Nuclear factor-κB activation in Schwann cells regulates regeneration and remyelination

Abstract

Schwann cells (SCs) are crucial for peripheral nerve development and regeneration; however, the intrinsic regulatory mechanisms governing postinjury responses are poorly understood. Activation and deacetylation of nuclear factor-κB (NF- κB) in SCs have been implicated as prerequisites for peripheral nerve myelination. Using GFAP-IκBα-dn mice in which NF- κB transcriptional activation is inhibited in SCs, we found no discernable differences in the quantity or structure of myelinated axons in adult facial nerves. Following crush injury, axonal regeneration was impaired at 31 days and significantly enhanced at 65 days in transgenic animals. Compact remyelination and Remak bundle organization were significantly compromised at 31 days and restored by 65 days post injury. Together, these data indicate that inhibition of NF-κB activation in SCs transiently delays axonal regeneration and compact remyelination. Manipulating the temporal activation of nuclear factor-κB in Schwann cells may offer new therapeutic avenues for PNS and CNS regeneration.

Figure 1

Naïve GFAP-IκBα-dn nerves display no developmental aberrations. (A) Naive, WT facial nerve schematic showing the buccal (*) and mandibular (upper(U) and lower (L)) branches. Arrow indicates the cranial nerve exit site from the stylomastoid foramen, caudal to trifurcation. (B) Transverse, Toluidine Blue stains and electron micrographs (lower panels) of the buccal branch 8mm rostral to trifurcation. Scale bar: 20, 10, and 5 μm respectively from top to bottom panels. (C) Quantification of myelin rings (n=4). (D) Expression of MBP and MPZ proteins determined by Western Blot analysis (n=3–7). Results were obtained using stereology followed by the unpaired Student’s t-test and are expressed as the mean ± SEM.

Figure 2

Transgenic inhibition of NF-κB activation in denervated SCs following facial nerve crush injury. (A) Schematic representation of facial nerve crush model. (B) Immunostains of GFAP, phospho-p65, and DAPI within distal nerves 1 day following crush injury. Scale Bar: 20 μm. (C) Injured and uninjured buccal nerve. Arrow indicates crush site in the buccal branch. Neurofilament (NF) staining (top panels) of injured (ipsilateral) and uninjured (contralateral) nerves 2 days post injury. Scale Bar: 20 μm. (C) Fluorogold labeled motor neurons (bottom panels) in the facial motor nucleus 2 days post injury. Scale Bar: 50 μm.

Figure 3

Minimal activation of astroglial NF-κB within the facial motor nucleus following crush injury. Representative immunostains of GFAP (green), phospho-p65 (red), and DAPI (blue) within WT and transgenic (IκBα-dn) nerves 1, 4, and 31 days post injury. Note: astrocytic activation is strongest at 4 dpi and activated NF-κB is robust at 1 and 31 dpi, but not in astrocytes, in both groups. Scale Bar: 20 μm.

Figure 4

Quantification of gene expression using Real-time RT-PCR on cDNA (normalized to 18S) generated from buccal nerve tissue distal to the injury site. X-axis: number of days post injury. N: naive. Data are expressed as the mean ± SEM of 3 mice/group.

Figure 5

NF-κB activation in denervated SCs significantly influences axonal regeneration. (A) Fluorogold (FG) labeling of motor neuron (MN) cell bodies in the facial motor nucleus (FMN) of normal (WT) and transgenic (IκBα-dn) mice 12, 31 and 65 days post crush injury (dpi). Scale Bar: 50 μm (B) Stereological quantification of the number of FG+ MNs in the injured FMN. Results are expressed as the mean ± SEM of 5–9 animals/group. *p < .05, **p < .01, unpaired Student’s t-test (WT cpmared to transgenic). #p < .05, ###p < .001, one-way ANOVA.

Figure 6

Glial NF-κB activation is required for efficient axonal regeneration but not motor neuron survival 31 days after facial nerve axotomy. (A) Retrogradely labeled facial motor neuron soma within the facial motor nucleus of the brainstem following FG administration. Co: uninjured side. Ax: injured side. Scale Bar: 100 μm. (B) Quantification of FG⁺ MNs. Results expressed as a ratio (axotomized vs. control). Quantification of vibrissae motor performance scores. (C) Quantification of the total number of Cresyl Violet MNs (ipsilateral/contralateral). Quantification of MN soma area (μm²). Data expressed as the mean ± SEM of 4–5 anmials/group; Student’s -test (*, p<.05).

Figure 7

Effects of NF-κB inhibition on Schwann cell re-myelination. (A) Electron micrographs on transverse, ultrathin sections of the buccal branch 4 mm rostral to the crush injury site. *: intact, myelinated axon. Arrowhead: Remak bundle. SC: Schwann cell nuclei. Scale Bar: 5 μm. Quantification of intact, myelinated axons (B), average axonal diameter (C), gRatio (D), organized Remak bundles (E), unmyelinated axons (F) and Schwann cell nuclei (G) before and after injury. Geometric measurements were taken on all myelin rings encasing an axon regardless of ring morphology. X-axis: number of days post injury (dpi). N: naïve. Data were obtained from electron micrographs and are expressed as the mean ± SEM of 3–4 animals/group. *p<.05, **p<.01, unpaired Student’s t-test (WT vs transgenic). ###p<.001, one-way ANOVA.

Figure 8

Myelin-associated protein expression is unaltered in Schwann cells lacking NF-κB activation following crush injury. (A) Western blots of myelin-associated transcription factor (cJun, Krox-20) and structural (MPZ, MBP) protein expression within the distal nerve of normal (WT) and transgenic mice before (N) and 4, 12, and 31 days after injury. Lamin A/C serves as a loading control. (B) Densitometric quantification of protein expression normalized to WT naïve (N). AU: arbitrary units. Results are expressed as the mean ± SEM of 3–7 animals/group. *p<.05, unpaired Student’s t-test.

Figure 9

Wallerian degeneration is unaltered by transgenic inhibition of NF-κB in denervated Schwann cells. (A) Schematic representation of facial nerve transection model. (B) Transverse, semi-thin sections stained for myelin with paraphenylenediamine (PPD) and toluidine blue (TB). Sections were collected 4 mm distal to transection at 4 and 12 days post injury (dpi). Scale Bar: 10 μm. Bar graphs represent the total number of de-myelinating and intact axons. Data expressed as the mean ± SEM of 4–8 animals/group. WT: wild type. IκBα-dn: transgenic. (C) CD11b immunostain of macrophages in the distal facial nerve following injury and a naïve sciatic nerve. Bar graph represents stereological quantification of CD11b+ macrophages. Scale Bar: 20 μm. X-axis: days post injury. Results expressed as the mean ± SEM of 3–6 animals/group. (D) Longitudinal facial nerves distal to injury stained with myelin protein zero. Naïve: uninjured sciatic nerve from an adult WT mouse. Scale Bar: 20 μm.