**3. Skeletal muscle injury models**

In order to study the process of muscle regeneration in a controlled and reproducible way, it was necessary to develop experimental models of muscle injury [1]. In this sense, a variety of experimental models that compromise skeletal muscle function or destroy this tissue is available. Each of the injury models can potentially have a different effect on the fate of resi‐ dent cells and circulating cells within the muscle bed after the trauma [10]. A large number of studies, involving a variety of experimental injuries, such as injection of myotoxic agents, crush, ischemia, denervation and muscular dystrophies, demonstrate the unique ability of skeletal muscle for regeneration, irrespectively the precise method used to induce the initial injury [15]. In this review we will focus on chemical and mechanical models of skeletal mus‐ cle injury, adding a new model of muscle injury based on surgical myectomy, developed in order to mimic severe losses of skeletal muscle mass. Other models, like exercise and dener‐ vation, will also be outlined. The latter is not a model of injury but else of skeletal muscle disuse but that also can be used to investigate skeletal muscle remodeling. There is a variety of other genetic models that are essential in studying diseases like Duchenne muscle dystro‐ phy (Mdx mouse is currently the most widely used in this case) but will not be discussed in this review.

#### **3.1. Chemical methods of skeletal muscle injury**

The use of myotoxins, such as bupivacaine (Marcaine), cardiotoxin (CTX), and notexin (NTX) is perhaps the easiest and most reproducible way to induce muscle injury and regen‐ eration. Myotoxins are also widely used to induce skeletal muscle injury because their inoc‐ ulation by intramuscular injection does not require complex surgery. Several chemical agents are known to produce skeletal muscle damage. Severe muscle fiber damage, like breakdown of sarcolemma and myofibrils, has been described after intramuscular injections of 0.75% bupivacaine, 2% mepivacaine, or 2% lidocaine associated to epinephrine [18]. While lidocaine can cause rapid destruction of skeletal muscle fibers, long-acting anesthet‐ ics, like bupivacaine, are more often used to cause skeletal muscle injury in rodents [19].

#### *3.1.1. Bupivacaine*

The bupivacaine injection procedure is simple and quick, does not involve extensive sur‐ gery, and induces a regeneration process which is qualitatively similar to that observed in other model systems. Doses of 1.5 and 1% wt/vol produce significant levels of muscle injury and subsequent regeneration, but these doses also produce large regions of ischemic muscle tissue. Doses of 0.75 and 0.5% bupivacaine are also effective in inducing regeneration and produce little or no ischemia [16]. Muscle fiber necrosis is extremely rapid after induced bu‐ pivacaine injury [14]. Injection of the drug into small skeletal muscles of rat or mouse leads to immediate and massive myonecrosis followed by phagocytosis of necrotic debris and a rapid and apparently complete regeneration of muscle fibers 3-4 wk after injection. The peak isometric twitch and tetanic tensions produced by rat fast-twitch *extensor digitorum longus* muscle injected with bupivacaine returns to normal values by 21 d after injection [17]. Mor‐ phological analysis has shown that many indexes of successful regeneration in healthy mus‐ cle can be completed within 2–3 weeks of recovery from injury [14]. The sequence of fiber breakdown induced by bupivacaine is similar to that of progressive muscular dystrophy [18] and it is also striking that the same types of muscle fibers are spared by both Duch‐ enne's muscular dystrophy and bupivacaine toxicity. It has been suggested that bupivacaine may disrupt Ca2+ homeostasis *in vivo*, triggering Ca2+-activated cellular death pathways that include proteolysis. This suggestion is supported by the findings that

**i.** bupivacaine affects sarcoplasmic reticulum function *in vitro*,


Extracellular Ca2+ plays a part in mediating the muscle damage caused by bupivacaine but other factors must also be involved [20]. For example macrophage invasion is necessary for complete degeneration of myofibrillar components [21]. Saito and Nonaka [22] injected 0,5ml of 0,5% bupivacaine after *soleus* muscle exposure of Wistar rats, and observed that SCs proliferation began at almost the same time as following muscle crush injuries. Bupivacaine is still commonly used for the purpose of studying the mechanisms of skeletal muscle regen‐ eration following injury [7, 14, 15, 22].

#### *3.1.2. Cardiotoxin and Notexin*

injury [15]. In this review we will focus on chemical and mechanical models of skeletal mus‐ cle injury, adding a new model of muscle injury based on surgical myectomy, developed in order to mimic severe losses of skeletal muscle mass. Other models, like exercise and dener‐ vation, will also be outlined. The latter is not a model of injury but else of skeletal muscle disuse but that also can be used to investigate skeletal muscle remodeling. There is a variety of other genetic models that are essential in studying diseases like Duchenne muscle dystro‐ phy (Mdx mouse is currently the most widely used in this case) but will not be discussed in

The use of myotoxins, such as bupivacaine (Marcaine), cardiotoxin (CTX), and notexin (NTX) is perhaps the easiest and most reproducible way to induce muscle injury and regen‐ eration. Myotoxins are also widely used to induce skeletal muscle injury because their inoc‐ ulation by intramuscular injection does not require complex surgery. Several chemical agents are known to produce skeletal muscle damage. Severe muscle fiber damage, like breakdown of sarcolemma and myofibrils, has been described after intramuscular injections of 0.75% bupivacaine, 2% mepivacaine, or 2% lidocaine associated to epinephrine [18]. While lidocaine can cause rapid destruction of skeletal muscle fibers, long-acting anesthet‐ ics, like bupivacaine, are more often used to cause skeletal muscle injury in rodents [19].

The bupivacaine injection procedure is simple and quick, does not involve extensive sur‐ gery, and induces a regeneration process which is qualitatively similar to that observed in other model systems. Doses of 1.5 and 1% wt/vol produce significant levels of muscle injury and subsequent regeneration, but these doses also produce large regions of ischemic muscle tissue. Doses of 0.75 and 0.5% bupivacaine are also effective in inducing regeneration and produce little or no ischemia [16]. Muscle fiber necrosis is extremely rapid after induced bu‐ pivacaine injury [14]. Injection of the drug into small skeletal muscles of rat or mouse leads to immediate and massive myonecrosis followed by phagocytosis of necrotic debris and a rapid and apparently complete regeneration of muscle fibers 3-4 wk after injection. The peak isometric twitch and tetanic tensions produced by rat fast-twitch *extensor digitorum longus* muscle injected with bupivacaine returns to normal values by 21 d after injection [17]. Mor‐ phological analysis has shown that many indexes of successful regeneration in healthy mus‐ cle can be completed within 2–3 weeks of recovery from injury [14]. The sequence of fiber breakdown induced by bupivacaine is similar to that of progressive muscular dystrophy [18] and it is also striking that the same types of muscle fibers are spared by both Duch‐ enne's muscular dystrophy and bupivacaine toxicity. It has been suggested that bupivacaine may disrupt Ca2+ homeostasis *in vivo*, triggering Ca2+-activated cellular death pathways that

include proteolysis. This suggestion is supported by the findings that

**i.** bupivacaine affects sarcoplasmic reticulum function *in vitro*,

this review.

*3.1.1. Bupivacaine*

**3.1. Chemical methods of skeletal muscle injury**

334 Advances in Biomaterials Science and Biomedical Applications

Snake venom is known for a long time to directly affect the skeletal muscle, producing fibril‐ lation, contractures and depolarization of the sarcolemma. Although initially ascribed to the phospholipase A content of this venom, muscle contracture and depolarization seem to be related to the cardiotoxic action of cobra venom [23]. Notexin (NTX) is a phospholipase A2 neurotoxin peptide extracted from snake venoms that blocks neuromuscular transmission by inhibition of acetylcholine release. Cardiotoxin (CTX) is also a peptide isolated from snake venoms, but it is a protein kinase C-specific inhibitor that appears to induce the depo‐ larization and contraction of muscle cells, disruption of membrane structure, and lysis of various cell types [1]. CTX is postulated to be neurotoxic as its injection destroys neuromus‐ cular junctions [24]. However, CTX might cause direct destruction of muscle tissues [25]. Snake CTX polypeptide is now known to be a potent inducer of muscle contracture with phospholipase A likely acting in accelerating the action of CTX rather than in augmenting it [23, 26]. Dantrolene antagonizes CTX-induced contractures, suggesting a role for Ca2+ de‐ rived from the sarcoplasmic reticulum in CTX action. CTX rapidly lowers the threshold for Ca2+-induced Ca2+ release in heavy sarcoplasmic reticulum fractions. The mechanism of ac‐ tion involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca2+ release by the sarcoplasmic reticulum) [27, 28].

A more recent study by Gutiérrez and Ownby [25] focused on the role of PLA2 as important myotoxic components in these venoms suggesting that myotoxic PLA2s binds to acceptors in the plasma membrane leading to its disruption and pronounced Ca2+ influx which, in turn, initiates a complex series of degenerative events associated with contracture, activation of calpains and cytosolic Ca2+-dependent PLA2s, and mitochondrial Ca2+ overload. Fourie et al. [30] already had suggested that the biological effects of CTX could be a consequence of in‐ hibition of plasma membrane (Ca2+ + Mg2+)-ATPases. The local myonecrosis is often associat‐ ed with other effects, such as hemorrhage, blistering and edema, in a complex pattern of local tissue damage. Apart from membrane-active CTXs, snake venom hemorrhagic metallo‐ proteinases also cause myonecrosis, but the mechanism involved is likely to be an indirect one, probably related to ischemia [25]. CTX is a useful model for muscle regeneration that does not influence muscle architecture like basal lamina or microvasculature, making the re‐ generation process less complicated than other models like crush, where for example, inade‐ quate blood supply might result in an increase of fibrosis. CTX injection also results in faster and more extensive muscle degeneration, and an earlier start of the reconstruction phase, than muscle crushing [24].

#### **3.2. Mechanical methods of skeletal muscle injury**

Crush injuries of the skeletal muscle can occur in considerable numbers following natural disasters or acts of war and terrorism. They can also occur sporadically after industrial accidents or following periods of unconsciousness from drug intoxication, anesthesia, trau‐ ma or cerebral events [31]. Crushing as a method of inducing muscle injury and regenera‐ tion was first described by Bassaglia and Gautron [32], and has since been used in several published research studies [24]. Muscle damage occurs at three distinct stages: at the time of the initial mechanical crushing force, during the period of ischemia and during the peri‐ od of reperfusion [31]. It has been hypothesized that ischemia is the primary instigator of local muscle damage following crush injuries [33]. However, studies have shown that al‐ though skeletal muscle tissue can survive circulatory ischemia for 4h, the mechanical force sustained in crushing, along with ischemia, causes skeletal muscle death in only 1 h. Stud‐ ies of enzyme release suggest that most damage to myocytes occurs during the reperfu‐ sion stage rather than the ischemic stage [31]. Animal models of muscle injury should closely mimic the clinical situation. Among these models open crush lesion have been used frequent‐ ly, allowing standardized evaluation of regeneration in a selected muscle. For application of the trauma, either forceps or custom-made devices have been used. There are two types of muscle-crush models described in the literature: the segmental crush and the complete crush, where only 4-6% of the fibers remain intact [34]. There are different forms to accom‐ plish the segmental crush model but most of them include the use of a surgical instru‐ ment (hemostatic clamp e.g.) to produce a standardized closing force in a specific area of a muscle causing a compression contusion injury [35]. One of the important steps of this procedure is denervation, which makes the initial steps of regeneration less painful for the animal. Skeletal muscle contusion can also be performed without skin incision by drop‐ ping a mass over a selected muscle. This technique was used by Iwata, Fuchioka [36] em‐ ploying a 640g mass dropped from a 25 cm height onto an impactor (diameter 10 mm) placed on the belly of the rat medial *gastrocnemius*. This procedure damaged around 47% of the entire cross-sectional area of both medial and lateral *gastrocnemius*. At day 2 postinjury, an intense inflammatory response and necrotized myofibers with infiltrated mono‐ nuclear cells were observed. No myotubes were found at this stage. However, a number of regenerative myotubes were detected at days 7, 14, and 21 days post-injury. This study also showed that normal locomotion recovers prior to isometric force and complete regenera‐ tion of the injured muscle [36]. The main disadvantage of the complete muscle crush is the potential damaging of myoneural junctions which triggers not only regeneration of mus‐ cle substance but also initial innervation deficits. These deficits always lead to impaired healing [34]. Histological analysis of muscle regeneration after crush injury shows an ini‐ tial phase of inflammation followed by SCs activation, myotube regeneration and fibrosis of the muscle. It has been shown that development of fibrotic tissue is one of the main factors affecting the recovery of muscle function after traumatic muscle injury [34]. In a qualitative assessment performed by our group we tested the open crush lesion in the *tibialis anterior* (TA) muscle of adult Sasco Sprague rats. Different standardized force intensities, durations of muscle compression (30 seconds and 1 minute) and time points (3, 8, 15 and 21 days post-surgery) were considered for the histological evaluation of skeletal muscle injury. Hematoxilin-eosin (HE) and Masson's trichrome staining were employed in this preliminary study. At day 3 post-surgery, myofiber damage was evident and the lymph nodes were reactive due to the active inflammatory process. The presence of fibrosis was evident only following 15 days from the initial injury. This evaluation revealed that the crush model was not the most appropriate for *in vivo* evaluation of cellular therapies for skeletal muscle regeneration aid, since the extent of this injury type did not present the magnitude required to accurately appreciate the biological effects of MSCs utilization [38].

#### **3.3. Myectomy and myotomy**

proteinases also cause myonecrosis, but the mechanism involved is likely to be an indirect one, probably related to ischemia [25]. CTX is a useful model for muscle regeneration that does not influence muscle architecture like basal lamina or microvasculature, making the re‐ generation process less complicated than other models like crush, where for example, inade‐ quate blood supply might result in an increase of fibrosis. CTX injection also results in faster and more extensive muscle degeneration, and an earlier start of the reconstruction phase,

Crush injuries of the skeletal muscle can occur in considerable numbers following natural disasters or acts of war and terrorism. They can also occur sporadically after industrial accidents or following periods of unconsciousness from drug intoxication, anesthesia, trau‐ ma or cerebral events [31]. Crushing as a method of inducing muscle injury and regenera‐ tion was first described by Bassaglia and Gautron [32], and has since been used in several published research studies [24]. Muscle damage occurs at three distinct stages: at the time of the initial mechanical crushing force, during the period of ischemia and during the peri‐ od of reperfusion [31]. It has been hypothesized that ischemia is the primary instigator of local muscle damage following crush injuries [33]. However, studies have shown that al‐ though skeletal muscle tissue can survive circulatory ischemia for 4h, the mechanical force sustained in crushing, along with ischemia, causes skeletal muscle death in only 1 h. Stud‐ ies of enzyme release suggest that most damage to myocytes occurs during the reperfu‐ sion stage rather than the ischemic stage [31]. Animal models of muscle injury should closely mimic the clinical situation. Among these models open crush lesion have been used frequent‐ ly, allowing standardized evaluation of regeneration in a selected muscle. For application of the trauma, either forceps or custom-made devices have been used. There are two types of muscle-crush models described in the literature: the segmental crush and the complete crush, where only 4-6% of the fibers remain intact [34]. There are different forms to accom‐ plish the segmental crush model but most of them include the use of a surgical instru‐ ment (hemostatic clamp e.g.) to produce a standardized closing force in a specific area of a muscle causing a compression contusion injury [35]. One of the important steps of this procedure is denervation, which makes the initial steps of regeneration less painful for the animal. Skeletal muscle contusion can also be performed without skin incision by drop‐ ping a mass over a selected muscle. This technique was used by Iwata, Fuchioka [36] em‐ ploying a 640g mass dropped from a 25 cm height onto an impactor (diameter 10 mm) placed on the belly of the rat medial *gastrocnemius*. This procedure damaged around 47% of the entire cross-sectional area of both medial and lateral *gastrocnemius*. At day 2 postinjury, an intense inflammatory response and necrotized myofibers with infiltrated mono‐ nuclear cells were observed. No myotubes were found at this stage. However, a number of regenerative myotubes were detected at days 7, 14, and 21 days post-injury. This study also showed that normal locomotion recovers prior to isometric force and complete regenera‐ tion of the injured muscle [36]. The main disadvantage of the complete muscle crush is the potential damaging of myoneural junctions which triggers not only regeneration of mus‐ cle substance but also initial innervation deficits. These deficits always lead to impaired

than muscle crushing [24].

**3.2. Mechanical methods of skeletal muscle injury**

336 Advances in Biomaterials Science and Biomedical Applications

The loss of a portion of a skeletal muscle poses a unique challenge for regeneration of mus‐ cle tissue and restoration of its normal structure and function [39]. In the event of large-scale soft tissue traumas, extensive loss of full-thickness native tissue architecture renders the wound site unable to support normal regeneration process. In severe tissue injuries the acute inflammatory response is followed by formation of a provisional fibrin matrix derived from trauma-associated blood clotting and this matrix is then infiltrated by type I collagenproducing fibroblasts [40]. In order to mimic those situations, new experimental models have been developed in which a defined portion of the muscle tissue is removed, creating a myectomy defect within the muscle. For example, Merrit et al. [39] removed a 0.5 x 1.0 cm or a 1.0 x 1.0 cm fraction of the *gastrocnemius* muscle of rats, creating a small and large defect respectively. This was accomplished lacerating the lateral side of the muscle with a #9 scal‐ pel blade. We have recently developed a novel experimental muscle injury model in the TA muscle of adult Sasco Sprague rat, by using a biopsy punch to create a standardized myecto‐ my defect. Sasco Sprague male rats with 250-300g were used and after a standardized 5 mm diameter myectomy lesion in the mid-belly of the *tibialis anterior* muscle, the defect was com‐ pletely filled with different vehicles and/or biomaterials, cellular suspensions containing 1x106 human MSCs isolated from Wharton's jelly and conditioned media (Figure 1). This concentrated media contains trophic factors secreted by MSCs during cell culture. In our re‐ search work, the myectomy model proved to be the most appropriate for a comprehensive and standard evaluation of the rat skeletal muscular regeneration ability. The regeneration process in other models of lesion, like simple muscle crush, did not present the magnitude required to accurately appreciate the biological effects of MSCs [38].

**Figure 1.** Biopsy punch for myectomy lesion creating a 5 mm Ø defect in the rat *tibialis anterior* (TA) muscle.

Another less invasive model of muscle injury has been used in a number of studies by pro‐ ducing a laceration injury (myotomy) [42-44]. In some cases this was obtained by a partial thickness (50%) cut of gastrocnemius muscles in mice at 60% of their length from their distal insertion, through 75% of their width and then sutured with a modified Kessler stitch and simple sutures using a PDS 7.0 wire (Ethicon, Somerville, New Jersey) [43]. Other studies used a full-thickness (100%) cut though 50% of the *gastrocnemius* muscle width [44]. The ad‐ vantages of this model are its reproducibility and the ability to apply consistently precise in‐ jections into the laceration site [43].

#### **3.4. Denervation (indirect model)**

Innervation regulates skeletal muscle mass and muscle phenotype and peripheral nerve in‐ jury in the rat is a widely used model to investigate nerve regeneration and can also be em‐ ployed as a model of muscle inactivity and muscle atrophy. Changes in the muscles may contribute to functional deficit after nerve injury [47]. Denervation induces muscle atrophy and 25 months post denervation muscle fibers cross sectional area of the *extensor digitorium longus* (EDL) muscle diminish to only 2.5% of control animals although their fascicular or‐ ganization is maintained [47]. The effect of denervation on muscle atrophy is both activitydependent and activity-independent since the degree of hindlimb muscle atrophy after spinal isolation (activity-independent nerve influence) is less when compared to the atrophy caused by removal of all nerve influences by transecting the sciatic nerve [9]. Two basic mechanisms are responsible for denervation-induced muscle atrophy. First, there is aug‐ mented activity of the ubiquitin-proteasome pathway and proteolysis [48]. Second, there is cell death and myonuclei apoptosis conjugated with decreased capacity of satellite cell-de‐ pendent reparative myogenesis [49]. Together with atrophy, denervated/reinnervated mus‐ cles undergo phenotypical changes and conversion between muscle fiber types [50]. The relative increase in type I or type II muscle fibers following denervation seems to depend on the type of muscle fibers predominant in the muscle, with type II muscle fibers (fast fibers) increasing in proportion in *soleus* (considered a slow muscle) and type I muscle fiber num‐ ber increasing in *gastrocnemius* and TA muscles [51]. Likewise, the degree of muscle fiber atrophy in short-term denervation (4 weeks) has been noticed to be greater in the muscle fi‐ ber type that is more abundant in the affected muscles [52]. Earlier studies suggested that it was possible that denervated muscles could have increased muscle plasticity due to acceler‐ ation in the early myoblastic stages of muscle regeneration. Nevertheless McGeachie and Grounds [53] data proved that very few precursors were proliferating in denervated muscle within 30 h after injury, and the onset of myogenesis at 30 h was essentially the same in de‐ nervated and innervated muscle. They compared the onset of DNA synthesis in muscle pre‐ cursors in denervated and innervated muscle of adult BALBc mice regenerating after a simple cut injury. This study concluded that although denervation of skeletal muscles caus‐ es an increase in SCs and connective tissue cell turnover, it does not "prime" the general population of muscle precursors to start synthesizing DNA more rapidly after injury than in innervated muscle [53]. After sciatic nerve transection at an adult age, electromyography (EMG) patterns in hindlimb muscles during locomotion remained highly abnormal even af‐ ter recovery periods lasting 15 or 21 weeks [54]. This may be a limitation when using dener‐ vated muscles as a model of muscle injury since regeneration might be affected for a very prolonged period. Like already mentioned, other models of skeletal muscle injury, like com‐ plete crush or myectomy, can be accompanied by denervation since these traumatic models may possibly damage peripheral nerves or myoneural junctions. This might also be an un‐ desirable occurrence in the standardization of these models of muscle injury. In fact, our preliminary work using TA myectomy showed that few animals developed severe muscle force deficit after 4 weeks recovery, suggesting that damaged of the supplying nerve occur‐ red in these animals subset.

**Figure 1.** Biopsy punch for myectomy lesion creating a 5 mm Ø defect in the rat *tibialis anterior* (TA) muscle.

jections into the laceration site [43].

338 Advances in Biomaterials Science and Biomedical Applications

**3.4. Denervation (indirect model)**

Another less invasive model of muscle injury has been used in a number of studies by pro‐ ducing a laceration injury (myotomy) [42-44]. In some cases this was obtained by a partial thickness (50%) cut of gastrocnemius muscles in mice at 60% of their length from their distal insertion, through 75% of their width and then sutured with a modified Kessler stitch and simple sutures using a PDS 7.0 wire (Ethicon, Somerville, New Jersey) [43]. Other studies used a full-thickness (100%) cut though 50% of the *gastrocnemius* muscle width [44]. The ad‐ vantages of this model are its reproducibility and the ability to apply consistently precise in‐

Innervation regulates skeletal muscle mass and muscle phenotype and peripheral nerve in‐ jury in the rat is a widely used model to investigate nerve regeneration and can also be em‐ ployed as a model of muscle inactivity and muscle atrophy. Changes in the muscles may contribute to functional deficit after nerve injury [47]. Denervation induces muscle atrophy and 25 months post denervation muscle fibers cross sectional area of the *extensor digitorium longus* (EDL) muscle diminish to only 2.5% of control animals although their fascicular or‐ ganization is maintained [47]. The effect of denervation on muscle atrophy is both activitydependent and activity-independent since the degree of hindlimb muscle atrophy after spinal isolation (activity-independent nerve influence) is less when compared to the atrophy caused by removal of all nerve influences by transecting the sciatic nerve [9]. Two basic mechanisms are responsible for denervation-induced muscle atrophy. First, there is aug‐ mented activity of the ubiquitin-proteasome pathway and proteolysis [48]. Second, there is cell death and myonuclei apoptosis conjugated with decreased capacity of satellite cell-de‐ pendent reparative myogenesis [49]. Together with atrophy, denervated/reinnervated mus‐ cles undergo phenotypical changes and conversion between muscle fiber types [50]. The

In our research group, standard peripheral nerve injuries in the rat sciatic nerve model have been performed [57-63] in order to evaluate different therapeutic approaches including sev‐ eral biomaterials and cellular systems to promote sensitive and functional recovery of the nerve. A standard crush injury is performed by a non-serrated clamp (Institute of Industrial Electronic and Material Sciences, University of Technology, Vienna, Austria), exerting a con‐ stant force of 54 N for a period of 30s, 10mm above the bifurcation into tibial and common peroneal nerves, inducing a 3mm axonotmesis lesion [57-63]. In order to induce a standard neurotmesis lesion in the rat sciatic nerve model, considered a more serious lesion, under deep anaesthesia, the right sciatic nerve is exposed through a skin incision extending from the greater trochanter to the distal mid-half followed by a muscle splitting incision. After nerve mobilisation, a transection injury is performed (neurotmesis) using straight microsur‐ gical scissors. The nerve is injured at a level as low as possible, in general, immediately above the terminal nerve ramification. To prevent autotomy, a deterrent substance should be daily applied to rat right foot [57-63]. Both experimental injuries induce severe motor def‐ icit and loss of sensory function, evaluated by measuring extensor postural thrust (EPT) and withdrawal reflex latency (WRL), respectively [57-63]. Sensory and motor deficit then pro‐ gressively decreased along the post-operative, depending on the therapeutic approach used. Very promising results were obtained with chitosan type III membranes and MSCs isolated from the umbilical cord matrix. In addition, we also perform kinematic analysis of the rat walk which is a more sensitive behavioral test. This analysis is increasingly being used to assess functional recovery in peripheral nerve research because of its higher accuracy and better relationship with histological outcome [57-63]. We should bare in mind that locomo‐ tion is also of higher functional relevance since it involves integrated function of both the motor and sensory systems and their respective components, such as skeletal muscles, sen‐ sory endings, efferent and afferent nerve fibers and integrative centers within the central nervous system. Muscles innervated by sciatic nerve branches include both dorsiflexors and plantarflexors and, although in our published studies we focused our kinematic analysis on‐ ly in the stance phase, we now prefer to include analysis of the ankle joint motion also dur‐ ing the swing phase in order to provide additional information [59]. Denervation can be a very useful model of skeletal muscle injury for some experimental studies but some limita‐ tions might be pointed out in studies that attempt to focus exclusively on the muscular re‐ generation process. Nevertheless and as demonstrated by several studies, this muscular regeneration process is highly dependent of the neural supply and the nerve regeneration itself can be influenced by the damaged muscle tissue.
