Hypoxic stress enhances extension and branching of dorsal root ganglion neuronal outgrowth

Abstract

It has been shown that painful intervertebral discs (IVDs) were associated with a deeper innervation. However, the effect of the disc's degenerative microenvironment on neuronal outgrowth remains largely unknown. The focus of this study was to determine the influence of hypoxia on dorsal root ganglion (DRG) neurite outgrowth. Toward this aim, the DRG‐derived cell line ND7/23 was either directly subjected to 2% or 20% oxygen conditions or exposed to conditioned medium (CM) collected from IVDs cultured under 2% or 20% oxygen. Viability and outgrowth analysis were performed following 3 days of exposure. Results obtained with the cell line were further validated on cultures of rabbit spinal DRG explants and dissociated DRG neurons. Results showed that hypoxia significantly increased neurite outgrowth length in ND7/23 cells, which was also validated in DRG explant and primary cell culture, although hypoxia conditioned IVD did not significantly increase ND7/23 neurite outgrowth. While hypoxia dramatically decreased the outgrowth frequency in explant cultures, it significantly increased collateral sprouting of dissociated neurons. Importantly, the hypoxia‐induced decrease of outgrowth frequency at the explant level was not due to inhibition of outgrowth branching but rather to neuronal necrosis. In summary, hypoxia in DRG promoted neurite sprouting, while neuronal necrosis may reduce the density of neuronal outgrowth at the tissue level. These findings may help to explain the deeper neo‐innervation found in the painful disc tissue.

Highlights

Hypoxia promoted elongation and branching of neurite outgrowth at single cell level, but reduced outgrowth density at tissue level, possibly due to hypoxia‐induced neuronal necrosis; these findings may help to explain the deeper neo‐innervation found in clinically painful tissues.

Abstract

Deeper innervation has been observed in degenerative and painful intervertebral disc (IVD). Ischemia‐related deprivation of oxygen (hypoxia) is among the multiple factors that are associated with IVD degeneration and pain. The study investigated the influence of hypoxia in both peripheral nerve (namely dorsal root ganglion DRG) and IVD on aberrant neurite sprouting at both cell and tissue explant levels. Although hypoxia in IVD did not seem to produce an IVD conditioned medium that promoted neurite sprouting, hypoxia in DRG directly induced a longer neurite outgrowth with higher collateral sprouting. Additionally, hypoxia leaded to DRG neuronal necrosis, which might reduce the neurite frequency at the tissue explant level. Protecting DRG from hypoxic stress may be of importance in the treatment of discogenic pain.