在健康和受伤的老鼠心脏中为三维纤维细胞组织进行精炼的清晰组织清除
Summary
开发并应用于成年小鼠心脏的组织清除方法。这种方法旨在清除密集的，自动荧光的心脏组织，同时保持标记成纤维细胞荧光归因于遗传报告器策略。
Abstract
心血管疾病是全世界最常见的死亡原因，通常以心脏纤维化加剧为标志，这种纤维化会导致心室僵硬性随着心脏功能的改变而增加。心脏心室纤维化的增加是由于活化了常驻成纤维细胞，尽管这些细胞在三维（3-D）心脏内、基线或激活后如何运作，尚不清楚。为了检查成纤维细胞如何导致心脏病及其在三维心脏中的动力学，开发了一种精致的基于CLARITY的组织清除和成像方法，显示整个小鼠心脏内荧光标记的心脏成纤维细胞。组织常驻成纤维细胞是使用Roas26-loxP-eGFP荧光记者小鼠与表达Tcf21-MerCreMer敲门线的心脏成纤维细胞交叉的基因标记。该技术用于观察健康小鼠和心脏病纤维小鼠模型中整个成人左心室的成纤维细胞定位动力学。有趣的是，在一个损伤模型中，在受伤的小鼠心脏中观察到心脏成纤维细胞的独特模式，这些模式跟随包裹的纤维带向收缩方向。在缺血伤害模型中，成纤维细胞死亡发生，随后从梗塞边界区重新填充。总的来说，这种精炼的心脏组织澄清技术和数字成像系统允许心脏成纤维细胞的三维可视化，而不受抗体穿透失败或以前因组织处理而失去荧光的问题的限制。
Introduction
虽然心肌细胞是心脏中体积最大的部分，但心脏成纤维细胞更丰富，并且严重参与调节该器官的基线结构和修复特征。心脏成纤维细胞具有高度的移动性、机械反应能力和表型范围，具体取决于其激活的程度。心脏成纤维细胞是维持细胞外基质（ECM）正常水平所必需的，这些细胞产生太少或过多的ECM可能导致疾病1、2、3。鉴于心脏成纤维细胞在疾病中的重要性，在确定新的治疗策略方面，特别是试图限制过度纤维化4、5、6、7方面，已成为研究的日益重要的课题。受伤后，成纤维细胞激活并分化成一种更合成的细胞类型，称为肌纤维细胞，它可以是增殖和分泌丰富的 ECM，以及具有收缩活性，有助于改造心室。
虽然心脏成纤维细胞在二维文化6、8、9、10中对其特性进行了广泛的评估，但更不了解其在三维活心脏中的特性和动态，无论是基线还是疾病刺激。在这里，描述了一种精致的方法，组织清除成年小鼠的心脏，同时保持与Roas26-loxP-eGFP x Tcf21-MerCreMer遗传报告系统标记的成纤维细胞的荧光。在心脏内，Tcf21是静默成纤维细胞4的相对特定的标记。在给予塔莫西芬以激活不可受诱惑的MerCreMer蛋白后，基本上所有静默成纤维细胞都会永久表达来自Roas26轨迹的增强绿色荧光蛋白（eGFP），从而允许它们在体内跟踪。
有许多完善的组织清除协议存在，其中一些已经应用于心脏11，12，13，14，15，16，17。然而，许多用于不同组织清除协议的试剂被发现淬灭内源性荧光信号18。此外，成人心脏是很难清除，由于丰富的血红蛋白组含有蛋白质，产生自流19。因此，本协议的目标是保持成纤维细胞标记荧光与同时抑制血红细胞自身荧光在受伤的成人心脏，以最佳的三维可视化体内12，13，14，16，17，20。
先前的研究试图检查心脏成纤维细胞在体内使用香水抗体来标记这些细胞，虽然这样的研究受到抗体渗透和心脏血管结构14，16，17，20的限制。虽然萨拉蒙等人已经显示出组织清除与维持新生儿心脏局部神经元荧光，和Nehrhoff等人已显示维持荧光标记骨髓细胞，维持内源性荧光通过整个心室壁尚未证明，包括成人心脏成纤维细胞在基线或受伤后13，20的可视化。此组织清除协议根据 CLARITY 方法（透明脂质交换丙烯酰胺-杂交硬成像/免疫染色/原位混合兼容组织水凝胶）和 PEGASOS（聚乙二醇 （PEG） 相关溶剂系统）优化了先前协议的混合物。这一精炼的协议允许对处于基线的老鼠心脏中的心脏成纤维细胞以及它们对不同类型的损伤的反应进行更有力的检查。该协议是直接和可重复的，将有助于描述心脏成纤维细胞在体内的行为。
Protocol
所有涉及小鼠的实验都得到了辛辛那提儿童医院医疗中心机构动物护理和使用委员会（IACUC）的批准。该机构还通过了AALAC（美国实验室动物护理认证协会）认证。老鼠通过颈椎脱位被安乐死，接受生存外科手术的小鼠得到疼痛缓解（见下文）。用于疼痛管理和安乐死的所有方法都基于美国兽医医学协会安乐死小组的建议。所有的老鼠都被安置在玉米棒床上用品单元里，随时有水和食物。老鼠被安…
Representative Results
心脏成纤维细胞对于心脏的基线功能以及心脏损伤的反应至关重要。以前试图了解这些细胞的排列和形态，主要是在二维设置中进行的。然而，一个精致的心脏组织清除（图2）和三维成像技术已经发布，这使得心脏成纤维细胞的先进，更详细的可视化。通过这种成像技术，发现成纤维细胞密集，在未受伤的心脏中有一个旋转形态（图3，补充视频…
Discussion
本文提出了一种精炼的组织清除方法，允许在内在心脏成纤维细胞的可视化，无论是在基线和受伤后，以特征和更好地了解成纤维细胞在小鼠的心脏。这个增强的协议解决了现有的组织清除协议的限制，试图确定特定的细胞类型在成人或新生儿心脏12，13，14，16，17，20。…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢CCHMC康福克斯成像核心在开发该模型的帮助和指导，以及来自临床工程的马特·巴蒂对所有3D打印部件的设计。德米特里亚·菲切瑟得到了国家卫生研究院（NHLBI，T32 HL125204）的培训补助金的支持，杰弗瑞·莫尔肯廷得到了霍华德·休斯医学研究所的支持。
Materials
| 4-0 braided silk | Ethicon | K871H | |
| 8-0 prolene | Ethicon | 8730H | |
| 40% Acrylamide Solution | Bio-Rad | 1610140 | |
| Angiotensin II | Sigma | A9525-50G | |
| Artificial Tear Ointment | Covetrus | 048272 | |
| DABCO (1,4-diazabicyclo[2.2. 2]octane) | Millipore Sigma | D27802-25G | |
| GLUture topical tissue adhesive | World Precision Instruments | 503763 | |
| Heparin | Sigma | H0777 | |
| Imaris Start Analysis Software | Oxford Instruments | N/A | |
| Micro-osmotic pumps | Alzet | Model 1002 | |
| Nikon Elements Analysis Software | Nikon | N/A | |
| Nikon A1R HD upright microscope | Nikon | N/A | |
| Normal autoclaved chow | Labdiet | 5010 | |
| Nycodenz, 5- (N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N'-bis (2, 3 dihydroxypropyl) isophthalamide |
CosmoBio | AXS-1002424 | |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| Phenylephrine Hydrochloride | Sigma | P6126-10G | |
| Photoinitiator | Wako Chemicals | VA-044 | |
| Rosa26-nLacZ [FVB.Cg-Gt(ROSA)26Sortm1 (CAG-lacZ,-EGFP)Glh/J] | Jackson Laboratories | Jax Stock No:012429 | |
| Sodium Azide | Sigma Aldrich | S2002-5G | |
| Sodium Chloride solution | Hospira, Inc. | NDC 0409-4888-10 | |
| Tamoxifen | Sigma Aldrich | T5648 | |
| Tamoxifen food | Envigo | TD.130860 | |
| Tween-20 | Thermo Fisher Scientific | BP337-500 | |
| Quadrol, N,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine, decolorizing agent | Millipore Sigma | 122262-1L | |
| X-Clarity electrophoretic clearing chamber | Logos Biosystems | C30001 | |
| X-Clarity electrophoretic clearing solution | Logos Biosystems | C13001 | |
| X-Clarity electrophoresis tissue basket | Logos Biosystems | C12001 | |
| X-Clarity electrophoresis tissue basket holder | Logos Biosystems | C12002 |
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Citazione di questo articolo
Fischesser, D. M., Meyer, E. C., Sargent, M., Molkentin, J. D. Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts. J. Vis. Exp. (171), e62023, doi:10.3791/62023 (2021).