Testing the effectiveness and the contribution of experimental supercharge (reversed) end-to-side nerve transfer

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OBJECTIVE

Supercharge end-to-side (SETS) transfer, also referred to as reverse end-to-side transfer, distal to severe nerve compression neuropathy or in-continuity nerve injury is gaining clinical popularity despite questions about its effectiveness. Here, the authors examined SETS distal to experimental neuroma in-continuity (NIC) injuries for efficacy in enhancing neuronal regeneration and functional outcome, and, for the first time, they definitively evaluated the degree of contribution of the native and donor motor neuron pools.

METHODS

This study was conducted in 2 phases. In phase I, rats (n = 35) were assigned to one of 5 groups for unilateral sciatic nerve surgeries: group 1, tibial NIC with distal peroneal-tibial SETS; group 2, tibial NIC without SETS; group 3, intact tibial and severed peroneal nerves; group 4, tibial transection with SETS; and group 5, severed tibial and peroneal nerves. Recovery was evaluated biweekly using electrophysiology and locomotion tasks. At the phase I end point, after retrograde labeling, the spinal cords were analyzed to assess the degree of neuronal regeneration. In phase II, 20 new animals underwent primary retrograde labeling of the tibial nerve, following which they were assigned to one of the following 3 groups: group 1, group 2, and group 4. Then, secondary retrograde labeling from the tibial nerve was performed at the study end point to quantify the native versus donor regenerated neuronal pool.

RESULTS

In phase I studies, a significantly increased neuronal regeneration in group 1 (SETS) compared with all other groups was observed, but with modest (nonsignificant) improvement in electrophysiological and behavioral outcomes. In phase II experiments, the authors discovered that secondary labeling in group 1 was predominantly contributed from the donor (peroneal) pool. Double-labeling counts were dramatically higher in group 2 than in group 1, suggestive of hampered regeneration from the native tibial motor neuron pool across the NIC segment in the presence of SETS.

CONCLUSIONS

SETS is indeed an effective strategy to enhance axonal regeneration, which is mainly contributed by the donor neuronal pool. Moreover, the presence of a distal SETS coaptation appears to negatively influence neuronal regeneration across the NIC segment. The clinical significance is that SETS should only employ synergistic donors, as the use of antagonistic donors can downgrade recovery.

ABBREVIATIONS CMAP = compound muscle action potential; DiI = 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; ETE = end-to-end; ETS = end-to-side; NIC = neuroma in continuity; SETS = supercharge ETS.

Article Information

Correspondence Mustafa Nadi: NSHA/Dalhousie University, Halifax, NS, Canada. mustafanadi@yahoo.com.

INCLUDE WHEN CITING Published online May 18, 2018; DOI: 10.3171/2017.12.JNS171570.

M.N. and S.R. share first authorship of this work.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A: Experimental groups. Group 1 (G1), treatment group, NIC with SETS. Group 2 (G2), injury control group, NIC without SETS. Group 3 (G3), positive controls. Group 4 (G4), SETS control group, transection with SETS. Group 5 (G5), negative control group. The tibial nerve was injured close to its origin, with NIC performed using a malleus nipper. SETS was performed from the peroneal nerve (green), through an epineural window 15 mm distal to the injured segment of the tibial nerve (pink). All stumps were capped following transection to prevent spurious regeneration. Distal black bars represent sections for histomorphometric analysis and the site for final introduction of the retrograde tracer. Nippers are used to create the NIC injuries, and the scalpel to create the neurotomies. B: SETS repair in the treatment group (G1). There is a double NIC injury in the proximal tibial nerve. SETS was performed 15 mm distal to the injured segment between the peroneal and tibial nerves using 10-0 sutures. Figure is available in color online only.

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    Mean ± SEM of CMAP amplitudes of the experimental groups. The sham tibial injury control group (group 3) exhibited a stable CMAP amplitude throughout the study that was significantly better (***p < 0.05) than that of all other groups from the week 2 to week 8 assessments. The other groups all showed a progressive CMAP recovery at the final end point, except the negative control (group 5), which remained poor throughout. Note that there was no statistically significant difference between the other 3 groups. Figure is available in color online only.

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    Serial behavioral assessment by ladder rung tests with slip ratios plotted over time. The graph represents the mean slip ratios ± SEM for each group. All groups demonstrate impaired locomotion throughout the study, with only modest improvement from week 4 to week 10 in the treatment group (group 1) and SETS control group (group 4), with no statistically significant difference between the other groups. Figure is available in color online only.

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    Representative images and counts of retrograde-labeled motor neurons in the lumbar spinal cord sectioned in the longitudinal coronal plane following distal tibial nerve exposure to the fast blue (FB) tracer. Labeling in the treatment group (group 1) is dense and widespread, presumably reflecting contribution from both tibial (right side of image; more caudal and medial) and peroneal (left side of image; more rostral and lateral) neuronal pools. More restricted distribution of labeled cells in the caudal part of the spinal cord lumbar area reflects tibial motor neuron pools in groups 2 and 3. Also, note the overall sparse, and less dense, labeling in the SETS control group (group 4), which represents the contribution from the peroneal nerve alone. Calibration bar is 500 µm (*p < 0.05). Figure is available in color online only.

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    Representative toluidine blue–stained cross sections of the distal tibial nerve of the various groups. Larger images correspond to ×40 magnification, whereas the inset images are ×400 magnification. The sham control group (group 3) contains a population of normally distributed, well-myelinated axons. All other groups show evidence of regenerated axons, which are somewhat smaller in caliber than the sham but well myelinated by week 10. Bar = 50 µm. Refer to Table 1 for quantitative summary of myelinated axon counts and other indices. Figure is available in color online only.

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    Representative images of retrograde-labeled motor neurons in the lumbar spinal cord sectioned in the longitudinal coronal plane after initial distal tibial nerve exposure to DiI (red fluorescence) prior to surgery and fast blue tracer (blue label) postsurgery. A: Representative low-power micrograph from group 1 demonstrating the distribution of the tibial (DiI labeled) and peroneal pools (fast blue labeled). B: Group 2 gives rise to the highest amount of double-labeled neurons, which can be noted by the coloration of the neurons (double-labeled are light/dark purple, with examples denoted by arrows, while single-labeled neurons are clearly either red or blue). Figure is available in color online only.

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    Phase II counts (mean ± SEM) from primary and secondary labeling of all experimental groups. A: The mean DiI labeling counts (D0) in groups 1, 2, and 4 represent the baseline number of motor neurons in the tibial pool. B: Total fast blue (FB) counts following secondary retrograde labeling from the distal tibial nerve at study termination. This is reflective of the collective labeling of all regenerated neurons and was found to be higher in group 1 than in group 2, suggestive of increased neuronal regeneration following SETS. The difference was statistically significant and is consistent with the findings in phase I. C: Exclusive FB (FB0) labeling count demonstrates that the group 1 labeling counts are higher than those in group 2 due to the presence of SETS. The group 4 counts are low, despite the presence of SETS, suggestive of better maturation of SETS in the presence of NIC compared with transection. The difference between groups 1 and 4 was statistically significant. D: The double-labeled count (FB+) shows that the counts are lower in group 1 than in group 2, suggestive of decreased tibial neuronal regeneration across the NIC in the presence of SETS. *p < 0.05; ***p < 0.005.

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