A High-throughput Analysis of Gene Expression in the Intestine in Severe Neonatal Anemia

Background: In recent years, most centers caring for premature and critically ill infants have adopted restrictive red blood cell (RBC) transfusion guidelines to minimize transfusions in asymptomatic infants unless the hematocrit drops below 20-22%.However, there is renewed awareness about the risks of anemia in neonates.We have shown that severely anemic human infants show higher intestinal permeability, bacterial translocation, and both local/systemic inflammation.In this study, we used an established mouse pup model to investigate the effect of severe anemia in the intestine using an open-ended, high-throughput approach using microarrays.Materials and methods: C57BL/6 mouse pups were rendered severely anemic (hematocrits = 20-24%, n = 14 in control and anemic arms) by serial phlebotomies on postnatal days 2-10.The ileocecal region was processed to extract mRNA, and gene expression was measured using open-ended, high-throughput microarray analyses, using a threshold for altered gene expression of ±1.5-fold.To ensure the effectiveness of the analyses and avoid unknown errors, the authors compiled their own robust bioinformatics pipelines for microarray data processing, principal component analysis (PCA), screening of differentially expressed genes (DEGs), hierarchical clustering analysis, gene-set enrichment analysis, and quantitative analysis.Results: We first conducted PCA using log-transformed gene expression data.The visualization of a few principal components (PCs) in intestinal tissue samples after batch-effect correction showed a distinct separation between control and anemia groups.Comparison of the anemic vs control samples showed 2,826 DEGs, with 1,041 upregulated and 1,825 downregulated genes.Enrichment analyses were conducted, targeting gene ontology (GO) for biological processes (BPs), molecular functions (MFs), and cellular components (CC).Two major pathway analyses were also used.The study showed that severe neonatal anemia has a significant biological impact in the gastrointestinal tract, particularly on the maintenance of epithelial cell integrity and gut leakiness, local innate immune responses, and inflammation.Conclusions/significance: We reviewed the upregulated and downregulated DEGs in this neonatal mouse model of severe anemia.However, even though many of these genes exert similar-looking pathophysiological effects, the reasons for some being induced and others being suppressed were not readily evident.Similarly, the patterns seen in altered expression of some genetic pathways identified in ontology analyses could not be explained.The stage of development, and in humans, transfused blood (composition of the hemoglobin with adult and fetal isoforms) can be important confounding factors and need study.Overall, severe anemia induces significant pathophysiological changes in gut barrier function, innate immune responses, and inflammation.