手术小鼠模型中骨关节炎的标准化形态学评价
1Center for Orthopedic Research and Translational Sciences, Department of Orthopedics and Rehabilitation,Pennsylvania State College of Medicine, 2Department of Biochemistry and Molecular Biology,Pennsylvania State College of Medicine, 3Department of Pharmacology,Pennsylvania State College of Medicine
Summary
目前的议定书建立了一个严格和可重复的方法，定量与骨关节炎的形态关节变化。该协议的应用对于监测骨关节炎的疾病进展和评估治疗干预具有价值。
Abstract
骨关节炎（OA）是美国最常见的关节疾病之一，其特征是关节软骨逐渐退化，主要发生在髋关节和膝关节，对患者的流动性和生活质量造成重大影响。迄今为止，目前还没有能够减缓或抑制软骨退化的OA治疗。目前，有广泛的正在进行的研究，以了解OA病理学，并发现新的治疗方法或代理，可以有效地减缓，停止，甚至反向OA。因此，关键是要有一个定量和可重复的方法，以准确评估OA相关的病理变化在关节软骨，软骨，和下骨骨。目前，OA严重性和进展主要使用国际骨关节炎研究学会（OARSI）或曼金评分系统进行评估。尽管这些评分系统非常重要，但它们是半定量的，并且可能受用户主观性的影响。更重要的是，在早期疾病状态或早期治疗阶段，它们未能准确评估软骨的微妙但重要的变化。我们在这里描述的协议使用计算机化半自动的形态学软件系统来建立标准化、严格和可重复的定量方法，用于评估OA的联合变化。该协议为现有系统提供了强大的补充，并可以更有效地检测关节中的病理变化。
Introduction
OA是美国最常见的关节疾病之一，其特征是关节软骨逐渐退化，主要在髋关节和膝关节，对患者的流动性和生活质量产生显著影响11，2，3。2,3关节软骨是二关节的专用结缔组织，旨在最大限度地减少摩擦、促进运动和承受关节压缩4。关节软骨由两个主要成分组成：软骨细胞和细胞外基质。软细胞是专门、代谢活性细胞，在细胞外基质4的开发、维护和修复中起着主要作用。软骨细胞肥大 （CH） 是 OA 发育的主要病理体征之一。其特征是细胞体积增加，蛋白酶产量减少，软骨基质降解酶的产量增加，最终导致软骨退化55，6，7。6,7此外，关节下骨和关节的病理变化在OA,9,10,11,发育和进展中起重要作用。迄今为止，目前还没有抑制软骨退化的疗法11、2、3、13、14。2,3,13,14因此，有广泛的正在进行的研究，旨在了解OA病理学和发现新的治疗方法，能够减慢，甚至停止OA。因此，越来越需要定量和可重复的方法，以便准确评估关节软骨、阴骨和下骨的OA相关病理变化。
目前，OA 严重性和进展主要使用 OARSI 或曼金评分系统15进行评估。然而，这些计分系统只是半定量的，并且可能受用户主观性的影响。更重要的是，它们未能准确评估在疾病期间关节或对基因操纵或治疗干预的反应的细微变化。文献中有零星的报告，描述软骨、阴囊或下骨的形态学分析16、17、18、19、20、21。16,17,18,19,20,21然而，对于所有这些关节成分进行严格和可重复的形态分析的详细协议仍然缺乏，这在现场造成了未满足的需求。
为了研究OA的病理变化，使用组织学分析，我们使用手术OA小鼠模型诱导OA通过中膜半月板（DMM）的不稳定。在已建立的鼠OA模型中，DMM被选作我们的研究，因为它涉及伤害的创伤性较小机制22、23、24、25、26。22,23,24,25,26与男性韧带损伤（MLI）或前十字韧带损伤（ACLI）手术相比，DMM促进OA的逐步进展，类似于OA在人类22，24，25，2624,25,26的发育22。小鼠在DMM手术后12周被安乐死，以评估关节软骨、下骨和阴离子的变化。
该协议的目标是建立一个标准化、严格和定量的方法，以评估随 OA 附带的联合更改。
Protocol
十二周大的雄性C57BL/6小鼠是从Jax实验室购买的。所有小鼠都被安置在每微隔离器笼子里3~5只老鼠的组，在一个12小时深沉的房间里。所有动物程序均根据国家卫生研究所（NIH）实验室动物护理和使用指南执行，并经宾夕法尼亚州立大学动物护理和使用委员会批准。 1. 创伤后骨关节炎（PTOA）手术模式 通过腹内注射，用氯胺酮（100毫克/千克）/xylazine（10mg/kg）组合…
Representative Results
DMM诱导OA导致关节软骨退化和软骨损失DMM引起的OA导致OARSI评分与假小鼠相比增加，其显著特征是表面侵蚀和软骨损失（图1A，D）。D这里详述的通体方案，发现多项OA相关变化，包括软骨总面积减少及未加垢软骨面积减少（图1A、B、E、G）;Figure 1ABEG减少总软骨素数;并且，重要的是，产生?…
Discussion
最近的骨关节炎研究加深了我们对关节内不同组织之间的交叉以及每个组织在疾病启动或进展中的作用的理解8、9、10、35、36。9,10,35,368因此，很明显，OA的评估不应局限于软骨分析，还应包括对下骨和软骨的分析。尽管如此，关节软骨一直是OA15，37，38,…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢比较医学部工作人员以及宾夕法尼亚州立大学米尔顿·赫希医疗中心的分子和组织病理学核心的帮助。资金来源：NIH NIAMS 1RO1AR071968-01A1（F.K.），ANRF关节炎研究补助金（F.K.）。
Materials
| 10% Buffered Formalin Phosphate | Fisher Chemical | SF100-20 | For sample fixation following harvest |
| Acetic Acid, Glacial (Certified A.C.S.) | Fisher Chemical | A38S-212 | For Decalcification Buffer preparation and acetic acid solution preparation for staining |
| Cintiq 27QHD Creative Pen Display | Wacom | https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch | For histomorphometric analysis and imaging |
| Cintiq Ergo stand | Wacom | https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch | For histomorphometric analysis and imaging |
| Ethylenediaminetetraacetic acid, tetrasodium salt dihydrate, 99% | Acros Organics | AC446080010 | For Decalcification Buffer preparation |
| Fast Green stain | SIGMA Life Sciences | F7258 | For sample staining |
| Fisherbrand Superfrost Plus Microscope Slides | Fisher | 12-550-15 | For sample section collection |
| HistoPrep Xylene | Fisherbrand | HC-700-1GAL | For sample deparrafinization and staining |
| Histosette II Tissue Cassettes – Combination Lid and Base | Fisher | 15-182-701A | For sample processing and embedding |
| HP Z440 Workstation | HP | Product number: Y5C77US#ABA | For histomorphometric analysis and imaging |
| Manual Rotary Microtome | Leica | RM 2235 | For sample sectioning |
| Marking pens | Leica | 3801880 | For sample labeling, cassettes and slides |
| OLYMPUS BX53 Microscope | OLYMPUS | https://www.olympus-lifescience.com/en/microscopes/upright/bx53f2/ | For histomorphometric analysis and imaging |
| OLYMPUS DP 73 Microscope Camera | OLYMPUS | https://www.olympus-lifescience.com/en/camera/color/dp73/ | For histomorphometric analysis and imaging (discontinued) |
| ORION STAR A211 pH meter | Thermo Scientific | STARA2110 | For Decalcification Buffer preparation |
| OsteoMeasure Software | OsteoMetrics | https://www.osteometrics.com/index.htm | For histomorphometric measurement and analysis |
| Perfusion Two Automated Pressure Perfusion system | Leica | Model # 39471005 | For mouse knee harvest |
| PRISM 7 Software | GraphPad | Institutional Access Account | Statistical Analysis |
| Safranin-O stain | SIGMA Life Sciences | S8884 | For sample staining |
| ThinkBoneStage – Rotating Microscope Stage | Think Bone Consulting Inc. – OsteoMetrics (supplier) | http://thinkboneconsulting.com/index_files/Slideholder.php | For histomorphometric analysis and imaging |
| Wacom Pro Pen Stylus | Wacom | https://www.wacom.com/en-es/products/pen-displays/cintiq-27-qhd-touch | For histomorphometric analysis and imaging |
| Weigerts Iron Hematoxylin A | Fisher | 5029713 | For hematoxylin staining |
| Weigerts Iron Hematoxylin B | Fisher | 5029714 | For hematoxylin staining |
References
- Ma, V. Y., Chan, L., Carruthers, K. J. Incidence, prevalence, costs, and impact on disability of common conditions requiring rehabilitation in the United States: stroke, spinal cord injury, traumatic brain injury, multiple sclerosis, osteoarthritis, rheumatoid arthritis, limb loss, and back pain. Archives of Physical Medicine and Rehabililation. 95 (5), 986-995 (2014).
- Hopman, W., et al. Associations between chronic disease, age and physical and mental health status. Journal of Chronic Diseases in Canada. 29 (3), 108-116 (2009).
- Lorenz, J., Grässel, S., Singh, S., Coppola, V. Experimental osteoarthritis models in mice. Mouse Genetics. Methods in Molecular Biology. 1194, 401-419 (2004).
- Sophia Fox, A. J., Bedi, A., Rodeo, S. A. The basic science of articular cartilage: structure, composition, and function. Journal of Sports Health. 1 (6), 461-468 (2009).
- Van der Kraan, P., Van den Berg, W. Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration. Osteoarthritis and Cartilage. 20 (3), 223-232 (2012).
- Hodsman, A. B., et al. Parathyroid hormone and teriparatide for the treatment of osteoporosis: a review of the evidence and suggested guidelines for its use. Endocrine Reviews. 26 (5), 688-703 (2005).
- Pitsillides, A. A., Beier, F. Cartilage biology in osteoarthritis-lessons from developmental biology. Nature Reviews Rheumatology. 7 (11), 654 (2011).
- Yuan, X., et al. Bone-cartilage interface crosstalk in osteoarthritis: potential pathways and future therapeutic strategies. Osteoarthritis and Cartilage. 22 (8), 1077-1089 (2014).
- Goldring, S. R., Goldring, M. B. Changes in the osteochondral unit during osteoarthritis: structure, function and cartilage-bone crosstalk. Nature Reviews Rheumatology. 12 (11), 632 (2016).
- Martel-Pelletier, J., et al. Osteoarthritis. Nature Reviews Disease Primers. 2 (1), 16072 (2016).
- Goldring, M. B., Otero, M. Inflammation in osteoarthritis. Current Opinion in Rheumatology. 23 (5), 471 (2011).
- Sellam, J., Berenbaum, F. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis. Nature Reviews Rheumatology. 6 (11), 625 (2010).
- Ma, H., et al. Osteoarthritis severity is sex dependent in a surgical mouse model. Osteoarthritis and Cartilage. 15 (6), 695-700 (2007).
- Katon, W., Lin, E. H., Kroenke, K. The association of depression and anxiety with medical symptom burden in patients with chronic medical illness. General Hospital Psychiatry. 29 (2), 147-155 (2007).
- Glasson, S., Chambers, M., Van Den Berg, W., Little, C. The OARSI histopathology initiative-recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis and Cartilage. 18, 17-23 (2010).
- Pastoureau, P., Leduc, S., Chomel, A., De Ceuninck, F. Quantitative assessment of articular cartilage and subchondral bone histology in the meniscectomized guinea pig model of osteoarthritis. Osteoarthritis and Cartilage. 11 (6), 412-423 (2003).
- O’Driscoll, S. W., Marx, R. G., Fitzsimmons, J. S., Beaton, D. E. Method for automated cartilage histomorphometry. Tissue Engineering. 5 (1), 13-23 (1999).
- Matsui, H., Shimizu, M., Tsuji, H. Cartilage and subchondral bone interaction in osteoarthrosis of human knee joint: a histological and histomorphometric study. Microscopy Research Technique. 37 (4), 333-342 (1997).
- Hacker, S. A., Healey, R. M., Yoshioka, M., Coutts, R. D. A methodology for the quantitative assessment of articular cartilage histomorphometry. Osteoarthritis and Cartilage. 5 (5), 343-355 (1997).
- Pastoureau, P., Chomel, A., DeCeuninck, F., Sabatini, M., Pastoureau, P. Methods for Cartilage and Subchondral Bone Histomorphometry. Cartilage and Osteoarthritis. Methods in Molecular Medicine. 101, 79-91 (2004).
- McNulty, M. A., et al. A comprehensive histological assessment of osteoarthritis lesions in mice. Cartilage. 2 (4), 354-363 (2011).
- Glasson, S., Blanchet, T., Morris, E. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis and Cartilage. 15 (9), 1061-1069 (2007).
- Singh, S. R., Coppola, V. . Mouse Genetics: Methods and Protocols. , (2004).
- Fang, H., Beier, F. Mouse models of osteoarthritis: modelling risk factors and assessing outcomes. Nature Reviews Rheumatology. 10 (7), 413 (2014).
- Culley, K. L., Westendorf, J., van Wijnen, A., et al. Mouse Models of Osteoarthritis: Surgical Model of Posttraumatic Osteoarthritis Induced by Destabilization of the Medial Meniscus. Osteoporosis and Osteoarthritis. Methods in Molecular Biology. 1226, 143-173 (2015).
- Van der Kraan, P. Factors that influence outcome in experimental osteoarthritis. Osteoarthritis and Cartilage. 25 (3), 369-375 (2017).
- Gage, G. J., Kipke, D. R., Shain, W. Whole animal perfusion fixation for rodents. Journal of Visualized Experiments. (65), e3564 (2012).
- Callis, G., Sterchi, D. Decalcification of bone: literature review and practical study of various decalcifying agents. Methods, and their effects on bone histology. Journal of Histotechnology. 21 (1), 49-58 (1998).
- Lajeunesse, D., Massicotte, F., Pelletier, J. P., Martel-Pelletier, J. Subchondral bone sclerosis in osteoarthritis: not just an innocent bystander. Modern Rheumatology. 13 (1), 0007-0014 (2003).
- Li, G., et al. Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes. Arthritis Research Therapy. 15 (6), 223 (2013).
- Kapoor, M., Martel-Pelletier, J., Lajeunesse, D., Pelletier, J. P., Fahmi, H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nature Reviews Rheumatology. 7 (1), 33 (2011).
- Scanzello, C. R., Goldring, S. R. The role of synovitis in osteoarthritis pathogenesis. Bone. 51 (2), 249-257 (2012).
- Benito, M. J., Veale, D. J., FitzGerald, O., van den Berg, W. B., Bresnihan, B. Synovial tissue inflammation in early and late osteoarthritis. Annals of the Rheumatic Diseases. 64 (9), 1263-1267 (2005).
- De Lange-Brokaar, B. J., et al. Synovial inflammation, immune cells and their cytokines in osteoarthritis: a review. Osteoarthritis and Cartilage. 20 (12), 1454-1499 (2012).
- Findlay, D. M., Kuliwaba, J. S. Bone-cartilage crosstalk: a conversation for understanding osteoarthritis. Bone Research. 4, 16028 (2016).
- Lories, R. J., Luyten, F. P. The bone-cartilage unit in osteoarthritis. Nature Reviews Rheumatology. 7 (1), 43 (2011).
- Pritzker, K. P., et al. Osteoarthritis cartilage histopathology: grading and staging. Journal of Osteoarthritis and Cartilage. 14 (1), 13-29 (2006).
- Hayami, T., et al. Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis. Bone. 38 (2), 234-243 (2006).
- Priemel, M., et al. mineralization defects and vitamin D deficiency: Histomorphometric analysis of iliac crest bone biopsies and circulating 25-hydroxyvitamin D in 675 patients. Journal of Bone and Mineral Research. 25 (2), 305-312 (2010).
- Yukata, K., et al. Continuous infusion of PTH 1–34 delayed fracture healing in mice. Scientific Reports. 8 (1), 13175 (2018).
- Kawano, T., et al. LIM kinase 1 deficient mice have reduced bone mass. Bone. 52 (1), 70-82 (2013).
- Zhang, L., Chang, M., Beck, C. A., Schwarz, E. M., Boyce, B. F. Analysis of new bone, cartilage, and fibrosis tissue in healing murine allografts using whole slide imaging and a new automated histomorphometric algorithm. Bone Research. 4, 15037 (2016).
- Wu, Q., et al. Induction of an osteoarthritis-like phenotype and degradation of phosphorylated Smad3 by Smurf2 in transgenic mice. Arthritis Rheumatism. 58 (10), 3132-3144 (2008).
- Hordon, L., et al. Trabecular architecture in women and men of similar bone mass with and without vertebral fracture: I. Two-dimensional histology. Bone. 27 (2), 271-276 (2000).
Citer Cet Article
Pinamont, W. J., Yoshioka, N. K., Young, G. M., Karuppagounder, V., Carlson, E. L., Ahmad, A., Elbarbary, R., Kamal, F. Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model. J. Vis. Exp. (159), e60991, doi:10.3791/60991 (2020).