The neuroprotective effect of cannabidiol in an in vitro model of newborn hypoxic–ischemic brain damage in mice is mediated by CB₂ and adenosine receptors

• A. Castillo^a,
• M.R. Tolón^a, b,
• J. Fernández-Ruiz^b, c,
• J. Romero^a, b,
• J. Martinez-Orgado^{a, b, d, ,} 

• ^a Laboratorio de Apoyo a la Investigación, Hospital Universitario Fundación Alcorcón, Spain
• ^b Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
• ^c Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Spain
• ^d Neonatología, Servicio de Pediatría, Hospital Universitario Puerta de Hierro, Madrid, Spain.

• http://dx.doi.org/10.1016/j.nbd.2009.10.023, How to Cite or Link Using DOI

• Permissions & Reprints 

  Abstract

  To investigate the mechanisms involved in cannabidiol (CBD)-induced neuroprotection in hypoxic–ischemic (HI) immature brain, forebrain slices from newborn mice underwent oxygen and glucose deprivation in the presence of vehicle, or CBD alone or with selective antagonists of cannabinoid CB₁ and CB₂, and adenosine A₁ and A₂ receptors. CBD reduced acute (LDH efflux to the incubation medium) and apoptotic (caspase-9 concentration in tissue) HI brain damage by reducing glutamate and IL-6 concentration, and TNFα, COX-2, and iNOS expression. CBD effects were reversed by the CB₂ antagonist AM630 and by the A_2A antagonist SCH58261. The A_1A antagonist DPCPX only counteracted the CBD reduction of glutamate release, while the CB₁ antagonist SR141716 did not modify any effect of CBD. In conclusion, CBD induces robust neuroprotection in immature brain, by acting on some of the major mechanisms underlying HI cell death; these effects are mediated by CB₂ and adenosine, mainly A_2A, receptors.

  Keywords

  • Cannabidiol;
  • Adenosine;
  • Neuroprotection;
  • Hypoxia–ischemia;
  • Newborn;
  • Mice

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  Fig. 1. Temporal profile of LDH efflux in brain slices from 7- to 10-day-old C57BL6 mice, maintained in oxygenated glucose-containing physiological solution (control group, Δ), or exposed to oxygen–glucose deprivation (OGD) for 30 min with vehicle (●) or cannabidiol 100 μM (○). Points represent the mean ± SEM of 14 to 30 experiments. SeeMethods for details. (⁎) ANOVA p < 0.05 vs. control, (†) ANOVA p < 0.05 vs. OGD + cannabidiol.

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  Fig. 2. LDH efflux to the incubation medium (area under the curve) (top) and tissue caspase-9 concentrations (bottom) in brain slices from 7- to 10-day-old C57BL6 mice, maintained in oxygenated glucose-containing physiological solution (CTL), or exposed to oxygen–glucose deprivation (OGD) for 30 min with vehicle (VEH), or cannabidiol (CBD) alone or with the CB₂ receptor antagonist AM630, the A_1A receptor antagonist DPCPX, or the A_2A receptor antagonist SCH58261 (all 100 μM). See Methods for details. Bars represent the mean ± SEM of 14 to 30 experiments. Different letters represent statistical differences (p < 0.05) by ANOVA with Newman–Keuls test for multiple comparisons.

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  Fig. 3. Concentrations of glutamate (Glu) in the incubating medium of brain slices from 7- to 10-day-old C57BL6 mice, maintained in oxygenated glucose-containing physiological solution (CTL), or exposed to oxygen–glucose deprivation (OGD) for 30 min with vehicle (VEH), or cannabidiol (CBD) alone or with the CB₂ receptor antagonist AM630, the A_1Areceptor antagonist DPCPX, or the A_2A receptor antagonist SCH58261 (all 100 μM). Glutamate concentration was measured at the end of the OGD or equivalent period (Glu0) or 30 min later (Glu30). See Methods for details. Bars represent the mean ± SEM of 14 to 30 experiments. Different letters represent statistical differences (p < 0.05) by ANOVA with Newman–Keuls test for multiple comparisons.

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  Fig. 4. Determinations of iNOS expression in brain slices from 7- to 10-day-old C57BL6 mice, maintained in oxygenated glucose-containing physiological solution (CTL), or exposed to oxygen–glucose deprivation (OGD) for 30 min with vehicle (VEH), or cannabidiol (CBD) alone or with the CB₂ receptor antagonist AM630, the A_1A receptor antagonist DPCPX, or the A_2A receptor antagonist SCH58261 (all 100 μM). See Methods for details. Bars represent the mean ± SEM of 14 to 30 experiments. Different letters represent statistical differences (p < 0.05) by ANOVA with Newman–Keuls test for multiple comparisons.

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  Fig. 5. Determinations of IL-6 concentration (top), and TNFα (medium), and COX-2 (bottom) expression in brain slices from 7- to 10-day-old C57BL6 mice, maintained in oxygenated glucose-containing physiological solution (CTL), or exposed to oxygen–glucose deprivation (OGD) for 30 min with vehicle (VEH), or cannabidiol (CBD) alone or with the CB₂ receptor antagonist AM630, the A_1A receptor antagonist DPCPX, or the A_2A receptor antagonist SCH58261 (all 100 μM). See Methods for details. Bars represent the mean ± SEM of 14 to 30 experiments. Different letters represent statistical differences (p < 0.05) by ANOVA with Newman–Keuls test for multiple comparisons.

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  Corresponding author. Neonatology. Pediatric Department, Hospital Universitario Puerta de Hierro, 28222-Majadahonda, Madrid, Spain. Fax: +34 917913023.

  Copyright © 2009 Elsevier Inc. All rights reserved.