Skip to main content
main-content

01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Neuroinflammation 1/2017

Inflammatory signature of cerebellar neurodegeneration during neonatal hyperbilirubinemia in Ugt1 -/- mouse model

Zeitschrift:
Journal of Neuroinflammation > Ausgabe 1/2017
Autoren:
Simone Vodret, Giulia Bortolussi, Jana Jašprová, Libor Vitek, Andrés F. Muro
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12974-017-0838-1) contains supplementary material, which is available to authorized users.

Abstract

Background

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo.

Methods

We used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult.

Results

We showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNFα and NFKβ as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression.
Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals.

Conclusions

This study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1 -/- mice.
Zusatzmaterial
Additional file 1: Table listing the oligonucleotides used. (XLSX 13 kb)
12974_2017_838_MOESM1_ESM.xlsx
Additional file 2: A) Left panel, representative Nissl staining of cerebellar layers at P8 of WT and Ugt1 -/- mice. Right panel, layer depth quantification (μm) at P8 and P10 of WT and Ugt1 -/- mice. Scale bar 100 μm. B) WB analysis of total cerebellum protein extracts using an anti-NeuN antibody at P8. β-tubulin was used as a loading control. Values represent mean ± SD. C) Representative fluorescent immunohistochemistry of cerebellar sections from WT and Ugt1 -/- mice using anti-NeuN antibody (green) to stain differentiated granule cells at P8. Hoechst (blue) was used to mark nuclei. Scale bar: 50 μm. For all the experiments the values represent the mean ± SD. Student t test, ns not significant; *p < 0.05, **p < 0.01. WT n = 4, Ugt1 -/- n = 4. EGL, external germinal layer; IGL internal granular layer; ML, molecular layer. (TIF 6110 kb)
12974_2017_838_MOESM2_ESM.tif
Additional file 3: A) Representative fluorescent immunohistochemistry of WT and Ugt1 -/- cerebellum sections using anti-GFAP antibody (red) to highlight astrocytes, co-stained with an anti-calbindin antibody (green) to highlight PCs. Hoechst (blue) was used to mark nuclei. Scale bar: 500 μm. Boxed areas indicate fields shown in Fig.  3b. IV, VI, VIb, VII and IX indicate the cerebellar fissures. B) mRNA expression levels of GFAP at P5, P8 and P10 in total RNA preparations of WT and Ugt1 -/- cerebella. For each gene, data were normalized according to the values of the WT samples at P5. Values represent the mean ± S.D. Two-way ANOVA, ** p < 0.01, *** p < 0.001. Number of WT and Ugt1 -/- was ≥3 in all the experiments. (TIF 11619 kb)
Additional file 5: A) Example of expression levels under the detection limit of the technique. PCR product of IL1β mRNA expression. As positive control dendritic cells (DC) were treated with LPS for 24 hs. (lane 2). B) Example of detectable mRNA: TNFα expression. C) List of mRNAs that were not detected by qRT-PCR in cerebellar total RNA extracts. D) TLR2 and E) iNOS mRNA relative expression levels in WT and Ugt1 -/- animals at the indicated time points. For each gene, data were normalized according to the values of the WT samples at P5. Values represent mean ± SD. Two-way ANOVA, ns not significant. For representative agarose gels, lane 1: 1Kb ladder; lane 2: PCR product from dendritic cells (DCs) treated with LPS for 24 h; lane 3: CB, Ugt1 -/- cerebellar total RNA extract; lane 4, blank. (TIF 2216 kb)
12974_2017_838_MOESM5_ESM.tif
Additional file 6: A) Relative mRNA expression of ER stress response genes showing no changes at the different time points. For each gene, data were normalized according to the values of the WT samples at P5. Values represent the mean ± S.D. Two-way ANOVA, ns not significant. Number of WT and Ugt1 -/- was ≥3 in all the experiments B) WB analysis and quantification of total cerebellum protein extracts of WT and Ugt1 -/- mice using an anti-CHOP antibody at the indicated time points. Actin was used as loading control. Student t test, ns not significant. C) Representative IF of cerebellar sections from Ugt1 -/- mice at P10 using an anti-CHOP antibody (red), co-stained with an anti-calbindin antibody (green) to highlight Purkinje cells; D) representative IF of cerebellar sections from WT and Ugt1 -/- mice at P10 using an anti-CHOP antibody (red), co-stained with an anti-NeuN antibody to highlight granule cells; E) representative IF of cerebellar sections from Ugt1 -/- mice at P10 using an anti-CHOP antibody (red), co-stained with an anti-Iba1 antibody to highlight microglia. For IF, Hoechst dye (blue) was used to mark nuclei. Scale bar: 50 μm. (TIF 24031 kb)
12974_2017_838_MOESM6_ESM.tif
Additional file 7: Relative mRNA expression of A) oxidative stress response genes and B) Cyp1a1 and Cyp2a5 showing no changes at the different time points. For each gene, data were normalized according to the values of the WT samples at P5. Values represent the mean ± S.D. Two-way ANOVA, ns not significant. Number of WT and Ugt1 -/- was ≥3 in all the experiments. C) Representative IF of cerebellar sections from WT and Ugt1 -/- mice at P5 using an anti-HO1 antibody (red), co-stained with anti-Iba1 antibody to highlight microglia. For IF, Hoechst dye (blue) was used to mark nuclei. Scale bar: 50 μm. (TIF 10064 kb)
12974_2017_838_MOESM7_ESM.tif
Literatur
Über diesen Artikel

Weitere Artikel der Ausgabe 1/2017

Journal of Neuroinflammation 1/2017 Zur Ausgabe

Neu in den Fachgebieten Neurologie und Psychiatrie

Meistgelesene Bücher in der Neurologie & Psychiatrie

  • 2016 | Buch

    Neurologie

    Das Lehrbuch vermittelt Ihnen das gesamte Neurologie-Prüfungswissen für Ihr Medizinstudium und bereitet auch junge Assistenzärzte durch detailliertes Fachwissen optimal auf die Praxis vor. Die komplett überarbeitete Auflage enthält sechs neue, interdisziplinäre Kapitel.

    Herausgeber:
    Werner Hacke
  • 2016 | Buch

    Komplikationen in der Neurologie

    Das Buch schildert Ereignisse im Rahmen der Neuromedizin, die während der Diagnostik und Therapie neurologischer Erkrankungen und Symptome auftreten können. Die Fallbeispiele sensibilisieren Sie für mögliche Risikofaktoren, um das Auftreten solcher Komplikationen zu vermeiden.

    Herausgeber:
    Frank Block
  • 2017 | Buch

    Facharztwissen Psychiatrie, Psychosomatik und Psychotherapie

    Leitsymptome, Untersuchungsmethoden, Krankheitsbilder, Notfälle & Co. – mit der Neuauflage des "Facharztwissens" sind Sie auf die Facharztprüfung in Psychiatrie und Psychotherapie optimal vorbereitet. In dieser 2. Auflage sind die Kapitel zu psychosomatischen Störungen deutlich ausgebaut.

    Herausgeber:
    Prof. Dr. Dr. Frank Schneider
  • 2019 | Buch

    Kompendium der Psychotherapie

    Für Ärzte und Psychologen

    Dieses Werk wendet sich an Ärzte und Psychologen, die an psychiatrischen und psychosomatischen Kliniken oder an Psychotherapeutischen Ausbildungsinstituten arbeiten und in den vorhandenen Lehrbüchern der Psychotherapie den Brückenschlag zur …

    Herausgeber:
    Tilo Kircher