Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture.

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. 2021 Feb 12;6(1):40-47.

doi: 10.1089/can.2019.0102. eCollection 2021.

Affiliations

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Jungnam Kim et al. Cannabis Cannabinoid Res. .

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.

Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.

FIG. 1.

Neurotoxicity of CBD to primary hippocampal neurons. The neurons were treated with CBD (0, 0.1, 1, 3, 5, 10, 15, 30, 50, and 100 μM) at 1 DIV, and the CLSM images were taken after 24 h of incubation. (a) CLSM images of hippocampal neurons (green: live, red: dead). (b) A graph of %viability versus log[CBD]. LC50 was calculated as the concentration at which the %viability was 50%. Data are expressed as mean±SE of n=3, each with three replicates. CBD, cannabidiol; CLSM, confocal laser-scanning microscope; DIV, days in vitro; LC50, lethal concentration 50; SE, standard error.

FIG. 2.

FIG. 2.

Effects of CBD on cell adhesion and morphology. Representative CLSM images of primary hippocampal neurons in CBD (10 μM) or 0.1% DMSO, after staining F-actin (green), cytoplasm (red), and nuclei (blue). DMSO, dimethyl sulfoxide.

FIG. 3.

FIG. 3.

Neurotoxicity of H2O2 to primary hippocampal neurons. The neurons were treated with H2O2 (0, 0.1, 1, 3, 5, 7, 10, 30, and 50 μM) at 1 DIV, and the CLSM images were taken after 24 h of incubation. (a) CLSM images of hippocampal neurons with different H2O2 concentrations. (b) A graph of %viability versus log[H2O2]. LC50 was calculated as the concentration at which the %viability was 50%. Data are expressed as mean±SE of n=3 each with three replicates. H2O2, hydrogen peroxide.

FIG. 4.

FIG. 4.

Neuroprotective effect of CBD against H2O2. Hippocampal neurons were treated with H2O2 (10 μM) and CBD (0, 0.1, 1, 2, 3, and 5 μM) at 1 DIV and incubated for 24 h. (a) CLSM images of primary hippocampal neurons after 24-h incubation with CBD and H2O2. (b) A graph of PR[CBD] versus the CBD concentration. Data are expressed as the mean %viabilities of the [+H2O2][+CBD] cases divided by the mean %viability of the [+H2O2][−CBD] case, repeated n=4 cultures, each with three replicates. The 0.1% DMSO was used for 0-μM CBD.

FIG. 5.

FIG. 5.

High-magnification CLSM images of hippocampal neurons after 24-h treatment of (middle) H2O2 (10 μM), and (right) H2O2 (10 μM), and CBD (5 μM). The cells were stained in green (F-actin), red (β-tubulin III), and blue (nuclei).

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