Cholecalciferol (vitamin D₃) improves myelination and recovery after nerve injury

Abstract

Previously, we demonstrated i) that ergocalciferol (vitamin D2) increases axon diameter and potentiates nerve regeneration in a rat model of transected peripheral nerve and ii) that cholecalciferol (vitamin D3) improves breathing and hyper-reflexia in a rat model of paraplegia. However, before bringing this molecule to the clinic, it was of prime importance i) to assess which form - ergocalciferol versus cholecalciferol - and which dose were the most efficient and ii) to identify the molecular pathways activated by this pleiotropic molecule. The rat left peroneal nerve was cut out on a length of 10 mm and autografted in an inverted position. Animals were treated with either cholecalciferol or ergocalciferol, at the dose of 100 or 500 IU/kg/day, or excipient (Vehicle), and compared to unlesioned rats (Control). Functional recovery of hindlimb was measured weekly, during 12 weeks, using the peroneal functional index. Ventilatory, motor and sensitive responses of the regenerated axons were recorded and histological analysis was performed. In parallel, to identify the genes regulated by vitamin D in dorsal root ganglia and/or Schwann cells, we performed an in vitro transcriptome study. We observed that cholecalciferol is more efficient than ergocalciferol and, when delivered at a high dose (500 IU/kg/day), cholecalciferol induces a significant locomotor and electrophysiological recovery. We also demonstrated that cholecalciferol increases i) the number of preserved or newly formed axons in the proximal end, ii) the mean axon diameter in the distal end, and iii) neurite myelination in both distal and proximal ends. Finally, we found a modified expression of several genes involved in axogenesis and myelination, after 24 hours of vitamin supplementation. Our study is the first to demonstrate that vitamin D acts on myelination via the activation of several myelin-associated genes. It paves the way for future randomised controlled clinical trials for peripheral nerve or spinal cord repair.

Figure 1. Assessment of Peroneal Functional Index (PFI) at Month+1, +2 and +3 post-surgery.

A. All animals (n = 6 per group) improved their hindlimb locomotion during the 12 weeks of experiment. B. A similar pattern was observed when 12 animals were included in the Control, Vehicle and D3–500 groups. Crosses (+) indicate that the response was significantly increased, when compared to the Vehicle group (+ p<0.05;++p<0.01).

Figure 2. Muscle mechanical properties.

Muscle contractions were obtained using peroneal nerve electrical stimulation. A. Twitches were analysed in terms of peak Amplitude (A) and Maximum Relaxation Rate (MRR), defined as the slope of a tangent drawn to the steepest portion of the relaxation curve. B. Electrical stimulation frequencies were used to reach the tetanus threshold. The experiment was first assessed in the 6 initial groups (n = 6 per group; right histograms) and then in the 3 final groups (n = 12 per group; left histograms). Crosses (+) indicate significant changes when compared to the Vehicle group (+ p<0.05;++p<0.01).

Figure 3. Vitamin D improves responses to muscle electrically-induced fatigue or to a chemical agent.

Ventilatory response of the animals after muscle stimulation (A) and response of metabosensitive afferent fibre activity after active muscle electrical stimulation (B) or intramuscular capsaicin injection (C). The experiment was first assessed in the 6 initial groups (n = 6 per group) (right histograms) and then in the 3 final groups (n = 12 per group) (left histograms). Crosses (+) indicate that the response was significantly increased, when compared to the Vehicle group (+ p<0.05;++p<0.01;+++p<0.001).

Figure 4. Histological analysis of the peroneal nerve, at 3 months post-surgery.

Peroneal nerves from the Control, Vehicle and D3–500 groups were either fixed with paraformaldehyde and included in paraffin (n = 6 per group) for counting axon numbers (A–D) or fixed with glutaraldehyde and included in resin (n = 6 per group) for assessing myelination (E–H). A,B. Representative pictures of nerve sections immunostained with an anti-neurofilament antibody in Vehicle (A) and D3–500 (B) groups. Quantitative analysis of axon numbers indicated that vitamin D3–500 induced a statistically significant doubling of axons in the proximal end (C) but not in the distal end (D). E. Low magnification view of a nerve section stained with p-phenyl-n-diamine (D3–500 group). F. High magnification view of a nerve section with arrows indicating how the G-ratio (the ratio between the diameter of the axon and the outer diameter of the myelinated fibre) was calculated (D3–500 group). Quantitative analysis indicates that vitamin D3–500 triggered myelination in the proximal (G) and the distal (H) ends of the nerve. Crosses (+) indicate significant changes when compared to the Vehicle group (+ p<0.05;++p<0.01). Scale bar:?

Figure 5. Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool.

A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells (A) or Schwann cells and dorsal root ganglion cells (B). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.

Figure 6. Main metabolic pathways associated to in vitro calcitriol supplementation.

A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes (Prx, Tspan2, IgF1, Spp1) involved in axogenesis and myelination.