White Matter Brain Development after Exposure to Circulating Cell-Free Hemoglobin and Hyperoxia in a Rat Pup Model

Abstract

Neonates born with critical congenital heart defects are at risk of diffuse white matter injuries and neurodevelopmental impairments. This study aimed to determine the impact of circulating cell-free hemoglobin and hyperoxia, both present during cardiopulmonary bypass circulation, on white matter brain development. Postnatal day 6 rat pups were injected intraperitoneally with cell-free Hb or vehicle and exposed to hyperoxia (fiO2 = 0.8) or normoxia (fiO2 = 0.21) for 24 h. We evaluated apoptosis, myelination, and oligodendrocyte maturation with immunohistochemistry, gene and protein analyses, and in vivo diffusion tensor magnetic resonance imaging (MRI). Consistent with previous studies, we found an increase in apoptosis of oligodendrocytes as determined by TUNEL+ staining in Olig2+ cells in white matter, cortex, thalamus, and hippocampus following exposure to hyperoxia with no additional effect of cell-free Hb. A transient increase in the mRNA expression of intercellular adhesion molecule 1 at 6 h was observed following combined exposure to cell-free Hb and hyperoxia. No indications of oligodendrocyte maturational delay or hypomyelination were observed after either insult, delivered separately or combined, as determined by immunohistochemistry, Western blot, and diffusion tensor MRI. In our model, exposure to circulatory cell-free Hb, with or without concomitant hyperoxia, did not significantly alter brain white matter development.

Keywords: Cardiopulmonary bypass; Cell-free hemoglobin; Hyperoxia; Oligodendrocyte; White matter.

Fig. 1

Diagram of the experimental setup. The time points of blood/tissue sampling and in vivoMRI are indicated with arrows. HH, intraperitoneal injection of cell-free Hb and exposure to hyperoxia; HN, intraperitoneal injection of cell-free Hb and exposure to normoxia; VH, intraperitoneal injection of vehicle and exposure to hyperoxia; VN, intraperitoneal injection of vehicle and exposure to normoxia; f_iO₂, fraction of inhaled oxygen; MRI, magnetic resonance imaging.

Fig. 2

TUNEL/Olig2 staining of rat pup brain sections after exposure to hyperoxia (VH), circulating cell-free Hb (HN), both insults (HH), or neither insult (VN). Representative images were acquired at P7 (24 h after injection) in all experimental groups. Scale bar, 50 µm. a DAPI staining of cell nuclei (blue). b Olig2+ cells (red). c TUNEL+ cells (green). d Merged image. White arrows indicate co-labeled cells. TUNEL+/Olig2+ cells were quantified in white matter (e), cortex (f), thalamus (g), and hippocampus (h) at P7. Results are presented as medians and interquartile ranges; n = 3–5. * p < 0.05, ** p < 0.01, *** p < 0.001. DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; P7, postnatal day 7; Olig2, oligodendrocyte transcription factor 2.

Fig. 3

Quantification of the total number of Olig2+ cells in different parts of the rat brain in groups exposed to hyperoxia (VH), circulating cell-free Hb (HN), both insults (HH), or neither insult (VN). Rats were sacrificed on postnatal day 11 (120 h after injection), and Olig2+ staining was evaluated in white matter (a), thalamus (b), and cingulum (c). Results are presented as medians and interquartile ranges; n = 5 (a–c). Olig2, oligodendrocyte transcription factor 2.

Fig. 4

Immunohistochemistry analysis of oligodendrocyte transcription factor (Olig)2+/adenomatous polyposis coli clone (CC1+) cells in rat pup brain sections after exposure to hyperoxia (VH), circulating cell-free Hb (HN), both insults (HH), or neither insult (VN). Representative images were acquired on postnatal day 11 (P11) (120 h after injection) in all experimental groups. Scale bar, 50 µm. a DAPI staining of cell nuclei (blue). b Olig2+ cells (red). c CC1+ cells (green). d Merged image. e–g Quantification of the percentage of CC1+ cells among Olig2+ cells in white matter (e), thalamus (f), and cingulum (g) on P11. Results are presented as medians and interquartile ranges; n = 5. * p < 0.05. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 5

MRI analysis of white matter microstructural organization in rat pup brain after exposure to hyperoxia (VH), circulating cell-free Hb (HN), both insults (HH), or neither insult (VN). RepresentativeMRI images show a rat brain on postnatal day 7 (P7). a Diffusion tensor imaging with the capsula interna dexter (c int dx) marked with a white line. b Corresponding T2-weighted image in the same animal. c Fractional anisotropy (FA) measured in the c int dx in all experimental groups at all time points. d T2 relaxation times measured in the c int dx in all experimental groups at all time points. c, d Median values (bars) and individual animals (symbols) are displayed. n =3–4 for each group.

Fig. 6

Immunohistochemistry analysis of myelin formation in rat pup brain after exposure to hyperoxia (VH), circulating cell-free Hb (HN), both insults (HH), or neither insult (VN). a–d Representative images show myelin basic protein (MBP) in brain sections on postnatal day 11 (P11) (120 h after injection) in all experimental groups. Scale bar, 50 µm. a DAPI staining of cell nuclei (blue). b Olig2+ cells (red). c MBP (green). d Merged image. e–g Quantification of MBP protein content displayed as a fraction of the region of interest area (%) in white matter (e), thalamus (f), and cingulum (g). h Western blot analysis of MBP content (MBP band 1 + 2) compared to endogenous β-actin (actB) in brain homogenates on P11. Results are presented as medians and interquartile ranges; n = 5 (a–c) and n = 8 (d). DAPI, 4′,6-diamidino-2-phenylindole; Olig2, oligodendrocyte transcription factor 2.

Fig. 7

qPCR array analysis of 4 different pathways of injury in pooled rat pup brain tissues after exposure to hyperoxia (VH), circulating cell-free Hb (HN), or both insults (HH). Oxidative stress, apoptosis, NFκB signaling pathway, and tight junction arrays were analyzed on postnatal day (P) 6 (6 h after injection), P7 (24 h after injection), and P11 (120 h after injection). Statistical analyses of intergroup comparisons were not performed as these analyses were performed on pooled material.

Fig. 8

qPCR analyses of ICAM-1 mRNA expression in individual rat pup brains after exposure to hyperoxia (VH), circulating cell-free Hb (HN), both insults (HH), or neither insult (VN). Relative expression levels were evaluated on postnatal day (P) 6 (6 h after injection) (a), P7 (24 h after injection) (b), and P11 (120 h after injection) (c). Fold changes are displayed as medians and interquartile ranges; n = 4–11. * p < 0.05. ICAM-1, intercellular adhesion molecule 1.