The previous stretching to resistance training promotes adaptations on the postsynaptic region in different myofiber types

This investigation revealed the postsynaptic morphological adaptations in static stretching, resistance training, and their association in adult male Wistar rats. We processed the soleus and plantaris muscle for histochemical (muscle hypertrophy) and postsynaptic region imaging techniques. We observed muscle hypertrophy in both groups submitted to resistance training, even though the cross-section area is larger when there is no previous static stretching. The soleus postsynaptic region revealed an increase in compactness and fragmentation index in combined exercise. The resistance training promotes high adaptations in the postsynaptic area of plantaris; moreover, the previous static stretching decreased this area. In conclusion, the neuromuscular system’s components respond according to the myofiber type even though it is the same physical exercise. Besides, static stretching (isolated or combined) plays a crucial role in neuromuscular adaptations.


Introduction
Exercise training promotes increased neuromuscular activity, and demonstes the plasticity of the neuromuscular junction (NMJ), resulting in improved endurance and resistance training in different ages 1,2 . Moreover, the NMJ presented a high potential in postsynaptic components adaptations. The increase in acetylcholine receptors (AChR) and endplate compactness, ensuring efficient communication between the pre-and postsynaptic region, and prolonged muscle fatigue 3,4,5 .
Resistance training (RT) has been used to promote chronic muscular adaptations in humans and demonstrated to be a principal modality for increasing muscle strength, endurance, power, hypertrophy, and motor performance 6,7 . These adaptations are predominantly due to increasing the muscle cross-section area through the differentiation and proliferation of satellite cells by the release of inflammatory cytokines and growth factors 8 . Also, RT is a method where manipulating their variables (series, repetitions, frequency, overload, and rest interval) is directly proportional to the progress of adaptive biological responses 9 .
Static stretching has been highly widespread among individuals of all physical activity levels, commonly prescribed during the warm-up of strength training, conditioning, and rehabilitation 10,11 . Also, static stretching does not require much time or effort, and has a low risk of injury in young adults, and has shown beneficial postural stability results 12,13 .
Evidence suggests that stretching performed before resistance training can directly influence strength production, reduce the number of repetitions, the total volume of training, and muscle hypertrophy 14,15,16 .
It is essential to understand the possible effects in the postsynaptic region of the NMJ caused by static stretching and resistance training, especially in their association. Therefore, we investigated the neuromuscular junction plasticity in 8 weeks of static stretching, resistance training and your association in adult male Wistar rats.

Cross-section area (CSA)
In the histochemical analysis, the Type I and II CSAs from the soleus muscle and the Type I, IIa, and IIx CSAs from plantaris of all experimental groups were obtained (Table 1).
Soleus: the CSA of Types I (p<0.05) and II fibers (p<0.0001) of soleus of the ST group were smaller compared to NT. The CSA of Type I fibers (p<0.0001) of RT was larger compared to NT. Additionally, the CSA of Type II fibers (p<0.01) of RT was smaller than that found in NT.
The CSA of Types I (p<0.05) and II (p<0.001) fibers of SRT were larger than NT group. The CSA of Types I (p=0.0760) and II (p<0.0001) fibers of SRT were larger than ST. Besides, the CSA of Type I (p<0.0001) fibers of SRT were smaller, and the Type II (p<0.0001) fibers were larger than that found in the RT group.
Plantaris: the CSA of Types I, IIa (p<0.0001), and IIx (p<0.001) fibers of plantaris of ST were both larger compared to NT. The CSA of Types I (p<0.0001), IIa (p<0.0001), and IIx (p<0.0001) fibers of RT were both larger compared to NT.
Furthermore, the CSA of Types I (p<0.0001) and IIa (p<0.0001) fibers of SRT were larger, and the Type IIx (p>0.9999) fibers were smaller than that found in NT.
In the SRT Group, the CSA of Types I (p<0.0001) and IIa (p<0,01) fibers were larger, and Type IIx (p<0.0001) fibers were smaller compared to ST. Additionally, the CSA of Types IIa (p<0.001) and IIx (p<0.0001) fibers of SRT were both smaller, and the Type I (p<0.0001) fibers were larger than that found in RT.

Postsynaptic regions
The   Nunes et al. 26 indicates that the stretching with external overload does a hypertrophic muscle effect, and changes in muscle size and architecture did not happen in the low-intensity stretch. Our results revealed a reduction in both myofibrillar types compared to the NT group in soleus, differently in plantaris that presented muscle hypertrophy. This fact suggests that this static stretching protocol is intense enough to make beneficial adaptations only in plantaris muscle fibers.
Regular stretching practice can be beneficial in maximum voluntary contraction, and running speed 27 . Our findings reveal that static stretching causes changes in the entire postsynaptic neuromuscular structure, and probably in its function.
Similar to resistance training, manipulating variables in static stretching training seems to play a key role in neuromuscular adaptations. In soleus Type I fibers, when static stretching is combinated with resistance training, we can observe a non-significant increase in CSA. In contrast, the highest CSA value was considered in Type II fibers after previous static stretching to resistance training. Probably, the hypertrophic response induced by stretching before resistance exercise seems to affect fast fibers, presumably due to the stretching of these fibers caused by static stretching and the tension generated through the load additional in resistance training.
The current literature suggests that low-intensity muscle stretching does not seem to respond significantly to muscle hypertrophy analyzed through CSA measurement; however, high-intensity stretching can cause possible adaptations that are still limited 28 . The practical implication of not performing the previous stretching is a shorter duration of the training sessions, improving adherence to exercise 28,29 .
In this study, changes in the postsynaptic region regarding static stretching and resistance training provided new data on the peripheral nervous system's plasticity. We About the fragmentation index, Prakash and Sieck 38 found the effects of aging on morphological adaptations of NMJs such as altered fragmentation. Although fragmentation may result from the redistribution of pre-synaptic components over a greater synaptic area, these alterations can also represent an increase in denervation and reinnervation, which may compromise motor innervation and contribute to muscle weakness 21 . As a result, these exercise-induced alterations need to be considered in training protocols or rehabilitation exercises to improve neuromuscular function 35 .

Conclusion
In conclusion, both groups submitted to resistance training showed similar muscle hypertrophy results, highlighting no previous static stretching. The musclespecific adaptation in the postsynaptic region occurs according to the training, in which the resistance training showed greater morphological remodeling. However, the previous static stretching changes this remodeling.

Animals
Thirty-two male Wistar rats with 60 days-old were divided into 4 Groups (n =

Resistance Training Protocol
The rats of RT and SRT Groups performed an 8-week (24 sessions) resistance training protocol (3x/week) in a vertical ladder (110x18cm, 2 cm grid, 80° incline). The sessions consisted of 4 to 9 progressive load climbs. They were allowed to rest for 120 seconds at the top of the ladder after each climb 40 .
The rats performed the first four climbs with 50%, 75%, 90%, and 100% of their body mass additional loads fixed to the tail's proximal region 41 . In the subsequent climbs, 30 g of the extra progressive load was added until the ninth climb or exhaustion occurred 18 . The rats performed the training protocols at the same time of the day across the experimental period.

Histochemistry
The belly samples of soleus and plantaris muscle (n = 5 in each) of experimental groups were dissected and cryofixed, then transverse sections were made (10 μm thickness) (Cryostat HM 505 E, MICROM™). The histochemical reaction was used for myosin adenosine triphosphatase (ATPase) and the pH lability to differentiate the fiber types (Type I and II in soleus/ Type I, IIa, and IIx in plantaris muscle) 20 .

Morphologic and morphometric analysis
The images were obtained by a Zeiss™ Axioskop (Jena, Germany) light microscope. After, the morphometry was performed in each pH by the ImageJ™ software. The cross-section area (CSA) of Type I and II of soleus myofibers and the CSA of Type I, IIa, and IIx of plantaris myofibers (n = 100/fiber type/group) were quantified using a 20X objective lens with 10X ocular magnification. Subsequently, we performed the normality test of the data and analyzed it using Kruskal-Wallis with Dunn's post hoc test (p < 0.05) 33 .

Tissue preparation
The

Morphometric analysis
The images (n = 20) for morphometric analysis were captured using a 100X objective lens with 10X ocular magnification by an Olympus BX61™ Fully Motorized Data availability: All relevant data are within the paper.