Histomorphological and functional contralateral symmetry in the gastrocnemius muscles of the laboratory rat

Abstract It is usual in anatomical and physiological research to assess the effects of some intervention on extremities (e.g., training programmes or injury recovery protocols) using one muscle for the intervention and its contralateral as control. However, the existence of laterality (left‐handedness or right‐handedness) in athletes of different specialities is widely recognized. In rats, gastrocnemius is one of the muscles most widely used because of its importance in locomotion and high relative limb mass. Since we have not found studies reporting laterality assessment on the morphology and function in rat gastrocnemius, our study aimed to evaluate the fibre histochemical, morphometrical and muscle force contractile properties between right and left gastrocnemius of the laboratory rat. Fibre‐type proportion, fibre morphometrical measurements, muscle capillarization and muscle force properties were analysed in the right and left gastrocnemius of six male rats. No statistically significant differences (p = 0.265) were found in gastrocnemius to body weight ratio (‰) between right (6.55 ± 0.40) and left (6.49 ± 0.40) muscles. The muscles analysed showed a great degree of heterogeneity in fibre type distribution, having three clearly distinguished regions named red, mixed and white. In the three regions, there were no statistical differences in fibre type proportions between right and left gastrocnemius, as is indicated by the p‐values (from 0.203 to 0.941) obtained after running t‐Student paired tests for each fibre type. When analysing fibre cross‐sectional area, individual fibre capillarization and fibre circularity, no significant differences between right and left gastrocnemius in any of these morphometrical parameters were found in any muscle region or fibre type. Most of the p‐values (70%) resulting from running t‐Student paired tests were higher than 0.400, and the lowest p‐value was 0.115. Seemingly, global capillary and fibre densities were not statistically different between right and left sides in all muscle regions with p‐values ranging from 0.337 to 0.812. Force parameters normalized to gastrocnemius mass (mN g−1) did not show any significant difference between right (PF = 74.0 ± 13.4, TF = 219.4 ± 13.0) and left (PF = 70.9 ± 10.7, TF = 213.0 ± 18.0) muscles with p = 0.623 (PF) and p = 0.514 (TF). Twitch time parameters (ms) also lacked significant differences between the two sides (CT: 43.4 ± 8.6 vs. 45.0 ± 14.3, p = 0.639; HRT: 77.6 ± 15.0 vs. 82.3 ± 25.3, p = 0.475). Finally, both muscles also showed similar (p = 0.718) fatigue properties. We did find an absence of laterality at the morphological and functional levels, which raises the possibility of using right and left gastrocnemius muscles interchangeably for experimental designs where one muscle is used to analyse data after a physiological intervention and its contralateral muscle plays the control role, thus allowing unbiased paired comparisons to derive accurate conclusions.


| INTRODUC TI ON
The unilateral leg model consists of the intervention of one leg or muscle group (e.g., subjected to exercise or ischemia, electrostimulated, injured or denervated), while the contralateral leg or muscle is used as an internal control (Contreras-Muñoz et al., 2017;Contreras-Muñoz et al., 2021;Masiero et al., 2009;Pereira et al., 2014). This experimental approach has been extensively used in the field of muscle morphological and physiological research (Guo & Zhou, 2003;Lexell & Taylor, 1991;Rab et al., 2000;Tarnopolsky et al., 2007), and is based on the assumption that, prior to the intervention, both legs or muscle pairs displayed similar morphological and physiological properties regarding the outcome of interest (Tarnopolsky et al., 2007) and on the assumption that the unilateral intervention has no impact on the contralateral leg.
Animals´ muscle symmetry between the right and left sides has been widely accepted from the morphological point of view. In fact, even reference works on muscle morpho-histology only include the muscles from one side of the body in their experimental design and analysis (Armstrong & Phelps, 1984). The similarity of the contralateral muscles has been also assumed when the influence of different factors was studied on the complex 3D muscle architecture.
For example, Wick et al. (2018) compared the muscle architecture of one isolated muscle versus its contralateral muscle surrounded by adjacent muscles, and Schenk et al. (2020) examined the influence of joint angle on 3D muscle architecture by comparing short left muscle and long right muscle. Obviously, these investigations are valid only if the right and left muscles were almost similar. In addition, in most studies, biochemical and physiological determinations are only performed on the muscles from one side of the sagittal line, considering that the paired muscle has a similar profile (Cornachione et al., 2011;Panisello et al., 2008;Rizo-Roca et al., 2017;Rizo-Roca et al., 2018).
Thus, considering the scarcity of experimental results, it is probably inappropriate to affirm that they are free of laterality (Guo & Zhou, 2003). However, at least humans show marked laterality or predisposition to preferential use in voluntary motor actions of parts of one side of the body over the other (Carpes et al., 2010;Kertesz et al., 1992). It is known that the asymmetric use of muscles can drive structural changes (Adam et al., 1998;Fugl-Meyer et al., 1982;Lexell & Taylor, 1991). Some studies have reported differences between contralateral muscles of human upper extremities, both on fibre type proportion and strength, these changes being dependent on the use and functional demands of each particular muscle (Adam et al., 1998;Aoki & Demura, 2008;Fugl-Meyer et al., 1982;Kubota & Demura, 2011;Venturelli et al., 2021). Regarding lower limbs, since they are usually used for symmetrical actions, less pronounced laterality than in upper limbs is observed (Maupas et al., 2002). There are very few studies investigating whether there is morphological or physiological symmetry between the muscles of the right and left parts of the lower body (Horwarth et al., 2021;Lexell & Taylor, 1991;Tarnopolsky et al., 2007).
For methodological and ethical reasons, the laboratory rat is one of the most widely used species in scientific research (Andersen & Tufik, 2016). An obvious question, which has been posed a few times in scientific literature, arises: could there be any structural, metabolic or functional difference between rats' right and left muscles? Among all rat muscles, the gastrocnemius is one of the most widely used in biomedical research due to its important role in locomotion and its high relative leg mass (Armstrong & Phelps, 1984;Cornachione et al., 2011). Moreover, its superficial location allows easy access for intervention, isolation and dissection. The aim of our study was to elucidate if there are morpho-functional differences between the right and left rat gastrocnemius muscles. The results will allow us to conclude if the experimental design of the unilateral leg model is correct and if the muscles from both sides can be used interchangeably.

| Animals
Fourteen male (9-week-old) Sprague-Dawley rats with a body mass of 289 ± 8 g (mean ± SD) were used in the study. Animals were housed at 25 ± 2°C and maintained on a 12-h light-dark cycle, with ad libitum access to water and food.  vs. 45.0 ± 14.3,p = 0.639;HRT: 77.6 ± 15.0 vs. 82.3 ± 25.3,p = 0.475). Finally, both muscles also showed similar (p = 0.718) fatigue properties. We did find an absence of laterality at the morphological and functional levels, which raises the possibility of using right and left gastrocnemius muscles interchangeably for experimental designs where one muscle is used to analyse data after a physiological intervention and its contralateral muscle plays the control role, thus allowing unbiased paired comparisons to derive accurate conclusions.

K E Y W O R D S
fibre types, histochemistry, laterality, muscle force

| Force measurements
After a week of quarantine, an in vivo determination of the gastrocnemius muscle contractile properties was performed. Animals were anaesthetized by an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg) and placed in a prone position on a dissection platform. Ketamine-xylazine anaesthetics were used following the current regulations by the local institutional Ethical Committee for Animal Experimentation (CEEA number 1899) and used in previous similar studies (Contreras-Muñoz et al., 2017. The gastrocnemius muscle was isolated from the surrounding musculature, leaving intact the blood supply and the nerves of the proximal insertion. The hind limb was immobilized by securing the knee and the ankle to the dissecting platform. The sciatic nerve was then exposed through a lateral incision on the thigh and connected to an electrode stimulator. Finally, the calcaneus tendon was cut at the distal insertion and tied to a force transducer (MLT 1030/D; ADInstruments) with an initial tension of 30 mN. To maintain optimal contraction conditions, the muscle was covered with a mineral oil solution (Sigma-Aldrich), which prevented it from drying out, and muscle temperature was maintained with a heat lamp (Daylight Basking Spot lamp 50 W; Exo Terra) which placed at 20 cm heated 33°C, according to manufacturer's specifications. Muscle temperature was checked at the beginning, during and at the end of the force measurement protocols by means of a non-contact infrared surface thermometer (PCE FIT-10; Spain), registering muscle temperature within a narrow range of variation (32-35°C). It has been described that this temperature range does not interfere with the muscle contractile properties (Ranatunga, 1982). Muscle contraction was elicited via supramaximal electrical sciatic nerve stimulation (3 mA, pulse width 0.05 ms) with the Stimulus isolator FE180 (ADInstruments). To set up the supramaximal response, the pulse width was fixed at 0.05 ms (according to manufacturer specifications) and the maximal twitch response was determined by increasing the intensity of the stimulus' current from 0.1 mA until further increases in stimulator intensity produced no further increase in the twitch amplitude. To ensure a supramaximal stimulation, the intensity producing the maximal response was multiplied by a factor of 1.5, resulting in 3 mA.
Before carrying out the different force measurements, the gastrocnemius muscle's optimal contraction length, at which its maximum twitch force is produced, was set with a series of twitch contractions. After that, the following stimulation protocol was applied to measure several muscle function parameters: (1) five isometric muscle twitches were recorded at a frequency of 1 Hz and the average of five consecutive isolated twitches was considered to obtain a single twitch peak force (PF, in mN), contraction time (CT, in ms) and half-relaxation time (HRT, in ms); (2) train of stimuli during 1 s at frequencies of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 and 200 Hz (with pauses of 1 min between trains to avoid muscle fatigue) were given to obtain force-frequency curve and the maximum tetanic force (TetF, in mN): (3) after 5 min of recovery (Allen et al., 2008), a fatigue test at low-frequency during 2 min was applied by continuous muscle stimulation at a frequency of 40 Hz and a fatigue index was calculated by measuring the area relative to baseline (force-time fatigue, in N s); and (4) step 1 was repeated and the percentage of post-fatigue PF with respect to the initial PF was calculated. The stimulation protocol was carried out on both gastrocnemius muscles of each rat, the order in which left and right muscles were tested was randomized, and the force recordings were normalized to the muscle mass. To measure and analyse all the parameters, the AD Instruments hardware (PowerLab/16SP) and software (LabCHart v7.3.7) were used. At the end of the force test, both gastrocnemius muscles were excised, weighed, and frozen at their resting sarcomere length in pre-cooled isopentane (Sigma-Aldrich) and finally stored at −80°C until further analysis.

| Histochemical procedures
Serial transverse cross-sections (14-16 μm) from the equatorial regions of the gastrocnemius muscle at 7 mm from the end of the distal myotendinous junction were perpendicularly cut in a cryostat at −20°C (Leica CM3050S). Muscle samples were placed in an orthogonal angle with respect to the fibres' orientation. Adjacent serial sections were mounted on gelatinized slides (0.02%) and processed with different histochemical techniques. First, all the sections were fixed for 5 min in formalin-sucrose solution to prevent shrinkage or wrinkle of the samples, and then they were incubated to: (1) succinate dehydrogenase staining (SDH), to recognize aerobic and anaerobic fibres (Nachlas et al., 1957), (2) alkaline myosin adenosine triphosphate (mATPase) after alkaline pre-incubation (pH 10.7), to differentiate between slow and fast-twitch fibres (Brooke & Kaiser, 1970), and (3) endothelial adenosine triphosphate (eATPase), to detect muscle capillaries (Fouces et al., 1993).

| Statistical analysis
Statistical analysis was performed using the statistical package  The only significant difference in circularity was found between SO and FOG fibres (p = 0.008) in the red region (Figure 4g), since no significant differences were found in either the mixed or the white region between fibre types (Figure 4h,i).

| Global regional capillary and fibre densities
No contralateral significant differences were evidenced in either reflecting the greater fibre sizes of the fibres from this region. The difference between red and mixed regions was also significant with p = 0.018.

| Muscle force and fatigue parameters
No statistical differences were shown between right and left gastrocnemius muscle mass (gastrocnemius/body mass) (right: 6.55 ± 0.40‰ vs. left: 6.49 ± 0.40‰, p = 0.265). Both right and left gastrocnemius muscles displayed similar mass-normalized peak (p = 0.664) and tetanic force (p = 0.493) after sciatic stimulation ( Figure 6a). This absence of significant differences was also evident for the twitch time parameters (contraction and half-relaxation times) (Figure 6b) and the two fatigue parameters measured (forcetime and percentage of peak force after the fatigue test) (Figure 6c).
Finally, the force-frequency curves of right and left muscles, obtained after sciatic stimulation at gradually increased frequencies, almost totally overlapped indicating similar muscle force production at each stimulation frequency (Figure 6d). In all cases, the tetanic force was obtained at a mean frequency of 90-100 Hz.

| DISCUSS ION
The body and organs of animals can be divided relative to different planes, i.e., imaginary flat surfaces that pass through them. The sagittal plane is a vertical plane that divides the bodies or organs into right and left sides (Kardong, 2019). In humans, some studies have attempted to corroborate the existence of symmetry between muscles on the right and left sides of the lower limb. Thus, it has been found that vastus lateralis and soleus muscles did not present systematic differences between right and left legs neither in the fibretype proportion nor in the area of the fibre (Fugl-Meyer et al., 1979;Horwarth et al., 2021;Lexell & Taylor, 1991;Tarnopolsky et al., 2007;Venturelli et al., 2021). Additionally, in the vastus lateralis, the absence of differences between both sides has also been reported in the number of myonuclei per fibre, the myonuclear domain, the transcriptome expression pattern and the isometric strength (Horwarth et al., 2021;Tarnopolsky et al., 2007). These studies have been carried out both in young adults (18-40 years) (Horwarth et al., 2021;Lexell & Taylor, 1991) and in older people (65 years) (Tarnopolsky et al., 2007), demonstrating that, despite age-related changes in skeletal muscle, the symmetry between muscles of both sides of the body remains similar. However, some discrepancies between muscle symmetry have been observed in resistance-trained men, who had differences between right and left vastus lateralis in fibre type proportion, although these differences did not translate into changes in strength (Arevalo et al., 2018;Lindström et al., 1995). This bilateral symmetry is not seen in athletes from unilateral sports either (Demura et al., 2010).
Regarding animal models, Lexell et al. (1994) analysed fibre type proportion and fibre density of right and left tibialis anterior and extensor digitorum longus in rabbits, finding no statistical differences between muscles of each leg. Later on, Rab et al. (2000) did not report differences between rabbit right and left rectus femoris, vastus medialis and adductor magnus muscles in fibre types and fibre areas. In rats, Guo and Zhou (2003) did not find differences between contralateral muscles in the concentration of some metabolites involved in lipid metabolism, such as glycerol, fatty acids and triglycerides, in muscles with mixed fibre composition (quadriceps, tibialis anterior and gastrocnemius), concluding that there is no metabolic laterality in the rat hind limb muscles (Guo & Zhou, 2003).
To the best of our knowledge, there are no histological or functional studies on rat gastrocnemius laterality. The gastrocnemius muscle is heterogeneous in its architecture, with three different regions within its cross-section according to the fibre-type proportion ( Figure 1) (Armstrong & Phelps, 1984). Our results did not show sta- The boxes represent the first and third quartiles separated by the median, the mean is represented with a black dot, and the whisker end points represent the minimum and maximum values. Data from the right gastrocnemius are shown in white boxes and data from the left gastrocnemius in grey boxes. p-values resulting from statistical comparisons between right and left muscles after running paired t-Student tests are shown for each muscle zone on the graph fibre length, pennation angle and curvature Stark & Schilling, 2010;Wick et al., 2018). This intramuscular architecture entails different fibre morphometrical measurements along the proximal, equatorial or distal muscle level (Torrella et al., 2000).
To avoid as much as possible biased results derived from changes in muscle orientation and muscle architecture characteristics, all muscles were cut in the same position at the equatorial (see Section 2), resulting in no changes observed in the FCSA and the SF of the fibres between right and left gastrocnemius muscles.
Taking into account that the different regions of the gastrocnemius muscle are recruited and used for different locomotion or postural activities (Kyröläinen et al., 2005), our results support the hypothesis that both gastrocnemius muscles are used equally for the same tasks or have a similar pattern of use.
It is well known that muscle force capacities are directly related to fibre type and muscle fibre size (Huard et al., 2002;Kardong, 2019). Thus, the absence of morphological differences between right and left gastrocnemius discussed above is in accordance with the lack of differences in muscle force production. All strength parameters measured in the present study were similar in the right and left gastrocnemius, showing no functional differences between legs ( Figure 6). Moreover, as shown in Figure 6d, the same force-frequency curves were observed in both muscles, suggesting that the Ca 2+ -handling and cross-bridge kinetics are similar. Since the structure and function are closely interdependent (Fugl-Meyer et al., 1982), the morpho-functional symmetry reported here lends additional support to a symmetrical use for both legs in the laboratory rat.
Thus, the morphological and physiological results derived from this study imply that, in further studies, one muscle could be used for intervention and the contralateral as a control. Moreover, right or left gastrocnemius could be used indistinctly for histological, F I G U R E 6 Force (a), twitch time (b) and fatigue parameters (c) in right and left rat gastrocnemius muscles. The boxes represent the first and third quartiles separated by the median, the mean is represented with a black dot, and the whisker end points represent the minimum and maximum values. Data from the right gastrocnemius are shown in white boxes and data from the left gastrocnemius in grey boxes. pvalues resulting from statistical comparisons between right and left muscles after running paired t-Student tests are shown on the graph. (d) Force-frequency curves for right and left rat gastrocnemius muscles expressed as a percentage of the tetanic force at different stimulation frequencies. Hollow and filled circles indicate the mean values and segments of standard deviation. All p-values resulting from statistical comparisons between right and left muscles after running paired t-student tests were p > 0.05 functional and, presumably, biochemical determinations. This is relevant because, in studies aimed at obtaining a representative value of a given parameter for the gastrocnemius muscle, it will not be necessary to analyse both sides of the muscle, thus reducing the total number of assays needed for the experimentation.
Furthermore, in research in which a large number of invasive experiments are carried out, which cannot all be applied in the same muscle, both contralateral muscles can be used, thus reducing the total number of animals (Böl et al., 2015). However, in some cases, further consideration is required and the use of the contralateral muscle as a control must be considered with caution in long-term experiments. For example, in studies involving unilateral interventions that cause compensatory mechanisms in the neuromuscular system, as is the case of investigations inducing denervation of one side, hypertrophy and shifts in the fibres of the contralateral non-intervened muscle have been described (Rab et al., 2000).
Moreover, denervation, immobilization and reinnervation could alter the animal's locomotion pattern, producing changes in the afferent activity, which in turn is translated into alterations in efferent activity in the contralateral non-intervened leg (Rab et al., 2000).

| CON CLUS ION
This is the first study in which morphological and functional lateral symmetry of the gastrocnemius muscle of the rat has been demonstrated. Our results report the absence of differences between right and left sides in muscle morphology (size and proportion of fibre types and muscle capillarization) and in muscle functional properties (twitch and tetanic force, twitch time parameters, fatigue parameters and force-frequency curves). These findings make it possible to state that the use of the unilateral leg model for the study of interventions in the gastrocnemius muscle is the correct method since it can be assumed that both gastrocnemius had the same morphofunctional profile before the intervention.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.