Dexmedetomidine attenuates the neurotoxicity of propofol toward primary hippocampal neurons in vitro via Erk1/2/CREB/BDNF signaling pathways

Abstract

Background: Propofol is a commonly used general anesthetic for the induction and maintenance of anesthesia and critical care sedation in children, which may add risk to poor neurodevelopmental outcome. We aimed to evaluate the effect of propofol toward primary hippocampal neurons in vitro and the possibly neuroprotective effect of dexmedetomidine pretreatment, as well as the underlying mechanism.

Materials and procedures: Primary hippocampal neurons were cultured for 8 days in vitro and pretreated with or without dexmedetomidine or phosphorylation inhibitors prior to propofol exposure. Cell viability was measured using cell counting kit-8 assays. Cell apoptosis was evaluated using a transmission electron microscope and flow cytometry analyses. Levels of mRNAs encoding signaling pathway intermediates were assessed using qRT-PCR. The expression of signaling pathway intermediates and apoptosis-related proteins was determined by Western blotting.

Results: Propofol significantly reduced cell viability, induced neuronal apoptosis, and downregulated the expression of the BDNF mRNA and the levels of the phospho-Erk1/2 (p-Erk1/2), phospho-CREB (p-CREB), and BDNF proteins. The dexmedetomidine pretreatment increased neuronal viability and alleviated propofol-induced neuronal apoptosis and rescued the propofol-induced downregulation of both the BDNF mRNA and the levels of the p-Erk1/2, p-CREB, and BDNF proteins. However, this neuroprotective effect was abolished by PD98059, H89, and KG501, further preventing the dexmedetomidine pretreatment from rescuing the propofol-induced downregulation of the BDNF mRNA and p-Erk1/2, p-CREB, and BDNF proteins.

Conclusion: Dexmedetomidine alleviates propofol-induced cytotoxicity toward primary hippocampal neurons in vitro, which correlated with the activation of Erk1/2/CREB/BDNF signaling pathways.

Keywords: brain-derived neurotrophic factor; cyclic AMP response element-binding protein; dexmedetomidine; extracellular signal-regulated MAP kinases; hippocampus; propofol.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1

Experimental groups and processing. Notes: Replace the culture medium in Group C with fresh maintenance medium. The cells in Group PDP, HDP, and KDP were preincubated with 25 µM PD98059 (inhibitor of Erk1/2 phosphorylation), 10 µM H89 (inhibitor of CREB phosphorylation), and 25 µM KG-501 (CREB inhibitor) for 30 minutes, respectively, and the cells in Group DMSO were preincubated with 0.25% DMSO, which served as a vehicle control. Then, cells in Group PDP, HDP, and KDP were treated with 10 µM dexmedetomidine and incubated for additional 30 minutes. Meanwhile, the cells in Group D and PD were pretreated with 10 µM dexmedetomidine for 30 minutes. Finally, the cells in Group P, PD, PDP, HDP, and KDP were treated with 100 µM propofol and incubated at 37°C for 3 hours. The cells in Group I were exposed to intralipid vehicle, which also served as a vehicle control. Abbreviations: DMSO, dimethyl sulfoxide; 8 DIV, 8 days in vitro.

Figure 2

Changes in the morphology of primary hippocampal neurons viewed under a light microscope. Notes: (A) Four hours after plating, most of the hippocampal neurons were clearly adherent, and the cells were spherical, elliptical, or irregularly cone-shaped, surrounded by obvious halos. Small dendrites of different lengths extended from some neurons, and connections were visible at the junctions. (B) After 24 hours, the cells were completely adherent, and most of the cells extended three to four dendrites. The dendrites extended and began to produce small branches. The cell bodies were translucent, showing a strong three dimensionality and refraction. (C, D) After 3–5 days, the hippocampal neurons exhibited various shapes, showing unipolar, bipolar, or multipolar processes. The cell body was highly refractive and displayed pronounced halos. The dendrites had further elongated and thickened. The cell bodies began to aggregate, and the dendrites connected to each other to form a sparse network. (E) After 7 days, the dendritic connections between hippocampal neurons were more closely linked, with interwoven protrusions that constitute a rich and dense neural network. The aggregation of neuronal cell bodies was more obvious. (F) After the ninth day, neurons gradually degenerated, the cell body shrunk, deformed and aggregated, the refractive index weakened, and the neural network became sparse.

Figure 3

Primary hippocampal neurons (8 DIV) were identified by anti-NSE immunocytochemical staining, and a purity analysis was performed. Notes: (A, B) Under the light microscope, the hippocampal neuronal cell bodies, dendrites, and axons were stained brown, and the axons and dendrites formed synaptic connections between the cells in a reticular pattern. Nuclei were stained blue with DAPI. Thus, rat hippocampal neuronal cells were successfully cultured in vitro. The proportion of NSE-positive hippocampal neurons among the total number of cells in the visual field was counted in 10 fields of view. The purity of hippocampal neurons was 95.2%±3.6% (n=10 filed). Abbreviation: 8 DIV, 8 days in vitro.

Figure 4

Dexmedetomidine attenuated propofol-induced apoptosis and increased neuronal viability. Notes: (A) The neurons displayed vacuolar degeneration, chromatin margination, cell shrinkage, and a large number of apoptotic bodies after propofol exposure. Pretreatment with 10 µM dexmedetomidine significantly alleviated the propofol-induced neuroapoptosis, with fewer degenerating vacuoles and apoptotic bodies observed under transmission electron microscope (TEM). (B) Propofol exposure significantly reduced the neuronal viability by 32.71% compared with Group C. Dexmedetomidine remarkably increased neuronal viability by 20.85% compared with cells exposed to propofol without the dexmedetomidine pretreatment (one-way ANOVA, *P<0.001, n=18). (C, D) Propofol markedly increased the percentage of apoptotic neurons compared with Group C (one-way ANOVA, *P<0.001, n=3). Dexmedetomidine pretreatment significantly rescued neurons from propofol-induced apoptosis (one-way ANOVA, *P<0.001 in comparing with Group P, n=3). (E–I) Propofol exposure significantly increased the levels of the cleaved-caspase3 protein and decreased the Bcl-2/Bax ratio (one-way ANOVA, *P<0.001 in comparing with Group C, n=9). The dexmedetomidine pretreatment evidently increased the Bcl-2/Bax ratio and decreased the levels of the cleaved-caspase3 protein in hippocampal neurons (one-way ANOVA, *P<0.001 in comparing with Group P, n=9).

Figure 5

Levels of the Erk1/2, CREB, and BDNF mRNAs in primary hippocampal neurons exposed to propofol with or without pretreatment with dexmedetomidine and with inhibitors. Notes: (A) Propofol markedly downregulated the expression of the BDNF mRNA (one-way ANOVA, *P<0.001 in comparing with Group C, n=9). The expression of the BDNF mRNA was notably upregulated by pretreatment with 10 µM dexmedetomidine for 30 minutes (one-way ANOVA, *P<0.001 in comparing with Group P, n=9) (B) and was then significantly downregulated by preincubation with inhibitors 30 minutes prior to dexmedetomidine pretreatment (one-way ANOVA, *P<0.001 in comparing with Group PD, n=9) (C). No significant differences were observed in the expression of the Erk1/2 and CREB mRNAs between groups (one-way ANOVA, P>0.05 in comparing with Group C, P, or PD, n=9).

Figure 6

Levels of the Erk1/2, p-Erk1/2, CREB, p-CREB, and BDNF proteins in primary hippocampal neurons exposed to propofol following pretreatment with or without dexmedetomidine as well as inhibitors. Notes: (A) ERK-activated RSK phosphorylates the transcription factor CREB at serine 133 (Ser-133), which promotes the recruitment of the coactivators CBP and p300 and strongly enhances CREB-dependent transcription. Additionally, the PKA complex is composed of two catalytic and two regulatory subunits; the dissociated catalytic subunits are active and capable of activating CREB by phosphorylating it at Ser-133. The phosphorylation of CREB at Ser-133 (p-CREB) regulates the transcription of pro-survival factors, including BDNF and Bcl-2. (B–E) Propofol significantly decreased levels of the p-Erk1/2, p-CREB, and BDNF proteins compared with Group C (one-way ANOVA, *P<0.001, n=9). Pretreatment with dexmedetomidine evidently rescued the propofol-induced decrease in levels of the p-Erk1/2, p-CREB, and BDNF proteins (one-way ANOVA, *P<0.001 in comparing with Group P, n=9). The inhibitors prevented the dexmedetomidine pretreatment from rescuing the propofol-induced decrease in levels of the p-Erk1/2, p-CREB, and BDNF proteins (one-way ANOVA, *P<0.001 in comparing with Group PD, n=9). Abbreviation: DMSO, dimethyl sulfoxide.

Figure 7

The neuroprotective effect of dexmedetomidine was partially abolished by PD98059, H89, and KG501. Notes: (A) Primary hippocampal neurons were preincubated with 25 µM PD98059, 10 µM H89, or 25 µM KG501 for 30 minutes before pretreatment with 10 µM dexmedetomidine. Compared with Group PD, preincubation with inhibitors significantly abolished the neuroprotective effects of dexmedetomidine, with more degenerating vacuoles and apoptotic bodies observed in propofol-treated neurons under the TEM compared with Group PD. In addition, inhibitors markedly reduced the neuronal viability (one-way ANOVA, *P<0.001 in comparing with Group PD, n=18) (B) and increased the percentage of apoptotic hippocampal neurons (one-way ANOVA, *P<0.001 in comparing with Group PD, n=3) (C, D). (E–I) Preincubation with inhibitors significantly increased the level of the cleaved-caspase3 protein and reduced the Bcl-2/Bax ratio in hippocampal neurons (one-way ANOVA, *P<0.001 in comparing with Group PD, n=9). Abbreviation: TEM, transmission electron microscope.