Dose- and sex-dependent effects of phlebotomy-induced anemia on the neonatal mouse hippocampal transcriptome

Abstract

Background: Phlebotomy-induced anemia (PIA) is universal and variable in degree among preterm infants and may contribute to neurodevelopmental risk. In mice, PIA causes brain tissue hypoxia, iron deficiency, and long-term sex-dependent neurobehavioral abnormalities. The neuroregulatory molecular pathways disrupted by PIA underlying these effects are unknown. Methods: Male and female pups were phlebotomized daily from postnatal day (P)3-P14 via facial venipuncture to target hematocrits of 25% (moderate, mPIA) and 18% (severe, sPIA). P14 hippocampal RNA from non-bled control and PIA mice was sequenced by Next-Generation Sequencing to identify differentially-expressed-genes (DEGs) that were analyzed using Ingenuity Pathway Analysis. Results: mPIA females showed the least DEGs (0.5% of >22,000 genes) whereas sPIA females had the most (8.6%), indicating a dose-dependent effect. mPIA and sPIA males showed similar changes in gene expression (5.3% and 4.7%, respectively), indicating a threshold effect at mPIA. The pattern of altered genes induced by PIA indicate sex-specific and anemia-dose-dependent effects with increased pro-inflammation in females and decreased neurodevelopment in males. Conclusion: These gene-expression changes may underlie the reduced recognition memory function in male and abnormal social-cognitive behavior in female adult mice following neonatal PIA. These results parallel clinical studies demonstrating sex-specific behavioral outcomes as a function of neonatal anemia.

Sex-Specific Effects of Early-Life Iron Deficiency and Prenatal Choline Treatment on Adult Rat Hippocampal Transcriptome

“…By assessing global changes in hippocampal gene expression, this study presents novel findings pertaining to long-term sex-specific hippocampal effects of early-life ID and prenatal choline supplementation on gene regulation and changes in pre-defined neurologic biomarkers. Consistent with our previous findings in a neonatal model of PIA (8), substantially more gene expression changes in the hippocampal transcriptome were found in female than male rodents, indicating a more robust gene regulatory response to early-life ID in female rats. Prenatal choline supplementation, a promising adjunct therapy that prevents some long-term behavioral abnormalities following early-life ID, led to even more DEGs in both sexes of iron-sufficient compared to iron-deficient groups, further supporting previous observations of the interaction between choline and iron status in regulating hippocampal gene expression (25, 28).…”

“…In the formerly ID male rats, activation of ESR1 is a novel discovery and may have a role in reducing hippocampal-dependent cognitive impairment (32,33). These male-specific effects on gene regulators could explain the sex-specific cognitive impairments associated with the interaction between early-life neuroinflammation and iron status in both preclinical models and human infants (7,8,34,35). In the formerly ID female rats, activation of FOXO3 and MECP2 gene network could improve the overall metabolic activity and neuronal differentiation via recruitment of relevant target genes, including EGR4, FOS, ENO2, and NPAS4, thereby compensating for the reduced capacity of iron-mediated cellular metabolism (36)(37)(38)(39)(40)(41).…”

“…In the clinical population, there is evidence supporting sexspecific outcomes for iron-deficient individuals or its treatment during early life, including differential effects on fecal microbiota (5), brain development (6), verbal fluency (7), and risks of cognitive disorders such as schizophrenia (2). Sex-specific physiologic and behavioral effects are also observed in preclinical models (8)(9)(10)(11). Early postnatal ID anemia causes sex-specific neuroinflammatory responses (8), seizure vulnerability (9), social and affective behavioral abnormality (10), and vasculature dysfunction (11).…”

Survey of transfusion practices in preterm infants in Europe

“…The reasons for the use of such low haemoglobin thresholds in 22% of units are unclear but may represent an underestimation of transfusion threshold, as we had to use wide ranges for several clinical scenarios as described previously (online supplemental table S1). Without further clinical trials, it is not known whether these low haemoglobin thresholds are safe for neonates, as various preclinical studies have suggested adverse outcomes following prolonged severe anaemia 21–23…”

“…Without further clinical trials, it is not known whether these low haemoglobin thresholds are safe for neonates, as various preclinical studies have suggested adverse outcomes following prolonged severe anaemia. [21][22][23]…”