PhD V.F. Noskova¹
PhD E.A. Dekunets^2
1St. Petersburg Scientific Research Institute of Physical Culture, St. Petersburg
²Federal Scientific Center for Physical Culture and Sports, Moscow

Keywords: skeletal muscle regeneration, growth factors, cytokines, macrophages.

Background. Skeletal muscle damage may lead to malfunctioning of the bodily systems responsible for movements, respiration, and postural control. Skeletal muscle injuries can be caused by dystrophy, mechanical effects, aging, and other factors. The issue of skeletal muscle damage is also relevant in sports physiology, as the potential for injury is typical for any type of active muscle work. Therefore, the molecular processes of recovery of the skeletal muscles are of interest to sports science.

The role of macrophages can change greatly during skeletal muscle regeneration, thus contributing to greater damage at one stage and facilitating their recovery at another [5]. The role of different growth drivers, cytokines, and chemokines involved in skeletal muscle regeneration should also be emphasized. These cells are mainly secreted by the active immune cells, damaged skeletal muscles, and activated macrophages that penetrate the necrosis area [3]. In general, changes in the number of cells, their phenotype, or the stage of regeneration at which they are involved may lead to different outcomes of the regeneration process [2].

Objective of the study was to determine the sequence of events and complex intercellular interactions involved in the complex biological process of skeletal muscle regeneration in mice.

Methods and structure of the study. Sampled for the experiment were the mice inbred line C57BL/6. C57BL/6 mice were divided into two groups: Control Group with the skeletal muscle damage (Group S) and Experimental Group with the muscle damage and macrophage depletion (Group T). After which the groups were divided into 6 subgroups, 8 mice each, depending on the time elapsed after the injury (12 hours, 1 day, 3 days, 5 days, 7 days and 14 days). The trauma was inflicted by dropping a ball with a diameter of 15.9 mm and a weight of 16.3 g into a 100 cm pipe to hit the hind leg (calf muscle) of the animal. 

For depletion of macrophages, the mice were intra-abdominally injected with 2 mg clodronate-containing liposomes 3 days before the injury, followed by 0.5 mg on the 0th, 3rd, 6th, 9th, and 12th days after the injury. Hematoxylin and eosin stainings were used for morphological analysis. The fibrous tissue was studied using Masson's trichrome staining.

The real-time method was used to estimate the level of mRNA expression using the StepOnePlus™PCR-Cycler (LifeTechnologies) device.

Results and discussion. The skeletal muscle injury was accompanied by damage to all components of the skeletal muscle tissue. On the 1st day after the injury, we observed a significant infiltration of the inflammatory cells in the damaged area. By the 3rd day after the injury, along with the inflammatory cells, primary myoblasts with centered nuclei appeared, indicating a shift from the pro-inflammatory phase of the immune response to the anti-inflammatory one, thus testifying to wound healing. By the 14th day after the injury, the CG subjects were found to have minimal signs of fiber inflammation or degeneration. In the EG, the number of collagen by the 14-day post-traumatic period was significantly higher than in the CG.

The release of inflammatory cytokines and chemokines was characterized by its specific dynamics, which manifested itself in the execution of reparative functions of macrophages and their distant effects [2].

The intensive production of cytokines, especially interleukin 1β (IL-1β) and 6 (IL-6), indicated the activation of macrophages with the M1 phenotype in response to injury at the initial stage of the immune response, as inactive macrophages do not produce IL-1β [5].

Cytokine mRNA expression in CG (black columns) and EG (white columns). Letters denote the level of significance of expression differences between the groups: c - p<0.05, cc - p<0.01

Another pro-inflammatory cytokine is a tumor necrosis factor alpha (TNF-α) produced in the early stages of the immune response after acute traumas of the skeletal muscles. In addition, this cytokine participates in the late stages of skeletal muscle tissue regeneration [6].

In our study, the TNF-α mRNA expression in the CG peaked on the 1st day after the injury, and then began to decrease, though not as rapidly as IL-1β; there was also a sharp increase in the mRNA expression levels in the first 12 hours after the injury (see the figure).

In the first hours after the injury, we observed a strong increase in the level of IL-6 mRNA expression, which is also important in the rapid formation of the body’s response to tissue damage after acute traumas of the skeletal muscles. On the other hand, IL-6 may contribute to the reduction of pro-inflammatory cytokine IL-1 and TNF-α due to its autocrine activity [2].

Interleukin 10 (IL-10) is a factor that modulates the function of macrophages and is characterized by a powerful anti-inflammatory activity [4]. The transforming growth factor beta (TGF-β) has multiple effects on a large number of cell types and is involved in the processes of regulation of growth, differentiation, and apoptosis of cells and modulation of the immune system [3]. In our study, the expression of these two cytokines in the CG increased gradually, peaking on the 3rd day after the injury (see the figure).

Therefore, we observed a standard immune response in the CG with a sharp increase in the concentration of pro-inflammatory cells followed by its gradual decrease, while the anti-inflammatory cells continued to grow in quantity. These results indicated a shift in the macrophage phenotypes from M1 to M2, reflecting the completion of pro-inflammatory processes and the beginning of the process of wound healing.

As a result of the macrophage depletion in the EG, the expression of all the cell populations was disrupted, so was the process of recovery of the muscle tissue.

Masson's trichrome staining enabled to determine the replacement of muscle tissue with fibrous one in the EG.

With the macrophage depletion, the level of IL-1β pro-inflammatory cytokine expression also increased in the 1st day after the injury, but its amount was significantly lower than the expression level, and at the late stages of skeletal muscle tissue regeneration, the expression level was higher than that in the CG. We observed a significant increase in the level of TNF-α pro-inflammatory cytokine in the late stages of recovery in the EG. Elevated levels of IL-1β and TNF-α in the late stages of skeletal muscle regeneration may indicate incomplete removal of necrotic debris and hence a malfunction of these pro-inflammatory cytokines.

Since the induction of IL-6 expression has a profound effect on muscle differentiation and plays a functional role in muscle growth, the decrease in the level of IL-6 mRNA expression disrupts the transition from the proliferative stage to the early stage of myogenic differentiation.

Conclusion. The macrophage depletion disrupts the microenvironment, causes changes in the cytokine functions, which may lead to cicatrization as a result of impaired muscle healing.

Although the study was mainly conducted on animals, these models provide an understanding of the cellular and molecular signaling pathways involved in the muscle degeneration and regeneration processes, and therefore potentially lead to clinical interventions and cellular therapy. Consequently, further research should seek to additionally and more fully define the molecular pathways and interactions that are necessary for effective skeletal muscle regeneration, which may contribute to the development of new methods of treatment in humans.

References

1. Chereshnev V.A., Gusev E.Yu. Immunology of inflammation: role of cytokines. Med. immunologiya.2001. no. 3. pp. 361-368.
2. Charge S., Rudnicki M. Cellular and molecular regulation of muscle regeneration. The American [J] Physiological society  2004. 84 (1). p. 209-38
3. Karalaki M., Fili S., Philippou A., Koutsilieris M. Muscle regeneration: cellular and molecular events. In vivo.  2009. 23. pp. 779-96
4. Mantovani A., Sica A., Sozzani S., Allavena P., Vecchi A., Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004. 25(12). pp. 677–686
5. Ten Broek R.W., Grefte S., Von Den Hoff J.W. Regulatory factors and cell populations involved in skeletal muscle regeneration. J. of Cellular physiology. 2010. 224. pp. 7-16
6. Warren G.L., Hulderman T., Jensen N., McKinstry M., Mishra M., Luster M., Simeonova P. Physiological role of tumor necrosis factor α in traumatic muscle injury.The FASEB Journal. 2002. 30(4). pp. 74-85.

Corresponding author: skorobey64@mail.ru

Abstract

Objective of the study was to determine the sequence of events and complex intercellular interactions involved in the complex biological process of skeletal muscle regeneration in mice.

Methods and structure of the study. Sampled for the experiment were the mice inbred line C57BL/6. C57BL/6 mice were divided into two groups: Control Group with the skeletal muscle damage (Group S) and Experimental Group with the muscle damage and macrophage depletion (Group T). The trauma was inflicted by dropping a ball with a diameter of 15.9 mm and a weight of 16.3 g into a 100 cm pipe to hit the hind leg (calf muscle) of the animal. For depletion of macrophages, the mice were intra-abdominally injected with 2 mg clodronate-containing liposomes 3 days before the injury, followed by 0.5 mg on the 0th, 3rd, 6th, 9th, and 12th days after the injury. Hematoxylin and eosin stainings were used for morphological analysis. The fibrous tissue was studied using Masson's trichrome staining.

Results and conclusions. The macrophage depletion disrupts the microenvironment, which causes changes in the regulatory influence of inflammatory and anti-inflammatory cells. This, in turn, disrupts the balance between Th1 and Th2 immune responses resulting in an impaired immune response, slower regeneration, and incomplete recovery of muscle tissue.