INTRODUCTION: Recent evidence indicates that NGF is increased in intervertebral discs (IVDs) after disc injury and that anti-NGF therapy improves low back pain in humans. M1 and M2 macrophage subtypes were recently reported to play a role in degenerative IVD injury. We examined the possible regulation of nerve growth factor (NGF) by M1 and M2 macrophages following IVD injury in mice. 
 
METHODS: Ten-week-old male C57BL/6J mice were used. All experimental protocols were approved by the Kitasato University School of Medicine Animal Care Committee (reference number: 2017-097). We divided the mice into two groups as follows: 20 of 90 mice comprised a control group in which IVDs were left intact (day 0); and the remaining 70 comprised a punctured IVD injury group in which coccygeal (Co) 5-6 and Co 6-7 IVDs were punctured 10 times with a 27-gauge needle. On days 0, 1, 7, 14, and 28, discs were extracted from the control and IVD injury groups for reverse transcriptionpolymerase chain reaction (RT-PCR) analysis (n=10 for each time point) and flow cytometric analysis (n=5 for each time point). On days 0, 1 and 28, discs were extracted from the control and IVD injury groups for enzyme-linked immunosorbent assay (ELISA) (n=5 for each time point). Statistical analyses were conducted in SPSS software version 11.0 (SPSS Inc, Chicago, IL, USA). Gene and protein expression was compared using a one-way analysis of variance with a Bonferroni’s post hoc multiple comparisons test. P < 0.05 was considered significant. 
 
RESULTS SECTION: Ngf mRNA expression increased 1 day after injury in injured compared to control mice(p<0.0001), and persisted for up to 28 days (p<0.0001, Figure 1A). NGF protein levels were increased only 28 days after IVD injury in injured compared to control mice (p=0.001). Flow cytometric analysis demonstrated that F4/80+CD11b+ cells were significantly increased 1 day after injury in IVD-injured compared to control mice (p=0.036). The increase in F4/80+CD11b+ cells was observed from 7 to 28 days (day 7, p<0.0001; day 14, p<0.0001; day 28, p=0.001). M1 macrophage markers Tnfa, Il1b, and Nos2 were significantly increased 1 day after injury in IVD-injured compared to control mice, after which expression gradually decreased (p<0.0001). At 28 days, there was no significant difference in the expression of M1 markers between injured and control mice (p=1.000). The M2a macrophage marker, Ym1, was only significantly increased 1 day after injury in IVD-injured compared to control mice (p<0.0001), and decreased thereafter to control levels. In contrast, the M2a and M2c macrophage marker Cd206 was significantly increased 7, 14, and 28 days after injury in IVD-injured compared to control mice (p<0.0001). The M2a and M2c macrophage-derived cytokine Tgfb was likewise significantly increased 7, 14, and 28 days after injury in IVD-injured compared to control mice (p<0.0001). RT-PCR analysis demonstrated that Ngf mRNA expression was significantly higher in disc cells after stimulation for 6 h with 2.5 or 25 ng/mL mrTNF-α or 1 or 10 ng/ml mrTGF-β compared to vehicle-treated cells (p=0.004, 0.004, 0.007, and p<0.0001, respectively). While Ngf mRNA expression was also significantly increased following 24 h stimulation with 25 ng/ml mrTNF-α or 10 ng/ml mrTGF-β compared to vehicletreated cells (p<0.0001 and p=0.008), expression was lower than that observed following 6 h stimulation with mrTNF-α or mrTGF-β, respectively (p=0.002 and p=0.003). NGF protein levels were also significantly higher following 6 h and 24 h stimulation with mrTNF-α or mrTGF-β compared to vehicle cells. In contrast to the mRNA findings, however, NGF protein levels were higher following 24 h stimulation with mrTNF-α or mrTGF-β than following 6 h stimulation (p=0.008 and p=0.002). 
 
DISCUSSION: These results suggest that M1 and M2 macrophages may differentially regulate NGF in both inflammatory and non-inflammatory states following IVD injury. We previously evaluated Ngf mRNA expression in the acute phase of injury and revealed that expression was increased 1 day after disc injury in mice,1 as we also observed in the present study. Notably, we observed here that the elevated Ngf expression persisted for up to 28 days after disc injury, despite a decrease in the expression of Tnfa and Il1b, inflammatory cytokines that are thought to regulate Ngf at 28 days after injury. Further, NGF protein was increased 28 days after injury but not earlier. The lack of an earlier increase may be due to the fact that protein synthesis takes time, and that transcript changes may not have been reflected in protein levels on day 1. Our results suggest that NGF is regulated not only by inflammatory factors but also anti-inflammatory factors in IVDs. Several studies have suggested that macrophages may be an important source of inflammatory factors in IVD. Coculturing IVD cells with macrophages increased IL-6 production. Interaction between IVD cells and macrophages induced mechanical hyperalgesia and cytokine production in a rat herniated disc model. In the present study, while there was a significant increase in the number of macrophages following IVD injury, specific temporal changes were observed in M1 and M2 macrophage marker expression after injury. M1 macrophages play an important role in inflammation, and increased M1 macrophage numbers have been observed in degenerative IVD, osteoarthritis and rheumatoid arthritis. A recent study showed that M1 macrophages were increased in degenerative human IVDs compared to healthy controls. M1 macrophages produce TNF-α, which induces NGF expression in human synovial fibroblasts and macrophages. Recombinant TNF-α stimulation increases NGF production in mouse and human disc cells.1 Here, the number of macrophages and TNF-α expression immediately increased following disc injury. In addition, TNF-α stimulation induced Ngf mRNA and NGF protein production. Taken together, the present and previous findings suggest that M1 macrophages may stimulate NGF production in the inflammatory phase after IVD injury. M2 macrophage subtypes suppress inflammation via the production of anti-inflammatory cytokines and their phagocytic function, as observed in the healing process. Like M1 macrophages, increases in M2c macrophages have been observed in degenerative IVDs in humans. Several studies have demonstrated that M2 macrophages, in particular the M2c subtype, produce TGF-β. In cultured human osteoarthritic articular cartilage, TGF-β induced higher NGF expression levels than IL-1β. Here, the increase in Tgfb expression was consistent with that of the M2a and M2c marker Cd206 but not the M2a marker Ym1, and TGF-β stimulation promoted Ngf mRNA expression and NGF protein production, suggesting that noninflammatory M2 macrophages, in particular the M2c subtype, may stimulate NGF production via TGF-β production. This study has some limitations. First, while we demonstrated that NGF was increased after IVD injury, further studies are needed to establish whether increased NGF levels are correlated with IVD pathology. Second, we did not examine whether macrophage depletion reduces NGF expression in vivo. Additional studies are also required to determine whether inhibition of TNF-α and TGF-β reduces NGF expression in vivo. In conclusion, M1 and M2c macrophages may stimulate NGF production via TNF-α and TGF-β, respectively, following IVD injury in inflammatory and non-inflammatory states.

0850 - Possible Regulation Of Nerve Growth Factor M1 And M2 Macrophages Following Intervertebral Disc Injury In Mice