Up-Regulation of Pain Behavior and Glial Activity in the Spinal Cord after Compression and Application of Nucleus Pulposus onto the Sciatic Nerve in Rats

Masaki Norimoto,Yoshihiro Sakuma,Miyako Suzuki,Sumihisa Orita,Kazuyo Yamauchi,Gen Inoue,Yasuchika Aoki,Tetsuhiro Ishikawa,Masayuki Miyagi,Hiroto Kamoda,Gou Kubota,Yasuhiro Oikawa,Kazuhide Inage,Takesh 대한척추외과학회 2014 Asian Spine Journal Vol.8 No.5

Study Design: Experimental animal study. Purpose: To evaluate pain-related behavior and changes in glial activity in the spinal dorsal horn after combined sciatic nerve compression and nucleus pulposus (NP) application in rats. Overview of Literature: Mechanical compression and inflammation caused by prostaglandins and cytokines at disc herniation sites induce pain. Structural changes and pain-associated cytokines in the dorsal root ganglia and spinal dorsal horn contribute to prolonged pain. Glial cells in the spinal dorsal horn may also function in pain transmission. Methods: The sciatic nerve was compressed with NP for 2 seconds using forceps in the NP+nerve compression group; the shamoperated group received neither compression nor NP; and the control group received no operation. Mechanical hyperalgesia was measured for 3 weeks using von Frey filaments. Glial activity in the spinal dorsal horn was examined 7 days and 14 days postsurgery using anti-glial fibrillary acidic protein and anti-Ionized calcium binding adaptor molecule-1 antibodies to detect astrocytes and microglia, respectively. Results: Mechanical hyperalgesia was detected throughout the 14-day observation in the NP+nerve compression group, but not in control or sham-operated groups (p <0.05). Both astrocytes and microglia were significantly increased in the spinal dorsal horn of the NP+nerve compression group compared to control and sham groups on days 7 and 14 (p <0.05). Conclusions: Nerve compression with NP application produces pain-related behavior, and up-regulates astrocytes and microglia in the spinal dorsal horn, suggesting that these glia may be related to pain transmission.

Evaluation of Behavior and Expression of Receptor Activator of Nuclear Factor-Kappa B Ligand in Dorsal Root Ganglia after Sciatic Nerve Compression and Application of Nucleus Pulposus in Rats

Yoshiyuki Matsuyama,Yoshihiro Sakuma,Miyako Suzuki,Sumihisa Orita,Kazuyo Yamauchi,Gen Inoue,Yasuchika Aoki,Tetsuhiro Ishikawa,Masayuki Miyagi,Hiroto Kamoda,Gou Kubota,Yasuhiro Oikawa,Kazuhide Inage,Ta 대한척추외과학회 2014 Asian Spine Journal Vol.8 No.5

Study Design: Experimental animal study. Purpose: To evaluate pain-related behavior and changes in nuclear factor-kappa B (NF-kB), receptor activator of NF-kB (RANK), and ligand (RANKL) in dorsal root ganglia (DRG) after combined sciatic nerve compression and nucleus pulposus (NP) application in rats. Overview of Literature: The pathological mechanisms underlying pain from lumbar-disc herniation have not been fully elucidated. RANKL are transcriptional regulators of inflammatory cytokines. Our aim was to evaluate pain-related behavior and RANKL expression in DRG after sciatic-nerve compression and application of NP in rats. Methods: Mechanical hyperalgesia and RANKL expression were assessed in three groups of rats: NP+sciatic nerve compression (2 seconds), sham-operated, and controls (n=20 each). Mechanical hyperalgesia was measured every other day for 3 weeks using von Frey filaments. RANKL expression in L5 DRGs was examined at five and ten days after surgery using immunohistochemistry. Results: Mechanical hyperalgesia was observed over the 12-day observation period in the NP+nerve compression group, but not in the control and sham-operated animal groups (p <0.05). RANKL immunoreactivity was seen in the nuclei of L5 DRG neurons, and its expression was significantly upregulated in NP+nerve compression rats compared with control and sham-operated rats (p <0.01). Conclusions: The exposure of sciatic nerves to mechanical compression and NP produces pain-related behavior and up-regulation of RANKL in DRG neurons. RANKL may play an important role in mediating pain after sciatic nerve injury with exposure to NP.

Evaluation of Histological Changes in Back Muscle Injuries in Rats over Time

Koki Abe,Kazuhide Inage,Yoshihiro Sakuma,Sumihisa Orita,Kazuyo Yamauchi,Miyako Suzuki,Go Kubota,Yasuhiro Oikawa,Takeshi Sainoh,Jun Sato,Kazuki Fujimoto,Yasuhiro Shiga,Hirohito Kanamoto,Kazuhisa Takaha 대한척추외과학회 2017 Asian Spine Journal Vol.11 No.1

Study Design: Animal model study. Purpose: The purpose of this study was to evaluate the histological variation in the injured muscle and production of calcitonin generelated peptide in rats over time. Overview of Literature: Vertebral surgery has been reported to cause atrophy of the back muscles, which may result in pain. However, few reports have described the time series histological variation in the injured muscle and changes in the dominant nerve. Methods: We used 30 male, 8-week-old Sprague-Dawley rats. The right and left sides of the paravertebral muscle were considered as the injured and uninjured sides, respectively. A 115 g weight was dropped from a height of 1 m on the right paravertebral muscle. Hematoxylin and eosin (H&E) staining of the muscle was performed 1–3 weeks after injury for histological evaluation. Fluoro-Gold (FG) was injected into the paravertebral muscle. The L2 dorsal root ganglia on both sides were resected 1, 2, and 3 weeks after injury, and immunohistochemical staining for calcitonin gene-related peptide was performed. Results: H&E staining of the paravertebral muscle showed infiltration of inflammatory cells and the presence of granulation tissue in the injured part on the ipsilateral side 1 week after injury. Muscle atrophy occurred 3 weeks after injury, but was repaired via spontaneous replacement of muscle cells/fibers. In contrast, compared with the uninjured side, the percentage of cells double-labeled with FG and calcitonin gene-related peptide in FG-positive cells in the dorsal root ganglia of the injured side was significantly increased at each time point throughout the study period. Conclusions: These results suggest that sensitization of the dominant nerve in the dorsal root ganglia, which may be caused by cicatrix formation, can protract injured muscle pain. This information may be helpful in elucidating the underlying mechanism of persistent pain after back muscle injury.

Inhibiting Vascular Endothelial Growth Factor in Injured Intervertebral Discs Attenuates Pain-Related Neuropeptide Expression in Dorsal Root Ganglia in Rats

Jun Sato,Kazuhide Inage,Masayuki Miyagi,Yoshihiro Sakuma,Kazuyo Yamauchi,Masao Koda,Takeo Furuya,Junichi Nakamura,Miyako Suzuki,Go Kubota,Yasuhiro Oikawa,Takeshi Sainoh,Kazuki Fujimoto,Yasuhiro Shiga 대한척추외과학회 2017 Asian Spine Journal Vol.11 No.4

Study Design: An experimental animal study. Purpose: To evaluate effects of anti-vascular endothelial growth factor (VEGF) on the content and distribution of the calcitonin generelated peptide (CGRP) in the dorsal ganglia in a rat model. Overview of Literature: Increased expression of VEGF in degenerative disc disease increases the levels of inflammatory cytokines and nerve ingrowth into the damaged discs. In animal models, increased levels of VEGF can persist for up to 2 weeks after an injury. Methods: Through abdominal surgery, the dorsal root ganglia (DRG) innervating L5/L6 intervertebral disc were labeled (FluoroGold neurotracer) in 24, 8-week old Sprague Dawley rats. The rats were randomly allocated to three groups of eight rats each. The anti- VEGF group underwent L5/6 intervertebral disc puncture using a 26-gauge needle, intradiscal injection of 33.3 μg of the pegaptanib sodium, a VEGF165 aptamer. The control-puncture group underwent disc puncture and intradiscal injection of 10 μL saline solution, and the sham-surgery group underwent labeling but no disc puncture. Two rats in each group were sacrificed on postoperative days 1, 7, 14, and 28 after surgery. L1–L6 DRGs were harvested, sectioned, and immunostained to detect the content and distribution of CGRP. Results: Compared with the control, the percentage of CGRP-positive cells was lower in the anti-VEGF group (p <0.05; 40.6% and 58.1% on postoperative day 1, 44.3% and 55.4% on day 7, and 42.4% and 59.3% on day 14). The percentage was higher in the control group compared with that of the sham group (p <0.05; sham group, 34.1%, 40.7%, and 33.7% on postoperative days 1, 7, and 14, respectively). Conclusions: Decreasing CGRP-positive cells using anti-VEGF therapy provides fundamental evidence for a possible therapeutic role of anti-VEGF in patients with discogenic lower back pain.

The Time Course Changes in Bone Metabolic Markers after Administering the Anti-Receptor Activator of Nuclear Factor-Kappa B Ligand Antibody and Drug Compliance among Patients with Osteoporosis

Kazuhide Inage,Sumihisa Orita,Kazuyo Yamauchi,Yoshihiro Sakuma,Go Kubota,Yasuhiro Oikawa,Takeshi Sainoh,Jun Sato,Kazuki Fujimoto,Yasuhiro Shiga,Kazuhisa Takahashi,Seiji Ohtori 대한척추외과학회 2015 Asian Spine Journal Vol.9 No.3

Study Design: Retrospective study. Purpose: We conducted a study to investigate the time course changes in bone metabolic markers after the administration of the anti-receptor activator of nuclear factor-kappa B ligand (RANKL) antibody and to assess drug compliance among osteoporotic patients. Overview of Literature: The anti-RANKL antibody is expected to provide an improvement in those with a bone metabolism disorder. However there are only a few clinical reports available on the effect of treatment. Methods: We included 40 post-menopausal osteoporotic patients who received the anti-RANKL antibody. To determine the time course changes in the bone metabolic markers, we measured the serum tartrate-resistant acid phosphatase 5b (TRACP 5b; a bone resorption marker) and the serum N-terminal propeptide of type 1 collagen (P1NP; a bone formation marker) levels prior to and 1 month after administrating the anti-RANKL antibody. To evaluable drug compliance, we assessed the dropout rate during treatment and at 6 months after treatment. Results: The average TRACP 5b level significantly decreased from 574.8 mU/dL before treatment to 153.2 mU/dL 1 month after treatment (p <0.05). There was no significant difference in the average P1NP level, which was 56.9 μG/L and 35.1 μG/L before and 1 month after treatment, respectively (p >0.05). As for drug compliance, we did not have any dropouts during the treatment or after 6 months (dropout rate: 0%). Conclusions: Our study suggests that anti-RANKL antibody treatment suppresses bone resorption and maintains bone formation.

Efficacy of Anti-NaV1.7 Antibody on the Sensory Nervous System in a Rat Model of Lumbar Intervertebral Disc Injury

Seiji Ohtori,Daisuke Nojima,Kazuhide Inage,Yoshihiro Sakuma,Jun Sato,Sumihisa Orita,Kazuyo Yamauchi,Yawara Eguchi,Nobuyasu Ochiai,Kazuki Kuniyoshi,Yasuchika Aoki,Junichi Nakamura,Masayuki Miyagi,Miyak 연세대학교의과대학 2016 Yonsei medical journal Vol.57 No.3

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.

Improvements in Intractable Lumbar and LowerExtremity Symptoms after Systemic Administration of Tocilizumab, an Anti-interleukin-6 Receptor Antibody

Sainoh Takeshi,Orita Sumihisa,Miyagi Masayuki,Suzuki-Narita Miyako,Sakuma Yoshihiro,Oikawa Yasuhiro,Kubota Go,Sato Jun,Shiga Yasuhiro,Fujimoto Kazuki,Eguchi Yawara,Koda Masao,Aoki Yasuchika,Akazawa Ts 대한척추외과학회 2022 Asian Spine Journal Vol.16 No.1

Study Design: Prospective cohort study (open-label, single-arm, and non-blinded).Purpose: This study aims to determine the effects of systemic administration of tocilizumab, an anti-interleukin-6 (IL-6) receptor antibody on refractory low back pain and leg symptoms.Overview of Literature: IL-6 overexpression is associated with neuropathic pain pathogenesis, which is potentially followed by chronic low back pain, including leg pain and numbness. This finding suggest that inhibition of IL-6 at the site of pain or in the transmission pathway could provide novel therapeutic targets for chronic low back pain.Methods: This prospective, single-arm study included 11 patients (eight men; mean age, 62.7 years) with ≥3-months’ chronic pain history due to lumbar disease. Subcutaneous TCZ injections were administered twice, at a 2-week interval. We evaluated low back pain, leg pain, and leg numbness using numeric rating scales and the Oswestry Disability Index (ODI; baseline and 6 months postinjection); serum IL-6 and tumor necrosis factor-α levels (baseline and 1 month postinjection); and clinical adverse events.Results: Intractable symptoms reduced after TCZ administration. Low back pain improved for 6 months. Improvements in leg pain and numbness peaked at 4 and 1 month, respectively. Improvements in ODI were significant at 1 month and peaked at 4 months. Serum IL-6 was increased at 1 month. IL-6 responders (i.e., patients with IL-6 increases >10 pg/mL) showed particularly significant improvements in leg pain at 2 weeks, 1 month, and 2 months compared with nonresponders. We observed no apparent adverse events.Conclusions: Systemic TCZ administration improved symptoms effectively for 6 months, with peak improvements at 1–4 months and no adverse events. Changing serum IL-6 levels correlated with leg pain improvements; further studies are warranted to elucidate the mechanistic connections between lumbar disorders and inflammatory cytokines.

Long-Term Outcomes of In Situ Fusion for Treating Dysplastic Spondylolisthesis

Kazuhide Inage,Sumihisa Orita,Kazuyo Yamauchi,Miyako Suzuki,Yoshihiro Sakuma,Go Kubota,Yasuhiro Oikawa,Takeshi Sainoh,Jun Sato,Kazuki Fujimoto,Yasuhiro Shiga,Koki Abe,Hirohito Kanamoto,Masahiro Inoue 대한척추외과학회 2017 Asian Spine Journal Vol.11 No.2

Study Design: Retrospective, observational, single-center study. Purpose: To investigate the long-term outcomes of in situ fusion procedures for treating dysplastic spondylolisthesis. Overview of Literature: In situ fusion performed in patients with dysplastic spondylolisthesis avoids the development of nerve complications. Methods: In total, 12 of 28 patients who underwent in situ fusion for treating dysplastic spondylolisthesis at Chiba University Hospital from 1974 to 2004 were followed up in August 2013. Surgical complications were evaluated. Low back pain and leg pain were assessed using a visual analog scale (VAS). Vertebral alignment, including the lumbosacral angle and lumbar lordosis angle measurement on radiographic images (profile view in the neutral standing position), was evaluated during preoperative, postoperative, and final examinations. Results: The mean follow-up duration, patient age at the final examination, and patient age at operation were 20.0±7.2, 42.3±13.3, and 22.3±11.4 years, respectively. No complications were reported. Mean VAS scores for low back pain and leg pain were significantly lower at the final examination than at the preoperative examination (p <0.05). At the preoperative, postoperative, and final examinations, the mean lumbosacral angle was 32.3°±14.2°, 33.7°±11.8°, and 36.5°±16.4°, while the mean lumbar lordosis angle was 51.0°±14.8°, 48.6°±18.8°, and 49.6°±15.5°, respectively. No significant differences were noted among these values across the different time periods (p <0.05). Conclusions: In situ fusion performed in patients with dysplastic spondylolisthesis avoids the development of nerve complications such as nerve paralysis that may occur after repositioning operation and maintains appropriate long-term sagittal alignment, even 20 years after operation.

Dose Optimization for Single Intradiscal Administration of the Tumor Necrosis Factor-α Inhibitor, Etanercept, in Rat Disc Injury Models

Kazuhide Inage,Sumihisa Orita,Kazuyo Yamauchi,Takane Suzuki,Miyako Suzuki,Yoshihiro Sakuma,Go Kubota,Yasuhiro Oikawa,Takeshi Sainoh,Jun Sato,Kazuki Fujimoto,Yasuhiro Shiga,Koki Abe,Hirohito Kanamoto,M 대한척추외과학회 2016 Asian Spine Journal Vol.10 No.4

Study Design: Experimental animal study. Purpose: We aimed to determine the optimal dose of a single direct injection of the tumor necrosis factor (TNF)-α inhibitor, etanercept, by using the rat model of degenerative intervertebral disc from injury. Overview of Literature: The pain-related peptide expression was suppressed in the etanercept (100 μg and 1,000 μg)-administered groups in a dose-dependent manner. Methods: The neurotracer FluoroGold (FG) was applied to the surfaces of L4/5 discs to label their innervating dorsal root ganglion (DRG) neurons (n=50). Ten rats were included in the nonpunctured disc sham surgery control group, whereas the other 40 were included in the experimental group in which intervertebral discs were punctured with a 23-gauge needle. Saline or etanercept (10 μg, 100 μg, or 1,000 μg) was injected into the punctured discs (n=10 for each treatment). After 14 days of surgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP). The proportion of FG-labeled CGRPimmunoreactive DRG neurons was evaluated in all the groups. Results: There were no significant differences between the puncture+saline group and the puncture+10-μg etanercept group (p >0.05). However, a significant decrease in the percentage of FG and CGRP double-positive cells in FG-positive cells was observed in the etanercept (100 μg and 1,000 μg)-administered groups in a dose-dependent manner (p <0.05). Conclusions: When a low dose of the TNF-α inhibitor (10 μg of etanercept) was directly administered to the rat intervertebral disc in the rat model of degenerative intervertebral disc from injury, no suppressive effect on the pain-related peptide expression was observed. However, when a higher dose of etanercept (100 μg and 1,000 μg) was administered, the pain-related peptide expression was suppressed in a dose-dependent manner.

Increase of TRPV1-Immunoreactivity in Dorsal Root Ganglia Neurons Innervating the Femur in a Rat Model of Osteoporosis

Kensuke Yoshino,Seiji Ohtori,Miyako Suzuki,Yuya Kawarai,Yoshihiro Sakuma,Gen Inoue,Sumihisa Orita,Kazuyo Yamauchi,Yasuchika Aoki,Tetsuhiro Ishikawa,Masayuki Miyagi,Hiroto Kamoda,Gou Kubota,Yasuhiro Oi 연세대학교의과대학 2014 Yonsei medical journal Vol.55 No.6

Purpose: Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselectivecation 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 innervatingthe 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 femursand 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 osteoporoticfemurs.