Transient effect of melatonin treatment after neonatal hypoxic-ischemic brain injury in rats

Melatonin has potential neuroprotective capabilities after neonatal hypoxia-ischemia (HI), but long-term effects have not been investigated. We hypothesized that melatonin treatment directly after HI could protect against early and delayed brain injury. Unilateral HI brain injury was induced in postnatal day 7 rats. An intraperitoneal injection of either melatonin or vehicle was given at 0, 6 and 25 hours after hypoxia. In-vivo MRI was performed 1, 7, 20 and 43 days after HI, followed by histological analysis. Forelimb asymmetry and memory were assessed at 12–15 and at 36–43 days after HI. More melatonin treated than vehicle treated animals (54.5% vs 15.8%) developed a mild injury characterized by diffusion tensor values, brain volumes, histological scores and behavioral parameters closer to sham. However, on average, melatonin treatment resulted only in a tendency towards milder injury on T2-weighted MRI and apparent diffusion coefficient maps day 1 after HI, and not improved long-term outcome. These results indicate that the melatonin treatment regimen of 3 injections of 10 mg/kg within the first 25 hours only gave a transient and subtle neuroprotective effect, and may not have been sufficient to mitigate long-term brain injury development following HI

Early metabolite changes after melatonin treatment in neonatal rats with hypoxic-ischemic brain injury studied by in-vivo 1H MR spectroscopy

Melatonin is a promising neuroprotective agent after perinatal hypoxic-ischemic (HI) brain injury. We used in-vivo 1H magnetic resonance spectroscopy to investigate effects of melatonin treatment on brain metabolism after HI. Postnatal day 7 Sprague-Dawley rats with unilateral HI brain injury were treated with either melatonin 10 mg/kg dissolved in phosphate-buffered saline (PBS) with 5% dimethyl sulfoxide (DMSO) or vehicle (5% DMSO and/or PBS) directly and at 6 hours after HI. 1H MR spectra from the thalamus in the ipsilateral and contralateral hemisphere were acquired 1 day after HI. Our results showed that injured animals had a distinct metabolic profile in the ipsilateral thalamus compared to sham with low concentrations of total creatine, choline, N-acetyl aspartate (NAA), and high concentrations of lipids. A majority of the melatonin-treated animals had a metabolic profile characterized by higher total creatine, choline, NAA and lower lipid levels than other HI animals. When comparing absolute concentrations, melatonin treatment resulted in higher glutamine levels and lower lipid concentrations compared to DMSO treatment as well as higher macromolecule levels compared to PBS treatment day 1 after HI. DMSO treated animals had lower concentrations of glucose, creatine, phosphocholine and macromolecules compared to sham animals. In conclusion, the neuroprotective effects of melatonin were reflected in a more favorable metabolic profile including reduced lipid levels that likely represents reduced cell injury. Neuroprotective effects may also be related to the influence of melatonin on glutamate/glutamine metabolism. The modulatory effects of the solvent DMSO on cerebral energy metabolism might have masked additional beneficial effects of melatonin

Early metabolite changes after melatonin treatment in neonatal rats with hypoxic-ischemic brain injury studied by <i>in-vivo</i> ¹H MR spectroscopy

<div><p>Melatonin is a promising neuroprotective agent after perinatal hypoxic-ischemic (HI) brain injury. We used <i>in-vivo</i> ¹H magnetic resonance spectroscopy to investigate effects of melatonin treatment on brain metabolism after HI. Postnatal day 7 Sprague-Dawley rats with unilateral HI brain injury were treated with either melatonin 10 mg/kg dissolved in phosphate-buffered saline (PBS) with 5% dimethyl sulfoxide (DMSO) or vehicle (5% DMSO and/or PBS) directly and at 6 hours after HI. ¹H MR spectra from the thalamus in the ipsilateral and contralateral hemisphere were acquired 1 day after HI. Our results showed that injured animals had a distinct metabolic profile in the ipsilateral thalamus compared to sham with low concentrations of total creatine, choline, <i>N</i>-acetyl aspartate (NAA), and high concentrations of lipids. A majority of the melatonin-treated animals had a metabolic profile characterized by higher total creatine, choline, NAA and lower lipid levels than other HI animals. When comparing absolute concentrations, melatonin treatment resulted in higher glutamine levels and lower lipid concentrations compared to DMSO treatment as well as higher macromolecule levels compared to PBS treatment day 1 after HI. DMSO treated animals had lower concentrations of glucose, creatine, phosphocholine and macromolecules compared to sham animals. In conclusion, the neuroprotective effects of melatonin were reflected in a more favorable metabolic profile including reduced lipid levels that likely represents reduced cell injury. Neuroprotective effects may also be related to the influence of melatonin on glutamate/glutamine metabolism. The modulatory effects of the solvent DMSO on cerebral energy metabolism might have masked additional beneficial effects of melatonin.</p></div

¹H spectrum of the ipsilateral thalamus.

<p>Acquired ¹H spectrum of the ipsilateral thalamus of an animal exposed to HI. Cr, creatine; DMSO, dimethyl sulfoxide; Gln, glutamine; Glu, glutamate; GPC, glycerophosphocholine; GSH, glutathione; Ins, myo-inositol; NAA, N-acetyl aspartate; PCh, phosphocoline; PCr, phosphocreatine; Tau, taurine.</p

Metabolic profiles of the ipsilateral thalamus of HI animals after 2 treatment injections.

<p>(a) The metabolic profiles of the HI animals treated with either melatonin (10 mg/kg) dissolved in PBS with 5% DMSO (MEL), PBS with 5% DMSO (DMSO) or only PBS (PBS) are displayed in the PCA score plot. The shaded area marks the animals treated with melatonin with higher levels of energy metabolites, neurotransmitters and macromolecules as shown in (b). (b) The corresponding loading plot shows the metabolic alterations related to the separation along PC 1. Glx, glutamine+glutamate; GPC glycerophosphocholine; HI, hypoxia-ischemia; NAA, N-acetyl aspartate; NAAG, N-acetyl aspartyl glutamate; MM, macromolecules; Lip, lipids; PC, principle component; PCA, principle component analysis; PCh, phosphocholine; PCr, phosphocreatine; ppm, parts per million.</p

Metabolite concentrations in the contralateral thalamus within 1 day after HI.

<p>Concentrations of (a) energy metabolites, (b) neurotransmitters, (c) anti-oxidants and osmolytes, (d) metabolites representing cell injury/viability and (e) lipids and macromolecules in HI animals exposed to PBS, DMSO or melatonin (MEL) treatment and sham animals (here presented as one group). Results are presented as mean ± SEM. * significant differences between groups. GPC glycerophosphocholine; HI, hypoxia-ischemia; Lip, lipids; MM, macromolecules; NAA, N-acetyl aspartate; ppm, parts per million; sham, sham-operated.</p

Metabolic profiles of the ipsilateral thalamus of HI and sham animals 1 day after HI.

<p>(a) The PCA score plot shows the metabolic profiles of all animals. The HI animals were separated from the sham animals with the corresponding loading plot of PC 1 (b) showing the metabolic alterations that caused this separation. The shaded area is drawn based on visual inspection and is meant to highlight the separation between the groups. Glx, glutamine+glutamate; GPC glycerophosphocholine; HI, hypoxia-ischemia; NAA, N-acetyl aspartate; NAAG, N-acetyl aspartyl glutamate; MM, macromolecules; Lip, lipids; PC, principle component; PCA, principle component analysis; PCh, phosphocholine; PCr, phosphocreatine; ppm, parts per million.</p