Korean J Pain.  2022 Apr;35(2):160-172. 10.3344/kjp.2022.35.2.160.

Insulin enhances neurite extension and myelination of diabetic neuropathy neurons

• Pham VM
• Thakor N

• KMID: 2527767
• DOI: http://doi.org/10.3344/kjp.2022.35.2.160

Abstract

Background
The authors established an In Vitro model of diabetic neuropathy based on the culture system of primary neurons and Schwann cells (SCs) to mimic similar symptoms observed in in vivo models of this complication, such as impaired neurite extension and impaired myelination. The model was then utilized to investigate the effects of insulin on enhancing neurite extension and myelination of diabetic neurons.
Methods
SCs and primary neurons were cultured under conditions mimicking hyperglycemia prepared by adding glucose to the basal culture medium. In a single culture, the proliferation and maturation of SCs and the neurite extension of neurons were evaluated. In a co-culture, the percentage of myelination of diabetic neurons was investigated. Insulin at different concentrations was supplemented to culture media to examine its effects on neurite extension and myelination.
Results
The cells showed similar symptoms observed in in vivo models of this complication. In a single culture, hyperglycemia attenuated the proliferation and maturation of SCs, induced apoptosis, and impaired neurite extension of both sensory and motor neurons. In a co-culture of SCs and neurons, the percentage of myelinated neurites in the hyperglycemia-treated group was significantly lower than that in the control group. This impaired neurite extension and myelination was reversed by the introduction of insulin to the hyperglycemic culture media.
Conclusions
Insulin may be a potential candidate for improving diabetic neuropathy. Insulin can function as a neurotrophic factor to support both neurons and SCs. Further research is needed to discover the potential of insulin in improving diabetic neuropathy.

Keyword

Cell Proliferation; Culture Techniques; Diabetic Neuropathies; Glucose; Hyperglycemia; Insulin; In Vitro Techniques; Motor Neurons; Myelin Sheath; Nerve Growth Factors; Neurites; Schwann Cells

Figure

• 

  Fig. 1 Schwann cell proliferation is attenuated in culture under hyperglycemia. (A) Representative images show changes in cell morphology and confluence under hyperglycemic culture. Scale bar: 300 µm. (B) Representative images of live and dead assay performed at 7 days of culture in control and hyperglycemic conditions. Scale bar: 100 µm. (C) The graph shows the number of cells at 1, 3, 5, and 7 days of culture in control medium (5.5 mM glucose) and in hyperglycemic medium containing 10, 30, and 60 mM glucose. At 1 day, n = 4 wells each group; non-repeated-measures ANOVA, not significant. At 3 days, n = 4 wells each group; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 12) = 6.327, **P < 0.01 vs. control group. At 5 days, n = 6 wells each group; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 20) = 16.09, *P < 0.05, **P < 0.01, and ****P < 0.0001 vs. control group. At 7 days, n = 7 wells each group; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 24) = 24.47, ****P < 0.0001 vs. control group. (D) The graph shows the percentage of dead cells at 7 days of culture in control medium (5.5 mM glucose) and in hyperglycemic media. n = 5 wells each group; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 16) = 43.92, *P < 0.05 and ****P < 0.0001 vs. control group.

• 

  Fig. 2 Schwann cell maturation is impaired under hyperglycemia. (A) Representative images of immunocytochemistry with p75NGFR and MBP performed at 7 days of culture in control and hyperglycemic conditions. Scale bar: 50 µm. (B) The graph shows the fluorescent intensity ratio of MBP and p75NGFR at 7 days of culture in control medium (5.5 mM glucose) and in hyperglycemic medium containing 10, 30, and 60 mM glucose. n = 6, *P < 0.05 vs. control group. n = 6; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 20) = 4.845, *P < 0.05 vs. control group.

• 

  Fig. 3 Hyperglycemia impairs neurite extension. (A, B) Representative images of sensory (A) and motor (B) neurons stained for NF-160 at 2 days of culture in control and hyperglycemic conditions. Scale bar: 50 µm. (C, D) The graph shows the significantly decreased average neurite length of sensory (C) and motor (D) neurons under hyperglycemia at 2 days of culture. (C) n = 3 wells each group, non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 8) = 32.57, ***P < 0.001 vs. control group. (D) n = 3 wells each group, non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 8) = 22.14, **P < 0.01 and ***P < 0.001 vs. control group.

• 

  Fig. 4 Hyperglycemia impairs myelination of sensory neurites. (A) Representative images of sensory neurons stained for NF-160 and MBP at 7 days of co-culture with Schwann cells in control and hyperglycemic conditions. Scale bar: 100 µm. (B) The graph shows the significantly decreased percentage of myelinated sensory neurites under hyperglycemia. n = 3 wells each group; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 8) = 50.10, **P < 0.01 and ****P < 0.0001 vs. control group.

• 

  Fig. 5 Hyperglycemia impairs myelination of motor neurites. (A) Representative images of motor neurons stained for NF-160 and MBP at 7 days of co-culture with Schwann cells in control and hyperglycemic conditions. Scale bar: 100 µm. (B) The graph shows the significantly decreased percentage of myelinated motor neurites under hyperglycemia. n = 3 wells each group; non-repeated-measures ANOVA followed by Dunnett’s post hoc test, F (3, 8) = 238.5, ****P < 0.0001 vs. control group.

• 

  Fig. 6 Insulin promotes neurite extension. (A, B) Representative images of sensory (A) and motor (B) neurons stained for NF-160 at 2 days of culture in neurobasal medium (25 mM glucose) supplemented with a series of insulin concentrations ranging from 0.01–2 µM. Scale bar: 50 µm. (C) The graph shows the significantly different average neurite length of sensory neurons under the effect of insulin. n = 3 wells each group; non-repeated-measures ANOVA followed by Tukey’s post hoc test, F (6, 14) = 3.970, *P < 0.05 vs. control group. (D) The graph shows the significantly different average neurite length of motor neurons under the effect of insulin. n = 3 wells each group; non-repeated-measures ANOVA followed by Tukey’s post hoc test, F (6, 14) = 47.26, ****P < 0.0001 vs. control group; #P < 0.05 and ##P < 0.01 vs. 0.01 µM; ιιιP < 0.001 and ιιιιP < 0.0001 vs. 0.05 µM; aP < 0.0001 vs. 0.1 µM.

• 

  Fig. 7 Insulin promotes sensory neurite myelination. (A) Representative images of sensory neurons stained for NF-160 and MBP at 7 days of co-culture with Schwann cells in hyperglycemic insult (60 mM glucose) supplemented with insulin at 0.1 µM. Scale bar: 100 µm. (B) The graph shows the significantly promoted percentage of myelinated sensory neurites in the group treated by insulin. n = 6; two-tailed Student’s t-test, t (4) = 8.135, ****P < 0.0001.

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