Inhibiting CSF1R signaling reduces disc degeneration and discogenic back pain

Inhibiting CSF1R signaling reduces disc degeneration and discogenic back pain | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Inhibiting CSF1R signaling reduces disc degeneration and discogenic back pain Yi Lu, Lei Liu, Jingdong Zhang, Yizheng Yao, Weixuan Yan, Songlin Zhou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5647673/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Graphical Abstract Abstract Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. Inflammation has been implicated in discogenic back pain and disc degeneration, however, the detailed molecular mechanisms remain unclear. Herein we demonstrate that Colony Stimulating Factor 1 Receptor (CSF1R) signaling plays an essential role in the development of IVD degeneration and discogenic back pain. Genetic deletion of CSF1R from microglia/macrophages or oral administration of a CSF1R competitive inhibitor, GW2580, decreased IVD degeneration as evidenced by serial magnetic resonance imaging (MRI) and histopathological analyses in adult mice following disc injury. CSF1R deletion or GW2580 administration inhibited pro-inflammatory cytokine release from injured discs and blocked dorsal root ganglion (DRG) macrophage and spinal cord dorsal horn microglia activation and in so doing, eliminated neuropathic pain secondary to disc injury. These results suggest a novel therapeutic strategy for the treatment of chronic low back pain secondary to IVD degeneration. Health sciences/Pathogenesis Biological sciences/Physiology/Neurophysiology Biological sciences/Physiology/Bone quality and biomechanics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). The prevalence of LBP continues to increase with an annual prevalence of 15–45% amongst adults in the United States, with significant health care and economic consequences 1 . Current medical treatment for disc degeneration and associated LBP is centered on activity modification, physical therapy, and non-steroid anti-inflammatory drugs (NSAIDs). The effect of those treatments is generally limited, and many patients progress to surgery despite medical treatment or suffer from lifelong chronic LBP. More importantly, no treatments so far have been shown to effectively mitigate the progression of the disc degeneration process itself 2 . Healthy IVDs are immune-privileged sites because the annulus fibrosus (AF) and cartilaginous endplate (CEP) isolate the nucleus pulposus (NP) from the immune system. Breakdown of the blood-NP barrier secondary to aging or injury exposes NP, which in turn initiates an immune response 3 . Proteins from the NP lead to macrophage activation and infiltration with secretion of inflammatory cytokines 4 . Inflammatory cells accelerate disc degeneration by decreasing the production of extracellular matrix (ECM) proteins (e.g., aggrecan and collagen II), while concurrently upregulating catabolic enzymes such as disintegrin, metalloproteinase with thrombospondin motifs (ADAMTS)-4 and − 5 and matrix metalloproteinases (MMPs) 5 . Inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-α stimulate the expression of angiogenic and neurotrophic factors 6 which trigger nociceptive nerve ingrowth into the CEPs and IVDs 7 , contributing to discogenic back pain 8 , 9 . Recent reports have defined the importance of macrophages and microglia in the initiation and maintenance of centralized pain after peripheral nerve injury(10,11,12). Specifically, dorsal root ganglion (DRG) macrophages have been shown to be critical contributors to both the initiation and maintenance of mechanical hypersensitivity which is a hallmark of neuropathic pain in mice 13 . The increased numbers of macrophages found within the DRGs of injured nerves corresponds to an increase in activation of spinal cord dorsal horn microglia 13 . Spinal cord microglia plays an important role in the centralization 14 and maintenance of neuropathic pain and the long-term potentiation of altered nociceptive pathways after peripheral nerve injury 15 . Macrophages in the DRG and microglia in the spinal cord appear to act synergistically and promote the transition from acute to chronic pain following peripheral nerve injury 16 . Colony stimulating factor 1 receptor (CSF1R) is expressed in macrophages, dendritic cells and microglia 17 and is a critical mediator of proliferation and activation 18 . Upregulation of CSF1R was observed in DRG macrophages and spinal cord dorsal horn microglia after peripheral nerve injury 19 . Upregulation of CSF1R expression on spinal microglia was also observed in a disc degeneration-induced back pain mouse model 20 . These findings hinted that signaling through CSF1R might play a role in discogenic back pain. Using human healthy and degenerate IVD single-cell transcriptome database, we identified increased macrophage appearance and increased csf1 and csf1r expression in degenerate IVDs compared to healthy IVDs. To test the role of CSF1 and CSF1R in IVD degeneration and discogenic back pain, we developed a murine disc injury model that elicited structural disc degeneration and discogenic pain–related behaviors similar to human clinical conditions. We demonstrated that deletion of CSF1R ablated disc degeneration and discogenic pain–related behaviors. Similarly, inhibition of CSF1R function using pharmacological approach decreased inflammation in IVD, decreased IVD degeneration, reduced expression of the spinal nociception markers c-Fos and alleviated discogenic pain-related behaviors. Results Characterization of csf1 in human IVDs To investigate cell population change and gene features of human NP during IVD degeneration, we used Single-cell RNA sequencing (scRNA-seq) database GSE199866 of human NP cells from degenerate and healthy IVDs of the same individual. Cells are clustered according to transcriptome similarity in 2D space (Fig. 1 A upper panel). UMAP plot revealed 13 clusters (0–12). In all transcriptionally distinct subpopulations, chondrocytes make up most of the cells in all samples based on cell lineage-specific marker gene expression. Chondrogenic marker gene SOX9 and chondrocyte-specific ECM genes ACAN were ubiquitously expressed in all chondrocyte clusters. Endothelial cells were identified by feature genes like PECAM1 expression (Supplementary Fig. 1C and D). Clusters 12, macrophage, is found only in NP from degenerate IVDs (Fig. 1 A lower panel). The cluster of cells were enriched with genes involved in macrophage inflammatory response like CD74 , TYROBP and LAPTMS (Supplementary Fig. 1D). We used GO analysis and identified the top15 enriched biological functional pathways of upregulated genes in degenerate NPs. The identified biological processes and molecular functions are directly related to myeloid leukocyte activation (Fig. 1 B). A combined analysis of the genes in degenerate NPs highlighted csf1 as one of the 8 top upregulated genes (Fig. 1 C). Furthermore, UMAP distribution of csf1 and its receptor expression in degenerate NPs were performed and shown in the scatter plot (Fig. 1 D and E). These data confirmed that expression of the csf1 is enriched in chondrocytes of degenerate NPs. csf1r is enriched only in macrophages. We also mapped human endplate database GSE153761 and found csf1 was 1 of 8 top upregulated proinflammatory factors in degenerate CEPs, as compared to healthy CEPs (Fig. 1 F and G). There is no difference between normal and degenerate endplate on the expression of csf1r and another ligand IL-34 (Fig. 1 F and G). These results identified CSF1/CSF1R pathway may be involved in macrophage activation and pathogenesis of disc degeneration. Experimental IVD injury reproducibly induces disc degeneration and discogenic pain To create a robust and reproducible model of IVD degeneration and discogenic back pain, we modified an existing IVD puncture procedure (see Methods and Supplemental Fig. 2A-C). These mice were then subjected to behavioral tests and serial MRIs. Histopathology of IVDs was measured at several endpoints (Fig. 2 A). Uninjured IVDs in mice that underwent sham surgeries demonstrated classical healthy histopathologic architecture (Fig. 1 B, Supplemental Fig. 2E). Safranin O and Fast Green staining (SO&FG) staining of IVD sections revealed the intact NP, AF and CEP (Fig. 1 B). A well-defined border between the AF and NP was also noted (Fig. 1 B, Supplemental Fig. 2E). The CEP was continuous with the AF and was separated from the vertebral body by a layer of growth plate (GP). The CEP contained layers of rounded chondrocyte-like cells in a proteoglycan rich matrix. A tidemark (TM, dotted line) was present between the GP and the CEP (Supplemental Fig. 2E). In mice that underwent intervertebral disc puncture (DP), at an early stage (14 days post injury), the CEP kept its continuity and morphology, but NP and AF lamellae became disorganized (Supplemental Fig. 2D). 3- and 6- months after DP, the NP progressively collapsed (Fig. 1 B, Supplemental Fig. 2E). In addition, the proteoglycan matrix (Safranin O positive, red) progressively decreased, the AF lamellae became disorganized, and the border between the NP and AF became less distinct (Fig. 1 B, Supplemental Fig. 2E). Further, the CEP was noted to be in discontinuity with the AF (Fig. 1 B) and the tidemark between the CEP and GP disappeared (Supplemental Fig. 2E). MRIs were used to serially follow IVD degeneration. The T2 bright area in the sham-treated IVDs remained consistent over the 6 month follow up period. In comparison, progressive loss in signal intensity was noted in IVDs at 3- and 6- months after DP. Representative T2-weighted, midsagittal images are shown in Fig. 1 C. T2 mapping revealed a 48% and 66% decrease in signal intensity at 3-month and 6-month post injury, respectively (Fig. 2 C and D). On MRI, IVDs with DP demonstrated height loss at both 3- and 6-months follow-ups compared to sham-treated mice (Fig. 2 E). In humans, mechanical back pain is subjectively quantified using the Visual analogue scale (VAS). In addition, several behavior functional scoring systems, such as the Oswestry disability index (ODI) have been used to quantify the severity of back pain 21 . In mice, analogous behavioral scoring metrics for pain include the commonly used von Frey assay 22 , 23 . The sham surgery group displayed a mildly decreased von-Frey test threshold 3 days after surgery but returned to normal by day 28. In contrast, in the DP group, the Von-Frey test threshold dramatically decreased at 3 days and remained so for up to 90 days post injury (Fig. 2 F). DP also led to cold allodynia (Fig. 2 G) and decreased thermal threshold (Supplemental Fig. 3A). The presence of pain induces behavioral changes in animals 24 . Burrowing has been reported to be reduced in the setting of chronic pain 25 , 26 . Mice that underwent DP displayed a significant impairment in burrowing compared to sham-treated group (Supplemental Fig. 3B). Motor function was also evaluated using both accelerating rotarod and treadmill tests. Maximal speed tolerance (Supplemental Fig. 3C) and performance on the rotarod (Supplemental Fig. 3D) were similar between the DP and sham-treated groups. Open field testing also showed similar results between the groups in terms of running velocity, distance travel, and time spent in pre-defined zones (Supplemental Fig. 3E, F and G). These results indicated that modified DP method leads to reproducible progressive IVD degeneration with reproducible discogenic back pain related behavior. Csf1r deletion decreases proinflammatory cytokines release after disc injury We then asked whether the CSF1/CSF1R pathway was involved in the inflammatory response to DP injury within the disc itself. Using Cx3cr1 +/GFP mice, macrophage infiltration was found in disc after DP (Fig. 3 A). We also used macrophage specific markers Iba1 and CSF1R for immunostaining of IVDs. After disc injury, abundant Iba1 + and CSF1R + cells were detected in different parts of the discs, including the NPs, AFs, and CEPs (Fig. 3 B, C, D and E). In comparison, there was no Iba1 or CSF1R positive staining in the discs in sham-injured mice (Fig. 3 B and C). We next asked whether CSF1R was involved in the release of disc inflammatory factors after disc injury. IVDs were collected and cultured from sham injured mice or from mice with disc injury 90 days after the injury (Fig. 3 F). A panel of inflammatory factors were examined. When compared to cultures from sham injury IVDs, those from injured IVDs displayed strong upregulation of 31 inflammatory mediators as measured by inflammation assays (Fig. 3 G). Among the upregulated cytokines noted were MMP2 and pro-MMP9, which are involved in catabolic pathways that contribute to matrix degradation 5 . Proinflammatory factors (including IL1-α, IL5, IL6, IL12-p40/p70, G-CSF/CSF3 and M-CSF/CSF1) and macrophage chemokine ligands (include MDC, Thymus CK-1, TROY, KC, MCP-1, MIP-1-alpha, MIP-1-gamma, MIP-2, MIP-3-beta, MIP-3-alpha and PF4) 8 , 27 were also increased. The increase of soluble TNFR, ICAM-1, lungkine and TPO was also observed and these are thought to be related to canonically activated macrophages 6 , 28 . Growth factors (bFGF, IGF1 and VEGF) and 3 IGF binding proteins (IGFBP2, 3 and 6) related to angiogenesis 29 , 30 were also upregulated. Consistent with findings from human IVD degeneration, osteoprotegerin (OPG) was also significantly upregulated 31 , 32 (Fig. 3 G and H, Supplemental Fig. 4A and B). Compared to vehicle control Cx3cr1 CreER/+ : Csf1r fl/fl littermates, the mice treated with tamoxifen (TAM) hence with CSF1R deletion, released significantly lower levels of many of these factors after DP. The release of proinflammatory factors (CSF1, CSF3, IL-1α and IL-6) and matrix degradation proteins pro-MMP-9 were eliminated compared to injured control IVDs. The upregulation of OPG was also eliminated with CSF1R deletion (Fig. 3 G and H, Supplemental Fig. 4B). These results suggested CSF1R signaling play an important role in inflammatory cytokine release after disc injury. Csf1r deletion reduces IVD degeneration after injury IVDs were collected from control vehicle or TAM treated Cx3cr1 CreER/+ : Csf1r fl/fl mice at indicated days post DP (Fig. 4 A). While IVDs from both control and CSF1R deleted mice exhibited gross evidence of AF disruption associated with IVD degeneration, AF architecture was better preserved in csf1r deleted (TAM group) as compared to the control (vehicle) mice (Fig. 4 B). Proteoglycan (Safranin O positive) in CEP was markedly reduced after DP in both groups but less destruction was apparent in the TAM treated group. To quantify these alterations in structure we measured CEP thickness (Fig. 4 C). These results indicated that while IVD degeneration still occurred in mice with csf1r deletion following disc injury, the process was significantly less destructive when compared to controls. Vehicle control and csf1r deletion (TAM) mice were serially followed using MRI. Figure 4 D presents the quantitative changes in the T2-weighted image intensity index (T2 mapping) at 90- and 180-days post-injury. Compared to the vehicle control which demonstrated a 66% decrease in T2 intensity at 180 days post-injury, IVDs in the csf1r deletion group displayed a 25% decrease at 180 days post-injury (Fig. 4 D). In addition, IVD height in csf1r deletion (TAM) mice was significantly increased as compared to controls (Fig. 4 E). The CSF1R antagonist GW2580 attenuates disc degeneration after injury Given that Csf1r genetic deletion decreased disc degeneration after DP, we next wanted to evaluate whether CSF1R antagonist would have similar effects, and therefore could potentially be used clinically for treating disc degeneration. We tested the selective CSF1R competitive inhibitor GW2580 given its high affinity and specificity for CSF1R and low toxicity. GW2580 prevents CSF1R activation without inhibiting related kinases such as c-Kit, FTL3 and PDGFRβ 33 , 34 . Importantly, contrary to other CSF1R inhibitors such as PLX3397, BLZ945, etc., GW2580 does not cause ablation of resident macrophage or microglial population 35 , 36 , 37 , 38 . This is important for treating benign conditions such as back pain. We administered GW2580 by oral gavage (80 mg/kg) 2 hours after the disc puncture surgery and every 24 hours thereafter until the animals were sacrificed (Fig. 5 A). At 90 and 180 days after disc injury, progressive loss in disc MRI T2 signal intensities were seen in both vehicle and GW2580 treated mice but GW2580 treated mice demonstrated a significantly less decrease in disc T2 signal intensities (Fig. 5 B and C). IVD height loss in GW2580 treated group was also significantly less compared to the vehicle treated control group (Fig. 5 D). Histological analysis demonstrated discs from GW2580 treated mice having better preserved disc architecture compared to the controls (Fig. 5 E and F). Finally, the infiltration of inflammation cells, demonstrated by Iba1 immunostaining (Fig. 5 G and H) or GFP + cells in CX3CR1 +/GFP reporter mice after disc injury, decreased with GW2580 treatment when compared to vehicle control (Supplemental Fig. 5). Disc injury induces DRG CSF1 expression and transportation to spinal cord The IVD is innervated by branches of the sinuvertebral nerve and derivatives from the ventral rami 39 . ECM degeneration, inflammatory mediators and neurotrophins that generate inflammatory conditions in the IVD result in membrane depolarization of peripheral nociceptive nerve endings. It has been reported that peripheral nerve injury induces CSF1 de novo synthesis in DRGs, which appears to be essential for spinal cord dorsal horn microglia activation and pain centralization after peripheral nerve injury, following transportation of expressed CSF1 along the dorsal root from DRG to the spinal cord 19 (Fig. 6 A). Here we ask whether DP triggers similar signaling activation in DRGs and spinal cord, resulting in centralized pain induction, and if so, whether it could be altered by CSF1R pathway inhibition. First, we examined DRGs and their peripheral branches and central branches. CSF1 was expressed in very few DRG neurons without disc injury. After DP, increase of CSF1 expression was found in DRG neurons and central branches, but not in the peripheral branches distal to the DRG (Fig. 6 B). The increase of CSF1 in DRG neurons persisted for at least 4 weeks after DP (Fig. 6 B, Supplemental Fig. 6A and B). CSF1 expression was not seen in Iba1 + cells, indicating its expression was not from DRG macrophages (Supplemental Fig. 6B). The expression of CSF1 in DRG was reduced in Csf1r deletion mice (TAM treated Cx3cr1 CreER/+ : Csf1r fl/fl group) compared to vehicle control treated group (Fig. 6 C). This is likely because of the decreased inflammation signal from the injured disc due to the CSF1R deletion. CSF-1/CSF-1R is essential for DRG macrophage and spinal cord microglia activation after disc injury Immunofluorescence analysis revealed an increased number of macrophages (Iba1 + cells) as well as active macrophages (CD68 + ) in the DRGs after DP (Supplemental Fig. 6C, 6D). Ki67 and GFP immunofluorescence analysis on CX3CR1 +/GFP mice DRG suggested the increase of macrophages in the DRGs after DP is likely from CX3CR1 + resident macrophages proliferation (Supplemental Fig. 6E). The increase of DRG Iba1 + was reduced in c sf1r deletion mice (TAM treated Cx3cr1 CreER/+ : Csf1r fl/fl group) (Supplemental Fig. 6F). In the spinal cord, CSF1R is only expressed in microglia 19 . After DP, CSF1R expression significantly increased in the dorsal spinal cord microglia as compared to sham injured controls (Supplemental Fig. 6G). The Csf1r deletion in the CX3CR1 CreER/+ :Csf1r fl/fl mice after TAM injection was confirmed, as shown with no CSF1R staining after disc injury (Supplemental Fig. 6H). The increase of Iba1 + and Cd68 + microglia cells in the dorsal spinal cord was abolished in Csf1r deletion DP mice (Fig. 6 D, E and F). These data suggest that CSF1R signaling is essential for the spinal cord dorsal horn microglia activation after DP. Csf1r deletion eliminates neuropathic pain after disc injury Given these findings we next sought to explore whether the decreased spinal cord microglia and DRG macrophage activation from CSF1R deletion alters pain behavior in mice after disc injury. Using the Von-Frey test, we discovered that mechanical hypersensitivity following disc injury was completely abolished in CX3CR1 CreER/+ :Csf1r fl/fl mice treated with tamoxifen but not in controls (Fig. 6 G). This phenotype was noted to last at least 90 days after disc injury. These results suggest that CSF1R signaling is critical to the development of discogenic neuropathic pain. GW2580 alleviates acute and chronic discogenic pain after disc injury Similar to Csf1r deletion, GW2580 reduced the CSF1 positive neurons in DRG (Fig. 7 A and B). The activation (CD68 + ) and proliferation (Iba1 + and Ki67 + ) of macrophage in DRG was also down regulated by GW2580 treatment (Supplemental Fig. 7A, B and C). GW2580 also markedly decreased inflammatory CD68 + in the spinal cord dorsal horn after disc injury (Fig. 7 C and D). Disc injury induced CSF1R upregulation in the spinal cord dorsal horn was also reduced by the GW2580 treatment (Fig. 7 C and E). These results indicate that GW2580 downregulated immune cell response in DRG and spinal cord. Peripheral nerve injury leads to the activation of the immediate early gene fos in the dorsal spinal cord pain circuits. As Fos itself has a very short half-life 40 , Targeted Recombination in Active Populations (TRAP) mice were used to detect the accumulated Fos activation during a certain period of time when tamoxifen was given 41 . This method utilizes Fos CreERT 2 : Rosa26 ChR 2 mice in which the tamoxifen-dependent recombinase CreERT2 is expressed in an activity-dependent manner from the Fos loci. The active neurons during the duration of tamoxifen presence are labeled with tdTomatos (Fig. 7 E). In TRAP mice that underwent sham control surgery, there was little or no Fos (tdTomato + cells) within spinal cord (Fig. 7 E) or DRG (Fig. 7 F). After disc injury, the increase of Fos(tdTomato) positive cells was detected in the DRGs and spinal cord (Fig. 7 E, F and G). Fos (tdTomato + ) was noted in lamina I/II of the dorsal horn, an area associated with nociceptive processing after DP (Fig. 7 E). The Fos(tdTomato) peaked at 4 weeks after DP, and at this time, an increase in Fos (tdTomato) positive cells was also seen in the spinal cord lamina III/IV (Fig. 7 H). GW2580 treatment significantly decreased the number of Fos (tdTomato) positive cells in both the spinal cord dorsal horn and DRGs after DP (Fig. 7 G and H). With Von Frey test, the GW2580 treated group showed an improved pain threshold compared to the control treated group, up to 90 days when the experiment ended (Fig. 7 I). In addition, GW2580 treated mice exhibited decreased acetone cold allodynia between 14- and 28-days after disc injury compared to vehicle treated controls (Supplemental Fig. 7D). The burrowing test showed GW2580 treated mice having less disturbed burrowing behavior compared to vehicle treated controls (Supplemental Fig. 7E). These results indicate that GW2580 treatment downregulated spinal nociception markers c-Fos expression and alleviated nociceptive behavior. Discussion IVD degeneration is a progressive process and is a major cause of LBP 2 , 42 . Herein, we demonstrated that the CSF1R signaling pathway plays an essential role in the development of IVD degeneration and discogenic back pain. Targeted deletion of Csf1r from microglia/macrophages or oral administration of the small molecular CSF1R inhibitor GW2580 decreased IVD degeneration. Furthermore, Csf1r deletion or GW2580 treatment inhibited DRG macrophage and spinal cord dorsal horn microglia activation and in so doing dramatically decreased neuropathic pain after disc injury. Proteins within the NP are recognized as non-self by the immune system and as such exposure of NP material from disc injury may elicit and propagate an immune response in a CSF1R dependent manner 3 . Inflammatory factors secreted by macrophages, together with neurotrophins (e.g., NGF, VEGF and substance P), drive angiogenesis and nerve ingrowth (i.e., the sinuvertebral nerve and sympathetic afferents) into degenerating IVDs 7 , 30 , 29 . The nerve in-growth in injured or degenerating IVDs may activate satellite glia within the DRGs which in turn increases DRG neuronal CSF1 expression 43 . CSF1 is then transported along the sensory axons to the dorsal horn of the spinal cord, where it activates microglia via CSF1R. Activated microglia then induces overexcitation of dorsal horn neurons (i.e. through BDNF signaling pathway) 44 , which induces neuropathic pain 45 , 46 . Generation of an IVD degeneration and discogenic back pain animal model Animal models are vital tools for IVD degeneration research. Given the complex pathobiology surrounding IVD degeneration and discogenic back pain, developing an appropriate and reliable animal model has been a challenge. Our modified disc puncture model displays morphological, biochemical, inflammatory and behavioral features similar to those found in humans with degenerative disc disease 47 . Previously, posterior approaches have been used to puncture discs to induce disc degeneration and pain. In the posterior approach model, one facet joint is removed, and the posterior column is disrupted which results in mechanical instability 48 , 49 . In addition to the inherent stability problems, it adds an additional cause of back pain, and therefore represents a major limitation 47 . Other approaches to disc puncture include an anterior transabdominal approach. This approach has risks of abdominal viscera injury which often leads to gastrointestinal distress and feeding impairment, thereby confounding experimental observations related to pain and associated behavioral assessments 50 , 51 . In our modified procedure, the lumbar spine was approached via a posterolateral angle on the right side. The L4/5 and L5/6 IVDs were accessed with minimal paraspinal muscle disruption using a 25G custom-made needle. This approach minimized disruption of normal spinal architecture, avoided damaging the transversing spinal nerve roots and circumvented abdominal injury. Endplate disruption was included in our disc puncture model. Endplate disruption allows the entry of inflammatory factors to the IVDs and likely plays an important role in the pathophysiology of discogenic back pain development in humans 52 , 53 , 54 . Using serial MRIs and histologic analyses, we found that our model produced consistent progressive IVD degeneration similar to human conditions. We also employed stimulus-evoked (i.e., mechanical, thermal) and non-stimulus evoked methods (i.e., burrowing assays, gait analysis, and/or automated behavioral analysis) to assess pain related behaviors 55 . Persistent and reproducible mechanical hyperalgesia was noted to last for at least 25 weeks after disc injury. In human subjects, back pain is mostly reported subjectively by the patient using VAS scale. It is challenging to subjectively determine the level of discogenic back pain in rodent models. Pain like behaviors indicative of a localized painful responses included decreased hind paw mechanical and thermal sensitivities, increased grooming, and altered walking gait patterns with longer stance phases and shorter swing phases 56 . Our mice model with degenerated IVDs had significantly reduced mechanical withdrawal thresholds and a trend towards shorter thermal withdrawal latency, like prior studies 57 . The mechanical test is more sensitive than the thermal test in assessing painful IVD degeneration in rodents 56 . Although not ideal, Von Frey test is most commonly used as an objective measure to determine discogenic back pain in rodent models 58 . Blocking CSF1R signaling slows down IVD degeneration The IVD is an immune-privileged site. Disruptions in the vertebral endplates expose the IVDs to the bone marrow which has a rich supply of immune cells and immune progenitors. Once exposed, recruitment and activation of immune cells within the IVD is initiated 3 . Early in the process, an inflammatory infiltrate comprised mostly of macrophages localizes proximal to the defects in the endplates 59 , 60 . IVD tissue (i.e., the NP) tends to polarize macrophages toward the pro-inflammatory M1-like profile which further perpetuates IVD degeneration 28 , 61 . Disc injury in our model confirmed the induction of robust disc macrophage infiltration with an increase in pro-inflammatory factors secretion. One such factor is CSF-1 which regulates macrophage survival and proliferation and modifies macrophage activation and recruitment. CSF-1 executes its myriad of functions through engagement with its cognate receptor CSF1R 17 . Here, we demonstrated that CSF1R expression is increased in infiltrating IVD macrophages after disc injury and that macrophage recruitment is inhibited by CSF1R deletion or GW2580 administration. It was reported that the levels of proinflammatory factors are higher in injured IVDs that cause pain than those from asymptomatic IVDs, suggesting that the levels of inflammation are related to pain 57 . Previous reports have demonstrated increased soluble TNF-α and MMP-3 in IVDs after exposure to macrophages 62 . In our study, pro-MMP9 was significantly upregulated in chronically injured IVDs, suggesting active ECM destruction during the course of disc degeneration 5 . TNF-α was not detected in either intact or injured IVDs, but up-regulation of sTNF RI and sTNF RII were noted in the injured IVD cultures. Such increases have previously been linked with increases in inflammation 63 . Similar to reported results from humans, levels of IL-6, CSF1, CSF3 were increased in chronically degenerating IVDs. Our work demonstrated that deletion of macrophage CSF1R decreased the secretion of pro-inflammatory factors from the chronically injured IVDs. During IVD degeneration, angiogenesis takes place and blood vessels can be seen growing into IVDs. Clinical studies have observed higher rates of angiogenesis within the inner regions of degenerated IVDs in patients experiencing LBP 30 . We detected an increase in several pro-angiogenic growth factors such as bFGF, IGF1 and VEGF in chronically injured IVDs. CSF1R deletion abolished the increase of pro-angiogenic growth factors after disc injury. Significant increases in the expression of the RANK/RANKL/OPG (osteoporotegerin) system have also been noted in advanced stages of IVD degeneration 31 . In our model, OPG upregulation with degenerating IVDs was also eliminated with CSF1R deletion. It is prudent to note that the condition of IVD culture may cause degeneration during the culture period and release inflammatory factors as a result 64 , 65 , 66 . However, in those studies that reported such phenomena, cultures were continued for a prolonged period of time (i.e., 20 days vs our 5-day culture period). Other reports have indicated that IVD units can remain healthy for up to 14 days in submersion culture medium supplemented with 10% FBS 64,65,66 . Our intact IVD control cultures generated very low levels of inflammation factors, suggesting that the increased inflammation factors in our chronically injured IVD cultures were reflective of the underlying pathobiology of disc degeneration, not a result of the culture condition. Discogenic pain is eliminated with inhibition of CSF1R signaling pathway It has been demonstrated that peripheral nerve injury induces de novo CSF1 expression in injured sensory neurons and that CSF1 is transported to the dorsal horn of the spinal cord afterwards 19 . DRG neuron derived CSF1 not only stimulates proliferation of surrounding macrophages but also induces spinal cord microglia proliferation and expression of a host of neuropathic pain–associated genes 46 . Both DRG macrophages and dorsal horn microglia have been shown to contribute to the initiation of pain and the transition from acute to persistent neuropathic symptoms 14 , 13 . The anterior annulus of IVD is innervated by nerves derived from the ventral and gray rami communicans of the autonomic nervous system. The posterior annulus is innervated by the sinuvertebral nerve, a branch of the spinal nerve at each associated intervertebral level 39 , 67 . Disc injury induced inflammation has been shown to stimulate sensory nerve fibers and DRG sensory neurons. Consistent with reports in other models, we showed that de novo CSF1 expression did in fact increase in the DRG neurons after disc injury 19 . CSF1 production in the DRG reached a high level 7 days after disc injury, and it persisted for at least 28 days. Other labs found that CSF1 was induced in CGRP-expressing DRG neurons 2 weeks after disc injury 20 . Such differences may be the result of variations between experimental models. Similar to responses after peripheral nerve injury, prominent microglia and macrophage activation in spinal cord dorsal horn and DRGs were observed after disc injury in our model. As CSF1R activation in spinal cord microglia induced pain behavior in peripheral nerve injury models 68 , 19 , we proposed that increases in CSF1R within spinal cord microglia after disc injury contribute to the development of discogenic back pain. Indeed, the alleviation of discogenic neuropathic pain after the genetic deletion of CSF1R or CSF1R specific inhibitor suggested an essential role of CSF1R signaling pathway in the initiation and maintenance of the discogenic neuropathic pain after disc injury. We also examined c-Fos , an immediate–early gene whose expression in the spinal cord has been extensively used as a marker for peripheral noxious stimulation 69 . We used TRAP mice to monitor c-fos gene expression related to pain in spinal cord neurons. c-Fos was highly expressed in the spinal cord dorsal horn after disc injury. Disc injury included c-Fos expression was largely eliminated following GW2580 treatment. GW2580 as a potential therapeutic agent for disc degeneration and discogenic pain A therapeutic approach centered on the modulation of CSF1R signaling needs to be effective at inhibiting microglial/macrophage activation, but not affecting basal mononuclear phagocyte survival, so that it does not lead to adverse effects in CNS homeostasis. This is particularly important for treating benign conditions such as back pain. CSF1R belongs to the type III class of growth factor receptors, which includes PDGFR, c-KIT, and FLT3 17 . An analysis of the activity and selectivity of several CSF1R inhibitors such as PLX3397, imatinib, BLZ945, Ki20227 or edicotinib demonstrated that they were also inhibitors of PDGFRβ and c-Kit 35 . For example, PLX3397 inhibits the survival of microglia within the healthy brain and concurrently inhibits c-Kit, FTL3 and PDGFRβ 70 . Loss of PDGFβ signaling impacts survival of NG2 pericytes and may therefore lead to blood–brain barrier damage and neurodegeneration. 18 PLX5622 also inhibits the survival of microglia 37 . GW2580 is a selective competitive inhibitor with a high affinity for CSF1R. It inhibits CSF1R activation without affecting related kinases or ablating the resident macrophage and microglial population 33 . It has been shown to decrease neurotoxicity in animal models of Alzheimer's disease, amyotrophic lateral sclerosis, and prion disease 33 , 38 , 71 . In a spinal cord injury model, GW2580 specifically inhibited microglia proliferation following spinal cord injury but did not perturb microglia responses and functions in control animals 72 . In the present study, we administered GW2580 orally to treat disc degeneration and discogenic neuropathic pain. GW2580 selectively inhibits spinal microglial proliferation and activation without affecting the survival of resident microglia. GW2580 did not change microglia numbers when the spinal cord or brain were examined in control animals. GW2580 inhibited pro-inflammatory activation of DRG macrophages but did not affect the overall number of resident macrophages. GW2580 also decreased macrophage infiltration into the disc AF and NP (Fig. 5 ). No effective medical treatments currently exist for the prevention of disc degeneration. While some experimental treatments have been delivered via local administration 2 , oral treatment might be superior, given that disc degeneration often occurs at multiple spine levels. Clinically, discogenic pain sometime is difficult to localize to a single level, even with advanced diagnostic methods such as discography 42 . While oral administration raises concerns of possible systemic adverse effects, GW2580 treatment had no discernable side effects in our studies. No effect on food intake, animal behavior or animal weight was noticed. No systemic adverse effects were observed on veterinary pathological autopsy. Overall, CSF1R specific inhibitor GW2580 presents a promising therapeutic avenue for the treatment of disc degeneration and discogenic back pain. With its outstanding oral bioavailability, safety profile and efficacy, GW2580 and its next-generation derivatives can be excellent candidates for potential medical treatments aimed at mitigating disc degeneration and alleviating chronic discogenic low back pain. Materials and Methods Animals Mice were housed under standard 12-hour light/12-hour dark conditions with ad libitum access to food/water. Animal care and handling procedures were congruent with those set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal protocols employed throughout this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the Brigham and Women’s Hospital, Harvard Medical School. Cx3cr1 +/GFP knock-in/knock-out mice (JaxMice; stock #: 005582) were used as a macrophage/monocyte reporter line. B6.Cg-Csf1r fl/fl mice (JaxMice; stock #: 02212) were crossed with the B6.129-Cx3cr1tm2.1 (CreER) (JaxMice; stock #: 020940). The resulting mice displayed tamoxifen inducible CRE activity specifically in mononuclear phagocytes leading to a non-functional CSF1R protein. Fos CreER mice (c-Fos Cre ERT2 (B6.129(Cg)- Fostm1.1(cre/ERT2)Luo /J JaxMice; stock #: 021882) and Ai14 (B6.Cg- Gt(ROSA)26Sortm14(CAG-tdTomato-)Hze /J, JaxMice; stock #: 007914) mice were obtained from the Jackson Laboratory and maintained as the provided guidelines. Genotyping : For Fos CreER mice, WT product: 215, mutant fragment:293. Common forward, CAC CAG TGT CTA CCC CTG GA; WT reverse, CGG CTA CAC AAA GCC AAA CT; mutant reverse, CGC GCC TGA AGA TAT AGA AGA. For Ai14 mice, WT fragment: 297 bp, mutant fragment: 196 bp. WT forward, AAG GGA GCT GCA GTG GAG TA; WT reverse, CCG AAA ATC TGT GGG AAG TC; mutant reverse, GGC ATT AAA GCA GCG TAT CC; mutant forward: TTC CTG TAC GGC ATG G. For Cx3cr1 GFP knock-in/knock-out mice, WT fragment: 410 bp, mutant fragment: 500 bp Wild type forward: GTC TTC ACG TTC GGT CTG GT; Common: CCC AGA CAC TCG TTG TCC TT; Mutant Forward: CTC CCC CTG AAC CTG AAA C. For B6.Cg-Csf1r fl/fl mice, WT fragment: 193 bp, mutant fragment: 273 bp. Forward: GGA CTA GCC ACC ATG TCT CC; Reverse: CAT GGC TGT GGC CTA GAG A. For B6.129-Cx3cr1 CreER mice, WT product:151bp, mutant fragment: 230bp. Wild type forward, AGC TCA CGA CTG CCT TCT TC; Common, ACG CCC AGA CTA ATG GTG AC; mutant forward, GTT AAT GAC CTG CAG CCA AG.) Tamoxifen preparation/administration Tamoxifen was dissolved in corn oil (Sigma-Aldrich cat# C8267) and 100% ethanol for 1 h at 37°C and was vortexed every 15 min. We used ∼75 mg tamoxifen/kg body weight and 100 µl tamoxifen/corn oil solution was administered via intraperitoneal injection for 14 consecutive days. GW2580 Treatment using Oral Gavage GW2580 (SML1047 Sigma-Aldrich) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 80 and was dosed orally at 80 mg/kg (0.2 ml per mouse). Mice were orally gavaged with 18G needles (Instech, Cat No. FTP-18-30-50). Surgical Procedure(s) The mouse model of IVD degeneration was established using the following procedure. Briefly, the mouse was fixed in a prone position after being anesthetized (Ketamine 100–120 mg/Kg and Xylazine 10 mg/Kg). The lumbar spine was approached via posterolateral angle on the right side. The L4/5 and L5/6 IVD was accessed/punctured through minimal paraspinal muscle using a 25G custom-made needle. Needles were inserted through the dorsal annulus, through the NP center and partially through the ventral annulus (controlled depth of 1.75 mm or 90% of the dorsoventral width) for 30 s and removed. It is then followed with rotating a custom-made tool inside the disc socket to ensure the damage of vertebrate endplates. Sham surgery consisted of an incision followed by exposure of L4/5 and L5/6 IVDs. Then the wound was sutured without receiving any needle puncture in IVDs. After the surgery, experimental animals had standard postoperative treatment. The animals were placed in individual cages after the operation. The animals’ lower extremity activity, puncture incision healing, and death were monitored. Lumbar magnetic resonance imaging scans and quantification Scans were performed using Paravision 5.1 (Bruker BioSpin Corporation, Billerica, MA) interfaced to a 7.0T Bruker BioSpec USR (Bruker BioSpin Corp.) using a custom –made 2cm volumetric coil in the Small Animal Imaging Laboratory (SAIL) at Brigham and Women’s Hospital. The sagittal and cross-section T2 weighted MR images(T2WIs) were qualitatively analyzed to evidence the degenerative changes. Parameters: repetition time 2500 ms, echo time 30 ms, RARE factor 4, averages 24, field of view 20mm*20mm, slice thickness = 0.6 mm, for a scan time of 25 minutes per mouse plus setup. Disc height measuring and Pfirrmann grade interpretating were analyzed from T2WIs using Bruker Topspin, version 3.2 and Case Viewer, version 2.3. Quantification of MRIs was performed for the lumbar discs using the series section of each disc. A series of mid-sagittal slices for T2 mapping (TE = I *8ms, I = 1,2,3,4,…25, 25 echo times in total) were obtained with an inplane resolution of 100um and a 0.6mm slice thickness. T2 maps of one mid-sagittal slice from each sample were then generated in Bruker Topspin. A region of interest (ROI) including cartilaginous endplates, annulus fibrosus (AF) and nucleus pulposus (NP), was drawn manually to calculate mean T2 values, structures outside the disc were carefully avoided. Data were expressed as percentages of the results obtained when using sham surgery control discs. Here, the control was defined from the value of adjacent uninjured IVD. All the image assessments were performed by two independent blind observers, and the quantitative data were presented as means of three evaluations. Histology After the MRI examinations, mice were fixed by transcardiac perfusion with 4% paraformaldehyde. Discs were post-fixed with 4% (w/v) paraformaldehyde for 48 hours and decalcified in Kristensen’s decalcifying solution for 2 weeks and then washed for 24 hours under running tap water and paraffin embedded. Discs were cut into transverse 5-µm sections and collected on Superfrost Plus slides and stored at room temperature until use. Staining was performed at room temperature. Mid-sagittal sections were dewaxed in xylene in the fume hood and rehydrated through 95%, 70%, and 50% ethanol washes for 2 min each. Safranin O-fast green staining. Briefly, sections were stained with Weigert’s iron hematoxylin working solution for 10 minutes followed by fast green (FCF) solution for 5 minutes. Then, sections were rinsed quickly with 1% acetic acid solution for 10 − 15 seconds followed by staining with 0.1% safranin O solution for 5 minutes. For H&E staining, sections were stained in Mayer’s hematoxylin for 6 min and washed under running tap water before staining in eosin for 2 min. After a quick rinse in tap water, sections were dehydrated through 50%, 70%, 95%, and absolute ethanol washes for 1 min each. All sections were cleared in two changes of xylene and covered with the distyrene-plasticizer-xylene (DPX) mounting medium and a coverslip. The sections were placed in an oven at 37°C to enable the mounting medium to solidify before imaging under a light microscope (Nikon). Histological classification of disc degeneration For each H&E classification type, the maximum points represent severe degeneration. The control was defined as the histological score of L3/4 IVD. Stained slides were graded with a validated histological grading system, as described previously 73 . Major anatomical structures of the AF and NP were included in this classification, resulting in four subcategories. Each item was graded as zero, one, or two on the H&E and safranin O-fast green sections, with zero representing nondegenerative characteristics, one representing mild degenerative characteristics, and two representing severe characteristics of degeneration. The total score was the sum of the four different scoring items, resulting in a minimum score of zero, corresponding to a healthy disc, and a maximum score of eight, corresponding to an entirely degenerated disc. All the histological assessments were performed by two independent blind observers, and the quantitative data were presented as the mean of three evaluations. Behavioral tests Adult male and female mice were used for behavioural assays. Before each assay, animals were acclimated to the experimental conditions for 3 days (once per day). Mice of each group were tested in a random and blinded fashion. Von Frey tests The von Frey test was carried out 2 days before surgery (day − 2) and on 1, 7, 14-, 21-, 28- and 42-days post-surgery. The mice were individually placed into six-compartment mice enclosure with wire mesh floors and lids with air holes (IITC Life Science) for a 20-min habituation period to minimize exploratory activity. Threshold responses to a mechanical (tactile) stimulus were measured by placing each subject in an elevated observation chamber with a wire mesh floor whereupon the plantar surface of the ipsilateral hindpaw could be stimulated with a graduated series of seven Von Frey filaments (Stoelting), ranging from 0.03–2.04 g, using the up/down method. Response percentages per 10 tries with 3 min intervals were also measured for each stimulus intensity. Acetone evaporation test Cold sensitivity was assessed by acetone evaporative cooling. Through the mesh floor a series of five applications of acetone (50 µl; application separated by at least 5 min) were gently applied to the bottom of the paw using a multidose syringe device. Individual responses were scored on a 0–2 scale, wherein 0 = no response or a rapid transient lifting or shaking of the hindpaw that subsides immediately; 1 = lifting, licking, and/or shaking of the hindpaw, which continues beyond the initial application, but subsides within 5 s; and 2 = protracted, repeated lifting, licking, and/or shaking of the hindpaw. Individual scores are averaged over the five applications. Alternatively, cumulative paw licking duration was recorded over 60 s for each of three acetone applications, with the average taken for each animal. Hot plate test The surface of a hot plate is heated to a temperature of 55 ± 1°C. The mouse is placed on the heated plate, and the latency for it to show a nociceptive response with paw lick, paw flick, or a jump is measured with the timer. The mouse is immediately removed when this response is observed. If the mouse does not display a response within 30 sec, the mouse is removed from the heated plate to prevent any tissue damage. Burrowing assay Mice were acclimatized to the specific burrowing tube once on the day before the burrowing assay was performed (for 1 h), and again on the day of testing (for 30 min). For acclimatization an empty tube was placed into the home cage, such that all five mice in the cage were exposed to the tube. All mice were observed to voluntarily enter the tube within 10–15 min. On the day of testing, a separate cage was prepared for each mouse to be tested individually. An acrylic tube was filled with 90 g of the same corncob bedding used in the home cage and placed in one corner, parallel to the long walls of the cage. Mice were then transferred to these cages. After test, mouse was returned to the home cage and the bedding remaining in the tube was weighed. The burrowing activity was calculated by subtracting the weight of bedding present at the end of the experiment from the starting weight and expressing the proportion of bedding that had been displaced as a percentage. To minimize any potentially confounding effects of differing olfactory cues, approximately 5 g of bedding from the home cage was transferred to the testing cage immediately prior to first testing. In addition, all bedding from a particular test cage was stored for re-use in a re-sealable plastic bag between tests. Treadmill gait For treadmill walking, mice were placed on the DigiGate at various speed. Speed tolerance was defined as the maximal speed a mouse can walk on the treadmill without falling. All trials were video recorded (Hotshot e64, 100 fps). Gait parameters were measured and repeated at post-operative days 3, 7, 14-, 28- and 42-days. Rotarod test The rotarod performance test involves forced motor activity by rodents on a rotating rod with accelerating speed. Rodents are trained for 2–3 days on a rotarod (Med Associates, Inc., St. Albans, VT) at varying speeds before the final test is conducted. The rotarod started from stationary and accelerated from 4 to 40 rpm over 5 min. In the test, a mouse is placed on a horizontally oriented, rotating cylinder (rod) suspended above a cage floor. The rod is low enough that the animal will not be injured if it falls but high enough to induce avoidance of fall. The length of time that a given animal stays on the rotating rod is a measure of their balance, coordination, physical condition, and motor-planning. The maximum end-speed was recorded when the mouse fell off the treadmill. Three trials were performed with a 20-min break between trials. This test was performed 1 day pre-surgery and days 3, 7, 14, 28, and 42 post-surgeries. Open field test The open field consists of a circular environment with 1.2 m diameter closed by a wall of 0.45 m high. The mice are allowed to move freely within the space, and the time spent in each region is quantified. The circular environment is virtually divided into regions so that there is a clear center region. The number of central regions visited, the time spent in the central region, and overall locomotion were quantified. The open field behavior is calculated by SMART 3 software. Both the number of central squares visited, and the time spent in the central squares are markers of exploratory behavior (80) . This test was performed 1 day pre-surgery and days 14 and 42 post-surgeries. Disc organ culture Soft tissues and posterior elements around discs were removed, and discs were rinsed in saline solution before being placed in culture plates. The suspension culture in which a sterile 8 µm transwell was first placed at the center of each well of 24-well plates, which were filled with 10% FBS DMEM medium. The bottom half of each column was first loosely stuffed with cotton balls semi-saturated with medium, and then the IVD tissue sample was placed at the center of the column, which was subsequently filled with further medium-semisaturated cotton balls. Experimental specimens were cultured in 5% CO2 and 37 o C. IVD culture medium was collected for Cytokine analysis. Cytokine analysis The concentration of cytokines in the IVD culture medium was quantified using mouse cytokine Array C1000 (AAM-CYT-1000) (RayBiotech Life, Inc. GA) according to manufacturers' instructions. Arrays were imaged with the provided enhanced chemiluminescence kit using an ImageQuant LAS4000 (GE Healthcare, Baie d'Urfe, QC, Canada). ImageQuant TL array analysis software (GE Healthcare) was used to analyze the blots. The relative quantity of each factor present in each media sample was calculated using the controls included in the protein arrays. Mean relative quantities of each factor for the degenerating and healthy discs were then calculated. Immunofluorescence and confocal imaging Immunofluorescence labeling was performed according to previously published method 19 . Primary antibodies used in this study are of the following sources and were used at the indicated dilutions: goat anti-iba1 (1:1000, Novus Biologicals, NB100-1028), rabbit anti-Ki67 (1:200, Abcam, ab15580), chicken anti-neurofilament H (NF-H, 1:500, EMD millipore, AB5539). Mouse M-CSF antibody (1:1000, AF416), anti-mouse CD115 (CSF-1R) antibody(1:1000, LS‑C130595). Anti-CD68 antibody (1:1000, ab53444). Fluorescent Nissl Stain (1:500, N21482). Alexa Fluor secondary antibodies from Jackson ImmunoResearch Laboratories (Alexa Fluor® 488 AffiniPure Donkey Anti-Goat IgG (H + L), AB_2340428, Alexa Fluor® 594 AffiniPure Donkey Anti-Mouse IgG (H + L) AB_2340855, Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H + L), AB_2492288, 1:250 dilution) were used for multicolor immunofluorescence imaging, whereas 4′,6-diamidino-2-phenylindole, dilactate (DAPI; 1 µg/ml, Thermo Fisher Scientific) was used for nuclear counterstaining. Sections were imaged on a Zeiss LSM 710 confocal microscope system equipped with 405, 488, 555, and 647 nm lasers. Confocal images were acquired using a Zeiss Axiocam 506 Mono camera and mosaics created using the Zen 2.3 software (Blue edition). For immunohistochemical staining of CSF1R and IBA1 in paraffin sections, 4 µm thin IVD tissue sections were dewaxed in xylene, acetone and Tris-buffered saline, followed by antigen retrieval using sodium citrate buffer at 95°C for 10 min. Primary antibodies were incubated over night at 4 o C. MACH 4 Universal HRP-Polymer (Biocare, M4U536) were used to detect either mouse or rabbit antibodies. After DAB staining, counterstain slides for 1 min with Mayer's Hematoxylin. Dehydrate slides by incubating for 3 min each in: 70% EtOH − 80% EtOH − 95% EtOH − 2x 100% EtOH − 2x Xylene. Sections were mounted with coverslips using a Xylol-based Fast Mounting Medium. Statistics Two-way ANOVA was utilized for time course studies to determine the interaction of genotype versus day of study. Differences between csf1r knockout genotypes on specific days were assessed by post hoc Bonferroni. One-way ANOVA was utilized to determine within-genotype differences by day of study with individual comparisons made using a post hoc Dunnett's multiple comparisons test with significance set at p < 0.05. GraphPad Prism 4 software was utilized for statistical analyses. Data Availability Statement Values for all data points in graphs are reported in the Supporting Data Values file. Declarations Acknowledgments We thank Professor Zhigang He (F.M. Kirby Neurobiology Center, Boston Children’s Hospital) for valuable comments. Funding: This work received funding from The Stepping Strong Innovator Awards program 2018 and The Stepping Strong Innovator Awards program 2020. Author contributions : Conceptualization: LL, HG, YL Methodology: LL, JZ, YY, WY, HG Investigation: LL, JZ, YY, WY, FT, BC, HG, YL Visualization: LL, JB, HG Writing – Original Draft: LL, HG Writing – Review & Editing: HG, JB, JC, YL Supervision – HG, YL Competing interests: Authors declare that they have no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. References Hicks, G. E., Morone, N. & Weiner, D. K. Degenerative lumbar disc and facet disease in older adults: prevalence and clinical correlates. 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Regulation of Microglial Proliferation during Chronic Neurodegeneration. J. Neurosci. 33, 2481 (2013). Gerber, Y. N. et al. CSF1R Inhibition Reduces Microglia Proliferation, Promotes Tissue Preservation and Improves Motor Recovery After Spinal Cord Injury. Frontiers in Cellular Neuroscience 12, 368 (2018). Ohnishi, T. et al. In Vivo Mouse Intervertebral Disc Degeneration Model Based on a New Histological Classification. PLOS ONE 11, e0160486 (2016). Additional Declarations There is no conflict of interest Supplementary Files SupfigureBoneres.docx supplemental figure file Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5647673","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":394939067,"identity":"1ee5c022-eeeb-41dd-acd5-8e76e13489ed","order_by":0,"name":"Yi Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYDACdgYGg48NDAkMEhA+YwNQAL8WZgaGwpkka/nMS5IWg8M8hpttdxzOY5BufvaZp+aebAN78zYJAlqMjXPPHC5mkDlmPJvnWLFxA8+xMkJazIxz224nNkgkGDPOYEsAMnLMCGkx/20J1pL+mXHGP6AW+TcEtRgYM4K15BgzfGwD2cKDX4vkYbYCw94z/xPbJHKKGT72JRi38aQVW+DTwne8eYPBzx1pif0S6ZsZEr4lyPazH954A58WhQNQBhsDOgMXkG8gpGIUjIJRMApGAQBcBUpTVF+9JwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8180-8578","institution":"Brigham and Women's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Lu","suffix":""},{"id":394939068,"identity":"5a6b57f1-b090-4ba9-b48a-292af4e95c96","order_by":1,"name":"Lei Liu","email":"","orcid":"","institution":"Zhongda Hospital, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Liu","suffix":""},{"id":394939069,"identity":"51b01d37-6e57-4735-a6b9-92353af02058","order_by":2,"name":"Jingdong Zhang","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Jingdong","middleName":"","lastName":"Zhang","suffix":""},{"id":394939070,"identity":"cb96fb39-70b8-4a01-9755-c50bf825fadc","order_by":3,"name":"Yizheng Yao","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yizheng","middleName":"","lastName":"Yao","suffix":""},{"id":394939071,"identity":"7d34560e-0a15-4827-a6cc-bf48b935adf4","order_by":4,"name":"Weixuan Yan","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weixuan","middleName":"","lastName":"Yan","suffix":""},{"id":394939072,"identity":"f30a5d30-b83f-4443-90f1-a7bbc9e4db09","order_by":5,"name":"Songlin Zhou","email":"","orcid":"","institution":"Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Songlin","middleName":"","lastName":"Zhou","suffix":""},{"id":394939073,"identity":"66d943f8-0ee4-4516-8ab3-7a87f02b6109","order_by":6,"name":"Joshua Bernstock","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Bernstock","suffix":""},{"id":394939074,"identity":"1371c1d4-0958-4868-9844-9a0a252e21b4","order_by":7,"name":"Joshua Chalif","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Chalif","suffix":""},{"id":394939075,"identity":"5666cff6-2040-44e8-8dd2-29ff6b814464","order_by":8,"name":"Zhimin Li","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhimin","middleName":"","lastName":"Li","suffix":""},{"id":394939076,"identity":"99847288-e798-4bfa-b9b8-1205d31e4f55","order_by":9,"name":"Feng Tian","email":"","orcid":"","institution":"Beth Israel Deaconess Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Tian","suffix":""},{"id":394939077,"identity":"5f0e3d14-9a19-4b85-81da-52f12cfaad89","order_by":10,"name":"Bo Chen","email":"","orcid":"","institution":"University of Texas Medical Branch","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Chen","suffix":""},{"id":394939078,"identity":"491a6708-1b19-4338-b312-a85cb3452364","order_by":11,"name":"Hong Guo","email":"","orcid":"","institution":"Brigham and Women's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2024-12-15 13:25:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5647673/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5647673/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72643215,"identity":"d87daa0b-3d1b-495b-9714-cdfa52fdc0d1","added_by":"auto","created_at":"2024-12-30 16:37:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":356942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecsf1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in the human IVD.\u003c/strong\u003e A)Uniform Manifold Approximation and Projection (UMAP) plot representing color-coded cell clusters identified in merged single-cell transcriptomes of healthy and degeneration cells from same patients. Note:cluster 12 is macrophage and appered in IVD with degeneration. B) GO analysis showing top15 enriched biological functional pathways of upregulated genes in degeneration NP. C) Volcano plot depicting differences of cluster abundance in healthy compared to degeneration plotting fold change (log2) against \u003cem\u003ep\u003c/em\u003e value (−log10). Dotted line indicates significance threshold. D)Feature plots depicting distribution of the expression of \u0026nbsp;\u003cem\u003ecsf1\u003c/em\u003e in NP cells. E) Dot plot depicting \u003cem\u003ecsf1\u003c/em\u003e and \u003cem\u003ecsf1r\u003c/em\u003e genes in cell clusters. Dot size encodes percentage of cells expressing the gene, color encodes the average per cell gene expression level. (F) Heat map depicting the expression patterns of the human inflammation genes in endplate. Red bars represent the normal and blue bars represent the degenerated IVDs. (G) Expression of CSF1. p\u0026lt;0.05. n=3. (C) Expression of IL34. no significant difference. n=3 (D) Expression of CSF1R. no significant difference. n=3.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/5dfd277a77f55875c9547f5c.png"},{"id":72645758,"identity":"44488c7f-4449-46c7-afd9-ba064afa5270","added_by":"auto","created_at":"2024-12-30 16:45:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisc injury induced persistent disc degeneration and allodynia. \u003c/strong\u003e(A) Schematic diagram of the experimental timeline. Mice received baseline behavioral measurement at P56, DP at P57. Behavioral measurement from P60–P240 before terminal histological analysis. (B)Representative histologic images of L4/5 IVDs sagittal Safranin-O/Fast Green stained sections from uninjured, 3 months after DP and 6 months after DP were shown. Scale bar = 200 µm in upper panel and 125 μm in lower panel magnified images. (C) Representative MRI images of IVDs (arrow: L4/5 and L5/L6 IVD) from uninjured mouse and mice 3 and 6 months after DP. (D) MRI T2 values. (n=8 for sham group, n = 6 for DP groups). Data was expressed as mean ± SEM. *p \u0026lt; 0.05. (E) Disc heights were measured in mice 3- and 6- month after DP (n = 10 for each group) and uninjured mice (n =9). (F) Mechanical allodynia was analyzed by von Frey test at indicated time after DP. n = 10. *p\u0026lt;0.05. (G) Cold hyperalgesia was analyzed by the plantar test at indicated time after DP. n = 10. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/f8d19a3f13397c6cbf65ec78.png"},{"id":72643216,"identity":"18b7bb8a-6a63-4919-8882-336ea1f147a6","added_by":"auto","created_at":"2024-12-30 16:37:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":458841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCSF1R signaling in IVD inflammation\u003c/strong\u003e. (A) Immunofluorescence of macrophage and quantification in disc. cx3cr1-GFP mouse intact and injured IVD were stained with GFP. Scale bar: 200µm. n=7 mice. (B and C) Intact and DP IVD stained with Iba1(B) and CSF1R(C). Scale bar: 20µm. (D-E) Quantification of CSF1R\u003csup\u003e+\u003c/sup\u003e (E) and Iba1\u003csup\u003e+\u003c/sup\u003e in(D) from intact and DP IVDs (n = 4 mice per group). *p\u0026lt;0.05.\u0026nbsp; (F) Schematic of the basic process of the IVD culture. Sham or DP mice were euthanized and the lumbar IVDs were dissected out. Individual IVD tissue contained the endplates on both sides. A sterile 8mm-diameter transwell was first placed at the center of each well of a 24-well plate, which was filled with complete medium. The bottom half of each transwell was loosely stuffed with medium saturated cotton balls. The IVD tissue sample was placed at the center of the transwell, which was then filled to the top with medium saturated cotton balls. (G and H) Cytokine production in IVDs from Cx3cr1\u003csup\u003eCreER/+\u003c/sup\u003e: Csf1r\u003csup\u003efl/fl\u003c/sup\u003e mice with sham surgery (uninjured), DP with vehicle or DP with TAM (Csf1r deletion).\u0026nbsp; Five days after culture, cytokine productions included were evaluated. *p \u0026lt; 0.05, n=6 discs for each group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/8632a8fe6f909309eaed6af2.png"},{"id":72645781,"identity":"5f3705af-f7a6-41d5-966b-f89163fd5af5","added_by":"auto","created_at":"2024-12-30 16:45:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCsf1r \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeletion alleviated IVD degeneration following DP.\u003c/strong\u003e(A)Schematic diagram of the experimental timeline.\u003cstrong\u003e \u003c/strong\u003e(B) Quantification of the MRI T2 signal in IVDs 90- and 180- days after surgery from Cx3cr1CreER: Csf1rfl/fl mice with sham surgery, with vehicle treatment after DP, or with TAM treatment after DP. (C) Quantification of the disc height 6 months after surgery. (D) Representative Safranin-O/Fast Green stained sagittal sections of the IVDs from Cx3cr1CreER: Csf1rfl/fl mice with sham surgery (uninjured) (left), with vehicle treatment 6 months after DP (middle), with TAM treatment 6 months after DP (right). Scale bar = 200 µm (upper panel) and 125 μm (lower panel). (E) Quantification of CEP thickness. *p \u0026lt; 0.05, n=6 for uninjured group. n=7 for vehicle and TAM group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/b994177cb3a92d2acf63101d.png"},{"id":72643217,"identity":"7309530e-4907-43e8-9675-960ee5558ee4","added_by":"auto","created_at":"2024-12-30 16:37:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":517255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of CSF1R pathway using GW2580 alleviated IVD degeneration following DP. \u003c/strong\u003e(A) An experimental diagram showing the timeline of drug treatments after DP. C57BL/6 mice with DP received GW2580 or vehicle after the surgery. Mice continued to receive GW2580 or vehicles until the end of the experiments. At indicated time points, mice were subjected to MRIs, behavioral tests and tissue immunostaining. (B) MRI images of IVDs from mice received GW2580 or vehicle 90 and 180 days after injury. Note the L4/L5 and L5/L6 IVDs (White arrow). (C) Quantification of the T2 signal in IVDs 90 days and 180 days after DP. *p \u0026lt; 0.05, n=7 for each group. (D) Quantification of the disc height 180 days after DP. *p \u0026lt; 0.05, n=5 for each group. (E) H\u0026amp;E staining of the sagittal sections of IVDs in the uninjured, DP with vehicle control and DP with GW2580 groups 180 days after injury. Left panel, scale bar = 150 μm. Right panel (magnification of left), scale bar = 30 μm. (F) Representative sagittal Safranin-O/Fast Green-stained sections of the IVDs of from mice with sham surgery (uninjured, left), mice treated with vehicle or GW2580 at 3 6- months after DP. Scale bar = 150 μm. (G) IBA1 staining of the sagittal sections of IVDs in DP mice with vehicle control and DP mice with GW2580 groups 180 days after injury. Scale bar = 15 μm. (H) Quantification of G *p \u0026lt; 0.05, n=5 for each group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/9f75925f41f9551f60422828.png"},{"id":72643239,"identity":"94a010d8-59c9-4381-a3d3-18123704f2fe","added_by":"auto","created_at":"2024-12-30 16:37:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":486674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCsf1r\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion prevented microglia activation and neuropathic pain development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustrating relevant neuroanatomy. Injured sensory neurons in dorsal root ganglia (DRG) release signals that communicate with and activate spinal cord microglia which contributes to the pain. (B) CSF1 and Nissl staining on DRG from uninjured and DP mice 7 days post injury. Peripheral branch (star). Central branch (namely the dorsal root, triangle). DRG (arrowhead). Note CSF1 expression in DRG neurons (Nissl) and dorsal nerve root (triangle). Scale bar: 100 μm; inset, 30 μm. Central branch CSF1 was quantified (right panel) n = 5 DRG per group. *p\u0026lt;0.05. (C)\u003cstrong\u003e \u003c/strong\u003eDP-induced CSF1 upregulation in the DRG was downregulated by \u003cem\u003eCsf1r\u003c/em\u003e deletion. Scale bar: 50µm. CSF1/Nissl ratio was quantified (right panel of C). n = 6-7 DRGs per group. *p\u0026lt;0.05.(D) Iba1 and CD68 (active microglia) in the dorsal spinal cord were downregulated with \u003cem\u003eCsf1r\u003c/em\u003edeletion. Scale bar: 50µm. (E-F) Quantification of Iba1(E) and CD68(F) in D (n = 7-10 mice per group). *p\u0026lt;0.05. (G) Mice with \u003cem\u003eCsf1r \u003c/em\u003edeletion showed a decrease in mechanical allodynia comparing with mice treated with vehicle control after DP. n=11 for uninjured, n=12 for DP with vehicle group and n = 10 mice for DP with TAM treated group. *p\u0026lt;0.05 compared with vehicle control.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/46b51c102ded66c4fc30cfe1.png"},{"id":72645759,"identity":"9502fade-7c6f-4536-acfb-9ebb2702ed91","added_by":"auto","created_at":"2024-12-30 16:45:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":489833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of CSF1R pathway using GW2580 prevented neuropathic pain development after DP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) DRG CSF1 expression at indicated days after DP. Scale bar: 50µm. (B) Quantification of CSF1/Nissl ratio. n = 6 DRGs per group. *p\u0026lt;0.05. (C) Iba1 and CD68 in the spinal cord dorsal horn(left). Iba1 and CSF1R expression in the spinal cord dorsal horn(right). Scale bar: 50µm. (D) Quantification of CD68 from (C). (n = 8-9 in each group). *p\u0026lt;0.05. (E) Quantification of CSF1R from (C). (n = 8-9 in each group). *p\u0026lt;0.05. (F) Schematic of the \u003cem\u003eTRAP\u003c/em\u003e system. In the presence of TAM and DP injury, CreERT2 recombination can occur in active cells and result in tdTomato (Fos) expression. Increased Fos (tdTomato) expression in lumbar dorsal horn after DP. Scale bar: 400 µm. Insets: high-magnification images of the boxed areas in the respective micrographs. Scale bar: 10 µm. (G) Fos (tdTomato) expression in DRGs in mice with sham surgery(uninjured), with DP surgery then treated with vehicle or GW2580. Scale bar: 250 µm. Insets: high-magnification images of the boxed areas in the respective micrographs. Quantification of Fos(tdTomato) positive neurons (right panel of G). Scale bar: 40 µm. *p\u0026lt;0.05. n=7-9 DRGs in each group. (H) Fos (tdTomato) expression in the lumbar dorsal horn of mice after DP, treated with vehicle or GW2580. Scale bar: 400 µm. Insets: high-magnification images of the boxed areas in the respective micrographs. Scale bar: 10 µm. Quantification of Fos(tdTomato) positive neurons in spinal cord dorsal horn(right panel of H). *p\u0026lt;0.05. n=5 in each group. (I) Von Frey behavioral assay showing mechanical allodynia in mice with sham surgery(uninjured), with DP surgery then treated with vehicle or GW2580. (Data represent mean ± s.e.m., n=9 for GW2580 group and 8 for sham and vehicle groups. *p\u0026lt;0.05.(J) A simplified illustration showing that CSF1 produced by DRG activates spinal microglia and subsequently modulates the ascending pain pathway. The inhibition of CSF1-CSF1R pathway by CSF1R deletion or GW2580 alleviated the pain.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/46ed855ef772e37d46f9cebd.png"},{"id":72643221,"identity":"e78060e1-492b-44e8-9e3d-15cb6ec7aefa","added_by":"auto","created_at":"2024-12-30 16:37:30","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":81180,"visible":true,"origin":"","legend":"Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. Inflammation has been implicated in discogenic back pain and disc degeneration, however, the detailed molecular mechanisms remain unclear. Herein we demonstrate that Colony Stimulating Factor 1 Receptor CSF1R) signaling plays an essential role in the development of IVD degeneration and discogenic back pain. Genetic deletion of from microglia/macrophages or oral administration of a CSF1R competitive inhibitor, GW2580, decreased IVD degeneration as evidenced by serial magnetic resonance imaging (MRI) and histopathological analyses in adult mice following disc injury. deletion or GW2580 administration inhibited pro-inflammatory cytokine release from injured discs and blocked dorsal root ganglion (DRG) macrophage and spinal cord dorsal horn microglia activation and in so doing, eliminated neuropathic pain secondary to disc injury. These results suggest a novel therapeutic strategy for the treatment of chronic low back pain secondary to IVD degeneration.","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/801859412b7c63a12e967555.png"},{"id":78175600,"identity":"a6687f6e-4e58-493b-a6f7-ba440b7735b0","added_by":"auto","created_at":"2025-03-10 15:29:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4471321,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/f1fcca6c-e4dd-42e3-b3e0-230dd1a27edf.pdf"},{"id":72643218,"identity":"fda44750-127b-4f57-b0a5-329f5c4a49ba","added_by":"auto","created_at":"2024-12-30 16:37:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6032868,"visible":true,"origin":"","legend":"supplemental figure file","description":"","filename":"SupfigureBoneres.docx","url":"https://assets-eu.researchsquare.com/files/rs-5647673/v1/7484dfefa4b3b6448a746071.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Inhibiting CSF1R signaling reduces disc degeneration and discogenic back pain","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). The prevalence of LBP continues to increase with an annual prevalence of 15\u0026ndash;45% amongst adults in the United States, with significant health care and economic consequences\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Current medical treatment for disc degeneration and associated LBP is centered on activity modification, physical therapy, and non-steroid anti-inflammatory drugs (NSAIDs). The effect of those treatments is generally limited, and many patients progress to surgery despite medical treatment or suffer from lifelong chronic LBP. More importantly, no treatments so far have been shown to effectively mitigate the progression of the disc degeneration process itself\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHealthy IVDs are immune-privileged sites because the annulus fibrosus (AF) and cartilaginous endplate (CEP) isolate the nucleus pulposus (NP) from the immune system. Breakdown of the blood-NP barrier secondary to aging or injury exposes NP, which in turn initiates an immune response\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Proteins from the NP lead to macrophage activation and infiltration with secretion of inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Inflammatory cells accelerate disc degeneration by decreasing the production of extracellular matrix (ECM) proteins (e.g., aggrecan and collagen II), while concurrently upregulating catabolic enzymes such as disintegrin, metalloproteinase with thrombospondin motifs (ADAMTS)-4 and \u0026minus;\u0026thinsp;5 and matrix metalloproteinases (MMPs)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-α stimulate the expression of angiogenic and neurotrophic factors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e which trigger nociceptive nerve ingrowth into the CEPs and IVDs\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, contributing to discogenic back pain\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent reports have defined the importance of macrophages and microglia in the initiation and maintenance of centralized pain after peripheral nerve injury(10,11,12). Specifically, dorsal root ganglion (DRG) macrophages have been shown to be critical contributors to both the initiation and maintenance of mechanical hypersensitivity which is a hallmark of neuropathic pain in mice\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The increased numbers of macrophages found within the DRGs of injured nerves corresponds to an increase in activation of spinal cord dorsal horn microglia\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Spinal cord microglia plays an important role in the centralization \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and maintenance of neuropathic pain and the long-term potentiation of altered nociceptive pathways after peripheral nerve injury\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Macrophages in the DRG and microglia in the spinal cord appear to act synergistically and promote the transition from acute to chronic pain following peripheral nerve injury\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eColony stimulating factor 1 receptor (CSF1R) is expressed in macrophages, dendritic cells and microglia \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and is a critical mediator of proliferation and activation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Upregulation of CSF1R was observed in DRG macrophages and spinal cord dorsal horn microglia after peripheral nerve injury\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Upregulation of CSF1R expression on spinal microglia was also observed in a disc degeneration-induced back pain mouse model\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These findings hinted that signaling through CSF1R might play a role in discogenic back pain.\u003c/p\u003e \u003cp\u003eUsing human healthy and degenerate IVD single-cell transcriptome database, we identified increased macrophage appearance and increased \u003cem\u003ecsf1\u003c/em\u003e and \u003cem\u003ecsf1r\u003c/em\u003e expression in degenerate IVDs compared to healthy IVDs. To test the role of CSF1 and CSF1R in IVD degeneration and discogenic back pain, we developed a murine disc injury model that elicited structural disc degeneration and discogenic pain\u0026ndash;related behaviors similar to human clinical conditions. We demonstrated that deletion of CSF1R ablated disc degeneration and discogenic pain\u0026ndash;related behaviors. Similarly, inhibition of CSF1R function using pharmacological approach decreased inflammation in IVD, decreased IVD degeneration, reduced expression of the spinal nociception markers \u003cem\u003ec-Fos\u003c/em\u003e and alleviated discogenic pain-related behaviors.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003ecsf1\u003c/b\u003e \u003cb\u003ein human IVDs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate cell population change and gene features of human NP during IVD degeneration, we used Single-cell RNA sequencing (scRNA-seq) database GSE199866 of human NP cells from degenerate and healthy IVDs of the same individual. Cells are clustered according to transcriptome similarity in 2D space (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA upper panel). UMAP plot revealed 13 clusters (0\u0026ndash;12). In all transcriptionally distinct subpopulations, chondrocytes make up most of the cells in all samples based on cell lineage-specific marker gene expression. Chondrogenic marker gene \u003cem\u003eSOX9\u003c/em\u003e and chondrocyte-specific ECM genes \u003cem\u003eACAN\u003c/em\u003e were ubiquitously expressed in all chondrocyte clusters. Endothelial cells were identified by feature genes like \u003cem\u003ePECAM1\u003c/em\u003e expression (Supplementary Fig.\u0026nbsp;1C and D). Clusters 12, macrophage, is found only in NP from degenerate IVDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA lower panel). The cluster of cells were enriched with genes involved in macrophage inflammatory response like \u003cem\u003eCD74\u003c/em\u003e, \u003cem\u003eTYROBP\u003c/em\u003e and \u003cem\u003eLAPTMS\u003c/em\u003e (Supplementary Fig.\u0026nbsp;1D). We used GO analysis and identified the top15 enriched biological functional pathways of upregulated genes in degenerate NPs. The identified biological processes and molecular functions are directly related to myeloid leukocyte activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A combined analysis of the genes in degenerate NPs highlighted \u003cem\u003ecsf1\u003c/em\u003e as one of the 8 top upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, UMAP distribution of \u003cem\u003ecsf1\u003c/em\u003e and its receptor expression in degenerate NPs were performed and shown in the scatter plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). These data confirmed that expression of the \u003cem\u003ecsf1\u003c/em\u003e is enriched in chondrocytes of degenerate NPs. \u003cem\u003ecsf1r\u003c/em\u003e is enriched only in macrophages. We also mapped human endplate database GSE153761 and found \u003cem\u003ecsf1\u003c/em\u003e was 1 of 8 top upregulated proinflammatory factors in degenerate CEPs, as compared to healthy CEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G). There is no difference between normal and degenerate endplate on the expression of \u003cem\u003ecsf1r\u003c/em\u003e and another ligand \u003cem\u003eIL-34\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and G). These results identified CSF1/CSF1R pathway may be involved in macrophage activation and pathogenesis of disc degeneration.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental IVD injury reproducibly induces disc degeneration and discogenic pain\u003c/h2\u003e \u003cp\u003eTo create a robust and reproducible model of IVD degeneration and discogenic back pain, we modified an existing IVD puncture procedure (see Methods and Supplemental Fig.\u0026nbsp;2A-C). These mice were then subjected to behavioral tests and serial MRIs. Histopathology of IVDs was measured at several endpoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Uninjured IVDs in mice that underwent sham surgeries demonstrated classical healthy histopathologic architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplemental Fig.\u0026nbsp;2E). Safranin O and Fast Green staining (SO\u0026amp;FG) staining of IVD sections revealed the intact NP, AF and CEP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A well-defined border between the AF and NP was also noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplemental Fig.\u0026nbsp;2E). The CEP was continuous with the AF and was separated from the vertebral body by a layer of growth plate (GP). The CEP contained layers of rounded chondrocyte-like cells in a proteoglycan rich matrix. A tidemark (TM, dotted line) was present between the GP and the CEP (Supplemental Fig.\u0026nbsp;2E). In mice that underwent intervertebral disc puncture (DP), at an early stage (14 days post injury), the CEP kept its continuity and morphology, but NP and AF lamellae became disorganized (Supplemental Fig.\u0026nbsp;2D). 3- and 6- months after DP, the NP progressively collapsed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplemental Fig.\u0026nbsp;2E). In addition, the proteoglycan matrix (Safranin O positive, red) progressively decreased, the AF lamellae became disorganized, and the border between the NP and AF became less distinct (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplemental Fig.\u0026nbsp;2E). Further, the CEP was noted to be in discontinuity with the AF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and the tidemark between the CEP and GP disappeared (Supplemental Fig.\u0026nbsp;2E).\u003c/p\u003e \u003cp\u003eMRIs were used to serially follow IVD degeneration. The T2 bright area in the sham-treated IVDs remained consistent over the 6 month follow up period. In comparison, progressive loss in signal intensity was noted in IVDs at 3- and 6- months after DP. Representative T2-weighted, midsagittal images are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. T2 mapping revealed a 48% and 66% decrease in signal intensity at 3-month and 6-month post injury, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). On MRI, IVDs with DP demonstrated height loss at both 3- and 6-months follow-ups compared to sham-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eIn humans, mechanical back pain is subjectively quantified using the Visual analogue scale (VAS). In addition, several behavior functional scoring systems, such as the Oswestry disability index (ODI) have been used to quantify the severity of back pain\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In mice, analogous behavioral scoring metrics for pain include the commonly used von Frey assay\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The sham surgery group displayed a mildly decreased von-Frey test threshold 3 days after surgery but returned to normal by day 28. In contrast, in the DP group, the Von-Frey test threshold dramatically decreased at 3 days and remained so for up to 90 days post injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). DP also led to cold allodynia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and decreased thermal threshold (Supplemental Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003eThe presence of pain induces behavioral changes in animals\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Burrowing has been reported to be reduced in the setting of chronic pain\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Mice that underwent DP displayed a significant impairment in burrowing compared to sham-treated group (Supplemental Fig.\u0026nbsp;3B). Motor function was also evaluated using both accelerating rotarod and treadmill tests. Maximal speed tolerance (Supplemental Fig.\u0026nbsp;3C) and performance on the rotarod (Supplemental Fig.\u0026nbsp;3D) were similar between the DP and sham-treated groups. Open field testing also showed similar results between the groups in terms of running velocity, distance travel, and time spent in pre-defined zones (Supplemental Fig.\u0026nbsp;3E, F and G). These results indicated that modified DP method leads to reproducible progressive IVD degeneration with reproducible discogenic back pain related behavior.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCsf1r\u003c/b\u003e \u003cb\u003edeletion decreases proinflammatory cytokines release after disc injury\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe then asked whether the CSF1/CSF1R pathway was involved in the inflammatory response to DP injury within the disc itself. Using \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/GFP\u003c/em\u003e\u003c/sup\u003e mice, macrophage infiltration was found in disc after DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We also used macrophage specific markers Iba1 and CSF1R for immunostaining of IVDs. After disc injury, abundant Iba1\u003csup\u003e+\u003c/sup\u003e and CSF1R\u003csup\u003e+\u003c/sup\u003e cells were detected in different parts of the discs, including the NPs, AFs, and CEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C, D and E). In comparison, there was no Iba1 or CSF1R positive staining in the discs in sham-injured mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003eWe next asked whether CSF1R was involved in the release of disc inflammatory factors after disc injury. IVDs were collected and cultured from sham injured mice or from mice with disc injury 90 days after the injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). A panel of inflammatory factors were examined. When compared to cultures from sham injury IVDs, those from injured IVDs displayed strong upregulation of 31 inflammatory mediators as measured by inflammation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Among the upregulated cytokines noted were MMP2 and pro-MMP9, which are involved in catabolic pathways that contribute to matrix degradation \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Proinflammatory factors (including IL1-α, IL5, IL6, IL12-p40/p70, G-CSF/CSF3 and M-CSF/CSF1) and macrophage chemokine ligands (include MDC, Thymus CK-1, TROY, KC, MCP-1, MIP-1-alpha, MIP-1-gamma, MIP-2, MIP-3-beta, MIP-3-alpha and PF4) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e were also increased. The increase of soluble TNFR, ICAM-1, lungkine and TPO was also observed and these are thought to be related to canonically activated macrophages\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Growth factors (bFGF, IGF1 and VEGF) and 3 IGF binding proteins (IGFBP2, 3 and 6) related to angiogenesis \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e were also upregulated. Consistent with findings from human IVD degeneration, osteoprotegerin (OPG) was also significantly upregulated \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H, Supplemental Fig.\u0026nbsp;4A and B).\u003c/p\u003e \u003cp\u003eCompared to vehicle control \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreER/+\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eCsf1r\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e littermates, the mice treated with tamoxifen (TAM) hence with CSF1R deletion, released significantly lower levels of many of these factors after DP. The release of proinflammatory factors (CSF1, CSF3, IL-1α and IL-6) and matrix degradation proteins pro-MMP-9 were eliminated compared to injured control IVDs. The upregulation of OPG was also eliminated with CSF1R deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H, Supplemental Fig.\u0026nbsp;4B). These results suggested CSF1R signaling play an important role in inflammatory cytokine release after disc injury.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCsf1r\u003c/b\u003e \u003cb\u003edeletion reduces IVD degeneration after injury\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIVDs were collected from control vehicle or TAM treated Cx3cr1\u003csup\u003eCreER/+\u003c/sup\u003e: Csf1r\u003csup\u003efl/fl\u003c/sup\u003e mice at indicated days post DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). While IVDs from both control and CSF1R deleted mice exhibited gross evidence of AF disruption associated with IVD degeneration, AF architecture was better preserved in csf1r deleted (TAM group) as compared to the control (vehicle) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Proteoglycan (Safranin O positive) in CEP was markedly reduced after DP in both groups but less destruction was apparent in the TAM treated group. To quantify these alterations in structure we measured CEP thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicated that while IVD degeneration still occurred in mice with \u003cem\u003ecsf1r\u003c/em\u003e deletion following disc injury, the process was significantly less destructive when compared to controls.\u003c/p\u003e \u003cp\u003eVehicle control and \u003cem\u003ecsf1r\u003c/em\u003e deletion (TAM) mice were serially followed using MRI. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD presents the quantitative changes in the T2-weighted image intensity index (T2 mapping) at 90- and 180-days post-injury. Compared to the vehicle control which demonstrated a 66% decrease in T2 intensity at 180 days post-injury, IVDs in the csf1r deletion group displayed a 25% decrease at 180 days post-injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In addition, IVD height in \u003cem\u003ecsf1r\u003c/em\u003e deletion (TAM) mice was significantly increased as compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe CSF1R antagonist GW2580 attenuates disc degeneration after injury\u003c/h3\u003e\n\u003cp\u003eGiven that \u003cem\u003eCsf1r\u003c/em\u003e genetic deletion decreased disc degeneration after DP, we next wanted to evaluate whether CSF1R antagonist would have similar effects, and therefore could potentially be used clinically for treating disc degeneration. We tested the selective CSF1R competitive inhibitor GW2580 given its high affinity and specificity for CSF1R and low toxicity. GW2580 prevents CSF1R activation without inhibiting related kinases such as c-Kit, FTL3 and PDGFRβ\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Importantly, contrary to other CSF1R inhibitors such as PLX3397, BLZ945, etc., GW2580 does not cause ablation of resident macrophage or microglial population\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This is important for treating benign conditions such as back pain. We administered GW2580 by oral gavage (80 mg/kg) 2 hours after the disc puncture surgery and every 24 hours thereafter until the animals were sacrificed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At 90 and 180 days after disc injury, progressive loss in disc MRI T2 signal intensities were seen in both vehicle and GW2580 treated mice but GW2580 treated mice demonstrated a significantly less decrease in disc T2 signal intensities (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). IVD height loss in GW2580 treated group was also significantly less compared to the vehicle treated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Histological analysis demonstrated discs from GW2580 treated mice having better preserved disc architecture compared to the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). Finally, the infiltration of inflammation cells, demonstrated by Iba1 immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H) or GFP\u003csup\u003e+\u003c/sup\u003e cells in CX3CR1\u003csup\u003e+/GFP\u003c/sup\u003e reporter mice after disc injury, decreased with GW2580 treatment when compared to vehicle control (Supplemental Fig.\u0026nbsp;5).\u003c/p\u003e\n\u003ch3\u003eDisc injury induces DRG CSF1 expression and transportation to spinal cord\u003c/h3\u003e\n\u003cp\u003eThe IVD is innervated by branches of the sinuvertebral nerve and derivatives from the ventral rami\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. ECM degeneration, inflammatory mediators and neurotrophins that generate inflammatory conditions in the IVD result in membrane depolarization of peripheral nociceptive nerve endings. It has been reported that peripheral nerve injury induces CSF1 de novo synthesis in DRGs, which appears to be essential for spinal cord dorsal horn microglia activation and pain centralization after peripheral nerve injury, following transportation of expressed CSF1 along the dorsal root from DRG to the spinal cord\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Here we ask whether DP triggers similar signaling activation in DRGs and spinal cord, resulting in centralized pain induction, and if so, whether it could be altered by CSF1R pathway inhibition.\u003c/p\u003e \u003cp\u003eFirst, we examined DRGs and their peripheral branches and central branches. CSF1 was expressed in very few DRG neurons without disc injury. After DP, increase of CSF1 expression was found in DRG neurons and central branches, but not in the peripheral branches distal to the DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The increase of CSF1 in DRG neurons persisted for at least 4 weeks after DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Supplemental Fig.\u0026nbsp;6A and B). CSF1 expression was not seen in Iba1\u003csup\u003e+\u003c/sup\u003e cells, indicating its expression was not from DRG macrophages (Supplemental Fig.\u0026nbsp;6B).\u003c/p\u003e \u003cp\u003eThe expression of CSF1 in DRG was reduced in \u003cem\u003eCsf1r\u003c/em\u003e deletion mice (TAM treated \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreER/+\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eCsf1r\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group) compared to vehicle control treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This is likely because of the decreased inflammation signal from the injured disc due to the CSF1R deletion.\u003c/p\u003e\n\u003ch3\u003eCSF-1/CSF-1R is essential for DRG macrophage and spinal cord microglia activation after disc injury\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence analysis revealed an increased number of macrophages (Iba1\u003csup\u003e+\u003c/sup\u003e cells) as well as active macrophages (CD68\u003csup\u003e+\u003c/sup\u003e) in the DRGs after DP (Supplemental Fig.\u0026nbsp;6C, 6D). Ki67 and GFP immunofluorescence analysis on CX3CR1\u003csup\u003e+/GFP\u003c/sup\u003e mice DRG suggested the increase of macrophages in the DRGs after DP is likely from CX3CR1\u003csup\u003e+\u003c/sup\u003e resident macrophages proliferation (Supplemental Fig.\u0026nbsp;6E). The increase of DRG Iba1\u003csup\u003e+\u003c/sup\u003e was reduced in c\u003cem\u003esf1r\u003c/em\u003e deletion mice (TAM treated \u003cem\u003eCx3cr1\u003c/em\u003e\u003csup\u003e\u003cem\u003eCreER/+\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003eCsf1r\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e group) (Supplemental Fig.\u0026nbsp;6F).\u003c/p\u003e \u003cp\u003eIn the spinal cord, CSF1R is only expressed in microglia\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. After DP, CSF1R expression significantly increased in the dorsal spinal cord microglia as compared to sham injured controls (Supplemental Fig.\u0026nbsp;6G). The \u003cem\u003eCsf1r\u003c/em\u003e deletion in the CX3CR1\u003csup\u003eCreER/+\u003c/sup\u003e:Csf1r\u003csup\u003efl/fl\u003c/sup\u003e mice after TAM injection was confirmed, as shown with no CSF1R staining after disc injury (Supplemental Fig.\u0026nbsp;6H). The increase of Iba1\u003csup\u003e+\u003c/sup\u003e and Cd68\u003csup\u003e+\u003c/sup\u003e microglia cells in the dorsal spinal cord was abolished in \u003cem\u003eCsf1r\u003c/em\u003e deletion DP mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E and F). These data suggest that CSF1R signaling is essential for the spinal cord dorsal horn microglia activation after DP.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCsf1r\u003c/b\u003e \u003cb\u003edeletion eliminates neuropathic pain after disc injury\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven these findings we next sought to explore whether the decreased spinal cord microglia and DRG macrophage activation from CSF1R deletion alters pain behavior in mice after disc injury. Using the Von-Frey test, we discovered that mechanical hypersensitivity following disc injury was completely abolished in CX3CR1\u003csup\u003eCreER/+\u003c/sup\u003e:Csf1r\u003csup\u003efl/fl\u003c/sup\u003e mice treated with tamoxifen but not in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). This phenotype was noted to last at least 90 days after disc injury. These results suggest that CSF1R signaling is critical to the development of discogenic neuropathic pain.\u003c/p\u003e\n\u003ch3\u003eGW2580 alleviates acute and chronic discogenic pain after disc injury\u003c/h3\u003e\n\u003cp\u003eSimilar to \u003cem\u003eCsf1r\u003c/em\u003e deletion, GW2580 reduced the CSF1 positive neurons in DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). The activation (CD68\u003csup\u003e+\u003c/sup\u003e) and proliferation (Iba1\u003csup\u003e+\u003c/sup\u003e and Ki67\u003csup\u003e+\u003c/sup\u003e) of macrophage in DRG was also down regulated by GW2580 treatment (Supplemental Fig.\u0026nbsp;7A, B and C). GW2580 also markedly decreased inflammatory CD68\u003csup\u003e+\u003c/sup\u003e in the spinal cord dorsal horn after disc injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D). Disc injury induced CSF1R upregulation in the spinal cord dorsal horn was also reduced by the GW2580 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and E). These results indicate that GW2580 downregulated immune cell response in DRG and spinal cord.\u003c/p\u003e \u003cp\u003ePeripheral nerve injury leads to the activation of the immediate early gene \u003cem\u003efos\u003c/em\u003e in the dorsal spinal cord pain circuits. As Fos itself has a very short half-life\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, Targeted Recombination in Active Populations (TRAP) mice were used to detect the accumulated Fos activation during a certain period of time when tamoxifen was given\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This method utilizes Fos\u003csup\u003eCreERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e: Rosa26\u003csup\u003eChR\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e mice in which the tamoxifen-dependent recombinase CreERT2 is expressed in an activity-dependent manner from the \u003cem\u003eFos\u003c/em\u003e loci. The active neurons during the duration of tamoxifen presence are labeled with tdTomatos (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In TRAP mice that underwent sham control surgery, there was little or no Fos (tdTomato\u003csup\u003e+\u003c/sup\u003e cells) within spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) or DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). After disc injury, the increase of Fos(tdTomato) positive cells was detected in the DRGs and spinal cord (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F and G). Fos (tdTomato\u003csup\u003e+\u003c/sup\u003e) was noted in lamina I/II of the dorsal horn, an area associated with nociceptive processing after DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). The Fos(tdTomato) peaked at 4 weeks after DP, and at this time, an increase in Fos (tdTomato) positive cells was also seen in the spinal cord lamina III/IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). GW2580 treatment significantly decreased the number of Fos (tdTomato) positive cells in both the spinal cord dorsal horn and DRGs after DP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and H). With Von Frey test, the GW2580 treated group showed an improved pain threshold compared to the control treated group, up to 90 days when the experiment ended (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). In addition, GW2580 treated mice exhibited decreased acetone cold allodynia between 14- and 28-days after disc injury compared to vehicle treated controls (Supplemental Fig.\u0026nbsp;7D). The burrowing test showed GW2580 treated mice having less disturbed burrowing behavior compared to vehicle treated controls (Supplemental Fig.\u0026nbsp;7E). These results indicate that GW2580 treatment downregulated spinal nociception markers \u003cem\u003ec-Fos\u003c/em\u003e expression and alleviated nociceptive behavior.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIVD degeneration is a progressive process and is a major cause of LBP\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Herein, we demonstrated that the CSF1R signaling pathway plays an essential role in the development of IVD degeneration and discogenic back pain. Targeted deletion of \u003cem\u003eCsf1r\u003c/em\u003e from microglia/macrophages or oral administration of the small molecular CSF1R inhibitor GW2580 decreased IVD degeneration. Furthermore, \u003cem\u003eCsf1r\u003c/em\u003e deletion or GW2580 treatment inhibited DRG macrophage and spinal cord dorsal horn microglia activation and in so doing dramatically decreased neuropathic pain after disc injury.\u003c/p\u003e \u003cp\u003eProteins within the NP are recognized as non-self by the immune system and as such exposure of NP material from disc injury may elicit and propagate an immune response in a CSF1R dependent manner\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Inflammatory factors secreted by macrophages, together with neurotrophins (e.g., NGF, VEGF and substance P), drive angiogenesis and nerve ingrowth (i.e., the sinuvertebral nerve and sympathetic afferents) into degenerating IVDs\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The nerve in-growth in injured or degenerating IVDs may activate satellite glia within the DRGs which in turn increases DRG neuronal CSF1 expression\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. CSF1 is then transported along the sensory axons to the dorsal horn of the spinal cord, where it activates microglia via CSF1R. Activated microglia then induces overexcitation of dorsal horn neurons (i.e. through BDNF signaling pathway)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, which induces neuropathic pain\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eGeneration of an IVD degeneration and discogenic back pain animal model\u003c/h3\u003e\n\u003cp\u003eAnimal models are vital tools for IVD degeneration research. Given the complex pathobiology surrounding IVD degeneration and discogenic back pain, developing an appropriate and reliable animal model has been a challenge. Our modified disc puncture model displays morphological, biochemical, inflammatory and behavioral features similar to those found in humans with degenerative disc disease\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Previously, posterior approaches have been used to puncture discs to induce disc degeneration and pain. In the posterior approach model, one facet joint is removed, and the posterior column is disrupted which results in mechanical instability\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In addition to the inherent stability problems, it adds an additional cause of back pain, and therefore represents a major limitation\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Other approaches to disc puncture include an anterior transabdominal approach. This approach has risks of abdominal viscera injury which often leads to gastrointestinal distress and feeding impairment, thereby confounding experimental observations related to pain and associated behavioral assessments\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our modified procedure, the lumbar spine was approached via a posterolateral angle on the right side. The L4/5 and L5/6 IVDs were accessed with minimal paraspinal muscle disruption using a 25G custom-made needle. This approach minimized disruption of normal spinal architecture, avoided damaging the transversing spinal nerve roots and circumvented abdominal injury. Endplate disruption was included in our disc puncture model. Endplate disruption allows the entry of inflammatory factors to the IVDs and likely plays an important role in the pathophysiology of discogenic back pain development in humans \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Using serial MRIs and histologic analyses, we found that our model produced consistent progressive IVD degeneration similar to human conditions. We also employed stimulus-evoked (i.e., mechanical, thermal) and non-stimulus evoked methods (i.e., burrowing assays, gait analysis, and/or automated behavioral analysis) to assess pain related behaviors\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Persistent and reproducible mechanical hyperalgesia was noted to last for at least 25 weeks after disc injury.\u003c/p\u003e \u003cp\u003eIn human subjects, back pain is mostly reported subjectively by the patient using VAS scale. It is challenging to subjectively determine the level of discogenic back pain in rodent models. Pain like behaviors indicative of a localized painful responses included decreased hind paw mechanical and thermal sensitivities, increased grooming, and altered walking gait patterns with longer stance phases and shorter swing phases\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Our mice model with degenerated IVDs had significantly reduced mechanical withdrawal thresholds and a trend towards shorter thermal withdrawal latency, like prior studies\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The mechanical test is more sensitive than the thermal test in assessing painful IVD degeneration in rodents\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Although not ideal, Von Frey test is most commonly used as an objective measure to determine discogenic back pain in rodent models \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eBlocking CSF1R signaling slows down IVD degeneration\u003c/h3\u003e\n\u003cp\u003eThe IVD is an immune-privileged site. Disruptions in the vertebral endplates expose the IVDs to the bone marrow which has a rich supply of immune cells and immune progenitors. Once exposed, recruitment and activation of immune cells within the IVD is initiated\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Early in the process, an inflammatory infiltrate comprised mostly of macrophages localizes proximal to the defects in the endplates\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. IVD tissue (i.e., the NP) tends to polarize macrophages toward the pro-inflammatory M1-like profile which further perpetuates IVD degeneration\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Disc injury in our model confirmed the induction of robust disc macrophage infiltration with an increase in pro-inflammatory factors secretion.\u003c/p\u003e \u003cp\u003eOne such factor is CSF-1 which regulates macrophage survival and proliferation and modifies macrophage activation and recruitment. CSF-1 executes its myriad of functions through engagement with its cognate receptor CSF1R\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Here, we demonstrated that CSF1R expression is increased in infiltrating IVD macrophages after disc injury and that macrophage recruitment is inhibited by CSF1R deletion or GW2580 administration.\u003c/p\u003e \u003cp\u003eIt was reported that the levels of proinflammatory factors are higher in injured IVDs that cause pain than those from asymptomatic IVDs, suggesting that the levels of inflammation are related to pain\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Previous reports have demonstrated increased soluble TNF-α and MMP-3 in IVDs after exposure to macrophages\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In our study, pro-MMP9 was significantly upregulated in chronically injured IVDs, suggesting active ECM destruction during the course of disc degeneration\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. TNF-α was not detected in either intact or injured IVDs, but up-regulation of sTNF RI and sTNF RII were noted in the injured IVD cultures. Such increases have previously been linked with increases in inflammation\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Similar to reported results from humans, levels of IL-6, CSF1, CSF3 were increased in chronically degenerating IVDs. Our work demonstrated that deletion of macrophage CSF1R decreased the secretion of pro-inflammatory factors from the chronically injured IVDs.\u003c/p\u003e \u003cp\u003eDuring IVD degeneration, angiogenesis takes place and blood vessels can be seen growing into IVDs. Clinical studies have observed higher rates of angiogenesis within the inner regions of degenerated IVDs in patients experiencing LBP\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. We detected an increase in several pro-angiogenic growth factors such as bFGF, IGF1 and VEGF in chronically injured IVDs. CSF1R deletion abolished the increase of pro-angiogenic growth factors after disc injury. Significant increases in the expression of the RANK/RANKL/OPG (osteoporotegerin) system have also been noted in advanced stages of IVD degeneration\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In our model, OPG upregulation with degenerating IVDs was also eliminated with CSF1R deletion.\u003c/p\u003e \u003cp\u003eIt is prudent to note that the condition of IVD culture may cause degeneration during the culture period and release inflammatory factors as a result \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. However, in those studies that reported such phenomena, cultures were continued for a prolonged period of time (i.e., 20 days vs our 5-day culture period). Other reports have indicated that IVD units can remain healthy for up to 14 days in submersion culture medium supplemented with 10% FBS\u003csup\u003e64,65,66\u003c/sup\u003e. Our intact IVD control cultures generated very low levels of inflammation factors, suggesting that the increased inflammation factors in our chronically injured IVD cultures were reflective of the underlying pathobiology of disc degeneration, not a result of the culture condition.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDiscogenic pain is eliminated with inhibition of CSF1R signaling pathway\u003c/h2\u003e \u003cp\u003eIt has been demonstrated that peripheral nerve injury induces de novo CSF1 expression in injured sensory neurons and that CSF1 is transported to the dorsal horn of the spinal cord afterwards\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. DRG neuron derived CSF1 not only stimulates proliferation of surrounding macrophages but also induces spinal cord microglia proliferation and expression of a host of neuropathic pain\u0026ndash;associated genes\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Both DRG macrophages and dorsal horn microglia have been shown to contribute to the initiation of pain and the transition from acute to persistent neuropathic symptoms\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe anterior annulus of IVD is innervated by nerves derived from the ventral and gray rami communicans of the autonomic nervous system. The posterior annulus is innervated by the sinuvertebral nerve, a branch of the spinal nerve at each associated intervertebral level\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Disc injury induced inflammation has been shown to stimulate sensory nerve fibers and DRG sensory neurons. Consistent with reports in other models, we showed that de novo CSF1 expression did in fact increase in the DRG neurons after disc injury\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. CSF1 production in the DRG reached a high level 7 days after disc injury, and it persisted for at least 28 days. Other labs found that CSF1 was induced in CGRP-expressing DRG neurons 2 weeks after disc injury\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Such differences may be the result of variations between experimental models.\u003c/p\u003e \u003cp\u003eSimilar to responses after peripheral nerve injury, prominent microglia and macrophage activation in spinal cord dorsal horn and DRGs were observed after disc injury in our model. As CSF1R activation in spinal cord microglia induced pain behavior in peripheral nerve injury models\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we proposed that increases in CSF1R within spinal cord microglia after disc injury contribute to the development of discogenic back pain. Indeed, the alleviation of discogenic neuropathic pain after the genetic deletion of CSF1R or CSF1R specific inhibitor suggested an essential role of CSF1R signaling pathway in the initiation and maintenance of the discogenic neuropathic pain after disc injury.\u003c/p\u003e \u003cp\u003eWe also examined \u003cem\u003ec-Fos\u003c/em\u003e, an immediate\u0026ndash;early gene whose expression in the spinal cord has been extensively used as a marker for peripheral noxious stimulation\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. We used TRAP mice to monitor c-fos gene expression related to pain in spinal cord neurons. c-Fos was highly expressed in the spinal cord dorsal horn after disc injury. Disc injury included \u003cem\u003ec-Fos\u003c/em\u003e expression was largely eliminated following GW2580 treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGW2580 as a potential therapeutic agent for disc degeneration and discogenic pain\u003c/h2\u003e \u003cp\u003eA therapeutic approach centered on the modulation of CSF1R signaling needs to be effective at inhibiting microglial/macrophage activation, but not affecting basal mononuclear phagocyte survival, so that it does not lead to adverse effects in CNS homeostasis. This is particularly important for treating benign conditions such as back pain. CSF1R belongs to the type III class of growth factor receptors, which includes PDGFR, c-KIT, and FLT3\u003csup\u003e17\u003c/sup\u003e. An analysis of the activity and selectivity of several CSF1R inhibitors such as PLX3397, imatinib, BLZ945, Ki20227 or edicotinib demonstrated that they were also inhibitors of PDGFRβ and c-Kit\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. For example, PLX3397 inhibits the survival of microglia within the healthy brain and concurrently inhibits c-Kit, FTL3 and PDGFRβ\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Loss of PDGFβ signaling impacts survival of NG2 pericytes and may therefore lead to blood\u0026ndash;brain barrier damage and neurodegeneration.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e PLX5622 also inhibits the survival of microglia\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGW2580 is a selective competitive inhibitor with a high affinity for CSF1R. It inhibits CSF1R activation without affecting related kinases or ablating the resident macrophage and microglial population\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. It has been shown to decrease neurotoxicity in animal models of Alzheimer's disease, amyotrophic lateral sclerosis, and prion disease\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. In a spinal cord injury model, GW2580 specifically inhibited microglia proliferation following spinal cord injury but did not perturb microglia responses and functions in control animals\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. In the present study, we administered GW2580 orally to treat disc degeneration and discogenic neuropathic pain. GW2580 selectively inhibits spinal microglial proliferation and activation without affecting the survival of resident microglia. GW2580 did not change microglia numbers when the spinal cord or brain were examined in control animals. GW2580 inhibited pro-inflammatory activation of DRG macrophages but did not affect the overall number of resident macrophages. GW2580 also decreased macrophage infiltration into the disc AF and NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNo effective medical treatments currently exist for the prevention of disc degeneration. While some experimental treatments have been delivered via local administration\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, oral treatment might be superior, given that disc degeneration often occurs at multiple spine levels. Clinically, discogenic pain sometime is difficult to localize to a single level, even with advanced diagnostic methods such as discography\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. While oral administration raises concerns of possible systemic adverse effects, GW2580 treatment had no discernable side effects in our studies. No effect on food intake, animal behavior or animal weight was noticed. No systemic adverse effects were observed on veterinary pathological autopsy.\u003c/p\u003e \u003cp\u003eOverall, CSF1R specific inhibitor GW2580 presents a promising therapeutic avenue for the treatment of disc degeneration and discogenic back pain. With its outstanding oral bioavailability, safety profile and efficacy, GW2580 and its next-generation derivatives can be excellent candidates for potential medical treatments aimed at mitigating disc degeneration and alleviating chronic discogenic low back pain.\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMice were housed under standard 12-hour light/12-hour dark conditions with \u003cem\u003ead libitum\u003c/em\u003e access to food/water. Animal care and handling procedures were congruent with those set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal protocols employed throughout this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the Brigham and Women\u0026rsquo;s Hospital, Harvard Medical School.\u003c/p\u003e\u003cp\u003eCx3cr1\u003csup\u003e+/GFP\u003c/sup\u003e knock-in/knock-out mice (JaxMice; stock #: 005582) were used as a macrophage/monocyte reporter line. B6.Cg-Csf1r\u003csup\u003efl/fl\u003c/sup\u003e mice (JaxMice; stock #: 02212) were crossed with the B6.129-Cx3cr1tm2.1 (CreER) (JaxMice; stock #: 020940). The resulting mice displayed tamoxifen inducible CRE activity specifically in mononuclear phagocytes leading to a non-functional CSF1R protein. Fos\u003csup\u003eCreER\u003c/sup\u003e mice (c-Fos Cre ERT2 (B6.129(Cg)-\u003cem\u003eFostm1.1(cre/ERT2)Luo\u003c/em\u003e/J JaxMice; stock #: 021882) and Ai14 (B6.Cg-\u003cem\u003eGt(ROSA)26Sortm14(CAG-tdTomato-)Hze\u003c/em\u003e/J, JaxMice; stock #: 007914) mice were obtained from the Jackson Laboratory and maintained as the provided guidelines.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGenotyping\u003c/em\u003e: For Fos\u003csup\u003eCreER\u003c/sup\u003e mice, WT product: 215, mutant fragment:293. Common forward, CAC CAG TGT CTA CCC CTG GA; WT reverse, CGG CTA CAC AAA GCC AAA CT; mutant reverse, CGC GCC TGA AGA TAT AGA AGA. For Ai14 mice, WT fragment: 297 bp, mutant fragment: 196 bp. WT forward, AAG GGA GCT GCA GTG GAG TA; WT reverse, CCG AAA ATC TGT GGG AAG TC; mutant reverse, GGC ATT AAA GCA GCG TAT CC; mutant forward: TTC CTG TAC GGC ATG G. For Cx3cr1\u003csup\u003eGFP\u003c/sup\u003e knock-in/knock-out mice, WT fragment: 410 bp, mutant fragment: 500 bp Wild type forward: GTC TTC ACG TTC GGT CTG GT; Common: CCC AGA CAC TCG TTG TCC TT; Mutant Forward: CTC CCC CTG AAC CTG AAA C. For B6.Cg-Csf1r\u003csup\u003efl/fl\u003c/sup\u003e mice, WT fragment: 193 bp, mutant fragment: 273 bp. Forward: GGA CTA GCC ACC ATG TCT CC; Reverse: CAT GGC TGT GGC CTA GAG A. For B6.129-Cx3cr1\u003csup\u003eCreER\u003c/sup\u003e mice, WT product:151bp, mutant fragment: 230bp. Wild type forward, AGC TCA CGA CTG CCT TCT TC; Common, ACG CCC AGA CTA ATG GTG AC; mutant forward, GTT AAT GAC CTG CAG CCA AG.)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTamoxifen preparation/administration\u003c/strong\u003e \u003cp\u003eTamoxifen was dissolved in corn oil (Sigma-Aldrich cat# C8267) and 100% ethanol for 1 h at 37\u0026deg;C and was vortexed every 15 min. We used \u0026sim;75 mg tamoxifen/kg body weight and 100 \u0026micro;l tamoxifen/corn oil solution was administered via intraperitoneal injection for 14 consecutive days.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGW2580 Treatment using Oral Gavage\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGW2580 (SML1047 Sigma-Aldrich) was suspended in 0.5% hydroxypropylmethylcellulose and 0.1% Tween 80 and was dosed orally at 80 mg/kg (0.2 ml per mouse). Mice were orally gavaged with 18G needles (Instech, Cat No. FTP-18-30-50).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSurgical Procedure(s)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe mouse model of IVD degeneration was established using the following procedure. Briefly, the mouse was fixed in a prone position after being anesthetized (Ketamine 100\u0026ndash;120 mg/Kg and Xylazine 10 mg/Kg). The lumbar spine was approached via posterolateral angle on the right side. The L4/5 and L5/6 IVD was accessed/punctured through minimal paraspinal muscle using a 25G custom-made needle. Needles were inserted through the dorsal annulus, through the NP center and partially through the ventral annulus (controlled depth of 1.75 mm or 90% of the dorsoventral width) for 30 s and removed. It is then followed with rotating a custom-made tool inside the disc socket to ensure the damage of vertebrate endplates. Sham surgery consisted of an incision followed by exposure of L4/5 and L5/6 IVDs. Then the wound was sutured without receiving any needle puncture in IVDs. After the surgery, experimental animals had standard postoperative treatment. The animals were placed in individual cages after the operation. The animals\u0026rsquo; lower extremity activity, puncture incision healing, and death were monitored.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLumbar magnetic resonance imaging scans and quantification\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eScans were performed using Paravision 5.1 (Bruker BioSpin Corporation, Billerica, MA) interfaced to a 7.0T Bruker BioSpec USR (Bruker BioSpin Corp.) using a custom \u0026ndash;made 2cm volumetric coil in the Small Animal Imaging Laboratory (SAIL) at Brigham and Women\u0026rsquo;s Hospital. The sagittal and cross-section T2 weighted MR images(T2WIs) were qualitatively analyzed to evidence the degenerative changes. Parameters: repetition time 2500 ms, echo time 30 ms, RARE factor 4, averages 24, field of view 20mm*20mm, slice thickness\u0026thinsp;=\u0026thinsp;0.6 mm, for a scan time of 25 minutes per mouse plus setup. Disc height measuring and Pfirrmann grade interpretating were analyzed from T2WIs using Bruker Topspin, version 3.2 and Case Viewer, version 2.3.\u003c/p\u003e \u003cp\u003eQuantification of MRIs was performed for the lumbar discs using the series section of each disc. A series of mid-sagittal slices for T2 mapping (TE\u0026thinsp;=\u0026thinsp;\u003cem\u003eI\u003c/em\u003e*8ms, \u003cem\u003eI\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1,2,3,4,\u0026hellip;25, 25 echo times in total) were obtained with an inplane resolution of 100um and a 0.6mm slice thickness. T2 maps of one mid-sagittal slice from each sample were then generated in Bruker Topspin. A region of interest (ROI) including cartilaginous endplates, annulus fibrosus (AF) and nucleus pulposus (NP), was drawn manually to calculate mean T2 values, structures outside the disc were carefully avoided. Data were expressed as percentages of the results obtained when using sham surgery control discs. Here, the control was defined from the value of adjacent uninjured IVD. All the image assessments were performed by two independent blind observers, and the quantitative data were presented as means of three evaluations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter the MRI examinations, mice were fixed by transcardiac perfusion with 4% paraformaldehyde. Discs were post-fixed with 4% (w/v) paraformaldehyde for 48 hours and decalcified in Kristensen\u0026rsquo;s decalcifying solution for 2 weeks and then washed for 24 hours under running tap water and paraffin embedded. Discs were cut into transverse 5-\u0026micro;m sections and collected on Superfrost Plus slides and stored at room temperature until use. Staining was performed at room temperature. Mid-sagittal sections were dewaxed in xylene in the fume hood and rehydrated through 95%, 70%, and 50% ethanol washes for 2 min each.\u003c/p\u003e\u003cp\u003eSafranin O-fast green staining. Briefly, sections were stained with Weigert\u0026rsquo;s iron hematoxylin working solution for 10 minutes followed by fast green (FCF) solution for 5 minutes. Then, sections were rinsed quickly with 1% acetic acid solution for 10 \u0026minus;\u0026thinsp;15 seconds followed by staining with 0.1% safranin O solution for 5 minutes. For H\u0026amp;E staining, sections were stained in Mayer\u0026rsquo;s hematoxylin for 6 min and washed under running tap water before staining in eosin for 2 min. After a quick rinse in tap water, sections were dehydrated through 50%, 70%, 95%, and absolute ethanol washes for 1 min each. All sections were cleared in two changes of xylene and covered with the distyrene-plasticizer-xylene (DPX) mounting medium and a coverslip. The sections were placed in an oven at 37\u0026deg;C to enable the mounting medium to solidify before imaging under a light microscope (Nikon).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistological classification of disc degeneration\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor each H\u0026amp;E classification type, the maximum points represent severe degeneration. The control was defined as the histological score of L3/4 IVD. Stained slides were graded with a validated histological grading system, as described previously\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Major anatomical structures of the AF and NP were included in this classification, resulting in four subcategories. Each item was graded as zero, one, or two on the H\u0026amp;E and safranin O-fast green sections, with zero representing nondegenerative characteristics, one representing mild degenerative characteristics, and two representing severe characteristics of degeneration. The total score was the sum of the four different scoring items, resulting in a minimum score of zero, corresponding to a healthy disc, and a maximum score of eight, corresponding to an entirely degenerated disc. All the histological assessments were performed by two independent blind observers, and the quantitative data were presented as the mean of three evaluations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAdult male and female mice were used for behavioural assays. Before each assay, animals were acclimated to the experimental conditions for 3 days (once per day). Mice of each group were tested in a random and blinded fashion.\u003c/p\u003e \u003cp\u003eVon Frey tests\u003c/p\u003e \u003cp\u003eThe von Frey test was carried out 2 days before surgery (day \u0026minus;\u0026thinsp;2) and on 1, 7, 14-, 21-, 28- and 42-days post-surgery. The mice were individually placed into six-compartment mice enclosure with wire mesh floors and lids with air holes (IITC Life Science) for a 20-min habituation period to minimize exploratory activity. Threshold responses to a mechanical (tactile) stimulus were measured by placing each subject in an elevated observation chamber with a wire mesh floor whereupon the plantar surface of the ipsilateral hindpaw could be stimulated with a graduated series of seven Von Frey filaments (Stoelting), ranging from 0.03\u0026ndash;2.04 g, using the up/down method. Response percentages per 10 tries with 3 min intervals were also measured for each stimulus intensity.\u003c/p\u003e \u003cp\u003eAcetone evaporation test\u003c/p\u003e \u003cp\u003eCold sensitivity was assessed by acetone evaporative cooling. Through the mesh floor a series of five applications of acetone (50 \u0026micro;l; application separated by at least 5 min) were gently applied to the bottom of the paw using a multidose syringe device. Individual responses were scored on a 0\u0026ndash;2 scale, wherein 0\u0026thinsp;=\u0026thinsp;no response or a rapid transient lifting or shaking of the hindpaw that subsides immediately; 1\u0026thinsp;=\u0026thinsp;lifting, licking, and/or shaking of the hindpaw, which continues beyond the initial application, but subsides within 5 s; and 2\u0026thinsp;=\u0026thinsp;protracted, repeated lifting, licking, and/or shaking of the hindpaw. Individual scores are averaged over the five applications. Alternatively, cumulative paw licking duration was recorded over 60 s for each of three acetone applications, with the average taken for each animal.\u003c/p\u003e \u003cp\u003eHot plate test\u003c/p\u003e \u003cp\u003eThe surface of a hot plate is heated to a temperature of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The mouse is placed on the heated plate, and the latency for it to show a nociceptive response with paw lick, paw flick, or a jump is measured with the timer. The mouse is immediately removed when this response is observed. If the mouse does not display a response within 30 sec, the mouse is removed from the heated plate to prevent any tissue damage.\u003c/p\u003e \u003cp\u003eBurrowing assay\u003c/p\u003e \u003cp\u003eMice were acclimatized to the specific burrowing tube once on the day before the burrowing assay was performed (for 1 h), and again on the day of testing (for 30 min). For acclimatization an empty tube was placed into the home cage, such that all five mice in the cage were exposed to the tube. All mice were observed to voluntarily enter the tube within 10\u0026ndash;15 min. On the day of testing, a separate cage was prepared for each mouse to be tested individually. An acrylic tube was filled with 90 g of the same corncob bedding used in the home cage and placed in one corner, parallel to the long walls of the cage. Mice were then transferred to these cages. After test, mouse was returned to the home cage and the bedding remaining in the tube was weighed. The burrowing activity was calculated by subtracting the weight of bedding present at the end of the experiment from the starting weight and expressing the proportion of bedding that had been displaced as a percentage. To minimize any potentially confounding effects of differing olfactory cues, approximately 5 g of bedding from the home cage was transferred to the testing cage immediately prior to first testing. In addition, all bedding from a particular test cage was stored for re-use in a re-sealable plastic bag between tests.\u003c/p\u003e \u003cp\u003eTreadmill gait\u003c/p\u003e \u003cp\u003eFor treadmill walking, mice were placed on the DigiGate at various speed. Speed tolerance was defined as the maximal speed a mouse can walk on the treadmill without falling. All trials were video recorded (Hotshot e64, 100 fps). Gait parameters were measured and repeated at post-operative days 3, 7, 14-, 28- and 42-days.\u003c/p\u003e \u003cp\u003eRotarod test\u003c/p\u003e \u003cp\u003eThe rotarod performance test involves forced motor activity by rodents on a rotating rod with accelerating speed. Rodents are trained for 2\u0026ndash;3 days on a rotarod (Med Associates, Inc., St. Albans, VT) at varying speeds before the final test is conducted. The rotarod started from stationary and accelerated from 4 to 40 rpm over 5 min. In the test, a mouse is placed on a horizontally oriented, rotating cylinder (rod) suspended above a cage floor. The rod is low enough that the animal will not be injured if it falls but high enough to induce avoidance of fall. The length of time that a given animal stays on the rotating rod is a measure of their balance, coordination, physical condition, and motor-planning. The maximum end-speed was recorded when the mouse fell off the treadmill. Three trials were performed with a 20-min break between trials. This test was performed 1 day pre-surgery and days 3, 7, 14, 28, and 42 post-surgeries.\u003c/p\u003e \u003cp\u003eOpen field test\u003c/p\u003e \u003cp\u003eThe open field consists of a circular environment with 1.2 m diameter closed by a wall of 0.45 m high. The mice are allowed to move freely within the space, and the time spent in each region is quantified. The circular environment is virtually divided into regions so that there is a clear center region. The number of central regions visited, the time spent in the central region, and overall locomotion were quantified. The open field behavior is calculated by SMART 3 software. Both the number of central squares visited, and the time spent in the central squares are markers of exploratory behavior \u003cem\u003e(80)\u003c/em\u003e. This test was performed 1 day pre-surgery and days 14 and 42 post-surgeries.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDisc organ culture\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSoft tissues and posterior elements around discs were removed, and discs were rinsed in saline solution before being placed in culture plates. The suspension culture in which a sterile 8 \u0026micro;m transwell was first placed at the center of each well of 24-well plates, which were filled with 10% FBS DMEM medium. The bottom half of each column was first loosely stuffed with cotton balls semi-saturated with medium, and then the IVD tissue sample was placed at the center of the column, which was subsequently filled with further medium-semisaturated cotton balls. Experimental specimens were cultured in 5% CO2 and 37\u003csup\u003eo\u003c/sup\u003eC. IVD culture medium was collected for Cytokine analysis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCytokine analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe concentration of cytokines in the IVD culture medium was quantified using mouse cytokine Array C1000 (AAM-CYT-1000) (RayBiotech Life, Inc. GA) according to manufacturers' instructions. Arrays were imaged with the provided enhanced chemiluminescence kit using an ImageQuant LAS4000 (GE Healthcare, Baie d'Urfe, QC, Canada). ImageQuant TL array analysis software (GE Healthcare) was used to analyze the blots. The relative quantity of each factor present in each media sample was calculated using the controls included in the protein arrays. Mean relative quantities of each factor for the degenerating and healthy discs were then calculated.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence and confocal imaging\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eImmunofluorescence labeling was performed according to previously published method \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Primary antibodies used in this study are of the following sources and were used at the indicated dilutions: goat anti-iba1 (1:1000, Novus Biologicals, NB100-1028), rabbit anti-Ki67 (1:200, Abcam, ab15580), chicken anti-neurofilament H (NF-H, 1:500, EMD millipore, AB5539). Mouse M-CSF antibody (1:1000, AF416), anti-mouse CD115 (CSF-1R) antibody(1:1000, LS‑C130595). Anti-CD68 antibody (1:1000, ab53444). Fluorescent Nissl Stain (1:500, N21482). Alexa Fluor secondary antibodies from Jackson ImmunoResearch Laboratories (Alexa Fluor\u0026reg; 488 AffiniPure Donkey Anti-Goat IgG (H\u0026thinsp;+\u0026thinsp;L), AB_2340428, Alexa Fluor\u0026reg; 594 AffiniPure Donkey Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) AB_2340855, Alexa Fluor\u0026reg; 647 AffiniPure Donkey Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), AB_2492288, 1:250 dilution) were used for multicolor immunofluorescence imaging, whereas 4\u0026prime;,6-diamidino-2-phenylindole, dilactate (DAPI; 1 \u0026micro;g/ml, Thermo Fisher Scientific) was used for nuclear counterstaining. Sections were imaged on a Zeiss LSM 710 confocal microscope system equipped with 405, 488, 555, and 647 nm lasers. Confocal images were acquired using a Zeiss Axiocam 506 Mono camera and mosaics created using the Zen 2.3 software (Blue edition).\u003c/p\u003e \u003cp\u003eFor immunohistochemical staining of CSF1R and IBA1 in paraffin sections, 4 \u0026micro;m thin IVD tissue sections were dewaxed in xylene, acetone and Tris-buffered saline, followed by antigen retrieval using sodium citrate buffer at 95\u0026deg;C for 10 min. Primary antibodies were incubated over night at 4\u003csup\u003eo\u003c/sup\u003eC. MACH 4 Universal HRP-Polymer (Biocare, M4U536) were used to detect either mouse or rabbit antibodies. After DAB staining, counterstain slides for 1 min with Mayer's Hematoxylin. Dehydrate slides by incubating for 3 min each in: 70% EtOH \u0026minus;\u0026thinsp;80% EtOH \u0026minus;\u0026thinsp;95% EtOH \u0026minus;\u0026thinsp;2x 100% EtOH \u0026minus;\u0026thinsp;2x Xylene. Sections were mounted with coverslips using a Xylol-based Fast Mounting Medium.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eTwo-way ANOVA was utilized for time course studies to determine the interaction of genotype versus day of study. Differences between \u003cem\u003ecsf1r\u003c/em\u003e knockout genotypes on specific days were assessed by post hoc Bonferroni. One-way ANOVA was utilized to determine within-genotype differences by day of study with individual comparisons made using a post hoc Dunnett's multiple comparisons test with significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GraphPad Prism 4 software was utilized for statistical analyses.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eValues for all data points in graphs are reported in the Supporting Data Values file.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Zhigang He (F.M. Kirby Neurobiology Center, Boston Children\u0026rsquo;s Hospital) for valuable comments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work received funding from The Stepping Strong Innovator Awards program 2018 and The Stepping Strong Innovator Awards program 2020.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: LL, HG, YL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: LL, JZ, YY, WY, HG\u003c/p\u003e\n\u003cp\u003eInvestigation: LL, JZ, YY, WY, FT, BC, HG, YL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVisualization: LL, JB, HG\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Original Draft: LL, HG\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Review \u0026amp; Editing: HG, JB, JC, YL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision \u0026ndash; HG, YL\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u0026nbsp;\u003c/strong\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHicks, G. 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PLOS ONE 11, e0160486 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5647673/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5647673/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. Inflammation has been implicated in discogenic back pain and disc degeneration, however, the detailed molecular mechanisms remain unclear. Herein we demonstrate that Colony Stimulating Factor 1 Receptor (CSF1R) signaling plays an essential role in the development of IVD degeneration and discogenic back pain. Genetic deletion of CSF1R from microglia/macrophages or oral administration of a CSF1R competitive inhibitor, GW2580, decreased IVD degeneration as evidenced by serial magnetic resonance imaging (MRI) and histopathological analyses in adult mice following disc injury. CSF1R deletion or GW2580 administration inhibited pro-inflammatory cytokine release from injured discs and blocked dorsal root ganglion (DRG) macrophage and spinal cord dorsal horn microglia activation and in so doing, eliminated neuropathic pain secondary to disc injury. These results suggest a novel therapeutic strategy for the treatment of chronic low back pain secondary to IVD degeneration.","manuscriptTitle":"Inhibiting CSF1R signaling reduces disc degeneration and discogenic back pain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 16:37:25","doi":"10.21203/rs.3.rs-5647673/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de1b5b0b-db81-4f62-9d3e-dad872f3c16b","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42084727,"name":"Health sciences/Pathogenesis"},{"id":42084728,"name":"Biological sciences/Physiology/Neurophysiology"},{"id":42084729,"name":"Biological sciences/Physiology/Bone quality and biomechanics"}],"tags":[],"updatedAt":"2025-03-10T15:20:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-30 16:37:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5647673","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5647673","identity":"rs-5647673","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}