Kim Hyunchul, W Caspar Tyler, Shah Sameer B, Hsieh Adam H
Fischell Department of Bioengineering, University of Maryland, College Park, Jeong H. Kim Engineering Building, College Park, MD 20742, USA.
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.
Spine J. 2015 Aug 1;15(8):1823-31. doi: 10.1016/j.spinee.2015.03.017. Epub 2015 Mar 20.
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.
The aim was to investigate the effects of proinflammatory cytokines on 3D neuronal growth into collagen matrices.
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.
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.
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).
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.
椎间盘退变常与腰痛以及源自背根神经节（DRG）的神经纤维浸润增加有关。退变的椎间盘还具有促炎细胞因子，这可能影响轴突生长。为了更好地理解DRG神经元在顺应性细胞外基质中的生长，我们开发了一种新型实验系统，用于测量成年大鼠腰段DRG神经元在三维（3D）胶原水凝胶中的轴突生长，并使用该系统研究白细胞介素1β（IL-1β）和肿瘤坏死因子（TNF）-α处理的效果。
旨在研究促炎细胞因子对DRG神经元在胶原基质中三维生长的影响。
这是一项关于成年大鼠腰段DRG神经元轴突向胶原凝胶生长以响应IL-1β和TNF-α的体外研究。
从成年Sprague Dawley大鼠获取腰段DRG，将其对半切开以暴露细胞体，并置于24孔Transwell小室中制备的胶原凝胶构建体上。然后将背根神经节用不含神经生长因子（NGF）的Neurobasal培养基（阴性对照）或含有0、1和10 ng/mL IL-1β和TNF-α的补充NGF的培养基处理。7天后，对胶原凝胶-DRG构建体进行免疫染色，以检测磷酸化神经丝，这是一种轴突标记物。使用Simple Neurite Tracer（Fiji/ImageJ）从共聚焦图像堆栈中定量三维轴突生长。使用单因素方差分析进行数据分析，并在p<0.05水平进行Tukey HSD事后校正。
免疫染色显示，所有经NGF处理的DRG均有大量轴突向胶原凝胶生长。阴性对照显示神经突极少且短。与仅含NGF的培养基相比，肿瘤坏死因子-α（1和10 ng/mL）显著抑制轴突生长（分别为p<0.026和p<0.02）。经IL-1β处理后，1 ng/mL时平均轴突长度降低10%，10 ng/mL时升高7.5%，但这些差异无统计学意义。然而，在细胞因子处理组中，IL-1β（10 ng/mL）组的平均轴突长度显著高于其他组（p<0.05）。
使用新型三维胶原凝胶培养系统研究调节神经元向内生长的因素。我们的结果表明，NGF是促进神经突向胶原凝胶生长所必需的。在促炎细胞因子存在的情况下，高浓度的IL-1β比TNF-α和低水平的IL-1β诱导的轴突生长显著更高。
