Neuron-Glia Crosstalk Plays a Major Role in the Neurotoxic Effects of Ketamine via Extracellular Vesicles

Abstract

Background: There is a compelling evidence from animal models that early exposure to clinically relevant general anesthetics (GAs) interferes with brain development, resulting in long-lasting cognitive impairments. Human studies have been inconclusive and are challenging due to numerous confounding factors. Here, we employed primary human neural cells to analyze ketamine neurotoxic effects focusing on the role of glial cells and their activation state. We also explored the roles of astrocyte-derived extracellular vesicles (EVs) and different components of the brain-derived neurotrophic factor (BDNF) pathway. Methods: Ketamine effects on cell death were analyzed using live/dead assay, caspase 3 activity and PARP-1 cleavage. Astrocytic and microglial cell differentiation was determined using RT-PCR, ELISA and phagocytosis assay. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell cultures. In addition, the role of isolated and secreted EVs in this cross-talk were studied. The expression and function of different components of the BDNF pathway were analyzed using ELISA, RT-PCR and gene silencing. Results: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted pro-inflammatory astrocyte (A1) and microglia (M1) phenotypes. Astrocytes and microglia enhanced the neurotoxic effects of ketamine on neuronal cells, whereas neurons increased oligodendrocyte cell death. Ketamine modulated different components in the BDNF pathway: decreasing BDNF secretion in neurons and astrocytes while increasing the expression of p75 in neurons and that of BDNF-AS and pro-BDNF secretion in both neurons and astrocytes. We demonstrated an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death and a role for EV-associated BDNF-AS in this effect. Conclusions: Ketamine exerted a neurotoxic effect on neural cells by impacting both neuronal and non-neuronal cells. The BDNF pathway and astrocyte-derived EVs represent important mediators of ketamine effects. These results contribute to a better understanding of ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact.

Keywords: BDNF; BDNF-AS; astrocytes; ketamine; microglia; neurotoxicity.

FIGURE 1

Neurotoxic effects of ketamine on human neuronal cells. Human neurons were treated with ketamine for 6 h. The medium was replaced with fresh medium and% dead cells was determined following 48 h using the live/dead assay (A), caspase 3 activity (B) and analysis of cleaved PARP by Western blot analysis (C). The results are the means ± SD of three different experiments analyzed in quadruplet (A,B) or a representative of three independent experiments (C) ***P < 0.001.

FIGURE 2

Ketamine induces activation of astrocytes and microglia cells. Human astrocytes were treated with ketamine and were analyzed after 48 h for the expression of A1 (C3) and the A2 (S100A10) markers using RT-PCR (A) and Western blot analysis (B). The expression of the glutamate transporter EAAT2 mRNA was analyzed using RT-PCR (C) and Western blot analysis (D). Human microglia cells were treated with ketamine and the relative expression of M1 and M2 markers was analyzed using RT-PCR (E). The ketamine treated microglia cells were also analyzed for secretion of IL-1β (F) and IL-13 (G) using ELISA and for phagocytosis using the pHrodo^TM assay (H). The results are the means ± SD of a three independent experiments (A,C,E,H) or are a representative of three independent experiments (B,D,F,G). *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 3

Ketamine inhibits cell proliferation and increases cell death in oligodendrocyte precursor cells. Human oligodendrocyte progenitor cells were treated with ketamine and analyzed 48 h later for cell proliferation (A) and cell death using the live/dead assay (B) and caspase 3 activity (C). The results are the means ± SD of four independent test analyzed in triplicates. *P < 0.05 and ***P < 0.001.

FIGURE 4

Ketamine regulates the BDNF pathway in neurons and astrocytes. Human neurons were treated with ketamine and the expression of BDNF mRNA was determined using RT-PCR (A) and BDNF secretion by ELISA (B). Pro-BDNF secretion was analyzed using ELISA (C) and the expression of p75^NTR and TrkB was analyzed using Western blot analysis (D). The effect of ketamine on BDNF (F) and pro-BDNF (G) secretion in astrocytes was analyzed using ELISA. The expression of the lncRNA BDNF-AS was analyzed using RT-PCR in both neurons (E) and astrocytes (H). The results are the means ± SD of three independent experiments analyzed in quadruplets or are a representative of three independent experiments (D). *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 5

The role of neuron-glia interactions in the neurotoxic effects of ketamine. Neurons and astrocytes (A), neurons and microglia (B) or neurons and oligodendrocytes (C) were plated alone or co-cultured in transwell plates with a 1-μm filter. The co-cultures were treated with ketamine and caspase 3 activity was determined for the cultured cells after 48 h. The results are the means ± SD of six independent experiments analyzed in triplicates. ***P < 0.001 (co-cultured neurons vs. neurons alone (A,B) or co-cultured oligodendrocytes vs. oligodendrocytes alone (C). **P < 0.01 and ***P < 0.001 (ketamine-treated cells vs. controls).

FIGURE 6

Astrocyte-secreted EVs mediate the increased neurotoxic effects of ketamine in neuron-astrocyte co-cultures. Cultured neurons were treated with EVs isolated from control or ketamine-treated astrocytes. Percent of dead cells was determined after 48 h (A). To further analyze the role of EVs secreted from astrocytes, these cells were pre-treated with GW4869 (20 μM) prior to ketamine treatment. The treated astrocytes were then co-cultured with neurons and treated with ketamine (50 μM). Neuronal cells death was analyzed after 48 h (B). A similar treatment of GW4869 was performed in neuronal cells cultured alone (B). The expression of BDNF-AS in EVs isolated from control and ketamine-treated astrocytes was analyzed by RT-PCR (C). EVs isolated from astrocytes that were silenced for BDNF-AS and then treated with ketamine were added to neuronal cultures. Cell death was determined after 48 h (D). The results are the means ± SD of six independent experiments analyzed in quadruplets. **P < 0.01 and ***P < 0.001.

FIGURE 7

A diagram summarizing the roles of neuron-glia interactions, the BDNF pathway and extracellular vesicles in ketamine’s effects. The effects of ketamine on various components of the BDNF pathway, the interactions of neuron and glial cells and the role of EVs as mediators of these interactions are depicted in this diagram.