Neuron-glia crosstalk plays a major role in the neurotoxic effects of ketamine via extracellular vesicles

Document Type

Conference Proceeding

Publication Date

5-1-2021

Publication Title

Anesth Analg

Abstract

INTRODUCTION: There is overwhelming evidence from animal studies that general anesthetics (GA) lead to neurodevelopmental abnormalities including cell death, cognitive and behavioral changes.1 Human studies have not been conclusive and are challenging since they must account for numerous confounding factors. There is now powerful evidence for non-cell autonomous mechanisms2 in almost every pathological condition in the brain, especially relevant to glial cells3, mainly astrocytes and microglia, that exhibit structural and functional contacts with neurons. These interactions were recently reported to occur via the secretion of extracellular vesicles (EVs) that play important roles in both physiological and pathological pathways4. EVs carry a specific cargo consisting of RNA molecules, proteins and lipids. Dysregulated EV-related cargo and communication have been implicated in a variety of pathological conditions5, including stroke, brain injury and neurodevelopmental disorders. Here, we employed primary human neural cells to analyze ketamine effects, focusing on the functions of glial cells and their polarization/differentiation state. We also explored the roles of extracellular vesicles (EVs) and different components of the BDNF pathway. METHODS: Ketamine effects on neuronal and glial cell death were analyzed using live/dead assay, caspase 3 activity and PARP-1 cleavage. Astrocytic (A1 vs. A2) and microglial (M1 vs. M2) cell differentiation were determined using RT-PCR and phagocytosis assays. The impact of the neuron-glial cell interactions in the neurotoxic effects of ketamine was analyzed using transwell cultures. The roles of the brain-derived neurotrophic factor (BDNF) pathway, including levels of BDNF, pro-BDNF, the lncRNA BDNF-AS and the receptors p75 and TrkB were analyzed using RT-PCR, ELISA western blot and gene silencing. EVs secreted by ketamine-treated cells were isolated, characterized and analyzed for their effects in neuron-glia cell interactions. The results are presented as the mean values ± SE. Data were analyzed using analysis of variance or a Student's t test with correction for data sets with unequal variances. RESULTS: Ketamine induced neuronal and oligodendrocytic cell apoptosis and promoted the expression of pro-inflammatory astrocytes (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 oligodendrocytes. In addition, ketamine treatment increased the lncRNA BDNF-AS levels and the secretion of pro-BDNF secretion in both neurons and astrocytes. We found an important role of EVs secreted by ketamine-treated astrocytes in neuronal cell death. Using knockdown experiments, we demonstrated that EVs secreted from ketamine-treated astrocytes expressed high levels of BDNF-AS and that silencing of the expression of this lncRNA in astrocytes abrogated the increased ketamine toxicity in neuron-astrocytes cocultures, indicating a role for EV-associated BDNF-AS in this effect. CONCLUSION: Ketamine exerted a complex neurotoxic effect on neural cells by impacting both neuronal and glial cells, therefore indicating that ketamine neurotoxicity involves both autonomous and non-cell autonomous mechanisms. We identified the role of different components of the BDNF pathway expressed by neurons and glial cells as major regulators of ketamine effects. Finally, we demonstrated for the first time a role of EVs as important mediators of ketamine effects by the delivery of specific non-coding RNAs between cells. These results may contribute to a better understanding of cellular and molecular mechanisms underlying ketamine neurotoxic effects in humans and to the development of potential approaches to decrease its neurodevelopmental impact. (Figure Presented).

Volume

132

Issue

5

First Page

558

Last Page

559

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