In this study we demonstrated that EREG have the ability to alter nociceptive signaling.
However, through which mechanisms EREG affects neurons remains to be investigated.
It would be interesting to examine which signaling pathways that are activated in spinal- and/or DRG neurons following administration of EREG. Moreover, one could study whether the effect of EREG on neuronal excitability is due to a local inflammation or by a direct alternation of nociceptive neurons. In addition, it would be interesting to see if EREG can alter expression of genes that are directly linked to neuronal excitability, like neurotransmitters and ligand-gated ion channels.
57
7 Conclusions
I) Our data showed that NP taken out of its natural environment may release EREG.
EREG may affect nearby cells by binding to its receptors.
II) Administration of EREG onto the dorsal nerve roots induced a decrease in C-fiber response. A loss in responsiveness to electrical stimuli applied onto the sciatic nerve was observed. Our findings suggest that EREG may be involved in sensory deficits in patients following a disc herniation.
III) Application of EREG onto the dorsal nerve roots induced a pronounced increase in spontaneous activity in nociceptive neurons in the dorsal horn. The increased excitability in nociceptive neurons lasted throughout the experiment of 180min.
Hence, EREG may be important for the long-lasting pain often seen in patients following a disc herniation.
IV) NP tissue exposed to the dorsal nerve roots for 180min induced a significant up-regulation of EREG in the DH. This up-up-regulation suggests that NP tissue has a pro-inflammatory effect and may activate microglia and astrocytes in the spinal cord, in addition to recruiting circulating macrophages.
V) We demonstrated an up-regulation of EREG receptors in NP tissue, DH tissue and DRG tissue. These findings suggest that NP tissue enhance EREG signaling and may thereby strengthen pathophysiological changes following a disc herniation.
58
59
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Appendices
Appendix 1 Procedure for protein analysis using Chemiluminescence immunoassay (SCB945Ra, Cloud-Clone Corp. USA)
Reagent preparation:
1. All kit components were kept at room temperature.
2. The Standard solution was reconstituted with 1.0mL of Standard Diluent, to get a start concentration of 2000pg/mL. The solution was incubated for 10 min at room temperature and shaken gently.
3. 300μl of standard solution was diluted with 300μl standard Diluent to get a stock concentration of 1000pg/mL. The standard dilution series was prepared as described in the table below.
Dilution series Standard Standard Diluent Concentration
Standard stock 600µL 1000pg/mL
1 300µL from stock + 600μL 333.33pg/mL Detection reagent B was mixed with 11.9mL of Assay Diluent B.
5. 300mL solution was prepared by diluting 10mL of the wash solution with 290mL distilled water.
6. The substrate working solution was prepared 15 min before assay by mixing 9900μl Stubstrat A with 100μl Substrat B.
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Assay preparation:
1. 100μL of each dilution of standard, blank and samples was added to each well, and the plate was covered with a plate sealer and incubated for 2hours at 37ᵒC.
1. 100μL of each dilution of standard, blank and samples was added to each well, and the plate was covered with a plate sealer and incubated for 2hours at 37ᵒC.