In our study, autograft facilitated the most sciatic nerve regeneration following segmental nerve surgery compared with collagen-based conduits. Significant group-level differences were only found with AD and axonal diameter distal to the surgical site, with autograft showing the greatest nerve regeneration, followed by hollow conduit and collagen-filled conduit, which may indicate that AD is potentially a viable DTI marker to detect group differences in axonal regeneration. Our results are corroborated by a prior ex-vivo rat study detecting a significant correlation of AD with axon diameter . A possible explanation for this is the sensitivity of eigenvalues to underlying changes in axonal swelling and flow , which is abundant in axonal regeneration  and could have a direct impact on AD. In theory, water diffusion parallel to the axon is decreased due to a restriction in flow during injury.
Previous studies have shown in-vivo DTI to be predictive of nerve degeneration and regeneration in rabbit [16,17,18, 20] and rat [8,9,10] sciatic nerve crush and laceration injury models. These studies demonstrated an initial FA decrease and RD increase in the sciatic nerve distal to the injury site immediately following sciatic nerve crush injury, presumably due to Wallerian degeneration. Thereafter, there was a period of significant FA increase and RD decrease due to regenerating myelin sheath [8, 10, 16,17,18, 20] until reaching preinjury levels at 10 weeks. In our study, we did not find a significant RD decrease by week 13, which may indicate that the regenerative phase was incomplete at this time. Previous animal studies have shown that FA and RD correlate with the ratio of myelinated axons and total number of myelinated axons , which concur with DTI degenerative studies in the brain [21, 22].
Several animal model studies have shown that bioabsorbable nerve conduits are efficacious in facilitating regeneration across nerve gaps [23,24,25,26,27,28]. Prior studies examined the best combination of biomaterials to replicate autograft properties . An ideal conduit should arguably include the presence of a basal lamina scaffold to serve as an adhesive and to promote dendrite elongation . A semipermeable inner structure facilitates the entry of growth factors, secretion of neurotrophic factors by nerve stumps, and the exchange of metabolic nutrients . The conduit should degrade at a rate long enough to warrant an environment in which regeneration and reorganization of the nerve is effective, but slow enough to deter scar tissue accumulation inhibiting longitudinal nerve growth [32, 33].
The Nerbridge™ collagen-filled conduit is a collagen-coated PGA conduit and porous collagen scaffold inner tube (collagen types I and III). It is biocompatible and commercially available for use in Japan. A previous rat study of a 1-mm transected laryngeal nerve bridged either with the collagen-filled conduit or direct suture assessed vocal fold mobility, nerve conduction velocity, morphology, and histology at 15 weeks following transection . No significant differences in vocal fold recovery or conduction velocity were found in either group. However, more clearly myelinated fibers and less laryngeal muscle atrophy were observed for the collagen-filled conduit, supporting its potential efficacy. As evidenced in prior studies using conduits composed of PGA (the material of the collagen-filled outer core of the conduit), the structure tended to resorb quicker than other synthetic conduits [23, 35], further evidenced by a fully dissolved collagen-filled conduit at 13 weeks postsurgery. PGA-based conduits were superior to autograft in sensory recovery assessments in nerve gaps of less than 4 mm and greater than 8 mm . A similar study in a rodent model involving much larger cohort sizes (without MRI) demonstrated overall similar results at the 12-week time point; however, analysis of a later 16-week time point revealed an accelerated rate of recovery in axonal density for collagen-filled conduit between 12 and 16 weeks postoperatively . This suggests that the collagen-filled conduit may show improved recovery over the course of a longer time point, which was not investigated here.
Several reasons may help explain significantly larger axonal diameter and AD in the hollow conduit compared with the collagen-filled conduit in our study. One of the theoretical benefits of nerve conduits is to impede negative growth factors (e.g., excessive scar tissue, edema, inflammation, necrosis) from the internal, regenerating environment. Faster degradation rates of the collagen-filled conduit may be detrimental to nerve regeneration, as evidenced by the two hollow conduits still intact by week 13 compared with the collagen-filled conduit, all of which had dissolved. This could perhaps be specific to rabbits, due to their reliance on hopping and hind limb muscle function  which may result in proportionally larger amounts of scar tissue generated, although results of various transplants and regenerative successes in a rabbit and rat model were similar . Rates of degradation as well as internal matrix morphology and pore size of conduits are areas that may warrant further exploration.
The extent of motor functional recovery was assessed by the change in maximum tetanic, CMAP, animal weight, ankle contracture, and muscle weight at the 13-week time point in the current study. Maximum tetanic is considered the most objective indicator of strength associated with the degree of muscle innervation by the nerve . However, in this investigation, tetanic contraction had a high variance but did show favorability towards the collagen-filled conduit and conflicted with AD and axonal diameter findings. Tetanic-induced contractions provide a purely functional response, which could be originating from smaller axons if they form a tighter connection with muscle tissue. Hence, axon diameter may not accurately reflect actual function.
Several limitations to this study should be considered. First, due to the small diameter of the rabbit sciatic nerve (approximately 2–3 mm) and a relatively low in-plane spatial resolution of the echo-planar sequence (1.7 mm), partial volume effects at the borders of the nerve may have biased DTI measurements by averaging of voxels that include both nerve and surrounding scar tissue/muscle and may have falsely elevated ADC and lowered FA. Second, the study could have benefitted from the inclusion of more rabbits in each surgical group due to borderline p values after Bonferroni correction. Third, unlike previous studies, our investigation did not evaluate continuous time points, but rather a single time point that we thought would afford enough time for significant axonal regeneration based on previous crush and laceration studies demonstrating nearly complete sciatic nerve remyelination for a rabbit sciatic nerve by weeks 8–10 [16,17,18]. In our study, axon density and diameter did not return to expected levels as compared with the nonoperated nerve by the 13-week time point, indicating the healing process is likely longer for nerve gap versus laceration/crush injuries. It may be prudent in future studies to examine multiple time points following segmental nerve surgery and at a longer follow-up time to accurately determine the longitudinal time course of nerve regeneration with MRI, histology, and functional testing. Fourth, CMAP and maximum tetanic force are commonly used in research studies measuring functional recovery in rats [26, 27, 41], but we are unaware of such studies in rabbits. Additional functional measurements including the Tarlov scale, which assesses locomotor ability , and the toe-spreading reflex, which assess the degree of toe spread , have been evaluated in other rabbit studies [16,17,18, 20] and may have been useful to assess functional recovery.
In summary, autograft facilitated greater sciatic nerve regeneration following segmental nerve surgery compared with collagen-based conduits, hollow conduit, and collagen-filled conduit. Hollow conduit performed significantly better with regards to the axonal diameter and the DTI parameter of axial diffusivity compared with collagen-filled conduit. However, collagen-filled conduit performed better with regards to the maximum tetanic force, which is the most objective measure of functionality. This investigation suggests that DTI may be a potential, noninvasive biomarker to assess peripheral nerve regeneration. Ultimately, larger studies will need to be conducted to determine its efficacy and role.