Toshinori Ito

Tumor necrosis factor-immunoreactive cells and PGP 9.5-immunoreactive nerve fibers in vertebral endplates of patients with discogenic low back Pain and Modic Type 1 or Type 2 changes on MRI.

Study Design. Immunohistochemistry for tumor necrosis factor (TNF) and protein gene product (PGP) 9.5 in vertebral endplates of patients with discogenic low back pain and Modic Type 1 or Type 2 endplate changes on MRI. Objectives. To examine whether inflammatory cytokines and nerve in-growth into the vertebral endplate are associated with discogenic low back pain. Summary and Background Data. Degenerated discs and endplate abnormalities can be a cause of discogenic low back pain. However, the presence of TNF-immunoreactive cells and PGP 9.5-immunoreactive nerve fibers has not been studied in patients with discogenic low back pain and endplate changes on MRI. Methods. Eighteen endplates showing either normal intensity signals on MRI (endplate change −), Modic Type 1 signals (low intensity on T1-weighted spin-echo images), or Modic Type 2 signals (high intensity) from patients with discogenic low back pain (n = 14) or controls requiring surgery for other back problems (n = 4; scoliosis and traumatic injury of vertebra) were harvested during surgery. Endplates were immunostained using antibodies to TNF and PGP 9.5 and immunostained cells and nerve fibers in the endplates were counted. Results. Vertebral endplates from patients with Modic Type 1 or Type 2 endplate changes on MRI had significantly more PGP 9.5-immunoreactive nerve fibers and TNF-immunoreactive cells in comparison with patients with normal endplates on MRI (P < 0.01). The number of TNF-immunoreactive cells in endplates exhibiting Modic Type 1 changes was significantly higher than in endplates exhibiting Modic Type 2 changes (P < 0.05). Conclusions. The results suggest that endplate abnormalities are related to inflammation and axon growth induced by TNF. TNF expression and PGP 9.5-positive nerve in-growth in abnormal endplates may be a cause of low back pain.

Existence of a Neuropathic Pain Component in Patients with Osteoarthritis of the Knee

Purpose Pain from osteoarthritis (OA) is generally classified as nociceptive (inflammatory). Animal models of knee OA have shown that sensory nerve fibers innervating the knee are significantly damaged with destruction of subchondral bone junction, and induce neuropathic pain (NP). Our objective was to examine NP in the knees of OA patients using painDETECT (an NP questionnaire) and to evaluate the relationship between NP, pain intensity, and stage of OA. Materials and Methods Ninety-two knee OA patients were evaluated in this study. Pain scores using Visual Analogue Scales (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), painDETECT, duration of symptoms, severity of OA using the Kellgren-Lawrence (KL) system, and amount of joint fluid were evaluated and compared using a Spearmans correlation coefficient by rank test. Results Our study identified at least 5.4% of our knee OA patients as likely to have NP and 15.2% as possibly having NP. The painDETECT score was significantly correlated with the VAS and WOMAC pain severity. Compared with the painDETECT score, there was a tendency for positive correlation with the KL grade, and tendency for negative correlation with the existence and amount of joint fluid, but these correlations were not significant. Conclusion PainDETECT scores classified 5.4% of pain from knee OA as NP. NP tended to be seen in patients with less joint fluid and increased KL grade, both of which corresponded to late stages of OA. It is important to consider the existence of NP in the treatment of knee OA pain.

Exposure of the nucleus pulposus to the outside of the anulus fibrosus induces nerve injury and regeneration of the afferent fibers innervating the lumbar intervertebral discs in rats.

Study Design. Using a retrograde tracing method and immunohistochemistry, we assessed the expression of activating transcription factor 3 (ATF3), a marker of nerve injury, and growth-associated protein 43 (GAP-43), a marker of axonal growth, in dorsal root ganglion (DRG) neurons innervating the lumbar intervertebral discs in rats. Objectives. To investigate ATF3 and GAP-43 expression in DRGs innervating the intervertebral discs after exposure of the nucleus pulposus to the outside of the anulus fibrosus. Summary of Background Data. Degeneration of lumbar intervertebral discs is considered as a cause of low back pain. We speculated that exposure of the nucleus pulposus to the outside of the anulus fibrosus may induce nerve injury and ingrowth into the disc. Methods. A neurotracer, Fluoro-Gold (F-G), was applied to the ventral aspect of L5–L6 intervertebral discs in 20 rats. The rats were classified into 2 groups: an NP group whose disc was punctured to expose the nucleus pulposus (n = 10) and a sham-operated group whose anulus fibrosus surface was scratched superficially (n = 10). Ten days after surgery, bilateral L1–L5 DRGs were processed for staining of ATF3 and GAP-43. Results. In the NP group, 13.9% ± 2.9% of the F-G-labeled neurons innervating the discs were positive for ATF3, while 19.3% ± 2.7% were positive for GAP-43. In contrast, in the sham-operated group, only 0.8% ± 0.4% of the F-G-labeled neurons were positive for ATF3 while 7.4% ± 1.7% were positive for GAP-43. The percentage of both ATF3-immunoreactive (IR) and GAP-43-IR neurons in the NP group was significantly higher than in the sham-operated group (P < 0.05). Conclusions. ATF3-IR and GAP-43-IR neurons were significantly increased in the NP group. These results suggested that exposure of the nucleus pulposus to the outside of the anulus fibrosus induced nerve injury and in growth into the discs. These findings may explain discogenic lower back pain in patients with lumbar disc degeneration.

Glial phosphorylated p38 MAP kinase mediates pain in a rat model of lumbar disc herniation and induces motor dysfunction in a rat model of lumbar spinal canal stenosis.

Study Design. Immunohistochemical and behavioral study using rat models of lumbar disc herniation and cauda equina syndrome. Objective. To investigate the expression of activated p38 mitogen-activated protein kinases (p38 MAP kinase; p38) in the spinal cord and to determine the effect of intrathecal administration of a specific p38 inhibitor on pain in a lumbar disc herniation model and on motor function and hypoalgesia in a spinal canal stenosis (SCS) model. Summary of Background Data. In pathologic lumbar disc herniation-induced neuropathic pain and compression of cauda equina-induced motor dysfunction and hypoalgesia caused by SCS, glia are activated and produce certain cytokines, including tumor necrosis factor-alpha (TNF-&agr;) and interleukins, which play a crucial role in the pathogenesis of nerve degeneration. p38 is phosphorylated by these cytokines, suggesting that it may play an important role in pain transmission and nerve degeneration. Here we have examined the role of p38 in rat models of lumbar disc herniation and SCS. Methods. Six-week-old male Sprague-Dawley rats were used. For the disc herniation model, autologous nucleus pulposus was applied to L5 nerve roots, which were then crushed. For the SCS model, a piece of silicon was placed under the lamina of the fourth lumbar vertebra. We assessed mechanical allodynia, hypoalgesia, and motor function using von Frey hairs, treadmill tests, and immunohistochemical localization of phosphorylated p38 (P-p38) in the cauda equina, dorsal root ganglion (DRG), and spinal cord, which were also double-stained with NeuN (neuronal marker), GFAP (astrocyte/Schwann cell marker), or isolectin B4 (IB4; microglia marker). We also examined the effects of intrathecal administration of a specific p38 inhibitor, FR167653, on nucleus pulposus-induced pain, hypoalgesia, and motor dysfunction following SCS. Results. We demonstrated that activated P-p38-immunoreactive cells in the spinal cord and cauda equina were not observed before nerve injury but appeared in the cauda equina, DRG, and spinal dorsal horn in the disc herniation and SCS models. Double-labeling revealed that most P-p38-immunoreactive cells were isolectin B4-labeled microglia and GFAP-immunoreactive Schwann cells. Intrathecal administration of the p38 inhibitor FR167653 decreased mechanical allodynia in the disc herniation model and improved hypoalgesia and intermittent motor dysfunction in the SCS model. Conclusions. Our findings suggest that activated p38 may play an important role in the involvement of microglia in the pathophysiology of pain following lumbar disc herniation and mechanical hypoalgesia, and motor nerve dysfunction of cauda equina following SCS.

Up-regulation of acid-sensing ion channel 3 in dorsal root ganglion neurons following application of nucleus pulposus on nerve root in rats.

Study Design. Immunocytochemistry for acid-sensing ion channel 3 (ASIC3) in neurons of rat dorsal root ganglions (DRGs) from animals exposed to a model of lumbar disc herniation. Objective. To examine expression of ASIC3 in DRGs and the effect of a sodium channel blocker applied to the nerve root in a rat model of lumbar disc herniation. Summary of Background Data. Radicular pain is a common symptom of lumbar disc herniation in human beings. A depolarizing sodium channel gated by protons during tissue acidosis, ASIC3, is specifically expressed in sensory neurons. It has been associated with cardiac ischemic and inflammatory pain. We often perform spinal nerve root block for radicular pain using a sodium channel blocker, such as lidocaine; however, it has been unclear whether the effective period of this treatment is usually longer than the expected duration of efficacy. Methods. For the lumbar disc herniation model, nucleus pulposus was harvested from the tail and applied to the L5 nerve root, and the nerve roots were pinched. We evaluated mechanical allodynia in sham-operated animals and a disc herniation model. Immunohistochemistry was used to examine ASIC3 expression in L5 DRGs. Finally, the effect of lidocaine on pain and ASIC3 expression in the disc herniation model was examined. Results. Animals exposed to the lumbar disc herniation model showed allodynia for 8 days, and ASIC3 immunoreactivity was up-regulated in DRG neurons. After administration of lidocaine to spinal nerve roots affected by disc herniation, ASIC3 immunoreactivity was down-regulated in DRG neurons, and the level of mechanical allodynia was significantly decreased for 8 days. Conclusions. Our results suggest that ASIC3 in DRG neurons may play an important role in nerve root pain caused by lumbar disc herniation. Lidocaine decreased ASIC3 expression in DRG neurons and pain associated with the disc herniation model.

TNF-alpha in nucleus pulposus induces sensory nerve growth: a study of the mechanism of discogenic low back pain using TNF-alpha-deficient mice.

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-&agr;-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-&agr; 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.

Nerve growth factor of cultured medium extracted from human degenerative nucleus pulposus promotes sensory nerve growth and induces substance p in vitro.

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)&bgr; 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-NGF&bgr; 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.

Evaluation of low back pain using the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire for lumbar spinal disease in a multicenter study: differences in scores based on age, sex, and type of disease

BackgroundThe Japanese Orthopaedic Association (JOA) has investigated the JOA Back Pain Evaluation Questionnaire (JOABPEQ) to evaluate several aspects of low back pain in patients. The score includes five categories (25 items) selected from the Roland Morris Disability Questionnaire and Short Form 36, and a visual analogue scale. Japanese physicians have recently used these scores to evaluate back pain; however, the efficacy has not been fully explored in large-scale studies. In the current study, we used the JOABPEQ to evaluate lumbar spinal disease in 555 patients (with lumbar disc herniation, lumbar spinal stenosis, and lumbar disc degeneration/spondylosis) in multiple spine centers and compared the results based on age, sex, and type of disease.MethodsA total of 555 patients who had low back or leg pain were selected in 22 hospitals in Chiba Prefecture. Spine surgeons diagnosed their disease type based on symptoms, physical examination, radiography images, and magnetic resonance imaging. In all, 486 patients were diagnosed with spinal stenosis (239 patients), disc degeneration/spondylosis (143 patients), or disc herniation (104 patients). The other 69 patients were diagnosed with spondylolysis (16 patients) or other diseases (53 patients). The pain score in all patients was evaluated using the JOABPEQ (from 0 to 100, with 0 indicating the worst pain).ResultsThe age of the patients was 56.1 ± 13.3 years (mean ± SD); the age of patients in the disc herniation and disc degeneration/spondylosis group was significantly lower than that in the spinal stenosis group. The average JOABPEQ scores in all patients were, for low back pain, 47.1; lumbar function, 53.6; walking ability, 54.8; social life function, 48.7; and mental health, 48.3. The low back pain score in men was significantly worse than that in women. In contrast, the mental health score in women was significantly higher than that in men. The low back pain score in patients <40 years old and the walking ability score in patients >65 years old were significantly lower than those scores in other patients. Based on the disease type, low back pain, lumbar function, social life function, and mental health scores for patients with disc herniation were significantly worse than for those with spinal stenosis.ConclusionJOABPEQ scores were evaluated for several lumbar diseases. The average of five categories of JOABPEQ scores in all patients was similarly distributed. However, the average scores in the five categories were significantly different depending on age, sex, and type of disease. Compared with prior mass data (baseline data on the observational cohort of the Spine Patient Outcomes Research Trial in the United States), many data were similar based on the type of disease in the current study. Furthermore, the JOABPEQ is easy to use compared with the SF-36. Hence, we concluded that the JOABPEQ could be used worldwide as a tool for evaluating low back pain.

The p75 receptor is associated with inflammatory thermal hypersensitivity.

Inflammatory pain, characterized by a decrease in the nociceptive threshold, arises through the actions of inflammatory mediators, and one of the key molecules is nerve growth factor (NGF). Here we report that the administration of neutralizing antibody to the neurotrophin receptor p75 (p75NTR) blocks hyperalgesia, which develops with complete Freunds adjuvant (CFA)‐induced inflammation or with an intraplantar injection of NGF. Although CFA injection results in the up‐regulation of calcitonin gene‐related peptide (CGRP) levels in the primary sensory neurons, blocking p75NTR abolishes this effect. We further demonstrate that pro‐NGF is the predominant ligand of p75NTR in vivo. Plasmin treatment, which is intended to decompose pro‐NGF, ameliorates CFA‐induced hyperalgesia. In addition, an intraplantar injection of pro‐NGF induces hyperalgesia. These data together suggest that pro‐NGF, as well as mature NGF, binding to p75NTR plays an important role in inflammation‐induced hyperalgesia. Interference in the binding may provide a therapeutic approach for the treatment of inflammatory pain.

Sensory innervation of lumbar vertebral bodies in rats.

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.