ENDURANCE TRAINING INDUCES STRUCTURAL AND MORPHOQUANTITATIVE CHANGES IN RAT VAGUS NERVE

1. Universidade São Judas Tadeu, Department of Physical Education, São Paulo, SP, Brazil. 2. Universidade Cruzeiro do Sul, Department of Physical Education, São Paulo, SP, Brazil. 3. Universidade de Guarulhos, Department of Physiotherapy, São Paulo, SP, Brazil. 4. Universidade São Judas Tadeu, Department of Physical Education, São Paulo, SP, Brazil and Universidade de São Paulo, Department of Anatomy, São Paulo, SP, Brazil.


INTRODUCTION
The quantitative changes promoted by endurance training on somatic nerves are well established.There is an increase in the area of myelin sheath (MA), unmyelinated axons (UA) and myelinated fibers. 1,2,35][6][7][8] Goldsmith et al. 4 and Iellano et al. 5 demonstrated that moderate endurance exercise increases the vagal tonus and diminishes the action of the sympathetic nerve, protecting the heart.
][17] It contains also a number of collagen fibers in the interstitial space among nerve fibers. 18n this study we have used electron microscopy to check the influence of aerobic training on quantitative aspects from myelinated and unmyelinated fibers of the vagus nerve using the Wistar rat as an animal model.The effects of treadmill training on vagus nerve structure were assessed by comparison of two sets of rats: sedentary animals of the same age and rats of the same body weight as the exercised cohort.Therefore, this study aimed to analyze the effect of endurance training on the vagus nerve morphology and ultrastructure of rats

MATERIALS AND METHODS
Male Wistar rats (6 months of age) were acclimated during one week before the initiation of the experiment.It received commercial food and water ad libitum, and housed in a light-and temperature--controlled room.After acclimatization, rats were randomly divided into two groups: control group (CG) (n=8) and aerobic trained group (AT) (n= 8), both sacrificed with 9 months of age.

Training protocol
AT group was submitted to a treadmill exercise training program according to previously described. 19Before training, rats were familiarized with the equipment by running for 15 min/day during 5 days.After these, rats performed the protocol 5 days/week during 12 weeks, with speed and duration progressively increased.AT rats began training at 12 m/min for 15 min/day.The speed was progressively increased such that by the end of the second week, the rats were running at 16 m/min for 60 min/day, 5 days/week.The duration was maintained but speed was increased 3-4 m/min each week.At week 10, the animals ran at 32 m/min for 60 min/day and this program was maintained until rats were sacrificed by the end of the protocol.Just to pass it by, animals from CG were placed on the treadmill one time per week for 10 min/session (speed= 5m/min) to provide an equivalent amount of stress.All animals were weighted one/month from beginning to end.All experimental procedures were approved by the Institutional Animal Care and Use Committee of Universidade São Judas Tadeu, São Paulo, Brasil.
At the end of the experiment, the rats were anaesthetized with sodium pentobarbital (60 mg/Kg, i.p.) and then euthanized.The hearts were excised in diastole, heparinized and the myocardium was perfused through the aorta using a 2.5% glutaradehyde diluted in 0.1 M cacodylate buffer.The left ventricle, including the septum, was then isolated and weighted.A perpendicular section to the long axis of the left ventricle including the entire thickness of the left ventricle (LV) was cut at the level of the papillary muscles.Then, the thickness of the LV wall and internal diameter were obtained by measuring the widths of four of uniformly spaced sites along the length of each section using a computerized program (Axio Vision, Carl Zeiss AG, Jena Germany).
Under a dissecting microscope, the vagus nerve trunk was exposed at mid-cervical level and bathed in fixative solution (2.5 % glutaraldehyde -2% paraformadehyde in cacodylate buffer 0.1 M, pH 7.4).Segments 5 mm in length of the vagus nerve were harvested, fixed (4 h, 4º C) and rinsed in buffer.Nerve segments were then cut, post-fixed in osmium tetroxide (2%, 2 h, 4º C), dehydrated through a graded series of ethanol and contrasted with uranyl acetate.In order to obtain transverse sections, the nerve was oriented longitudinally in a silicone mold and embedded in Araldite.
To determine the transversal section area of the nerve, semi-thin sections of 0.3 µm were obtaining, stained in 1 % toluidine blue and evaluated with a light microscope combined with an image analyzing system (Axion Vision, Zeiss).
The density of Schwann cells (number/µm 2 ), the cross-sectional area of myelinated axon (µm 2 ) and the thickness of myelin sheath (µm) in each section was obtained directly on electron micrographs at a magnification of x4000 on 10 fields chosen by systematic random sampling of squares 13 , covering representative parts of the nerve profile.The ratio of axon diameter to the total fiber diameter (g ratio diameter -a measure of degree of myelination) was also calculated for the two groups.
Only myelinated axon (MA) and Schwann cell (SCs) whose contour was completely within each field was counted.In addition, the number of unmyelinated axons (UMs) present in each section was obtained with electron micrographs at a magnification of x6000 on 10 randomly chosen fields per animal.The density of MA (number/µm 2 ) of the nerve was also determined.
Five electron photomicrographs from myelinated fibers per animal were taken (x50) and neurotubules and neurofilaments were counted over a representative unit rectangular sampling area (2.16 µm 2 ).

Statistical analysis
The results were expressed as means ± standard deviation.Between groups data was compared statistically by ANOVA and Tukey's test for group comparisons.The level of significance was set at p≤0.05 (figure 1).

Effects of endurance training on body weight and cardiac hypertrophy.
The ratio Left ventricle weight-body weight (LVW/BW) increased 19% in AT group (p < 0.01) compared to the CG group.The LV internal diameter increased 20% in the AT group (p < 0.05) compared with the CG group.No significant difference was observed in the body weight and LV wall thickness between the CG and AT groups (table 1).months of endurance training, both, thickness of myelin sheath and of the unmyelinated axon were significantly larger than CG group (p<0.05).The average value of the g ratio was 0.6 for CG and 0.7 for AT.However, statistical difference was not significant (p > 0.05).The mean number of microtubules and neurofilaments per unit area was greater in myelinated axons in the AT rats than in the controls (table 2).
Figure 4 shows the histograms for the myelinated axon diameters in the two groups of rats.Myelinated axon diameters were unimodally distributed for both groups of rats and a right-ward shift of the distribution was observed for the AT group of rats.

Qualitative Morphological changes
Photomicrographs of representative semithin sections of the vagus nerves obtained from CG and AT group rats appear in figure 2. Transverse sections from CG and AT showed nerves enveloped by a well-defined Perineurium.The endoneural space contained numerous nerve fibers and several blood vessels.The diameter of the nerve seems to be greater in AT than in CG rats.
Electron photomicrographs of cross sections of the nerves showed myelinated fibers of various diameters, intermingled with unmyelinated ones and several capillaries within the endoneural space.Other endoneural space components were Schwann cells, interstitial tissue and fibroblasts (figure 3).
The density of myelinated fibers (number/µm 2 ) in CG and AT rats was 18 ±3 and 17 ± 2, respectively.The cross-sectional area of the vagus nerve increased significantly in the trained group in relation to the CG group (p < 0.05).The axon diameter of myelinated axon in AT group was significantly larger than that of CG rats (p < 0.05).After 3

DISCUSSION
There are reports on the effects of endurance training on peripheral nerves in rats, but the vagus nerve has never been investigated from this point of view. 20,21The importance of examining the effects of training on the right vagus nerve resides on the fact that the right vagus nerve innervates mainly the sinoatrial node which is of particular interest in regard to heart rate control during training.
There are two major findings in the present work.First, rats subjected to chronic endurance training for 12 weeks exhibited a significant increase in the diameter of the axon and of the myelin sheath of the vagus nerve fibers compared to sedentary controls.The diameter of the unmyelinated fibers increased significantly in trained rats.Second, an increase in the number of microtubules and neurofilaments per unit area was detected in myelinated axons from trained rats.
In the present study, no significant differences were observed between body weight of sedentary and trained rats and AT animals have larger LV internal diameter compared to CG.These results were similar to those described in the literature. 22During aerobic training, in addition to the increased cardiac output, the blood pressure increases.Consequently, the LV must adapt to both volume and pressure loads.The LV responds by increasing its internal diameter. 23,24Therefore, the aerobic-trained heart develops eccentric hypertrophy. 25he results of the present study show that in rats submitted to endurance training, the diameter of the axon and of the myelin sheath of the vagus nerve fibers increases.The diameter of the unmyelinated fibers also increases.Furthermore, the histograms of the frequency distribution of axon diameter of the vagus nerve showed a unimodal distribution for both groups of rats with a right-ward shift of the distribution for the AT group of rats.No significant difference was observed between the average values of the g ratio for the two groups.The quantitative nerve changes observed in the present study could be induced by the increase of the vagus nerve function promoted by training.Increase in the nerve function with training is related to factors such as increased production of growth factors and nucleic acids, which result in a series of chemical events leading to gradual increase in neuron sizes. 26he exposure to training probably induces production of proteins, lipids Brain-derived neurotrophic factor, also known as BDNF.This is a secreted protein that, in humans, is encoded by the BDNF gene. 27DNF is a member of the "neurotrophin" family of growth factors and was the second neurotrophic factor to be characterized after nerve growth factor (NGF).These factors are found in the brain and the periphery; they support the survival of neurons and the growth of new ones.Exercise has been shown to increase the secretion of BDNF as a myokine at the mRNA and protein levels in the rodent hippocampus, suggesting the potential increase of this neurotrophin after exercise in humans. 26he present work showed correlation between the myelin sheath and the diameter of the respective axon, i.e. there was no significant difference between the groups about the g ratio.This means that trai-ning promotes a similar growth in myelin sheath and axon.According to Rushton 28 values between 0.6 and 0.7 would be the g ratio for the best and maximum conduction velocity of a myelinated fiber but Fazan et al. 29 have shown that small myelinated fibers as observed in baroreflex afferents have g ratios between 0.5 and 0.6.
In the present study, not all types of nerve fibers were affected by training in the same way.The increase of myelinated axon diameter by aerobic training involved particularly the medium sized axons, the small diameter nerve fibers remaining virtually largely intact.The cause is not known.
In the present study, number of neurotubules and neurofilaments per area also increased in myelinated axons from trained rats.According to Hoffman et al. 30 , Komiya 31 and McQuarrie et al. 32 an important determinant of axon size is the rate of transport of neurofilaments.It is possible that the progressive increase in the rate of neurofilament transport during training promoted an increased diameter of the axons.This hypothesis could explain the increased axonal diameter in training rats observed in this work.

Figure 2 .
Figure 2. Semithin transverse sections of the Vagus nerves of CG (A) and AT (B) groups of rats.Note the cervical vagus nerves are enveloped by a well-defined perineurium (arrowheads).The endoneural space contains hundreds fibers and several blood vessels (arrows) accompanying the longitudinal axis of the fascicles.Diameter difference was observed between CG and AT rats.Toluidine blue stained.

Figure 3 .
Figure 3. Electron photomicrographs of cross sections of vagus nerve from CG (A) and AT rats (B) showing a number of myelinated (M) and unmyelinated (U) fibers of various sizes and various nuclei of Schwann cells (arrows).

Figure 4 .
Figure 4. Histograms showing the frequency distribution of axon diameter of the vagus nerve.These histograms include all nerves measured: a total of 120 fibers of CG rats and 120 fibers in AT group of rats.Note the unimodal distribution for both groups of rats and the right-ward shift of the distribution for the AT group of rats.

Table 1 .
Body weight (BW), Left ventricle weight/body weight (LVW/BW) ratio, Left ventricle wall thickness and Left ventricle internal diameter in sedentary (CG) and trained rats (AT).
Values are the means ± SEM. *Significant vs. CG.

Table 2 .
Quantitative analysis of structural changes in number of fibers, axon diameter, myelin sheath sizes, g ratio and number of microtubules and neurofilaments in the vagus nerve of rats from CG and AT groups.