Axonal Growth Potential of Lumbar Dorsal Root Ganglion Neurons in an Organ Culture System

Study design The axonal growth potential of dorsal root ganglion (DRG) neurons in an organ culture system was investigated. Objective To examine the effects of neuronal injury and tumor necrosis factor-alpha (TNF-alpha) on the axonal growth potential of 2 types of nociceptive DRG neurons: nerve growth factor (NGF)-sensitive and glial cell line-derived neurotrophic factor (GDNF)-sensitive neurons. Summary of background data Nerve ingrowth into the disc is recognized to be one of the causes of discogenic pain. Almost all of these disc-innervating neurons are NGF-sensitive. The axonal growth potential of NGF-sensitive neurons has not been investigated. Methods Adult Sprague-Dawley rats were used for immunohistochemistry (n = 7) and cell viability studies (n = 6). Bilateral L3-L5 DRGs, which were successfully removed without damage, were noncultured or cultured in serum-free medium containing TNF-alpha at 0, 0.01, 0.1, and 1 ng/mL for 48 hours (n = 5, each treatment). The DRGs were then immunostained for activating transcription factor 3 (ATF3, a marker for injured neurons) or double-stained for growth-associated protein 43 (GAP-43, a marker for axonal growth) with calcitonin gene-related peptide (CGRP, a marker for NGF-sensitive neurons) or isolectin B4 (IB4, a marker for GDNF-sensitive neurons). Cell viability was assessed by a lactate dehydrogenase (LDH) assay and an MTS assay (n = 6, each treatment). Results Immunoreactive evidence of injured neurons (ATF3 positive) was frequently observed in cultured DRGs, but never in noncultured DRGs. The percentage of neurons exhibiting axonal growth potential (GAP-43 immunoreactive) was significantly higher for NGF-sensitive neurons than for GDNF-sensitive neurons at any concentration of TNF-alpha. More than 95% of the cultured neurons were viable. Conclusions The results suggest that the cultured DRG neurons exhibit pathologic changes similar to those found in injured neurons. NGF-sensitive neurons, which include disc-innervating neurons, may have a greater potential to extend their axons in response to neuronal injury under pathologic conditions in the presence of TNF-alpha than GDNF-sensitive neurons.