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. 2021 Mar 23;22(6):3275.
doi: 10.3390/ijms22063275.

Neonatal Mesenchymal Stem Cell Treatment Improves Myelination Impaired by Global Perinatal Asphyxia in Rats

Andrea Tapia-Bustos  1   2 , Carolyne Lespay-Rebolledo  1 , Valentina Vío  1 , Ronald Pérez-Lobos  1 , Emmanuel Casanova-Ortiz  1 , Fernando Ezquer  3 , Mario Herrera-Marschitz  1 , Paola Morales  1   4
Affiliations

Neonatal Mesenchymal Stem Cell Treatment Improves Myelination Impaired by Global Perinatal Asphyxia in Rats

Andrea Tapia-Bustos et al. Int J Mol Sci. .

Abstract

The effect of perinatal asphyxia (PA) on oligodendrocyte (OL), neuroinflammation, and cell viability was evaluated in telencephalon of rats at postnatal day (P)1, 7, and 14, a period characterized by a spur of neuronal networking, evaluating the effect of mesenchymal stem cell (MSCs)-treatment. The issue was investigated with a rat model of global PA, mimicking a clinical risk occurring under labor. PA was induced by immersing fetus-containing uterine horns into a water bath for 21 min (AS), using sibling-caesarean-delivered fetuses (CS) as controls. Two hours after delivery, AS and CS neonates were injected with either 5 μL of vehicle (10% plasma) or 5 × 104 MSCs into the lateral ventricle. Samples were assayed for myelin-basic protein (MBP) levels; Olig-1/Olig-2 transcriptional factors; Gglial phenotype; neuroinflammation, and delayed cell death. The main effects were observed at P7, including: (i) A decrease of MBP-immunoreactivity in external capsule, corpus callosum, cingulum, but not in fimbriae of hippocampus; (ii) an increase of Olig-1-mRNA levels; (iii) an increase of IL-6-mRNA, but not in protein levels; (iv) an increase in cell death, including OLs; and (v) MSCs treatment prevented the effect of PA on myelination, OLs number, and cell death. The present findings show that PA induces regional- and developmental-dependent changes on myelination and OLs maturation. Neonatal MSCs treatment improves survival of mature OLs and myelination in telencephalic white matter.

Keywords: apoptosis; hypomyelination; mesenchymal stem cells; myelination; neonatal asphyxia/ischemia; neuroinflammation; oligodendrocyte; periventricular leukomalacia; rat brain; telencephalon.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effect of perinatal asphyxia (PA) and neonatal development on myelination at P1, P7, and P14, measured in external capsule (A); corpus callosum (B); cingulum (C) and fimbriae of hippocampus (D) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red) and DAPI (blue; nuclei)-positive cells in external capsule (1A); corpus callosum (1B); cingulum (1C); and fimbriae of hippocampus (1D) from control (CS) and asphyxia-exposed (AS) rat neonates. Microphotographs show MBP, indicating both myelinated fibers and mature oligodendrocytes (OLs). Scale bar: 20 μm. At P1, no MBP immunoreactivity was observed in any of the analyzed regions and experimental conditions. The density of MBP increased significantly along development. At P7, the density of MBP fibers (white head arrows) was low, letting us visualize individual mature OL (white arrows). In corpus callosum and fimbriae of hippocampus some individual OL can also be seen, showing long and branched processes. In AS, there was a decrease in the density of MBP in external capsule, corpus callosum, and cingulum compared to that in CS. No differences could be seen in fimbriae of hippocampus at P7. At P14 a dense network of MBP fibers can be seen in all regions, but no independent OLs soma can be distinguished.
Figure 1
Figure 1
Effect of perinatal asphyxia (PA) and neonatal development on myelination at P1, P7, and P14, measured in external capsule (A); corpus callosum (B); cingulum (C) and fimbriae of hippocampus (D) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red) and DAPI (blue; nuclei)-positive cells in external capsule (1A); corpus callosum (1B); cingulum (1C); and fimbriae of hippocampus (1D) from control (CS) and asphyxia-exposed (AS) rat neonates. Microphotographs show MBP, indicating both myelinated fibers and mature oligodendrocytes (OLs). Scale bar: 20 μm. At P1, no MBP immunoreactivity was observed in any of the analyzed regions and experimental conditions. The density of MBP increased significantly along development. At P7, the density of MBP fibers (white head arrows) was low, letting us visualize individual mature OL (white arrows). In corpus callosum and fimbriae of hippocampus some individual OL can also be seen, showing long and branched processes. In AS, there was a decrease in the density of MBP in external capsule, corpus callosum, and cingulum compared to that in CS. No differences could be seen in fimbriae of hippocampus at P7. At P14 a dense network of MBP fibers can be seen in all regions, but no independent OLs soma can be distinguished.
Figure 2
Figure 2
Effect of perinatal asphyxia (PA) on glial cells at P7, measured in external capsule (A); corpus callosum (B); cingulum (C); and fimbriae of hippocampus (D) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red), glial fibrillary acidic protein (GFAP; red), ionized calcium binding adaptor molecule 1 (Iba-1; green) and DAPI (blue; nuclei)-positive cells in external capsule (2A); corpus callosum (2B); cingulum (2C); and fimbriae of hippocampus (2D), from control (CS) and asphyxia exposed (AS) rat neonates. White arrows show mature oligodendrocyte (OL), astrocyte, and microglia phenotype. Scale bar: 20 μm. At P7, the number of MBP-DAPI+ cells/mm3 decreased after PA in external capsule, corpus callosum, and cingulum, but not in fimbriae of the hippocampus.
Figure 2
Figure 2
Effect of perinatal asphyxia (PA) on glial cells at P7, measured in external capsule (A); corpus callosum (B); cingulum (C); and fimbriae of hippocampus (D) of rat neonates. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red), glial fibrillary acidic protein (GFAP; red), ionized calcium binding adaptor molecule 1 (Iba-1; green) and DAPI (blue; nuclei)-positive cells in external capsule (2A); corpus callosum (2B); cingulum (2C); and fimbriae of hippocampus (2D), from control (CS) and asphyxia exposed (AS) rat neonates. White arrows show mature oligodendrocyte (OL), astrocyte, and microglia phenotype. Scale bar: 20 μm. At P7, the number of MBP-DAPI+ cells/mm3 decreased after PA in external capsule, corpus callosum, and cingulum, but not in fimbriae of the hippocampus.
Figure 3
Figure 3
Effect of MSCs treatment on cell death induced by perinatal asphyxia (PA), measured at P7 in external capsule (A); corpus callosum (B); and cingulum (C) of rat neonates. Representative microphotographs obtained by confocal microscopy showing TUNEL (green), DAPI (nuclei, blue)-positive cells in external capsule (3A); corpus callosum (3B); and cingulum (3C) from vehicle- and MSCs-treated control (CS) and asphyxia-exposed (AS) neonates. Scale bar: 20 μm. (A–C). The number of TUNEL-DAPI cell/mm3 is increased when comparing vehicle-treated AS versus CS neonates, but the number of TUNEL-DAPI cell/mm3 is decreased in MSCs- versus vehicle-treated AS rat neonates in all evaluated regions.
Figure 3
Figure 3
Effect of MSCs treatment on cell death induced by perinatal asphyxia (PA), measured at P7 in external capsule (A); corpus callosum (B); and cingulum (C) of rat neonates. Representative microphotographs obtained by confocal microscopy showing TUNEL (green), DAPI (nuclei, blue)-positive cells in external capsule (3A); corpus callosum (3B); and cingulum (3C) from vehicle- and MSCs-treated control (CS) and asphyxia-exposed (AS) neonates. Scale bar: 20 μm. (A–C). The number of TUNEL-DAPI cell/mm3 is increased when comparing vehicle-treated AS versus CS neonates, but the number of TUNEL-DAPI cell/mm3 is decreased in MSCs- versus vehicle-treated AS rat neonates in all evaluated regions.
Figure 4
Figure 4
Effect of MSCs treatment on myelination and mature oligodendrocyte (OLs) injury induced by perinatal asphyxia (PA), measured at P7 in external capsule (A), and cingulum (B) of control (CS) and (AS) rats. Representative microphotographs obtained by confocal microscopy showing myelin basic protein (MBP; red) and DAPI (blue; nuclei)-positive cells in external capsule (4A); and cingulum (4B) from control (CS) and asphyxia-exposed (AS) rat neonates, including vehicle and MSCs treated groups. Microphotographs show MBP, indicating myelinated fibers (white head arrows) and mature oligodendrocytes (OL) (white arrows). Scale bar: 20 μm. The density of MBP and number of MBP-DAPI cells/mm3 is increased in MSCs- versus vehicle-treated AS neonates in all evaluated regions.
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