基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价
Summary
本文描述了一种基于微 CT 和荧光分子层析成像的无创多模成像方法, 用于对博莱霉素双气管灌注诱导的小鼠肺纤维化模型进行纵向评价。
Abstract
特发性肺纤维化 (IPF) 是一种致命的肺部疾病的特点是渐进和不可逆转的破坏肺结构, 这导致严重恶化的肺功能和随后死亡的呼吸衰竭。
博莱霉素在实验动物模型中的发病机制已被注射。本文研究了双气管内注射博莱霉素诱导的森林小组样小鼠模型。用于研究肺纤维化的标准组织学评估是侵入性终端程序。这项工作的目的是通过非侵入性成像技术, 如荧光分子层析和微 CT 监测肺纤维化。通过组织学研究证实的这两种技术可以代表一种革命性的功能性方法, 用于对森林小组疾病的严重程度和进展进行实时无创监测。不同方法的融合是进一步了解森林小组疾病的一步, 在这种情况下, 在病理条件下发生的分子事件可以用裂变材料观察到, 随后的解剖变化可以通过微 CT 监测。
Introduction
特发性肺纤维化 (IPF) 是慢性肺病, 肺功能逐渐减少, 不幸的是, 在四年的诊断中往往是致命的1。森林小组的主要特点是胞外基质沉积和成纤维细胞增殖, 但尚未完全了解发病机制。最受支持的假说是, 多循环的肺损伤导致肺泡上皮细胞的破坏, 导致间充质细胞周期增殖的改变, 成纤维细胞和肌的夸大积累, 以及增加了矩阵的生成。在人类森林小组或博莱霉素诱导的动物模型中, 失调在纤维化发展中发现了基质金属蛋白酶等过程中所涉及的调解人。无控制的基质生产导致在肺间质和肺泡空间内的胶原沉积不平衡, 模仿异常伤口修复 1, 2.
药物发现和发展的主要障碍之一是可利用的老鼠模型, 模仿人类发病机制和疾病表型。不同的药物已被用来诱导动物模型中的肺纤维化: 辐照损伤, 石棉和二氧化硅的管理, fibrinogenic 细胞因子的管理和博莱霉素3,4;然而, 博莱霉素是最常用的老鼠, 老鼠, 豚鼠, 仓鼠, 兔子5或在大型动物 (非人类灵长类, 马, 狗和反刍动物)6,7。博莱霉素是一种抗生素, 由细菌链霉菌 verticillus8 , 并用作抗癌剂9。肺纤维化是药物的常见副作用, 因此, 它被用于实验动物模型中, 诱发肺纤维化。
博莱霉素诱导的肺纤维化模型, 在博莱霉素管理后 14-21 天发生纤维性病变。在所提出的工作中, 我们采用了新的协议来诱导小鼠肺纤维化的双博莱霉素气管灌注。博莱霉素小鼠模型是非常耗时的, 因为新的药物需要评估已建立的纤维化病变, 并测试, 以区分其抗纤维化的效果与抗炎作用。
对胶原含量、形态测量学和组织学分析的生化测定是基于死后分析, 限制了在同一动物中跟踪疾病发病机制的可能性。虽然这些参数被认为是纤维化评价的金标准, 但它们没有提供任何时间或空间分布的纤维性病变, 并排除了一个方法来调查疾病进展的过程。10
最近, 非侵入性影像技术已被应用于监测小鼠气道重塑、炎症和纤维化进展: 磁共振成像 (MRI), 微计算机断层扫描 (微 CT), 荧光分子层析成像 (裂变材料) 和生物发光 (BLI)11,12,13,14,15,,16,17,18 ,20,21。在博莱霉素挑战22之后, 我们提出一种非侵入性的影像学方法来监测纵向肺纤维化进展的裂变材料和微 CT 在不同的时间点。
许多途径参与了纤维化的建立和发展, 而不多的是已知的。只有对这些过程有更深的了解, 才能转化为可能转移到诊所的更多药物靶点。利用荧光分子层析成像技术对基质金属蛋白酶的活化进行纵向监测, 并通过显微 CT 检测肺实质变化, 可用于今后对治疗的临床反应。
Protocol
Representative Results
Discussion
尽管许多研究小组专注于开发新药来治疗森林小组, 但目前只有两个患者可以使用。有迫切的医疗需要找到更有效的治疗方法7 , 因为只有肺 transplantationis 能够延长4-5 年26的生存。转化医学和新药开发的基本前提是能提供一种模仿森林小组特点的动物模型, 而介入研究对临床的成功有预测意义。然而, 现有的肺纤维化动物模型的效用仍然有争议的27</s…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢丹妮拉 Pompilio 博士和罗伯塔 Ciccimarra 的技术帮助。
Materials
| FMT 2500 Fluorescence Tomography System | Perkin Elmer Inc. | Experimental Builder | |
| MMPsense 680 | Perkin Elmer Inc. | NEV10126 | Protect from light, store the probe at 4 °C |
| TrueQuant software | Perkin Elmer Inc. | ||
| Female inbred C57BL/6 | San Pietro NatisoneHorst, The Netherlands (UD), | Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions | |
| Isoflurane | ESTEVE spa | 571329.8 | Do not inhale |
| Automated cell counter | Dasit XT 1800J | Experimental Builder | |
| Saline Solution, 0.9% Sodium Chloride (NaCl) | Eurospital | 15A2807 | |
| Quantum FX Micro-CT scanner | Perkin Elmer Inc. | ||
| Bleomycin sulphate from Streptomyces Verticillus | Sigma | B2434 | |
| Automatic tissue Processor | ATP700 Histo-Line Laboratories | ATP700 | |
| Embedding system | EG 1160 Leica Biosystems | EG 1160 | |
| Rotary microtome | Slee Cut 6062 | ||
| Digital slide scanner | NanoZoomer S60, Hamamatsu Photonics | ||
| NIS-AR image analysis software | Nikon | ||
| Masson’s Trichrome Staining | Histo-Line Laboratories | ||
| 10% neutral-buffered formalin | Sigma | HT5012-1CS | |
| Penn-century model DP-4M Dry power insufflator | Penn-century | DPM-EXT | |
| PE190 micro medical tubing | 2biological instruments snc | BB31695-PE/8 | |
| Syringe without needle 5 mL | Terumo | SS*05SE1 | Cut the boards of the piston by scissors |
| Hamilton 0.10 mL (model 1710) | Gastight | 81022 | |
| Discofix 3-way Stopcock | Braun | 4095111 | |
| Syringe with needle 1 mL | Pic solution | 3,071,260,300,320 | Use without needle |
| Plastic feeding tubes 18 ga x 50 mm | 2biological instruments snc | FTP-18-50 | Cut obliquely the tip |
References
- Wynn, T. A. Integrating mechanisms of pulmonary fibrosis. J. Exp. Med. 208 (7), 1339-1350 (2011).
- Wynn, T. A., Ramalingam, T. R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 18 (7), 1028-1040 (2012).
- Moore, B. B. Animal models of fibrotic lung disease. Am J Respir Cell Mol Biol. 49 (2), 167-179 (2013).
- Ackermann, M., et al. Effects of nintedanib on the microvascular architecture in a lung fibrosis model. Angiogenesis. , (2017).
- Moore, B. B., Hogaboam, C. M. Murine models of pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol. 294 (2), 152-160 (2008).
- Organ, L., et al. A novel segmental challenge model for bleomycin-induced pulmonary fibrosis in sheep. Exp Lung Res. 41 (3), 115-134 (2015).
- Organ, L., et al. Structural and functional correlations in a large animal model of bleomycin-induced pulmonary fibrosis. BMC Pulm Med. 15, 81 (2015).
- Shen, B., Du, L., Sanchez, C., Edwards, D. J., Chen, M., Murrell, J. M. Cloning and characterization of the bleomycin biosynthetic gene cluster from Streptomyces verticillus ATCC15003. J Nat Prod. 65 (3), 422-431 (2002).
- Yu, Z., et al. Targeted Delivery of Bleomycin: A Comprehensive Anticancer Review. Curr Cancer Drug Targets. 16 (6), 509-521 (2016).
- Ashcroft, T., Simpson, J. M., Timbrell, V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 41 (4), 467-470 (1988).
- Stellari, F., et al. In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin. J Transl Med. 13, 251 (2015).
- Stellari, F., et al. In vivo monitoring of lung inflammation in CFTR-deficient mice. J Transl Med. 14 (1), 226 (2016).
- Stellari, F. F., et al. In vivo imaging of transiently transgenized mice with a bovine interleukin 8 (CXCL8) promoter/luciferase reporter construct. PloS one. 7 (6), 39716 (2012).
- Stellari, F. F., et al. Enlightened Mannhemia haemolytica lung inflammation in bovinized mice. Vet Res. 45, 8 (2014).
- Tassali, N., et al. MR imaging, targeting and characterization of pulmonary fibrosis using intra-tracheal administration of gadolinium-based nanoparticles. Contrast Media Mol Imaging. 11 (5), 396-404 (2016).
- Ma, X., et al. Assessment of asthmatic inflammation using hybrid fluorescence molecular tomography-x-ray computed tomography. J Biomed Opt. 21 (1), 15009 (2016).
- Van de Velde, G., et al. Longitudinal micro-CT provides biomarkers of lung disease that can be used to assess the effect of therapy in preclinical mouse models, and reveal compensatory changes in lung volume. Dis Model Mech. 9 (1), 91-98 (2016).
- Stellari, F. F., et al. Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice. Front Immunol. 8, 199 (2017).
- Hellbach, K., et al. X-ray dark-field radiography facilitates the diagnosis of pulmonary fibrosis in a mouse model. Sci Rep. 7 (1), 340 (2017).
- Zhou, Y., et al. Noninvasive imaging of experimental lung fibrosis. Am J Respir Cell Mol Biol. 53 (1), 8-13 (2015).
- Ruscitti, F., et al. Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice. Multidiscip Respir Med. 12, 8 (2017).
- Stellari, F., et al. Monitoring inflammation and airway remodeling by fluorescence molecular tomography in a chronic asthma model. J Transl Med. 13, 336 (2015).
- . . Guide for the Care and Use of Laboratory Animals, 8th ed. , (2011).
- Meganck, J. A., Liu, B. Dosimetry in Micro-computed Tomography: a Review of the Measurement Methods, Impacts, and Characterization of the Quantum GX Imaging System. Mol Imaging Biol. , (2016).
- Hubner, R. H., et al. Standardized quantification of pulmonary fibrosis in histological samples). BioTechniques. 44 (4), 507-514 (2008).
- King, T. E., Pardo, A., Selman, M. Idiopathic pulmonary fibrosis. Lancet. 378 (9807), 1949-1961 (2011).
- De Langhe, E., et al. Quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography. PloS one. 7 (8), 43123 (2012).
