Toshiya Tachibana

Differential Activation of Extracellular Signal-Regulated Protein Kinase in Primary Afferent Neurons Regulates Brain-Derived Neurotrophic Factor Expression after Peripheral Inflammation and Nerve Injury

To investigate the intracellular signal transduction pathways involved in regulating the gene expression of brain-derived neurotrophic factor (BDNF) in primary afferent neurons, we examined the activation of extracellular signal-regulated protein kinase (ERK) in dorsal root ganglion (DRG) neurons after peripheral inflammation and sciatic nerve transection. Peripheral inflammation induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase A-containing small-to-medium-diameter DRG neurons. The treatment of the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126 reversed the pain hypersensitivity and the increase in phosphorylated-ERK (p-ERK) and BDNF in DRG neurons induced by complete Freunds adjuvant. On the other hand, axotomy induced the activation of ERK mainly in medium-and large-sized DRG neurons and in satellite glial cells. U0126 suppressed the axotomy-induced autotomy behavior and reversed the increase in p-ERK and BDNF. The intrathecal application of nerve growth factor (NGF) induced an increase in the number of p-ERK-and BDNF-labeled cells, mainly small neurons, and the application of anti-NGF induced an increase in p-ERK and BDNF in some medium-to-large-diameter DRG neurons. The activation of MAPK in the primary afferents may occur in different populations of DRG neurons after peripheral inflammation and axotomy, respectively, through alterations in the target-derived NGF. These changes, including the changes in BDNF expression, might be involved in the pathophysiological changes in primary afferent neurons.

VR1, but not P2X3, increases in the spared L4 DRG in rats with L5 spinal nerve ligation

&NA; We investigated the expression of two candidate transducers of noxious stimuli in peripheral tissues, the vanilloid receptor subtype 1 (VR1) and the P2X3, a subunit of the ionotropic P2X receptor for ATP, in spared L4 DRG neurons following L5 spinal nerve ligation, a neuropathic pain model. VR1 mRNA expression increased in the small‐ and medium‐sized DRG neurons from the first to 28th day after injury, and this up‐regulation corresponded well with the development and maintenance of thermal hyperalgesia of the hind paw. The increase in VR1‐immunoreactive (ir) neurons was confirmed at the third day after surgery. In contrast, there was no change in expression of P2X3 mRNA over 4 weeks after ligation, or in the percentage of P2X3‐ir neurons observed 3 days after surgery. Our data suggests that increased VR1 in the spared L4 DRG may contribute to the exaggerated heat response observed in this neuropathic pain model. Taken together with the previous reports that P2X3 expression increases in the spared DRG neurons in other neuropathic pain models, there appears to be differences in the phenotypic changes and pathomechanisms of the various neuropathic pain models.

Change in mRNAs for neuropeptides and the GABAA receptor in dorsal root ganglion neurons in a rat experimental neuropathic pain model

&NA; We examined two possible mechanisms of neuropathic pain: contribution of adjacent intact nerves and decrease in presynaptic inhibition at the central terminal of the injured primary afferent. To this end, we examined the effects of unilateral L5 spinal nerve ligation, which causes mechanical allodynia and heat hyperalgesia in the ipsilateral hind paw, on gene expression in L4 and L5 dorsal root ganglion (DRG) neurons using in situ hybridization (ISH). Specifically, we examined changes in the expression of messenger RNAs (mRNAs) for neuropeptides which have been reported to be up‐ or down‐regulated in the axotomized DRG neurons and for &ggr;‐aminobutyric acid (GABA)A receptor (GABAA‐R) subunits which contribute to presynaptic inhibition at the primary afferent terminals. Seven days following ligation, ISH demonstrated an increase in signal intensity for calcitonin gene‐related peptide (CGRP) mRNA in the subpopulation of small‐ to medium‐sized L4 DRG neurons ipsilateral to the ligation which were not directly injured as compared to the contralateral side, although the overall percentages and the size distribution of positively labelled neurons for CGRP mRNA were not different between the bilateral L4 DRGs. This suggests that the L4 DRG neurons which express CGRP mRNA constitutively up‐regulated the gene expression and the functional importance of these neurons has increased following L5 spinal nerve ligation. However, the mRNAs for other neuropeptides such as preprotachykinin (PPT), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and galanin (GAL), were not different between the bilateral L4 DRGs. The mRNA for the GABAA‐R&ggr;2 subunit was significantly down‐regulated in the medium‐ to large‐sized L5 DRG neurons ipsilateral to the ligation as compared to the contralateral side. GABAA‐R&agr;2 subunit mRNA also decreased in the ipsilateral L5 DRG neurons but did not reach statistical significance. There was no difference in mRNAs between the bilateral L4 DRGs. These data suggest that the presynaptic disinhibition of the ipsilateral L5 primary afferent terminals may be explained at least partly by the down‐regulation of GABAA‐R following L5 spinal nerve ligation. Thus, both the up‐regulation of CGRP in adjacent intact nerves and the decrease in presynaptic inhibition at the central terminal of the injured primary afferent could cause the hyper‐excitability of dorsal horn neurons and contribute to the molecular mechanisms of this neuropathic pain model.

The role of ERK signaling and the P2X receptor on mechanical pain evoked by movement of inflamed knee joint

Abstract Pain during inflammatory joint diseases is enhanced by the generation of hypersensitivity in nociceptive neurons in the peripheral nervous system. To explore the signaling mechanisms of mechanical hypersensitivity during joint inflammation, experimental arthritis was induced by injection of complete Freund’s adjuvant (CFA) into the synovial cavity of rat knee joints. As a pain index, the struggle threshold of the knee extension angle was measured. In rats with arthritis, the phosphorylation of extracellular signal‐regulated kinase (ERK), induced by passive joint movement, increased significantly in dorsal root ganglion (DRG) neurons innervating the knee joint compared to the naïve rats that received the same movement. The intrathecal injection of a MEK inhibitor, U0126, reduced the phosphorylation of ERK in DRG neurons and alleviated the struggle behavior elicited by the passive movement of the joint. In addition, the injection of U0126 into the joint also reduced the struggle behavior. These findings indicate that the ERK signaling is activated in both cell bodies in DRG neurons and peripheral nerve fibers and may be involved in the mechanical sensitivity of the inflamed joint. Furthermore, the phosphorylated ERK‐positive neurons co‐expressed the P2X3 receptor, and the injection of TNP‐ATP, which antagonizes P2X receptors, into the inflamed joint reduced the phosphorylated ERK and the struggle behavior. Thus, it is suggested that the activation of the P2X3 receptor is involved in the phosphorylation of ERK in DRG neurons and the mechanical hypersensitivity of the inflamed knee joint.

Responses of dorsal column nuclei neurons in rats with experimental mononeuropathy

&NA; To examine the functional role of dorsal column nuclei in neuropathic pain, electrophysiological properties of low‐ and high‐threshold dorsal column nuclei neurons in neuropathic and normal rats were examined. Single‐neuronal activities were recorded from the gracile nucleus (GN) in rats at 10–14 days after application of four loose ligatures around the sciatic nerve (chronic constriction nerve injury; CCI). A total of 190 units were recorded from the GN in naive and CCI rats. The largest population of low‐threshold mechanoreceptive (LTM) neurons recorded from the GN of naive rats were classified as rapidly‐adapting (RA) LTM neurons, whereas those from CCI rats were slowly‐adapting (SA) neurons. Mean orthodromic latencies of GN neurons ipsilateral to the CCI after sciatic nerve stimulation were significantly longer than those of naive animals and those of GN neurons without receptive fields were significantly longer than any other type of neurons. One hundred and eight of 190 GN neurons were also antidromically activated following electrical stimulation of the ventro‐lateral posterior nucleus of the thalamus. Furthermore, when stronger stimuli were applied to the sciatic nerve, some GN neurons also responded with long latencies. GN neurons of sham‐operated naive rats and those contralateral to the CCI had mechanical receptive fields on the paw, whereas 8.0% of the GN neurons in the CCI side did not have any detectable mechanical receptive field. Receptive field size was not significantly different between neurons ipsilateral or contralateral to the CCI and those of naive rats. Spontaneous activity of GN neurons from the ipsilateral side was significantly higher than those from the contralateral side. On the other hand, spontaneous activity of GN neurons both ipsilateral and contralateral to the CCI were significantly higher than those from naive rats. Furthermore, afterdischarges of GN neurons ipsilateral and contralateral to the CCI were significantly higher than those of naive rats. The present data suggest that the dorsal column pathway is involved in CCI‐produced sensory abnormalities by conveying their hyperactivity to thalamic neurons.

C-5 palsy after cervical laminoplasty with instrumented posterior fusion

OBJECT Postoperative C-5 palsy is known as a common complication after cervical laminoplasty. The authors of this article have encountered postoperative C-5 palsy more often when laminoplasty was combined with instrumented posterior spinal fusion than when it was performed alone. The purpose of this clinical study was to examine the incidence of fifth cervical nerve root palsy (C-5 palsy) and surgical results in patients with cervical myelopathy who had undergone laminoplasty with or without instrumented spinal fusion. METHODS The authors retrospectively studied patients with cervical myelopathy who had undergone laminoplasty with or without instrumented posterior spinal fusion. RESULTS Clinical data on 58 patients were evaluated and analyzed. Preoperative diagnoses were cervical spondylotic myelopathy or ossification of the posterior longitudinal ligament of the cervical spine. Twenty-four patients with spondylolisthesis or kyphosis underwent laminoplasty combined with posterior spinal fusion using instrumented lateral mass fixation (fusion group), while the remaining 34 patients underwent laminoplasty without posterior spinal fusion (no-fusion group). In the fusion group, C-5 palsy developed in 6 patients; in the no-fusion group, it occurred in only 1 patient. There was a significant difference in the rate of this complication between the 2 groups. In the fusion group, local kyphosis and spondylolisthesis level were reduced at the fusion level, and all patients with C-5 palsy underwent C4-5 spinal fusion. CONCLUSIONS The incidence of postoperative C-5 palsy is significantly higher after laminoplasty when it is combined with spinal fusion. Correction of kyphosis and spondylolisthesis using posterior instrumentation may be a risk factor for iatrogenic intervertebral foraminal stenosis leading to C-5 palsy.

Changes in phosphorylation of ERK and Fos expression in dorsal horn neurons following noxious stimulation in a rat model of neuritis of the nerve root

Mechanical compression and chemical inflammation of the spinal nerve root are considered major sensory pathologies secondary to a lumbar disc herniation. In order to elucidate the dorsal horn responsiveness to noxious stimulation to the peripheral tissue in the neuritis model of the nerve root, we examined extracellular signal-regulated kinase (ERK) phosphorylation and Fos expression in spinal cord dorsal horn neurons. Male Sprague-Dawley rats received hemilaminectomies and the implantation of disc tissue that was obtained from coccygeal intervertebral discs. Three or 7 days after surgery, rats were perfused after receiving noxious mechanical stimulation of the plantar surface of the hind paw using a hemoclip, and the L4/5 spinal cord was processed for immunohistochemistry with antibodies for phospho-ERK and Fos. The number of Fos-immunoreactive (Fos-LI) neurons and phospho-ERK-immunoreactive (phospho-ERK-LI) neurons in the neuritis group after the noxious stimulation significantly increased compared to the sham-treated group at 3 and 7 days after surgery. The change in number of phospho-ERK-LI and Fos-LI neurons occurred mainly in the superficial dorsal horn. The number of Fos-LI neurons observed when the MEK inhibitor, U0126, was administered was significantly suppressed compared to the DMSO- (vehicle control) administered group. The increase in ERK phosphorylation and Fos expression in the spinal cord dorsal horn neurons indicates that responses/activation by the noxious stimulation applied to the periphery were elevated in spinal cord neurons in this neuritis model of the lumbar nerve root. Moreover, the increase in the Fos expression in the spinal cord dorsal horn may have been the result of the activation of the MAP kinase cascade.

Heme oxygenase-1 expression after spinal cord injury: the induction in activated neutrophils.

Tissue damage and neurological dysfunction after spinal cord injury may result, in part, from delayed or secondary mechanisms that appear to involve several endogenous factors. Among them, neutrophils are known to play important roles in the pathomechanisms of the secondary injury, that is, neutrophils are activated by an interaction with the endothelial cells, migrate into the damaged tissue and release several kinds of proteases or oxygen radicals. In the present study, we examined heme oxygenase-1 expression in the damaged spinal cord. The administration of an inhibitor of heme oxygenase-1 in vivo produced a delayed recovery of motor function after spinal cord injury, suggesting that heme oxygenase-1 may play roles as an endogenous anti-inflammatory enzyme and protective gene in the damaged and inflammatory tissue. We found that many neutrophils expressing heme oxygenase-1 mRNA and protein were recruited into the damaged spinal cord with extensive hemorrhages during early stage of spinal cord injury. In an in vitro study, neutrophils incubated with proinflammatory cytokines, such as interleukin-1, 6 or interferon-gamma, expressed heme oxygenase-1 mRNA and protein. Based on these findings we conclude that the activated neutrophils can express heme oxygenase-1 in the injured spinal cord tissue, perhaps expecting modulatory and neuroprotective actions in the inflammatory response to spinal cord injury.

Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy.

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Suppression of neuropeptides' mRNA expression by herbal medicines in a rat model of peripheral inflammation

The traditional Chinese medicines have been used clinically for a long time in some Asian countries, however, very few studies have been done to demonstrate the working mechanisms of these medicines using recently developed biochemical methodologies. In this study, we examined the anti-inflammatory effect of Huang-Lian-Jie-Du-Tang (HLJDT), a combination of herbs used in traditional Chinese medicine, on paw edema, thermal hyperalgesia and the mRNA increase of neuropeptides in spinal dorsal horn and hypothalamic neurons using a rat model of peripheral inflammation and hyperalgesia. The rats that received HLJDT from 3 days before the injection of complete Freunds adjuvant (CFA) into the plantar had significantly less edema and reduced thermal hyperalgesia compared to control rats that received CFA injection. The up-regulation of preprodynorphin mRNA in L4-5 dorsal horn neurons 8 hours after CFA injection that was observed in control rats, was also decreased in the HLJDT-treated rats. Moreover, there was a significant decrease in mRNA level of corticotropin-releasing factor in the paraventricular hypothalamic nucleus in the HLJDT-treated rats. These data demonstrate that HLJDT is anti-inflammatory, and produces changes in mRNA expression in dorsal horn and hypothalamic neurons. This is the first demonstrated that a traditional Chinese medicine can affect the excitability of neurons through an anti-inflammatory action.