成年雄性小鼠经尿道灌注术
Summary
经尿道灌注是一项具有挑战性的手术, 在文献中没有得到很好的描述。本手稿的目的是描述一种技术, 经尿道插入导尿管膀胱灌注液体的活性物质进入膀胱和/或前列腺的成年雄性小鼠。
Abstract
经尿道灌注可用于提供不同的解决方案与活性成分 (例如,药物, 化学药品, 细菌和病毒) 当地进入膀胱, 要么诱发动物模型膀胱病理或评估膀胱灌注治疗的有效性。大多数啮齿动物模型的下尿路 (查找) 病理是诱导雌性小鼠, 由于容易膀胱灌注的物质通过女性尿道。然而, 由于女性与男性之间的解剖差异, 经尿道灌注的雄性小鼠被认为是一个非常具有挑战性的过程, 并没有事先描述。在这份手稿中, 我们提供了一个详细的描述如何准备聚乙烯 (PE) 油管后插入到尿道的雄性鼠标。此外, 我们还讨论了理想的 PE 管的类型, 要根据所需的接种地点使用。此外, 我们还描述了如何准备一个成功的经尿道灌注的动物, 以避免对尿道的伤害, 并确保提供的解决方案的理想地点。手术的开始是通过缩回包皮和头来暴露尿道口的开口。其次, 用钝 non-crushing 钳抓住阴蒂, 稳定小弟弟和 PE 管。PE 导管首先插入到与动物身体平行的尿道道中, 然后通过倾斜导管来调整它的角度来操纵它以遵循尿道的自然曲率。这项技术可用于诱导小鼠膀胱病理模型和/或评估的有效性, 在雄性老鼠的治疗。
Introduction
在以往的研究中, 采用经尿道灌注法作为建立膀胱疾病模型的方法之一1,2,3,4 , 可用于评估当地提供的治疗小鼠的效果。虽然动物模型不能完全重述人类的病症, 但在动物研究中的基本机制的识别更能更好地理解人类的查找障碍, 如间质性膀胱炎/膀胱疼痛综合症, 神经原膀胱炎, 自身免疫性膀胱炎, 前列腺炎症5。
在成年雄性小鼠身上进行的经尿道灌注手术比在成年雌性老鼠6中的膀胱灌注更具有技术挑战性。男性尿道的自然弯曲解剖和它的小直径使它在技术上难以完成经尿道插入导管。因此,详细说明对经尿道感染的小鼠泌尿道传染病的诱导6和查找源性炎症7,8,910,12,13,14,15通过膀胱插入导管之前只对雌性小鼠进行了概述。这份手稿的目的是提供一个 step-by 一步的描述的技术, 在男性小鼠经尿道注入的物质, 包括视频剪辑, 图像和插图。经尿道灌注手术的成年雄性小鼠可以执行的变量接种类似于以前所描述的雌性鼠标7和合并的其他技术, 如肌电图记录visceromotor 响应 (VMR)16。
Protocol
Representative Results
Discussion
本手稿详细介绍了经尿道灌注法在成年雄性小鼠中的应用。采用不同直径的 PE 管材, 可达到差异主播种面积。PE-50 是建议成功达到膀胱腔, 而较大的直径 PE-10 是用来达到 AP 和 SV (图 3A-b)。除了选择 PE 管的大小和靶器官的接种外, 其他因素, 如注入量的接种, 也发挥了作用的最终位置和分布的解决方案。我们的实验证明, 经尿道灌注100µL 的蓝色染料使用 PE-10 或 PE-50…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者没有任何致谢。
Materials
| BD INTRAMEDIC Polyethylene Tubing/ PE 10 | BD Medical | 63019-004 | |
| BD INTRAMEDIC Polyethylene Tubing/ PE 50 | BD Medical | 63019-048 | |
| BD Insyte-N Autoguard/ Angiocath | BD Medical | 311411 | |
| Ethanol (EtOH) | Fisher Scientific | BP2818500 | |
| BD Needle Only 22 Gauge (G) 1 inch | Becton Dickson | 205155 | |
| BD Needle Only 27 Gauge (G) 1/2 inch | Becton Dickson | 305129 | |
| Luer stub, 22 G X 0.5 in / Blunt Needle | Instech | LS22 | |
| Luer stub, 27 G X 0.5 in / Blunt Needle | Instech | LS27 | |
| 6" Long x 2" Wide x 1" Thick, Aluminum Oxide Sharpening Stone / Oil Stone | Norton | 40204034 | |
| Surgical Lubricant Sterile Bacteriostatic Folilpac | Surgilube | 281020543 | |
| Isoflurane | Fluriso | 13985-528-60 | |
| SomnoSuite Low-Flow Anesthesia System | Kent Scientific | SS-01 | |
| Dissecting Forceps, Curved | vwr | 82027-392 | |
| BD 1 ml Syringe | BD Medical | 309626 | |
| India Ink Solution., 0.2% in PBS buffer | Alfa Aesar | J61007 | |
| 8~12 weeks old male C57B/6 Mice | Jackson Lab | #000664 |
Riferimenti
- Lee, S., Yang, G., Bushman, W. Prostatic inflammation induces urinary frequency in adult mice. PLoS One. 10 (2), e0116827 (2015).
- Wong, L., Hutson, P. R., Bushman, W. Prostatic inflammation induces fibrosis in a mouse model of chronic bacterial infection. PLoS One. 9 (6), e100770 (2014).
- Elkahwaji, J. E., Zhong, W., Hopkins, W. J., Bushman, W. Chronic bacterial infection and inflammation incite reactive hyperplasia in a mouse model of chronic prostatitis. Prostate. 67 (1), 14-21 (2007).
- Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. Acute bacterial inflammation of the mouse prostate. Prostate. 72 (3), 307-317 (2012).
- Bjorling, D. E., Wang, Z. Y., Bushman, W. Models of inflammation of the lower urinary tract. Neurourol Urodyn. 30 (5), 673-682 (2011).
- Thai, K. H., Thathireddy, A., Hsieh, M. H. Transurethral induction of mouse urinary tract infection. J Vis Exp. (42), (2010).
- Bjorling, D. E., Wang, Z. Y., Boldon, K., Bushman, W. Bacterial cystitis is accompanied by increased peripheral thermal sensitivity in mice. J Urol. 179 (2), 759-763 (2008).
- Hopkins, W. J., Gendron-Fitzpatrick, A., Balish, E., Uehling, D. T. Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains. Infect Immun. 66 (6), 2798-2802 (1998).
- Dinis, P., Charrua, A., Avelino, A., Cruz, F. Intravesical resiniferatoxin decreases spinal c-fos expression and increases bladder volume to reflex micturition in rats with chronic inflamed urinary bladders. BJU Int. 94 (1), 153-157 (2004).
- Cayan, S., et al. Botulinum toxin type A may improve bladder function in a rat chemical cystitis model. Urol Res. 30 (6), 399-404 (2003).
- Jerde, T. J., Bjorling, D. E., Steinberg, H., Warner, T., Saban, R. Determination of mouse bladder inflammatory response to E. coli lipopolysaccharide. Urol Res. 28 (4), 269-273 (2000).
- Saban, M. R., et al. LPS-sensory peptide communication in experimental cystitis. Am J Physiol Renal Physiol. 282 (2), F202-F210 (2002).
- Schilling, J. D., Mulvey, M. A., Vincent, C. D., Lorenz, R. G., Hultgren, S. J. Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism. J Immunol. 166 (2), 1148-1155 (2001).
- Saban, M. R., et al. Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG). BMC Immunol. 8, 6 (2007).
- Bjorling, D. E., et al. Acute acrolein-induced cystitis in mice. BJU Int. 99 (6), 1523-1529 (2007).
- Sadler, K. E., Stratton, J. M., Kolber, B. J. Urinary bladder distention evoked visceromotor responses as a model for bladder pain in mice. J Vis Exp. (86), (2014).
