Peripheral Nerve Regeneration Is Independent From Schwann Cell p75NTR Expression

Abstract

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75^NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75^NTR knockout mouse models and cannot dissect the specific role of p75^NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75^NTR expression in Schwann cells was generated, where p75^NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75^NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75^NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75^NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75^NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75^NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75^NTR in remyelination most likely depending on axonal/neuronal p75^NTR and/or mutual glial-axonal interactions.

Keywords: Schwann cells; myelination; nerve injury; p75NTR; regeneration.

FIGURE 1

P75^NTR inactivation in Schwann cells. (A) Double-label immunofluorescence microscopy of p75^NTR (green) and βIII-tubulin (pink) in P11 sciatic nerve of SC-p75^NTR-KO and WT littermates. mCherry emitting fluorescence was identified in channel 568. Nuclei are labeled with Hoechst (blue). The overlap of tubulin and p75^NTR reflects axonal p75^NTR (white). Note that in the WT, there is a noticeable amount of p75^NTR staining that does not overlap with tubulin (green color in the merge), possibly due to p75^NTR expression in Schwann cells. Images represent n = 4 mice/group; scale bar 25 μm. (B) Fluorescence images of cells that were primary cultured from adult mice sciatic nerves, fixed and stained with an antibody against p75^NTR (green). mCherry 568 fluorescence was observed in Schwann cells from SC-p75^NTR-KO but not WT littermates. In the merge panel, it is clear that p75^NTR was only present in Schwann cells and not fibroblasts from the WT nerves (cells with bigger nuclei displayed with Hoechst, blue), being completely absent in the SC-p75^NTR-KO. Scale bar 10 μm; n = 16 mice/group. (C) P75^NTR was determined by immunoblot analysis in extracts of sciatic nerves isolated from adult mice. Quantification of p75^NTR ratios by densitometry against levels of β-actin. Data represent the mean ± SEM for six mice/genotype (^∗∗∗p < 0.001 compared with littermate controls). (D) Immunofluorescence microscopy of Caspr (green) in naïve P11 sciatic nerves. Note similar intensity and distribution of immunoreactivity throughout the tissue in both genotypes. Nuclei are identified with DAPI (blue) and mCherry visualized in the red channel in SC-p75^NTR-KO mice. Scale bar, 50 μm; n = 4 mice/group.

FIGURE 2

Sensorimotor phenotypic analysis before and after sciatic nerve crush injury. (A) Mechanical nociceptive functional recovery evaluated using the Von Frey filaments before and 1, 7, 14, 21, and 28 days after nerve injury. Significant difference noticed between contralateral sham and injured paw at 1- and 21-days post-lesion in the SC-p75^NTR-KO group, as consequence of nerve injury (^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). (B) Nociception evaluated with the Hargreaves test at baseline (time = 0), 1, 7, 14, 21, and 28 days after nerve crush injury; (^*p < 0.05, ^∗∗p < 0.01). (C) Locomotor function recovery assessed by walking tract analysis and calculation of SFI at pre-treatment (time = 0), 1, 7, 14, 21, and 28 days after nerve crush injury. Nerve conduction velocity analysis of sural (sensory) (D) and sciatic (motor) (E) nerves in naïve SC-p75^NTR-KO and control littermates (n = 6 mice per genotype). (F) Nerve conduction velocity measurements in both genotypes 29 days after injury (^∗∗∗p < 0.001; n = 6 SC-p75^NTR-KO and n = 8 littermates).

FIGURE 3

Ablation of p75^NTR in Schwann cells has no impact on re-myelination after nerve injury. (A) Representative light microscopy images of toluidine-blue stained semithin transverse sections of sham contralateral and distal stumps from crush injured nerves, 15- and 29-days post-lesion. Scale bar 20 μm. (B–G) Histograms obtained from the morphometrical analysis of the myelinated fibers. (B) Total number of myelinated fibers, (C) axon diameter, (D) fiber diameter, (E) myelin thickness, and (F) g-ratio. Scatter plots showing g-ratio of individual myelinated axons as a function of axon diameter in contralateral shams (G) or injured nerves at time points of 15 (H) and 29 days post lesion (I). Numbers for quantifications were as follows: n = 11 sham, n = 6 at 15 days, and n = 6 at 29 days for SC-p75^NTR-KO; n = 9 sham, n = 3 at 15 days, and n = 6 at 29 days for littermates.

FIGURE 4

Trend toward decreased C-fiber density in injured nerves from mice lacking Schwann cell p75^NTR expression. (A) Representative images of ultrathin transverse sciatic nerve sections from sham contralateral nerves (left panel) and 29 days post injury distal stumps (right panel) of the two experimental groups. Arrowheads identify C-fibers. Scale bar 2 μm. (B) Density of C-fibers expressed as number of unmyelinated axons per square millimeter (n/mm²) Numbers for quantifications were as follows: n = 12 sham, n = 6 at 15 days, and n = 4 at 29 days for SC-p75^NTR-KO; n = 9 sham, n = 3 at 15 days, and n = 4 at 29 days for littermates. (C) Electron micrographs showing re-myelinated fibers (asterisk) in both SC-p75^NTR-KO and control WT littermates, 15 days after sciatic nerve crush injury. (D) Representative pictures of segmental demyelination observed in both strains after sciatic nerve injury, both at internodal and paranodal regions. Scale bar 5 μm.