Netrin-1 mediates nerve innervation and angiogenesis leading to discogenic pain

Abstract

Objective: Discogenic low back pain (LBP) is associated with nociceptive nerve fibers that grow into degenerated intervertebral discs (IVD) but the etiopathogenesis of disease is not fully understood. The purpose of this study was to clarify the role of Netrin-1 in causing discogenic LBP.

Methods: The level of nociceptive nerve innervation was examined in disc degenerative patients and rat needle-punctured models by immunohistochemistry. Nucleus pulposus (NP) cells were isolated from IVD tissues of rats and induced degeneration by interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα). The candidate genes related to neuron outgrowth and migration were selected by Next-generation sequencing (NGS). CRISPR/Cas9 was used to knockdown Netrin-1 in NP cells. The impact of Netrin-1 on nerve innervation were evaluated with P2X2、NF200 staining and microfluidics assay. Meanwhile the CD31 staining and transwell assay were used to evaluate the impact of Netrin-1 in angiogenesis. The proteins and RNA extracted from NP cells related to catabolism and anabolism were examined by western blot assay and RT-qPCR experiment. ChIP and luciferase experiments were used to assess the intracellular transcriptional regulation of Netrin-1. Further, a needle-punctured rat model followed by histomorphometry and immunofluorescence histochemistry was used to explore the potential effect of Netrin-1 on LBP in vivo.

Results: The level of nerve innervation was increased in severe disc degenerative patients while the expression of Netrin-1 was upregulated. The supernatants of NP cells stimulated with IL-1β or TNFα containing more Netrin-1 could promote axon growth and vascular endothelial cells migration. Knocking down Netrin-1 or overexpressing transcription factor TCF3 as a negative regulator of Netrin-1 attenuated this effect. The needle-punctured rat model brought significant spinal hypersensitivity, nerve innervation and angiogenesis, nevertheless knocking down Netrin-1 effectively prevented disc degeneration-induced adverse impacts.

Conclusion: Discogenic LBP was induced by Netrin-1, which mediated nerve innervation and angiogenesis in disc degeneration. Knocking down Netrin-1 by CRISPR/Cas9 or negatively regulating Netrin1 by transcription factor TCF3 could alleviate spinal hypersensitivity.

The translational potential of this article: This study on Netrin-1 could provide a new target and theoretical basis for the prevention and treatment for discogenic back pain.

Keywords: Discogenic back pain; Intervertebral disc degeneration; Nerve innervation; Netrin-1.

Figure 1

Nerve innervation in degenerative intervertebral disc tissue. a Immunohistochemistry to detect P2X2⁺ cells in mild, moderate and sever degenerative IVD tissues. Scale bar, 20 ​μm ​b Percentage of P2X2⁺ cells in IVD (n ​= ​6, N ​= ​2–4, each group). ∗∗p ​< ​0.01 compared with mild group. c Representative images of Safranin O (second row), AlcianBlue (third row), NF200 staining (fourth row) and P2X2 staining (sixth row) of needle-punctured IVD after 2 weeks and 4 weeks. Scale bar, 80 ​μm ​d Histological grading scores at different time points after needle puncture. ∗∗∗p ​< ​0.001 compared with sham group, ###p ​< ​0.001 compared with 2-week time point. Results are shown as means ​± ​SD.

Figure 2

Netrin-1 expression level increases after inducing intervertebral disc degeneration. a Gene set enrichment analysis (GSEA) for GO_Neuron_Migration pathway enrichment analysis of cultured rat nucleus pulposus cells treated with IL-1β (10 ​ng/ml) or b TNFα (50 ​ng/ml) for 48 ​h. c, d Heatmaps of differential genes in GSEA set. e, f Volcano plots (Fold change versus P value). g A Venn diagram showing the intersection of differential gene sets of IL-1β (red circle) and TNFα (bule circle) groups. h qPCR analysis for representative differential genes Netrin-1 of degenerative nucleus pulposus cells (n ​= ​6, each group). i Immunoblots showing Netrin-1 and Col2, ADAMTS5, SOX9, MMP13 in nucleus pulposus cells treated with IL-1β or TNFα for 48 ​h. j Quantification of the blot (n ​= ​3, each group). k Representative immunohistochemistry of Netrin-1 in human IVD tissues. Scale bar, 80 ​μm (left column), 40 ​μm (right column). l Summarized analysis of percentage of positive staining (red arrow) cells (n ​= ​4, each group). Results are shown as means ​± ​SD. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 3

Netrin-1 induces axonal growth and vascular endothelia cells' migration. a The protein expression levels of Netrin-1 in Western blot of nucleus pulposus cells transfected with CRISPR/Cas9-ctrl gRNA or NTN1 gRNA1 and NTN1 gRNA2. b Quantification of the blot (n ​= ​3). c Schematic drawing of the design for the conditioned medium (CM) model. d Expression of Netrin-1 in the conditioned mediums of groups as detected by ELISA (n ​= ​5). e Representative photomicrographs of DRG neuron axonal outgrowth in microfluidics assay treated with nucleus pulposus cells transfected with CRISPR/Cas9-ctrl gRNA or NTN1 gRNA conditioned medium induced by IL-1β or TNFα for 48 ​h (n ​= ​4). Scale bar, 100 ​μm f Analysis of the relative length of axons. g Transwell migration assay of human umbilical cord endothelial cells (HUVECs) co-cultured with nucleus pulposus cells transfected with CRISPR/Cas9-ctrl gRNA or NTN1 gRNA for 14 ​h (n ​= ​4). Scale bar, 1 ​mm ​h Quantified results of mean migrate cells. Results are shown as means ​± ​SD. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 compared with ctrl groups, #p ​< ​0.05, ##p ​< ​0.01 ###p ​< ​0.001 compared with groups transfected with CRISPR/Cas9-ctrl gRNA.

Figure 4

Animal spinal hypersensitivity pain behavior in needle-punctured rats. a Representative MRI images of L4/5 needle-punctured rats after 3 ​weeks ​b Analysis of guarding behavior (n ​= ​8). Score scale: 0 - no guarding, 1 - mild shift of weight away from paw; 2 - unequal weight bearing with some part of foot not touching the floor; 3 - paw licking, foot completely raised. c 50% paw withdrawal threshold (PWT) of sham, AAV-Null and AAV-NTN1 gRNA groups in the ipsilateral and d contralateral right paws tested by von Frey filament. Results are shown as means ​± ​SD (n ​= ​8, each group). ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 compared with sham group, #p ​< ​0.05, ##p ​< ​0.01 ###p ​< ​0.001 compared with AAV-Null group at the corresponding time points. e Representative images of immunofluorescence showing DAPI (blue), CGRP (green) of sham and needle-punctured rats transfected with AAV-Null or AAV-NTN1 gRNA at 4 weeks and 8 weeks (n ​= ​8). Scale bar, 60 ​μm f Quantification of the CGRP ​⁺ ​area. g Representative images of immunofluorescence showing CD31 (red) and h quantification of CD31⁺ area of sham and needle-punctured rats transfected with AAV-Null or AAV-NTN1 gRNA at 4 weeks and 8 weeks (n ​= ​8). Scale bar, 60 ​μm. Results are shown as means ​± ​SD (n ​= ​8, each group). ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 compared with sham group, #p ​< ​0.05, ##p ​< ​0.01 ###p ​< ​0.001 compared with AAV-Null group at the corresponding time points. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 5

Nerve innervation in L4/5 IVD traced by retrograde and anterograde marker. a A graph of experiment pattern. Rats were inject with AAV-Null or AAV-NTN1 gRNA at L4/5 pre-surgery, and 1 week after that needle-punctured operation was performed through right-sided approach using 21G needle. Dil was injected into L4/5 IVD at 8 weeks after surgery. b Representative images of Dil⁺ (red) neurons and DAPI (blue) nuclei staining at both sides of T12 - L6. Scale bar, 80 ​μm c Quantification of the Dil ​⁺ ​area, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001 compared with sham group, #p ​< ​0.05, ##p ​< ​0.01 ###p ​< ​0.001 compared with AAV-Null group at the corresponding segements (n ​= ​4, each group). Results were means ​± ​SD. d Representative images of Dil ​⁺ ​area at L4/L5 IVD and e quantification of the Dil ​⁺ ​area, after injected Dil in the right L1/2 DRGs at 8weeks. Scale bar, 500 ​μm (upper panel), 80 ​μm (lower panel). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 6

TCF3 negatively regulates Netrin-1 transcription. a Relative mRNA expression level of Netrin-1 in nucleus pulposus cells transfected with or without TCF3 overexpression (n ​= ​6). b Protein expression of Netrin-1 transfected with or without TCF3 overexpression and c quantification of the blot (n ​= ​3). d Putative TCF3-binding sites within the 2200 bp region of Netrin-1 promoter. e Netrin-1 promoter activity was assessed using luciferase assay in HEK-293 ​cells of P0, P1, P2, P3 and P4 segment (n ​= ​3). f TCF3-binding sites of Netrin-1 promoter was detected by ChIP assay. g Mutation in the putative TCF3-binding site of the P0 and P1 segment. h P0 (left), P1 (right) and its mutant activity was analyzed by luciferase assay in HEK-293 ​cells (n ​= ​3). i The mRNA level of TCF3 in nucleus pulposus cells treated with IL-1β or TNFα (n ​= ​6). j Protein expression of TCF3 in nucleus pulposus cells treated with IL-1β or TNFα and k quantification of the blot (n ​= ​3). ∗∗∗p ​< ​0.001 compared ctrl group. All data were shown as means ​± ​SD.