Comparative Study of Injury Models for Studying Muscle Regeneration in Mice.
Comparative Study of Injury Models for Studying Muscle Regeneration in Mice.
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DOI：
10.1371/journal.pone.0147198
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Chrétien F
中科院分区：
文献类型：
--
作者：
Hardy D;Besnard A;Latil M;Jouvion G;Briand D;Thépenier C;Pascal Q;Guguin A;Gayraud-Morel B;Cavaillon JM;Tajbakhsh S;Rocheteau P;Chrétien F
再生医学的一个长期目标是在损伤或疾病后重建功能组织或器官。注意力集中在组织特异性干细胞的鉴定和相对贡献的再生过程。关于生理过程是如何被其他组织成分调节的，我们知之甚少。许多损伤模型被用于研究组织再生，然而，这些模型往往知之甚少。具体而言，对于骨骼肌再生，文献中报道了几种模型，但对肌肉生理学和不同细胞类型的相对影响尚未得到广泛表征。我们使用转基因Tg：Pax 7 nGFP和Flk 1GFP/+小鼠模型，通过共聚焦显微镜分别计数肌肉干（卫星）细胞（SC）的数量和血管的数量/形状。我们进行了组织学和免疫染色，以评估关键再生步骤的差异。通过Luminex®在体内评估免疫细胞的浸润、趋化因子和细胞因子产生。比较了冷冻损伤（FI）、氯化钡（BaCl 2）、Notexin（NTX）和心脏毒素（CTX）4种最常用的损伤模型。FI是最具破坏性的。在该模型中，高达96%的SC与其周围环境（基底层和脉管系统）一起被破坏，留下缺乏活细胞的“死区”。再生过程本身在所有4种模型中都得到了实现，除了FI模型外，损伤后28天几乎没有纤维化。炎性细胞在CTX、BaCl 2中恢复到基础水平，但在FI和NTX模型中在损伤后1个月仍然显著高。有趣的是，SC的数量仅在损伤后1个月的FI中恢复正常，而在其他模型中，SC在诱导损伤后3个月仍在循环。我们的研究表明，损伤模型的性质应根据实验设计和预期结果仔细选择。虽然在所有模型中，肌肉都能完全再生，但再生过程的轨迹差异很大。此外，我们发现，组织学参数并不完全足以宣布再生是完全的，因为分子改变（例如循环SC，细胞因子）可能具有重大的持续影响。
A longstanding goal in regenerative medicine is to reconstitute functional tissus or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®. We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a “dead zone” devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models. Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.