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.

Expression of neurotrophic factors in the dorsal root ganglion in a rat model of lumbar disc herniation.

A variety of molecules released by inflammatory reactions in the dorsal root and dorsal root ganglion (DRG) may play important roles in the pathology of neuronal abnormalities in lumbar disc herniation. In order to elucidate the pathophysiological mechanisms of painful radiculopathy, secondary to lumbar disc herniation, we evaluated pain-related behavior and the change of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in the DRG and dorsal root using a rat model of lumbar disc herniation. In the nucleus pulposus (NP) group, the left L4/5 nerve roots were exposed after hemilaminectomies and autologous intervertebral discs, which were obtained from coccygeal intervertebral discs, were implanted on each of the exposed nerve roots without mechanical compression. Rats in the NP group, but not the sham-operated rats, developed mechanical allodynia on the ipsilateral hind paw for 1 day after surgery and showed a significant increase in the number of NGF-immunoreactive (IR) cells in the nerve root and DRG. NGF-IR cells in the nerve root and DRG included macrophages and Schwann cells, because these cells were labeled for NGF and ED-1 or glial fibrillary acid protein by dual immunostaining. A significant increase in the percentage of BDNF-IR neurons in the DRG was observed in the NP group at 3 days after surgery and the increase in BDNF mRNA expression was confirmed using in situ hybridization histochemistry and reverse transcription-polymerase chain reaction. We also injected NGF into the endoneurial space of the normal rat spinal nerve root and found that the NGF injection produced dose-dependent mechanical allodynia on the ipsilateral hind paw at 1 day after surgery and an increase in the percentage of BDNF-IR neurons in the DRG at 3 days after surgery compared to the group receiving saline injection. These findings suggest that in the lumbar disc herniation model, i.e. neuritis of the nerve root, increased NGF produced by the inflammatory responses in the dorsal root and DRG tissues may affect the production of BDNF in the DRG and may play important roles in the modulation of the dorsal horn neurons. These changes in neurotrophic factors in the primary afferents may be involved in the pathophysiological mechanisms of neuropathic pain produced by lumbar disc herniation.

Differential regulation of P2X(3) mRNA expression by peripheral nerve injury in intact and injured neurons in the rat sensory ganglia.

The P2X(3) receptor is a ligand-gated cation channel activated by the binding of extracellular adenosine 5'-triphosphate (ATP), an agent that has been suggested to have a role in the nociceptive pathway after tissue and nerve injury. After peripheral nerve injury, both down regulation and up regulation of the P2X(3) receptor in sensory ganglion neurons have been observed. The purpose of this study was to examine the precise regulation of P2X(3) mRNA expression in primary sensory neurons after nerve injury. We used two nerve injury models in the rat, the transection of the tibial and common peroneal nerves and the transection of the infraorbital nerve, and observed dorsal root ganglion (DRG) and trigeminal ganglion neurons, respectively. P2X(3) mRNA in both neuron populations was detected by in situ hybridization with an oligonucleotide probe that was confirmed by Northern blot analysis. To identify axotomized neurons, we examined the expression of activating transcription factor 3 (ATF3), which is regarded as a neuronal-injury marker, using immunohistochemistry. In the DRG, the mean percentage of P2X(3) mRNA-labeled neurons relative to the total number of neurons increased from 32.7% in the naive rats to 42.7% at 3 days after injury. The mean percentage of P2X(3) mRNA-labeled neurons in ATF3 immunoreactive (ir) neurons was 29.5% at 3 postoperative days, which gradually decreased to 11.2% at 28 days after injury. In the trigeminal ganglion, the mean percentage of P2X(3) mRNA-labeled neurons was 36.9% at 3 days after injury, versus 26.0% in the naive rats. In the ATF3-ir neurons, the mean percentage of P2X(3) mRNA-labeled neurons was 25.3% at 1 postoperative day and was reduced to 6.1% at 28 postoperative days. The finding that P2X(3) mRNA in ATF3-ir neurons decreased significantly after injury indicates that axotomized neurons decreased the expression of P2X(3) mRNA, despite the increase in P2X(3) mRNA relative to the total number of sensory ganglion neurons. These data strongly suggest that P2X(3) mRNA expression increases in intact neurons and that P2X(3) mRNA in intact neurons may play a role in the pathomechanism of post-nerve injury in primary sensory neurons.