Donor nerve axotomy and axonal regeneration after end-to-side neurorrhaphy in a rodent model

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OBJECTIVE

In this study, the authors used a surgical model of end-to-side neurorrhaphy between a nerve graft and a donor tibial nerve in adult rats to investigate the optimal conditions for axonal regeneration induced by the donor nerve. They also assessed the importance of a more favorable pathway using a predegenerated nerve graft to attract regenerating axons to regrow into the graft and then directing and improving their growth toward the target in comparison with results obtained with a fresh nerve graft.

METHODS

End-to-side neurorrhaphy was performed between a nerve graft and a donor tibial nerve. The nerve graft was obtained from the left tibial nerve, which was either freshly removed or predegenerated 1 week prior to neurorrhaphy. The donor right tibial nerve was injured by epineurium removal alone, injured by epineurium removal with cross section of 20% or 50% of the total axons at the coaptation site, or left intact. The animals were followed postoperatively for a 6-week period, and outcomes were evaluated by optical microscopy and retrograde labeling to detect the regenerated primary sensory neurons located in the lumbar dorsal root ganglia and spinal motor neurons located in the lumbar spinal ventral horn.

RESULTS

At the end of the follow-up period, no regenerating axons were observed in the nerve grafts when the donor nerve was left intact, and very few axons were detected when the donor nerve was injured by epineurium removal alone. However, numerous regenerating axons appeared in the grafts when the donor nerve was axotomized, and the greatest number was achieved with a 50% cross section axotomized nerve. In the rats with a 50% cross section of the donor nerve, better nerve-like morphology of the grafts was observed, without connective adhesions. When a predegenerated nerve graft was used, more regenerating axons were attracted and elongated with a more regular shape and improved myelination.

CONCLUSIONS

Axonal regrowth into a nerve graft depends on axotomy of the donor nerve after end-to-side neurorrhaphy. More efficient attraction and an improved structure of the regenerating axons were achieved when a predegenerated nerve graft was used. Furthermore, a nerve graft may require a certain number of regenerating axons to maintain a nerve-like morphology.

ABBREVIATIONS CTB = cholera toxin subunit B; DRG = dorsal root ganglion; FG = FluoroGold (hydroxystilbamidine); SE = standard error.

Article Information

Correspondence Song Liu: U 1195, INSERM, Université Paris-Sud and Université Paris-Saclay, Le Kremlin-Bicêtre, France. song.liu@inserm.fr.

INCLUDE WHEN CITING Published online February 16, 2018; DOI: 10.3171/2017.8.JNS17739.

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.

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Figures

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    A–C: Schematics showing the end-to-side neurorrhaphy procedures between a nerve graft and a donor tibial nerve. After exposing the right tibial nerve in situ as a donor nerve, end-to-side neurorrhaphy was performed (A) with a fresh or predegenerated nerve graft from the left tibial nerve at a location 3 mm distal to the bifurcation of the tibial and common peroneal nerves. The proximal end of the nerve graft was cut into 2 flaps (B) to create a fish mouth and then surgically bridged to the coaptation site of the donor tibial nerve. Four conditions (C) were created at the coaptation site of the donor tibial nerve: no injury (intact), epineurium removal alone, epineurium removal with a quarter cross section of the donor nerve corresponding to approximately 20% of the total axons (20% axotomy), and epineurium removal with a half cross section of the donor nerve corresponding to approximately 50% of the total axons (50% axotomy). For epineurium removal alone, a 3-mm-diameter region of the epineurium was carefully removed from the tibial nerve using microscissors to open a window and expose the axons while avoiding injury. D: Flow diagram of this study showing the timing of the surgical procedures and evaluations.

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    Retrograde labeling of lumbar spinal cord motoneurons or DRG sensory neurons was performed by applying the retrograde tracer CTB-Alexa 555 at the level of the nerve graft 5 mm distal to the coaptation site. Either fresh nerve grafts (FNG) or predegenerated nerve grafts (PNG) were used to perform neurorrhaphy with the donor tibial nerve. The tibial donor nerve remained intact (noninjured), was injured by removing the epineurium alone to open an epineurium window (EW), or was injured by removing the epineurium along with the cross section of approximately 20% or 50% of axons. A: Global analysis of the data using 2-way ANOVA revealed significant effects at 2 weeks after neurorrhaphy. Spinal cord motoneurons (left): effect of the graft type (F[1,24] = 7.16, p ≤ 0.05), effect of the donor nerve condition (F[3,24] = 188.3, p ≤ 0.001), and a significant interaction between the 2 factors (F[3,24] = 4.6, p ≤ 0.05). DRG sensory neurons (right): effect of the graft type (F(1,24) = 5.29, p ≤ 0.05), effect of the donor nerve condition (F(3,24) = 100.3, p ≤ 0.001), and a significant interaction between the 2 factors (F[3,24] = 6.97, p ≤ 0.01). B: At 6 weeks after surgery, 2-way ANOVA revealed similar main effects and a significant interaction between the graft type and the donor nerve condition. Spinal cord motoneurons (left): effect of the graft type (F[1,24] = 8.73, p ≤ 0.01), effect of the donor nerve condition (F[3,24] = 159, p ≤ 0.001), and a significant interaction between the 2 factors (F[3,24] = 3.79, p ≤ 0.05). DRG sensory neurons (right): effect of the graft type (F[1,24] = 7.01, p ≤ 0.05), effect of donor nerve condition (F[3,24] = 32.04, p ≤ 0.001), and a significant interaction between the 2 factors (F[3,24] = 5.8, p ≤ 0.01). Following global 2-way ANOVA, between-group differences were analyzed by Tukey’s post hoc tests. ****p ≤ 0.0001; ***p ≤ 0.001; *p ≤ 0.01; *p ≤ 0.05. Notably, results of comparisons between EW and noninjured groups were always nonsignificant.

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    Representative photomicrographs showing retrograde labeling of motoneurons in the right ventral horn of the lumbar spinal cord at 6 weeks after surgery. A: Neurons (red) that had grown axons into the nerve graft and were retrogradely labeled with CTB-Alexa 555. The tracer was applied to the nerve graft at a location 5 mm distal to the coaptation site. B: Neurons (blue) with axons projecting into the distal part of the tibial donor nerve that were retrogradely labeled with FG. The tracer was applied to the donor tibial nerve at a location 5 mm distal to the coaptation site. C: Double-labeled neurons (purple, indicated by arrows) after merging. These neurons correspond to regenerating neurons projecting 2 axon branches, one into the nerve graft and one into the distal part of the tibial nerve. Bar = 50 µm.

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    Representative photomicrographs showing myelinated axons in thionin-stained semi-thin sections of the nerve graft at 6 weeks after the operation. Nerve grafts were either fresh (left column) or predegenerated (right column). No regenerating axons were observed in the fresh (A) or predegenerated (B) nerve grafts when the donor nerve was left intact for end-to-side neurorrhaphy. Very few scattered axons were observed in the fresh (C) and predegenerated (D) nerve grafts when the donor nerve was injured by epineurium removal alone. An evident increase in the number of myelinated axons was observed in the fresh (E) or predegenerated (F) nerve grafts when the donor nerve was cross-sectioned by 20% at the coaptation site, but no significant differences in the numbers of axons present in these nerve grafts were observed. Abundant myelinated axons were observed in the fresh (G) and predegenerated (H) nerve grafts when the donor nerve was cross-sectioned by 50%. Note the size and regular shape of regenerating axons and their surrounding myelin sheaths when a predegenerated nerve graft was used (H). Bar = 50 µm. Quantitative analysis of the number of myelinated axons within the fresh nerve grafts (FNG) or predegenerated nerve grafts (PNG) (I) 6 weeks after neurorrhaphy. At the coaptation site of neurorrhaphy, the donor nerve was noninjured, injured by removing the epineurium alone to open an epineurium window (EW), or injured by removing the epineurium along with the cross section of approximately 20% or 50% of axons. Two-way ANOVA revealed significant effects of the graft type (F[1,16]) = 6.2, p ≤ 0.05) and donor nerve condition (F[3,16] = 109.8, p ≤ 0.001) and a significant interaction between the 2 factors (F[3,16] = 5.1, p ≤ 0.05). Between-group differences were analyzed using Tukey’s post hoc tests. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. Figure is available in color online only.

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    Graphs showing the frequency distribution of the cross-sectional areas of myelinated axons in fresh (A) or predegenerated (B) nerve grafts at 6 weeks after end-to-side neurorrhaphy with donor tibial nerves that were cross-sectioned by 50%. The cross-sectional areas of myelinated axons in rats receiving a fresh nerve graft ranged from 1 to 30 µm2, which was smaller than the range of 3–70 µm2 for rats receiving a predegenerated nerve graft.

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    Representative electron micrographs showing myelinated axons in ultra-thin sections of nerve graft at 6 weeks after the operation. Nerve grafts were either fresh or predegenerated. Myelinated axons within a fresh nerve graft (FNG) were small and grouped into fascicles, likely reflecting axonal sprouting. In contrast, myelinated axons within a predegenerated nerve graft (PNG) appeared individually and were large and surrounded by thick myelin sheaths. Bar = 5 µm.

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