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.HighlightsHypoxia 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.

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia

“…A cell with more processes displays a higher ‘Transformation Index’, while a higher ‘Elliptical Form Factor’ indicates an elongated cell shape. Unlike microglia, in vitro cultured astrocytes show a complex pattern of outgrowth, so the ‘Sholl’ analysis was only applied to astrocytes to evaluate branching of the outgrowth arbors [ 67 , 68 ]. In the ‘Sholl’ analysis, the number of branches (represented as number of intersections of outgrowth with circled samplings) at different distance from the soma was evaluated ( Figure 5 G).…”

Controversies in spine research: Organ culture versus in vivo models for studies of the intervertebral disc

“…However, even in ex vivo work, different results can be obtained depending on the study design. For example, differential effects of hypoxic stress on neurite outgrowth in DRGs were reported between the single cell and tissue levels 77 . Therefore, the study of neural activity in context of IVDD in vivo may yield contrasting results from those obtained ex vivo.…”

“…These pathophysiological mechanisms of pain are not fully replicated in all in vivo models of LBP, which, combined with limited validated methodologies to accurately measure pain in such models, limits the relevance of investigation in vivo. Meanwhile, pain‐related molecular factors can be studied in organ culture models; for example, neurotrophic factor expression, which reduces the need to provoke pain behavior in animal models, in alignment with the 3Rs 77 . In addition, these cellular and signaling mechanisms in organ culture models can be deterministically attributed to the IVD, and the results are specific to the biology of the IVD.…”

“…Meanwhile, pain‐related molecular factors can be studied in organ culture models; for example, neurotrophic factor expression, which reduces the need to provoke pain behavior in animal models, in alignment with the 3Rs. 77 In addition, these cellular and signaling mechanisms in organ culture models can be deterministically attributed to the IVD, and the results are specific to the biology of the IVD.…”

Screening for Growth-Factor Combinations Enabling Synergistic Differentiation of Human MSC to Nucleus Pulposus Cell-Like Cells

“…Alternatively, the outer collagen layers, collectively termed the annulus fibrosus (AF), can similarly lose their integrity, thereby enabling disc bulging or complete AF rupture. Finally, in reaction to these degenerative changes, the native IVD cells secrete inflammatory factors, potentially stimulating nerve ingrowth, vascularization, and immunogenic cell influx in the normally avascular and un-innervated discs or sensitizing nearby nerve structures [8][9][10]. Collectively, these changes can trigger nociception and in severe cases, might result in debilitating pain and disability in patients [11,12].…”