小鼠涎腺功能的测定方法
Summary
涎腺功能是自身免疫性疾病和放射治疗的常见后果。小鼠涎腺功能的重现性评价这些疾病的模型是一个技术挑战。在这里, 一个简单的方法, 准确和重复的测量唾液生产的小鼠描述。
Abstract
干燥氏综合征是一种影响外分泌腺体的自身免疫性疾病, 它会发展涎腺炎症并减少唾液的产生。同样, 在接受放疗治疗头颈部癌症的患者中, 唾液的分泌也受到严重损害。啮齿动物模型, 开发, 以模仿这些临床条件, 促进了解疾病的发病机制, 并允许发展新的治疗策略。因此, 在动物模型中准确、性和反复测量涎腺功能的能力是至关重要的。根据文献中描述的程序, 建立了符合这些标准的方法, 并用于评价小鼠唾液腺功能。这种新方法的另一个优点是它易于掌握, 并具有极少的互操作性变化。唾液腺功能是评估的数量 (重量或体积) 或速率 (毫升/分钟) 的反应产生的匹罗卡品刺激。收集的唾液是分析蛋白质含量、免疫球蛋白浓度和其他生物分子的好来源。
Introduction
唾液腺产生唾液, 以响应各种神经和机械刺激1。刺激是通过交感神经和副交感神经系统的肾上腺素和胆碱受体在腺体。匹罗卡品是一种胆碱能交感神经剂, 主要作用于毒蕈受体。在唾液腺, 它通过作用于毒蕈乙酰胆碱受体 M31来诱导唾液的产生。在匹罗卡品管理的唾液生产是一个指标的能力, 唾液腺反应刺激和通常使用作为衡量唾液腺功能。
唾液生产的准确测量是研究涎腺疾病的关键, 包括干燥氏综合征2和头颈癌治疗后的辐射损伤3。几种不同的方法已经被开发, 以测量唾液生产啮齿动物。这些包括排泄涎腺导管的直接插管4, 从口腔中收集的唾液在真空下的5, 并收集使用玻璃毛细管6或微7,8,9. 唾液腺导管的直接插管提供了最准确和纯净的唾液。然而, 这是一个技术上具有挑战性的过程, 和潜在的造成导管损伤排除重复唾液收集来自同一动物。在真空中收集唾液会导致因唾液从管子中干燥而产生的变化。这种损失是进一步夸大小鼠与减少流涎。玻璃毛细管允许收集唾液进行后续分析, 但是, 在患病状态下分泌的唾液粘度的变化防止毛细血管的有效充填。此外, 试图清扫口腔, 以收集残留的唾液可能导致伤害。吸管方法允许完全收集, 对于同一个算子, 它在实验之间是非常一致的。但是, 由于未知的原因, 此方法在不同运算符之间显示出显著的差异。因此, 要允许进行适当的比较, 同样的操作者必须执行与特定项目相关的所有实验。显然, 这是一个实验室的主要劣势。
为了克服这些问题, 制定了一个程序, 将唾液收集方法用于人类和啮齿动物。用棉签法测定匹罗卡品引起的唾液体积简单、重现性好, 不受操作者影响, 可在同一动物中重复进行。此外, 它允许收集唾液, 随后分析蛋白质, 免疫球蛋白, 或其他生物分子。
Protocol
下文所述的议定书得到了机构动物保育和使用委员会的批准, 并遵循了国家卫生研究院制定的道德准则。本报告所列的所有数据都是使用10-12 周龄的雌性小鼠产生的。使用的小鼠如下: C57BL/6, balb/c, DBA1, 129S, 和 (B6XA/J) F1。所有小鼠都被安置在屏障笼 (每笼5只动物) 的特定无病原体条件, 并提供饲料和水 ad 随意。 1. 准备 要准备一份盐酸匹罗卡品的溶液, 重10毫克的化合物, ?…
Representative Results
在实验小鼠模型系统中, 以匹罗卡品刺激产生唾液的能力被用作唾液腺功能的指标。用拭子法测定了11周老 C57BL/6 雌性小鼠的唾液分泌。在图 1A中, 结果被表示为在增加的匹罗卡品之后收集的唾液量 (mg)。在1毫克/千克体重的剂量, 一些小鼠开始表现出窘迫 (不由自主的颤抖和颤抖)。因此, 在本研究中没有对1毫克/千克体重的匹罗卡品进行测试。在<strong c…
Discussion
唾液生产是一个复杂的过程, 受多种因素的影响。因此, 测定实验动物涎腺功能是一个挑战。另一项挑战是, 在同一动物中反复测量唾液腺功能, 以确定疾病的发病或在治疗后证明恢复。
本报告中描述的拭子方法在技术上是简单的, 可以使用多个选项来表示数据。所得到的结果是可重现的, 但很少有互操作性变化。该方法可检测不同模型涎腺功能障碍的唾液分泌减少。一个重要?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国家卫生研究院、国家牙科和颅面研究研究所 (DE025030) 的资助。
Materials
| Chemicals for saliva collection | |||
| Pilocarpine hydrochloride | Alfa Aesar, Tewksbury, MA, USA | B21410 | Dilute in sterile isotonic saline. Store single use aliquots of 100X stock at -80oC |
| Name | Company | Catalog Number | Comments |
| Anesthesia Mix solution | Mix 1 mL ketamine hydrochloride + 0.5 mL xylazine + 8.5 mL sterile isotonic saline in a sterile vial. Can be used for 3 months. | ||
| Zetamine (Ketamine hydrochloride) | Vet One, Boise, Idaho, USA | C3N VT1 | Stock is 100 mg/mL |
| Anased (Xylazine) | Med-Vet International, Mettawa, IL, USA | RXANASED-20 | Stock is 20 mg/mL |
| Isotonic sterile saline | Vet One, Boise, Idaho, USA | 501032 | Used as diluent |
| Artificial tears (lubricant ophthalmic ointment) | Henry Schien, Dublin, OH, USA | 48272 | Used to prevent eyes from drying during the procedure |
| Name | Company | Catalog Number | Comments |
| Materials for saliva collection | |||
| SalivaBio Children's swab | Salimetrics, LLC Carlsbad, CA, USA | N/A | Individually wrapped swabs |
| 50 mL polypropylene tubes | VWR, Radnor, PA, USA | 89004-364 | Cut off the bottom 1 cm of the tube. Make sure that the cut edge is smooth. |
| Microcentrifuge tubes 0.6 mL | VWR, Radnor, PA, USA | 87003-290 | Make holes in the bottom of tube with heated 18 gauge needles |
| Microcentrifuge tubes 2 mL | VWR, Radnor, PA, USA | 87003-298 | |
| Insulin Syringes with permanently attached needles | Becton Dickinson and Company, Franklin Lakes, NJ, USA | 324702 | |
| 18 gauge regular bevel needles | Becton Dickinson and Company, Franklin Lakes, NJ, USA | 305195 | |
| Sterile scalpel blade #11 | Integra York Inc PA, USA | 4-311 | |
| Microdissecting forceps | Roboz, Gaithersburg, MD, USA | RS-5139 | Serrated angular 0.8 mm tip, 4" length |
| Label tape | Santa Cruz Biotechnology, Dallas, TX, USA | sc-224487 | |
| 3 channel timer | Amazon.com, Seattle, WA, USA | B06W2KCYVN | |
| Analytical Balance | Mettler Toledo, Columbus OH, USA | ||
| Name | Company | Catalog Number | Comments |
| Chemicals/ reagents for inducing salivary dysfunction | |||
| LPS | Invivogen, San Deigo, CA, USA | tlrl-b5lps | Dissolve in endotoxin free water, and store stock solution at 5 mg/mL . dilute in sterile HBSS 100 ug/mL – inject 100 uL/ moue ip |
| Imject Alum adjuvant | Thermo Scientific | 77161 | Dilute 1:1 in sterile saline. Inject intraperitoneally 0.1 mL/mouse |
| Rabbit anti-Ro52 antiserum | Generated in lab | Immunization of rabbits with recombinant mouse Ro52 | |
| Name | Company | Catalog Number | Comments |
| Chemicals/ Kits for saliva analyses | |||
| Salivary lysozyme estimation | |||
| EnzChek Lysozyme Assay Kit | Molecular Probes, Eugene, OR, USA | E-22013 | Used as per manufacturer's instructions |
| Name | Company | Catalog Number | Comments |
| Salivary IgA estimation by sandwich ELISA | |||
| Mouse IgA | Southern Biotech, Birmingham, AL, USA | 0106-01 | Standards for sandwich ELISA – range 30 ng/mL to 0.5 ng/mL |
| Goat anti- mouse IgA unlabeled | Southern Biotech, Birmingham, AL, USA | 1040-01 | Coat at 1 ug/mL in bicarbonate buffer |
| Goat anti- mouse IgA HRP | Southern Biotech, Birmingham, AL, USA | 1040-05 | Detection antibody used at 1:4000 dilution |
| TMB substrate | Becton Dickinson and Company, Franklin Lakes, NJ, USA | 555214 | Used as per manufacturer's instructions |
| Immulon 4HBX Microtiter 96 well plates | Thermo Scientific, Rochester, NY, USA | 3855 | |
| Name | Company | Catalog Number | Comments |
| Salivary protein estimation | |||
| Bio-Rad Protein Assay Dye Reagent Concentrate | BioRad, Hercules, CA, USA | 5000006 | For protein estimation as per manufacturer's instructions |
Referencias
- Proctor, G. B. The physiology of salivary secretion. Periodontol 2000. 70 (1), 11-25 (2016).
- Fox, R. I. Sjögren’s syndrome. Lancet. 366 (9482), 321-331 (2005).
- Eisbruch, A., Kim, H. M., Terrell, J. E., Marsh, L. H., Dawson, L. A., Ship, J. A. Xerostomia and its predictors following parotid-sparing irradiation of head-and-neck cancer. Int J Radiat Oncol Biol Phys. 50 (3), 695-704 (2001).
- Marmary, Y., Fox, P. C., Baum, B. J. Fluid secretion rates from mouse and rat parotid glands are markedly different following pilocarpine stimulation. Comp Biochem Physiol A Comp Physiol. 88 (2), 307-310 (1987).
- Lin, A. L., Johnson, D. A., Wu, Y., Wong, G., Ebersole, J. L., Yeh, C. K. Measuring short-term gamma-irradiation effects on mouse salivary gland function using a new saliva collection device. Arch Oral Biol. 46 (11), 1085-1089 (2001).
- Scofield, R. H., Asfa, S., Obeso, D., Jonsson, R., Kurien, B. T. Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren’s syndrome. J Immunol. 175 (12), 8409-8414 (2005).
- Deshmukh, U. S., Nandula, S. R., Thimmalapura, P. R., Scindia, Y. M., Bagavant, H. Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function. J Oral Pathol Med. 38 (1), 42-47 (2009).
- Deshmukh, U. S., Ohyama, Y., Bagavant, H., Guo, X., Gaskin, F., Fu, S. M. Inflammatory stimuli accelerate Sjögren’s syndrome-like disease in (NZB x NZW)F1 mice. Arthritis Rheum. 58 (5), 1318-1323 (2008).
- Szczerba, B. M., et al. Interaction between innate immunity and Ro52-induced antibody causes Sjögren’s syndrome-like disorder in mice. Ann Rheum Dis. 75 (3), 617-622 (2016).
- Takakura, A., Moreira, T., Laitano, S., De Luca Júnior, L., Renzi, A., Menani, J. Central muscarinic receptors signal pilocarpine-induced salivation. J Dent Res. 82 (12), 993-997 (2003).
- Yao, C., et al. Lipopolysaccharide-induced elevation and secretion of interleukin-1beta in the submandibular gland of male mice. Immunology. 116 (2), 213-222 (2005).
- Knudsen, J., Nauntofte, B., Josipovic, M., Engelholm, S. A., Hyldegaard, O. Effects of isoflurane anesthesia and pilocarpine on rat parotid saliva flow. Radiat Res. 176 (1), 84-88 (2011).
- Kohrs, R., Durieux, M. E. Ketamine: teaching an old drug new tricks. Anesth Analg. 87 (5), 1186-1193 (1998).
- Kozaki, T., Hashiguchi, N., Kaji, Y., Yasukouchi, A., Tochihara, Y. Effects of saliva collection using cotton swab on cortisol enzyme immunoassay. Eur J Appl Physiol. 107 (6), 743-746 (2009).
Citar este artículo
Bagavant, H., Trzeciak, M., Papinska, J., Biswas, I., Dunkleberger, M. L., Sosnowska, A., Deshmukh, U. S. A Method for the Measurement of Salivary Gland Function in Mice. J. Vis. Exp. (131), e57203, doi:10.3791/57203 (2018).