title: Effects of proinflammatory cytokines on axonal outgrowth from adult rat lumbar dorsal root ganglia using a novel three-dimensional culture system.
abstract: BACKGROUND
  Degeneration of the intervertebral disc is often associated with low back pain and increased infiltration of nerve fibers originating from dorsal root ganglia (DRG). The degenerated disc is also characterized by the presence of proinflammatory cytokines, which may influence axonal outgrowth. Toward an improved understanding of the growth of DRG neurons into compliant extracellular matrices, we developed a novel experimental system to measure axonal outgrowth of adult rat lumbar DRG neurons within three-dimensional (3D) collagen hydrogels and used this system to examine the effects of interleukin 1β (IL-1β) and tumor necrosis factor (TNF)-α treatment.
  
  
  OBJECTIVE
  The aim was to investigate the effects of proinflammatory cytokines on 3D neuronal growth into collagen matrices.
  
  
  METHODS
  This was an in vitro study of neurite outgrowth from adult rat lumbar DRG into collagen gels in response to IL-1β and TNF-α.
  
  
  METHODS
  Lumbar DRG were obtained from adult Sprague Dawley rats, bisected to expose cell bodies and placed onto collagen gel constructs prepared in 24-well Transwell inserts. Dorsal root ganglia were then treated with nerve growth factor (NGF)-free Neurobasal media (negative control) or NGF-supplemented media containing 0, 1, and 10 ng/mL of IL-1β and TNF-α. After 7 days, collagen gel-DRG constructs were immunostained for phosphorylated neurofilament, an axonal marker. Simple Neurite Tracer (Fiji/ImageJ) was used to quantify 3D axonal outgrowth from confocal image stacks. Data were analyzed using one-way analysis of variance, with Tukey HSD post hoc correction at a level of p<.05.
  
  
  RESULTS
  Immunostaining showed robust axonal outgrowth into collagen gels from all NGF-treated DRG. The negative control demonstrated very few and short neurites. Tumor necrosis factor-α (1 and 10 ng/mL) significantly inhibited axonal outgrowth compared with NGF-only media (p<.026 and p<.02, respectively). After IL-1β treatment, average axon length was 10% lower at 1 ng/mL and 7.5% higher at 10 ng/mL, but these differences were not statistically significant. Among cytokine treatments, however, average axon length in the IL-1β (10 ng/mL) group was significantly higher than that in the other groups (p<.05).
  
  
  CONCLUSIONS
  A novel 3D collagen gel culture system was used to investigate factors modulating neuronal ingrowth. Our results showed that NGF was necessary to promote neurite growth into collagen gels. In the presence of proinflammatory cytokines, high concentrations of IL-1β induced significantly higher axonal outgrowth than TNF-α and low levels of IL-1β.
authors: Fischell Department of Bioengineering, University of Maryland, College Park, Jeong H. Kim Engineering Building, College Park, MD 20742, USA.
authors: Hyunchul
authors: H
authors: Kim
authors: Fischell Department of Bioengineering, University of Maryland, College Park, Jeong H. Kim Engineering Building, College Park, MD 20742, USA.
authors: Tyler
authors: T
authors: W Caspar
authors: Department of Orthopaedic Surgery, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
authors: Sameer B
authors: SB
authors: Shah
authors: Fischell Department of Bioengineering, University of Maryland, College Park, Jeong H. Kim Engineering Building, College Park, MD 20742, USA; Department of Orthopaedics, University of Maryland, Baltimore, 22 S. Greene Street, Baltimore, MD 21201, USA. Electronic address: hsieh@umd.edu.
authors: Adam H
authors: AH
authors: Hsieh
mesh_terms: D000818:Animals; D001369:Axons / Q000187:drug effects; D018929:Cell Culture Techniques; D003094:Collagen; D016207:Cytokines / Q000494:pharmacology*; D005727:Ganglia, Spinal / Q000187:drug effects* / Q000254:growth & development; D020100:Hydrogels; D053583:Interleukin-1beta / Q000494:pharmacology*; D008161:Lumbosacral Region; D023421:Models, Animal; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects*; D018373:Peripheral Nervous System Agents / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016207:Cytokines; D020100:Hydrogels; D053583:Interleukin-1beta; D018373:Peripheral Nervous System Agents; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen; D020932:Nerve Growth Factor
keywords: Axonal outgrowth; Collagen gel; Dorsal root ganglia; Explant culture; IL-1β; Neurite extension; TNF-α