Morphological Evidence of Telocytes in Skeletal Muscle Interstitium of Exercised and Sedentary Rodents

Skeletal muscle atrophy, resulting from states of hypokinesis or immobilization, leads to morphological, metabolic, and functional changes within the muscle tissue, a large variety of which are supported by the stromal cells populating the interstitium. Telocytes represent a recently discovered population of stromal cells, which has been increasingly identified in several human organs and appears to participate in sustaining cross-talk, promoting regenerative mechanisms and supporting differentiation of local stem cell niche. The aim of this morphologic study was to investigate the presence of Telocytes in the tibialis anterior muscle of healthy rats undergoing an endurance training protocol for either 4 weeks or 16 weeks compared to sedentary rats. Histomorphometric analysis of muscle fibers diameter revealed muscle atrophy in sedentary rats. Telocytes were identified by double-positive immunofluorescence staining for CD34/CD117 and CD34/vimentin. The results showed that Telocytes were significantly reduced in sedentary rats at 16 weeks, while rats subjected to regular exercise maintained a stable Telocytes population after 16 weeks. Understanding of the relationship between Telocytes and exercise offers new chances in the field of regenerative medicine, suggesting possible triggers for Telocytes in sarcopenia and other musculoskeletal disorders, promoting adapted physical activity and rehabilitation programmes in clinical practice.


Introduction
The skeletal muscle is a highly dynamic and plastic tissue that promptly responds to physical activity and sedentary behavior [1]. Skeletal muscle disuse atrophy (SMDA) refers to biochemical, morphological, and functional changes in skeletal muscle that may result from states of hypokinesis or immobilization, e.g., following fractures or elective orthopedic surgery, and represents a major topic in the fields of regenerative and rehabilitation medicine [2,3]. On the contrary, both resistance and aerobic training induce metabolic changes within the muscle by altering protein synthesis, muscle proteolysis, therefore inducing molecular and cellular adaptations that regulate homeostasis and hypertrophy [4][5][6]. A large variety of stromal cells are involved during physiological processes following physical exercise, in order to sustain remodeling and regeneration [7,8]. More specifically, the involvement of Pax+ cells, side population cells (SP cells), pericytes, fibro/adipogenic progenitor cells (FAPs), and PW1+/Pax7-interstitial progenitor cells (PICs), has received It is noteworthy to mention that this discovery raised skepticism and controversy, since ICLCs were also described merely as CD34-positive stromal cells acting as stem cells during regeneration processes [39,40], or the term was used interchangeably to describe fibroblasts [41]. Therefore, there is a need for new evidence that can discriminate the different populations hosted in the stem cell niche of the tissues, not only for their morphol- It is noteworthy to mention that this discovery raised skepticism and controversy, since ICLCs were also described merely as CD34-positive stromal cells acting as stem cells during regeneration processes [39,40], or the term was used interchangeably to describe fibroblasts [41]. Therefore, there is a need for new evidence that can discriminate the different populations hosted in the stem cell niche of the tissues, not only for their morphology but also for their function. TCs are typically described as cells with small bodies, reported as pear-, spindle-, triangular-shaped, and very long cytoplasmic processes, up to hundreds of micrometers but only approximately 0.2 µm thick [42,43]. The identification of TCs, via transmission electron microscopy, showed characteristic features [33,44] (Table 1). Although it is still unclear, the role of TCs seems to participate in sustaining cross-talk communication between stromal cells through signaling transmission via exosomes [13,45], secreting vascular endothelial growth factor (VEGF) and, broadly, promoting myofibers regenerative mechanisms by supporting local stem cell niche differentiation, vasculogenesis, and preventing fibrosis [46]. The presence of this population has been observed, under physiological conditions, in numerous organs and tissues [47,48], and also following pathologic situations, such as musculoskeletal injuries [49], suggesting their role in healing processes and their function for regenerative medicine strategies [50][51][52][53]. As already mentioned, physical inactivity and activity have been extensively studied in relation to stem cell niche and, more recently, are also attracting attention with regard to TCs [49,54,55].
Finally, the aim of this morphologic study was to investigate the presence/absence of TCs in tibialis anterior muscle of healthy rats who underwent a protocol of endurance training for either 4 weeks or 16 weeks in comparison to sedentary rats who were inactive, i.e., not engaging in any physical exercise, throughout the duration of the experiment.

Ethical Approval
All the procedures involving alive animals were performed at the Center for Ad-

Animals: Housing and Breeding
Two-month-old healthy female Wistar outbred rats, with a bodyweight of 200 ± 20 g, were purchased from Charles River Laboratories, Milan, Italy and bred in the animal facilities at the University of Catania. Rats were maintained and kept in polycarbonate cages (10.25 W × 18.75 D × 8 H) in stable hygrometric and thermic conditions (20-23 • C) on 12 h light/dark cycle with ad libitum access to water and food, throughout the whole period of the experiment. It was used a standard rat chow: carbohydrates (40%), proteins containing all essential amino acids (30%), and lipids (30%). Lipids were a mixture of neutral fatty acids, saturated fatty acids, and unsaturated fatty acids. Diets were provided by Laboratorio Dottori Piccioni, Gessate (Milan), Italy. Twenty rats were used in this study equally divided into two groups, sedentary and undergoing physical exercise, respectively CTRL and PA, sacrificed at two different time points, 4 and 16 weeks: CTRL4W, control sedentary rats sacrificed at 4 weeks; PA4W, rats performing physical exercise sacrificed at 4 weeks; CTRL16W, control sedentary rats sacrificed at 16 weeks; PA16W, rats performing physical exercise sacrificed at 4 weeks. All animals were randomly distributed to groups. All efforts were made to minimize the number of mice, according to the principles of the 3Rs and the resource equation approach, and reduce their suffering [56]. Throughout the whole period of the experiment, the animals were free to move in the cages and their wellness was monitored through objective observation and daily checks (weight, claudication, fur and eyes appearance, consumption of food and water, lethargy) [57]. The animals were sacrificed by carbon dioxide (CO2) overdose, at the established time points. After euthanasia, tibialis anterior muscles were explanted and processed for the planned experiments since the high responsivity of this muscle to exercise [58].

Treadmill Training
Two groups of rats (PA4W and PA16W) performed physical activity in the form of running on a treadmill (2Biological instrument, Varese, Italy) ( Figure 2 and Supplementary Video S1). Rats were made familiar with the instrument for 1 week prior to surgery, at a speed of 10 m/min (type of exercise: interval training, between mild and moderate) for 5 min daily. This type of exercise is used to stimulate the muscles, joints, and bones in the work of flexion-extension of the limbs. The rats exercised 3 days a week and, in order to adapt the settings to the time-dependent ability of the rats to perform the exercise, the speeds and the durations were gradually incremented from 10-15 m/min for 5 min to 20-30 m/min for 15 min (from week 1 to week 4), from 10-15 m/min for 5 min to 30-40 m/min for 20 min (from week 1 to week 8), from 10-15 m/min for 5 min to 40-50 m/min for 25 min (from week 1 to week 16). The treadmill was gradually inclined between 2 • and 6 • degrees. A minimal electric shock (0.2 mA) was used to avoid the rat to stop running, if distracted, to stimulate the walking and to instruct the rats in the first place. All-electric shock bouts were closely monitored in real-time and acquired by the embedded data acquisition software (2Biological instrument, Varese, Italy). Rats that exceeded the number of five electric shocks, in one session, were suspended from the exercise.

Histology Analysis
Tibialis anterior muscle samples were washed in phosphate-buffered saline (PBS, Bio-Optica, Milano, Italy), fixed in 10% buffered-formalin (Bio-Optica, Milan, Italy) for 24 h at room temperature. Afterwards, the samples were dehydrated in graded ethanol (Bio-Optica, Milan, Italy), cleaned in xylene (Bio-Optica, Milan, Italy) and paraffin-embedded (Bio-Optica, Milan, Italy), being careful to preserve the desired anatomical orientation. Slides of 5 µm thickness were cut from the obtained paraffin blocks and hematoxylin and eosin-stained (H&E, Bio-Optica, Milan, Italy) following a protocol described elsewhere [59]. The samples were then examined in triplicate for morphological evaluation with a Zeiss Axioplan light microscope (Carl Zeiss, Oberkochen, Germany) and by a digital camera (AxioCam MRc5, Carl Zeiss, Oberkochen, Germany), used to take images. 6° degrees. A minimal electric shock (0.2 mA) was used to avoid the rat to stop if distracted, to stimulate the walking and to instruct the rats in the first place. A shock bouts were closely monitored in real-time and acquired by the embedded quisition software (2Biological instrument, Varese, Italy). Rats that exceeded the of five electric shocks, in one session, were suspended from the exercise.

Histology Analysis
Tibialis anterior muscle samples were washed in phosphate-buffered sal Bio-Optica, Milano, Italy), fixed in 10% buffered-formalin (Bio-Optica, Milan, Ita h at room temperature. Afterwards, the samples were dehydrated in graded etha Optica, Milan, Italy), cleaned in xylene (Bio-Optica, Milan, Italy) and paraffin-em (Bio-Optica, Milan, Italy), being careful to preserve the desired anatomical or Slides of 5 μm thickness were cut from the obtained paraffin blocks and hemato eosin-stained (H&E, Bio-Optica, Milan, Italy) following a protocol described e [59]. The samples were then examined in triplicate for morphological evaluatio Zeiss Axioplan light microscope (Carl Zeiss, Oberkochen, Germany) and by a dig era (AxioCam MRc5, Carl Zeiss, Oberkochen, Germany), used to take images.

Histomorphometric Analysis
Each H&E stained muscle cross section was subjected in triplicate to morp analysis by calculating the area of twenty muscle fibers of five randomly selec with a total area of about 35.000 μm2, using a software for image acquisition (Ax Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany) [ were then expressed as diameter mean ± standard deviation (SD). Statistical sig of results was thus accomplished. Three investigators (two anatomical morpholo one histologist) made the morphological assessment. If disputes occurred, a un

Histomorphometric Analysis
Each H&E stained muscle cross section was subjected in triplicate to morphometric analysis by calculating the area of twenty muscle fibers of five randomly selected fields with a total area of about 35.000 µm 2 , using a software for image acquisition (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany) [60]. Data were then expressed as diameter mean ± standard deviation (SD). Statistical significance of results was thus accomplished. Three investigators (two anatomical morphologists and one histologist) made the morphological assessment. If disputes occurred, a unanimous agreement was reached after section re-evaluation and before proceeding with data interpretation.

Double Immunofluorescence Analysis
Paraffin-embedded muscle tissue sections of 5 µm thickness were subjected in triplicate to double immunofluorescence (IF) combining anti-mouse and anti-rabbit goat secondary antibodies with either mouse or rabbit primary antibodies. Muscle sections were deparaffinized with xylene and rehydrated in graded ethanol scale. Afterwards, the slides were cleaned for 20 min with phosphate-buffered saline (PBS; Bio-Optica, Milan, Italy) and unmasked in citrate buffer (pH 6.0; Bio-Optica, Milan, Italy), or in ethylenediaminetetraacetic acid-Tris buffer (Tris-EDTA pH 8.0, Bio-Optica, Milan, Italy) for the antigenic retrieval and incubated in 0.3% H 2 O 2 /PBS, for 30 min, to block endogenous peroxidase activity. Non-specific antibody binding sites were blocked by applying a solution of 1% bovine serum albumin (BSA; Sigma-Aldrich, Saint Louis, MO, USA) in PBS 1X for 1 h at room temperature. Tissue slides were washed in PBS and, then, incubated overnight at +4 • C with a mixture of mouse and rabbit primary antibodies at appropriate dilution in antibody dilution buffer: mouse monoclonal anti-CD34  Table 2. Negative controls were performed by replacing primary antibodies with non-immune serum, while cross reactivity of secondary antibodies was verified by omitting primary antibodies. Immunolabeled samples were rinsed in PBS and mounted using an anti-fade mounting medium containing 4 ,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining (Vectashield, Vector Laboratories, Burlingame, CA, USA) and sealed with nail polish.

Computerized Densitometric Measurements and Image Analysis
Digital micrographs of double immunofluorescence sections were taken using a confocal laser scanning microscopy (CLSM, Zeiss LSM700, Carl Zeiss, Oberkochen, Germany) with ZEN-2010 software. In order to detect the fluorophore signal, three lasers with 405, 488, and 555 nm wavelengths were used for the analysis of blue, green, and red signals, respectively. Image analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany), which quantifies the level of double positive staining of anti-CD34/anti-CD117 and anti-CD34/anti-Vimentin immunolabeling, was used to calculate the densitometric count in five fields, 10× magnification, randomly selected from each section. Statistical results are expressed as densitometric count (pixel 2 )/(pixel 2 ) of double immunostaining on muscle tissue. Three blinded investigators (two anatomical morphologists and one histologist) made the evaluations that were assumed to be correct if values have not statistically significant difference. If disputes concerning interpretation occurred, unanimous agreement was reached after sample re-evaluation.

Statistical Analysis
Statistical analysis was performed using GraphPad Instat ® Biostatistics version 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The sample size calculation for this study was established using the resource equation approach, including minimum and maximum sample sizes, because it was not possible to assume standard deviation and effect size [56]. Data were tested for normality with the Kolmogorov-Smirnov and Shapiro-Wilk test. All variables were normally distributed. Differences between experimental groups were evaluated by using one-way ANOVA (histomorphometric and immunofluorescence analysis) and two-way ANOVA (weights) followed by Tukey's multiple comparison post hoc test. For all experiments, p-values of less than 0.05 (p < 0.05) were considered statistically significant; p values of less than 0.01 (p < 0.01) were considered to be highly statistically significant. The data are presented as the mean value ± SD. Cohen's κ was applied to measure the agreement between the three blinded observers and averaged to evaluate overall agreement.

Body Weight
Body weights and food and drink consumption were monitored throughout the experiment, 3 days per week, for a total of 48 time points. A physiological increase in body weight during the weeks in all groups was observed since the differences between groups, for each time point, are never significant (p > 0.05), as expected (Figure 3). At the start of the experiment, the mean ± SD body weight of all rats was 209.4 ± 13.52 g, at the end of the fourth week, it was 240.95 ± 10.95 g, reaching 290.7 ± 15.97 g, at the end of the sixteenth week, for the remaining animals.

Histology and Histomorphometry
Histological analysis with H&E were examined to highlight the possible alterations in muscle tissue of all experimental groups. No cytological altera tected in the muscle fibers of all groups. The morphometric analysis of the diam (mean ± SD) of the muscle fibers highlights a significant hypertrophy of t PA16W (30. 46

Immunohistochemistry
Skeletal muscle sections showed CD34+ cells at the periphery of the fibers, within the interstitium between muscle fibers (Figure 7). These cells appear to exhibit morphological features of TCs, i.e., spindle nuclei, approximately 5-10 μm in diameter, and multiple long cytoplasmic processes, approximately 10-25 μm in length and 0.1-0.2 μm in thickness, identifiable with telopodes. Comparison between PA16W and CTRL16W highlights a highly significant higher expression of TCs in exercised rats at 16 weeks (** p < 0.01). No other comparisons show to be significant. Data were tested for normality with the Kolmogorov-Smirnov and Shapiro-Wilk test, and differences between experimental groups were evaluated by using one-way ANOVA, followed by Tukey's multiple comparison post hoc test. The slides are scanned by confocal laser scanning microscopy (CLSM; Zeiss LSM700, Oberkochen, Germany) at 200× magnification. Scale bars: 50 µm. CTRL4W, control sedentary rats sacrificed at 4 weeks; PA4W, rats performing physical exercise sacrificed at 4 weeks; CTRL16W, control sedentary rats sacrificed at 16 weeks; PA16W, rats performing physical exercise sacrificed at 4 weeks.

Immunohistochemistry
Skeletal muscle sections showed CD34+ cells at the periphery of the fibers, within the interstitium between muscle fibers (Figure 7). These cells appear to exhibit morphological features of TCs, i.e., spindle nuclei, approximately 5-10 µm in diameter, and multiple long cytoplasmic processes, approximately 10-25 µm in length and 0.1-0.2 µm in thickness, identifiable with telopodes.

Discussion
TCs have been largely identified, over the last ten years, as populating the stromal compartments of a variety of organs, belonging to the tissue stem cell niche [33,49,65]. Within the skeletal muscle tissue, TCs are distributed throughout the perimysium and endomysium and could reach long-distances through their telopodes which allow these cells to make contact with myofibers, nerve terminals, blood vessels, and other stromal populations, including SCs, sited beneath the surrounding basal lamina of the myofiber.
In the present morphological study, the presence of TCs in tibialis anterior muscle of healthy rats who underwent a protocol of endurance training for either 4 weeks or 16 weeks was investigate in relation to sedentary rats who were inactive, i.e., not engaging in any physical exercise, throughout the duration of the experiment.
H&E staining shows no cytological alteration in the muscle tissue of all groups, although the morphometric analysis of the size of the muscle fibers highlights a significant

Discussion
TCs have been largely identified, over the last ten years, as populating the stromal compartments of a variety of organs, belonging to the tissue stem cell niche [33,49,65]. Within the skeletal muscle tissue, TCs are distributed throughout the perimysium and endomysium and could reach long-distances through their telopodes which allow these cells to make contact with myofibers, nerve terminals, blood vessels, and other stromal populations, including SCs, sited beneath the surrounding basal lamina of the myofiber.
In the present morphological study, the presence of TCs in tibialis anterior muscle of healthy rats who underwent a protocol of endurance training for either 4 weeks or 16 weeks was investigate in relation to sedentary rats who were inactive, i.e., not engaging in any physical exercise, throughout the duration of the experiment. H&E staining shows no cytological alteration in the muscle tissue of all groups, although the morphometric analysis of the size of the muscle fibers highlights a significant atrophy, defined as a decrease in the size of myofibers, of the sedentary control group at 16 weeks (CTRL16W) when compared to physically active rats undergoing treadmill training for both 16 weeks (** p < 0.0001) and 4 weeks (* p < 0.001) (Figure 4). Muscle morphological adaptations to active/inactive styles were not, however, accompanied by statistically significant differences (p > 0.05) in body weights in all groups, indicating a physiological growth of the rats during the weeks, as expected ( Figure 3).
Whether atrophy may affect muscle stem cells numbers or behavior is still controversial and should be further elucidated to assess the role of daily mechanical stress administered through exercise [66]. Mitchell et al. [67] reported that the number of stem cells in hindlimb muscles of mice was reduced after 2 weeks of hind limb suspension, and Verdijk et al. [68] observed similar decline in human vastus lateralis muscle following sarcopenia, reversed by resistance training which increased satellite cell content and type II muscle fiber size. Conversely, aerobic and resistance training have been reported to be a stimulus for the formation of new muscle fibers and maintain their homeostasis [4,5].
Although SCs are the main characters of renewal programme in skeletal muscle, other tissue residents and recruited stromal cells, e.g., fibroblasts, fibro-adipogenic progenitors, endothelial cells, perycites, and macrophages, are paramount supporting players [16,22,69]. Among these cell-cell interactions, TCs and SCs seem to interact by juxtacrine and paracrine intercellular signaling, in order to support in a concerted manner [13] the network mediating new tissue organization [70]. However, the interplay between these two cell types, especially in skeletal muscle injury [49], has yet to be elucidated in depth since cue-based investigations rely mainly on knowledge of the close proximity of TCs to SCs and their ability to communicate.
This study provides a novel finding that interstitial TCs are decreased in the muscles of sedentary rats compared to exercised rats, supporting the recent reports that TCs are, otherwise, increased in exercise-induced cardiac growth [55], participate in early moderate exercise-induced remodeling after acute myocardial infarct [71] and in eccentric contractioninduced skeletal muscle injury, in rodents. Since their discovery is relatively recent, TCs are not yet defined by specific antigenic markers, although CD34 is the most commonly used antigen to characterize their presence [48,72]. Vimentin, CD117/c-kit, PDGFR-β (plateletderived growth factor receptor β), and SMA (smooth muscle actin) are the antigens most frequently associated with TCs [47,62,73,74]. Bei et al. analyzed immunofluorescence double staining for cardiac TCs and fibroblasts in vitro. CD34/CD117, CD34/vimentin, and CD34/PDGFR-β were positive for TCs, whereas fibroblasts showed positivity only for vimentin and PDGFR-β [75].
In this work, double positivity for CD34+/CD117+ and CD34+/vimentin+ were used to individuate TCs and to discriminate this population from fibroblasts which are CD34-/CD117-/vimentin+. CD34 is a sialomucin mainly expressed in hematopoietic stem cells (HSCs) surface but it has also been found in other tissue-specific stem cells [76]. Vimentin is cytoskeletal type III intermediate filament protein found in mesenchymal-derived cells that provides an architectural network for organelles anchoring to cytoplasm [73]. CD117 is a transmembrane receptor tyrosine kinase widely used for TCs identification which is active in proliferation and differentiation [77]. Based on two distinct double-immunostainings for CD34/CD117 and CD34/Vimentin, skeletal muscle TCs were identified in all groups. CD34/CD177 statistical analysis indicates a significant difference in the higher level of TCs in exercised rats at 16 weeks in relation to their control group (* p < 0.05) ( Figure 5). This result is also more significantly supported by CD34/VIM double-immunostaining (** p < 0.01) ( Figure 6). These results indicate a potential targeting of TCs, as belonging to muscle stem cell niche, in cell dysfunction associated with atrophic condition [67]. This latter negatively affects stem cells via presence of catabolic factors such as myostatin [78] and tumor necrosis factor α [79] as well as decreases in trophic factors [80]. In contrast, in this work, rats subjected to regular physical training for 16 weeks maintained a stable TCs population, although not statistically increased compared to rats who performed exercise for only 4 weeks.
Finally, immunohistochemistry showed CD34+ cells, within the interstitium between muscle fibers (Figure 7), with characteristic features of TCs and telopodes, i.e., spindle nuclei and multiple long cytoplasmic processes. These observations are in support with current evidence suggesting TCs' role in intercellular signaling through their strategic position and organized network of telopodes with local cellular neighborhood, nerves, and capillaries [33,81,82]. Homo-and heterocellular communication seems to be carried out via small molecules and shedding microvesicles carrying various molecules like proteins, RNAs, and microRNA [49,83,84]. These mechanisms of transmission allow rapid cell engagement in the tissue milieu, in order to approach adaptations to biochemical and physical changes. The use of TEM would have been useful to determine the ultrastructural features of TCs in exercised rats and this should be noticed as a limitation of this study.

Conclusions
These findings herein are intended to encourage knowledge about TCs population and their role in the stem cells niche of skeletal muscles. Further studies investigating TCs in response to different types of exercise (resistance, aerobic, isotonic, flexibility), sedentary behavior, ageing, and pathophysiological conditions, as well as studies on the possibility of triggering TCs through exercise to reverse atrophic conditions, would be scientifically valuable.
This study may be framed in a field still in its infancy, although it is rapidly attracting the attention of the scientific community. The understanding of the above-mentioned mechanisms between the cells involved in the tissue remodeling process could offer new chances in regenerative tissue strategies and insights about finding possible triggers for TCs in sarcopenia and other musculoskeletal disorders, in clinical medicine.
Finally, since exercise training has been shown to exert protective effects against sedentary-induced atrophy, rather by sustaining muscle remodeling and maintenance of TCs, this finding might promote further skeletal muscle adapted physical activity and rehabilitation programmes for humans.