Peripheral nerve injuries represent a clinical problem and cause a profound and permanent impact on the patients’ life quality by altering their ability to perform activities of daily living, as well as their return to work [
The most common causes of peripheral nerve injuries are motor vehicle accidents, lacerations with sharp objects, fractures, and firearm or weapon injuries, among others [
In cases of peripheral nerve lesions in the lower limbs, the most commonly affected nerve is the sciatic [
Although the peripheral nervous system (PNS) has an intrinsic ability to regenerate, the treatment options available, in compression severe cases, produce unsatisfactory results, and currently the treatment of choice is microsurgical repair [
The exercise can contribute to the peripheral nerve injury treatment, at least in part, because it induces the synthesis of neurotrophic factors, which stimulate nerve growth. Therefore, therapeutic exercise could form a useful means for the stimulation of growth and regeneration of peripheral axons [
Furthermore, the literature shows that physical exercise could alleviate neuropathic pain, which may cause the release of endogenous opioids in the body, which consequently leads to reduced pain [
This study is characterized as an experimental research, with quantitative and random nature, which was developed after approval by the Ethics Committee on Animal Use (CEUA) of Universidade Estadual do Oeste do Paraná (Unioeste).
The sample consisted of 24 Wistar rats, males, mean age 10 weeks, weighing on average 352 g (375 g after the experiment), kept in standard boxes of polypropylene, in environment of
The animals were randomized into four groups consisting of six animals each. Control group (CG): the animals were not injured or submitted to the physical exercise protocol. Exercise group (GE): the animals were uninjured and perform resistance physical exercise protocol. Injury group (LG): animals were injured. Lesion exercise group (LEG): animals were injured and underwent resistance physical exercise.
The animals were subjected to sciatic nerve constriction injury, previously weighed, and anesthetized with ketamine (95 mg/kg) and xylazine (12 mg/kg) intraperitoneally. After verification of the animal’s state of consciousness, through the clamping of the tail and interdigital folds, trichotomy of the right posterior thigh and disinfecting the area with the use of povidone were held. Then, an incision parallel to the biceps femoris fibers to expose the sciatic nerve and subsequent compression of it was taken with a hemostat for 30 seconds in order to reproduce chronic pain on nerve path [
The clamping pressure was standardized for all animals, using as reference the second tooth rack hemostat, being performed by the same researcher. After the clamping, a tag at the lesion site was performed by means of graft suture using nylon filament 10.0 in order to facilitate the location of the injured nerve collecting region [
To carry out the treatment with resistance exercise, a vertical wooden ladder was used, which has 67 steps of iron, height of 1.18 m, width of 20.5 cm, and tilt 60°. At the top of the stairs, a dark box with a height of 18.5 cm and width of 15 cm, in which the animals were resting between one exercise series and another, was placed [
The procedure consists in the following: the animal performs two sets of 10 climbs of the ladder with overhead 100 grams adapted to its tail and with one-minute interval between one series and another. Treatment with climb stairs resistance exercise started from the third day after surgery (3rd PS—postsurgery) and they were performed 5 days per week, during 3 weeks, and they had 2 days break every 5 days. To perform the exercise on the stairs, all the animals were previously acquainted with the equipment for two weeks prior to surgery.
The method used to analyze and to measure allodynia was the paw withdrawal threshold with the aid of a digital analgesy-meter, filament type Von Frey. The equipment consists of an arm with a disposable polypropylene tip, with the ability to measure between 0.1 and 1000 grams, and an amplifier connected to box, that is, a pressure transducer adapted to a digital counterforce expressed in grams [
The filament was applied to the plantar region of the right hindlimb paw. Therefore, the animal was kept in high wooden box, with screen floor and the filament positioned at 90 degrees with the animal’s paw; pressure was applied with gradual increase in this region, until the animal withdraws its limb, by noting then the value of the withdrawal threshold indicated by the apparatus. To facilitate the adaptation of animals to this instrument in the three days prior to the injury, the simulation was carried out for this evaluation [
The evaluations related to allodynia occurred at the following times: a first assessment of baseline latency (BL) was before injury; the second evaluation (EV2) was on the 3rd PS before resistance exercise training (RET) and EV3 was on the same day, immediately after RET; the EV4 was on postoperative day 7 after RET; EV5 was on the 10th PS before RET; EV6 was on the 14th PO after RET; EV7 was on 21st PO after the last RET; and the last evaluation (no resistance physical exercise), EV8, occurred in 22nd PO, prior to euthanasia of animals.
In the end of the 22nd PS, the animals were weighed and again anesthetized with ketamine and xylazine, and 2 cm fragments were dissected from the right sciatic nerve distal. This had been compressed to carry out the morphological and molecular analyses. Then, the animals were euthanized by decapitation.
A part of the collected nerve fragments was set at bouin, embedded in paraffin, and subjected to cross sections of 5
In histology analysis, the features shown on the slide collection were observed and described, as the following structures: epineurium, perineurium and endoneurium, nerve fiber, inflammatory infiltrate, Schwann cells, fibroblasts, and blood vessels. The results were presented descriptively, from a detailed observation of the slide collection and illustrated in a histological board mounted with photomicrograph images of each group.
From the histological slides prepared, a section of each was selected which was photographed by Olympus BX 50 device. Using 100x objective, images were captured in 4 visual fields by systematically being located in the upper left quadrant, upper right, lower right, and bottom left, following the recommendations of Geuna et al. [
Based on the photomicrographs images were performed to measure the diameter of the nerve fiber (DNF), the axon (DAX), the thickness of the myelin sheath (TMS), and the ratio G (the ratio of the DAX/DNF). These measurements were made at 100 axons by nerve, 25 in each quadrant in order to obtain an equivalent number for comparison.
The results were expressed using descriptive statistics. The normality of the data was analyzed using the Shapiro-Wilk test. Then the results of functional evaluation were submitted to inferential statistics by ANOVA mixed measures with Bonferroni posttest; the results of morphometric analysis by one-way ANOVA with post-
Analyzing the data of the eight evaluations, a significant difference (
Comparing the groups, there was significant difference between CG and the others, in which it showed lower results than EG (
Data of mechanical allodynia measurements in different groups of study (mean ± standard deviation in grams) according to evaluation (baseline: BL; evaluation: EV).
Evaluations | Groups | |||
---|---|---|---|---|
CG | EG | LG | LEG | |
BL• | 62.99 ± 15.69 | 81.33 ± 23.31 | 66.52 ± 6.33 | 70.04 ± 19.21 |
EV2 | 50.55 ± 8.21 | 59.45 ± 16.03 | 33.00 ± 11.75 | 28.25 ± 8.68 |
EV3 | 48.50 ± 13.10 | 52.45 ± 19.40 | 26.47 ± 9.36 | 29.95 ± 15.04 |
EV4•• | 42.94 ± 12.02 | 50.79 ± 10.29 | 27.95 ± 9.77 | 19.50 ± 5.85 |
EV5 | 52.05 ± 11.97 | 61.04 ± 10.75 | 22.09 ± 9.14 | 25.05 ± 9.60 |
EV6 | 59.50 ± 11.00 | 50.87 ± 15.17 | 31.28 ± 7.30 | 25.33 ± 7.01 |
EV7 | 49.44 ± 12.36 | 59.41 ± 6.49 | 45.57 ± 15.71 | 29.00 ± 15.63 |
EV8 | 45.94 ± 14.71 | 65.58 ± 10.21 | 51.71 ± 14.08 | 28.33 ± 7.01 |
Mean | 51.48 ± 12.38 | 60.11 ± 13.95 |
38.07 ± 10.43 |
31.93 ± 11.32 |
Histological analysis of the sciatic nerve CG revealed that the nerve fibers were of normal range, with different diameters, and revealed the presence of Schwann cell nuclei at the periphery of the myelin sheath and fibroblast nuclei in the endoneurium. Perineurium consists of connective tissue modeled; it performed involving the whole nerve, forming concentric layers around the same; fibroblast nuclei were also visualized. Adjacent to the perineurium was observed epineurium which also consists of connective tissue but not modeled (Figures
Photomicrographs of the sciatic nerve of Wistar rats in cross section, and the 1st column images are stained with hematoxylin and eosin and the 2nd column with osmium tetroxide. ((a) and (b)) Control group of nerve fibers intact (
In the EG, the morphological approaches to what was observed in the control group are that the nerve fibers are presented in an organized manner, and no inflammatory cells or nerve fiber degeneration was found (Figures
However, at the LG, there was disruption of the nervous tissue with nerve fiber degeneration, increasing the number of Schwann cell nuclei, large amount of inflammatory infiltrate, with the presence of macrophages, forming giant cell clumps, increase in the amount of fibroblasts, and nerve fibers of smaller diameter compared to the control group. Additionally, the myelin sheath is made thinner and in some cases absent (Figures
In the LEG, very heterogeneous nerve fibers were noted, with being morphologically similar to the injury group, but other nerve fibers presented similar to that displayed in the control group, progressing to degree of normality (Figures
Regarding the nerve fibers diameter yielded significant differences (
Graphical representation of results for morphometry. (a) Nerve fiber diameter, which is smaller in injured groups; (b) axon diameter, which was lower in the group only exercised and injured; (c) thickness of the myelin sheath, which was also lower in lesioned groups; and (d) G ratio, which was higher in the control group compared to the others.
The diameter of the axon yielded significant differences (
As regards the thickness of the myelin sheath, the result was similar to nerve fiber diameter (
About the G ratio the results were significant (
About allodynia results in the plantar region of the animals right hindlimb, it was observed that, with the passing of the evaluations, the values are not matching the assessment before injury, which is in all cases greater, indicating that the exercise was not effective to increase the nociceptive threshold. However, only EV4 was significantly smaller than the other two. This indicates that, with the passage of time, there was a small increase in the nociceptive threshold.
The study by Galdino et al. [
Checking the comparison between the groups it was observed that the nonlesioned group showed significantly superior results to injured. However, the control group achieved a lower result than did only exercise, showing that although the exercise was not significantly effective in promoting increased nociceptive threshold, a sedentary lifestyle, represented in CG, it is not beneficial, since, in EG, the exercise promoted increased nociceptive threshold. That is, the climb stairs resistance exercise increased the nociceptive threshold in healthy animals but was not enough to influence the degree of neuropathic pain in the group of injured and treated animals.
Corroborating these findings, Mazzardo-Martins et al. [
Another factor to be highlighted is the time it took for the animals to be reevaluated after the exercise, which may have influenced the results of this study. According Galdino et al. [
In human studies, the effect of exercise in relation to pain has also been observed. Hoffman et al. [
Regarding the histological analysis of the sciatic nerve, it was observed that the injury model performed in this study was effective because it caused significant changes in the sciatic nerve of the animals. However, climb stairs resistance exercise on the proposed parameters failed to accelerate the healing process of all nerve fibers analyzed, but some fibers of the LEG showed up with normal features, like CG, showing a slight improvement of nervous tissue.
Corroborating this study, Raducan et al. [
On this same theme, Sta et al. [
Ilha et al. [
Similar to this study, Teodori et al. [
In the study, Ilha et al. [
As regards limitations of this study, it may be emphasized that the overload was similar in all the rats, not individualizing for each animal. And furthermore, evaluation of nociception occurred about 20 minutes after the exercise.
It is concluded from this study that in the proposed parameters the climb stairs resistance exercise was not effective in speeding the nerve regeneration process.
The authors declare that they have no competing interests.
The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for granting master’s scholarship.