Hideo Doya

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA–Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.

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.

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.

Rho‐kinase inhibition enhances axonal regeneration after peripheral nerve injury

Abstract  In injured adult neurons, the process of axonal regrowth and reestablishment of the neuronal function have to be activated. We assessed in this study whether RhoA, a key regulator of neurite elongation, is activated after injury to the peripheral nervous system. RhoA is activated in motoneurons but not in Schwann cells after mouse sciatic nerve injury. To examine whether the activation of RhoA and its effector, Rho‐kinase, retards axon regeneration of injured motoneurons, we employed a Rho‐kinase inhibitor, fasudil. Amplitudes of distally evoked compound muscle action potentials are increased significantly faster after axonal injury in mice treated with fasudil compared with controls. Histological analysis shows that fasudil treatment increases the number of regenerating axons with large diameter, suggesting that axon maturation is facilitated by Rho‐kinase inhibition. In addition, fasudil does not suppress the myelination of regenerating axons. These findings suggest that RhoA/Rho‐kinase may be a practical molecular target to enhance axonal regeneration in human peripheral neuropathies.

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.

Up-regulation of TNFα in DRG satellite cells following lumbar facet joint injury 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 ganglia (DRG). Sensory fibers from the L1 and L2 DRG are reported to non-segmentally innervate the paravertebral sympathetic trunks, while those from the L3 to L5 DRGs segmentally innervate the L5/6 facet joint. Tumor necrosis factor alpha (TNFα) is a mediator of peripheral and central nervous system inflammatory response and plays a crucial role in injury and its pathophysiology. In the current study, change in TNFα in sensory DRG neurons innervating the L5/6 facet joint following facet joint injury was investigated in rats using a retrograde neurotransport method and immunohistochemistry. Neurons innervating the L5/6 facet joints, retrogradely labeled with fluoro-gold (FG), were distributed throughout DRGs from L1 to L5. Most DRG FG-labeled neurons innervating L5/6 facet joints were immunoreactive (IR) for TNFα before and after injury. In the DRG, glial fibrillary acidic protein (GFAP)-IR satellite cells emerged and surrounded neurons innervating L5/6 facet joints after injury. These satellite cells were also immunoreactive for TNFα. The numbers of activated satellite cells and TNFα-IR satellite cells were significantly higher in L1 and L2 DRG than in L3, L4, and L5 DRG. These data suggest that up-regulation of glial TNFα may be involved in the pathogenesis of facet joint 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.

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.