Canna~Fangled Abstracts

Mechanisms of cannabidiol neuroprotection in hypoxic-ischemic newborn pigs: role of 5HT₁A and CB2 receptors.

By August 6, 2013No Comments

elsevierMechanisms of cannabidiol neuroprotection in hypoxic–ischemic newborn pigs: Role of 5HT1A and CB2 receptors

  • a Experimental Unit, Pediatric Department, University Hospital Puerta de Hierro Majadahonda, 28222 Madrid, Spain
  • b Neonatal Unit, Pediatric Department, University Hospital Puerta de Hierro Majadahonda, 28222 Madrid, Spain
  • c Experimental Perinatal Physiopathology Research Unit, Gurutzetako Ospitalea, 48903 Bizkaia, Spain
  • d Applied Medical Research Center (CIMA), University of Navarra, 31009 Pamplona, Spain
  • e Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain
  • f Department of Physiology and Pharmacology, University of Cantabria, Cantabria Biomedicine and Biotechnology Institute (UC-CSIC_SODERCAN), 39005 Santander, Spain
  • g Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Institute of Health Carlos III, Madrid, Spain
  • h Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
  • i Laboratory for Research Support, University Hospital Fundación Alcorcón, 28922 Madrid, Spain

Highlights

•

CBD administration prevents neuron and astrocyte damage after hypoxia–ischemia.

•

CBD modulates excitotoxicity, inflammation and oxidative stress.

•

5HT1A and CB2 receptors are involved in CBD neuroprotection.

•

CB2 involvement in CBD effects is not because of endocannabinoid levels enhancement

•

5HT1A receptors form heteromers with CB2 receptors in living cells

 

Abstract

The mechanisms underlying the neuroprotective effects of cannabidiol (CBD) were studied in vivo using a hypoxic–ischemic (HI) brain injury model in newborn pigs. One- to two-day-old piglets were exposed to HI for 30 min by interrupting carotid blood flow and reducing the fraction of inspired oxygen to 10%. Thirty minutes after HI, the piglets were treated with vehicle (HV) or 1 mg/kg CBD, alone (HC) or in combination with 1 mg/kg of a CB2 receptor antagonist (AM630) or a serotonin 5HT1A receptor antagonist (WAY100635). HI decreased the number of viable neurons and affected the amplitude-integrated EEG background activity as well as different prognostic proton-magnetic-resonance-spectroscopy (H±-MRS)-detectable biomarkers (lactate/N-acetylaspartate and N-acetylaspartate/choline ratios). HI brain damage was also associated with increases in excitotoxicity (increased glutamate/N-acetylaspartate ratio), oxidative stress (decreased glutathione/creatine ratio and increased protein carbonylation) and inflammation (increased brain IL-1 levels). CBD administration after HI prevented all these alterations, although this CBD-mediated neuroprotection was reversed by co-administration of either WAY100635 or AM630, suggesting the involvement of CB2 and 5HT1A receptors. The involvement of CB2 receptors was not dependent on a CBD-mediated increase in endocannabinoids. Finally, bioluminescence resonance energy transfer studies indicated that CB2 and 5HT1A receptors may form heteromers in living HEK-293T cells. In conclusion, our findings demonstrate that CBD exerts robust neuroprotective effects in vivo in HI piglets, modulating excitotoxicity, oxidative stress and inflammation, and that both CB2 and 5HT1A receptors are implicated in these effects.

Keywords

  • Hypoxia–ischemia;
  • Brain;
  • Neuroprotection;
  • Cannabidiol;
  • Cannabinoids;
  • Serotonin;
  • Newborn;
  • Pigs

Abbreviations

  • AEA, arachidonylethanolamide;
  • 2-AG, 2-arachidonoylglycerol;
  • aEEG, amplitude-integrated EEG;
  • CBD,cannabidiol;
  • CO, cardiac output;
  • HI, hypoxic–ischemic;
  • NHIE, newborn HI encephalopathy;
  • OEA,oleoylethanolamide;
  • OGD, oxygen-glucose deprivation;
  • PEA, palmitoylethanolamide

Figures and tables from this article:

Full-size image (36 K)
Fig. 1. CBD-induced recovery of brain activity after HI was reversed by 5HT1A or CB2 receptor antagonists. Brain activity studied by continuous aEEG recording in 1-to-2 day-old piglets after sham operation (SHM) or after hypoxic–ischemic (HI) insult and treatment with vehicle (HV), CBD (HC), CBD + AM630 (HCA) or CBD + WAY100630 (HCW). Top: Changes of mean amplitude of aEEG trace throughout the experiment. Bottom: Qualitative assessment of aEEG background activity by a neurological activity score. Results are expressed as means ± SEM of 6–10 animals. (*)p < 0.05 vs. SHM. (§) p < 0.05 vs HC. See Section 2.1 for details.
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Fig. 2. CBD-induced prevention of neuronal and astroglial death after HI was abolished by 5HT1A or CB2 receptor antagonists. Representative light microphotographs of Nissl (top) or GFAP (bottom) stained brain sections, obtained after from 1-to-2 day-old piglets after sham operation (SHM) or after hypoxic–ischemic (HI) insult and treatment with vehicle (HV), CBD (HC), CBD + AM630 (HCA) or CBD + WAY100630 (HCW). In brain from HV there is an increase in number of pyknotic cells and a decrease in number of viable neurons and GFAP + cells (arrows). Administration of CBD reduced the presence of pyknotic cells and the loss of viable neurons and GFAP + cells. Original magnification ×200, bar: 100 μm. Results are expressed as means ± SEM of 6–10 animals. (*) p < 0.05 vs. SHM.
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Fig. 3. CBD-induced improvement of H+-MRS biomarkers after HI was reversed by 5HT1A or CB2 receptor antagonists. Top: Representative brain H+-MRS spectrum from a normal piglet, showing the peaks of the different metabolites studied. Bars represent the results, expressed as means ± SEM, of different metabolite ratios obtained from studies performed in brain samples from 1-to-2 day-old piglets after sham operation (SHM) or after hypoxic–ischemic (HI) insult and treatment with vehicle (HV), CBD (HC), CBD + AM630 (HCA) or CBD + WAY100630 (HCW). Cho: Choline; Cr: Creatine; Lac: Lactate; Glu: Glutamate; GSH: Reduced glutathione; NAA: N-acetylaspartate. (*) p < 0.05 vs. SHM.
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Fig. 4. CBD-induced reduction of brain protein carbonylation and IL-1 production after HI was reversed by 5HT1A or CB2receptor antagonists. (A) Top: Representative Western blot probed with antibody to derived protein carbonyl side groups (OxyBlot), carried out in brain samples from 1-to-2 day-old piglets after sham operation (SHM) or after hypoxic-ischemic (HI) insult and treatment with vehicle (HV), CBD (HC), CBD + AM630 (HCA) or CBD + WAY100630 (HCW). Bottom: Densitometric analysis of relative protein carbonyl contents. The levels of protein oxidation were normalized by total protein loading (Red Ponceau staining) and expressed by the OxyBlot/Red Ponceau ratio (see Section 2.5 for details). (B) Brain concentration of IL-1 quantified by microarrays in samples from the aforementioned groups. Bars represent the mean ± SEM of 6–8 experiments. (*) p < 0.05 vs. SHM.
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Fig. 5. HI-induced increase of brain endocannabinoid levels was prevented by CBD. Brain concentration of endocannabinoid level was quantified by liquid chromatography–mass spectrometry in samples from 1- to 2-day-old piglets after sham operation (SHM) or after hypoxic-ischemic (HI) insult and treatment with vehicle (HV) or CBD (HC). See Section 2.7 for details. Bars represent the mean ± SEM of 6–8 experiments. AEA: Arachinodoylethanolamide; 2-AG: 2-Arachidonoylglycerol; OEA: Oleylethanolamide; PEA: Palmitoylethanolamide. (*) p < 0.05 vs. SHM.
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Fig. 6. 5HT1A receptors form heteromers with CB2 receptors in living cells. BRET saturation experiments showing CB2R/5HT1AR heteromerization were performed using HEK-203 cells transfected with 0.5 μg of cDNA corresponding to 5HT1AR-Rluc and increasing amounts of cDNA (0–2 μg cDNA) corresponding to CB2R–YFP (circles). As negative control, cells transfected with cDNA corresponding to 5HT1AR-Rluc (0.5 μg) and to D4,2R–YFP (0–4 μg cDNA) were also used (squares). Both fluorescence and luminescence for each sample were measured before every experiment to confirm similar donor expressions (approximately 100,000 bioluminescence units) while monitoring the increase in acceptor expression (up to 70,000 net fluorescence units). The relative amount of BRET is given as the ratio between the net fluorescence of the acceptor (YFP) and the luciferase activity of the donor (Rluc). BRET data are expressed as means ± SEM. Of 4–8 different experiments grouped as a function of the amount of BRET acceptor. See Section 2.8 for details.
Table 1. Cardiorespiratory parameters.
Values obtained from 1- to 2-day-old piglets after sham operation (SHM) or after hypoxia–ischemia and treatment with vehicle (HV), CBD (HC), CBD + AM630 (HCA) or CBD + WAY100630 (HCW).B: basal; D: drug; E: end; CO: cardiac output (mL/min/kg); MBP: mean blood pressure (mmHg). (*) p < 0.05 vs SHM. (#) p < 0.05 vs B. (§) p < 0.05 vs. HC.
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Corresponding author contact information
Corresponding author. Neonatal Unit, Department of Pediatrics, University Hospital Puerta de Hierro Majadahonda, Joaquin Rodrigo, 1. 28222 Majadahonda, Madrid, Spain. Tel.: +34 629356330; fax: +34 917913023.

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