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Degenerate human nucleus pulposus cells promote neurite outgrowth in neural cells.

Richardson SM, Purmessur D, Baird P, Probyn B, Freemont AJ, Hoyland JA - PLoS ONE (2012)

Bottom Line: Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells.Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells.Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Regenerative Medicine, Institute of Inflammation and Repair, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, United Kingdom.

ABSTRACT
Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.

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Schematic overview of experimental setup and analysis.(A) Diagram illustrating the co-culture model. (B) Measurement of neurite outgrowth from SH-SY5Y cells. Neurites were traced (red lines) and the mean neurite length measured for the total number of cells in each field of view. The mean percentage number of neurite expressing cells in each field of view was also calculated.
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pone-0047735-g001: Schematic overview of experimental setup and analysis.(A) Diagram illustrating the co-culture model. (B) Measurement of neurite outgrowth from SH-SY5Y cells. Neurites were traced (red lines) and the mean neurite length measured for the total number of cells in each field of view. The mean percentage number of neurite expressing cells in each field of view was also calculated.

Mentions: Alginate beads containing NP cells (n = 6 [3 non-degenerate (histological grade <3; age range 37–61 years) and 3 degenerate samples (histological grade >7; age range 27–79 years)]) were then added to each insert with serum-free SH-SY5Y cell media containing 20 ng/ml PDGF both within and surrounding the inserts (figure 1A). Co-cultures were conducted for 48 hrs at 37°C, 5% CO2. SH-SY5Y cells cultured in serum-free SH-SY5Y cell media containing 20 ng/ml PDGF with cell-free alginate beads served as controls (N = 6).


Degenerate human nucleus pulposus cells promote neurite outgrowth in neural cells.

Richardson SM, Purmessur D, Baird P, Probyn B, Freemont AJ, Hoyland JA - PLoS ONE (2012)

Schematic overview of experimental setup and analysis.(A) Diagram illustrating the co-culture model. (B) Measurement of neurite outgrowth from SH-SY5Y cells. Neurites were traced (red lines) and the mean neurite length measured for the total number of cells in each field of view. The mean percentage number of neurite expressing cells in each field of view was also calculated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472988&req=5

pone-0047735-g001: Schematic overview of experimental setup and analysis.(A) Diagram illustrating the co-culture model. (B) Measurement of neurite outgrowth from SH-SY5Y cells. Neurites were traced (red lines) and the mean neurite length measured for the total number of cells in each field of view. The mean percentage number of neurite expressing cells in each field of view was also calculated.
Mentions: Alginate beads containing NP cells (n = 6 [3 non-degenerate (histological grade <3; age range 37–61 years) and 3 degenerate samples (histological grade >7; age range 27–79 years)]) were then added to each insert with serum-free SH-SY5Y cell media containing 20 ng/ml PDGF both within and surrounding the inserts (figure 1A). Co-cultures were conducted for 48 hrs at 37°C, 5% CO2. SH-SY5Y cells cultured in serum-free SH-SY5Y cell media containing 20 ng/ml PDGF with cell-free alginate beads served as controls (N = 6).

Bottom Line: Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells.Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells.Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Regenerative Medicine, Institute of Inflammation and Repair, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, United Kingdom.

ABSTRACT
Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.

Show MeSH
Related in: MedlinePlus