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

Krock E, Rosenzweig DH, Chabot-Doré AJ, Jarzem P, Weber MH, Ouellet JA, Stone LS, Haglund L - J. Cell. Mol. Med. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Orthopeadic Research Laboratory, Division of Orthopedic Surgery, McGill University, Montreal, QC, Canada; McGill Scoliosis and Spine Research Group, Montreal, QC, Canada.

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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.
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fig04: 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.

Mentions: To determine if NGF released from degenerating IVDs is contributing to the increase in CGRP immunoreactivity, experiments using an anti-NGF antibody were conducted. Like in PC12 cultures, 100 pg/ml of NGF was used as an additional control in this set of experiments. Following treatment with 100 pg/ml of NGF, 26 ± 1% of neurons were CGRP immunoreactive, which was not significantly different from 10 ng/ml NGF-treated neurons (P = 0.97). Incubating 100 pg/ml NGF media with normal IgG did not significantly alter CGRP immunoreactivity (P = 0.99). In contrast, incubating media containing 100 pg/ml of NGF with an anti-NGF antibody, CGRP expression was significantly reduced to 13 ± 3% (P = 0.038). Similarly, addition of anti-NGF antibody to degenerating, painful IVD media significantly reduced CGRP expression to 20 ± 2% (P = 0.048) when compared to degenerating media without the antibody (Fig.4, n = 2 IVD samples in each group).


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

Krock E, Rosenzweig DH, Chabot-Doré AJ, Jarzem P, Weber MH, Ouellet JA, Stone LS, Haglund L - J. Cell. Mol. Med. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: 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.
Mentions: To determine if NGF released from degenerating IVDs is contributing to the increase in CGRP immunoreactivity, experiments using an anti-NGF antibody were conducted. Like in PC12 cultures, 100 pg/ml of NGF was used as an additional control in this set of experiments. Following treatment with 100 pg/ml of NGF, 26 ± 1% of neurons were CGRP immunoreactive, which was not significantly different from 10 ng/ml NGF-treated neurons (P = 0.97). Incubating 100 pg/ml NGF media with normal IgG did not significantly alter CGRP immunoreactivity (P = 0.99). In contrast, incubating media containing 100 pg/ml of NGF with an anti-NGF antibody, CGRP expression was significantly reduced to 13 ± 3% (P = 0.038). Similarly, addition of anti-NGF antibody to degenerating, painful IVD media significantly reduced CGRP expression to 20 ± 2% (P = 0.048) when compared to degenerating media without the antibody (Fig.4, n = 2 IVD samples in each group).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Orthopeadic Research Laboratory, Division of Orthopedic Surgery, McGill University, Montreal, QC, Canada; McGill Scoliosis and Spine Research Group, Montreal, QC, Canada.

Show MeSH
Related in: MedlinePlus