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

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

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

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

Litter differences in DTI metrics.

<p>Fractional anisotropy (a); mean (b), radial (c) and axial diffusivity (d) in white matter areas of litter A (grey columns, n = 13), B (black columns, n = 9) and controls (white columns, n = 8) on postnatal day 14. Mean, axial and radial diffusivity are shown in units of mm²/s. Data are presented as mean ± 95% confidence interval. Differences between litters are marked where significant (<i>p</i> < 0.05). Abbreviations: wm: all of white matter; bcc: corpus callosum (body); ec: external capsule; ic: internal capsule; IHH, intermittent hyperoxia-hypoxia; hf: hippocampal fimbriae. </p

Litter differences in T₂.

<p>T₂-relaxation time in the brain at P14 in litter A (grey columns, n = 13), litter B (black columns, n = 9) and C (white columns, n = 8) in the cortex (a), hippocampus (b), putamen (c) and thalamus (d). T₂-relaxation times are shown in milliseconds. Differences between litters are marked where significant (<i>p</i> < 0.05). Data are presented as mean ± 95% confidence interval. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P14: postnatal day 14; P28: postnatal day 28. </p

Vascular density.

<p>(a) Vascular density as % of controls (white columns, n = 4) in IHH animals at P14 (grey columns; IHH: n = 10) and P28 (black columns; IHH: n = 12). Data are expressed as mean ± 95% confidence intervals. (b-e) x400 magnification of lectin-stained endothelium in IHH (upper row) and control (lower row) at P14 (b & d) and P28 (c & e). *<i>p</i> = 0.005 IHH vs. control. Scale bar = 50 μm. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P14, postnatal day 14; P28, postnatal day 28; pvwm, periventricular white matter. </p

Albumin.

<p>Albumin immunoreactivity (brown stain) at P14 (a-b) and P28 in controls (c-d) and IHH (e-f). Note positive immunoreactivity for albumin in both experiment groups at P14 and several spots of albumin immunoreactivity in the cortex and thalamus of the IHH animal at P28. Albumin is present in intracellular vesicles in neuroependymal cells in the ventricles (b & d). (g) The sum of positive scores for albumin leakage in the neuropil of the respective brain areas are presented as fractions of the maximum possible score in each experiment group at P28. (a, c & e): scale bar = 2 mm (b, d & f): scale bar = 50 μm at x400 magnification. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P14, postnatal day 14; P28, postnatal day 28. </p

Retina.

<p>(a-d): H&E retinal slices from control (a) and IHH animals at P14 (b-d) showing haemorrhage in the ganglion cell layer (b), inner nuclear layer (c) and outer nuclear cell layer (d). (e-h): Retinal wholemounts stained with endothelial-specific Biotinylated <i>Griffonia</i> (<i>Bandeiraea</i>) <i>Simplicifolia</i> Lectin I Isolectin B4 from controls at P28 with a mature vascular bed (e) and no vasculature extending beyond the ora serrata (f). Retinal vascular bed in IHH animal at P28 with less remodelling (g) and areas of vascularization beyond the ora serrata (h). (a-d): x400 magnification; scale bar = 50 μm. (e-h): x100 magnification; scale bar = 200 μm. Abbreviations, IHH, intermittent hyperoxia-hypoxia; P14, postnatal day 14; P28, postnatal day 28.</p