Research ArticleNEUROSCIENCE

Peripheral nerve transfers change target muscle structure and function

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Science Advances  02 Jan 2019:
Vol. 5, no. 1, eaau2956
DOI: 10.1126/sciadv.aau2956


  • Fig. 1 Experimental nerve transfer model.

    A high-capacity multifascicular donor nerve originating from a different spinal topography was surgically transferred to selectively reinnervate a single target muscle instead of its original motor branch. The contralateral untreated side was used as control. The nerve transfer successfully reinnervated the target muscle, and no additional reinnervation was observed during dissection or electrophysiological testing. MCN, musculocutaneous nerve.

  • Fig. 2 Structural hyper-reinnervation of the targeted muscle was indicated by retrograde labeling.

    (A) The number of motor units reinnervating the targeted muscle increased significantly (unpaired Student’s t test, P = 0.006), from 29.33 ± 10.01 in control muscles (n = 6) to 50.56 ± 13.58 (n = 9) by 172.35% at 12 weeks after the nerve transfer. The donor ulnar nerve contained 280.50 ± 25.87 (n = 6) motor neurons, but only 18.02% reinnervated the targeted muscle (unpaired Student’s t test, P < 0.0001). ***P < 0.01. (B) Example of the ulnar nerve’s motor neuron column in the spinal cord of a control rat (scale bar, 50 μm; longitudinal section, rat spinal cord).

  • Fig. 3 Confocal and multiphoton imaging of muscular reinnervation after the nerve transfer.

    (A and B) The high-capacity donor nerve contained a higher axonal load than the original motor branch. This led to the formation of a neuroma at the muscular insertion point. The typically severe pain or consecutive relieving posture following neuroma formation was not present in any animal. (C) The nerve transfer reinnervated the muscle following the route of the original motor branch. Axonal architecture showed the typical arborization of axons as they approached neuromuscular junctions in nerve transfer muscles (n = 6). In the six targeted muscles, a total of 2120 neuromuscular junctions were analyzed (353.33 ± 133.55) per animal. Denervated (n = 1, 0.16%) or polyinnervated (n = 17, 2.79%) neuromuscular junctions were present only in one animal. All other animals did not show any denervated or polyinnervated neuromuscular junction. (D) Axons innervated multiple muscle fibers proximal or adjacent to each other. Innervation of neuromuscular junctions by reinnervating axons was confirmed by matching fluorescence expression of axons (green) and neuromuscular junctions (red).

  • Fig. 4 Nerve transfers changed muscle fiber populations.

    These were analyzed using immunohistochemistry against MHC proteins. (A) Control muscle samples (n = 10) of the biceps’ lateral head showed predominantly fast MHC-IIb fibers (red; 63.30%, 4445.10 ± 1787.08) gathering around a core of intermediate MHC-IIa fibers (green; 32.63%, 2291.70 ± 995.99) and fewer slow MHC-I fibers (golden; 4.07%, 285.80 ± 204.51). (B) After the nerve transfer, populations changed substantially as MHC-IIb fibers (red; 46.44%, 2622.30 ± 953.35) were reduced, while MHC-IIa (green; 44.84%, 2531.70 ± 1566.45) and MHC-I fibers (golden; 8.72%, 492.10 ± 313.53) increased (n = 10). Above the dashed line is the medial head of the biceps whose innervation remained unchanged, containing mainly MHC-IIB fibers (red). The overall number of muscle fibers between the nerve transfer and control muscles did not statistically differ at 12 weeks [analysis of covariance (ANCOVA), P = 0.43], suggesting no significant muscle fiber loss due to denervation. (C) Lumbrical muscles innervated by the donor ulnar nerve physiologically show a similar fiber pattern as the biceps after the nerve transfer (B), with exclusively slow MHC-I and intermediate MHC-IIa fibers.

  • Fig. 5 Functional and structural muscle analyses.

    Muscle weight, force, and motor unit number estimations (MUNEs) significantly increased between weeks 3 and 12 (ANCOVA, P < 0.0001), indicating a progressive reinnervation of the target muscle after the nerve transfer. Muscle force was low (34.98 ± 15.45%, n = 10) compared with the contralateral control side at 3 weeks, but significantly increased to 75.87 ± 24.39% (n = 11) at 6 weeks (ANCOVA, ***P < 0.001) and to 92.57 ± 10.15% (n = 9) from 6 to 12 weeks (ANCOVA, **P = 0.006). Muscle mass initially decreased to 81.38 ± 6.52% because of denervation compared with control at 3 weeks (n = 14) but increased to 88.25 ± 6.40% (n = 13) at 6 weeks and to 98.18 ± 2.95% (n = 14) at 12 weeks. The differences between weeks 3 and 6 and between weeks 6 and 12 were both significant (ANCOVA, *P = 0.014 and ***P < 0.0001, respectively). Motor units increased from 56.62 ± 14.87% (n = 10) compared with control at 3 weeks, to 111.22 ± 44.58% (n = 11) at 6 weeks, and 116.31 ± 24.50% (n = 9) at 12 weeks. Differences between weeks 3 and 6 were statistically significant (ANCOVA, ***P < 0.0001).

  • Fig. 6 Electrophysiological muscle analyses.

    Histograms of the muscle fiber conduction velocity (MFCV) and motor unit action potential amplitude for the reinnervated (A and B) and control (C and D) sides. Data from nine animals are pooled together (100 and 70 motor units were detected from the nerve transfer and control sides, respectively; no motor unit action potentials were observed for two animals in the control side). Greater conduction velocity was observed following the nerve transfer [2.50 ± 0.86 m/s versus 2.25 ± 0.75 m/s; P = 0.017, mixed model analysis, Gaussian distribution (A and C)]. For both groups, the amplitude distribution was skewed (B and D), and the linear mixed model was applied to the logarithmic values of the amplitude. The amplitude was not affected by the nerve transfer (P = 0.15, linear mixed model analysis). (E) Epimysial high-density EMG signals were recorded with multichannel electrode arrays with 16 oval detection sites (140 μm by 40 μm) with 1-mm intersite distance from the biceps lateral head muscle. Three arrays were applied over the epimysium along the muscle fibers, and asynchronous motor unit activity was induced by sequential crushes of the ulnar nerve in the reinnervated side, as represented in this figure (or the musculocutaneous nerve in the control side). (F) Action potentials generated by the same motor unit (gray traces) were averaged (black traces) and characterized by the conduction velocity. Two to four channels with clear propagating components (red traces) were selected to calculate the propagation delay. Conduction velocity was obtained as the ratio between the intersite distance and the propagation delay. In the representative example shown in this figure, the conduction velocity calculated from the three selected channels from the central electrode was 2.01 m/s. MU, motor unit.

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