Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture

Abstract

Introduction: Reports on the neurotoxic and neuroprotective effects of cannabidiol (CBD) have not been in complete accord, showing different and somewhat contradictory results depending upon the brain cell types and experimental conditions employed. This work systematically examines the neuroprotective capability of CBD against oxidative stress (i.e., hydrogen peroxide [H₂O₂]) as well as its toxicity profile in the in vitro culture platform of primary hippocampal neurons. Materials and Methods: The low cell-density (100 neurons per mm²) culture was used for analyzing the viability and morphology of neurons at a single-cell level with a confocal laser-scanning microscope (CLSM). Primary neurons were obtained from the hippocampal tissues of embryonic day-18 (E18) Sprague-Dawley rat pups and treated with CBD (0.1-100 μM) and/or H₂O₂ (0.1-50 μM) at 1 DIV (days in vitro). Results: The lethal concentration 50 (LC₅₀) value (the concentration causing 50% cell death) of CBD was calculated to be 9.85 μM after 24 h of incubation, and that of H₂O₂ was 2.46 μM under the same conditions. The neuroprotection ratio of CBD against H₂O₂ ([H₂O₂]=10 μM) was 2.40 with 5 μM of CBD, increasing the cell viability to 57% from 24%. The CLSM analysis suggested that the cell-death mechanisms were different for CBD and H₂O₂, and CBD did not completely rescue the morphological alterations of primary hippocampal neurons caused by H₂O₂, such as neurite degeneration, at least in the in vitro neuron culture. Conclusion: Although CBD showed both neurotoxic and neuroprotective effects on hippocampal neurons in the in vitro setting, the use of low-concentrated (i.e., 5 μM) CBD, not causing toxic effects on the neurons, significantly rescued the neurons from the oxidative stress (H₂O₂), confirming its neuroprotection capability.

Keywords: cannabidiol; cell culture; hydrogen peroxide; neuroprotection; primary hippocampal neurons.