Background: I Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Nerve growth factor (NGF) concentrations increase in IVD after disc injury, and anti-NGF therapy has been shown to suppress LBP in humans. Elevated concentrations of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in degenerative IVD and in vitro studies suggest that these factors promote NGF production. However, it remains unclear whether these factors regulate NGF in vivo. Therefore, we studied the regulation of NGF in a mouse model of IVD injury.
Methods: After induction of IVD injury, mRNA levels of Tnfa, Tgfb, and Ngf in IVD of control and IVD-injured mice were examined for 7 days. To this end, magnetic cell separation techniques were used to isolate CD11b(+) (enriched in macrophages) and CD11b(-) (enriched in IVD cells) cell fractions from the injured IVDs. to examine the effect of TNF-α on Ngf expression, C57BL/6 J and Tnfa knockout (KO ) mice (C57BL/6 J background), Ngf expression in damaged IVDs was examined. to examine the effect of TGF-β on Ngf expression, TGF-β inhibitor SB431542 or DMSO solution (vehicle) was injected intraperitoneally into C57/BL6J mice 1 day and 2 days before IVDs were harvested. Intraperitoneal injections were made.
Results: Tnfa, Tgfb, and Ngf mRNA expression was significantly increased in injured IVDs; Tnfa was expressed predominantly in the CD11b(+) fraction and Tgfb in the CD11b(-) fraction; Ngf expression was similar in CD11b(+) and CD11b(-) fractions and on days 1, 3, and 7 after injury in It was equivalent in wild-type and Tnfa-KO mice.SB431542 suppressed TGF-β-mediated Ngf expression and NGF production in vitro. Furthermore, treatment with SB431542 significantly reduced Ngf expression in the IVD to levels below those observed in vehicle-treated mice at PID3 and PID7.
Discussion: A number of reports have demonstrated that NGF levels rise locally at sites of inflammation. TNF-α is a major factor in IVD inflammation, TNF-α was thought to regulate NGF in IVD injury. However, we found that although Tnfa and Ngf expression was immediately elevated in injured IVDs in wild-type mice at PID1, Tnfa deficiency did not alter Ngf expression in injured IVDs. Thus, elevated TNF-α levels may not play a major role in regulating NGF expression after IVD injury. Some reports indicate that TGF-β is also produced by macrophages in the setting of injury, while others indicate that TGF-β levels are elevated in chondrocyte-like cells in degenerated discs. TGF-β expression has been observed in many cells. In the present study, TGF-β was more significantly expressed in intervertebral disc cells. Furthermore, Ngf expression was predominantly suppressed by TGF-β inhibitor. Our results indicate TGF-β regulates NGF during IVD degeneration and that changes in Ngf expression may be due to increased autocrine/paracrine activity of IVD cell-derived TGF-β.