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. 2016 Jul 12;10:323.
doi: 10.3389/fnins.2016.00323. eCollection 2016.

Effects of Cannabidiol and Hypothermia on Short-Term Brain Damage in New-Born Piglets After Acute Hypoxia-Ischemia

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Free PMC article

Effects of Cannabidiol and Hypothermia on Short-Term Brain Damage in New-Born Piglets After Acute Hypoxia-Ischemia

Hector Lafuente et al. Front Neurosci. .
Free PMC article

Abstract

Hypothermia is a standard treatment for neonatal encephalopathy, but nearly 50% of treated infants have adverse outcomes. Pharmacological therapies can act through complementary mechanisms with hypothermia improving neuroprotection. Cannabidiol could be a good candidate. Our aim was to test whether immediate treatment with cannabidiol and hypothermia act through complementary brain pathways in hypoxic-ischemic newborn piglets. Hypoxic-ischemic animals were randomly divided into four groups receiving 30 min after the insult: (1) normothermia and vehicle administration; (2) normothermia and cannabidiol administration; (3) hypothermia and vehicle administration; and (4) hypothermia and cannabidiol administration. Six hours after treatment, brains were processed to quantify the number of damaged neurons by Nissl staining. Proton nuclear magnetic resonance spectra were obtained and analyzed for lactate, N-acetyl-aspartate and glutamate. Metabolite ratios were calculated to assess neuronal damage (lactate/N-acetyl-aspartate) and excitotoxicity (glutamate/Nacetyl-aspartate). Western blot studies were performed to quantify protein nitrosylation (oxidative stress), content of caspase-3 (apoptosis) and TNFα (inflammation). Individually, the hypothermia and the cannabidiol treatments reduced the glutamate/Nacetyl-aspartate ratio, as well as TNFα and oxidized protein levels in newborn piglets subjected to hypoxic-ischemic insult. Also, both therapies reduced the number of necrotic neurons and prevented an increase in lactate/N-acetyl-aspartate ratio. The combined effect of hypothermia and cannabidiol on excitotoxicity, inflammation and oxidative stress, and on cell damage, was greater than either hypothermia or cannabidiol alone. The present study demonstrated that cannabidiol and hypothermia act complementarily and show additive effects on the main factors leading to hypoxic-ischemic brain damage if applied shortly after the insult.

Keywords: cannabidiol; hypothermia; hypoxic-ischemic encephalopathy; neonatal brain; neurodevelopment; neuroprotection; newborn animal.

Figures

Figure 1
Figure 1
Mean arterial blood pressure and amplitude-integrated electroencephalography (aEEG) in experimental groups of piglets. Line draws represent the mean arterial blood pressure (A) and the mean aEEG (B), obtained from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia (shaded symbol) or hypothermia (empty symbol) and administered with either vehicle (square) or cannabidiol (triangle). Data are represented as mean ± SEM. (*) P < 0.05 vs. NV group by Kruskall-Wallis one-factorial analysis of variance; (**) P < 0.05 vs. both normothermic groups (NV and NC) by Kruskall-Wallis one-factorial analysis of variance; (***) P < 0.05 vs. all groups by Kruskall-Wallis one-factorial analysis of variance.
Figure 2
Figure 2
Effect of the treatments on the characteristics of excitotoxicity. (A) A representative brain spectra from a hypoxic-ischemic animal managed with normothermia (NV group). Arrows show the metabolite peaks of glutamate (Glu), N-acetylaspartate (NAA) and lactate (Lac). (B) Bars represent the results of Glu/NAA ratio (mean ± SEM), in brain samples from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia or hypothermia and administered with either vehicle or cannabidiol. (*) P < 0.05 vs. NV group by Kruskall-Wallis one-factorial analysis of variance; (***) P < 0.05 vs. all groups by Kruskall-Wallis one-factorial analysis of variance.
Figure 3
Figure 3
Effect of the treatments on the characteristics of oxidative stress. (A) A representative image of immunoblotting indicating protein carbonyl formation (Oxyblot) using anti-DNPH antibody. Samples were obtained using brain tissue from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia or hypothermia and administered with either vehicle or cannabidiol. (B) Relative protein carbonyl levels were quantified by densitometric analyses of the blots. The content of oxidized proteins was normalized by total Red Ponceau-stained protein loading and expressed as OxyBlot/Red Ponceau ratio (see Materials and Methods for details). Bars represent mean ± SEM of 6–8 experiments. (a.u.) arbitrary units; (*) P < 0.05 vs. NV group by Kruskall-Wallis one-factorial analysis of variance; (***) P < 0.05 vs. all groups by Kruskall-Wallis one-factorial analysis of variance.
Figure 4
Figure 4
Effect of the treatments on the characteristics of neuroinflammation. (A) A representative image of immunoblotting using anti-TNFα antibody, carried out on the samples from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia or hypothermia and administered with either vehicle or cannabidiol. (B) Densitometric analysis of relative TNFα contents. β-actin was used to normalize for the differences in protein loading between lanes in the blot. Bars represent mean ± SEM of 6-8 experiments. (a.u.) arbitrary units; (*) P < 0.05 vs. NV group by Kruskall-Wallis one-factorial analysis of variance; (***) P < 0.05 vs. all groups by Kruskall-Wallis one-factorial analysis of variance.
Figure 5
Figure 5
Effect of the treatments on the characteristics of brain lesion. (A) Representative light microphotographs of Nissl-stained brain sections, obtained from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia or hypothermia and administered with either vehicle or cannabidiol. The number of pyknotic cells was significantly higher in the NV group (arrows), while it was lower in the hypothermia and cannabidiol-treated groups. Original magnification x200, white bar: 100 μm. (B) Top: A representative image of immunoblotting using an anti-Caspase-3 antibody, carried out on the samples from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia, or hypothermia and administered with either vehicle or cannabidiol. Bottom: Densitometric analysis of relative caspase-3 contents. β-actin was used to normalize for differences in protein loading between lanes of the blot. (C) Changes in Lac/NAA ratio obtained by H+-MRS analysis of brain samples from 1- to 2-day-old piglets after hypoxic-ischemic insult, treated with normothermia or hypothermia and administered with either vehicle or cannabidiol. Lac: lactate; NAA: N-acetylaspartate. In all figures, bars represent mean ± SEM of 6–10 experiments. (a.u.) arbitrary units. (*) P < 0.05 vs. NV group by Kruskall-Wallis one-factorial analysis of variance; (**) P < 0.05 vs. both normothermic groups (NV and NC) by Kruskall-Wallis one-factorial analysis of variance; (***) P < 0.05 vs. all groups by Kruskall-Wallis one-factorial analysis of variance.

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