Assessing the developmental effects of fentanyl and impacts on lipidomic profiling using neural stem cell models

Abstract

Fentanyl is a potent and short-acting opioid that is often given to pediatric patients during surgery to relieve pain and as an adjunct to anesthesia. Its effects on the developing brain are yet to be determined. In the present study, commercially available human neural stem cells (NSCs) were used to model the effects of fentanyl on the developing human brain. We determined the dose dependent effects and temporal relationships between fentanyl exposures and NSC health, viability, and differentiation. Markers of mitochondrial health [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT)] and cell death/damage [lactate dehydrogenase (LDH)] were monitored to determine the dose response effects of fentanyl on NSC viability. In addition, lipidomics analysis was conducted to investigate lipid profile changes in differentiated neural cells treated with fentanyl. Fentanyl did not cause a significant increase in LDH release, nor MTT reduction after 24-h exposure at concentrations of 0.5, 1.0, 3.0, 10, or 100 μM, for both NSCs and differentiated neural cells. Lipidomics data showed the top 15 most variable important in projection (VIP) lipid species (the higher the VIP scores, the bigger changes in treated groups vs. controls), including lysophosphatidylcholines (LPCs), lysophosphatidylethanolamines (LPEs), ceramides (CER), cholesterol esters (ChEs) and sphingosine (SPH). The lipidomic data indicate that LPC (16:0), LPC (16:1), LPC (18:1), CER (d18:0_22:0), CER (d18:2_18:0), CER(d18:2_24:1) were significantly increased, and only ChE (24:5) and SPH (d18:1) were significantly decreased in the highest dose group versus control. These data indicated that fentanyl exposure (24-h) did not induce detectable cell death. However, a lipidomic analysis indicated that fentanyl may affect immature neural cell functions through modifying lipid composition and lipid metabolism. These data indicated that despite the absence of clear neurodegeneration, fentanyl may still have a negative impact on the developing brain.

Keywords: anesthetics; development; fentanyl; lipidomic analysis; neurotoxicity.

FIGURE 1

NSCs are multipotent cells in the nervous system. They have the features of being able to self-renew and give rise to differentiated progenitor cells to generate lineages of neurons as well as glia, such as astrocytes and oligodendrocytes. Human NSCs were seeded at a cell density of 1 × 10⁶/mL. When the cultures were maintained in the growth medium, the bipolar NSCs continuously proliferated.

FIGURE 2

NSCs differentiated into neural cells when the cultures were maintained in neural differentiation medium. It is shown that most cells are differentiated with multiple processes and a neural network is formed. Based on their morphology, ^ represent a typical neuron with an axon and multiple dendrite processes; * represents a differentiated oligodendrocyte and # indicates an astrocyte.

FIGURE 3

To determine the dose response effects and temporal relationships of fentanyl on cell viability, NSCs were exposed to fentanyl for 24 h at concentration of 0.5, 1.0, 3.0, 10 or 100 µM. Fentanyl exposure resulted in a slight dose-related increase (not significant) in the release of LDH (A) into the cell culture medium, compared with controls. No reduction in MTT (B) was observed in the fentanyl exposed group, compared with controls.

FIGURE 4

Exposure of differentiated cells to fentanyl for 24 h did not lead to changes in LDH release (A) or MTT reduction (B). Each treatment condition was assessed at least in triplicate, and experiments were repeated three times independently.

FIGURE 5

Principal component analysis (PCA) score plots (A), the top 15 variable importance in projection (VIP) plot (B) of lipidomics data from differentiated neural cells (derived from NSCs) of control (Ctr), treated with fentanyl 1 µM (FL), fentanyl 10 µM (FM) or fentanyl 100 µM (FH), for 24 h. Dark red indicates that the normalized level is higher than the average; dark blue indicates that the normalize level is lower. C = control; Fentanyl 1 µM (FL); Fentanyl 10 µM (FM); or Fentanyl 100 µM (FH). Bar graphs of the ratio of AcCa (16:0) (C), an intermediate metabolite of fatty acid β-oxidation in mitochondrial. Fentanyl 1 µM (FL); Fentanyl 10 µM (FM) or Fentanyl 100 µM (FH).

FIGURE 6

Bar graphs of the ratios of LPC (A), Hex2Cer (B), ChE (C), SPH (D) and CER (E) lipids (treated/Ctr). Ratios of LPC, CER and Hex2Cer had a dose-dependent increase, while ratios of ChE and SPH lipids had dose-dependent decreases. Ratios of CER had a dose-dependent increase (E). * Indicating ratios are significantly changed (vs. Control). Fentanyl 1 µM (FL, blue bar); Fentanyl 10 µM (FM, red bar) or Fentanyl 100 µM (FH, grey bar).

FIGURE 7

Summary illustration of the identified lipids involved in the ceramide pathways after fentanyl exposure, for 24 h. The scheme of the pathway is simplified and shows only relevant lipids. The bar graph shows the ratio of total lipid class level [(Sum (treated)/Sum (Control)]. The green (down-regulation) and pink (up-regulation) represent the changes in the total lipid class levels following fentanyl treatment. *Indicating ratios are significantly changed (vs. Control). C = control; Fentanyl 1 µM (FL); Fentanyl 10 µM (FM); or Fentanyl 100 µM (FH).