皮下给药的毒蕈碱受体拮抗剂和上睑提的Auris在小鼠体内长肌的三重免疫染色
Summary
我们描述的毒蕈碱受体信号抑制剂反复提的Auris长肌（LAL）年轻的成年小鼠的肌肉和管理程序，其神经肌肉接头在随后的免疫wholemounts（NMJs）。鲎的肌肉已经露出独特的优势<em>在体内</emNMJs>的药理作用。
Abstract
经常用于在哺乳动物NMJs的形成和维护必不可少的信号体内的药理研究啮齿类动物，如腓肠肌和胫前，后肢肌肉。然而，药物渗透到这些肌肉，皮下或肌肉注射后往往是不完整或不平坦的，很多NMJs可以不受影响。虽然与设备，如微型泵全身给药可改善的时空效应，这种方法的入侵性可以导致混淆的炎症反应和/或直接的肌肉损伤。此外，完整的分析后肢肌肉NMJs是具有挑战性的，因为它需要耗费时间的连续切片和广泛的免疫。
鼠标鲎是一种细长，肌肉平板位于颈部背部表面。这是一个快速肌功能移动耳廓。它包含延髓和尾鳍部分来自颅骨中线和横向延伸到每个耳廓软骨部分。肌肉是提供项目尾端，因为它退出茎乳孔面神经的一个分支。我们和其他人发现鲎是一种非常方便的准备，提供的短期和长期在体内药物的影响NMJs和肌肉的调查优势。首先，其表面的位置，方便多个本地应用程序，在浅麻醉药物。其次，它的薄（2-3层肌纤维）允许几乎所有的肌肉内NMJs的可视化和分析。第三，易于解剖与神经完好，其支配的格局， 允许在体外9,5补充电生理分析。最后，也许是最重要的是，一个小的应用量（〜50μL）轻松地覆盖了整个的肌肉表面，提供了一个其所有NMJs的统一和长时间暴露在药物和消除需要一个系统性的方法1,8。
Protocol
1。皮下给药毒蕈碱型乙酰胆碱受体拮抗剂（mAChR） 在无菌条件下适当的剂量，mAChR拮抗剂（参表）准备通过溶解在无菌生理盐水1.5ml的反应管中的药物。以下拮抗剂：阿托品，Methoctramine，4阻尼，AFDX – 116，AFDX – 384吨7。 绘制成a1cc胰岛素注射器的解决方案50μL，并为每个鼠标使用单独的注射器。还准备只包含每个对照组小鼠生理盐水的注射器。置于冰上直到准备注射器注入。 ?…
Discussion
这里介绍的方法，允许调查亚型特异性信号的稳定性和维护哺乳动物NMJs mAChR先前未确认的角色。此方法也将是有用的测试神经营养因子及药理制剂的影响。例如，我们的实验室发现，几乎所有的成年小鼠的鲎神经末梢引起睫状神经营养因子（CNTF ）发芽1。这与睫状神经营养因子治疗后肢肌肉，报告中度CA发芽前研究的结果对比。 13-33％的臀大肌，外侧腓肠肌路口3 9％。我们相信这种?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了肌肉萎缩症协会，国立卫生研究院（NS062320）。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
|---|---|---|---|
| ketamine | Hospira | NDC0409-2051-05 | Dose: 120mg/kg |
| xylazine | Lloyd Laboratories | LA33806 | Dose: 8mg/kg |
| atropine | Sigma-Aldrich | A0132 | (>98% purity); Dose: 0.2mg/kg – 20mg/kg |
| atropine | Voigt Global Distribution | AT105 | Pharmaceutical grade |
| Methoctramine | Sigma-Aldrich | M105 | Dose: 100 – 400M |
| 4-DAMP | Sigma-Aldrich | D142 | Dose: 2.5mg/kg |
| AFDX-116 | Tocris Bioscience | 1105 | 250M |
| AFDX-384 | Tocris Bioscience | 1345 | 50M – 500M |
| MT 7 | Peptides International | PMT-4340-s | 0.1M – 1M |
| 1X Phosphate Buffered Saline, pH 7.4 | Invitrogen | 10010049 | |
| Paraformaldehyde | Fisher | T353-500 | Make 10% solution first by dissolving 10g/100mL de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4 |
| Sodium pentobarbitol | Virbac Animal Health | NDC-051311-050-01 | Dose: 390mg/kg |
| Sylgard | Dow Corning | Part # 184 | Follow instructions that come with kit, can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs |
| 0.1M Glycine | Sigma-Aldrich | G-7126 | Add 0.185g to 25mL of 2% BSA/PBS |
| 2% Bovine serum albumin (2% BSA) | Sigma-Aldrich | A3059-100g | Dissolve 2g BSA into 100mL of 1X PBS |
| 0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer) | Sigma-Aldrich | T9284-100mL | Dissolve 0.2ml/100mL 2% BSA/PBS |
| α-bungarotoxin | Invitrogen | T1175 | Use at concentration of 1:200 |
| SMI-312 | Sternberger Monoclonals | SMI312 | Use at concentration of 1:1000 |
| SV2 | Developmental Studies Hybridoma Bank | SV2-Supernatant | Use at concentration of 1:10 |
| S100 | Dako | Z0311 | Use at concentration of 1:400 |
| FITC- goat anti-mouse IgG1 | Roche | 03117731001 | Use at concentration of 1:200, but if background is high, try 1:400 |
| Alexa-Fluor 647 conjugated goat anti-rabbit | Invitrogen | A21244 | Use at concentration of 1:200 |
| Vectashield fluorescent mounting media | Vector laboratories | H-1000 | This is not a hard-set media, you will need to secure the cover slip with clear nail polish. |
| Small Spring Scissors | Fine Science Tools | 15002-08 | |
| Dissection forceps | Fine Science Tools | 11295-51 |
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Tags
Subcutaneous AdministrationMuscarinic AntagonistsTriple-immunostainingLevator Auris Longus MuscleMiceHind Limb MusclesIn Vivo Pharmacological StudiesNMJsDrug PenetrationSubcutaneous AdministrationIntramuscular AdministrationSystemic AdministrationMini-pumpsInflammatory ResponsesDirect Muscle DamageHind Limb Muscle AnalysisSerial SectioningExtensive ImmunostainingMouse LAL MusclePinna MovementFacial Nerve BranchStylomastoid Foramen
Citer Cet Article
Wright, M., Kim, A., Son, Y. Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice. J. Vis. Exp. (55), e3124, doi:10.3791/3124 (2011).