Introduction
Regular physical exercise causes extensive molecular, cellular, and tissue adaptations in the human body, especially in the musculoskeletal system and the nerve-to-muscle junction. Studies have shown that the nerve-to-muscle junction has the ability and capacity for morphological changes as a result of muscle activity [1]. Exercise and physical activity prohibit damage and destructive changes in the peripheral nervous system [2, 3]. In this regard, it was found that in the axonal terminals, the expression of a group of axonal conduction syndromes, called Semaphorins can affect neuromuscular connections [4, 5]. SEMA3A is a type of Semaphorin that is expressed in muscle fibers and can cause axons to be expelled from the target tissue [5, 6]. Deschenes et al. (2011) investigated the effect of aging on the adaptation of neuromuscular junction (NMJ) to endurance training. The results showed that NMJ changes and nerve damage in muscle fibers occur before their atrophy. Exercise, on the other hand, delays nerve damage as we age [7]. Ghadiri et al. (2017) investigated the effects of a high-intensity exercise period on SEMA3A (semaphorin-3A) gene expression in the muscles of elderly mice. It was shown that with increasing age, SEMA3A gene expression in the long extensor muscle of the toes increases; however, exercise reduced its expression in both adult and elderly groups [8]. Another important factor involved in axonal growth is the neural adhesion molecules called NCAM. These molecules belong to the large family of immunoglobulins and are expressed only in nerve cells [9]. Recent evidence suggests that NCAM expression in muscle fibers increases following exercise [10]. Caldow et al. reported a significant increase in NCAM expression by examining early myogenic responses to exercise after 12 weeks of resistance exercise in young men [10]. Considering that, to date, the role of axonal conduction proteins, especially SEMA3A and NCAM as a stimulating factor in neurodevelopment in NMJ following sports adaptations at different ages has not been determined. Therefore, the present study aims to investigate the effects of 6 weeks of resistance aerobics training on the expression of SEMA3A and NCAM genes and proteins in the soleus muscle of male rats in three age groups.
Methods
This was an experimental study. A total of 30 male Wistar rats in three age groups of 2 weeks (100-150 g), 6 weeks (220-250 g), and 96 weeks (28-320 g) were kept in an environment with a temperature of 23°C ± 2°C, the humidity of 45% to 55%, and a 12 h dark cycle. During the research, standard food, pellets, and water were provided to the rats with no limits. After being adopted to the laboratory environment, the animals were divided into 3 groups of infants, young, and elderly in terms of age group, and then randomly divided into 2 groups (control and exercise) in terms of intervention.
Resistance training
The exercises included 3 training sessions per week (Saturday, Monday, and Wednesday) for 6 weeks, which included 3 sessions and each session included 4 times climbing a special ladder to a height of 1 m with 26 steps of 4 cm. There was a 30 s break for the rats between each exercise session. After attaching the weight to their tails, the rats were forced to climb the ladder. In the first week, the weight attached to the rats’ tails was 30% of their body weight, and gradually from the second week it increased to 70%, and in the third week, the weight was 100% of their body weight. In the fourth week, the weight was increased to 120% of their body weight [11].
Aerobic exercise
At first, the rats were subjected to a program of familiarization with how to work on the turntable for 3 days. Exercises were performed for 3 sessions per week (Sunday, Tuesday, and Thursday) and 3 days of resistance exercises on alternating days for 6 weeks. The intensity of the training in the first week was equal to 25% of the maximum speed of the subjects, and then it reached 50% of the maximum speed in the sixth week [12]. The rats were euthanized in a completely sterile environment 48 h after the last training session. Using a surgical razor, an incision was made in the back of their legs, and the soleus muscle was extracted, and immediately frozen in liquid nitrogen. This was applied to evaluate the expression of genes and proteins. A light microscope was used to measure SEMA3A and NCAM proteins in the muscle fibers. The quantitative polymerase chain reaction (QPCR) method was used to evaluate the expression of SEMA3A and NCAM genes. Meanwhile, β-actin was used as a control gene. One-way analysis of variance (ANOVA) and the Tukey post hoc test were used to determine the significance of the differences between the groups. All statistical studies were performed using the Prism 5 software (Table 1). 


Results
The results of this study showed that the expression of the SEMA3A gene was lower in the training groups compared to the control group in all three age groups; however, this difference was not significant. The results also showed a significant decrease in SEMA3A protein in young and mid-age exercise groups compared to the control group (P≤0.001). In the elderly group, the exercise showed a significant increase compared to the control. Regarding NCAM gene expression, the results showed a significant decrease in all three age groups after the exercise. NCAM protein expression was significantly reduced in the exercise groups of mid (P≤0.001) and elderly (P≤001); however, in the young group, this decrease was not significant (Figure 1).  Discussion
The present study investigated the effects of resistance aerobic training on the expression of SEMA3A and NCAM genes and proteins as two effective factors in altering the nerve fiber supply at NMJ in different age groups. SEMA3A as a repellent protein in various conditions, such as destructive diseases of the central and peripheral nerves and the aging process, destroys motor neurons, especially in the fast-twitch fibers [4]. Therefore, the results of the present study showed that although there was an insignificant decrease in SEMA3A gene expression in all three age groups, its protein levels were significantly reduced in both young and mid-age groups. A significant increase in the expression of this protein was shown in the elderly group. However, Ghadiri et al. (2017) reported a significant decrease in SEMA3A expression in the long extensor muscle of the fingers in older male rats after 4 weeks of intense intermittent exercise. The reason for this inconsistency may be explained by the type of exercise and the type of studied muscles, as it has been reported that different sports exercises cause different adaptations and results in the soleus muscle [8]. Accordingly, based on the findings, it can be stated that the expression of SEMA3A protein is related to the amount of neuromuscular activity. Following resistance aerobic training, the expression of this protein decreased significantly in the young and mid-age groups. One of the significant findings of the present study was the lack of change in NCAM gene expression in all three age groups and also a significant decrease in its protein expression in both mid-age and elderly groups. In the study of Taybi et al. (2019), which examined the effects of intense intermittent training on the cross-sectional area of muscle fibers and the expression of muscle-destroying genes in the plantaris muscle of elderly rats, they found that aging significantly reduces NCAM, and exercise increased the muscle cross-section and NCAM, which contradicts the findings of the present study [13].
The results of the present study showed that the expression of nerve repellent and healing proteins because of exercise is different at different ages. The expression of SEMA3A in the soleus muscle of infant and young group rats decreased and no significant difference was observed in the elderly. Exercise also decreased NCAM expression in the soleus muscle of young and elderly group rats, but these changes were not significant in the infant group rats.  

Ethical Considerations
Compliance with ethical guidelines
All the ethical principles of the present study were approved in accordance with the principles of working with laboratory animals, approved by the Ethics Committee of the Rasht Branch, Islamic Azad University, with the Ethics Code IR.IAU.RASHT.REC.1399.029.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
Study concept and design: Mohammad Mahdi Bagherpour Tabalvandani, Mohammad Reza Fadaei Chafy, Alireza Elmieh; Data acquisition, analysis, or interpretation: Mohammad Mahdi Bagherpour Tabalvandani, Mohammad Reza Fadaei Chafy, Alireza Elmieh; Drafting of the manuscript: Mohammad Mahdi Bagherpour Tabalvandani; Critical revision of the manuscript for important intellectual content: Mohammad Reza Fadaei Chafy; Statistical analysis: Mohammad Mahdi Bagherpour Tabalvandani, Mohammad Reza Fadaei Chafy, Alireza Elmieh; Study supervision: Mohammad Reza Fadaei Chafy, Alireza Elmieh. 

Conflicts of interest
The authors of this article had no conflict of interest.

Acknowledgements
The authors of this article consider it necessary to express their sincere thanks and appreciation to the management and staff of the Pasargad Institute of Tissue and Genetics Knowledge-Based Institute.


References

1. Deschenes MR, Kressin KA, Garratt RN, Leathrum CM, Shaffrey EC. Effects of exercise training on neuromuscular junction morphology and pre-to post-synaptic coupling in young and aged rats. Neuroscience. 2016; 316:167-77. [DOI:10.1016/j.neuroscience.2015.12.004] [PMID] [PMCID]
2. Nishimune H, Stanford JA, Mori Y. Role of exercise in maintaining the integrity of the neuromuscular junction. Muscle & Nerve. 2014; 49(3):315-24. [DOI:10.1002/mus.24095] [PMID] [PMCID]
3. Li Y, Thompson WJ. Nerve terminal growth remodels neuromuscular synapses in mice following regeneration of the postsynaptic muscle fiber. The Journal of Neuroscience. 2011; 31(37):13191-203. [DOI:10.1523/JNEUROSCI.2953-11.2011] [PMID] [PMCID]
4. Raper J, Mason C. Cellular strategies of axonal pathfinding. Cold Spring Harbor Perspectives in Biology. 2010; 2(9):a001933. [DOI:10.1101/cshperspect.a001933] [PMID] [PMCID]
5. De Winter F, Vo T, Stam FJ, Wisman LA, Bär PR, Niclou SP, et al. The expression of the chemorepellent semaphorin 3A is selectively induced in terminal schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease. Molecular and Cellular Neuroscience. 2006; 32(1-2):102-17. [DOI:10.1016/j.mcn.2006.03.002] [PMID]
6. Venkova K, Christov A, Kamaluddin Z, Kobalka P, Siddiqui S, Hensley K. Semaphorin 3A signaling through neuropilin-1 is an early trigger for distal axonopathy in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Journal of Neuropathology & Experimental Neurology. 2014; 73(7):702-13. [DOI:10.1097/NEN.0000000000000086] [PMID] [PMCID]
7. Vo TT. Studies on semaphorin 3A in the neuromuscular junction and in perineuronal nets [PhD dissertation]. Amsterdam: Vrije Universiteit; 2011. [Link]
8. Gyorkos AM, McCullough MJ, Spitsbergen JM. Glial cell line-derived neurotrophic factor (GDNF) expression and NMJ plasticity in skeletal muscle following endurance exercise. Neuroscience. 2014; 257:111-8. [DOI:10.1016/j.neuroscience.2013.10.068] [PMID] [PMCID]
9. Smith MB, Mulligan N. Peripheral neuropathies and exercise. Topics in Geriatric Rehabilitation. 2014; 30(2):131-47. [DOI:10.1097/TGR.0000000000000013]
10. Deschenes MR, Roby MA, Glass EK. Aging influences adaptations of the neuromuscular junction to endurance training. Neuroscience. 2011; 190:56-66. [DOI:10.1016/j.neuroscience.2011.05.070] [PMID] [PMCID]
11. Ghadiri Hormati L, Aminaei M, Dakhili AB. [The effect of high-intensity exercise training on gene expression of semaphorin 3A in extensor digitorum longus muscles of aged C57bl/6 mice (Persian)]. Scientific Journal of Ilam University of Medical Sciences. 2017; 25(1):92-102. [DOI:10.29252/sjimu.25.1.92]
12. Franz CK, Rutishauser U, Rafuse VF. Intrinsic neuronal properties control selective targeting of regenerating motoneurons. Brain. 2008; 131(6):1492-505. [DOI:10.1093/brain/awn039] [PMID]
13. Caldow MK, Thomas EE, Dale MJ, Tomkinson GR, Buckley JD, Cameron-Smith D. Early myogenic responses to acute exercise before and after resistance training in young men. Physiological Reports. 2015; 3(9):e12511. [DOI:10.14814/phy2.12511] [PMID] [PMCID]
14. Deschenes M, Tenny K, Wilson M. Increased and decreased activity elicits specific morphological adaptations of the neuromuscular junction. Neuroscience. 2006; 137(4):1277-83. [DOI:10.1016/j.neuroscience.2005.10.042] [PMID]
15. Valdez G, Tapia JC, Kang H, Clemenson GD, Gage F, Lichtman JW, et al. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proceedings of the National Academy of Sciences. 2010; 107(33):14863-8. [DOI:10.1073/pnas.1002220107] [PMID] [PMCID]
16. Tayebi SM, Siahkouhian M, Keshavarz M, Yousefi M. The effects of high-intensity interval training on skeletal muscle morphological changes and denervation gene expression of aged rats. Montenegrin Journal of Sports Science and Medicine. 2019; 8(2):39-45. [DOI:10.26773/mjssm.190906]
17. Sengupta P. A scientific review of age determination for a laboratory rat: How old is it in comparison with human age. Biomed International. 2011; 2(2):81-9. [Link]
18. Kim H-J, So B, Son JS, Song HS, Oh SL, Seong JK, et al. Resistance training inhibits the elevation of skeletal muscle derived-BDNF level concomitant with improvement of muscle strength in zucker diabetic rat. Journal of Exercise Nutrition & Biochemistry. 2015; 19(4):281-8. [DOI:10.5717/jenb.2015.15112402] [PMID] [PMCID]
19. Tsumiyama W, Oki S, Tamaru M, Ono T, Shimizu ME, Otsuka A. Evaluation of the lactate threshold of rats using external jugular vein catheterization. Journal of Physical Therapy Science. 2012; 24(11):1107-9. [DOI:10.1589/jpts.24.1107]
20. Deschenes MR, Judelson DA, Kraemer WJ, Meskaitis VJ, Volek JS, Nindl BC, et al. Effects of resistance training on neuromuscular junction morphology. Muscle & Nerve. 2000; 23(10):1576-81. [DOI:10.1002/1097-4598(200010)23:103.0.CO;2-J]
21. Aagaard P, Suetta C, Caserotti P, Magnusson SP, Kjær M. Role of the nervous system in sarcopenia and muscle atrophy with aging: Strength training as a countermeasure. Scandinavian Journal of Medicine & Science in Sports. 2010; 20(1):49-64. [DOI:10.1111/j.1600-0838.2009.01084.x] [PMID]
22. Eken T, Elder GC, Lømo T. Development of tonic firing behavior in rat soleus muscle. Journal of Neurophysiology. 2008; 99(4):1899-905. [DOI:10.1152/jn.00834.2007] [PMID]
23. Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiological Reviews. 2011; 91(4):1447-531. [DOI:10.1152/physrev.00031.2010] [PMID]
24. Torabimehr F, Kordi MR, Nouri R, Ai J, Bakhtiari Moghadam B, Shirian S. [The effect of voluntary and forced exercise on the expression level of NCAM-PSA protein in the neuromuscular junction of soleus muscle in a mice experimental autoimmune encephalomyelitis model (Persian)]. The Neuroscience Journal of Shefaye Khatam. 2020; 8(2):39-46. [DOI:10.29252/shefa.8.2.39]