小鼠涎腺照射后通过匹属刺激进行唾液功能评估
Summary
我们提出了一个详细的方法来执行唾液收集, 包括小鼠气管切开和隔离三大涎腺。
Abstract
Hyposalivation 通常观察到 Sjögren 综合征的自身免疫反应或继放射损伤的主要涎腺。在这些情况下, 有关疾病发病机制和有效干预的问题仍然存在。一种优化的技术, 允许对涎腺的功能性评估是非常宝贵的研究外分泌腺生物学, 功能障碍和治疗。在这里, 我们提出一步一步的方法来执行的匹凡刺激唾液分泌, 包括气管切开和解剖三大鼠涎腺。我们还详细介绍了适当的小鼠头部和颈部解剖学在这些技术的访问。这种方法是可伸缩的, 允许多个鼠标同时处理, 从而提高了工作流的效率。我们的目的是提高这些方法的重现性, 每一个都有进一步的应用领域内。除了唾液收集, 我们还讨论了量化和规范化这些组织的功能能力的指标。有代表性的数据包括从颌下腺功能下降2周后, 分光辐射 (4 剂量 6.85)。
Introduction
涎腺疾病包括失调或分泌障碍的症状, 导致生产过剩 (sialorrhea) 或减产 (口干症和 hyposalivation) 的唾液1。在这两种情况下, 都有兴趣提高我们对涎腺生物学的认识, 迈向治疗发展的最终目标2。
涎腺是高度敏感器官, 并且经常被损坏在头颈癌放疗期间, 导致永久干嘴 (口干症)3, 4.与其他敏感组织不同, 涎腺的更替率相对较低, 分泌性损失的机制不太了解5,6。在这种独特的损伤设置, 组织再生和防护策略需要唾液功能评估。实验中, 小鼠唾液收集是评估辐射和治疗药物的腺体反应的一个特别有价值的工具。
在这里, 我们提出了一个方法, 以执行和量化刺激唾液分泌使用的匹毒蕈, 一个强有力的7激动剂。匹凡刺激自主神经系统诱发腺体分泌8,9。为了适当地完成这项试验, 需要进行气管切开, 以确保老鼠在整个过程中保持一个专利气道, 并减少口腔内的分泌物窒息和吸入的风险10。
这是一个终端程序, 最终清除三大涎腺: 腮腺 (PG), 颌下腺 (SMG) 和舌下 (SLG)。在功能性研究中, 腺体重量被记录下来, 通常用来规范化唾液测量11,12,13。这些数据在辐射研究中尤为重要, 其中腺体萎缩是预期结果14,15
在文献中有可变性的关于如何刺激唾液分泌被执行和报告的16。例如, 在文学范围内的匹比剂量至少三级17,18,19,20,21,22,23。在这里, 我们提出了一个优化的高剂量匹凡协议, 目的是提高方法执行的重现性, 以及提供一个模块化的技术平台 (气管切开, 唾液收集和腺体解剖), 可以适应需要。
除了协议演示外, 我们还包括有代表性的2周内唾液流动的功能数据 (4 剂量 6.85) 到 SMG 地区。
Protocol
下文概述的所有体内程序均经罗切斯特罗切斯特大学动物资源大学委员会批准。纽约。 1. 准备 使用分析天平, 重20毫克的匹。将其溶于2毫升的无菌盐水中, 离心管。注意: 由于匹属轻敏感, 并且随着时间的推移而失去活动, 这个解决方案应该在注射之日准备好, 并在被管理之前保护光线。 使用分析平衡, 权衡和识别所有收集管和玻璃毛细血管。收集管和?…
Representative Results
当执行高剂量的匹比刺激唾液收集, 重要的是保持呼吸道, 以防止吸入或窒息的分泌物在口腔。提供了气管切开的示意图 (图 1)。气管切开后, 气孔必须保持清晰的组织和液体。 为了加强唾液收集过程中的毛细血管作用, 应将小鼠的头部置于45°角度向下定位。这些步骤可以在 > 1 鼠标上执行, 但不建议一次?…
Discussion
本文提出一种评价涎腺功能的多步法方法, 可用于研究腺损伤和治疗。我们的程序包括气管切开, 唾液收集和腺体解剖, 每个实验应用都能支持涎腺生物学的综合研究。例如, 小鼠气管切开术在阻塞口腔的过程中被用于一般的气道管理。
正确的解剖和气管切开是需要的, 以刺激唾液分泌100毫克/千克。另一种方法是, 减少的匹比, 可以避免对气管切开的需要, 因此用于纵向评估唾液…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该出版物中报告的研究得到国家牙科和颅面研究研究所 (NIDCR) 和国立卫生研究院全国癌症研究所 (R56 DE025098、UG3 DE027695 和 F30 CA206296) 的支持。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。这项工作也得到了 NSF DMR 1206219 和 IADR 口腔护理奖 (2016) 创新的支持。
我们要感谢 Eri 圆山博士和安德鲁 Hollomon 为他们提供的唾液收集帮助。我们要感谢培伦翁协助进行腺体解剖。我们要感谢马修·格尔斯在图准备方面的协助。我们要感谢伊莲 Smolock 博士和艾米莉. 吴对这篇手稿的批判性阅读。
Materials
| Pilocarpine hydrochloride | Sigma Aldrich | P6503 | Pilocarpine |
| Student Vannas Spring Scissors | Fine Science Tools | 91500-9 | Spring Scissors for Tracheostomy |
| Sterile Saline Solution | Medline | RDI30296H | Saline |
| Dumont #7 Forceps | Fine Science Tools | 11274-20 | Curved Forceps |
| Dumont #5 Forceps | Fine Science Tools | 11251-10 | Straight Forceps |
| Standard Pattern Forceps | Fine Science Tools | 11000-12 | Blunt Forceps |
| Fine Scissors- Tungsten Carbide | Fine Science Tools | 14568-09 | Dissection Scissors |
| Microhematocrit Heparinized Capillary Tubes | Fisher Scientific | 22362566 | Capillary tubes |
| Lubricant Eye Ointment | Refresh | N/A | Refresh Lacri-Lube |
| Goat polyclonal anti-Nkcc1 | Santa Cruz Biotech | SC-21545 | Nkcc1 Antibody |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | DAPI |
| GraphPad Prism | GraphPad | ver6.0 | Statistical Software |
| Cotton tipped applicator | Medline | MDS202000 | Applicator for eye ointment |
| 0.5cc Insulin Syringe, 29G x 1/2" | BD | 7629 | Syringe for intraperitoneal injection |
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Citazione di questo articolo
Varghese, J. J., Schmale, I. L., Hansen, M. E., Newlands, S. D., Benoit, D. S., Ovitt, C. E. Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation. J. Vis. Exp. (135), e57522, doi:10.3791/57522 (2018).