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Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats
Published on: November 20, 2015
Deficient brain RNA polymerase and altered nucleolar structure persists until day 8 after perinatal asphyxia of the
Philomena Kastner1, Wilhelm Mosgoeller, Susanne Fang-Kircher
1Department of Pediatrics, University of Vienna, Vienna, Austria.
Insights
Perinatal asphyxia (PA) significantly reduces RNA polymerase I (POL I) activity and causes nucleolar disintegration in the brain for up to 8 days. This persistent POL derangement impairs RNA and protein synthesis following birth asphyxia.
Area of Science:
- Biochemistry
- Neuroscience
- Molecular Biology
Background:
- RNA polymerases (POL) are crucial for protein synthesis, with POL I transcribing ribosomal RNA.
- Perinatal asphyxia (PA) is known to affect the POL system, impacting brain protein synthesis.
Purpose of the Study:
- To investigate the persistent effects of global perinatal asphyxia (PA) on RNA polymerase I (POL I) activity, mRNA, protein levels, and nucleolar morphology in a rodent model up to 8 days post-insult.
- To determine the long-term consequences of PA on brain protein synthesis machinery.
Main Methods:
- Assessed nuclear POL I activity using a radiochemical method.
- Evaluated mRNA and protein levels of the POL I/III subunit RPA40 via blotting.
- Determined the POL I subunit polymerase activating factor-53 (PAF-53) using immunohistochemistry.
- Examined nucleolar morphology via silver staining and transmission electron microscopy.
Main Results:
- Nuclear POL I activity decreased with PA severity up to 8 days post-insult.
- mRNA and protein levels of RPA40 remained unchanged.
- PAF-53 levels were significantly reduced in several brain regions.
- Significant nucleolar morphological changes, including disintegration, were observed.
Conclusions:
- Perinatal asphyxia leads to persistent decreases in POL I activity and nucleolar disintegration in the brain.
- Reduced PAF-53 and nucleolar damage suggest impaired RNA and protein synthesis post-PA.
- Severe acidosis and/or protein kinase C deficiency during PA may underlie these persistent effects.
Abstract:
RNA polymerases (POL) are integral constituents of the protein synthesis machinery, with POL I and POL III coding for ribosomal RNA and POL II coding for protein. POL I is located in the nucleolus and transcribes class I genes, those that code for large ribosomal RNA. It has been reported that the POL system is seriously affected in perinatal asphyxia (PA) immediately after birth. Because POL I is necessary for protein synthesis and brain protein synthesis was shown to be deranged after hypoxic-ischemic conditions, we aimed to study whether POL derangement persists in a simple, well-documented animal model of graded global PA at the activity, mRNA, protein, and morphologic level until 8 d after the asphyctic insult. Nuclear POL I activity was determined according to a radiochemical method; mRNA steady state and protein levels of RPA4O-an essential subunit of POL I and III-were evaluated by blotting methods; and the POL I subunit polymerase activating factor-53 was evaluated using immunohistochemistry. Silver staining and transmission electron microscopy were used to examine the nucleolus. At the eighth day after PA, nuclear POL I decreased with the length of the asphyctic period, whereas mRNA and protein levels for RPA4O were unchanged. The subunit polymerase activating factor-53, however, was unambiguously reduced in several brain regions. Dramatic changes of nucleolar morphology were observed, the main finding being nucleolar disintegration at the electron microscopy level. We suggest that severe acidosis and/or deficient protein kinase C in the brain during the asphyctic period may be responsible for disintegration of the nucleolus as well as for decreased POL activity persisting until the eighth day after PA. The biologic effect may be that PA causes impaired RNA and protein synthesis, which has been already observed in hypoxic-ischemic states.
