性寨卡综合征 - 已经导致对来自动物模型的预后数据的需求。 我们开发了羊膜内注射小鼠模型,其允许寨卡病毒(ZIKV)感染的小鼠生长至青春期。 在此系统中,ZIKV被注入怀孕小鼠的羊水中,然后感染胚胎。 ZIKV感染的小鼠显示出临床'先天性Zika综合征'的几种症状,包括脑容积减少和视网膜错层。 我们还评估了这些ZIKV感染小鼠的几种行为功能,例如,在小鼠到达青春期后,它们具有视觉和运动缺陷。 这种技术可用于筛选和评估候选药物,并有助于评估感染性神经发育疾病的预后。

【背景】自第一例人感染ZIKV病例以来,已有60多年的历史,但2007年仅记录了14例ZIKV感染的人类病例。 预后数据远远落后于2015年最近爆发的ZIKV。有证据表明小鼠模型可能对预后研究有效,因为之前的报道证明ZIKV在小鼠中的神经毒性。 因此,ZIKV感染的各种方法包括静脉注射,腹腔注射,足垫注射和脑注射已被用于研究胚胎期或婴儿期期间个体的先天性寨卡综合征。 这里给出的羊膜内注射模型具有两个有利的特征。 首先,可以使用野生型小鼠(C57 BL / 6J),并且研究不限于具有免疫缺陷的小鼠。 其次,ZIKV感染的小鼠可长成青春期,这对试图评估预后的研究是有益的。

关键字:羊膜内, 胚胎, 病毒, 感染

材料和试剂

  1. 玻璃移液器(世界精密仪器,目录号:4878)
  2. 无菌纱布(Winner Medical Group,目录号:016935)
  3. 棉花球(Winner Medical Group,产品目录编号:50401050)
  4. 1毫升注射器(KDL,目录号:60017031)

  5. 50 ml离心管(Corning,目录号:430828)
  6. 滴管(上海百千生物技术有限公司,产品目录号:J00082)
  7. 缝合针(宁波医用针业有限公司,7/0)
  8. 怀孕小鼠(E15)(上海益盛生物技术公司,菌株:C57 BL / 6J)
  9. 75%酒精(国药集团化学试剂,目录号:80176960)
  10. 氧气(上海绿敏燃气公司,纯度> 99%)
  11. 氰基丙烯酸酯(Pattex®,产品目录号:PSK12CT-2)
  12. 异氟醚(RWD Life Science,目录号:R510-22)
  13. Iodophor(上海立康消毒高科技,产品目录号:310100)
  14. 利多卡因(MP Biomedicals,目录号:190111)
  15. 氨苄青霉素(Inalco,目录号:1758-9314)
  16. 达尔伯克改良伊格尔培养基(DMEM)(Corning,目录号:10-013-CV)
  17. 10%胎牛血清(FBS)(Biological Industries,目录号:04-001-1ACS)
  18. Pen-Strep溶液(青霉素:10,000U / ml,链霉素:10mg / ml)(Biological Industries,ISRAEL,目录号:03-031-1B)
  19. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  20. 磷酸氢二钠二水合物(Na 2 HPO 4·2H 2 O)(Sigma-Aldrich,Fluka,目录号:71645) >
  21. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  22. 0.1M磷酸盐缓冲溶液(PBS)(见食谱)
  23. 氨苄青霉素溶液(见食谱)
  24. 培养基(见食谱)
  25. 1%利多卡因溶液(见食谱)

设备

  1. 火焰/棕色微量拉拔器(Sutter仪器,型号:P-97)
  2. 麻醉机(RWD Life Science,目录号:R610)
  3. 水浴(景洪实验设备,型号:XMID-8222)
  4. 镊子(VETUS,目录号:ST-11)
  5. 剪刀(RWD生命科学,产品目录号:S12003-09)
  6. 剃须刀(Codos,目录号:KP-3000)

程序

  1. 消毒:通过在121°C,0.12 MPa或75%酒精的高压灭菌处理消毒所有将用于此手术的工具。
  2. 分别用PBS和氨苄青霉素填充两个50ml离心管。使用前,将这些溶液在45°C水浴中加热。
  3. 移液器准备:我们将玻璃移液器与1 ml注射器连接以注入大量溶液(图1)。移液管尖端的直径约为50μm。如果吸头直径过大,羊水会溢出,这可能会影响胎儿的发育。如果移液器吸头的直径太小,移液器可能会很容易堵塞,这会增加胚胎暴露的时间(图2)。


    图1.玻璃移液器和注射器连接在一起


    图2.具有不同直径的玻璃吸管尖端 A.尖端太细。 B.提示很好。 C.提示太厚。

  4. 麻醉怀孕小鼠(E15)与1.5%异氟醚氧气在诱导室内,呼吸频率为每分钟60次。
    注:如果胚胎的年龄大于E17或小于E14,小鼠的存活率会下降。
  5. 从感应室中取出怀孕的小鼠。将小鼠的头部放入氧气中注入0.5-1%异氟醚的呼吸面罩。保持呼吸频率在每分钟80到100次之间,以确保胚胎不会达到缺氧状态,并且怀孕的小鼠在手术过程中完全被麻醉。

  6. 用75%酒精和碘伏消毒的剃须刀剃掉小鼠腹部。
  7. 将无菌纱布覆盖腹部,并做一个2厘米长的缝隙以暴露腹部切口。
  8. 在子宫周围的腹部皮肤和腹膜上切一个2厘米长的切口,注意从外面切到里面。
    注意不要损伤腹膜上的毛细血管,因为受损血管的渗血会导致伤口感染。
  9. 用镊子握住子宫壁并拔出子宫内的胚胎。注意不要损伤子宫壁上的血管,特别是那些靠近胎盘的血管。
    注射过程中使用无菌PBS保持小鼠胚胎表面潮湿(图3)。


    图3.注射前的胚胎

  10. 一只手拿着镊子,轻轻抬起子宫壁。另一方面,握住注射器在靠近镊子的位置注射0.1ml培养基(参见食谱)(注射速度:约10μl/ sec)到羊膜中(视频1)。

    视频1
  11. 注射其他类似体积和注射速度的胚胎。
    胚胎的存活率通常约为85%。

  12. 注射后用镊子将胚胎重新插入腹腔
  13. 用2 ml 0.1 mg / ml温热氨苄青霉素的PBS填充腹腔以预防感染。
    在腹部表面用纱布除去多余的溶液。

  14. 仔细缝合腹膜和腹部皮肤
  15. 为了缓解疼痛,应用1%利多卡因溶液(见食谱)在腹部皮肤上的伤口。完成手术所需的时间(步骤6至步骤15)不应超过半小时。
    如果手术超过30分钟,胚胎存活率会下降 注:步骤6-15的具体操作如视频2所示。

    视频2
  16. 将怀孕的小鼠移回家中,并用37℃的加热垫保温3-5小时,直到它从麻醉中恢复并可以自由移动。
  17. ZIKV感染的小鼠表现出行走缺陷或瘫痪。展示此行为的ZIKV鼠标的视频可在此处找到: http:/ /www.ebiomedicine.com/article/S2352-3964(17)30184-6/addons

数据分析

ZIKV感染小鼠视觉和运动缺陷的行为测试分析包括开放式测试,尾部悬挂测试,旋转棒测试,时装表演和高架十字迷宫,可以在线查看:http://www.ebiomedicine .com / article / S2352-3964(17)30184-6 /全文#s0010“target =”_ blank“> http://www.ebiomedicine.com/article/S2352-3964(17)30184-6 /全文#s0010 。

笔记

  1. 缝合针的直径应该约为120μm。线迹的直径应该约为70微米。缝合针和针的直径越大,创伤越大,特别是对于腹部伤口。用于缝合的细针将有助于愈合。
  2. 为了确保将培养基注入羊水中,在注入羊水之前,需要在无菌纱布附近注入约5μl的培养基。如果尖端未被阻塞,您将看到培养基流动。同样,检查以确保在注入羊水后尖端没有被堵塞。
  3. 如果怀孕的小鼠一直躺在腹部,需要再次将1%利多卡因溶液涂在伤口上,并在颈部注射0.3ml 0.9%氯化钠溶液以避免水痘。

食谱

  1. 0.1 M PBS溶液(pH 7.4)
    0.02M磷酸二氢钾(KH 2 PO 4)
    0.04M磷酸氢二钠(Na 2 HPO 4)

    0.1567 M氯化钠(NaCl)
  2. 氨苄西林溶液

    1 ml 0.1 M PBS中100μg氨苄青霉素
  3. 培养基
    包含以下内容的DMEM:
    10%FBS
    100单位/毫升青霉素
    100μg/ ml链霉素
  4. 1%利多卡因溶液
    1克利多卡因在100毫升0.9%氯化钠溶液中
    向溶液中加入一滴盐酸(〜40μl)以帮助溶解

致谢

作者声明没有竞争利益。该协议改编自Cui em et al。(2017)。 J.Z.感谢下列资助机构支持本工作:中国国家科学基金会(31771195,81790640),中国青年科技计划和中华人民共和国科技部(2015AA020512)。 L. L.感谢下列资助机构支持这项工作:中华人民共和国科学技术部(2015AA020930和2016YFC1202901)。

参考

  1. Cui,L.,Zou,P.,Chen,E.,Yao,H.,Zheng,H.,Wang,Q.,Zhu,JN,Jiang,S.,Lu,L.and Zhang,J.(2017) )。 先天性寨卡病毒感染成年小鼠视觉和运动障碍。 EBioMedicine 20:193-201。
  • English
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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: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cui, L., Zou, P., Meng, Q., Lu, L. and Zhang, J. (2018). Intra-amniotic Injection of Mouse Embryos. Bio-protocol 8(10): e2854. DOI: 10.21769/BioProtoc.2854.
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