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Bioengenharia

使用压力表谱对毛质生殖系统的双轴基底音和被动测试

Published: August 13, 2019
doi:
10.3791/60125
, , , , ,
1Department of Biomedical Engineering,Tulane University

Summary

该协议使用市售的压力子宫内科系统对鼠阴道和子宫颈进行压力阴唇测试。利用有钙和无钙的介质,在估计的生理条件下,对器官分离出平滑肌细胞(SMC)基底色和被动细胞外基质(ECM)的贡献。

Abstract

女性生殖器官,特别是阴道和子宫颈,由各种细胞成分和独特的细胞外基质(ECM)组成。平滑肌细胞在阴道和宫颈壁内表现出收缩功能。根据生化环境和器官壁的机械分裂,平滑肌细胞改变收缩条件。平滑肌细胞在基线生理条件下的贡献被归类为基底音。更具体地说,基础色调是在没有荷尔蒙和神经刺激的情况下平滑肌细胞的基线部分收缩。此外,ECM 为器官壁提供结构支持,并充当生化线索的储层。这些生化线索对各种器官功能至关重要,例如促进生长和维持平衡。每个器官的ECM主要由胶原纤维(主要是胶原蛋白I、III和V型)、弹性纤维和糖甘油糖/蛋白酶组成。ECM 的组成和组织决定了每个器官的机械特性。ECM成分的变化可能导致生殖疾病的发展,如骨盆器官脱垂或过早的宫颈重塑。此外,ECM微结构和刚度的变化可能会改变平滑肌细胞活动和表型,从而导致收缩力的丧失。

在这项工作中,报告的协议用于评估非怀孕的鼠阴道和宫颈在4-6个月大时在estrus的基础色调和被动机械特性。这些器官安装在市售的压力表上,并进行了压力直径和力长测试。包括用于生殖器官机械表征的样本数据和数据分析技术。这些信息可能有助于构建数学模型和合理设计治疗妇女健康病理的干预措施。

Introduction

阴道壁由四层组成,即上皮、拉米那皮、肌肉和新发。上皮主要由上皮细胞组成。拉米纳蛋白片有大量的弹性和纤维胶原纤维。肌肉也由乳原和胶原纤维组成,但有增加的平滑肌肉细胞量。Adventiia 由电子莱他、胶原蛋白和成纤维细胞组成,尽管与前一层相比浓度降低。平滑肌细胞对生物力学动机的研究组感兴趣,因为它们在器官的收缩性质中发挥作用。因此,量化平滑肌细胞面积分数和组织是理解机械功能的关键。先前的研究表明,阴道壁内的平滑肌含量主要组织在圆周和纵轴。组织学分析表明,1号墙近端和远端的平滑肌面积分数约为35%。

子宫颈是一种高度胶原蛋白的结构,直到最近,被认为有最小的平滑肌细胞含量2,3。然而,最近的研究表明,平滑肌细胞在子宫颈4,5中可能具有更大的丰度和作用。子宫颈显示平滑肌细胞的梯度。内部 os 包含 50-60% 平滑肌肉细胞,其中外部 os 仅包含 10%。然而,小鼠研究报告子宫颈由10-15%平滑肌肉细胞和85-90%纤维结缔组织组成,而没有提到区域差异6,7,8。鉴于鼠标模型不同于经常报告的人类模型,需要进一步调查小鼠子宫颈。

该协议的目的是阐明鼠阴道和子宫颈的机械特性。这是通过使用压力表线仪实现,该装置能够同时评估圆周和轴向的机械性能,同时保持原生细胞基质相互作用和器官几何。器官安装在两个定制管上,用丝质6-0缝合线固定。围绕估计的生理轴向拉伸进行压力直径测试,以确定符合性和切线莫杜利9。进行了力长度测试,以确认估计的轴向拉伸,并确保机械性能在生理范围内量化。实验方案在4-6个月大的非怀孕的鼠阴道和子宫颈上进行。

该协议分为两个主要的机械测试部分:基音和被动测试。基底色调被定义为平滑肌细胞的基线部分收缩,即使在没有外部局部,荷尔蒙和神经刺激10。阴道和子宫颈的这种基线收缩性质产生典型的机械行为,然后通过压力阴骨系统测量。被动特性通过去除维持收缩基线状态的细胞间钙进行评估,从而放松平滑肌细胞。在被动状态下,胶原蛋白和乳胶纤维为器官的机械特性提供主要贡献。

鼠模型被广泛用于研究妇女生殖健康的病理学。小鼠提供了几个优势,以量化ECM和免疫系统11,12,13,14中ECM和机械特性之间的演变关系。这些优势包括周期短、特征好、成本相对较低、操作方便、妊娠时间相对较短15。此外,实验鼠的基因组绘制良好,转基因小鼠是检验机械假说16、17、18的宝贵工具。

市售的压力表线系统被广泛用于量化各种组织和器官的机械反应。一些值得注意的结构分析的压力骨髓系统包括弹性动脉19,20,21,22,静脉和组织工程血管移植23,24,食道25和大肠26。压力显影技术允许同时评估轴向和圆周方向的特性,同时保持原生细胞-ECM相互作用和体内几何体。尽管在软组织和器官力学中广泛使用了阴唇系统,但以前还没有为阴道和子宫颈开发过利用压力性阴唇技术的协议。先前对阴道和子宫颈的机械特性的调查被评估为单轴27,28。然而,这些器官在体内经历多轴负荷29,30,因此量化其双轴机械反应是重要的。

此外,最近的研究表明,平滑肌细胞可能在软组织病理学5,28,31,32中起到潜在的作用。这为利用压力肌图技术提供了另一个吸引力,因为它保留了原生细胞基质的相互作用,从而允许划定平滑肌细胞在生理和病理生理学中所起作用的贡献条件。在此,我们提出了一个协议,以量化阴道和子宫颈在基础音和被动条件下的多轴机械性能。

Protocol

本研究使用雌性C57BL6J小鼠(29.4~6.8克)进行本研究。所有程序都得到杜兰大学动物护理和使用研究所委员会的批准。分娩后,小鼠在安乐死前适应一周,并处于标准条件下(12小时光/暗周期)。 1. 在埃斯特鲁斯的老鼠牺牲 确定周期:根据以前的研究15,33,34,通过视觉评估监测的周期。周期由四个阶段组成:孕原…

Representative Results

女性生殖器官的机械特性的成功分析取决于适当的器官解剖、可分离和测试。有必要将子宫角切除阴道,没有任何缺陷(图1)。根据器官类型,管状大小会有所不同(图2)。必须进行排泄,使器官在实验期间不能移动,但不得在手术过程中损坏器官的墙壁(图3)。任一步骤的失败都会导致容器无法保持压力。测试程序标准化对于协议的成功至关重要,以便产生…

Discussion

本文提供的方案提出了一种测定鼠阴道和子宫颈的机械特性的方法。该协议分析的机械特性包括器官的被动和基底色调条件。被动和基底色调条件是通过改变器官被淹没的生化环境引起的。对于该协议,参与基础测试的介质含有钙。测试基础色调条件允许分离平滑肌细胞机械贡献在女性生殖器官54,55。执行被动机械测试时,介质不含钙。钙的缺乏会抑制平?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作由NSF CAREER奖授予#1751050资助。

Materials

2F catheter Millar SPR-320 catheter to measure cervical pressure
6-0 Suture Fine Science Tools 18020-60 larger suture ties
CaCl2 (anhydrous) VWR 97062-590 HBSS concentration: 140 mg/ mL
CaCl2-2H20 Fischer chemical BDH9224-1KG
KRB concentration: 3.68 g/L
Dextrose (D-glucose) VWR 101172-434 HBSS concentration: 1000 mg/mL
KRB concentration: 19.8 g/L
Dumont #5/45 Forceps Fine Science Tools 11251-35 curved forceps
Dumont SS Forceps Fine Science Tools 11203-25 straight forceps
Eclipse Nikon E200 microscope used for imaging
Flow meter Danish MyoTechnologies 161FM flow meter within the testing apparatus
Force Transducer – 110P Danish MyoTechnologies 100079 force transducer
ImageJ SciJava ImageJ1 used to measure volume
Instrument Cases Fine Science Tools 20830-00 casing to hold dissection tools
KCl Fisher Chemical 97061-566 HBSS concentration: 400 mg/ mL
KRB concentration: 3.5 g/L
KH2PO4 G-Biosciences 71003-454 HBSS concentration: 60 mg/ mL
MgCl2 VWR 97064-150
KRB concentration: 1.14 g/L
MgCl2-6H2O VWR BDH9244-500G HBSS concentration: 100 mg/ mL
MgSO4-7H20 VWR 97062-134 HBSS concentration: 48 mg/ mL
Mircosoft excel Microsoft 6278402 program used for spreadsheet
Na2HPO4 (dibasic anhydrous) VWR 97061-588 HBSS concentration: 48 mg/mL
KRB concentration: 1.44 g/L
NaCl VWR 97061-274 HBSS concentration: 8000 mg/mL
KRB concentration: 70.1 g/L
NaHCO3 VWR 97062-460 HBSS concentration: 350 mg/ mL
KRB concentration: 21.0 g/L
Pressure myograph systems Danish MyoTechnologies 110P and 120CP Pressure myograph system:
prorgram, cannulation device,
and controller unit
Pressure Transducer Danish MyoTechnologies 100106 pressure transducer
Student Dumont #5 Forceps Fine Science Tools 91150-20 straight forceps
Student Vannas Spring Scissors Fine Science Tools 91500-09 micro-scissors
Tissue dye Bradley Products 1101-3 ink to measure in vivo stretch
Ultrasound transducer FujiFilm Visual Sonics LZ-550 ultrasound transducer used; 256 elements, 40 MHz center frequency
VEVO2100 FujiFilm Visual Sonics VS-20035 ultrasound used for imaging
Wagner Scissors Fine Science Tools 14069-12 larger scissors
DOWNLOAD MATERIALS LIST

Referências

  1. Capone, D., et al. Evaluating Residual Strain Throughout the Murine Female Reproductive System. Journal of Biomechanics. 82, 299-306 (2019).
  2. Danforth, D. The fibrous nature of the human cervix, and its relation to the isthmic segment in gravid and nongravid uteri. American Journal of Obstetrics and Gynecology. 53 (4), 541-560 (1947).
  3. Hughesdon, P. The fibromuscular structure of the cervix and its changes during pregnancy and labour. Journal of Obstetrics and Gynecology of the British Commonwealth. 59, 763-776 (1952).
  4. Bryman, I., Norstrom, A., Lindblo, B. Influence of neurohypophyseal hormones on human cervical smooth muscle cell contractility in vitro. Obstetrics and Gynecology. 75 (2), 240-243 (1990).
  5. Joy, V., et al. A New Paradigm for the Role of Smooth Muscle Cells in the Human Cervix. Obstetrics. 215 (4), e471-e478 (2016).
  6. Xu, X., Akgul, Y., Mahendroo, M., Jerschow, A. Ex vivo assessment of mouse cervical remodeling through pregnancy via Na (23) MRS. NMR Biomedical. 23 (23), 907-912 (2014).
  7. Leppert, P. Anatomy and Physiology of cervical ripening. Clinical Obstetrics and Gynecology. 43 (43), 433-439 (2000).
  8. Schlembach, D., et al. Cervical ripening and insufficiency: from biochemical and molecular studies to in vivo clinical examination. European Journal of Obstetrics, Gynecology, and Reproductive Biology. 144, S70-S79 (2000).
  9. Stoka, K., et al. Effects of Increased Arterial Stiffiness on Atherosclerotic Plaque Amounts. Journal of Biomechanical Engineering. 140 (5), (2018).
  10. Mohram, D., Heller, L. Ch. 7. Cardiovascular Physiology. , (2006).
  11. Yoshida, K., et al. Quantitative Evaluation of Collagen Crosslinks and Corresponding Tensile Mechanical Properties in Mouse Cervical Tissue during Normal Pregnancy. PLoS One. 9, e112391 (2014).
  12. Mahendroo, M. Cervical remodeling in term and preterm birth: insight from an animal model. Society for Reproduction and Fertility. 143 (4), 429-438 (2012).
  13. Elovitz, M., Miranlini, C. Can medroxyprogesterone acetate alter Toll-like receptor expression in a mouse model of intrauterine inflammation?. American Journal of Obstetrics and Gynecology. 193 (3), 1149-1155 (2005).
  14. Ripperda, C., et al. Vaginal estrogen: a dual-edged sword in postoperative healing of the vaginal wall. North American Menopause Society. 24 (7), 838-849 (2017).
  15. Nelson, J., Felicio, P., Randall, K., Sims, C., Finch, E. A Longitudinal Study of Estrous Cyclicity in Aging C57/6J Mice: Cycle, Frequency, Length, and Vaginal Cytology. Biology of Reproduction. 27 (2), 327-339 (1982).
  16. Ferruzzi, J., Collins, M., Yeh, A., Humphrey, J. Mechanical assessment of elastin integrity in fibrillin-1-deficient carotid arteries: implications for Marfan Syndrome. Cardiovascular Research. 92 (2), 287-295 (2011).
  17. Mariko, B., et al. Fribrillin-1 genetic deficiency leads to pathological ageing of arteries in mice. The Journal of Pathology. 224 (1), 33-44 (2011).
  18. Rahn, D., Ruff, M., Brown, S., Tibbals, H., Word, R. Biomechanical Properties of The Vaginal Wall: Effect of Pregnancy, Elastic Fiber Deficiency, and Pelvic Organ Prolapse. American Urogynecological Society. 198 (5), (2009).
  19. Caulk, A., Nepiyushchikh, Z., Shaw, R., Dixon, B., Gleason, R. Quantification of the passive and active biaxial mechanical behavior and microstructural organization of rat thoracic ducts. Royal Society Interface. 12 (108), 20150280 (2015).
  20. Amin, M., Le, V., Wagenseil, J. Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult. Journal of Visualized Experiments. (60), e3733 (2012).
  21. Sokolis, D., Sassani, S., Kritharis, E., Tsangaris, S. Differential histomechanical response of carotid artery in relation to species and region: mathematical description accounting for elastin and collagen anisiotropy. Medical and Biological Engineering and Computing. 49 (8), 867-879 (2011).
  22. Kim, J., Baek, S. Circumferential variations of the mechanical behavior of the porcine thoracic aorta during the inflation test. Journal of Biomechanics. 44 (10), 1941-1947 (2011).
  23. Faury, G., et al. Developmental adaptation of the mouse cardiovascular system to elastin haploinsufficency. Journal of Clinical Investigation. 11 (9), 1419-1428 (2003).
  24. Naito, Y., et al. Beyond Burst Pressure: Initial Evaluation of the Natural History of the Biaxial Mechanical Properties of Tissue-Engineered Vascular Grafts in the Venous Circulation Using a Murine Model. Tissue Engineering. 20, 346-355 (2014).
  25. Sommer, G., et al. Multaxial mechanical response and constitutive modeling of esophageal tissues: Impact on esophageal tissue engineering. Acta Biomaterialia. 9 (12), 9379-9391 (2013).
  26. Sokolis, D., Orfanidis, I., Peroulis, M. Biomechanical testing and material characterization for the rat large intestine: regional dependence of material parameters. Physiological Measurement. 32 (12), 1969-1982 (2011).
  27. Martins, P., et al. Prediction of Nonlinear Elastic Behavior of Vaginal Tissue: Experimental Results and Model Formation. Computational Methods of Biomechanics and Biomedical Engineering. 13 (3), 317-337 (2010).
  28. Feola, A., et al. Deterioration in Biomechanical Properties of the Vagina Following Implantation of a High-stiffness Prolapse Mesh. BJOG: An International Journal of Obstetrics and Gynaecology. 120 (2), 224-232 (2012).
  29. Huntington, A., Rizzuto, E., Abramowitch, S., Prete, Z., De Vita, R. Anisotropy of the Passive and Active Rat Vagina Under Biaxial Loading. Annals of Biomedical Engineering. 47, 272-281 (2018).
  30. Tokar, S., Feola, A., Moalli, P., Abramowitch, S. Characterizing the Biaxial Mechanical Properties of Vaginal Maternal Adaptations During Pregnancy. ASME 2010 Summer Bioengineering Conference, Parts A and B. , 689-690 (2010).
  31. Feloa, A., et al. Impact of Pregnancy and Vaginal Delivery on the Passive and Active Mechanics of the Rat Vagina. Annals of Biomedical Engineering. 39 (1), 549-558 (2010).
  32. Baah-Dwomoh, A., Alperin, M., Cook, M., De Vita, R. Mechanical Analysis of the Uterosacral Ligament: Swine vs Human. Annual Biomedical Engineering. 46 (12), 2036-2047 (2018).
  33. Champlin, A. Determining the Stage of the Estrous Cycle in the Mouse by the Appearance. Biology of Reproduction. 8 (4), 491-494 (1973).
  34. Byers, S., Wiles, M., Dunn, S., Taft, R. Mouse Estrous Cycle Identification Tool and Images. PLoS One. 7 (4), e35538 (2012).
  35. McLean, A. Performing Vaginal Lavage, Crystal Violet Staining and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification. Journal of Visualized Experiments. 67, e4389 (2012).
  36. Bugg, G., Riley, M., Johnston, T., Baker, P., Taggart, M. Hypoxic inhibition of human myometrial contractions in vitro: implications for the regulation of parturition. European Journal of Clinical Investigation. 36 (2), 133-140 (2006).
  37. Taggart, M., Wray, S. Hypoxia and smooth muscle function: key regulatory events during metabolic stress. Journal of Physiology. 509, 315-325 (1998).
  38. Yoo, K., et al. The effects of volatile anesthetics on spontaneous contractility of isolated human pregnant uterine muscle: a comparison among sevoflurane, desflurane, isoflurane, and halothane. Anesthesia and Analgesia. 103 (2), 443-447 (2006).
  39. de Souza, L., et al. Effects of redox disturbances on intentional contractile reactivity in rats fed with a hypercaloric diet. Oxidative Medicine and Cellular Longevity. , 6364821 (2018).
  40. Jaue, D., Ma, Z., Lee, S. Cardiac muscarinic receptor function in rats with cirrhotic cardiomyopathy. Hepatology. 25, 1361-1365 (1997).
  41. Xu, Q., Shaffer, E. The potential site of impaired gallbladder contractility in an animal mode of cholesterol gallstone disease. Gastroenterology. 110 (1), 251-257 (1996).
  42. Rodriguez, U., et al. Effects of blast induced Neurotrauma on pressurized rodent middle cerebral arteries. Journal of Visualized Experimentals. (146), e58792 (2019).
  43. Rubod, C., Boukerrou, M., Brieu, M., Dubois, P., Cosson, M. Biomechanical Properties of Vaginal Tissue Part 1: New Experimental Protocol. Journal of Urology. 178, 320-325 (2007).
  44. Robison, K., Conway, C., Desrosiers, L., Knoepp, L., Miller, K. Biaxial Mechanical Assessment of the Murine Vaginal Wall Using Extension-Inflation Testing. Journal of Biomechanical Engineering. 139 (10), 104504 (2017).
  45. Van loon, P. Length-Force and Volume-Pressure Relationships of Arteries. Biorheology. 14 (4), 181-201 (1977).
  46. Fernandez, M., et al. Investigating the Mechanical Function of the Cervix During Pregnancy using Finite Element Models Derived from High Resolution 3D MRI. Computational Methods Biomechanical and Biomedical Engineering. 19 (4), 404-417 (2015).
  47. House, M., Socrate, S. The Cervix as a Biomechanical Structure. Ultrasound Obstetric Gynecology. 28 (6), 745-749 (2006).
  48. Martins, P., et al. Biomechanical Properties of Vaginal Tissue in Women with Pelvic Organ Prolapse. Gynecologic and Obstetrics Investigation. 75, 85-92 (2013).
  49. Rada, C., Pierce, S., Grotegut, C., England, S. Intrauterine Telemetry to Measure Mouse Contractile Pressure In vivo. Journal of Visualized Experiments. (98), e52541 (2015).
  50. Lumsden, M. A., Baird, D. T. Intra-uterine pressure in dysmenorrhea. Acta Obstectricia at Gynecologica Scandinavica. 64 (2), 183-186 (1985).
  51. Milsom, I., Andersch, B., Sundell, G. The Effect of Flurbiprofen and Naproxen Sodium On Intra-Uterine Pressure and Menstrual Pain in Patients With Primary Dysmennorrhea. Acta Obstetricia et Gynecologica Scandinavica. 67 (8), 711-716 (1988).
  52. Park, K., et al. Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. International Journal of Impotence Journal. 9 (1), 27-37 (1997).
  53. Bulletti, C., et al. Uterine Contractility During Menstrual Cycle. Human Reproduction. 15, 81-89 (2000).
  54. Kim, N. N., et al. Effects of Ovariectomy and Steroid Hormones on Vaginal Smooth Muscle Contractility. International Journal of Impotence Research. 16, 43-50 (2004).
  55. Giraldi, A., et al. Morphological and Functional Characterization of a Rat Vaginal Smooth Muscle Sphincter. International Journal of Impotence Research. 14, 271-282 (2002).
  56. Gleason, R., Gray, S. P., Wilson, E., Humphrey, J. A Multiaxial Computer-Controlled Organ Culture and Biomechanical Device for Mouse Carotid Arteries. Journal of Biomechanical Engineering. 126 (6), 787-795 (2005).
  57. Swartz, M., Tscumperlin, D., Kamm, R., Drazen, J. Mechanical Stress is Communicated Between Different Cell Types to Elicit Matrix Remodeling. Proceedings of the National Academy of Sciences of the United States of America. 98 (11), 6180-6185 (2001).
  58. Rachev, A. Remodeling of Arteries in Response to Changes in their Mechanical Environment. Biomechanics of Soft Tissue in Cardiovascular Systems. 441, 221-271 (2003).
  59. Lee, E. J., Holmes, J., Costa, K. Remodeling of Engineered Tissue Anisotropy in Response to Altered Loading Conditions. Annals of Biomedical Engineering. 36 (8), 1322-1334 (2008).
  60. Akintunde, A., et al. Effects of Elastase Digestion on the Murine Vaginal Wall Biaxial Mechanical Response. Journal of Biomechanical Engineering. 141 (2), 021011 (2018).
  61. Griffin, M., Premakumar, Y., Seifalian, A., Butler, P., Szarko, M. Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing. Journal of Visualized Experiments. (118), e54872 (2016).
  62. Myers, K., Socrate, S., Paskaleva, A., House, M. A Study of the Anisotripy and Tension/Compression Behavior of Human Cervical Tissue. Journal of Biomechanical Engineering. 132 (2), 021003 (2010).
  63. Murtada, S., Ferruzzi, J., Yanagisawa, H., Humphrey, J. Reduced Biaxial Contractility in the Descending Thoracic Aorta of Fibulin-5 Deficent Mice. Journal of Biomechanical Engineering. 138 (5), 051008 (2016).
  64. Berkley, K., McAllister, S., Accius, B., Winnard, K. Endometriosis-induced vaginal hyperalgesia in the rat: effect of estropause, ovariectomy, and estradiol replacement. Pain. 132, s150-s159 (2007).
  65. van der Walt, I., Bø, K., Hanekom, S., Rienhardt, G. Ethnic Differences in pelvic floor muscle strength and endurance in South African women. International Urogynecology Journal. 25 (6), 799-805 (2014).

Tags

Keywords: Biaxial TestingPressure MyographReproductive SystemCervixVaginaSmooth MusclePassive MatrixMechanical PropertiesWomen’s HealthBiomechanics
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White, S. E., Conway, C. K., Clark, G. L., Lawrence, D. J., Bayer, C. L., Miller, K. S. Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph. J. Vis. Exp. (150), e60125, doi:10.3791/60125 (2019).
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