Axonal growth potential of lumbar dorsal root ganglion neurons in an organ culture system: response of nerve growth factor-sensitive neurons to neuronal injury and an inflammatory cytokine.
METHODS
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.


BACKGROUND
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.
Aoki
Yasuchika
Y
Department of Orthopedic Surgery, Rush Medical College at Rush University Medical Center, Chicago, IL 60612, USA.
An
Howard S
HS
Takahashi
Kazuhisa
K
Miyamoto
Kei
K
Lenz
Mary Ellen
ME
Moriya
Hideshige
H
Masuda
Koichi
K
D051700:Activating Transcription Factor 3 / Q000378:metabolism; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019922:GAP-43 Protein / Q000378:metabolism; D005727:Ganglia, Spinal / Q000187:drug effects / Q000293:injuries / Q000473:pathology; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology*; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000294:innervation; D008159:Lumbar Vertebrae; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009475:Neurons, Afferent / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D051700:Activating Transcription Factor 3; C497108:Atf3 protein, rat; D019922:GAP-43 Protein; D051100:Glial Cell Line-Derived Neurotrophic Factor; D014409:Tumor Necrosis Factor-alpha; D020932:Nerve Growth Factor