Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors

Abstract

Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-α, interleukin-1β, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo.

Keywords: CGRP; discogenic pain; human; inflammatory cytokines; intervertebral disc degeneration; nerve growth factor.

Figure 1

Tumour necrosis factor-α (TNF-α; A), nerve growth factor (NGF; B) and brain derived neurotrophic factor (BDNF; C) mean concentrations in media from healthy pain-free or degenerating, painful intervertebral disc (IVDs). n = 8 in degenerate, painful group and n = 11 in healthy, pain-free group for TNF-α and NGF ±95% CI, unpaired t-test. n = 7 in degenerating, painful group and n = 9 in healthy, pain-free group for BDNF, ±95% CI, Mann–Whitney test. **P < 0.01, ***P < 0.001.

Figure 2

PC12 neurite growth after 48 hrs of culture. Representative phase contrast image of untreated cultures (A), cultures treated with 2.5 ng/ml nerve growth factor (NGF; B), 2.5 ng/ml NGF and normal IgG (B'), 2.5 ng/ml NGF and anti-NGF antibody (B”), 100 pg/ml NGF (C), 100 pg/ml NGF and normal IgG (C'), 100 pg/ml NGF and anti-NGF antibody (C”), healthy pain-free intervertebral disc (IVD) media conditioned media (D), degenerating, painful IVD conditioned media (D') and degenerating, painful IVD conditioned media treated with anti-NGF antibody (D”). Scale bar: 62.5 μm. (E) Quantification of the proportion of cells with neurites after 48 hrs of culture. Untreated cultures, cultures with 2.5 ng/ml NGF, 100 pg/ml NGF, IgG and anti-NGF antibodies in different combinations were quantified as indicated. Neurite sprouting in Control cultures healthy pain-free IVD media (HD) cultures, degenerating, painful IVD media cultures DD and degenerating, painful IVD media cultures treated with anti-NGF (DD Ab) were quantified as indicated. Fold changes of marker genes compared to –NGF control for PC12 neuronal differentiation and growth measured by qRT-PCR after 24 hrs (F) and 48 hrs (G). n = 3 in each IVD media group, n = 2 for each control in 24 hr cultures. n = 6 in each IVD media group, n = 3 for each control for 48 hr cultures. n = 3 for DD Ab and n = 2 for 2.5 ng/ml and 100 pg/ml NGF and NGF IgG. Error bars; ±95% CI, one-way anova. *P < 0.05, **P < 0.01, ***P < 0.001. #P < 0.001 when compared to 2.5 ng/ml NGF control.

Figure 3

Calcitonin gene-related peptide (CGRP) immunoreactivity in mouse dorsal root ganglion neurons after 48 hrs of culture. (A) Representative fluorescent images of neuronal cultures that were untreated (row 1), treated with 10 ng/ml nerve growth factor (NGF; row 2), maintained in healthy, pain-free intervertebral disc (IVD) conditioned media (row 3) or in degenerating, painful IVD media (row 4). PGP 9.5 (green) is a general neuronal marker and CGRP (red) is nociceptive neuropeptide. PGP 9.5 and CGRP are overlaid in merged images. White arrows indicate CGRP-immunoreactive neurons; scale bar: 200 μm. (B) Quantification of CGRP immunoreactivity for each group. −NGF; untreated media, +NGF; media supplemented with NGF, HD; healthy disc conditioned media, DD, degenerating, painful IVD conditioned media. n = 3 samples per group, tested in duplicate, with five fields counted per duplicate totalling 10 fields counted per condition, error bars; ±95% CI, one-way anova. **P < 0.01. #P < 0.001 when compared to −NGF control.

Figure 4

Calcitonin gene-related peptide (CGRP) expression in mouse dorsal root ganglion neurons after 48 hrs of culture. (A) Representative fluorescent images of neuronal cultures treated with 100 pg/ml nerve growth factor (NGF; +NGF, Row 1), 100 pg/ml NGF and normal IgG (+NGF IgG, Row 2), 100 pg/ml NGF and anti-NGF antibody (+NGF Ab, Row 3), media conditioned by degenerating, painful intervertebral disc (IVDs; DD, Row 4), or degenerating IVD media with anti-NGF antibody (DD Ab, Row 5). PGP 9.5 (green) is a general neuronal marker and CGRP (red) is pain neurotransmitter. PGP 9.5 and CGRP are overlaid in merged images. White arrows indicate CGRP-immunoreactive neurons; scale bar: 200 μm. (B) Quantification of CGRP immunoreactivity for each group. n = 2 samples per group, tested in duplicate, with five fields counted per duplicate totalling 10 fields counted per condition, error bars; ±95% CI, one-way anova. *P < 0.05.

Figure 5

Comparison of factors released by healthy, pain-free and degenerating, painful intervertebral disc (IVDs) measured by protein arrays. The mean relative quantity of each factor released by healthy pain-free IVDs (grey bars) and degenerating, painful IVDs (black bars) are presented (A–D). Factors in blue have not been previously associated with disc degeneration or pain (A), factors in red have been associated with pain, but not disc degeneration (B), factors in black have been associated with disc degeneration (C) and factors in purple have been associated with both disc degeneration and pain (D). (E) Mean relative quantities of select factors involved in nociception are plotted to show individual variation between donors. n = 8 in degenerating, painful group and n = 11 in healthy, pain-free group, SEM for A–D, ±95% CI for E, unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.