Reduced Myelination and Increased Glia Reactivity Resulting from Severe Neonatal Hyperbilirubinemia

Abstract

Bilirubin-induced neurologic dysfunction (BIND) and kernicterus has been used to describe moderate to severe neurologic dysfunction observed in children exposed to excessive levels of total serum bilirubin (TSB) during the neonatal period. Here we use a new mouse model that targets deletion of the Ugt1 locus and the Ugt1a1 gene in liver to promote hyperbilirubinemia-induced seizures and central nervous system toxicity. The accumulation of TSB in these mice leads to diffuse yellow coloration of brain tissue and a marked cerebellar hypoplasia that we characterize as kernicterus. Histologic studies of brain tissue demonstrate that the onset of severe neonatal hyperbilirubinemia, characterized by seizures, leads to alterations in myelination and glia reactivity. Kernicterus presents as axonopathy with myelination deficits at different brain regions, including pons, medulla oblongata, and cerebellum. The excessive accumulation of TSB in the early neonatal period (5 days after birth) promotes activation of the myelin basic protein (Mbp) gene with an accelerated loss of MBP that correlates with a lack of myelin sheath formation. These changes were accompanied by increased astroglial and microglial reactivity, possibly as a response to myelination injury. Interestingly, cerebellum was the area most affected, with greater myelination impairment and glia burden, and showing a marked loss of Purkinje cells and reduced arborization of the remaining ones. Thus, kernicterus in this model displays not only axonal damage but also myelination deficits and glial activation in different brain regions that are usually related to the neurologic sequelae observed after severe hyperbilirubinemia.

Fig. 1.

Generation and characterization of UFP and UAC mice. (A) The targeting construct Ugt1a1^{loxP[FRTneoFRT]loxP} was electroporated into embryonic stem cells. The chimera mice were out-crossed with wild-type C57BL/6 mice, and then in-bred to generate mice carrying the homozygous Ugt1a1^{loxP[FRTneoFRT]loxP} allele (UFP mice). UFP mice were further bred into transgenic Albumin-Cre mice to generate Ugt1a1^F/F/albumin-Cre mice (UAC mice). (B) RT-PCR of mouse Ugt1a1 gene expression in liver tissue from mice with different genetic backgrounds. The primers crossed exon 1 to exon 5, generating a 1052-bp band for the intact Ugt1a1 gene, and a 788-bp band for the Ugt1a1 gene with exons 3 and 4 deleted as a result of Cre recombination. (C) Mouse liver microsomes (MLM) were prepared from mice at 14 days old. Western blot analysis demonstrated that MLM from UFP mice had lower UGT1A1 protein expression levels, in comparison with wild-type (wt) MLM samples. No detectable UGT1A1 expression was observed in UAC livers. (D) Bilirubin glucuronidation analysis was performed (mean ± S.E.M., ****p<0.0001, Student’s t test) by using MLM. (E) The lethality associated with neonatal UAC mice during different developmental stages was studied. GraphPad Prism was used to prepare the survival curve and the statistical analysis. (F) Blood samples were collected from both UFP and UAC mice at different developmental stages. Total serum bilirubin (TSB) was determined by using a bilirubinometer. Mice from at least three different cages were included at each time point. Student’s t test was used to determine the statistical significance (*P < 0.05, **P < 0.01). (G) Brains were collected from UFP and UAC neonates at 15 days after birth. Yellow color as a result of bilirubin accumulation was observed in the brain from UAC mice.

Fig. 2.

Kernicterus mice present axonal loss and decreased myelination. (A) Serial sagittal images of control and kernicterus mice. Each image represents a montage of 100–150 images at 10× magnification. Brain sections (3 μm) of each animal were immunolabeled to identify neuronal axons (neurofilaments, green, middle panel) and myelin basic protein (MBP, red, bottom panel). Top panel represents the superposition of neurofilaments, MBP and DAPI staining, the last used for nuclei counterstain (blue). Scale bar, 700 μm. (B) Expression of MBP in cerebellum. RNA was used for real-time PCR analysis and total cellular protein was used for Western blot analysis.

Fig. 3.

Reduced myelination and increased glial reactivity. Representative images from cerebellum, medulla and corpus callosum from control and kernicterus mice immunolabeled to identify (A) neurons (neurofilament, red) and myelin basic protein (MBP, green), (B) microglia (Iba-1+ staining, red) and MBP (green), and (C) astrocytes (GFAP+ staining, red) and MBP (green). Nuclei were counterstained with DAPI dye (blue).

Fig. 4.

Reduced myelinated fibers and increased microglia and astroglia burden in the kernicterus mice. Quantification of percentage of myelinated fibers (A) and the number of microglia (B) and astrocytes (C) per field in cerebellum, medulla, pons and corpus callosum from control and kernicterus mice. Results are mean ± S.E.M. from two control mice and three kernicterus mice performed in triplicate. *P < 0.05 versus respective control, **P < 0.01 versus respective control.

Fig. 5.

Myelination and glial reactivity changes in kernicterus mice. (A) Three different regions were specified, Region 1 (white matter nodes), region 2 (middle of white matter tracts), and region 3 (white matter terminals). Sections from control and kernicterus mice were immunolabeled to identify (B) neurons (neurofilament, red) and myelin basic protein (MBP, green), (C) microglia (Iba-1+ staining, red) and MBP (green), and (D) astrocytes (GFAP+ staining, red) and MBP (green). Nuclei were counterstained with DAPI dye (blue). Representative images from the three white mater regions are shown. Scale bar represents 25 µm.

Fig. 6.

Effect of kernicterus on myelination, microglia, and astrocyte density throughout white matter. Graph bars represent the quantification of percentage of (A) total area occupied by neurofilaments, (B) myelinated fibers, (C) the number of microglia, and (D) astrocytes per field in 3-different white matter regions (A, B, and C) in the cerebellum from control (UFP) and kernicterus (UAC) mice. Results are mean ± S.E.M. from three control mice and four kernicterus mice performed in triplicate. **P < 0.01 versus respective control.

Fig. 7.

Kernicterus mice present cerebellum atrophy and loss of Purkinje cells. (A) Three-micrometer sections of each animal were immunolabeled with adenomatous polyposis coli (CC-1) to identify Purkinje cells, and serial sagittal images were acquired from cerebellum of control and kernicterus mice. Each image represents a montage of 25–50 images at 10× magnification. Nuclei were counterstained with DAPI dye (blue). Scale bar, 700 μm. (B) Representative images of Purkinje cells morphology in control and kernicterus mice. Scale bar, 700 μm.