**3. The Dynamic and the Regulation of Regenerative Phases are Altered in Pathologic Conditions: The Case of Muscular Dystrophy**

The physiologic sequence of reparative phases, upon muscle injury, generally leads to the complete rescue of tissue morpho-functional properties. Unfortunately, the endogenous regenerative potential of skeletal muscle is not always sufficient to guarantee tissue restoration and/or maintenance. Pathologic conditions, including muscular dystrophies, are known to raise alterations in the dynamic and efficiency of regenerative steps. For this reason, complementary to physiologic regeneration, valuable information to clarify regenerative mechanisms can derive from models in which tissue healing is compromised because of cell-intrinsic and extrinsic defects. A well-characterized model of muscle wasting and regenerative impairment is the dystrophic mdx mouse, a classical model of Duchenne Muscular Dystrophy (DMD) pathology [118]. DMD is a degenerative disease in which the absence of the dystrophin protein leads to sarcolemma instability and fragility. The genetic defect is associated with the extensive damage of myofibers upon contraction which cannot be rescued by newly regenerated myotubes, being itself dystrophin deficient. This means that the degenerative stage of muscle healing, which is generally restricted to the first day after injury in wild-type mouse models, persists in dystrophic muscles throughout the necrotic stage of the pathology (mainly from 3 to 6 weeks of age) [119]. In this stage, EBD-injected mdx mice show high muscle permeability and dye uptake within damaged myofibers [11,120,121]. This is also associated with a sensible increase of serum CK and circulating myomiRs, further confirming the intense muscle damage in dystrophic muscle [11,120,122–128].

The continuous degeneration of dystrophin-deficient fibers represents a persistent stimulus to both inflammation and regeneration, thus inducing the alteration of the dynamic of both the inflammatory and regenerative stages. Indeed, it has been extensively described that dystrophic muscles are chronically dominated by inflammation, which can induce muscle fiber death through NO-mediated and perforin-dependent/independent mechanisms, respectively [68,119,129,130]. In accordance, Wehling and colleagues observed an improved membrane integrity upon antibody depletion of macrophages [131]. On the other hand, it has been recently reported that the local and transient depletion of macrophages in dystrophic muscle affected the balance between SC proliferation and differentiation, associated with defects in the formation of mature myofibers, inducing an exacerbation of the dystrophic phenotype [132]. Conflicting results can be associated with the technical approaches used in the studies, with reference to the persistence of the depletion and the stage of pathology in which the intervention acted. Indeed, Wehling and colleagues treated mdx mice with an anti-F4/80 antibody beginning at 1 week of age and continuing to 4 weeks of age, whereas Madaro and coworkers acted during the regenerative stage of the disease, that peaks between 9 to 12 weeks of age in mdx mice [131–133]. These observations, conflicting at first glance, provided intriguing insights about the complex impact of inflammatory events on the different stages of muscle regeneration. Thus, a better

understanding of the inflammatory process in the dystrophic muscle and of the mediators involved might open novel therapeutic perspectives.

Inflammatory cells are responsible for the secretion not only of trophic factors but also of elevated levels of inflammatory mediators, influencing SC behavior. Among them, the enhanced expression of IL-6 is thought be involved in the alteration of the muscle stem cell pool by promoting the proliferation of SCs and the impairment of myoblast differentiation [11,134,135]. Interestingly, blockade of IL6 activity, using a neutralizing antibody against the IL6 receptor, conferred robustness to dystrophic muscle, impeded the activation of a chronic inflammatory response, significantly reduced necrosis, and activated the circuitry of muscle differentiation and maturation. This resulted in a functional homeostatic maintenance of dystrophic muscle. [121,136].

It is also worth to report that, in addition to maladaptive environmental signals, the altered SC behavior can be intrinsically dictated by the absence of dystrophin protein, since a defective compartmentalization of factors during asymmetric division in dystrophin deficient SCs can alter the daughter cell fate [40].

In addition, the delicate interaction between FAPs and SCs is altered in dystrophic muscles. FAPs desist from their supportive role and turn into fibro-adipocytes, which mediate fat deposition and fibrosis, contributing to the exacerbation of the dystrophic hostile microenvironment [44].

A recent study also uncovers the Wingless-related integration site (WNT)/GSK3/β-catenin axis as a new and previously unexplored pathway contributing to control FAP adipogenesis and muscle fatty degeneration, thus contributing to develop strategy to counteract intramuscular fat infiltrations in myopathies [137].

The persistence of degeneration, chronic inflammation, and defective myogenesis contribute to the alteration of the final phases of muscle healing in dystrophic muscles, resulting in the continuous attempt of defective regeneration. Altogether, these alterations lead to the progressive exhaustion of the SC pool, to accumulation of fatty/fibrotic tissue, and thus to the loss of muscle mass and functionality.

#### **4. Technical Approaches to Induce Experimental Muscle Damage and Regeneration**

The adaptative response of skeletal muscle to damage can differ in relation with the type of insult, and this is exactly coherent with the elevated plasticity of skeletal muscle tissue. Indeed, although the phases of muscle regeneration are closely interrelated and their time-dependent sequence is highly conserved in different vertebrates, the kinetic and amplitude of these procedural steps can vary depending on the organism or the extent/quality of damaging events. Indeed, a traumatic event can lead to the lesion of a single myofiber or of a localized segment of a fascicle. Furthermore, an insult can induce the degeneration of an entire fiber or can pertain to a number of myofibers dispersed throughout uninjured tissue [138,139]. Furthermore, an increasing body of evidence suggests that the events starting early after the injury profoundly influence the dynamic of tissue regeneration, affecting at various degree the different muscle components [140]. These heterogeneous events can contribute to the occurrence of doubts and difficulties in the field of clinical and experimental pathology [138]. Thus, a comprehensive understanding of the mechanisms underlining skeletal muscle adaptation to different insults can contribute to extending the current knowledge about muscle physiology and can allow the development of specific pro-regenerative therapies. To this aim, animal models of muscle injury represent a valuable and powerful tool to monitor and study muscle response to damage.

#### *4.1. Models of Physical Injury*

Murine models of acute muscle injury have been extensively studied to investigate molecular mechanisms underlining regenerative events characterizing each phase of muscle regeneration. Since the induction of an injurious assault is a prerequisite for muscle regeneration, with the necrotic phase being the first step of muscle restoration, the choice of a proper model of injury is critical for a correct interpretation of data and for the dissection of molecular mechanisms taking place in damaged muscles. A wealth of experimental procedures, adopted to induce muscle damage, have been

developed and described over time, and qualitative/quantitative differences in the tissue response have been reported. Among them, the most commonly used methods are physical and chemical procedures. Most of the protocols of physical injury, which include freeze injury and crush models, are highly invasive and present technical complexities.

#### 4.1.1. Freeze Injury

The freeze injury (FI) method mainly consists of a skin incision to expose the target muscle, and a single or repetitive action of freeze-thawing by applying for a prefixed time (10–15 s) a liquid nitrogen or dry ice cooled metallic rod [140–142]. Using this protocol, the operator can induce a diffuse necrosis in the treated muscle but the extent of muscle damage can vary not only depending on the number of freezing cycles but also with the pressure applied to the tissue with the cooled probe. The operator-dependent variability can limit the reproducibility of the data and the homogeneous use of the protocol in different laboratories. However, Hardy and colleagues, in a comparative study of muscle damage and repair, highlighted how the freeze-injury method induced a severe necrosis at the site of the lesion, destroying muscle cell components such as myofibers and satellite cells, along with basal lamina and vascular bed [140]. This can allow the study of the dynamics of infiltrating cells. Indeed, the region of damage, the so-called "dead zone", is well marked by the absence of viable cells, and the activation of regenerative events particularly requires the migration inside the lesion not only of inflammatory cells and non-myogenic supportive cells but also of myogenic progenitors [142]. Thus, viable cells infiltrating the lesion are easily identifiable since they appeared as directionally displaced from the spared tissue into the dead zone, allowing the dissection of regenerative events. In particular, the first invading population 18 h after FI is comprised of neutrophils and their presence is accompanied by an early increase of monocyte chemoattractant protein 1 (MCP-1) IL-6. The peak of expression of MCP-1 and IL-6 forewarns the second wave of cellular infiltration, characterized by F4/80pos. macrophages and myogenic cells, since both macrophages and muscle precursor cells express the CCR2 and are thus responsive to MCP-1 [141]. IL-6 is a pro-inflammatory cytokine with important regulatory actions on muscle stem cell functions; thus, the heightened expression of this pleiotropic factor can participate in both inflammatory and regenerative processes. This is consistent with the observation of a regenerative front of myoblasts at the periphery of the death zone at a few days after the neutrophilic peak since neutrophils are also a pivotal source of the soluble isoform of the IL-6 receptor alpha (IL6R) necessary to amplify IL-6 signal transduction [1,120,140,143]. Of note, the levels and activity of IL-6 must be finely tuned, since circulating levels of the proinflammatory cytokine IL-6 can also perturb the physiologic redox balance in skeletal muscle and can contribute to exacerbating muscle disease [143,144].

It is worth to report that the extent of tissue damage in FI model is highly elevated, and this event can profoundly affect the behavior of satellite cells from early after trauma. Indeed, it has been reported that there is a dramatic delay of satellite cells early after freeze injury (18 h), which has been quantified as about 90% of devoid cells compared to uninjured muscles. The regeneration of the damaged site is largely accomplished by progenitors deriving from outside the lesion; thus, the proliferation of SCs occurs days after damage and cycling SCs have been reported until one month after FI [140–142]. Although the histological retrieval of muscle architecture appeared complete one month after damage, the total number of SCs in FI mice returned to control levels three months after freezing.

The features of skeletal muscle response to FI were useful to study cellular actors of muscle regeneration and to clarify the involvement of the different cell populations. For instance, in 1986, Shultz and colleagues used the FI technique on the entire extensor digitorum longus (EDL) muscle to verify whether myogenic cells could migrate from adjacent muscles or could be delivered through the bloodstream [145]. Their results highlighted how muscle regeneration mainly depends on the activity of the local population of satellite cells [145]. Although extrinsic myogenic cells, such as migrating myoblasts and CD133pos. mononucleated cells identified in adult peripheral blood, can potentially reach the lesion, they would not be sufficient to regenerate the entire muscle [145,146].

#### 4.1.2. Crush Injury and Ischemia-Reperfusion Damage

A muscle-crush injury occurs when high pressure is applied to skeletal muscle, which undergoes blood flow interruption, inducing the damage of myofibers. The combination of mechanical force and ischemia is known to cause an acute rhabdomyonecrosis since the profound alteration of the pressure balance can impair the volume regulation of myocytes, along with their permeability, leading to cell swelling [147].

Several experimental procedures have been developed over time to induce muscle-crush injury in rodents as a model of common trauma in humans and to study acute muscle inflammation and regeneration [148–153]. Among them, one of the most used is the opened model in which the muscle of the animal, generally the pelvic limb muscle, is surgically exposed and a force is applied by using a clamp.

Considering the invasiveness of the methodology and the needs of technical skills to perform the experiments guaranteeing the reproducibility of the data, closed noninvasive protocols have been tested. However, most of them involved dropping of weights upon the interested muscle region and such procedures can result in unwanted bone fractures. The fine-tuning of the procedure is still ongoing in order to minimize additional tissue damage induced by surgical interventions in opened models and to reduce the incidence of fractures due to dropping weights in closed models. For instance, Dobek and colleagues [148] proposed a sustained-force model of lower-extremity crush injury able to induce an acute inflammatory response, thereby reducing the extent of bone fractures. The proposed method, which has been described as a refinement of previous models, involved the use of a crush injury device platform. An air compressor activated a piston, situated in direct contact with the area to be injured, providing a contained force to the selected muscle. They reported that, although the force imposed was smaller than that applied in other studies (about 30 N in comparison with about 250 N), it was sufficient to induce muscle damage and the expected acute inflammatory response. Accordingly, it has been reported that a violent crush can destroy muscle tissue; however, even when the force is insufficient to directly wreck myofibers, the combination of mechanical force and ischemia will rapidly induce tissue degeneration [147]. Indeed, as a physiologic response to tissue injury, it has been reported that neutrophils, identified as 1A8, 7/4, and granulocyte antigen 1 (Gr1)-positive cells, rapidly invade the crushed muscle at the site of the lesion and then decreased from 24 to 48 h after injury [148]. CD68pos. and F4/80pos. macrophages followed the neutrophilic invasion increasing from 24 to 48 h after injury. However, it is plausible that the controlled force applied to the muscle would induce a mild tissue degeneration, useful to study the kinetic of inflammatory cell infiltration but probably with limitations regarding the study of regenerative events under critical conditions.

On the other hand, Criswell and colleagues [154] proposed and described a procedure of muscle crush in rats, which was able to induce tissue degeneration and to mimic the compartment syndrome (CS), a severe consequence of intense crush injuries frequently occurring in humans. Of note, the compartment syndrome occurs when the pressure within muscle fascicles dramatically increase due to posttraumatic ischemic swelling. This results in both ischemic and reperfusion insults, destroying the vasculature and the neural network and inducing the extensive necrosis of muscle tissue. In this protocol, a controlled compression of the rat hindlimb, proximal to the EDL muscle, has been cleverly obtained by using a neonatal blood pressure cuff in order to constantly maintain the pressure in a range of 120–140 mmHg for3h[154]. The persistent compression resulted in a composite injury of the muscular, vascular, and neural compartments. Tissue edema and disorganization were early observed 24 h after injury, and within the first 4 days, 50% of the muscle fibres underwent degeneration. Immune cell infiltration, as described in other models, occurred within 2 days after damage and persisted throughout the first week, with a peak at day four [154–156]. Following the acute inflammatory response, fibroblast and myofibroblast growth resulted in enhanced collagen deposition, also supporting the formation of newly regenerating fibers. Indeed, although early markers of satellite cell activation such as Pax7 and MyoD were observed early after damage (2 days), regenerating myofibers were detected 7 days after injury in correspondence with the phase of collagen deposition

(Figure 1) [154]. The extensive damage induced by muscle compression was also highlighted by signs of denervation, such as the dispersed localization of acetylcholine receptors around crushed myofibers and of vasculature alterations, including neo-angiogenesis preceded by the presence of enlarged vessels and hemorrhagic areas [154,156,157].

#### *4.2. Chemical Damage Induced by Myotoxic Agents*

The injection of myotoxic agents, such as the snake venom-derived toxins notexin or cardiotoxin, is one of the most frequently used methods to experimentally induce muscle damage and to study the subsequent regeneration. This is because the degeneration induced by these agents has been described as rapid, vigorous, and reproducible [139,158]. Moreover, venom toxins have been recognised as quite specific toxic agents on muscle fibers, without undermining blood vessels, basal lamina, and thus the activity of satellite cells [159–164]. This quite specific action can allow the dissection of regenerative events in a simplified model of muscle injury and study of the behavior of cellular actors and the profile of molecular players in muscle regeneration.

#### 4.2.1. Cardiotoxin Injection

Cardiotoxins (CTX) are small polypeptides made of 60–63 amino acid residues acting as protein kinase C-specific inhibitor. Over 40 homologous cardiotoxins have been isolated and sequenced over time [165]. The purified toxin derived from the venom of the Indian cobra snake *Naja naja* or from the *Naja mossambica mossambica* is the most widely used myotoxic agent in protocols of experimental injury [140]. Although protocols can vary among laboratories, the method mainly consists of an intramuscular injection of about 20–50 μL of a 10 μM CTX working solution in sterile phosphate buffered saline (PBS). The most frequently treated muscles in murine models are hindlimb muscles such as the tibialis anterior muscle (TA) or gastrocnemius [140,166,167]. The myolytic activity of CTX involves the alteration of ion fluxes, induced by membrane depolarization, and is accompanied by the loss of protein content and organelle breakdown. Muscle degeneration occurs early after the injection, and the injured tissue is rapidly invaded by inflammatory mononucleated cells (Figure 2). Enlarged necrotic fibers in CTX-treated muscles are reached firstly by neutrophils and, later on, by macrophages which have been also described as penetrating swollen fibers [168]. Hardy and colleagues, in a benchmark work on different models of muscle injury, reported that the inflammatory response stimulated by CTX-induced necrosis was not exuberant, and it has been described that, although the kinetic of infiltrating cells is maintained, the phases are more defined and staggered if compared to other models of injury [140]. Furthermore, pro- and anti-inflammatory mediators, except for IL-6 levels showing a significant heightening, undergo a weak induction at early stages to then return to basal levels. The persistence of elevated levels of IL-6 have been detected one month after damage, possibly explaining the highly increased number of satellite cells in completely regenerated CTX-injected muscles [140]. It is worth to report that a significant myofiber hypertrophy and an increased muscle weight has been observed in muscle regenerated after CTX injection [167,169]. On the other hand, it has been postulated that CTX itself may have chemotactic properties enhancing both macrophages and satellite cell activity, thus inducing an early and efficient tissue regeneration [139,167]. Accordingly, newly regenerating myotubes with central nuclei can be observed 4 days after damage; 7 days after injury, the inflammatory response declines and the diameter of centrally nucleated fibers considerably increase [168]. Although it has been extensively described that CTX toxic action does not directly influence microvasculature, a complete destruction of the capillary network has been reported in a 3D study on CTX-injected muscles derived from Flk1GFP/+ mice [140]. However, the initial vasculature breakdown was followed by active angiogenesis, and 1 month after the injection, the vessel network was restored [140]. The retrieval of a proper blood supply in injured muscle contributes to the efficient regeneration, without the occurrence of tissue fibrosis [164].

The ability of CTX to induce myofiber degeneration sparing the integrity of both basal lamina and satellite cells and thus inducing a controlled and rapid process of tissue reconstruction has been extensively used over time to study the role of molecular and cellular interactors in muscle regeneration. For instance, the specific action of cardiotoxin in inducing myofiber necrosis but not satellite cells (SCs) death in combination with a model of local Pax7pos. cell depletion contributed to clarifying the role of Pax7pos. cells in adult myogenesis [170]. Sambasivan and colleagues, inducing muscle damage in the presence or absence of satellite cells and monitoring the subsequent regenerative events, not only reported evidence about the essential role of SCs in skeletal muscle regeneration but also suggested the intriguing possibility that a threshold number of Pax7pos. cells would be required to obtain an efficient tissue reconstruction. This action would be associated not only with the proliferative rate of activated SCs but also with the potential ability of satellite cell to orchestrate the pro-regenerative action of non-myogenic cells in damaged muscle tissue. Furthermore, the cardiotoxin method was largely employed to dissect the action of inflammatory mediators in the stem cell niche after injury and to study the behaviour of satellite cell under pathologic conditions in which regeneration is known to be impaired [99,166,170–173].

#### 4.2.2. Notexin Injection

Notexin (NTX) is a myotoxic agent contained in the venom of the *Notechis scutatus*, the Australian tiger snake, and has been described as having a more toxic effect than cardiotoxin (four times more toxic than CTX) [174]. This phospholipase A2 presents an elevated myolytic impact, through the hydrolyzation of sarcolemma lipids, inducing the alteration of ionic fluxes, hypercontraction, and thus the degeneration of myofibers. Tissue insult provoked by notexin has been described as highly degenerative, causing myofiber breakdown and the loss of skeletal muscle functionality three days after the injection [175]. Moreover, a comparative study performed by Plant et al. reported that the maximum force of contraction recovered by notexin-injected muscles was reduced in comparison with other experimental models of muscle injury, with a percentage of force retrieval of only 10% seven days after damage and 39% after 10 days [175]. These data, along with the results from others benchmark studies, suggested that the extent and the modality of damage can influence the entity and the velocity of muscle recovery and thus the study of regenerative events [140,175–177]. For instance, myofiber regeneration, highlighted by the presence of centrally positioned nuclei, has been observed in the entire muscle injected with the toxin only 7 days after injury in contrast with the earlier observation reported in other models such as CTX-injury [140,167]. Despite the recognised action of notexin in inducing a generalized muscle damage, it is worth to report that fibers have a differential sensitivity to phospholipase A depending on the metabolism, with oxidative fibers showing an elevated susceptibility to NTX-induced damage [139].

Another important difference in the muscle response to notexin injection is the inflammatory response. Indeed, it has been described that there is a granulomatous inflammatory reaction to NTX-induced degeneration. Although the extensive necrosis produced by the myotoxic action of notexin did not activate an immediate inflammatory cell invasion, the immune response occurs with cell infiltration 4 days after damage. Instead of a typical kinetic of acute inflammatory response directed to the resolution, 12 days after injury in notexin-injected muscle, it was possible to observe multifocal calcium deposits, remains of necrotic myofibers, as a midpoint of a granulomatous reaction [140].

Interestingly, these chronically inflamed foci persist even when muscle tissue is quite completely regenerated 3–6 months after the experimental injury, potentially contributing to the establishment of an altered immune milieu. Furthermore, it has been reported that NTX can exert a neurotoxic action by blocking acetylcholine release, thereby altering the neuromuscular junctions, which must be restored for a functional tissue retrieval [178].

#### 4.2.3. Bupivacaine Administration

Another agent used to induce reversible muscle damage with the purpose to study regenerative events is Bupivacaine (BPVC). Bupivacaine is a local anesthetic that, thanks to its highly lipophilic properties, can efficiently penetrate the sarcolemma. Although the precise mechanisms have to be fully clarified, it has been described that Bupivacaine can induce muscle degeneration by perturbing the homeostasis of mitochondria and sarcoplasmic reticulum (SR), producing a dramatic calcium efflux and a simultaneous block of calcium reuptake by the SR. This action results in the hypercontraction and rapid death of myofibers along with the mitochondria membrane depolarization and sarcoplasmic reticulum alterations, which can further induce muscle degeneration [116,179,180]. Despite the intense impact recognized on rat skeletal muscle which has been reported, Bupivacaine can induce only a faint degeneration when injected in murine muscles. Indeed, the degenerative potential of this anesthetic has been described as limited, if compared to notexin or cardiotoxin, since its injection causes the degeneration only of a 45 percentage of fibers [175]. In accordance with the low degree of muscle degeneration induced by Bupivacaine, it has been reported that the force-generating capability of injected muscle was reduced to 42% of control muscles three days after damage and that this impairment was quite completely restored ten days after the injection [175]. However, the analysis of injected muscle cross sections performed by Plant and colleagues revealed that bupivacaine can spread throughout the muscle, equally affecting both inner and peripheral regions of the muscle. In accordance with the low degree of muscle damage induced by BPVC in murine muscle, a rapid inflammatory response and regenerative phase has been described. Three days after the injection of 50–100 μL of 0.5% BPVC, a robust inflammatory infiltrate can be observed surrounding necrotic fibers, whereas small regenerating fibers appearing on day 5 seem to quite completely regenerate the lesion by day 14, when inflammatory cells are significantly reduced [181].

#### **5. Conclusions**

Muscle regeneration is one of the most important homeostatic processes of adult tissue and, as such, must be finely regulated to guarantee functional recovery and to avoid muscle alteration and diseases [182]. Skeletal muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and/or restore a proper muscle structure and function. Although the main actors of the entire process are satellite cells, a heterogenous group of other cells cooperate to reestablish muscle homeostasis after damage. Indeed, each stage of the stepwise muscle healing is dominated by a peculiar combination of cell agents and molecular signals playing a specific role in the complex framework of regeneration. However, the multifaceted nature of the regenerative process has to be still completely unveiled, and a number of pathologic conditions impairing muscle regeneration still lack an effective therapy. Thus, the comprehensive understanding of healing mechanisms still deserves further research to identify novel reliable biomarkers and to develop advanced techniques supporting the future innovation of regenerative studies.

**Author Contributions:** A.M. conceptualized the study; A.M. and L.F. wrote the original draft; L.F. and M.C. wrote/reviewed and edited the text and figures. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by ASI, Ricerca Finalizzata (RF-2016-02364503), Ateneo-Sapienza.

**Acknowledgments:** We apologize to colleagues whose studies were not cited. The authors are thankful to Carmine Nicoletti and Carmen Miano for technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



