Seiji Ohtori

The degenerated lumbar intervertebral disc is innervated primarily by peptide-containing sensory nerve fibers in humans

Study Design. Immunohistochemical study of the sensory innervation of the human lumbar intervertebral disc. Objective. To determine the type of sensory fibers innervating human degenerated lumbar intervertebral discs. Summary of Background Data. Sensory neurons involved in pain perception related to inflammation in rats are typically small, peptide-containing neurons immunoreactive for calcitonin gene-related peptide (CGRP). Small non-peptide-containing neurons binding to isolectin B4 (IB4) may also be involved in pain states, such as nerve injury pain. The character of such sensory neurons in humans has not been clarified. Methods. A degenerated, painful lumbar intervertebral disc was harvested from each of 8 patients during surgery. Sections were immunostained for protein gene product 9.5 (PGP 9.5, a general neuronal marker), CGRP, and IB4. The numbers of PGP 9.5- and CGRP-immunoreactive, and IB4-binding nerve fibers in the discs were counted. Results. PGP 9.5-immunoreactive fibers were observed in all discs. Nerve fibers immunoreactive for CGRP were also observed in 6 of 8 cases. IB4-binding nerve fibers were not found in any case. Conclusions. Almost all of the nociceptive nerve fibers in the human intervertebral disc are peptide-containing nerve fibers, similar to the rat disc, suggesting that nerve fibers related to inflammation may transmit pain originating from human degenerated intervertebral discs.

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.

Expression and co-expression of VR1, CGRP, and IB4-binding glycoprotein in dorsal root ganglion neurons in rats : Differences between the disc afferents and the cutaneous afferents

Study Design. The expression of vanilloid receptor 1 (VR1), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4)-binding glycoprotein in dorsal root ganglion (DRG) neurons innervating the lumbar disc and the plantar skin was investigated. Objective To characterize the DRG neurons innervating lumbar discs and those innervating cutaneous tissue in rats. Summary and Background Data. Small nociceptive DRG neurons are divided into nerve growth factor (NGF) sensitive and glial cell line-derived neurotrophic factor (GDNF)-sensitive neurons. CGRP and IB4-binding glycoprotein are recognized as specific markers for NGF and GDNF-sensitive neurons, respectively. VR1 is localized in small DRG neurons. Methods. Using histochemical staining and retrograde tracing methods, the expression of VR1, CGRP, and IB4-binding glycoprotein in DRG neurons innervating the L5–L6 disc and the plantar skin was examined in rats. Results. DRG neurons innervating the disc showed positive staining as: 23.4% VR1, 54.4% CGRP, and 1.0% IB4-binding glycoprotein. The following distribution was found for DRG neurons innervating the skin: 35.1% VR1, 41.1% CGRP, and 19.5% IB4-binding glycoprotein. Percentages of neurons positive for VR1 and IB4-binding glycoprotein were significantly lower in DRG neurons innervating the disc than in DRG neurons innervating the skin (P < 0.05), while no significant difference was observed in the percentage of neurons positive for CGRP. Conclusions. VR1 is less abundant in lumbar disc than in cutaneous tissue. Our data suggest that nociceptive information from the disc is transmitted mostly by NGF-sensitive neurons, while that from the cutaneous tissue is transmitted by both NGF-sensitive and GDNF-sensitive neurons.

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.

Characteristics of sensory DRG neurons innervating the lumbar facet joints in rats.

The rat L5/6 facet joint, from which low-back pain can originate, is multisegmentally innervated from the L1 to L5 dorsal root ganglions (DRGs). Sensory fibers from the L1 and L2 DRGs are reported to non-segmentally innervate the paravertebral sympathetic trunks, whilst those from the L3 to L5 DRGs segmentally innervate the L5/6 facet joint. In the current study, characteristics of sensory DRG neurons innervating the L5/6 facet joint were investigated in rats, using a retrograde neurotransport method, lectin affinity- and immuno-histochemistry. We used four markers: (1) calcitonin gene-related peptide (CGRP) as a marker of small peptide containing neurons, (2) the glycoprotein binding the isolectin from Griffonia simplicifolia (IB4) or (3 the purinergic P2X3 receptor for small, non-peptide containing neurons, and (4) neurofilament 200 (NF200) for small and large myelinated fibers. IB4-binding and CGRP and P2X3 receptor containing neurons are typically involved in pain sensation, whereas NF200 is associated with pain and proprioception. Neurons innervating the L5/6 facet joints, retrogradely-labeled with fluoro-gold (FG), were distributed throughout DRGs from L1 to L5. Of FG-labeled neurons, the ratios of NF200 immunoreactive (IR) neurons and CGRP-IR neurons were 37% and 35% respectively. The ratio of IB4-binding and P2X3 receptor-IR neurons was 10%, significantly less than the ratio of CGRP-IR neurons to FG-labeled neurons. The ratios of IB4-binding and P2X3 receptor-IR neurons were significantly higher, and that of CGRP-IR neurons was significantly less in L1 and L2 DRGs than those in L3, L4 or L5 DRGs. Under physiological conditions in rats, DRG neurons transmit several types of sensations, such as proprioception or nociception of the facet joint. Most neurons transmitting pain are CGRP-IR peptide-containing neurons. They may have a more significant role in pain sensation in the facets via peptidergic DRG neurons.

Change of dorsal horn neurochemistry in a mouse model of neuropathic cancer pain

&NA; We investigated some neurochemical changes that take place in the spinal cord dorsal horn in a mouse model of neuropathic cancer pain. The model was produced by inoculation of Meth‐A sarcoma cells to the vicinity of the sciatic nerve, which resulted in growth of a tumor mass embedding the nerve. Hind paw‐lifting, a behavioral sign of spontaneous pain, was at maximum on Day 18, but decreased thereafter. The decrease was likely caused by progression of motor paralysis. On Day 18, thermal and mechanical pain thresholds of the affected paw were significantly increased. Histologically, the sciatic nerve presented damages to both unmyelinated and myelinated fibers on Day 18, which were more pronounced on Day 25. In the spinal cord, c‐Fos‐positive cells were significantly increased in the superficial and deep layers on Day 18. The number of c‐Fos‐positive cells in the superficial layer correlated with the duration of paw‐lifting. The increase in c‐Fos‐positive cells was still present on Day 25 despite decreased paw‐lifting. Substance P and calcitonin gene‐related peptide were up‐regulated on Day 18 but down‐regulated on Day 25. A marked up‐regulation of dynorphin A (DynA) was present on Day 18 and persisted through Day 25. Our model caused progressive damage to the sciatic nerve and presented spontaneous pain‐behavior while the paw became hyposensitive to mechanical and thermal stimuli. Since the up‐regulation of DynA in the dorsal horn persisted and paralleled the increase in c‐Fos‐positive cells, the release of DynA may be associated with spontaneous pain in our model.

Extracorporeal shockwaves induce the expression of ATF3 and GAP-43 in rat dorsal root ganglion neurons.

Although extracorporeal shockwave has been applied in the treatment of various diseases, the biological basis for its analgesic effect remains unclear. Therefore, we investigated the dorsal root ganglion neurons of rats following shockwave exposure to the footpad to elucidate its effect on the peripheral nervous system. We used activating transcription factor 3 (ATF3) and growth-associated phosphoprotein (GAP-43) as markers for nerve injury and axonal regeneration, respectively. The average number of neurons immunoreactive for ATF3 increased significantly in the treated rats at all experimental time points, with 78.3% of those neurons also exhibiting immunoreactivity for GAP-43. Shockwave exposure induced injury of the sensory nerve fibers within the exposed area. This phenomenon may be linked to the desensitization of the exposure area, not the cause of pain, considering clinical research with a particular absence of painful adverse effect. Subsequent active axonal regeneration may account for the reinnervation of exposed area and the amelioration of the desensitization.

Extracellular signal-regulated kinase mitogen-activated protein kinase activation in the dorsal root ganglion (DRG) and spinal cord after DRG injury in rats.

Study Design. We investigated the extracellular signal-regulated kinase (ERK) activation by immunohistochemically detecting phosphorylated ERK (pERK) in the dorsal root ganglion (DRG) and spinal cord. Objective. To clarify the ERK activation in the rat nervous system following DRG injury. Summary of Background Data. Radicular pain is known to be associated with DRG injury caused by intervertebral disc herniation. ERK is activated by phosphorylation in the DRG and spinal cord by noxious stimuli, which are related to pain hypersensitivity. Methods. From 2 minutes to 24 hours after the left L4 DRG crush injury, L4 DRGs and spinal cords were resected to prepare serial sections, which were investigated immunohistochemically. Results. In the DRG, ERK activation was detected in neurons and satellite cells at 2 minutes; the former was maintained at increased levels for 20 minutes, and the latter for 4 hours. At 30 minutes, pERK immunoreactivity was observed in Schwann cells, which continued for up to 24 hours. In the spinal cord, pERK-positive neurons were detected at 2 minutes, and the pERK levels were maintained at increased levels for 20 minutes. Conclusions. Profiles of pERK induction in neurons after DRG injury were similar between the DRG and spinal cord, whereas pERK induction in the satellite cells was more long lasting. The pERK induction in Schwann cells in the DRG was late onset and the most long lasting.

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.

Injection of nuclear factor-kappa B decoy into the sciatic nerve suppresses mechanical allodynia and thermal hyperalgesia in a rat inflammatory pain model.

Study Design. In vitro and in vivo study of a rat inflammatory pain model using nuclear factor-kappa B decoy. Objectives. To investigate transduction efficiency of nuclear factor-kappa B decoy into dorsal root ganglion, both in vivo and in vitro, and to assess the suppression of inflammatory pain by nuclear factor-kappa B decoy. Summary of Background Data. Transcription factor nuclear factor-kappa B is reported to play a crucial role in regulating pro-inflammatory cytokine gene expression. We hypothesized that inhibiting nuclear factor-kappa B gene expression with nuclear factor-kappa B decoy may suppress inflammatory pain. Methods. Nuclear factor-kappa B decoy-fluorescein isothiocyanate (FITC) was induced in explant culture, endoneurally injected into the sciatic nerve, and its transduction efficiency into dorsal root ganglion measured. For behavioral testing, 12 rats received plantar injections of complete Freunds adjuvant and were divided into 3 groups: decoy group, single endoneural injection of 10 &mgr;L of nuclear factor-kappa B decoy (n = 4); saline group, single endoneural injection of 10 &mgr;L of saline (n = 4); and naïve group, untreated (n = 4). Behavioral testing was performed using von Frey filaments and a Hargreaves device with a heat source. Results. Total transduction efficiency of nuclear factor-kappa B decoy-FITC was 53.6% in vitro and 20.5% in vivo. No statistical differences were observed with respect to types of cell size distributions of all FITC-positive neurons. In behavioral testing, withdrawal latencies or thresholds significantly differed between the decoy group and the saline group from 2 to 14 days after surgery in the mechanical allodynia experiments, and from 2 to 3 days after surgery in the thermal hyperalgesia experiments. Conclusions. Nuclear factor-kappa B decoy was conveyed and transduced into dorsal root ganglion both in vivo and in vitro. Additionally, nuclear factor-kappa B decoy reduced mechanical allodynia and thermal hyperalgesia in the rat inflammatory pain model, suggesting that inhibition of nuclear factor-kappa B with nuclear factor-kappa B decoy may represent a key mechanism for mediating inflammation or reducing inflammatory pain.