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

Lafuente H, Pazos MR, Alvarez A, Mohammed N, Santos M, Arizti M, Alvarez FJ, Martinez-Orgado JA - Front Neurosci (2016)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Neonatology Research Group, Biocruces Health Research Institute Bizkaia, Spain.

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.

No MeSH data available.


Related in: MedlinePlus

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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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.

Mentions: HI-induced brain damage and treatment-induced effects were both observed in the histological and biochemical analyses. At the end of the experiment, about a quarter of neurons in the cortex of the NV group animals appeared to be necrotic (Figure 5A). Either hypothermia treatment (HV group) or CBD administration (NC group) alone reduced this percentage to a similar extent. Administration of both therapies together produced an additive effect bringing the per cent of necrotic neurons in the HC group down compared to the NC or HV animals.


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

Lafuente H, Pazos MR, Alvarez A, Mohammed N, Santos M, Arizti M, Alvarez FJ, Martinez-Orgado JA - Front Neurosci (2016)

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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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.
Mentions: HI-induced brain damage and treatment-induced effects were both observed in the histological and biochemical analyses. At the end of the experiment, about a quarter of neurons in the cortex of the NV group animals appeared to be necrotic (Figure 5A). Either hypothermia treatment (HV group) or CBD administration (NC group) alone reduced this percentage to a similar extent. Administration of both therapies together produced an additive effect bringing the per cent of necrotic neurons in the HC group down compared to the NC or HV animals.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Neonatology Research Group, Biocruces Health Research Institute Bizkaia, Spain.

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.

No MeSH data available.


Related in: MedlinePlus