Background

Muscle injuries are a common type of injury that can result from various causes, from exertion to blast trauma. These injuries lead to a complex cascade of inflammatory and regenerative responses that result in the repair of the injured tissues. To characterize muscle injuries and the following responses and to evaluate the efficacy of potential treatments, various experimental models have been developed to replicate the process of muscle injury. In vitro approaches offer some practical advantages, and the advancement in 3D culture technique has enabled bioengineering of human cell-derived muscle tissue surrogates that may be used in muscle injury studies (Mills et al., 2017). In vivo models, however, remain imperative to understanding muscular injuries, as the complex processes involving the local and systemic responses that govern the overall regenerative process and the accompanying functional impairments/recovery can only be observed in a whole system of organisms. Rodent models of muscle injury are well-established and the most used systems for studying muscle regeneration today.

There are various modalities of injuries and their models, including laceration, crushing, blast injuries, volumetric muscle loss, myotoxins, and freezing. While all the modalities of injury go through common phases of healing (Huard et al., 2002; Jarvinen et al., 2005), some heterogeneity exists in the extent and quality of repair that follows regarding susceptibility/responsiveness of muscle stem cells to injury, tissue remodeling, inflammatory response, re-vascularization, and re-innervation (Caldwell et al., 1990; Lefaucheur and Sebille, 1995). Cryoinjuries, or traumatic injuries due to freezing, create localized injuries accompanied by substantial inflammatory and vascular responses (Warren et al., 2007), the extent of which can be adjusted and standardized according to the duration of the contact with the freezing instrument (Oh et al., 2016; Sinha et al., 2017; Endo et al., 2020). This makes it an easy and reliable method for generating reproducible outcomes, in contrast with the use of various myotoxins that lack comparative information needed for standardization. A step of skin incision is, however, required for the cryoinjury model, which could be considered a disadvantage over the use of injectable myotoxins. The cryoinjury model is applicable to a wide range of muscle injuries and is generally an appropriate choice for studying injuries without a substantial loss of muscle volume. In particular, cryoinjuries can simulate non-traumatic and mild traumatic injuries in striated muscles, including crushing injuries in skeletal muscle and myocardial infarction in cardiac muscles. Regenerative responses following cryoinjury can be identified histologically with clearly demarcated borders between injured and uninjured areas. Here, we describe a method of generating cryoinjuries in mouse skeletal muscle using dry ice and quantifying the regenerative response in myofibers.