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Monocular Deprivation in Mice
小鼠单眼剥离试验   

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Abstract

Monocular deprivation is an experimental technique to study the ocular dominance plasticity during critical period (Hubel and Wiesel, 1963). Generally one eye of an animal is sutured during critical period, and the sutured eye is re-opened after either less than three days (short term) or more than three days (long term). Here we describe a detailed protocol for short-term and long-term monocular deprivation in mouse (Ma et al., 2013).

Materials and Reagents

  1. Cotton
  2. 75% Ethanol
  3. Ketamine hydrochloride (100 mg/ml) (Ketaject, Phoenix Pharmaceuticals, NDC number: 57319-542-02 )
  4. Xylazine hydrochloride (100 mg/ml) (AnaSed Injection, LLOYD, NADA number: 139-236 )
  5. Xylocaine (lidocaine hydrochloride) 2% Jelly (AstraZeneca, NDC number: 0186-0330-01 )
  6. Bacitracin zinc ointment (USP 500 U/g) (Fougera Pharmaceuticals, NDC number: 0168-0011-31 )
  7. Saline (0.9% sodium chloride injection USP)

Equipment

  1. Hood
  2. Autoclave
  3. Aluminum foil
  4. Dumont #5 forcep (Fine Science Tools, catalog number: 11254-20 )
  5. Dumont #3c forcep (Fine Science Tools, catalog number: 11231-20 )
  6. Cohan-Vannas spring scissor (Fine Science Tools, catalog number: 15000-02 )
  7. Fine scissor (Fine Science Tools, catalog number: 14060-09 )
  8. UNIFY Silk surgical suture [small (P-3) 13 mm, reverse cutting 3/8 circle needle. 4-0 18"/45 cm thread] (AD Surgical)
  9. Alcohol pad (PDI, catalog number: B603 )
  10. Gloves
  11. Microscope
  12. Illuminator (AmScope, catalog number: Model HL250-AY )
  13. Heating pad (Harvard Apparatus, catalog number: 507220F )

Procedure

  1. One day before the surgery
    1. Sterilize the hood with ultraviolet germicidal irradiation for 1 h.
    2. Autoclave the surgical tools wrapped with aluminum foil, some cotton wrapped with aluminum foil and another N+1 (N is the number of mice to be sutured) aluminum foil. Surgical tools include two Dumont #5 forceps, one Dumont #3c forcep, one Cohan-Vannas spring scissor and one fine scissor.
    3. Sterilize the suture needle and thread with 75% ethanol one day before the surgery.

  2. On the day of surgery
    1. Put all the surgical tools along with some alcohol pads in the sterilized hood.
    2. Anesthetize the mouse with the mixture of 100 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride intraperitoneally.
    3. Clean the mouse with alcohol pads, and then put it on one sterilized aluminum foil. Put all the tools on another foil. Spray the gloves with 75% ethanol.
    4. Under microscope and illuminator, suture the eyelids using sterilized needle and thread with the help of Dumont #5 forceps as follow: make 2-3 stitches, tighten the thread, make two knots, and cut the thread with the fine scissor as short as possible. For monocular deprivation of more than three days, cut the eyelashes and the edge of eyelids using the spring scissors before suturing. In case of bleeding, stop it with the cotton before continuing.
    5. Apply a thin layer of xylocaine 2% Jelly and bacitracin zinc ointment to the sutured eyelids.
    6. Put the mouse on the heating pad.
    7. Spray the surgical tools with 75% ethanol, clean it with alcohol pads, and put it back on the aluminum foil. Dispose the dirty aluminum foil used for the mouse.
    8. Repeat procedure 2-7 for other mice.
    9. Put all the mice back into the holding cages after making sure they are fully recovered.
    10. Clean all the surgical tools and the hood with 75% ethanol. Sterilize the hood with ultraviolet germicidal irradiation for at least an hour.

  3. After monocular deprivation
    1. At the end of the deprivation period, mice were anesthetized, stitches were removed, and lid margins were separated. Eyes were flushed with sterile saline and checked for clarity under a microscope. Only mice without corneal opacities or signs of infection were used.

Acknowledgments

This protocol is adapted from a published paper: Ma et al. (2013). This work was supported by grants to H.W.T. from the National Institutes of Health (EY019049 and EY022478) and the Kirchgessner Foundation.

References

  1. Hubel, D. H. and Wiesel, T. N. (1963). Receptive fields of cells in striate cortex of very young, visually inexperienced kittens. J Neurophysiol 26(6): 994-1002.
  2. Ma, W. P., Li, Y. T. and Tao, H. W. (2013). Downregulation of cortical inhibition mediates ocular dominance plasticity during the critical period. J Neurosci 33(27): 11276-11280.

简介

单眼剥夺是研究关键期眼部优势可塑性的实验技术(Hubel和Wiesel,1963)。 通常在关键时期缝合一只动物的眼睛,缝合眼睛在短短三天(短期)或超过三天(长期)之后重新开放。 这里我们描述了一个关于小鼠短期和长期单眼剥夺的详细方案(Ma et al。,2013)。

材料和试剂

  1. 棉花
  2. 75%乙醇
  3. 盐酸氯胺酮(100mg/ml)(Ketaject,Phoenix Pharmaceuticals,NDC编号:57319-542-02)
  4. 甲苯噻嗪盐酸盐(100mg/ml)(AnaSed注射液,LLOYD,NADA数:139-236)
  5. 舒舒卡因(盐酸利多卡因)2%果冻(AstraZeneca,NDC编号:0186-0330-01)
  6. 杆菌肽锌软膏(USP 500U/g)(Fougera Pharmaceuticals,NDC编号:0168-0011-31)
  7. 盐水(0.9%氯化钠注射液USP)

设备

  1. 敞篷
  2. 高压灭菌器
  3. 铝箔
  4. Dumont#5 forcep(Fine Science Tools,目录号:11254-20)
  5. Dumont#3c forcep(Fine Science Tools,目录号:11231-20)
  6. Cohan-Vannas弹簧剪(Fine Science Tools,目录号:15000-02)
  7. 精细剪刀(Fine Science Tools,目录号:14060-09)
  8. UNIFY丝绸手术缝[小(P-3)13毫米,反向切割3/8圆针。 4-0 18"/45厘米螺纹](AD手术)
  9. 酒精垫(PDI,目录号:B603)
  10. 手套
  11. 显微镜
  12. 照明器(AmScope,目录号:型号HL250-AY)
  13. 加热垫(Harvard Apparatus,目录号:507220F)

程序

  1. 手术前一天
    1. 用紫外线杀菌照射灭菌罩1小时。
    2. 高压灭菌包裹铝箔的外科工具,一些棉包裹铝箔和另一N + 1(N是待缝合的小鼠的数量)铝箔。 手术工具包括两个Dumont#5镊子,一个Dumont#3c镊子,一个Cohan-Vannas弹簧剪刀和一个精细剪刀。
    3. 在手术前一天用75%乙醇消毒缝合针和线。

  2. 手术当天
    1. 将所有的手术工具和一些酒精垫放在消毒罩中
    2. 用100mg/kg氯胺酮盐酸盐和10mg/kg盐酸赛拉嗪的混合物腹膜内麻醉小鼠。
    3. 用酒精垫清洁老鼠,然后把它放在一个灭菌的铝箔。将所有的工具放在另一个箔。用75%乙醇喷涂手套。
    4. 在显微镜和照明器下,使用无菌针和线在Dumont#5镊子的帮助下缝合眼睑如下:做2-3针,拧紧线,做两个结,并用细剪刀切割线尽可能短。对于超过三天的单眼剥夺,在缝合之前使用弹簧剪刀剪下睫毛和眼睑的边缘。如果出血,在继续之前先用棉花停止。
    5. 在缝合的眼睑上涂上薄层的xylocaine 2%果冻和杆菌肽锌软膏。
    6. 将鼠标放在加热垫上。
    7. 用75%乙醇喷洒手术工具,用酒精垫清洁,并将其放回铝箔。 处理用于鼠标的脏铝箔。
    8. 对其他小鼠重复步骤2-7
    9. 将所有的小鼠放回笼子里,确保它们完全恢复。
    10. 清洁所有的手术工具和罩用75%乙醇。 用紫外线杀菌照射灭菌至少一小时。

  3. 单眼剥夺后
    1. 在剥夺期结束时,麻醉小鼠,取出缝线,分离盖边缘。 用无菌盐水冲洗眼睛,并在显微镜下检查透明度。 只要 使用没有角膜混浊或感染迹象的小鼠。

致谢

该协议改编自已发表的文章:Ma等人。(2013)。 这项工作得到了H.W.T. 来自国立卫生研究院(EY019049和EY022478)和Kirchgessner基金会。

参考文献

  1. Hubel,D.H。和Wiesel,T.N。(1963)。 非常年轻,视觉缺乏经验的小猫在条纹皮质中的细胞接受区域。 J Neurophysiol 26(6):994-1002。
  2. Ma,W. P.,Li,Y. T.和Tao,H. W.(2013)。 皮层抑制的下调在关键时期介导眼部优势可塑性。 Neurosci 33(27):11276-11280。
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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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Li, Y., Chou, X. and Tao, H. W. (2014). Monocular Deprivation in Mice. Bio-protocol 4(2): e1024. DOI: 10.21769/BioProtoc.1024.
  2. Ma, W. P., Li, Y. T. and Tao, H. W. (2013). Downregulation of cortical inhibition mediates ocular dominance plasticity during the critical period. J Neurosci 33(27): 11276-11280.
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