Reticulocyte hemoglobin content as an early predictive biomarker of brain iron deficiency.

BACKGROUND: Fetal and neonatal brain iron content is compromised at the time of anemia, suggesting that screening for iron deficiency by measuring hemoglobin is inadequate to protect the brain. Reticulocyte hemoglobin (Ret-He) reflects iron-deficient (ID) erythropoiesis prior to anemia. METHODS: At postnatal day (P), 10 and 20 iron-sufficient rat pups were fostered to ID dams to produce a postnatal ID (PNID) group, which was compared to 20 iron-sufficient (IS) pups fostered by IS dams. Pups were assessed from P13 to P15 for hemoglobin, hematocrit, reticulocyte count, and Ret-He. Hippocampal iron status was assessed by transferrin receptor-1 (Tfrc-1) and divalent metal transporter-1 (Slc11a2) mRNA expression. RESULTS: At P13, brain iron status was similar between groups; only Ret-He was lower in the PNID group. At P14, the PNID group had lower Ret-He, hematocrit, mean corpuscular volume (MCV), and reticulocyte percentage (RET%). Tfrc-1 expression was increased, consistent with brain iron deficiency. Both Ret-He and MCV correlated with brain iron status at P14 and P15. CONCLUSIONS: Ret-He was the only red cell marker affected prior to the onset of brain ID. The clinical practice of using anemia as the preferred biomarker for diagnosis of iron deficiency may need reconsidering.

Neonatal mouse hippocampus: phlebotomy-induced anemia diminishes and treatment with erythropoietin partially rescues mammalian target of rapamycin signaling.

BackgroundPhlebotomy-induced anemia (PIA) is common in premature infants and affects neurodevelopment. PIA alters hippocampal metabolism in neonatal mice through tissue hypoxia and iron deficiency. The mammalian target of rapamycin (mTOR) pathway senses the status of critical metabolites (e.g., oxygen, iron), thereby regulating hippocampal growth and function. We determined the effect of PIA and recombinant human erythropoietin (rHuEpo) treatment on mTOR signaling and expression of genes related to mTOR pathway functions.MethodsMice receiving an iron-supplemented diet were phlebotomized from postnatal day (P)3 to a target hematocrit of <25% by P7. Half were maintained at <25% until P14; half received rHuEpo from P7 to increase the hematocrit to 25-28%. Hippocampal phosphorylated to total protein ratios of four key mTOR pathway proteins were measured by western blotting at P14 and compared with non-phlebotomized, non-anemic control mice. mRNA levels of genes regulated by mTOR were measured by quantitative PCR.ResultsPIA suppressed phosphorylation of all mTOR proteins. rHuEpo restored AMP-activated protein kinase (AMPK) and AKT status, and partially rescued the mTOR output protein S6K. PIA and rHuEpo treatment also altered the expression of genes regulated by S6K.ConclusionPIA compromises and rHuEpo treatment partially rescues a pathway regulating neuronal DNA transcription, protein translation, and structural complexity.

Phlebotomy-induced anemia alters hippocampal neurochemistry in neonatal mice.

BACKGROUND: Phlebotomy-induced anemia (PIA) is common in preterm infants. The hippocampus undergoes rapid differentiation during late fetal/early neonatal life and relies on adequate oxygen and iron to support oxidative metabolism necessary for development. Anemia shortchanges these two critical substrates, potentially altering hippocampal development and function. METHODS: PIA (hematocrit <25%) was induced in neonatal mice pups from postnatal day (P)3 to P14. Neurochemical concentrations in the hippocampus were determined using in vivo (1)H NMR spectroscopy at 9.4T and compared with control animals at P14. Gene expression was assessed using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: PIA decreased brain iron concentration, increased hippocampal lactate and creatine concentrations, and decreased phosphoethanolamine (PE) concentration and the phosphocreatine/creatine ratio. Hippocampal transferrin receptor (Tfrc) gene expression was increased, while the expression of calcium/calmodulin-dependent protein kinase type IIα (CamKIIα) was decreased in PIA mice. CONCLUSION: This clinically relevant model of neonatal anemia alters hippocampal energy and phospholipid metabolism and gene expression during a critical developmental period. Low target hematocrits for preterm neonates in the neonatal intensive care unit (NICU) may have potential adverse neural implications.

Postnatal morphine administration alters hippocampal development in rats.

Morphine is frequently used as an analgesic and sedative in preterm infants. Adult rats exposed to morphine have an altered hippocampal neurochemical profile and decreased neurogenesis in the dentate gyrus of the hippocampus. To evaluate whether neonatal rats are similarly affected, rat pups were injected twice daily with 2 mg/kg morphine or normal saline from postnatal days 3 to 7. On postnatal day 8, the hippocampal neurochemical profile was determined using in vivo (1)H NMR spectroscopy. The mRNA and protein concentrations of specific analytes were measured in hippocampus, and cell division in dentate gyrus was assessed using bromodeoxyuridine. The concentrations of γ-aminobutyric acid (GABA), taurine, and myo-insotol were decreased, whereas concentrations of glutathione, phosphoethanolamine, and choline-containing compounds were increased in morphine-exposed rats relative to control rats. Morphine decreased glutamic acid decarboxylase enzyme levels and myelin basic protein mRNA expression in the hippocampus. Bromodeoxyuridine labeling in the dentate gyrus was decreased by 60-70% in morphine-exposed rats. These results suggest that recurrent morphine administration during brain development alters hippocampal structure.

Prenatal alcohol-related alterations in maternal, placental, neonatal, and infant iron homeostasis.

BACKGROUND: Prenatal alcohol exposure (PAE) is associated with postnatal iron deficiency (ID), which has been shown to exacerbate deficits in growth, cognition, and behavior seen in fetal alcohol spectrum disorders. However, the mechanisms underlying PAE-related ID remain unknown. OBJECTIVES: We aimed to examine biochemical measures of iron homeostasis in the mother, placenta, neonate, and 6.5-month-old infant. METHODS: In a prenatally recruited, prospective longitudinal birth cohort in South Africa, 206 gravidas (126 heavy drinkers and 80 controls) were interviewed regarding alcohol, cigarette, and drug use and diet at 3 prenatal visits. Hemoglobin, ferritin, and soluble transferrin receptor (sTfR) were assayed twice during pregnancy and urinary hepcidin:creatinine was assayed once. Infant ferritin and hemoglobin were measured at 2 weeks and 6.5 months and sTfR was measured at 6.5 months. Histopathological examinations were conducted on 125 placentas and iron transport assays (iron regulatory protein-2, transferrin receptor-1, divalent metal transporter-1, ferroportin-1, and iron concentrations) were conducted on 63. RESULTS: In multivariable regression models, prenatal drinking frequency (days/week) was related to higher maternal hepcidin and to sequestration of iron into storage at the expense of erythropoiesis in mothers and neonates, as evidenced by a lower hemoglobin (g/dL)-to-log(ferritin) (ug/L) ratio [mothers: raw regression coefficient (ß) = -0.21 (95% CI: -0.35 to -0.07); neonates: ß = -0.15 (95% CI: -0.24 to -0.06)]. Drinking frequency was also related to decreased placental ferroportin-1:transferrin receptor-1 (ß = -0.57 for logged values; 95% CI: -1.03 to -0.10), indicating iron-restricted placental iron transport. At 6.5 months, drinking frequency was associated with lower hemoglobin (ß = -0.18; 95% CI: -0.33 to -0.02), and increased prevalences of ID (ß = 0.09; 95% CI: 0.02-0.17) and ID anemia (IDA) (ß = 0.13; 95% CI: 0.04-0.23). In causal inference analyses, the PAE-related increase in IDA was partially mediated by decreased neonatal hemoglobin:log(ferritin), and the decrease in neonatal hemoglobin:log(ferritin) was partially mediated by decreased maternal hemoglobin:log(ferritin). CONCLUSIONS: In this study, greater PAE was associated with an unfavorable profile of maternal-fetal iron homeostasis, which may play mechanistic roles in PAE-related ID later in infancy.