Fellow University of Nebraska College of Medicine Omaha, Nebraska, United States
Background: Anemia is the common comorbiditity in premature infants. RBC transfusion remains the treatment of severe anemia that improves oxygen carrying capacity and promotes growth in the premature infants. In contrast anemia, blood transfusion and brain inflammation have correlation in clinical and animal studies. Severe anemia has shown hippocampal injury in animal study, but the effects of RBC transfusion on brain inflammation after anemia has not yet been evaluated. Objective: To identify the inflammatory changes in the neonatal mouse brain of phlebotomy-induced anemia and RBC transfusion. Design/Methods: C57BL/6 pups were studied in 4 groups namely naïve control, RBC transfused, severe anemia and severe anemia with RBC transfusion. Severe anemia was induced by facial vein phlebotomy on postnatal day (P) 2, 4, 6, 8, and 10. RBC transfusion consisted of 20 ml/kg of leukoreduced and stored packed RBC from allogeneic adult FVB mice donors, administered intravenously into the retro-orbital plexus on P11. Whole brain tissue was extracted 24 hr post-tranfusion, and total protein concentration was estimated by BCA Bradford assay. Milliplex Map Mouse cytokine/chemokine multiplex assay were used to quantify the cytokines and chemokines in brain tissue homogenate. Immunostaining was performed to localize the circulating monocyte recruitment and microglial activation by using antibodies for Ly6c and Iba-1. The recruitment of monocytes in anemic-transfused brain were investigated by flow cytometry. Results: The inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α and IL-12p70 were significantly increased in the brain of anemic transfused mouse compared to transfused controls. There were no significant changes on Il4 and IL5 levels. The chemokines (C-C motif ligands) MCP1 [CCL2], MIP1-α [CCL3], MIP1-β [CCL4], RANTES [CCL5] and Eotoxin [CCL11] were significantly increased in anemic-transfused mouse pups. Immunostaining of anemic-transfused brain showed presence of increase number of Ly6C+ monocytes than transfusion control and confirmed that anemic-transfusion related brain inflammation was associated with increased number of Ly6C+ monocytes and activated microglia (Iba1+ with round amoeboid shape) in the brain. Flow cytometry also confirmed the presence of circulating monocyte phenotype with CD11b+CD45+Ly6C+ markers in anemic-transfused brain.
Conclusion(s): RBC transfusions increase the inflammatory cytokines and monocyte attractant chemokines in murine pups brain with severe anemia. Circulating monocytes were recruited in the anemic-transfused brain that exacerbate anemia-induced inflammation.
