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, 1 (12), 955-67

Lactoferrin During Lactation Protects the Immature Hypoxic-Ischemic Rat Brain

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Lactoferrin During Lactation Protects the Immature Hypoxic-Ischemic Rat Brain

Yohan van de Looij et al. Ann Clin Transl Neurol.

Abstract

Objective: Lactoferrin (Lf) is an iron-binding glycoprotein secreted in maternal milk presenting anti-inflammatory and antioxidant properties. It shows efficient absorption into the brain from nutritional source. Brain injury frequently resulting from cerebral hypoxia-ischemia (HI) has a high incidence in premature infants with ensuing neurodevelopmental disabilities. We investigated the neuroprotective effect of maternal nutritional supplementation with Lf during lactation in a rat model of preterm HI brain injury using magnetic resonance imaging (MRI), brain gene, and protein expression.

Methods: Moderate brain HI was induced using unilateral common carotid artery occlusion combined with hypoxia (6%, 30 min) in the postnatal day 3 (P3) rat brain (24-28 weeks human equivalent). High-field multimodal MRI techniques were used to investigate the effect of maternal Lf supplementation through lactation. Expression of cytokine coding genes (TNF-α and IL-6), the prosurvival/antiapoptotic AKT protein and caspase-3 activation were also analyzed in the acute phase after HI.

Results: MRI analysis demonstrated reduced cortical injury in Lf rats few hours post-HI and in long-term outcome (P25). Lf reduced HI-induced modifications of the cortical metabolism and altered white matter microstructure was recovered in Lf-supplemented rats at P25. Lf supplementation significantly decreased brain TNF-α and IL-6 gene transcription, increased phosphorylated AKT levels and reduced activation of caspase-3 at 24 h post-injury.

Interpretation: Lf given through lactation to rat pups with cerebral HI injury shows neuroprotective effects on brain metabolism, and cerebral gray and white matter recovery. This nutritional intervention may be of high interest for the clinical field of preterm brain neuroprotection.

Figures

Figure 1
Figure 1
Lactoferrin nutritional supplementation reduces cortical damage. (A) Typical T2W images of rat pups treated with lactoferrin (Lf) (upper panel) or an isocaloric diet (Iso) (lower panel) 3–6 hours (P3) (left) or 22 days (P25) (right) after HI. Cerebral edema early after HI (gray arrows) as well as cortical loss 22 days after HI (white arrows) are obvious for both groups and are smaller in Lf-treated animals. (B) Histograms of the percentage of injured cortex at P3 and of cortical loss at P25 for both groups, showing a significant reduction in cortical damage in HI-Lf animals: HI-Lf (gray) and HI-Iso (black) (*P < 0.05). Values are mean ± SD. (C) Correlations between the percentage of injured cortex at P3 and the percentage of cortical loss at P25 for both groups: HI-Lf (gray) and HI-Iso (black). (D) Histograms of the mean values of diffusivities (MD, D// and D) of the Sham (white), HI-Lf (gray) and HI-Iso (black) groups measured in the cortex. Values are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. HI, hypoxia-ischemia.
Figure 2
Figure 2
Lactoferrin treatment reduces cortical metabolism alterations induced by perinatal hypoxia-ischemia (HI). (A) Representative 9.4 T spectrum in the ipsilateral cortex of a rat pup treated with lactoferrin (Lf), 22 days (P25) after hypoxia-ischemia (HI). (B) Histogram of the concentrations of the metabolites which are significantly modified at P25 by HI in rat pups treated with an isocaloric diet (HI-Iso) (black) compared to Sham. The Lf-treated (HI-Lf) (gray) group present less change, showing only a decreased NAA concentration compared to the sham group. Values are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Mac, macromolecules; Asc, ascorbate; bhB, beta-hydroxybutyrate; PCho, phosphorylcholine; Cr, creatine; PCr, phosphocreatine; GABA, γ-aminobutyric acid; Glc, glucose; Glu, glutamate; Gln, glutamine, myo-Ins, myo-inositol; Lac, lactate; NAA, N-acetylaspartate; NAAG, N-acetylaspartylglutamate; PCr, phosphocreatine; PE, phosphoethanolamine; Tau, taurine.
Figure 3
Figure 3
Protective effect of lactoferrin on white matter damage revealed by high-field DTI and phase contrast. Typical images of Sham (A–E), HI-Lf (F–J) or HI-Iso (K–O) rats 22 days (P25) after P3 cerebral hypoxia-ischemia showing white matter damage in direction-encoded color maps (A, F, and K) and altered track of the eigenvectors in the HI-Iso group in zoom on the EC (circles in B, G, and L). T2*W images (C, H, and M) revealing less severe cortical lesions in an HI-Lf rat. Phase contrast images (D, I, and N) and zoom on the EC (E, J, and O) illustrating the differences in contrast on phase images between the Sham, HI-Lf and HI-Iso groups (E, J, and O, respectively; contrast between black delineated zone in EC and adjacent gray matter). Histograms of the mean diffusion tensor imaging-derived parameters values: diffusivity values MD, D// and D (P, R) and fractional anisotropy (FA) (Q, S) of the Sham, HI-Lf and HI-Iso groups measured in the EC (P, Q) and the CC (R, S). Values are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. Histogram of the mean values of frequency shift over the EC and corpus callosum for the Sham, HI-Lf and HI-Iso groups (T, values are mean ± SEM). HI, hypoxia-ischemia.
Figure 4
Figure 4
Nutritional supplementation with lactoferrin decreased TNF-α and IL-6 genes transcription. Quantitative RT-PCR on cortical extracts 24 h after cerebral hypoxia-ischemia (HI) on P3 rats inducing (A) TNF-α and (B) IL-6 genes. Both sham groups (Sham-Iso and Sham-Lf) are shown despite being similar. Values are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Lactoferrin treatment decreased activation of caspase-3. Representative immunoblots and the corresponding quantifications of cleaved caspase-3 (A) and fractin (B) demonstrating an activation of caspase-3 24 h after hypoxia-ischemia (HI) reduced by lactoferrin (Lf) treatment. Both sham groups (Sham-Iso and Sham-Lf) are shown despite being similar. Values are mean ± SEM and are expressed as a percentage of the Sham-Iso operated-animal value, *P < 0.05, ** P < 0.01, ***P < 0.001.
Figure 6
Figure 6
HI-induced AKT deactivation is less under lactoferrin treatment. Representative immunoblots and the corresponding quantifications of active phosphorylated AKT (P-AKT) and pan-AKT showing a dephosphorylation of AKT 24 h after hypoxia-ischemia (HI) reduced by lactoferrin (Lf) treatment. Both sham groups (Sham-Iso and Sham-Lf) are shown despite being similar. Values are mean ± SEM and are expressed as a percentage of the Sham-Iso operated-animal value, *P < 0.05, **P < 0.01, ***P < 0.001.

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