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.
(−)-Cannabidiol (CBD, IUPAC name: 2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol, C21H30O2), the main nonpsychoactive compound among 100+ phytocannabinoids extracted from the genus Cannabis, anecdotally—if not clinically—shows potential therapeutic benefits in various health care applications, including epilepsy, inflammation, cancer, and neurodegenerative diseases.1–5 The year of 2018 witnessed the first approval of Cannabis-derived CBD (Epidiolex®) for the treatment of epileptic seizures associated with the Lennox–Gastaut or Dravet syndrome in patients 2 years of age and older by the U.S. Food and Drug Administration (FDA).6–8
In addition to the therapeutic effect on epileptic seizures, previous reports suggest that CBD is neuroprotective. The earliest research involved the neuroprotective action of CBD in the rat cortical neuron culture exposed to toxic levels of glutamate and tert-butyl hydroperoxide.9 The bidirectional Ca2+ regulation capability of CBD for primary hippocampal cells (neuron:glia=2:1) was reported, indicating the regulatory role of CBD in Ca2+ homeostasis10–12: for example, the intracellular calcium concentration ([Ca2+]i) was reduced by about 25% of basal Ca2+ level in both neurons and glia by CBD application to a HEPES-based buffer solution with double K+ concentration, while CBD caused the [Ca2+]i elevation, with no significant cell death, by about 45% of basal Ca2+ level under physiological conditions. The neuroprotective properties of CBD against oligomycin, an inhibitor of ATP synthetase,10 and ammonium acetate and ethanol in the hippocampal neuron culture also have been reported.13
Oxidative stress is thought to cause pathological conditions in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotropic lateral sclerosis.14,15 The cytoprotective effect of CBD against hydrogen peroxide (H2O2), a crucial metabolic molecule in the oxidative stress,16 was reported for cerebellar granule cells17 and oligodendrocyte progenitor cells,18 but there have been no reports on its effect against H2O2 in the neurons that are more relevant to the brain function, such as hippocampal neurons.19 H2O2 has been used as a model molecule to study oxidative stress-induced neural death and also to screen neuroprotective agents on the cultured neurons.16,20–25 As our on-going research program on neurochemistry and neurophysiology,26–31 this study investigates the neuroprotective effect of CBD against H2O2 in the hippocampal neuron culture.
In addition to the neuroprotective property, CBD has also been reported to show different toxicity profiles on different brain cell types. For example, even 0.1 μM of CBD increased [Ca2+]i and induced cell death in the case of optic nerve oligodendrocytes after 24 h of incubation, while 10 μM of CBD did not affect the viability of cortical neurons (CBD exposure time: 20–30 min).32 However, no cytotoxicity was observed with 1 μM of CBD for oligodendrocyte progenitor cells (CBD exposure time: 24 h).18 Furthermore, CBD of below 4 μM did not significantly increase hypodiploid apoptotic cells for primary microglial cells, but CBD of above 8 μM induced apoptosis in a time- and concentration-dependent manner, confirmed by increase in hypodiploid cells and DNA-strand breaks, and activation of capase-8 and -9 (CBD exposure time: 24 h).33 Considering the previous somewhat contradictory results, we first systematically evaluated the in vitro neurotoxic effect of CBD to hippocampal neurons with different CBD concentrations. (Read Full Report)