Background

The final goal of regenerative medicine is to reconstitute tissue and organ functionality after injury or disease (Mao and Mooney, 2015). Tissue regeneration is a physiological process orchestrated by different infiltrating and tissue-resident cell types. Skeletal muscle is one of the most dynamic and plastic tissues of the human body and represents approximately 40% of total body weight in humans (Frontera and Ochala, 2015). Skeletal muscle not only controls posture maintenance and locomotion but plays a fundamental role in the maintenance of metabolic homeostasis, being involved in heat production and carbohydrate and amino acid storage (Schiaffino et al., 2013). In this context, muscle degeneration due to acute and chronic conditions leads to reduced mobility and strength, and to metabolic disorders (Schiaffino et al., 2013). Regeneration of skeletal muscle relies on a tightly regulated gene expression program and on the activation of specific signaling pathways that characterize muscle embryonic development (Chargé and Rudnicki, 2004). The ability of muscle to regenerate is principally due to a specific population of normally quiescent muscle stem cells called satellite cells that are strictly associated with muscle fibers (Chargé and Rudnicki, 2004). Satellite cells are myogenic precursor cells (MPCs) that reside in a quiescent state beneath the basal lamina that surrounds muscle fibers (Chargé and Rudnicki, 2004). In detail, soon after muscle damage, satellite cells escape quiescence and start to proliferate. A subset of these activated cells proliferate and differentiate, whereas others return to quiescence to reconstitute the pool of satellite cells that will be crucial for further rounds of skeletal muscle regeneration (Chargé and Rudnicki, 2004).

Although satellite cells play a key role in muscle regeneration, their presence is not sufficient for efficient skeletal muscle repair, and many additional cell types play an active role in promoting tissue repair (Joe et al., 2010; Uezumi et al., 2010). Among them, key players in the complex scenario of skeletal muscle regeneration are inflammatory cells that invade muscle soon after injury and, together with satellite cells, contribute to a complete muscle regeneration (Tidball, 2017). Among the inflammatory cells, monocytes/macrophages play a major role in the repair process (Wang and Zhou, 2022). For example, macrophages release cytokines and growth factors that contribute to satellite cell activation, proliferation, and differentiation, according to their polarization state (Saclier et al., 2013). In particular, soon after muscle damage, macrophages adopt a pro-inflammatory profile, releasing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukine-6 (IL-6) and insulin-like growth factor (IGF-1) that, in turn, promote satellite cell proliferation (Tidball, 2017). Starting from three to five days post injury, these macrophages skew from a pro- to an anti-inflammatory profile, releasing anti-inflammatory cytokines that not only dampen the inflammatory response but also play trophic function by promoting satellite cell differentiation and fusion, thus contributing to the formation of new myofibers (Tidball, 2017). To date, different mouse models of acute muscle injury are available, as they represent an attractive system for investigating interactions between the immune system and satellite cells during skeletal muscle regeneration. Indeed, the onset of tissue damage is well defined and the time course of inflammation and regeneration predictable (Tidball, 2017). The most used injury models are freeze injury (FI), barium chloride injection, notexin (NTX) injection, and cardiotoxin (CTX) injection. The last one provides a useful model for sterile inflammation and, importantly, induces homogeneous damage to the whole muscle. Furthermore, it triggers the infiltration of many monocytes and macrophages participating in muscle repair, regeneration, and growth (Rigamonti et al., 2014). Here, we describe a protocol to induce muscle sterile injury by CTX injection. This method allowed us to study the influence of mitochondrial calcium uptake on macrophage metabolic profiles and on the skeletal muscle regeneration process (Feno et al., 2021).

Myonecrosis is induced by intramuscular injection of CTX in tibialis anterior (TA) muscles, as performed in many other laboratories (Mounier et al., 2013; Perdiguero et al., 2011; Saclier et al., 2013). Compared with other protocols, our method has the great advantage of avoiding muscle laceration (Feno et al., 2021). Specifically, we can directly inject CTX in TA muscles without exposing them, thus avoiding surgery and consequent damage. Muscles are collected for analysis at different time points after injury (at 3, 7, and 14 days) to precisely characterize the time course of skeletal muscle regeneration. After muscle collection, regenerating TA muscles are frozen, and muscle sections are characterized by hematoxylin and eosin (H&E) staining to evaluate the efficiency of the CTX- induced skeletal muscle regeneration process.

Materials and Reagents

1. 8 weeks C57BL6N male mice (Charles River)

2. Isoflurane 1000 mg/g (Piramal Critical Care, catalog number: 803249)

3. 500 μM CTX (Merk, catalog number: 217503-1MG)

4. 9% NaCl₂ (Merk, catalog number: S3014)

5. UltraPure DNase/RNase-Free distilled water (Thermo Fisher Scientific, catalog number: 10977049)

6. 70% v/v ethanol (Merk, catalog number: 64-17-5)

7. Isopentane (2-Methylbutane anhydrous ≥99%-1l) (Merck, catalog number: 277258)

8. Liquid nitrogen

9. Hematoxylin & Eosin (H&E) kit staining (Bio-Optica, catalog number: 04-061010)

10. Optimal cutting temperature compound (O.C.T.) (Tissue-Tek, catalog number: 4583)

Equipment

1. VetFlo veterinary anesthesia apparatus

2. Dissection tools:

   Fine scissors (Fine Science Tools, catalog number: 14160-10)

   Fine forceps (Fine Science Tools, catalog number: 11412-11)

   Standard forceps (Fine Science Tools, catalog number: 11150-10)

3. Microcentrifuge tubes 1.5 mL (Sarstedt, catalog number: 72706)

4. Cryovials (Sarstedt, catalog number: 72.694.006)

5. Glass slides (Vetrotecnica, catalog number: 01.4230.34)

6. 500 μL syringe 30 G (Vetrotecnica, catalog number: 11.3525.45)

7. Plastic scoop

8. Dewar bottle for liquid nitrogen

9. Cryostat (Leica, model: CM1850)

Procedure