Carnosic Acid Shows Higher Neuroprotective Efficiency than Edaravone or Ebselen in In Vitro Models of Neuronal Cell Damage

Abstract

This study compared the neuroprotective efficacy of three antioxidants—the plant-derived carnosic acid (CA), and two synthetic free radical scavengers: edaravone (ED) and ebselen (EB)—in in vitro models of neuronal cell damage. Results showed that CA protected mouse primary neuronal cell cultures against hydrogen peroxide-induced damage more efficiently than ED or EB. The neuroprotective effects of CA were associated with attenuation of reactive oxygen species level and increased mitochondrial membrane potential but not with a reduction in caspase-3 activity. None of the tested substances was protective against glutamate or oxygen-glucose deprivation-evoked neuronal cell damage, and EB even increased the detrimental effects of these insults. Further experiments using the human neuroblastoma SH-SY5Y cells showed that CA but not ED or EB attenuated the cell damage induced by hydrogen peroxide and that the composition of culture medium is the critical factor in evaluating neuroprotective effects in this model. Our data indicate that the neuroprotective potential of CA, ED, and EB may be revealed in vitro only under specific conditions, with their rather narrow micromolar concentrations, relevant cellular model, type of toxic agent, and exposure time. Nevertheless, of the three compounds tested, CA displayed the most consistent neuroprotective effects.

Protective Effects of Cannabidiol (CBD) against Qxidative Stress, but Not Excitotoxic-Related Neuronal Cell Damage—An In Vitro Study

“…The OGD model is commonly used for in vitro experimental modeling of stroke where excitotoxic processes are predominant [25][26][27]. In such a model in primary neuronal cell culture, we observed neuroprotective effects of NMDA receptor antagonist MK-801 (1 µM, given before + after OGD) at the level of cell viability (Figure 3C) as well as the LDH release (Figure 3D).…”

“…The intracellular ROS level was measured with 5-(and 6-)-chloromethyl-2 ′ ,7 ′ -dichl orodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) as described previously [27]. The cells were washed with pre-warmed FluoroBrite™ DMEM and loaded with 5 µM CM-H 2 DCFDA dissolved in FluoroBrite™ DMEM and located for 10 min in an incubator.…”

“…The tetramethylrhodamine ethyl ester (TMRE) was used for MMP measurement, as described previously [27]. The cells were pretreated for 30 min with CBD (0.01-5 µM) followed by 6 h exposure to H 2 O 2 (0.2 mM).…”

“…Primary neuronal cell cultures growing in 96-well format were treated with CBD (0.01-5 µM) and H 2 O 2 (0.2 mM) for 9 h. Caspase-3 inhibitor, Ac-DEVD-CHO (20 µM), was used as a positive control for the measurement. Caspase-3 activity was measured in cell lysates using the fluorogenic substrate Ac-DEVD-AMC (50 µM) as described previously [27]. The data were normalized to vehicle-treated cells (100%) and presented as the mean ± SEM from 4 independent experiments with 3-5 replicates each.…”

“…Primary neuronal cell cultures growing on poly-L-ornithine (0.05 mg/mL)-covered round cover glasses (diameter 11 mm) in 24-well plate format after 30 min pretreatment with CBD (0.1-1 µM) followed by 24 h exposure to H 2 O 2 (0.2 mM) were fixed with 4% paraformaldehyde and immunostained with neuronal (mouse anti-MAP-2 antibody) and astrocyte (rabbit anti-GFAP antibody) markers as described previously [27]. After staining with fluorescently labeled secondary antibodies (goat anti-mouse IgG (H+L) AlexaFluor ® 488 and donkey anti-rabbit IgG (H+L) AlexaFluor ® 568), the cells were counterstained with nuclear dye Hoechst 33342 and mounted in ProLong ® Gold antifade reagent).…”