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Purpose: The pathophysiology of discogenic low back pain is not fully understood. Tetrodotoxin-sensitive voltage-gated sodium (NaV) channels are associated with primary sensory nerve transmission, and the NaV1.7 channel has emerged as an analgesic target. Previously, we found increased NaV1.7 expression in dorsal root ganglion (DRG) neurons innervating injured discs. This study aimed to examine the effect of blocking NaV1.7 on sensory nerves after disc injury., Materials and Methods: Rat DRG neurons innervating the L5/6 disc were labeled with Fluoro-Gold (FG) neurotracer. Twenty-four rats underwent intervertebral disc puncture (puncture group) and 12 rats underwent sham surgery (non-puncture group). The injury group was divided into a saline infusion group (puncture+saline group) and a NaV1.7 inhibition group, injected with anti-NaV1.7 antibody (puncture+anti-NaV1.7 group); n=12 per group. Seven and 14 days post-surgery, L1 to L6 DRGs were harvested and immunostained for calcitonin gene-related peptide (CGRP) (an inflammatory pain marker), and the proportion of CGRP-immunoreactive (IR) DRG neurons of all FG-positive neurons was evaluated., Results: The ratio of CGRP-IR DRG neurons to total FG-labeled neurons in the puncture+saline group significantly increased at 7 and 14 days, compared with the non-puncture group, respectively (p<0.05). Application of anti-NaV1.7 into the disc significantly decreased the ratio of CGRP-IR DRG neurons to total FG-labeled neurons after disc puncture at 7 and 14 days (40% and 37%, respectively; p<0.05)., Conclusion: NaV1.7 antibody suppressed CGRP expression in disc DRG neurons. Anti-NaV1.7 antibody is a potential therapeutic target for pain control in patients with lumbar disc degeneration.

Purpose: Nuclear factor-κB (NF-κB), receptor activator of NF-κB (RANK), and RANK ligand (RANKL) are transcriptional regulators of inflammatory cytokines. RANKL expression in dorsal root ganglion (DRG) neurons is elevated in animal models of pain or intervertebral disc herniation. We sought to evaluate the effect of anti-RANKL antibodies on sensory nerves innervating injured intervertebral discs., Method: We labeled DRG neurons innervating L5-6 discs with FluoroGold (FG). The L5-6 discs of 36 rats were punctured using a 23-gage needle and 18 rats underwent sham surgery without disc puncture. The puncture group was evenly subdivided into a group in which 10 μl saline was administered to the injured disc and a group in which 10 μl of anti-RANKL antibody was administered. Seven and 14 days postsurgery, DRGs at L2 level were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP). The proportion of CGRP-immunoreactive (IR) DRG neurons of all FG-positive neurons was determined. Amount of tumor necrosis factor (TNF)-α and interleukin(IL)-6 was measured within the intervertebral discs in each group at 7 and 14 days after surgery using an enzyme-linked immunosorbent assay (ELISA)., Results: The proportion of CGRP-IR DRG neurons to total FG-labeled neurons innervating injured intervertebral discs and amount of TNF-α and IL-6 in the injured discs in the saline control group was significantly increased compared with that found in rats from the sham surgery group (P < 0.05). However, application of anti-RANKL antibody to the injured discs significantly decreased the proportion of CGRP-IR DRG neurons to total FG-labeled neurons and amount of TNF-α and IL-6 in the injured discs (P < 0.05)., Conclusions: TNF-α and IL-6 in the injured discs increased and CGRP expression increased in DRG neurons innervating injured discs, and antibodies to RANKL could suppress this increased TNF-α, IL-6, and CGRP expression. RANKL may be a therapeutic target for pain control in patients with lumbar disc degeneration.

Purpose: Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel, which can be activated by capsaicin and other noxious stimuli. Recently, an association between bone pain and TRPV1 has been reported. However, the influence of osteoporosis on TRPV1 in the sensory system innervating the femur has not been reported., Materials and Methods: TRPV1-immunoreactive (ir) in dorsal root ganglia (DRG) neurons labeled with neurotracer [Fluoro-Gold (FG)] innervating the femurs of Sprague Dawley rats were examined in control, sham, and ovariectomized (OVX) rats. We evaluated osteoporosis in the femurs and compared the proportion of TRPV1-ir DRG neurons innervating femur between the 3 groups of rats., Results: OVX rats showed osteoporotic cancellous bone in the femur. FG labeled neurons were distributed from L1 to L6 DRG, but there was no significant difference in the proportion of labeled neurons between the 3 groups (p>0.05). The proportions of FG labeled TRPV1-ir DRG neurons were 1.7%, 1.7%, and 2.8% of DRG neurons innervating the femur, in control, sham-operated, and OVX rats, respectively. The proportion of TRPV1-ir neurons in DRG innervating the femur in OVX rats was significantly higher than that in control and sham-operated rats (p<0.05)., Conclusion: Under physiological conditions, DRG neurons innervating femurs in rats contain TRPV1. Osteoporosis increases the numbers of TRPV1-ir neurons in DRG innervating osteoporotic femurs in rats. These findings suggest that TRPV1 may have a role in sensory perception of osteoporotic femurs.

Study Design: Animal study., Objective: To investigate pain-related expression of NaV1.7 in dorsal root ganglia (DRG) innervating intervertebral discs., Summary of Background Data: The pathophysiology of discogenic low back pain is not fully understood. Prostaglandins and cytokines produced by degenerated discs can cause pain, but nonsteroidal anti-inflammatory and steroid medications are often ineffective at pain reduction. Tetrodotoxin-sensitive, voltage-gated sodium (NaV) channels are associated with sensory transmission in primary sensory nerves, and the NaV1.7 channel has emerged as an attractive analgesic target. The purpose of this study was to investigate pain-related expression of NaV1.7 in DRG innervating intervertebral discs., Methods: Using a rodent model of disc puncture, we labeled DRG neurons innervating L5-L6 discs with FluoroGold neurotracer (n = 20). Half of the rats (n = 10) underwent intervertebral disc puncture using a 23-gauge needle (puncture group), and the other half underwent non-puncture sham surgery (non-puncture group). Seven and 14 days after surgery, DRGs from the L1 to L6 levels were harvested, sectioned, and immunostained for NaV1.7, and the proportion of NaV1.7-immunoreactive DRG neurons was evaluated., Results: NaV1.7 was expressed in DRG neurons innervating intervertebral discs from L1 to L5. The ratio of NaV1.7-expressing DRG neurons to total FG-labeled neurons was 7.2% and 7.6% at 1 and 2 weeks after surgery, respectively, in the non-puncture group and 16.2% and 16.3% at 1 and 2 weeks, respectively, in the puncture group. The upregulation of NaV1.7 after puncture was significant at both 1 and 2 weeks after surgery (P < 0.01)., Conclusion: We found that disc injury increases NaV1.7 expression in DRG neurons innervating injured discs. NaV1.7 may be a therapeutic target for pain control in patients with lumbar disc degeneration.

Purpose: The pathomechanisms of pain resulting from lumbar disc herniation have not been fully elucidated. Prostaglandins and cytokines generated at the inflammatory site produce associated pain; however, non-steroidal anti-inflammatory drugs and steroids are sometimes ineffective in patients. Tetrodotoxin-sensitive voltage-gated sodium (NaV) channels are related to sensory transmission in primary sensory nerves. The sodium channel NaV1.7 has emerged as an attractive analgesic target. The purpose of this study was to evaluate pain-related behavior and expression of NaV1.7 in dorsal root ganglia (DRG) after combined sciatic nerve compression and nucleus pulposus (NP) application in rats., Methods: Rats were divided into three groups and underwent either sciatic nerve compression with NP for 2 s using forceps (n = 20), sham operation with neither compression nor NP (n = 20), or no operation (controls, n = 20). Mechanical hyperalgesia was measured every second day for three weeks using von Frey filaments. NaV1.7 expression in L5 DRG was examined 7 and 14 days after surgery using immunohistochemistry. The number of neurons immunoreactive for NaV1.7 was compared among the three groups., Results: Mechanical hyperalgesia was found over the 14-day observation in the nerve compression plus NP application group, but not in the sham-operated or control groups (P < 0.05). NaV1.7 expression in L5 DRG was up-regulated in the nerve compression plus NP application group, compared with sham-operated and control rats (P < 0.01)., Conclusions: Our results indicate that nerve compression plus NP application produces pain-related behavior. We conclude that NaV1.7 expression in DRG neurons may play an important role in mediating pain from sciatic nerves after compression injury and exposure to NP.

Background: The dorsal root ganglia (DRG) neuron is an invaluable tool in axon growth, growth factor regulation, myelin formation and myelin-relevant researches. The purification of DRG neurons is a key step in these studies. Traditionally, purified DRG neurons were obtained in two weeks after exposure to several rounds of anti-mitotic reagent., Methods and Results: In this report, a novel, simple and efficient method for DRG purification is presented. DRG cultures were treated once with a high-dose anti-mitotic reagent cocktail for 72 hours. Using this new method, DRG neurons were obtained with 99% purification within 1 week. We confirmed that the neurite growth and the viability of the purified DRG neurons have no difference from the DRG neurons purified by traditional method. Furthermore, P0 and MBP expression was observed in myelin by immunocytochemistry in the DRG/SC co-culture system. The formation of mature node of Ranvier in DRG-Schwann cell co-culture system was observed using anti-Nav 1.6 and anti-caspr antibody., Conclusion and Significance: The results indicate that this high dose single treatment did not compromise the capacity of DRG neurons for myelin formation in the DRG/SC co-culture system. In conclusion, a convenient approach for purifying DRG neurons was developed which is time-saving and high-efficiency.

Study Design: Animal model of intravertebral disc (IVD) degeneration., Objective: To examine production of inflammatory mediators in IVDs and neuropeptides in dorsal root ganglia (DRGs) in rat models of IVD compression and injury., Summary of Background Data: Sensory nerve fibers in IVDs and inflammatory mediator responses have been verified in animal models of IVD injury. However, the IVD injury in animals incompletely models degenerated human IVDs causing discogenic low back pain, because human IVDs are also subject to compression., Methods: Experimental groups (controls, IVD injury, IVD compression, and their combination) of Sprague Dawley rats were prepared. Fluoro-Gold (FG; Fluorochrome, Denver, CO) was applied into coccygeal IVDs. Inflammatory mediators in IVDs, including nerve growth factor, tumor necrosis factor α, interleukin 1β, and interleukin 6, were quantified using enzyme-linked immunosorbent assays. DRGs were immunostained for calcitonin gene-related peptide, activating transcription factor 3, and growth-associated phosphoprotein 43., Results: The upregulation of inflammatory mediators was transient in the IVD injury group but delayed and long-lasting in the IVD compression group. When the IVD injury and compression were combined, the upregulation of inflammatory mediators was long-lasting through 8 weeks. The proportion of calcitonin gene-related peptide-immunoreactive neurons among Fluoro-Gold-labeled neurons remained significantly higher in the IVD injury, compression, and combination groups than in the controls. In contrast, increases in the proportions of activating transcription factor 3-immunoreactive or growth-associated phosphoprotein 43-immunoreactive neurons in the IVD injury group animals were transient but long-lasting in the compression and combination groups compared with controls., Conclusion: Disc injury in rats produces persistent increases in neuropeptides in DRGs but only transient increases in inflammatory mediators in IVDs. On the contrary, disc compression in rats produces a long-lasting increase in inflammatory mediators in IVDs and neuropeptides in DRGs. Moreover, disc compression induces persistent nerve injury and regeneration of the afferent fibers innervating IVDs.

Study Design: Immunohistological analysis of dichotomizing sensory nerve fibers projecting to the lumbar multifidus muscles and intervertebral disc (IVD), facet joint (FJ), or sacroiliac joint (SIJ) in rats., Objective: To elucidate dichotomizing sensory nerve fibers projecting to the lumbar multifidus muscles and to IVDs, FJs, or SIJs., Summary of Background Data: Clinically, the origin of low back pain remains unknown. Multiple studies have identified lumbar muscles, IVDs, FJs, and SIJs as sources of low back pain. Pain may originate directly from lumbar muscles or be referred from the spine, or both. Dorsal root ganglion (DRG) neurons with dichotomizing axons have been reported in several species and are thought to be related to referred pain., Methods: We used 2 neurotracers, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) and fluorogold (FG), in this double-labeling study involving 30 Sprague Dawley rats. DiI was applied to lumbar multifidus muscles in all rats. Simultaneously, FG was applied to the anterior left portion of L5-L6 IVDs in the IVD group (n = 10), to the left L5-L6 FJs in the FJ group (n = 10), and to the left SIJs in the SIJ group (n = 10). Fourteen days after surgery, left DRGs from L1 to L6 were harvested, sectioned, and observed under a fluorescence microscope., Results: We verified the existence of double-labeled DRG neurons (i.e., dichotomizing sensory nerve fibers) projecting to lumbar multifidus muscles and to IVDs, FJs, or SIJs, depending on the group. The proportion of double-labeled cells in all DiI-labeled DRG neurons was higher in the FJ group (6.8%) and SIJ group (7.1%) than in the IVD group (3.1%) (P < 0.05)., Conclusion: Our results document the presence of dichotomizing sensory nerve fibers projecting to lumbar multifidus muscles and to IVDs, FJs, and SIJs. Referred low back muscle pain may reflect disorders of lumbar posterior structures, such as FJs and SIJs, rather than disorders of lumbar anterior structures, such as IVDs.