Research ArticleNERVE REGENERATION

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates

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Science Translational Medicine  22 Jan 2020:
Vol. 12, Issue 527, eaav7753
DOI: 10.1126/scitranslmed.aav7753
  • Fig. 1 Study design and characterization of the PCL/GDNF nerve guide.

    (A) Schematic depicting experimental design. (B) Photograph of the 5.2-cm PCL/GDNF nerve guide. (C) SEM of the nerve guide cross section embedded with double-walled microspheres. Mag, magnification. (D) Diagram of the PCL/GDNF nerve guide cross section. (E) SEM of a bisected double-walled PLGA/PLA microsphere. (F) SEM of microsphere adhesion to the initial PCL layer during the manufacturing process. (G) Higher magnification of a cross section of a double-walled PLGA/PLA microsphere embedded in the PCL wall [rectangle in (C)]. EHT, electron high tension; WD, working distance.

  • Fig. 2 The gross morphological differences between treatment groups at the time of graft implantation and 1-year postoperative (explantation).

    Photographs of (A) exposed native nerve, (B) PCL/GDNF conduit explanted after 1 year, (C) implanted PCL/Empty conduit, (D) PCL/Empty conduit explanted after 1 year, (E) implanted PCL/GDNF conduit, and (F) autograft explanted after 1 year.

  • Fig. 3 Results and assessment technique for the analysis of NHP functional recovery.

    (A) Modified Klüver board with varying well diameters used for functional training and assessment. Well 1 has a diameter of 2.5 cm, and well 2 has a diameter of 0.5 cm. (B) Photograph of the correct pinching motion. (C) Photograph of the incorrect pinching motion. (D) Normalized functional bar graph comparing the NHPs’ 50-week functional recovery to their preoperative baselines. n.s., not significant. (E) Linear regression plot assessing functional recovery over 50 weeks for all treatment groups. n = 30 measurements per time point per NHP. Means represented with +SE/−SD. Adjusted P values presented as: **P < 0.01; ***P < 0.001 (select comparisons shown).

  • Fig. 4 Summary of the electrophysiological findings for the regenerated nerves in all the groups.

    Voltage waveforms over time in (A) native, (B) autograft, (C) PCL/Empty, and (D) PCL/GDNF groups. For each group, the top waveform, as indicated by “CNAP,” represents compound nerve action potential; and the bottom waveform, as indicated by “CMAP,” represents compound muscle action potential for the APB muscle. (E) The absolute median NCVs between native, autograft, PCL/Empty, and PCL/GDNF groups. (F) Absolute preoperative and postoperative median nerve conduction velocities between autograft, PCL/Empty, and PCL/GDNF groups. (G) The absolute median nerve–stimulated APB muscle amplitude changes between native, autograft, PCL/Empty, and PCL/GDNF groups. (H) Absolute preoperative and postoperative median nerve–stimulated APB amplitude changes between PCL/Empty and PCL/GDNF groups. n = 10 measurements per treatment group. Means represented with +SE/−SD. Adjusted P values presented as: *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5 Brightfield microscopy images of distal cross sections of the explanted native and regenerated nerves stained with Masson’s trichrome.

    All images were obtained from the distal nerve segment. (A) Native nerve, (B) autograft, (C) PCL/Empty, and (D) PCL/GDNF at ×100 magnification (scale bars, 500 μm). (E) Native nerve, (F) autograft, (G) PCL/Empty, and (H) PCL/GDNF at ×400 magnification. Scale bars, 100 μm.

  • Fig. 6 Fluorescent micrographs and quantification of Schwann cells in distal cross sections of the explanted native and regenerated nerves.

    Sections were stained with S-100 (red, indicating Schwann cells) and 4′,6-diamidino-2-phenylindole (DAPI) (blue, indicating nuclei). (A) Native nerve, (B) autograft, (C) PCL/Empty, and (D) PCL/GDNF (×400 magnification). (E) Average Schwann cell area fraction between native, autograft, PCL/Empty, and PCL/GDNF treatment groups. (F) Average Schwann cell perimeter between native, autograft, PCL/Empty, and PCL/GDNF treatment groups. n = 400 measurements per sample. Means represented with +SE/−SD. Adjusted P values presented as: *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 100 μm.

  • Fig. 7 Fluorescent micrographs and quantification of neurons in distal cross sections of the explanted native and regenerated nerves.

    Sections were stained with neurofilament (green, indicating neurons) and DAPI (blue, indicating cell nuclei). (A) Native nerve, (B) autograft, (C) PCL/Empty, and (D) PCL/GDNF (×400 magnification). (E) Average axonal area between native, autograft, PCL/Empty, and PCL/GDNF treatment groups. (F) Average axonal radius between native, autograft, PCL/Empty, PCL/GDNF treatment groups. n = 400 measurements per sample. Means represented with +SE/−SD. Adjusted P values presented as: ***P < 0.001. Scale bars, 100 μm.

  • Fig. 8 Degree of myelination as determined by g-ratio.

    (A) Native nerve, (B) autograft, (C) PCL/Empty, and (D) PCL/GDNF cross sections were stained with osmium tetroxide for contrast and viewed under brightfield microscopy at ×400 magnification. Red rings indicate the myelin, whereas green indicates the axon fiber. (E) Average g-ratio area between native, autograft, PCL/Empty, and PCL/GDNF treatment groups. (F) Average g-ratio perimeter between native, autograft, PCL/Empty, PCL/GDNF treatment groups. n = 60 measurements per sample. Means represented with +SE/−SD. Adjusted P values presented as: *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars, 100 μm.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/527/eaav7753/DC1

    Materials and Methods

    Fig. S1. CMAP amplitude for APB after ulnar stimulation.

    Fig. S2. Schematic of the histology study design.

    Movie S1. Preoperative functional assessment.

    Movie S2. Postoperative functional assessment at day 15.

    Movie S3. Postoperative functional assessment at week 50 after autograft.

    Movie S4. Postoperative functional assessment at week 50 after PCL/Empty scaffold.

    Movie S5. Postoperative functional assessment at week 50 after PCL/GDNF treatment.

    Data file S1. Primary data (Excel file).

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. CMAP amplitude for APB after ulnar stimulation.
    • Fig. S2. Schematic of the histology study design.
    • Legends for movies S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Preoperative functional assessment.
    • Movie S2 (.mp4 format). Postoperative functional assessment at day 15.
    • Movie S3 (.mp4 format). Postoperative functional assessment at week 50 after autograft.
    • Movie S4 (.mp4 format). Postoperative functional assessment at week 50 after PCL/Empty scaffold.
    • Movie S5 (.mp4 format). Postoperative functional assessment at week 50 after PCL/GDNF treatment.
    • Data file S1. Primary data (Excel file).

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