Notochordal Cell Conditioned Medium Inhibits Neurite Outgrowth and Maintains Neural Cell Viability

Abstract

Introduction Minimally invasive therapies to treat disk degeneration focus on analgesia rather than reducing the source of pain. Painful human disks demonstrate extension of neurons that are responsive to nerve growth factor (NGF) and substance P, into the annulus fibrosus (AF) and nucleus pulposus (NP).1,2 Understanding and recapitulating the developmental processes that occur during early patterning of the spine may help to formulate symptom-modifying treatments for painful disk degeneration. The chick notochord produces chemorepulsive factors including semaphorin 3A and CS proteoglycans that inhibit axonal elongation of dorsal root ganglia.3 We suggest that notochordal cells (NCs), which play an essential role in the formation of the avascular and aneural NP, produce soluble factors that can inhibit neural extension into the disk and limit painful symptoms. We hypothesize that conditioned media (CM) from NC tissue can inhibit neuronal growth through assessment of neurite-expressing cells, neurite length, and expression of neuronal outgrowth proteins (GAP43) while maintaining viability. Materials and Methods Notochordal cells (NC) CM from tissue (NCT) was generated from NC-rich NP tissue from six pig spines (age 2 to 8 months), soaked separately in basal media (DMEM + 1% ITS) and conditioned for 4 days in hypoxia (1% O2, 5% CO2, 37°C). Media was filtered and the filtrate retained, resuspended in EMEM:F12 serum-free (sf) media and stored at −20°C. Human SH-SY5Y neuroblastoma cells were seeded at a density of 50,000 cells per well in a 6-well poly-d-lysine plate and incubated with EMEM:F12 + 15% FBS for 24 hours at normoxia 5% CO2 37°C. SH-SY5Y cells were then incubated for 48 hours in NCT at doses of 100 and 10% and 50% in basal media (EMEM:F12 sf media +40 ng/mL PDGF) including a basal control group. After 48 hours, images at 10× were taken per group and the percentage of neurite expressing cells and mean neurite length were calculated per field. Cell number was assessed by counting total cells per field of view, and viability was assessed using the live or dead assay. Cell images were quantified using ImageJ software. qRT-PCR was assessed using TaqMan assays for Enolase, GAP43, neurofilament, NPY, and NRP. Immunohistochemical assessment of semaphorin 3A expression was performed on porcine NC tissue from which NCT was generated (Abcam: ab45376). Statistics involved a one-way ANOVA and Tukey posthoc test with p < 0.05 considered significant. Results NCT significantly decreased the percentage of neurite-expressing cells at doses of 10, 50, and 100% compared to basal (p < 0.05) (Fig. 1A). NCT also reduced mean neurite length from SH-SY5Y cells and this was significant at all doses compared to basal (p < 0.05) (Fig. 1B). Cell viability remained high with no differences between groups (Fig. 1C). Significant increases in cell number were observed at doses of 10 and 50% (102.1 ± 5.2 and 117.1 ± 5.9) compared to basal (79.5 ± 3.1) and 50% compared to 100% (93.5 ± 5.9). No significant difference in cell number was ...