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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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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.
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fig02: 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.

Mentions: As degenerating, painful IVDs release increased amounts of NGF and BDNF ex vivo, the conditioned media were tested for the ability to stimulate neurite growth in PC12 cells. After 48 hrs of culture, 20 ± 3% of untreated cells, 78 ± 4% of NGF-treated cells, 29 ± 2% of cells cultured in healthy, pain-free IVD media and 64 ± 2% of cells cultured in degenerating, painful IVDs had neurites (Fig.2A and B; n = 6 IVDs for each conditioned media group). A significantly greater proportion of cells treated with NGF extended neurites when compared to untreated cells (P < 0.0001). There was no significant difference in the proportion of cells with neurites between untreated and healthy IVD media groups (P = 0.25). NGF cultures had a greater proportion of cells compared to degenerating media groups (P < 0.01). However, degenerating, painful IVD media induced neurite sprouting in a significantly greater percentage of cells compared to cells cultured in healthy, pain-free IVD media (P = <0.0001) (Fig.2) indicating that painful, degenerating IVDs produce factors that promote neurite growth.


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)

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.
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fig02: 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.
Mentions: As degenerating, painful IVDs release increased amounts of NGF and BDNF ex vivo, the conditioned media were tested for the ability to stimulate neurite growth in PC12 cells. After 48 hrs of culture, 20 ± 3% of untreated cells, 78 ± 4% of NGF-treated cells, 29 ± 2% of cells cultured in healthy, pain-free IVD media and 64 ± 2% of cells cultured in degenerating, painful IVDs had neurites (Fig.2A and B; n = 6 IVDs for each conditioned media group). A significantly greater proportion of cells treated with NGF extended neurites when compared to untreated cells (P < 0.0001). There was no significant difference in the proportion of cells with neurites between untreated and healthy IVD media groups (P = 0.25). NGF cultures had a greater proportion of cells compared to degenerating media groups (P < 0.01). However, degenerating, painful IVD media induced neurite sprouting in a significantly greater percentage of cells compared to cells cultured in healthy, pain-free IVD media (P = <0.0001) (Fig.2) indicating that painful, degenerating IVDs produce factors that promote neurite growth.

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