Korean J Pain 2022; 35(2): 160-172
Published online April 1, 2022 https://doi.org/10.3344/kjp.2022.35.2.160
Copyright © The Korean Pain Society.
1Singapore Institute for Neurotechnology, National University of Singapore, Singapore
2Department of Biotechnology, Ho Chi Minh City University of Food Industry, Ho Chi Minh City, Vietnam
3Department of Biomedical Engineering, National University of Singapore, Singapore
4Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA
Correspondence to:Vuong M. Pham
Department of Biotechnology, Ho Chi Minh City University of Food Industry, 140 Le Trong Tan Street, Tan Phu District, Ho Chi Minh City 70000, Vietnam
Handling Editor: Sang Hun Kim
Received: February 3, 2022; Revised: March 3, 2022; Accepted: March 9, 2022
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Keywords: Cell Proliferation, Culture Techniques, Diabetic Neuropathies, Glucose, Hyperglycemia, Insulin, In Vitro Techniques, Motor Neurons, Myelin Sheath, Nerve Growth Factors, Neurites, Schwann Cells.
Diabetic peripheral neuropathy is one of the most common complications of both type 1 and type 2 diabetes , affecting 6%–51% of diabetic patients . The spectrum of diabetic neuropathy is wide with many varieties. More than one cell type is affected at the same time, which results in disruptions of cellular interactions . Many types of neurons, including pain fibers, motor neurons, and autonomic neurons are reported to be affected by this complication . In addition to neurons, Schwann cells (SCs) lose their ability not only to supply energy during diabetes for both myelinated and unmyelinated axons but also to transfer toxic lipid species to the axons they have contact with . Peripheral axons and SCs are dependent on each other for vitality and maintenance of the differentiated cellular phenotype. Thus, attempts to repair peripheral nerve damage in diabetic neuropathy should include approaches targeting both SCs and neurons .
Many models of diabetic neuropathy have been investigated for a better understanding of its mechanism and progress. In this regard,
Primary sensory and motor neurons were isolated from E15 rat embryos obtained from pregnant Sprague Dawley (SD) rats (InVivos Pte Ltd, Singapore) in compliance with the ethical guidelines and approved by the Institutional Animal Care and Use Committee (Protocol No.: R14-1635) at the National University of Singapore. All efforts were made to minimize animal suffering and the number of animals in this study. The isolation procedure was carried out under sterile conditions. In brief, the pregnant SD rats were completely euthanized by CO2 gas. The E15 embryos were then collected and placed in a cold L15 medium (11415064, Gibco, Grand Island, NY). Spinal cords were dissected in an L15 medium. Dorsal root ganglia (DRG) were carefully cut off the spinal cords. The membrane of the spinal cord was then removed and the whole spinal cords were transferred to a small Petri dish containing L15 medium supplemented with 1% penicillin–streptomycin (10378016, Gibco) and cut into small pieces. Both sensory and motor neurons were dissociated by 0.05% trypsin/EDTA (25300120, Gibco). Then, the dissociated neurons were counted and seeded into 96-well plates coated with Poly-D-Lysine (50 µg/mL) (A3890401, Gibco) and Laminin (10 µg/mL) (23017-015, Invitrogen, Waltham, MA). Particularly, the cells were seeded at the density of 10,000 cells/well and 5,000 cells/well for single cultures and co-cultures with mouse SCs, respectively, in 96-well plates.
In this study, SC medium (1701, ScienCell, Carlsbad, CA) and NeurobasalTM Medium (21103049, Gibco), with the respective basal glucose concentrations 5.5 mM and 25 mM, were used. The complete neurobasal medium was prepared by supplementing 2% B-27TM Supplement (50X) (17504044, Gibco) and 0.5 mM GlutaMAXTM Supplement (35050061, Gibco). The complete SC medium was prepared by adding 25 mL of fetal bovine serum (FBS), 5 mL of Schwann Cell Growth Supplement, and 5 mL of penicillin/streptomycin solution. Hyperglycemic media was prepared by adding glucose (47829, Sigma-Aldrich, Singapore) to these complete media. In particular, the glucose level of the SC medium was adjusted to 10, 30, and 60 mM and that of the Neurobasal Medium was adjusted to 35, 45, and 60 mM to produce hyperglycemic insults. These media were used at the cell time of seeding. For culture experiments lasting for more than 2 days, media was changed every 2 days to maintain the hyperglycemic condition during the experiments. For co-culture studies, SCs (ScienCell) were seeded into a 96-well plate at the density of 5,000 cells/well (for coculture in hyperglycemic insults alone) or 3,000 cells/well (for coculture in hyperglycemic insult supplemented with insulin) and induced to the mature phase in the complete SC medium supplemented with vitamin C (50 μg/mL) for 4 days prior to seeding neurons on top of the SC layers. The complete Neurobasal Medium with the assigned glucose concentrations was used for co-culture after seeding the neurons. All hyperglycemia cultures were performed in a randomized manner.
For the single culture of neurons, insulin (I9278, Sigma-Aldrich, Saint Louis, MO) ranging from 0.01–2 µM was introduced to the complete neurobasal medium at the seeding time and incubated for 2 days. For the experiment evaluating the effect of insulin on myelination, the treatment was carried out in a blinded manner. The complete neurobasal medium (adjusted to 60 mM glucose) supplemented with or without insulin at a dose of 0.1 µM was used after seeding neurons on top of the SC layers and replaced every two days of culture.
After removing the culture media, the cells were rinsed by PBS 3 times for 3 minutes each time. The cells were then fixed using 4% paraformaldehyde (158127, Sigma-Aldrich, Singapore) for 30 minutes at room temperature (RT), followed by PBS rinsing. After that, the cells were incubated with blocking solution containing 3% BSA (A7906, Sigma-Aldrich, Singapore) and 0.25% TritonX-100 (X100, Sigma-Aldrich, Singapore) for 1 hour at RT. Then, the cells were incubated overnight at 4°C with the appropriate primary antibodies. In particular, mouse anti-MBP (ab62631, 1:500) and rabbit anti-p75NGFR (ab227509, 1:500) antibodies were applied for the single culture of SCs. For the single culture of neurons, mouse anti-NF160 antibody (ab7794, 1:500) was used. For the co-culture of SCs and neurons, mouse anti-NF160 and rabbit anti-MBP (ab40390, 1:200) antibodies were used. Anti-MBP antibodies react with myelin basic proteins that are common participants in myelin sheaths. Anti-p75NGFR reacts with nerve growth factor receptor found on both neuronal cells and glial cells. In glial cells, p75NGFR is a positive modulator for myelination during development. Anti-NF160 antibody reacts with both phosphorylated and non-phosphorylated forms of medium neurofilament protein. All primary antibodies were purchased from Abcam (Cambridge, UK). After washing by PBS, the cells were incubated for 1 hour at RT with Alexa 488 goat anti-mouse (A-11017, 1:1,000, Molecular Probes, Eugene, OR) and Alexa 546 goat anti-rabbit (A-11072, 1:500, Molecular Probes) as the secondary antibodies. After that, the cells were washed by PBS 3 times, each time for 3 minutes.
The effect of hyperglycemia on the survival of SCs was assayed at 7 days of culture in control medium (5.5 mM glucose) and hyperglycemic media (10, 30, and 60 mM) by LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (L3224, Molecular Probes). The protocol was performed according to the instruction of the manufacturer. In brief, the media culture was discarded and the cells were washed gently by PBS to remove FBS remaining in the cell culture dish. The working solution was prepared by diluting calcein AM and EthD-1 in SC medium to the final concentrations of 2 and 4 µM, respectively. A sufficient working solution was added into the experimental wells so that all cells were covered with solution. The well plate was then covered by aluminum foil and transferred to the incubator for 30 minutes. After removing the working solution, the cells were washed twice by PBS and the images were obtained with a laser scanning confocal microscope (LSM 800; Carl Zeiss, Jena, Germany). Each well was divided into five areas: top, bottom, left, right, and center areas. Two photos were acquired randomly at each area.
The proliferation rate of SCs under hyperglycemic culture was evaluated by alamarBlue® (AB) (BUF012B, Bio-Rad, Hercules, CA) staining at 1, 3, 5, and 7 days of culture. SCs were seeded into a 96-well plate at a density of 1,000 cells/well and were grown in SC medium containing 5.5 mM (control), 10 mM, 30 mM, and 60 mM of glucose at the seeding time. After removing the culture medium, the cells were washed three times by PBS (–). AB was then diluted by FBS-free SC medium (1:10) and added into the experimental wells (250 µL each well). After that, the culture plate was protected from light and transferred into the cell incubator for 2 hours. Then, the incubated AB of each well was transferred to 4 wells of a new 96-well plate (50 µL each well) for measurement of the fluorescence density using a microplate reader at the excitation and emission wavelengths of 570 nm and 600 nm, respectively. The number of viable cells correlates with the percentage of AB reduction that was calculated according to the manufacturer’s instruction:
In the formula, εOX and εRED are constants representing the molar extinction coefficient of AB oxidized form (BLUE) and of AB reduced form (RED), respectively. A and A’, respectively, represent the absorbance of test wells (at λ1 = 570 nm and λ2 = 600 nm) and negative control wells that contain SC medium and AB without cells.
All immunofluorescent images were acquired on a laser scanning confocal imaging system LSM 800 (Carl Zeiss).
For evaluation of the de-differentiation of SCs under hyperglycemia, area, mean fluorescence, and several adjacent background readings of MBP- and p75NGFR-single channel of each image were first measured by FIJI (https://fiji.sc/). Then, the total corrected cellular fluorescence (TCCF) was calculated by a formula referred to in a previous paper by McCloy et al. : (TCCF) = integrated density – (area of selected cell × mean fluorescence of background readings). Each well was divided into five areas: top, bottom, left, right, and center areas. Two photos were acquired randomly at each area.
For measurement of neurite length, Simple Neurite Tracer, which is an open plugin of FIJI, was used as described previously by More et al. . At least 4 wells per group were analyzed. In the co-culture of SCs and neurons, the neurites that were positive for both NF-160 and MBP antibodies were considered myelinated neurites. The ones that were only positive for NF-160 antibody were counted as unmyelinated neurites. All neurons that were positive for NF-160 in each well were taken and analyzed.
All experiments were carried out in at least triplicate. The results were analyzed using one-way non-repeated ANOVA followed by Dunnett’s post hoc test (Figs. 1-5), one-way non-repeated ANOVA followed by Tukey’s post hoc test (Fig. 6), and a two-tailed Students
Unlike neurons, SCs can divide indefinitely throughout life . They have no ability to transmit synaptic messages; however, they play important roles in myelinating axons, guiding neurons, and eliminating cellular debris [25,26]. In this study, given that hyperglycemia, a key contributor to the progress of diabetic neuropathy , attenuates the proliferation of SCs, we evaluated the proliferation rate of SCs under hyperglycemic culture. Hyperglycemia was simulated by adjusting the glucose concentration of the SC medium from the basal level 5.5 mM to 10, 30, and 60 mM. These modified media were introduced to SCs at the seeding time and changed every two days to maintain the hyperglycemic condition during the experiments. The cells grown in hyperglycemic insults also presented decreased confluence (Fig. 1A, B). The proliferation of SCs was then assayed by alamarBlue staining at 1, 3, 5, and 7 days of culture under the hyperglycemic condition and compared with that in the normal medium. No major change in the cell density was found in any group after 1 day of culture. However, the cell proliferation in high glucose groups was significantly decreased by 3, 5, and 7 days of culture compared to the control group. Particularly, by 7 days, the number of cells in the control group was 3,076 ± 32.47
In addition, the viability of cells was examined by the LIVE/DEAD® Viability/Cytotoxicity Assay Kit at 7 days. As shown in Fig. 1B, live cells and dead cells fluoresced bright green and red-orange, respectively. In the basal level of glucose, the cells showed a higher density, and only a few dead cells (1.28 ± 0.19%) were found. In contrast, the cells grown in hyperglycemia media had a low density and a significantly higher percentage of dead cells (2.58 ± 0.54%, 3.57 ± 0.59%, and 8.61 ± 0.51%, respectively, in the 10, 30, and 60 mM glucose-treated groups) (F3, 16 = 43.92,
These results showed that hyperglycemia significantly attenuated the proliferation and viability of SCs.
SC myelination plays a crucial role not only in regulating peripheral nerve function and conduction velocity but also in maintaining the architecture of axons [9,27]. In diabetic neuropathy, loss of myelinated fibers is one of the most common reported histologic changes . In addition, previous studies showed that hyperglycemia triggers abnormal signaling pathways in the proliferation and differentiation of SCs [9,10]. In this study, to examine the changes in the differentiation of SCs under hyperglycemia, we analyzed the expression of immature (p75NGFR) and mature markers (MBP) of SCs at 7 days of culture under hyperglycemia. The ratio of fluorescent intensity of MBP and p75NGFR in the control group (3.748 ± 0.1774) was significantly higher than that in the high glucose concentration groups (2.643 ± 0.1433, F3, 20 = 4.845,
Unlike other cell types in our body, neurons have a unique morphology with axons that extend their branches over long distances to form connections with other cells . In addition, neurons have dendrites that play an important role in receiving input from other neurons and cells in the environment . Any abnormalities in the morphology of neurons will result in reduced neuronal activity and increased neurodegeneration. The biological properties, especially neurite outgrowth, were previously reported to be altered under diabetic conditions .
To examine the morphological changes of neurons cultured under conditions mimicking hyperglycemia, we measured the average neurite length of neurons by using an open plugin of FIJI named Simple Neurite Tracer, as described previously by More et al. . As shown in Fig. 3, the average neurite length of both sensory and motor neurons cultured in media containing high concentrations of glucose was significantly shorter than that in the control group. The average neurite length of the sensory neurons in the control group was 2,179.9 ± 106.7 µm, which was significantly longer than in the 45 mM- (1,655.5 ± 43.2 µm, F3, 8 = 32.57,
Formed by SCs in the peripheral nervous system and oligodendrocytes in the central nervous system, myelin is a unique plasma membrane with high lipid content (~70%). Myelin plays a crucial role in accelerating conduction velocity for the rapid conductivity of action potentials along neurons [34,35]. Our results first show that the proliferation and differentiation of SCs are impaired under diabetic conditions (Figs. 1, 2), suggesting that the neurite myelination by SCs will also be affected during diabetes. Indeed, our results showed that compared with the control groups, both sensory and motor neurons co-cultured with SCs under hyperglycemia showed an attenuated percentage of myelinated neurites. In the sensory neuron-SC co-culture, compared to the control group (90.35 ± 0.72%), the percentage of myelinated neurites was significantly impaired in the 35 mM group (85.86 ± 0.39%, F3, 8 = 50.10,
To examine the effect of insulin on neurite extension, a series of insulin solution ranging from 0.01–2 µM was supplemented to the culture media of primary neurons at the seeding time. At two days of culture, neurons were stained with NF-160, and neurite length was measured as described above. In the sensory neurons culture, insulin remarkably increased the neurite length at all examined concentrations, with significant enhancement found at the concentrations 0.01 (F6, 14 = 3.970,
Based on the results of the
The results showed that compared to the untreated group (55.86 ± 1.37%), insulin significantly enhanced neurite myelination to 63.33 ± 0.78%, t (4) = 8.135, ****
Many animal models of diabetic neuropathy have been successfully established by us, with a neuropathy complication developed from type-2 non-obese diabetes , and by others [37–40]. These models showed remarkable contributions to studies on the mechanism and progression of the neuropathy complication of diabetes and elucidated approaches for the treatment of this complication. However, the onset and progression of neuropathy in these animal models has not been totally understood . Understanding of the mechanisms that trigger diabetic neuropathy at the cellular and molecular levels still remains unclear . Thus, researchers have used combined
In this regard, findings achieved from
The difference in the vulnerability level between sensory and motor neurons to diabetes-mediated injury has been noticed in both human diabetic patients and animal models [3,44]. Particularly, sensory neurons are more vulnerable [3,44] and are involved in earlier stages of diabetes . This notion was also observed in our non-obese diabetes model. Seventeen weeks after co-injected by streptozotocin and nicotinamide, mice showed impaired sensory nerve conduction and the number of intraepidermal nerve fibers was significantly decreased. In contrast, impairment of the innervation of motor neurons was not found .
Several reasons have been proposed to explain why sensory neurons are more vulnerable to diabetic neuropathy. Hameed  reported that sensory axons express more distinct voltage gated sodium channels (Nav1.6, 1.7, 1.8, and 1.9) with different biophysical characteristics compared to those in motor axons (Nav1.6). The expression of these sodium channels possesses distinct electrophysiological properties in sensory axons. Specifically, Nav1.7 and 1.9 produce a persistent sodium current near the resting membrane potential. In addition, due to their smaller diameter than motor axons, sensory axons have a higher surface area to volume ratio, resulting in changes in sodium and calcium current and rendering sensory axons more vulnerable to injury . Furthermore, sensory axons are more susceptible to diabetes because of their physiological environment. Sensory neurons are protected by the blood–ganglion barrier, which is more permeable to macromolecules and other constituents than the blood–nerve barrier that protects motor neurons . Thus, DRG sensory neurons are more easily exposed to blood-borne toxins and more dependent on trophic support provided by SCs which are also remarkably affected by diabetes. Sensory neurons also have higher metabolic demand, supported by local blood flow, leaving them at greater risk from diabetic nutrient blood flow . In the present
Insulin, a member of the insulin-like superfamily, in addition to being widely known as a glucose homeostasis regulating hormone, has been identified recently as a neurotrophic factor that plays a role in synthesizing critical structural proteins of neurons . In diabetes, the insulin signaling is disrupted due to insulinopenia (type 1) or insulin resistance (type 2) , as well as insulin deficiency found in both type 1 and type 2 diabetes  that may lead to a delay in nerve regeneration . Previous studies demonstrated that insulin receptors (IRs) are expressed in many neural cell types such as SCs  and DRG . Some interesting questions and hypotheses have been raised about the role of IRs in neural cells because the majority of neurons are not dependent on insulin to take up glucose . Furthermore, the expression of IR in SCs during development parallels the myelin structural protein P0 expression (at both mRNA and protein levels) and the growth of the myelin sheath . These observations suggest that insulin may play a role in myelination and peripheral nerve support. In this study, the authors focused on examining the effect of insulin on myelination in their established
In conclusion, the
The authors would like to thank Dr. Luo Baiwen for helping in the initial set up of isolation of rat embryonic neurons. We also would like to thank Dr. Kazuhiko Nishida and Dr. Shinji Matsumura for a helpful discussion about the method to measure neurite length. We thank Dr. Aishwarya Bandla, Alexis Lowe, and Elizabeth Redmond for reading and giving comments on the manuscript.
Vuong M. Pham: Investigation; Nitish Thakor: Supervision.
No potential conflict of interest relevant to this article was reported.
No funding to declare.