Effect of applying p75NTR saporin to a punctured intervertebral disc on calcitonin gene-related peptide expression in rat dorsal root ganglion neurons.

BACKGROUND: Recent studies have revealed that the low-affinity nerve growth factor receptor, p75 neurotrophin receptor (p75NTR), is important in inflammatory pain. Moreover, p75NTR immunoreactive sensory nerve and dorsal root ganglion (DRG) neurons have been found to innervate lumbar intervertebral discs. The purpose of the current study was to investigate the effect of p75NTR saporin, a toxin used to destroy p75NTR, on calcitonin gene-related peptide (CGRP), an inflammatory neuropeptide associated with pain, in DRG neurons innervating punctured intervertebral discs in rats. METHODS: The neurotracer fluorogold (FG) was applied to the surfaces of L5/6 discs to label their innervating DRG neurons (n = 30). Of 30 rats, 10 were in a nonpunctured disc sham surgery control group (nonpuncture group), and the other 20 were in experimental groups in which intervertebral discs were punctured with a 23-gauge needle. p75NTR saporin was applied to the discs of 10 rats (puncture + p75NTR saporin group) and the other 10 received the same volume of saline (puncture + saline group). At 14 days after surgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for CGRP, and the proportions of CGRP-immunoreactive DRG neurons was evaluated. RESULTS: Of the FG-labeled neurons innervating the L5/6 disc, the proportion of CGRP-immunoreactive neurons was 32% +/- 6% (mean +/- SE) in the nonpuncture group, 47.2% +/- 8% in the puncture + saline group, and 34.6% +/- 9% in the puncture + p75NTR saporin group. The proportion of CGRP-immunoreactive neurons was significantly greater in the puncture + saline group compared with the nonpuncture and puncture + p75NTR saporin groups (P < 0.01). CONCLUSIONS: Half of the DRG neurons innervating the discs were positive for CGRP in the puncture + saline group. CGRP is important for mediating inflammatory and nerve-injured pain and may be important in discogenic pain. However, p75NTR saporin suppressed CGRP expression in DRG neurons. Therefore, p75NTR may be an important receptor for mediating discogenic pain via CGRP expression.

Direct application of the TNF-alpha inhibitor, etanercept, does not affect CGRP expression and phenotypic change of DRG neurons following application of nucleus pulposus onto injured sciatic nerves in rats.

STUDY DESIGN: Immunohistological and behavioral analysis of the effect of a tumor necrosis factor alpha (TNF-alpha) inhibitor in an injured-nerve model. OBJECTIVE: To examine the effect of direct application of a TNF-alpha inhibitor (etanercept) on injured-nerve pain caused by nucleus pulposus. SUMMARY AND BACKGROUND DATA: TNF-alpha is thought to play a crucial role in radicular pain. Calcitonin gene-related peptide (CGRP) is an inflammatory neuropeptide found in small sensory neurons. We have reported that CGRP appears in medium and large dorsal root ganglion (DRG) neurons that transmit proprioception in physiologic conditions. The purpose of the current study was to examine the change in behavior and phenotypic change of CGRP-immunoreactive DRG neurons by the TNF-alpha inhibitor, etanercept, in a disc herniation model. METHODS: For the injured-nerve model, nucleus pulposus was applied to the sciatic nerve and the sciatic nerve pinched. Saline (10 microL; n = 10), as a control, or etanercept (150 microg: n = 10) were applied to sciatic nerves simultaneously. Mechanical allodynia was examined. Immunohistochemistry was used to examine CGRP expression in L5 DRGs. RESULTS: Significant mechanical allodynia for 10 days was seen in the injured-nerve group compared with sham-operated animals. Etanercept ameliorated the mechanical allodynia slightly on day 2; however, there was no effect on other days. CGRP immunoreactivity was upregulated in the L5 DRG neurons of injured-nerve groups compared with the sham-operated group (P < 0.01). However, etanercept did not affect CGRP expression after nerve injury (P > 0.05). Proportions of CGRP- immunoreactive medium and large neurons were not significantly different in the nerve injury + saline group compared with the injury + etanercept group (P > 0.05). CONCLUSION: Our results indicate that direct application of a TNF-alpha inhibitor had a small effect on acute pain behavior and may not be effective for suppression of inflammatory peptides in the current disc-herniation model.

Existence of nerve growth factor receptors, tyrosine kinase a and p75 neurotrophin receptors in intervertebral discs and on dorsal root ganglion neurons innervating intervertebral discs in rats.

STUDY DESIGN: We evaluated 2 types of nerve growth factor (NGF) receptors on dorsal root ganglion (DRG) cells and nerve fibers innervating rat lumbar intervertebral discs. OBJECTIVE: To examine the NGF receptors, tyrosine kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) on DRG cells and nerve fibers innervating rat lumbar intervertebral discs using immunohistochemistry and a retrograde neurotracing method. SUMMARY OF BACKGROUND DATA: Nerve innervation of intervertebral discs is thought to be a pathology of discogenic low back pain. NGF is also important for mediating inflammatory pain from intervertebral discs via the high affinity receptor, TrkA. Recent research has also revealed that the low affinity NGF receptor, p75NTR plays an important role in inflammatory pain. However, the presence of TrkA and p75NTR-immunoreactive (NTR-IR) DRG neurons innervating the rat L5/6 intervertebral disc, and p75NTR-IR nerve fibers in rat intervertebral discs, has not been explored. METHODS: The Fluoro-gold neurotracer was applied to rat L5/6 intervertebral discs to determine the DRG neurons innervating the discs (n = 20). Fourteen days after surgery, bilateral DRG from the L1-L6 levels were harvested, sectioned, and immunostained for TrkA and p75NTR. The percentages of TrkA and p75NTR-IR DRG neurons were counted, and p75NTR-IR nerve fibers in L5/6 discs evaluated. RESULTS: p75NTR-IR nerve fibers were found in superficial layers in the annulus fibrosus in L5/6 intervertebral discs. Fluoro-gold-labeled neurons innervating the L5/6 discs were distributed throughout DRG from the L1-L6 levels. The percentage of TrkA-immunoreactive (TrkA-IR) neurons was 75.1% +/- 3.9% (mean +/- SE) and that of p75NTR-IR neurons was 75.8% +/- 5.1%. These percentages were similar for each level. CONCLUSION: Rat L5/6 intervertebral discs were innervated by multisegmental levels of DRG. Most DRG neurons innervating the discs were positive for 2 types of NGF receptors.