Effect of Perinatal Asphyxia on Protein Expression in Rat Prefrontal Cortex during Postnatal Development

Background:   Human epidemiological studies have linked labor and delivery complications leading to hypoxia/ischemia at birth with increased risk of neurodevelopmental disorders such as schizophrenia and autism. This can be modeled experimentally by exposing rat pups to 15 min of intrauterine anoxia during Caesarean section birth causing behavioral anomalies relevant to neurodevelopmental disorders such as autism. This model also affords us the opportunity to directly examine brain cellular mechanisms modified by this perinatal assault. 

Objectives:  To carry out a direct experimental test of the impact of perinatal asphyxia on protein expression in the postnatal prefrontal cortex.

Methods:  We used two dimensional differential in-gel electrophoresis and mass spectrometry, with targeted western blot analyses for confirmation, to quantify the proteomic profile of the prefrontal cortex - a region strongly implicated in autism - in 6 (adolescent) and 12 (adult) week old rats previously exposed to perinatal asphyxia. Control groups included pups delivered by C-section without anoxia and pups delivered vaginally. Group differences in global protein expression were examined using a partial least squares discriminative analysis ( SIMCA 9.0 software, Umetrics, Sweden; PLS-DA) and the expression differences of individual proteins examined using Analysis of Variance run on Progenesis software (initial screen across all groups, significance threshold set at p <0.1; post-hoc analysis of group contrasts, significance threshold set at p <0.05). Finally western blot was used to confirm gel-differences in selected proteins. 

Results:   Multivariate analyses of protein spots present in at least 75% of 60 images indicated a clear separation on the basis of protein expression between 6 and 12 week old animals and between the 3 experimental groups. Proteomic profiling revealed a significant up-regulation of mitochondrial enzymes such as isocitrate dehydrogenase, cytosolic malate dehydrogenase, and NADH dehydrogenase and significant  down-regulation of signaling proteins such as 14-3-3 and structural proteins such as neurofilament light polypeptide (NEFL) in the perinatally asphyxiated group.  Western blot confirmed the expression of isocitrate dehydrogenase was significantly up-regulated  whereas, 14-3-3 zeta and NEFL were significantly down-regulated, in the asphyxia group across all age groups (p<0.05). 

Conclusions:  The differences elicited by perinatal asphyxia are consistent with emerging evidence for mitochondrial dysregulation in neurodevelopmental disorders, and may in time contribute novel biomarkers and fresh targets for prevention or treatment.