In this study, we at first observed muscle atrophy followed by compensatory regrowth in uncontrolled, wild mice after severe cutaneous thermal injury. Immediately post-burn, we observed an inflammatory response characterized by the presence of intramuscular MPO-positive cells and the pro-inflammatory marker NF-κ B. Many studies have shown an accumulation of neutrophils in skeletal muscle tissue after injury, which reaches peak concentration at 1 day and returns to control levels at 7 times post-injury [ 39 – 41 ]. These studies also indicate that will neutrophils worsen muscle wasting. The correlation of MPO and NF-κ B expression may be due to MPO-positive tissues attaining NF-κ B activity during the hypermetabolic response, that is associated with muscle atrophy. In our study, we show co-localization of MPO cells with NF-κ B via immunofluorescence (Fig. 4a ). Most likely the recruitment of neutrophils results in nuclear translocation associated with NF-κ B as shown in Figs. 3 and 4 . This may lead to the secretion of cytokines that may change the microenvironment of skeletal muscle resulting in a good activation of the muscle atrophy marker Murf1 [ 26 , 27 ] as well as a decrease in myofiber size. Further studies to investigate the instrumental relationship between MPO-positive cells and NF-κ B appearance are warranted based on their correlation shown by this particular study. NF-κ B response elements are required for Murf1 gene activation during skeletal muscle atrophy in several illnesses [ 9 , 42 ], thus explaining why the protein appearance of NF-κ B and Murf1 mirror each other within our study (Fig. 5b ).
Prior to this particular study, the role of NF-κ B in muscles wasting post-burn was limited. A study found low amounts of NF-κ B in burn patients [ 43 ]. This really is expected since NF-κ B signaling in pro-inflammatory cellular material has been implicated in muscle atrophy and degeneration [ 44 , 45 ], whereas downregulation of NF-κ B is related to enhanced resistance to atrophy and regeneration of muscle materials [ 46 ]. The exact mechanisms by which NF-κ B encourages muscle wasting remain ambiguous. In cancer-induced cachexia, NF-κ B expression is associated with Pax7 dysregulation and muscle mass wasting [ 47 ]. A decreased prevalence of Pax7-positive satellite television cells has been associated with myofiber dysfunction and muscle throwing away muscle atrophy [ 48 , 49 ], whereas their proliferation is usually associated with muscle regeneration and myofiber synthesis [ 50 , 51 ]. Interestingly, our findings reveal that NF-κ B appearance coincides with the reduction in muscle fiber cross-sectional area as well as the downregulation of Pax7 expression at 2 days post-burn, an occurrence that is similar to that in cancer-induced cachexia. As the level of MPO and NF-κ B decreases from 7 and 14 days post-burn, we observed the shift into a pro-regenerative muscle niche characterized by proliferation associated with Pax7-positive satellite stem cells and subsequent muscle restitution and hypertrophy.
Typically, in burn and cancer-induced cachexia there is a systemic effect that promotes muscle wasting for a prolonged time period. In cancer-induced cachexia, NF-κ B activation coincides with the increase in Pax7 expression [ 47 ]. In contrast, our research found that NF-κ B coincides with a decrease in Pax7 expression, suggesting that the function of NF-κ B varies between a burn injury and cancer-induced cachexia. Within our burn study, we observed similar mechanisms of muscle tissue atrophy; however , these mechanisms were only observed transiently and locally. Although Pax7 cells have been shown to be essential for muscle hypertrophy [ 11 ], the relation between Pax7 activation and muscle protein synthesis is not clear because the activation and proliferation of satellite cells does not always lead to increased protein synthesis in skeletal muscle.
Reduced satellite cellular abundance was recently observed in pediatric burn patients; as a result, our rodent data supports clinical findings [ 52 ]. The reduction of satellite cells following burn injuries most likely effects the recovery of lean muscle mass and probably contributes to the muscle wasting that is characteristic of burn off patients. In contrast to our results, a recent paper has shown improved Pax7 mRNA expression at 1, 3, and 7 days post-burn in mice [ 53 ]. The difference within the induction of Pax7 expression between the studies can be described by differences between the burn protocols utilized. Our process involves burning 20% TBSA by immersing the dorsum of the mice in 98 ° C water just for 10s, whereas the other protocol involves injuring 12. 5% TBSA on the dorsum for 10s followed by a twelve. 5% TBSA on the ventrum for only 2 s i9000, totaling 25% TBSA. Therefore , differences in the severity associated with burn injury inflicted may result in a delayed induction associated with Pax7-positive cells. Moreover, the other study showed induction associated with Pax7 expression that was unable to overcome muscle atrophy. This is often explained by their protocol which involves burning the ventral surface area of the mice; this may affect mice mobility and exercise post-burn. Another key difference is the technique used to determine Pax7. The other authors examined Pax7 mRNA expression through quantitative polymerase chain reaction (qPCR), whereas we looked into Pax7 localization and protein level via immunohistochemistry plus Western blot. The Pax7 mRNA measured by RT-PCR does not necessarily predict its protein level. Finally, there exists a difference in the choice of muscle examined; the other study analyzed the gluteus maximus which has a significantly higher proportion associated with type IIB fast-twitch fibers than gastrocnemius muscle [ 54 ]. Type IIB fibers have been shown to be highly vunerable to muscle wasting, such as in aging [ 55 ] and cachexia [ 56 , 57 ], which may explain the overall atrophic response to burn observed in their study.
Keeping mice unrestrained may have inspired the fast muscle recovery and subsequent regrowth seen in this study. Although a burn affecting 20% TBSA can be life threatening to humans, our group provides previously reported that rodents in our protocols 24 l post-burn are highly active and able to resume giving normally [ 22 ]. Moreover, it is plausible that rodents become agitated in our burn model leading to more motion in comparison to sham mice. This is noteworthy considering the positive a result of mobilization on muscle growth. Muscle hypertrophy is not generally seen in human burn patients. The quick erosion associated with lean muscle mass in burn and cancer patients can be additional exacerbated by prolonged bed rest in the intensive treatment unit (ICU). Prolonged immobilization results in loss of body nitrogen and a decrease in skeletal muscle protein synthesis [ 58 , 59 ]. In contrast, assisting patients to move and exercise during in-hospital care may ameliorate the detrimental effects of bed sleep on muscle tissue [ 60 ].