山内 かづ代

STUDY DESIGN: We investigated the mechanism of discogenic low back pain using an in vitro model. OBJECTIVE: To evaluate the axonal growth and induction of a painful neuropeptide, substance P (SP), using rat dorsal root ganglion (DRG) neurons and degenerated human disc cells in vitro. SUMMARY OF BACKGROUND DATA: Degeneration of the lumbar intervertebral disc is a cause of low back pain. The pathologic mechanism is thought to be sensory nerve ingrowth into the inner layers of the degenerated intervertebral disc; however, the precise patho-mechanism has not been clarified. METHODS: The nucleus pulposus (NP) and annulus fibrosus (AF) of human intervertebral discs were harvested from patients with discogenic low back pain. Extracted medium from human degenerative intervertebral discs was cultured with neurons of rat DRGs. We evaluated the promotion of axonal growth and SP induction of DRG neurons in extracted medium from the NP and AF using immunocytochemistry. RESULTS: The average length of growing axons in the NP and AF was significantly longer than that in the control (P < 0.005). That in the NP was significantly longer than that in the AF. The average length of growing axons in the NP was significantly shortened after anti-nerve growth factor (NGF)beta treatment (P < 0.005); however, that in the AF was not (P > 0.05). The percentage of SP-immunoreactive cells with growing axons was significantly higher only in the NP group compared with the control and AF groups (P < 0.005), and anti-NGFbeta treatment decreased the expression of SP in the NP group (P < 0.05). CONCLUSION: Extracted medium from the NP and AF promoted axonal growth. Furthermore, NGF from the NP promoted axonal growth and induced SP. These in vitro results may suggest that NGF from the NP promotes the growth of sensory nerve fibers innervating the degenerated intervertebral disc and may induce SP related with pain transmission.

STUDY DESIGN: The effect of proopiomelanocortin (POMC) gene transfer with radial shock waves (RSW) was investigated in vitro and in vivo rat pain models. OBJECTIVE: To examine the efficacy of POMC gene transfer with RSW, efficiency of beta-endorphin production in transfected cells, and its effects and side effects in pain models. SUMMARY OF BACKGROUND DATA: Opioids have been used to treat chronic pain originating from knee osteoarthritis and the lower back; however, several side effects have been reported. Endogenous opioids are safe, but they are not used for clinical treatment because their metabolism is very fast. METHODS: POMC plasmid was produced from pretransformed rat brain cDNA. POMC gene was added to the muscle of rat in vitro and in vivo with RSWs. We assessed beta-endorphin activity using immunohistochemistry. For assessment of pain behavior, we evaluated change in pain score and the level of the inflammatory neuropeptide, calcitonin gene-related peptide (CGRP), after transfection of the POMC gene in an adjuvant induced pain model for 28 days after treatment. RESULTS: POMC transfected using RSW expressed beta-endorphin at a significantly increased level in muscle cells compared with non-RSW transfection and controls in vitro and in vivo (P < 0.05).Animals showed significant pain sensitivity and increased CGRP expression in dorsal root ganglia neurons in this model; however, these findings decreased for 14 days after transfection of POMC into muscle. There was no significant difference in side effects, such as a change in the level of food pellet intake or constipation, between POMC-treated animals and untreated animals. CONCLUSION: POMC transfection with RSW increased beta-endorphin expression in muscle for 14 days, and suppressed pain behavior and CGRP expression in dorsal root ganglia neurons without side effects. This suggested that transfer of POMC by RSW is an effective treatment for inflammatory pain.

UNLABELLED: Elderly patients with osteoporosis sometimes experience lumbar vertebral fracture and may feel diffuse nonlocalized pain in the back, the lateral portion of the trunk, and the area surrounding the iliac crest. The pattern of sensory innervation of vertebral bodies remains unclear. Some sensory nerves from the L2 and L5 vertebral bodies may enter the paravertebral sympathetic trunks and reach the L2 dorsal root ganglion. Our randomized controlled study was to clarify the effect of L2 spinal nerve block on low back pain originating from acute osteoporotic lumbar vertebral fracture. Patients with low back pain originating from acute L3 or L4 osteoporotic vertebral fractures received a spinal nerve root block (L2 block group, n = 30) or subcutaneous injection (control, n = 30). Both groups received 1.5 mL of 1% lidocaine. The visual analog scale score, Roland Morris Disability Questionnaire, and Short Form questionnaire were examined before and after treatment. In both groups, spinal nerve blocks were significantly effective in alleviating low back pain (P < .05). One hour, 1 week, and 2 weeks after treatment, the visual analog scale score improved more in the L2 block group than in the control group (P < .05). From 1 month to 4 months after treatment, there were no significant differences in the pain scores between groups (P > .05). We conclude that L2 spinal nerve block for acute L3 or L4 osteoporotic vertebral body fracture was effective for 2 weeks, but it had no long-term effects on pain and social function. PERSPECTIVE: L2 spinal nerve block treatment for L3 or L4 osteoporotic vertebral body fracture was effective. This results suggest that the L2 dorsal root ganglion may innervate the L3 and L4 vertebral bodies in humans. L2 spinal nerve block for lumbar osteoporotic vertebral fracture may be a useful strategy to treat acute low back pain.

Nuclear factor-kappa B (NF-kappaB) is a gene transcriptional regulator of inflammatory cytokines. We investigated the transduction efficiency of NF-kappaB decoy to dorsal root ganglion (DRG), as well as the decrease in nerve injury, mechanical allodynia, and thermal hyperalgesia in a rat lumbar disc herniation model. Forty rats were used in this study. NF-kappaB decoy-fluorescein isothiocyanate (FITC) was injected intrathecally at the L5 level in five rats, and its transduction efficiency into DRG measured. In another 30 rats, mechanical pressure was placed on the DRG at the L5 level and nucleus pulposus harvested from the rat coccygeal disc was transplanted on the DRG. Rats were classified into three groups of ten animals each: a herniation + decoy group, a herniation + oligo group, and a herniation only group. For behavioral testing, mechanical allodynia and thermal hyperalgesia were evaluated. In 15 of the herniation rats, their left L5 DRGs were resected, and the expression of activating transcription factor 3 (ATF-3) and calcitonin gene-related peptide (CGRP) was evaluated immunohistochemically compared to five controls. The total transduction efficiency of NF-kappaB decoy-FITC in DRG neurons was 10.8% in vivo. The expression of CGRP and ATF-3 was significantly lower in the herniation + decoy group than in the other herniation groups. Mechanical allodynia and thermal hyperalgesia were significantly suppressed in the herniation + decoy group. NF-kappaB decoy was transduced into DRGs in vivo. NF-kappaB decoy may be useful as a target for clarifying the mechanism of sciatica caused by lumbar disc herniation.

STUDY DESIGN: Randomized, controlled study. OBJECTIVE: To evaluate the diagnosis of discogenic low back pain (LBP) with discography and discoblock. SUMMARY OF BACKGROUND DATA: Discogenic LBP is usually diagnosed by magnetic resonance imaging and discography. However, the reliability of discography is controversial. Previously, we reported the usefulness of discoblock with bupivacaine for diagnosis, and discoblock improved the results of anterior interbody fusion surgery. However, that study was not a randomized, controlled study. Therefore, the purpose of the current study was to compare the results of surgery after diagnosis of LBP by discography and discoblock. METHODS: Patients (n = 42) with severe LBP showing L4-L5 or L5-S1 disc degeneration on magnetic resonance imaging were evaluated by discography (1.5 mL of contrast medium) or discoblock (intradisc injection of 0.75 mL of 0.5% bupivacaine). We randomized the patients in turn. Anterior discectomy and interbody fusion were performed in patients who responded to the diagnostic procedures. The visual analogue scale score (0, no pain; 100, worst pain), Japanese Orthopedic Association Score (0, worst pain; 3, no pain), Oswestry Disability Index, and patient satisfaction before and 3 years after surgery were recorded and compared between groups. RESULTS: Twelve patients did not show pain provocation by discography or pain relief by discoblock and were excluded. Fifteen patients who showed pain provocation by discography and 15 patients who experienced pain relief with discoblock were evaluated. Rates of improvement in the visual analogue scale score, Japanese Orthopedic Association Score, and Oswestry Disability Index score in the discoblock group were significantly higher than those in the discography group (P < 0.05) from baseline to 3 years after surgery. Three patients were dissatisfied with surgery after discography compared with one patient after discoblock. CONCLUSION: Pain relief after injection of a small amount of bupivacaine into the painful disc was a useful tool for the diagnosis of discogenic LBP compared with discography.

STUDY DESIGN: Comparative study of differing durations of antibiotics for spinal surgery. OBJECTIVE: To compare rate of infection, duration of hospital stay, days until normal body temperature, and a panel of blood tests after surgery between long-term and short-term administration of antibiotics for spinal surgery using instrumentation. SUMMARY OF BACKGROUND DATA: Recent studies have reported that long-term administration of intravenous antibiotics is not necessary to avoid superficial and deep infections. We therefore changed the duration of administration from 9 to 2 days in our lumbar surgery patients. METHODS: We examined 135 patients (average age, 64.9 years) who underwent lumbar spinal surgery to insert a pedicle screw system to treat spinal canal stenosis. We administered 2 g of cefotiam daily to 60 patients for 9 days after surgery and to 75 patients for 2 days after surgery. Surgical time, loss of blood, rate of infection, duration of hospital stay, days until normal body temperature, and data from blood analysis (white blood cell count, and C-reactive protein [CRP] level) were statistically compared between the 2 groups. RESULTS: No significant differences in intraoperative measures of surgical invasion were observed between the 2 groups (surgical time, 209 vs. 220 minutes; blood loss, 530 vs. 576 mL; blood transfusion, 344 vs. 380 mL for the long-term and short-term groups, respectively). No acute infections occurred in either group. However, the duration of hospital stay (20.7 days), time until normal body temperature (5.1 days), and CRP level (2.23 mg/dL) at day 7 after surgery were significantly less in the short-term group than those in the long-term group (27.9 days, 6.8 days, and 3.13 mg/dL, respectively; P < 0.05). DISCUSSION: These results indicate that short-term intravenous administration of antibiotics did not elevate the infection rate after spinal surgery using instrumentation. However, long-term administration of antibiotics prolonged the duration of hospital stay, inhibited normalization of body temperature, and elevated CRP levels. Long-term administration of antibiotics may suppress normal, beneficial bacteria, thereby having an adverse effect on patient recovery.

STUDY DESIGN: Immunohistological and behavioral analysis of the effect of a tumor necrosis factor alpha (TNF-alpha) inhibitor in an injured-nerve model. OBJECTIVE: To examine the effect of direct application of a TNF-alpha inhibitor (etanercept) on injured-nerve pain caused by nucleus pulposus. SUMMARY AND BACKGROUND DATA: TNF-alpha is thought to play a crucial role in radicular pain. Calcitonin gene-related peptide (CGRP) is an inflammatory neuropeptide found in small sensory neurons. We have reported that CGRP appears in medium and large dorsal root ganglion (DRG) neurons that transmit proprioception in physiologic conditions. The purpose of the current study was to examine the change in behavior and phenotypic change of CGRP-immunoreactive DRG neurons by the TNF-alpha inhibitor, etanercept, in a disc herniation model. METHODS: For the injured-nerve model, nucleus pulposus was applied to the sciatic nerve and the sciatic nerve pinched. Saline (10 microL; n = 10), as a control, or etanercept (150 microg: n = 10) were applied to sciatic nerves simultaneously. Mechanical allodynia was examined. Immunohistochemistry was used to examine CGRP expression in L5 DRGs. RESULTS: Significant mechanical allodynia for 10 days was seen in the injured-nerve group compared with sham-operated animals. Etanercept ameliorated the mechanical allodynia slightly on day 2; however, there was no effect on other days. CGRP immunoreactivity was upregulated in the L5 DRG neurons of injured-nerve groups compared with the sham-operated group (P < 0.01). However, etanercept did not affect CGRP expression after nerve injury (P > 0.05). Proportions of CGRP- immunoreactive medium and large neurons were not significantly different in the nerve injury + saline group compared with the injury + etanercept group (P > 0.05). CONCLUSION: Our results indicate that direct application of a TNF-alpha inhibitor had a small effect on acute pain behavior and may not be effective for suppression of inflammatory peptides in the current disc-herniation model.

STUDY DESIGN: We evaluated 2 types of nerve growth factor (NGF) receptors on dorsal root ganglion (DRG) cells and nerve fibers innervating rat lumbar intervertebral discs. OBJECTIVE: To examine the NGF receptors, tyrosine kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) on DRG cells and nerve fibers innervating rat lumbar intervertebral discs using immunohistochemistry and a retrograde neurotracing method. SUMMARY OF BACKGROUND DATA: Nerve innervation of intervertebral discs is thought to be a pathology of discogenic low back pain. NGF is also important for mediating inflammatory pain from intervertebral discs via the high affinity receptor, TrkA. Recent research has also revealed that the low affinity NGF receptor, p75NTR plays an important role in inflammatory pain. However, the presence of TrkA and p75NTR-immunoreactive (NTR-IR) DRG neurons innervating the rat L5/6 intervertebral disc, and p75NTR-IR nerve fibers in rat intervertebral discs, has not been explored. METHODS: The Fluoro-gold neurotracer was applied to rat L5/6 intervertebral discs to determine the DRG neurons innervating the discs (n = 20). Fourteen days after surgery, bilateral DRG from the L1-L6 levels were harvested, sectioned, and immunostained for TrkA and p75NTR. The percentages of TrkA and p75NTR-IR DRG neurons were counted, and p75NTR-IR nerve fibers in L5/6 discs evaluated. RESULTS: p75NTR-IR nerve fibers were found in superficial layers in the annulus fibrosus in L5/6 intervertebral discs. Fluoro-gold-labeled neurons innervating the L5/6 discs were distributed throughout DRG from the L1-L6 levels. The percentage of TrkA-immunoreactive (TrkA-IR) neurons was 75.1% +/- 3.9% (mean +/- SE) and that of p75NTR-IR neurons was 75.8% +/- 5.1%. These percentages were similar for each level. CONCLUSION: Rat L5/6 intervertebral discs were innervated by multisegmental levels of DRG. Most DRG neurons innervating the discs were positive for 2 types of NGF receptors.

STUDY DESIGN: Changes in behavior and the immunohistochemistry of dorsal root ganglion (DRG) neurons were examined using a mouse model of radicular pain. OBJECTIVE: To examine the effects of TNF-alpha in the nucleus pulposus (NP) on nerve roots. SUMMARY OF BACKGROUND DATA: Radicular pain is induced by mechanical compression and inflammation of nerve roots. Many authors have reported that following disc herniation, producing TNF-alpha plays a major role in neuropathic pain. Their findings suggest that TNF-alpha contained in the NP is significant in the development of pain and nerve root degeneration, but it has not been clearly demonstrated. METHODS: Wild-type NPs or TNF-KO NPs, which were harvested from C57BL/6 mice (wild-type NP) or TNF-knock-out mice (TNF-KO NP), were applied to the left sciatic nerves of 30 wild-type mice, and the nerves were pinched. Production of hind paw mechanical allodynia, activating transcription factor 3, and calcitonin gene- related peptide (CGRP) were assessed. RESULTS: Animals receiving a NP application demonstrated significant mechanical allodynia compared to the pinch-only and the control groups. The degree of mechanical allodynia was greater in the wild-type than in the TNF-KO group. The number of activating transcription factor 3 immunoreactive neurons was significantly higher in the wild-type than in the TNF-KO group. The number of CGRP-immunoreactive neurons was higher in the wild-type and TNF-KO than in the control groups. However, no significant difference in activity was observed between both CGRP positive groups. CONCLUSION: In this study TNF-alpha contained in the NP was important for the production of radicular pain accompanied by long-lasting degeneration of DRG neurons. However, other cytokines in the NP and nerve compression may also play important roles in pain transmission. In this model system, TNF-alpha in the NP appears to mediate pain, but not cause an increase in CGRP in the DRG neurons.

STUDY DESIGN: We used retrograde neurotracing with fluoro-gold to investigate the relationship between tumor necrosis factor (TNF-á) and nerve growth into the nucleus pulposus (NP) of wild-type and TNF-alpha-deficient mice. OBJECTIVE: To clarify mechanisms underlying nerve growth into the NP and the role of TNF-á in this process. SUMMARY OF BACKGROUND DATA: Degeneration of lumbar intervertebral discs is a cause of low back pain. Pathogenesis may involve sensory nerve ingrowth into the inner layers and NP of degenerating discs. We hypothesized that TNF-á in the NP is a major inducer of nerve ingrowth and investigated this hypothesis in vivo using wild-type and TNF-á-deficient mice. METHODS: NP was harvested at the L4/5 level from 10 wild-type and 10 TNF-deficient mice. These 20 samples of wild-type NP or TNF-deficient NP were mixed with fluoro-gold and injected into the left quadriceps muscle of 20 other wild-type mice (1 sample per mouse). Five control mice underwent sham operations in which they received similar injections of NP-free fluoro-gold into their left quadriceps muscles to detect whether neurons innervating the muscle establish contact with injected NP. Seven and 14 days after surgery, left L4 dorsal root ganglions were removed and incubated with antibodies against growth-associated protein 43 (GAP43), a marker of axonal growth. We evaluated fluoro-gold-labeled and GAP43-immunoreactive dorsal root ganglions neurons. RESULTS: Within the set of fluoro-gold-labeled neurons, 10% were positive for GAP43 in sham-operated animals, 22% positive in the TNF-deficient NP group, and 38% positive in the wild-type NP group. These intergroup differences in the percentage of GAP43-positive neurons were statistically significant (sham vs. TNF-deficient NP group: P = 0.009; TNF-deficient NP group vs wild-type NP group: P = 0.026). CONCLUSION.: The percentage of fluoro-gold-labeled GAP43-immunoreactive neurons significantly increased after injections of NP harvested from both mouse types. Furthermore, the percentage of GAP43-immunoreactive neurons was significantly higher in mice receiving wild-type NP compared with mice receiving TNF-deficient NP. These findings suggest that TNF-alpha acts as an inducer of axonal growth into degenerated discs, as evidenced by decreased GAP-43 immunoreactivity in mice receiving TNF-deficient NP injections and even lower GAP-43 immunoreactivity in control mice receiving NP-free fluoro-gold injections.

The rat L5/6 facet joint is multisegmentally innervated from the L1 to L6 dorsal root ganglia (DRG). Tumor necrosis factor (TNF) is a known mediator of inflammation. It has been reported that satellite cells are activated, produce TNF and surround DRG neurons innervating L5/6 facet joints after facet injury. In the current study, changes in TNF receptor (p55) expression in DRG neurons innervating the L5/6 facet joint following facet joint injury were investigated in rats using a retrograde neurotransport method followed by immunohistochemistry. Twenty rats were used for this study. Two crystals of Fluorogold (FG; neurotracer) were applied into the L5/6 facet joint. Seven days after surgery, the dorsal portion of the capsule was cut in the injured group (injured group n = 10). No injury was performed in the non-injured group (n = 10). Fourteen days after the first application of FG, bilateral DRGs from T13 to L6 levels were resected and sectioned. They were subsequently processed for p55 immunohistochemistry. The number of FG labeled neurons and number of FG labeled p55-immunoreactive (IR) neurons were counted. FG labeled DRG neurons innervating the L5/6 facet joint were distributed from ipsilateral L1 to L6 levels. Of FG labeled neurons, the ratio of DRG neurons immunoreactive for p55 in the injured group (50%) was significantly higher than that in the non-injured group (13%). The ratio of p55-IR neurons of FG labeled DRG neurons was significantly higher in total L1 and L2 DRGs than that in total L3, 4, 5 and 6 DRGs in the injured group (L1 and 2 DRG, 67%; L3, 4, 5 and 6 DRG, 37%, percentages of the total number of p55-IR neurons at L1 and L2 level or L3-6 level/the total number of FG-labeled neurons at L1 and L2 level or L3-6 level). These data suggest that up-regulation of p55 in DRG neurons may be involved in the sensory transmission from facet joint injury. Regulation of p55 in DRG neurons innervating the facet joint was different between upper DRG innervated via the paravertebral sympathetic trunks and lower DRG innervated via other direct routes.

STUDY DESIGN: Using a retrograde neurotracing method with Fluoro-Gold (FG), the level at which dorsal root ganglions (DRGs) innervate the L2 and L5 vertebral bodies and the innervation pathways were investigated in rats. OBJECTIVE: To clarify the levels at which DRGs innervate the lumbar vertebral bodies and to determine the pathways from the L2 and L5 vertebral bodies to DRGs. SUMMARY OF BACKGROUND DATA: Elderly patients with osteoporosis sometimes experience lumbar vertebral fracture and may also feel diffuse nonlocalized pain in the back, lateral portion of the trunk, and area surrounding the iliac crest. However, the pattern of sensory innervation of vertebral bodies remains unclear. METHODS: Forty female Sprague-Dawley rats were used. FG crystals were applied to the L2 (L2 vertebra group) or L5 (L5 vertebra group) vertebral bodies via an anterior approach, and numbers of labeled neurons in DRGs from T10 to L6 were counted. To determine sensory pathways, bilateral sympathectomy was performed. RESULTS: In nonsympathectomy animals, FG-labeled neurons were present in DRGs from T11 through L3 in the L2 vertebra group and from T13 through L6 in the L5 vertebra group. The number of labeled neurons following sympathectomy was not significantly different in L1, L2, and L3 DRGs in the L2 vertebra group or in L3, L4, L5, and L6 DRGs in the L5 vertebra group from those in nonsympathectomy animals. In contrast, fewer labeled DRG neurons were present in sympathectomy animals at T11, T12, and T13 in the L2 vertebra group, and at T13, L1, and L2 in the L5 vertebra group than in nonsympathectomy animals (P < 0.01). CONCLUSION: Sensory nerve fibers in the L2 and L5 vertebral bodies are derived from the T11-L3 and T13-L6 DRGs, respectively. Some sensory nerves from the L2 and L5 vertebral bodies enter the paravertebral sympathetic trunks and reach the DRGs at multisegmental levels. The present findings regarding multisegmental innervation to vertebral bodies may explain the diffuse pain that originates within osteoporotic vertebral fractures in elderly patients.

UNLABELLED: Characteristics of sensory dorsal root ganglia (DRG) neurons innervating the L5 vertebral body were investigated in rats by using a retrograde neurotransport method, lectin affinity- and immuno-histochemistry to further elucidate the causes of diffuse pain suffered by some elderly patients in their back, lateral trunk, and iliac crest, after lumbar osteoporotic vertebral fracture. We used calcitonin gene-related peptide (CGRP) as a marker of small peptide-containing neurons and the glycoprotein binding the isolectin from Griffonia simplicifolia (IB4) as a marker of small non-peptide-containing neurons. Neurons innervating the L5 vertebral bodies, retrogradely labeled with fluoro-gold (FG), were distributed throughout DRGs from T13 to L6. The proportion of CGRP-immunoreactive (IR) FG-labeled neurons was 32%. The proportion of IB4-binding FG-labeled neurons was significantly smaller, at 4%. Other neurons that were non-CGRP-IR and non-IB4-binding were mostly large neurons, and they may transmit proprioception from vertebral bodies. Most neurons transmitting pain are CGRP-IR peptide-containing neurons. They may have a more significant role in pain sensation in the vertebral bodies as peptidergic DRG neurons. PERSPECTIVE: This article shows that vertebral bodies are innervated by CGRP-IR neurons. CGRP-IR neurons may play a role in pain sensation through peptidergic DRG neurons. These findings contribute to an understanding of pain associated with the vertebral body such as tumor, infection, or osteoporotic fracture.