In this study, neurapraxia or axonotmesis by Seddon [16] and Sunderland [17] classification was simulated by traction injury with spring-balance device. Nerve injuries by traction or compression result in ischemia, intrafascicular edema, or demyelination. Usually, nerve damage showed slower recovery than other tissues and may take weeks to months for recovery [18]. In 2008, Mazzer et al. introduced a nerve injury device capable of applying constant and uniform loads in different quantities and varying lengths which is an important factor in inducing reproducible injuries [19]. However, traction injury of sciatic nerve was not simulated by Mazzer’s device. In this experiment, we could develop traction injury model of the sciatic nerve with various traction power, which might be utilized in the clinical situations.
Analyzing the sensory reaction of the rats was very challenging since they struggled during the test. To make it easy to examine the pinprick and toe-spreading tests, we waited 5 min to calm the animals down after gait analysis. The pinprick results showed no statistical difference between groups A and B. One of the possible explanations might be the traction power of 0.7N was reversible without sensory damage. Traction power over 1.0N could damage the sensory nerve irreversibly and impair function for long time. Because this study was terminated in 2 weeks after traction injury, long-term observation study is required to vindicate the reversal capacity of the traction damaged nerve.
In the walking track analysis, it is crucial to let the rats walk along the walking pathway freely before recording the steps. When the animal is placed in the pathway, they often tend to stop while pressing the entire footpad and heel-down, creating a false, untypical long print length. Also before entering the darkened pathway, the rat may stand up, putting all its weight onto its hind limbs and create a false long footprint [20]. A corridor was tailor-made to minimize the known errors, which was composed of a walking pathway, start-floor area, and end-floor area. The floors prevented false prints, and the rats were exposed to the printing paper only while walking down the pathway. Moreover, obtaining clear footprints might be challenging due to contractures, autotomy, smearing of the print, and dragging of the tail [14, 21]. The SFI was quite poor in group C which meant severe damage of the motor nerve.
In the previous study, partial weight bearing was started during the second week and the spreading of the toes during the third week post-injury [22]. Therefore, recognition and precisely marking the key points were reported to be very challenging in the first 2 weeks. Hence, in the present study, the test was performed 2 weeks post-injury. The results showed no significant difference between groups A and B, and groups B and C, which may be explained by the short recovery period after the nerve injury.
Kobayashi et al. reported that the gastrocnemius and extensor digitorum longus muscles were suitable to evaluate denervation effect of the experimental animals 6 months after damage [23]. However, denervation leads to adipose and fibrous tissue formation which increases the total mass, so it is hard to dissect the muscle only [24]. Therefore, in this study, samples were collected within the minimal healing period after the injury which minimizes fibrosis and adipose tissue formation. Moreover, the muscles were excised very carefully, eliminating any other tissues from the samples in an effort to improve the reliability of the results, which showed significant difference among the three groups.
Mazzer et al. showed that large diameter fibers were damaged first and more intensely following an injury, and small diameter fibers were relatively preserved [19]. However, after a certain threshold, the small fibers are also broken, and the damage increases rapidly in proportion to the load applied. The lower range of stretch which produces histological changes has been reported to be between 4 and 50% of the initial length [25, 26]. In our previous pilot experiment for this study, rupture of the sciatic nerve started at 3~6 N tensile strength from which point the forces went directly inside the nerve and causing complete disruption. The difference of the myelin thickness showed that fibers undergoing axon atrophy predominated over fibers undergoing demyelinization, thus indicating that the predominant type of injury produced by tension is axonotmesis. The differences of the myelin thickness and G-ratio in the three groups showed that more tensile strength results to more severe damage, which as a result, it changed the myelin thickness and G-ratio. Moreover, separation gaps were observed in the experimental groups which can be explained by the disruption of the vessels and discontinuity of nerve fiber due to the tension injury. Also, disruption on the vessels will cause hematoma, and as a result, narrow partial areas and separation gaps were observed in the longitudinal sections. Nonetheless, same pattern of damage was observed to some extent in the control group, which may be attributed to nerve handling, tissue harvesting, and histological preparation artifacts. Irregular round-shaped axons with irregular thicknesses of myelin were observed in sciatic nerve samples which made it difficult to estimate the diameter of axons and the total number of the axons. In this study, four different cross sections of the samples were used to measure the total number of axons and axon density. Some investigators applied the three-window sampling method to measure the total number of axons [27]. In this technique, all fascicles that could contain three separate rectangular windows of 0.012 mm2 were included. One of these three windows was placed in the center, whereas the other two were randomly positioned on both sides at the periphery of the fascicle in the opposite direction. In the present study, both techniques were used and compared. The results showed no significant difference between the two methods. Nonetheless, Cai et al. reported significant differences between the data obtained by the three-window sampling methods and the conventional method [28]. In this study, the tension injury decreased the total number and the density of axons, which was negatively correlated with the amount of the tensile strength applied.