Fetal asphyctic preconditioning alters the transcriptional response to perinatal asphyxia.

BACKGROUND: Genomic reprogramming is thought to be, at least in part, responsible for the protective effect of brain preconditioning. Unraveling mechanisms of this endogenous neuroprotection, activated by preconditioning, is an important step towards new clinical strategies for treating asphyctic neonates.Therefore, we investigated whole-genome transcriptional changes in the brain of rats which underwent perinatal asphyxia (PA), and rats where PA was preceded by fetal asphyctic preconditioning (FAPA). Offspring were sacrificed 6 h and 96 h after birth, and whole-genome transcription was investigated using the Affymetrix Gene1.0ST chip. Microarray data were analyzed with the Bioconductor Limma package. In addition to univariate analysis, we performed Gene Set Enrichment Analysis (GSEA) in order to derive results with maximum biological relevance. RESULTS: We observed minimal, 25% or less, overlap of differentially regulated transcripts across different experimental groups which leads us to conclude that the transcriptional phenotype of these groups is largely unique. In both the PA and FAPA group we observe an upregulation of transcripts involved in cellular stress. Contrastingly, transcripts with a function in the cell nucleus were mostly downregulated in PA animals, while we see considerable upregulation in the FAPA group. Furthermore, we observed that histone deacetylases (HDACs) are exclusively regulated in FAPA animals. CONCLUSIONS: This study is the first to investigate whole-genome transcription in the neonatal brain after PA alone, and after perinatal asphyxia preceded by preconditioning (FAPA). We describe several genes/pathways, such as ubiquitination and proteolysis, which were not previously linked to preconditioning-induced neuroprotection. Furthermore, we observed that the majority of upregulated genes in preconditioned animals have a function in the cell nucleus, including several epigenetic players such as HDACs, which suggests that epigenetic mechanisms are likely to play a role in preconditioning-induced neuroprotection.

Fetal brain genomic reprogramming following asphyctic preconditioning.

BACKGROUND: Fetal asphyctic (FA) preconditioning is effective in attenuating brain damage incurred by a subsequent perinatal asphyctic insult. Unraveling mechanisms of this endogenous neuroprotection, activated by FA preconditioning, is an important step towards new clinical strategies for asphyctic neonates. Genomic reprogramming is thought to be, at least in part, responsible for the protective effect of preconditioning. Therefore we investigated whole genome differential gene expression in the preconditioned rat brain. FA preconditioning was induced on embryonic day 17 by reversibly clamping uterine circulation. Male control and FA offspring were sacrificed 96 h after FA preconditioning. Whole genome transcription was investigated with Affymetrix Gene1.0ST chip. RESULTS: Data were analyzed with the Bioconductor Limma package, which showed 53 down-regulated and 35 up-regulated transcripts in the FA-group. We validated these findings with RT-qPCR for adh1, edn1, leptin, rdh2, and smad6. Moreover, we investigated differences in gene expression across different brain regions. In addition, we performed Gene Set Enrichment Analysis (GSEA) which revealed 19 significantly down-regulated gene sets, mainly involved in neurotransmission and ion transport. 10 Gene sets were significantly up-regulated, these are mainly involved in nucleosomal structure and transcription, including genes such as mecp2. CONCLUSIONS: Here we identify for the first time differential gene expression after asphyctic preconditioning in fetal brain tissue, with the majority of differentially expressed transcripts being down-regulated. The observed down-regulation of cellular processes such as neurotransmission and ion transport could represent a restriction in energy turnover which could prevent energy failure and subsequent neuronal damage in an asphyctic event. Up-regulated transcripts seem to exert their function mainly within the cell nucleus, and subsequent Gene Set Enrichment Analysis suggests that epigenetic mechanisms play an important role in preconditioning induced neuroprotection.

Fetal asphyctic preconditioning protects against perinatal asphyxia- induced apoptosis and astrogliosis in neonatal brain.

Hypoxic-ischemic preconditioning is an endogenous mechanism in which exposure to a sublethal episode of hypoxia-ischemia protects against a subsequent more severe episode. Although several postnatal models of hypoxic-ischemic preconditioning have been established, hardly any perinatal models exist. Therefore, the objective of this study is to validate a new rodent model. We investigate whether mild fetal asphyxia (FA) as a preconditioning stimulus, protects against severe perinatal asphyxia (PA) when looking at neonatal brain histology. FA was induced at embryonic day 17 (E17) by temporarily clamping the uterine circulation. A caesarean section was performed at E21/22 and PA was induced by submersing the uterine horns, still containing the fetuses, in a water bath. Brains were examined for histological changes at either postnatal day 7 or 14. We used terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to detect apoptotic cell death and a glial fibrillary acidic protein (GFAP) staining to detect reactive astrocytes. Interestingly, the preconditioned group showed significantly less perinatal mortality than non-preconditioned groups. Furthermore, preconditioned animals had significantly less TUNEL-positive cells and less GFAP-positive cells in striatum, prefrontal cortex and hippocampus compared to the non-preconditioned animals that underwent PA. Consequently, mild FA might cause neuroprotection by inducing anti-apoptotic mechanisms and attenuating astrogliosis. Considering the morphological findings in the neonatal brain from this study, together with previously reported long-term behavioral outcomes in this model, we can conclude that this is a suitable experimental model to investigate mechanisms of endogenous neuroprotection in the fetal brain. Identifying these endogenous neuroprotective mechanisms will provide novel potential targets for future pharmacological intervention in asphyctic newborns.