Deletion of Nrf2 impairs functional recovery, reduces clearance of myelin debris and decreases axonal remyelination after peripheral nerve injury

Abstract

Oxidative stress is generated in several peripheral nerve injury models. In response to oxidative stress, the transcription factor Nrf2 is activated to induce expression of antioxidant responsive element (ARE) genes. The role of Nrf2 in peripheral nerve injury has not been studied to date. In this study, we used a sciatic nerve crush model to examine how deletion of Nrf2 affects peripheral nerve degeneration and regeneration. Our study demonstrated that functional recovery in the Nrf2(-/-) mice were impaired compared to the wild type mice after sciatic nerve crush. Larger myelin debris were present in the distal nerve stump of the Nrf2(-/-) mice than in the wild type mice. The presence of larger myelin debris in the Nrf2(-/-) mice coincides with less macrophages accumulation in the distal nerve stump. Less accumulation of macrophages may have contributed to slower clearance of myelin and thus resulted in the presence of larger myelin debris. Meanwhile, axonal regeneration is comparatively lower in the Nrf2(-/-) mice than in the wild type mice. Even after 3months post the injury, more thinly myelinated axon fibers were present in the Nrf2(-/-) mice than in the wild type mice. Taken collectively, these data support the concept of therapeutic intervention with Nrf2 activators following nerve injury.

Fig. 1

Ablation of Nrf2 delays motor function recovery but does not affect nociceptive reception. (A) Sciatic functional index (SFI) from walking track analysis before surgery, 2, 7, 14, and 21 days post surgery (n=7 for the sham groups and n=8 for the crush groups). Statistics performed by two-way ANOVA is shown in supplementary table S1. (B) Withdrawal reflex latency from analgesia hot plate analysis at 1-week post surgery (n=8). Sh: sham; cr: crush; con: contralateral hind foot; ip: ipsilateral hind foot. Statistics performed by two-way ANOVA is shown in supplementary table S2. **: p<0.01; ***: p<0.001.

Fig. 2

Neuromuscular junction (NMJ) innervation pattern after nerve crush. (A) NMJ was stained with α-bungarotoxin (red), synaptophysin (green) and neurofilament M (green). Three innervation patterns were observed: innervated (A-1 to A-3), partially innervated (A-4 to A-6) and denervated (A-7 to A-9). (B) Percentage of innervated, partially innervated and denervated NMJ endplate at 1 day, 1 week, 2 weeks and 3 weeks after nerve crush in the contralateral and ipsilateral tibialis anterior muscle from WT mice (n=4). Statistics performed by one-way ANOVA is shown in supplementary table S3. (C) Percentages of innerveted, partially innervated and denervated NMJ endplate at 1 week after crush from WT and KO mice (n=6). Statistics performed by two-way ANOVA is shown in supplementary table S4. *: p<0.05; **: p<0.01; ***: p<0.001.

Fig. 3

Axonal morphology 1-week post sciatic nerve injury. (A) Light microscopy images of semi-thin sciatic nerve sections stained with Methylene Blue/Azure II. The proximal end of the sciatic nerve mostly remains intact after crush, whereas the distal end is degenerated and most fibers are lost 1-week after injury. Bigger myelin debris seems to be present in the ipsilateral distal end nerve in the KO mice than in the WT mice (examples are given by ellipse). Scale bar: 20 μm. (B) Ultra-structural morphology of sciatic nerve by EM. The proximal end from both WT and KO remain well myelinated. Macrophages (denoted by arrow) infiltrate to the injured nerve and help to clear up myelin debris (denoted by stars). Bigger myelin debris exists in the distal end in the KO mice than in the WT mice. Scale bar: 5 μm. (C) Quantification for the number of myelin debris in the distal end of WT and KO mice (n=3). Statistics performed by t-test. P value is 0.209. (D) Quantification for size of myelin debris in the distal end of WT and KO mice (n=3). Statistics performed by t-test. P value is 0.004. **: p<0.01.

Fig. 4

Clearance of myelin debris by macrophages in the nerve. (A) MAG (myelin associated glycoprotein) immunohistochemistry in contralateral and ipsilateral nerves (conN and ipN) 1 week after injury for WT and KO mice. Green: MAG. Scale bar: 10 μm. (B) MAC2 (red) and CD68 (green) immunohistochemistry in conN and ipN one week after injury for WT and KO mice. Nerves were counterstained with Hoechst. Scale bar: 10 μm.

Fig. 5

Expression of macrophage markers after sciatic nerve crush. (A) Quantification of CD68 positive cells in the distal nerve stump of WT and KO mice at 1 week post injury. Statistics performed by t-test. P value is 0.048. *: p<0.05. (B) Protein levels of CD11b and MAC2 in contralateral nerve (conN) and ipsilateral nerve (ipN) at 3 days, 1 week and 2 weeks post injury for WT and KO mice. (C-D) Quantification of CD11b (C) and MAC2 (D) protein levels in conN and ipN at 3 days, 1 week and 2 weeks post injury (n=3). Statistics performed by two-way ANOVA is shown in supplementary table S5 and S6. *: p<0.05; **: p<0.01; ***: p<0.001.

Fig. 6

Remaining fibers, sprouting axons and deposits of myelin debris at 1-week after nerve crush. (A) Remaining fibers in the distal end of WT and KO mice at 1-week post injury (denoted by arrows). Scale bar: 5 μm. (B) New axonal sprouts appear in the distal end in both WT and KO mice (denoted by stars). Myelin debris present in the distal end of both WT and KO mice (denoted by pound key #). Scale bar: 5 μm. (C) Quantification of remaining fibers in the injured nerve of WT and KO mice at 1-week post injury (n=4). Statistics performed by t-test. P value is 0.147. (D) Quantification of axonal sprouts in the injured nerve of WT and KO mice at 1-week post injury (n=3). Statistics performed by t-test. P value is 0.144. (E) Protein levels for the neuron marker NF-M and axonal regeneration marker GAP43 in the contralateral and ipsilateral nerve (conN and ipN) 1-week after crush for WT and KO mice. (F) Quantification of NF-M and GAP43 protein levels at 1-week after crush for WT and KO mice. Statistics performed by two-way ANOVA is shown in supplementary table S7. *: p<0.05; **: p<0.01; ***: p<0.001.

Fig. 7

Axonal morphology and myelination after injury. (A) Light microscopy images of semi-thin sciatic nerve sections for contralateral nerve (conN) and ipsilateral nerves (ipN) at 1-week, 3-week and 6-week after injury in WT mice. Scale bar: 20 μm. (B) Light microscopy images of semi-thin sciatic nerve sections for contralateral and ipsilateral nerves (conN and ipN) at 3-month after injury from WT and KO mice. Scale bar: 10 μm. (C) G-ratio for regenerated axon fibers in the distal end of the sciatic nerve from WT and KO mice at 3-month after injury (n=3). Statistics performed by two-way ANOVA is shown in supplementary table S8. *: p<0.05; **: p<0.01; ***: p<0.001.