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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) 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.
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fig03: 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.

Mentions: Calcitonin gene-related peptide is a neurotransmitter that acts as a pain modulator and increased production can cause hyperexcitability and sensitization. To determine the effect of degenerating, painful IVD media on CGRP expression, DRG neurons were exposed to this media and compared healthy pain-free IVD media. After 48 hrs, neuronal cultures were analysed for the expression of the general neuronal marker PGP 9.5 (green) and CGRP (red; Fig.3A). The proportion of neurons that showed CGRP expression was analysed for each treatment. 16 ± 1% of untreated neurons, and 30 ± 2% of 10 ng/ml NGF-treated neurons expressed CGRP. Similar to untreated controls, 18 ± 2% of neurons cultured in healthy, pain-free IVD media expressed CGRP. Similar to NGF-treated controls, 29 ± 2% of neurons cultured in degenerating, painful IVD media were CGRP immunoreactive. 10 ng/ml NGF-treated neurons had a significantly higher percentage of CGRP-immunoreactive cells compared to untreated controls (P = 0.0045). There was no difference in CGRP immunoreactivity between non-treated and healthy, pain-free media groups (P = 0.9999) or between NGF-treated and degenerating, painful media groups (P > 0.9999). A significantly greater proportion of cells cultured in degenerating, painful IVD were CGRP immunoreactive compared to neurons cultured in healthy, pain-free IVD media (P = 0.007, Fig.3B).


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) 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.
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fig03: 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.
Mentions: Calcitonin gene-related peptide is a neurotransmitter that acts as a pain modulator and increased production can cause hyperexcitability and sensitization. To determine the effect of degenerating, painful IVD media on CGRP expression, DRG neurons were exposed to this media and compared healthy pain-free IVD media. After 48 hrs, neuronal cultures were analysed for the expression of the general neuronal marker PGP 9.5 (green) and CGRP (red; Fig.3A). The proportion of neurons that showed CGRP expression was analysed for each treatment. 16 ± 1% of untreated neurons, and 30 ± 2% of 10 ng/ml NGF-treated neurons expressed CGRP. Similar to untreated controls, 18 ± 2% of neurons cultured in healthy, pain-free IVD media expressed CGRP. Similar to NGF-treated controls, 29 ± 2% of neurons cultured in degenerating, painful IVD media were CGRP immunoreactive. 10 ng/ml NGF-treated neurons had a significantly higher percentage of CGRP-immunoreactive cells compared to untreated controls (P = 0.0045). There was no difference in CGRP immunoreactivity between non-treated and healthy, pain-free media groups (P = 0.9999) or between NGF-treated and degenerating, painful media groups (P > 0.9999). A significantly greater proportion of cells cultured in degenerating, painful IVD were CGRP immunoreactive compared to neurons cultured in healthy, pain-free IVD media (P = 0.007, Fig.3B).

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