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. 2017 Apr;55(4):570-581.
doi: 10.1002/mus.25370. Epub 2016 Dec 28.

Effect Of combining FK506 and neurotrophins on neurite branching and elongation

Pratima Labroo  1 , Jill Shea  2 , Himanshu Sant  1 , Bruce Gale  1 , Jayant Agarwal  2
Affiliations

Effect Of combining FK506 and neurotrophins on neurite branching and elongation

Pratima Labroo et al. Muscle Nerve. 2017 Apr.

Abstract

Introduction: There is a clinical need to improve the outcomes of peripheral nerve regeneration and repair after injury. In addition to its immunosuppressive effects, FK506 (tacrolimus) has been shown to have neuroregenerative properties. To determine biologically relevant local FK506 and growth factor concentrations, we performed an in vitro bioassay using dorsal root ganglion (DRG) from chicken embryos.

Methods: Neurite elongation and neurite branching were analyzed microscopically after addition of FK506, glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF), each alone and in combination.

Results: FK506 induced modest neurite elongation (∼500-800 µm) without improving neurite branching significantly. The combination of FK506 with NGF, GDNF, or both, exerted a potentiating or competitive effect on neurite elongation (∼700-1100 µm) based on dosage and competitive effect on neurite branching (∼0.2-0.4).

Conclusions: These results strongly suggest that the interaction of FK506 with GDNF and NGF mediates distinct enhancement of neurite growth. Muscle Nerve 55: 570-581, 2017.

Keywords: FK506; GDNF; NGF; Neurite growth in vitro; dosage; nerve regeneration; potentiation.

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Figures

FIGURE 1
FIGURE 1
Phase contrast images of DRGs treated with different concentrations of FK506: control (left), 1 ng/ml (center), 10 ng/ml (right) (a); NGF: control (left), 1 ng/ml (center), 10 ng/ml (right) (b); GDNF: control (left), 1 ng/ml (center), 10 ng/ml (right) (c). Neurite outgrowth can be seen based on different doses of FK506, NGF, and GDNF. Scale = 500 μm.
FIGURE 2
FIGURE 2
Plots showing effect of single drug treatments on axonal elongation. (a) FK506 Dosage/length. (b) NGF Dosage/length. (c) GDNF Dosage/length. The bars represent mean ± SD for n = 4 (* statistically different from 0 ng/ml; P < 0.05).
FIGURE 3
FIGURE 3
Phase contrast images of DRGs treated with growth factor combinations (starting from top left). (a) control (0 ng/ml). (b) NGF 1 ng/ml + FK506 1 ng/ml. (c) NGF 1 ng/ml + FK506 10 ng/ml. (d) GDNF 1 ng/ml + FK506 1 ng/ml. (e) GDNF 1 ng/ml + FK506 10 ng/ml. (f) NGF 1 ng/ml + GDNF 1 ng/ml + FK506 1 ng/ml. (g) NGF 1 ng/ml + GDNF 1 ng/ml + FK506 10 ng/ml. (h) NGF 10 ng/ml + GDNF 10 ng/ml + FK506 10 ng/ml. (i) NGF 1 ng/ml + GDNF 1 ng/ml. Neurite outgrowth is increased with combined treatment of FK506 with growth factors. Scale = 500 μm.
FIGURE 4
FIGURE 4
Surface plots showing effect of combined drug treatments on axonal elongation. (a) NGF+FK506 combined dosage. (b) GDNF+FK506 combined dosage. (c) NGF+GDNF combined dosage. (Shaded region with dashed lines specifies the concentration range which produced significantly different axonal elongation response from 0 ng/ml, P < 0.05).
FIGURE 5
FIGURE 5
Fluorescence images of DRGs stained with β-tubulin. (a) control (0 ng/ml). (b) NGF 1 ng/ml + FK506 10 ng/ml. (c) GDNF 1 ng/ml + FK506 10 ng/ml. (d) NGF 1 ng/ml + GDNF 1 ng/ml + FK506 1 ng/ml. (e) NGF 1 ng/ml + GDNF 1 ng/ml. The images show enhanced neurite elongation with the specified treatments.
FIGURE 6
FIGURE 6
Plots showing the effect of single drug treatment on axon density. (a) FK506 Dosage/density. (b) NGF dosage/density. (c) GDNF dosage/density. The bars represent mean ± SD for n = 4 (*statistically different from 0 ng/ml; P < 0.05).
FIGURE 7
FIGURE 7
Surface Plots showing the effect of combined drug treatment on axon density. (a) NGF+FK506 combined dosage. (b) GDNF+FK506 combined dosage. (c) NGF+GDNF combined dosage. Shaded region with dashed lines specifies the concentration range that produced significantly different axonal branching responses when compared with 0 ng/ml, P < 0.05.
FIGURE 8
FIGURE 8
Kinetics of axonal elongation of DRGs. (a) treated with FK506 alone (10 ng/ml), NGF alone (10 ng/ml), combined NGF/FK506 (10 ng/ml each), combined NGF/GDNF/FK506 (10 ng/ml each), and untreated (control). (b) treated with GDNF alone (10 ng/ml), FK506 alone (10 ng/ml), combined GDNF/FK506 (10 ng/ml each), combined NGF/GDNF/FK506 (10 ng/ml each), and untreated (control). Bars = mean ± SD of n = 3. All treatment groups were significantly different (P < 0.05) from control (not indicated with asterisks).
FIGURE 9
FIGURE 9
Plots showing protein expression of DRG lysates treated with different drug treatments. The bars indicate mean ± SD for n = 3 obtained from densitometry of western blot scans.
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