The mean internal fascicular areas, number of axons, minimum axonal diameters, and axonal densities of the sham and SET groups are given in Table 1. The greater density of unmyelinated fibers in the SET group is explained by reduced fascicular area and increased variability in the number of unmyelinated axons. Figure 5 presents the frequency distribution for minimum axonal diameter in both groups. The axon diameters of the normal fibular nerve ranged from 0.2 to 2.8 μm. The minimum diameter of axons in the SET group ranged from 0.2 to 2.6 μm. There were more unmyelinated axons between 1.0 and 1.2 µm in the sham group, whereas there were more axons with minimum diameters between 0.4 and 0.6 µm in the SET group (Fig. 5).
The unmyelinated fibers in this study traversed the 5-mm gap created by SET and reached the distal segment of the fibular nerve. A literature search did not reveal previous experimental studies on unmyelinated fibers with SEN, either with or without tubulization from the normal tibial nerve to the fibular stump of a previously divided fibular nerve, which is the most common experimental model for SEN. We conducted tubulization using a rigid silicon tube with the intention that future studies can build on our work and observe the actions of nerve growth promoters. Although both rigid and nonrigid tubes have been used in previous studies, they have not previously been used in studies of unmyelinated fibers with SEN.33 , 34 , 37
The study evidence a difference between fascicular areas in half of the SET group that was lower than in the sham group. At this time, 70 days post surgery, data describing nerves area in current developing at axonal phase, referring to the regenerating process not completed yet.44 Data about perineurial layers difference between groups support this report, supposing that at the final regeneration process, the perineurial layers will be condensed in a unique multilayered cellular membrane.45
Although some previous studies have demonstrated that a perineurial window induces greater collateral fibers growth,46 , 47 we chose not to use an epi-perineurial window, due to work of Viterbo et al.24 not revealing any differences between end-to-side neurorrhaphies with and without perineurial window concerning the morphological features. Our findings demonstrate profuse growth of unmyelinated fibers, which were often more numerous than the average number of fibers in normal nerves. Future studies are needed to compare the growth of sensory and motor fibers this tubulization experimental model.
Unmyelinated fibers in the fibular nerve are generally classified as 73% afferent (sensory) and 27% efferent (sympathetic).48 The majority of quantitative studies of unmyelinated fibers in peripheral nerves are conducted on the sural nerve.
Unmyelinated fibers or C fibers carry thermal and pain sensations; therefore, the sural nerve is commonly studied because 92% of its axons conduct sensory information.6 A previous SEN study used the sural nerve as the donor nerve at the distal stump of the fibular nerve and evaluated myelinated and unmyelinated fibers. After 56 days, there were <800 axons observed 4 mm from the neurorrhaphy site in the fibular nerve.39 This SEN procedure was conducted without tubulization of the sural-fibular nerves; therefore, more profuse growth of unmyelinated fibers was expected. In the present study, we found a mean of 2,011 unmyelinated axons (median = 1,349.5) in the distal end of the tubulized fibular nerve. Despite the prominent axonal growth in our research, even applying a tubular scaffold on a gap comparing with the study with SEN above cited, we can’t assert that tibial nerve are better donor nerve than sural nerve, because the experimental models approaches are different.
We studied adult rats (130 days) because the sciatic nerve has more axons during development than in adult rats. Axons are eliminated during the maturation process.49 Furthermore, by using the tibial nerve as a source of unmyelinated fibers, we avoided the possibility of transitory sprouts that might experience further elimination, because axon growth appears to be stable in the tibial nerve. After axotomy or nerve crush injury in adult rats, the number of unmyelinated axons transiently increases before returning to normal numbers.50 A previous experiment by Kovacic found that coaptating the rat sural nerve as a lateral donor to the fibular nerve resulted in stabilization of unmyelinated fiber growth at 2–4 months after surgery.39 This leads us to suppose that our research time (70th day) contemplates the same situation.
Lack of those modalities of sensations can be potential causes of pressure ulcers and accidental injuries, as occur in some predominantly sensory neuropathies, such as leprosy and hereditary sensory neuropathies. So, it is recommended in the clinical setting, not only a clinical evaluation but a quantitative sensory test evaluation of those sensations of the skin supposedly reinnervated after nerve regeneration surgical procedure.
The present study demonstrates plasticity of unmyelinated axons with SET, using the tibial nerve as a donor. Long-term studies are needed to study changes in thermal and pain sensation that result from fibular nerve sectioning and to evaluate the stability of the unmyelinated fiber growth accomplished by SET. It is possible and even probable that in SEN without tubulization, even greater fiber growth occurs. The effect of nerve growth enhancers should also be studied, and SET is an appropriate model for these studies.
The authors thank Mr. Antonio Renato Meirelles e Silva, Mrs. Aracy Edwirges Vieira da Silva Dias, Mrs. Maria Teresa Picinoto Maglia, and Mr. José Augusto Maulin for their technical assistance, as well as Mr. Geraldo Cássio dos Reis for his assistance with the statistical analyses.
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