Effects of chronic Helicobacter pylori strain PMSS1 infection on whole brain and gastric iron homeostasis in male INS-GAS mice.

Iron deficiency, the most common micronutrient deficiency in humans, is associated with long-term deficits in cognition and memory if left untreated. Infection with the gastric pathogen Helicobacter pylori has been linked to iron deficiency anemia (IDA). The H. pylori virulence factor cytotoxin-associated gene A (cagA) is proposed to be especially pertinent in iron deficiency. Male INS-GAS/FVB mice were infected with the CagA+ strain pre-murine Sydney strain 1 (PMSS1) for 12-13 or 27-29 weeks to investigate the role of chronic H. pylori infection in iron deficiency and neurological sequelae. Mice at both timepoints demonstrated significantly elevated gastric histopathology scores and inflammatory cytokines compared to sham-dosed controls. However, only mice at 27-29 weeks post infection had changes in hematological parameters, with significantly decreased erythrocyte count, hematocrit, serum hemoglobin, and increased serum total iron binding capacity. Gastric transcription of iron-regulatory genes Hamp and Bmp4 were significantly downregulated at both timepoints. In the brain, iron-dependent myelingergic and synaptic markers were significantly downregulated at 27-29 weeks. These results indicated that long-term infection of the CagA + PMSS1 strain of H. pylori in this study caused anemia, altered gastric iron homeostasis, and neurological changes similar to those reported in other rodent H. pylori CagA- strain infection models.

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.

Early-Life Iron Deficiency Alters Glucose Transporter-1 Expression in the Adult Rodent Hippocampus.

BACKGROUND: Early-life iron deficiency (ID) impairs hippocampal energy production. Whether there are changes in glucose transporter (GLUT) expression is not known. OBJECTIVE: The aim of this study was to investigate whether early-life ID and the treatment iron dose alter brain regional GLUT expression in adult rats and mice. METHODS: In Study 1, ID was induced in male and female Sprague Dawley rat pups by feeding dams a 3-mg/kg iron diet during gestation and the first postnatal week, followed by treatment using low-iron [3-10 mg/kg; formerly iron-deficient (FID)-10 group], standard-iron (40-mg/kg; FID-40 group), or high-iron (400-mg/kg; FID-400 group) diets until weaning. The control group received the 40 mg/kg iron diet. GLUT1, GLUT3, hypoxia-inducible factor (HIF)-1α, and prolyl-hydroxylase-2 (PHD2) mRNA and protein expression in the cerebral cortex, hippocampus, striatum, cerebellum, and hypothalamus were determined at adulthood. In Study 2, the role of hippocampal ID in GLUT expression was examined by comparing the Glut1, Glut3, Hif1α, and Phd2 mRNA expression in adult male and female wild-type (WT) and nonanemic hippocampal iron-deficient and iron-replete dominant negative transferrin receptor 1 (DNTfR1-/-) transgenic mice. RESULTS: In Study 1, Glut1, Glut3, and Hif1α mRNA, and GLUT1 55-kDa protein expression was upregulated 20-33% in the hippocampus of the FID-10 group but not the FID-40 group, relative to the control group. Hippocampal Glut1 mRNA (-39%) and GLUT1 protein (-30%) expression was suppressed in the FID-400 group, relative to the control group. Glut1 and Glut3 mRNA expression was not altered in the other brain regions in the 3 FID groups. In Study 2, hippocampal Glut1 (+14%) and Hif1α (+147%) expression was upregulated in the iron-deficient DNTfR1-/- mice, but not in the iron-replete DNTfR1-/- mice, relative to the WT mice (P < 0.05, all). CONCLUSIONS: Early-life ID is associated with altered hippocampal GLUT1 expression in adult rodents. The mouse study suggests that tissue ID is potentially responsible.

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 hyperglycemia induces CXCL10/CXCR3 signaling and microglial activation and impairs long-term synaptogenesis in the hippocampus and alters behavior in rats.

BACKGROUND: Hyperglycemia is common in extremely low gestational age newborns (ELGAN) and is associated with increased mortality and morbidity, including abnormal neurodevelopment. Hippocampus-mediated cognitive deficits are common in this population, but the specific effects of hyperglycemia on the developing hippocampus are not known. METHODS: The objective of this study was to determine the acute and long-term effects of hyperglycemia on the developing hippocampus in neonatal rats using a streptozotocin (STZ)-induced model of hyperglycemia. STZ was injected on postnatal day (P) 2, and littermates in the control group were injected with an equivalent volume of citrate buffer. The acute effects of hyperglycemia on markers of oxidative stress, inflammatory cytokines, microglial activation, and reactive astrocytosis in the hippocampus were determined in the brain tissue collected on P6. The long-term effects on hippocampus-mediated behavior and hippocampal dendrite structure were determined on P90. RESULTS: On P6, the transcript and protein expression of markers of oxidative stress and inflammatory cytokines, including the CXCL10/CXCR3 pathway, were upregulated in the hyperglycemia group. Histological evaluation revealed microglial activation and astrocytosis. The long-term assessment on P90 demonstrated abnormal performance in Barnes maze neurobehavioral testing and altered dendrite structure in the hippocampus of formerly hyperglycemic rats. CONCLUSIONS: Neonatal hyperglycemia induces CXCL10/CXCR3 signaling, microglial activation, and astrocytosis in the rat hippocampus and alters long-term synaptogenesis and behavior. These results may explain the hippocampus-specific cognitive deficits common in ELGAN who experience neonatal hyperglycemia.

Neonatal hyperglycemia alters the neurochemical profile, dendritic arborization and gene expression in the developing rat hippocampus.

Hyperglycemia (blood glucose concentration >150 mg/dL) is common in extremely low gestational age newborns (ELGANs; birth at <28 week gestation). Hyperglycemia increases the risk of brain injury in the neonatal period. The long-term effects are not well understood. In adult rats, hyperglycemia alters hippocampal energy metabolism. The effects of hyperglycemia on the developing hippocampus were studied in rat pups. In Experiment 1, recurrent hyperglycemia of graded severity (moderate hyperglycemia (moderate-HG), mean blood glucose 214.6 ± 11.6 mg/dL; severe hyperglycemia (severe-HG), 338.9 ± 21.7 mg/dL; control, 137.7 ± 2.6 mg/dL) was induced from postnatal day (P) 3 to P12. On P30, the hippocampal neurochemical profile was determined using in vivo 1 H MR spectroscopy. Dendritic arborization in the hippocampal CA1 region was determined using microtubule-associated protein (MAP)-2 immunohistochemistry. In Experiment 2, continuous hyperglycemia (mean blood glucose 275.3 ± 25.8 mg/dL; control, 142.3 ± 2.6 mg/dL) was induced from P2 to P6 by injecting streptozotocin (STZ) on P2. The mRNA expression of glycogen synthase 1 (Gys1), lactate dehydrogenase (Ldh), glucose transporters 1 (Glut1) and 3 (Glut3) and monocarboxylate transporters 1 (Mct1), 2 (Mct2) and 4 (Mct4) in the hippocampus was determined on P6. In Experiment 1, MRS demonstrated lower lactate concentration and glutamate/glutamine (Glu/Gln) ratio in the severe-HG group, compared with the control group (p < 0.05). Phosphocreatine/creatine ratio was higher in both hyperglycemia groups (p < 0.05). MAP-2 histochemistry demonstrated longer apical segment length, indicating abnormal synaptic efficacy in both hyperglycemia groups (p < 0.05). Experiment 2 showed lower Glut1, Gys1 and Mct4 expression and higher Mct1 expression in the hyperglycemia group, relative to the control group (p < 0.05). These results suggest that hyperglycemia alters substrate transport, lactate homeostasis, dendritogenesis and Glu-Gln cycling in the developing hippocampus. Abnormal neurochemical profile and dendritic structure due to hyperglycemia may partially explain the long-term hippocampus-mediated cognitive deficits in human ELGANs.

Metabolomic analysis of CSF indicates brain metabolic impairment precedes hematological indices of anemia in the iron-deficient infant monkey.

OBJECTIVES: Iron deficiency (ID) anemia leads to long-term neurodevelopmental deficits by altering iron-dependent brain metabolism. The objective of the study was to determine if ID induces metabolomic abnormalities in the cerebrospinal fluid (CSF) in the pre-anemic stage and to ascertain the aspects of abnormal brain metabolism affected. METHODS: Standard hematological parameters [hemoglobin (Hgb), mean corpuscular volume (MCV), transferrin (Tf) saturation, and zinc protoporphyrin/heme (ZnPP/H)] were compared at 2, 4, 6, 8, and 12 months in iron-sufficient (IS; n = 7) and iron-deficient (ID; n = 7) infant rhesus monkeys. Five CSF metabolite ratios were determined at 4, 8, and 12 months using 1H NMR spectroscopy at 16.4 T and compared between groups and in relation to hematologic parameters. RESULTS: ID infants developed ID (Tf saturation < 25%) by 4 months of age and all became anemic (Hgb < 110 g/L and MCV < 60 fL) at 6 months. Their heme indices normalized by 12 months. Pyruvate/glutamine and phosphocreatine/creatine (PCr/Cr) ratios in CSF were lower in the ID infants by 4 months (P < 0.05). The PCr/Cr ratio remained lower at 8 months (P = 0.02). ZnPP/H, an established blood marker of pre-anemic ID, was positively correlated with the CSF citrate/glutamine ratio (marginal correlation, 0.34; P < 0.001; family wise error rate = 0.001). DISCUSSION: Metabolomic analysis of the CSF is sensitive for detecting the effects of pre-anemic ID on brain energy metabolism. Persistence of a lower PCr/Cr ratio at 8 months, even as hematological measures demonstrated recovery from anemia, indicate that the restoration of brain energy metabolism is delayed. Metabolomic platforms offer a useful tool for early detection of the impact of ID on brain metabolism in infants.

Characterization of the concurrent metabolic changes in brain and plasma during insulin-induced moderate hypoglycemia using 1H NMR spectroscopy in juvenile rats.

Treatment of hypoglycemia in children is currently based on plasma glucose measurements. This approach may not ensure neuroprotection since plasma glucose does not reflect the dynamic state of cerebral energy metabolism. To determine whether cerebral metabolic changes during hypoglycemia could be better characterized using plasma metabolomic analysis, insulin-induced acute hypoglycemia was induced in 4-week-old rats. Brain tissue and concurrent plasma samples were collected from hypoglycemic (N=7) and control (N=7) rats after focused microwave fixation to prevent post-mortem metabolic changes. The concentration of 29 metabolites in brain and 34 metabolites in plasma were determined using 1H NMR spectroscopy at 700MHz and examined using partial least squares-discriminant analysis. The sensitivity of plasma glucose for detecting cerebral energy failure was assessed by determining its relationship to brain phosphocreatine. The brain and plasma metabolite profiles of the hypoglycemia group were distinct from the control group (brain: R2=0.92, Q2=0.31; plasma: R2=0.95, Q2=0.74). Concentration differences in glucose, ketone bodies and amino acids were responsible for the intergroup separation. There was 45% concordance between the brain and plasma metabolite profiles. Brain phosphocreatine correlated with brain glucose (control group: R2=0.86; hypoglycemia group: R2=0.59; p<0.05), but not with plasma glucose. The results confirm that plasma glucose is an insensitive biomarker of cerebral energy changes during hypoglycemia and suggest that a plasma metabolite profile is superior for monitoring cerebral metabolism.

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.

Helicobacter pylori infection and low dietary iron alter behavior, induce iron deficiency anemia, and modulate hippocampal gene expression in female C57BL/6 mice.

Helicobacter pylori (H.pylori), a bacterial pathogen, is a causative agent of gastritis and peptic ulcer disease and is a strong risk factor for development of gastric cancer. Environmental conditions, such as poor dietary iron resulting in iron deficiency anemia (IDA), enhance H.pylori virulence and increases risk for gastric cancer. IDA affects billions of people worldwide, and there is considerable overlap between regions of high IDA and high H.pylori prevalence. The primary aims of our study were to evaluate the effect of H.pylori infection on behavior, iron metabolism, red blood cell indices, and behavioral outcomes following comorbid H. pylori infection and dietary iron deficiency in a mouse model. C57BL/6 female mice (n = 40) were used; half were placed on a moderately iron deficient (ID) diet immediately post-weaning, and the other half were maintained on an iron replete (IR) diet. Half were dosed with H.pylori SS1 at 5 weeks of age, and the remaining mice were sham-dosed. There were 4 study groups: a control group (-Hp, IR diet) as well as 3 experimental groups (-Hp, ID diet; +Hp, IR diet; +Hp,ID diet). All mice were tested in an open field apparatus at 8 weeks postinfection. Independent of dietary iron status, H.pylori -infected mice performed fewer exploratory behaviors in the open field chamber than uninfected mice (p<0.001). Hippocampal gene expression of myelination markers and dopamine receptor 1 was significantly downregulated in mice on an ID diet (both p<0.05), independent of infection status. At 12 months postinfection, hematocrit (Hct) and hemoglobin (Hgb) concentration were significantly lower in +Hp, ID diet mice compared to all other study groups. H.pylori infection caused IDA in mice maintained on a marginal iron diet. The mouse model developed in this study is a useful model to study the neurologic, behavioral, and hematologic impact of the common human co-morbidity of H. pylori infection and IDA.

Recurrent Moderate Hypoglycemia Suppresses Brain-Derived Neurotrophic Factor Expression in the Prefrontal Cortex and Impairs Sensorimotor Gating in the Posthypoglycemic Period in Young Rats.

Recurrent hypoglycemia is common in infants and children. In developing rat models, recurrent moderate hypoglycemia leads to neuronal injury in the medial prefrontal cortex. To understand the effects beyond neuronal injury, 3-week-old male rats were subjected to 5 episodes of moderate hypoglycemia (blood glucose concentration, approx. 30 mg/dl for 90 min) once daily from postnatal day 24 to 28. Neuronal injury was determined using Fluoro-Jade B histochemistry on postnatal day 29. The effects on brain-derived neurotrophic factor (BDNF) and its cognate receptor, tyrosine kinase receptor B (TrkB) expression, which is critical for prefrontal cortex development, were determined on postnatal day 29 and at adulthood. The effects on prefrontal cortex-mediated function were determined by assessing the prepulse inhibition of the acoustic startle reflex on postnatal day 29 and 2 weeks later, and by testing for fear-potentiated startle at adulthood. Recurrent hypoglycemia led to neuronal injury confined primarily to the medial prefrontal cortex. BDNF/TrkB expression in the prefrontal cortex was suppressed on postnatal day 29 and was accompanied by lower prepulse inhibition, suggesting impaired sensorimotor gating. Following the cessation of recurrent hypoglycemia, the prepulse inhibition had recovered at 2 weeks. BDNF/TrkB expression in the prefrontal cortex had normalized and fear-potentiated startle was intact at adulthood. Recurrent moderate hypoglycemia during development has significant adverse effects on the prefrontal cortex in the posthypoglycemic period.

Helicobacter pylori Infection Induces Anemia, Depletes Serum Iron Storage, and Alters Local Iron-Related and Adult Brain Gene Expression in Male INS-GAS Mice.

Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world's population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6-9 weeks of age, and were necropsied at 27-29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use of Hp SS1-infected INS-GAS mice will be an appropriate animal model for further study of the effects of concurrent H. pylori infection and anemia on iron homeostasis and adult iron-dependent brain gene expression.

Recurrent hypoinsulinemic hyperglycemia in neonatal rats increases PARP-1 and NF-κB expression and leads to microglial activation in the cerebral cortex.

BACKGROUND: Hyperglycemia is a common metabolic problem in extremely low-birth-weight preterm infants. Neonatal hyperglycemia is associated with increased mortality and brain injury. Glucose-mediated oxidative injury may be responsible. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in DNA repair and cell survival. However, PARP-1 overactivation leads to cell death. NF-κB is coactivated with PARP-1 and regulates microglial activation. The effects of recurrent hyperglycemia on PARP-1/NF-κB expression and microglial activation are not well understood. METHODS: Rat pups were subjected to recurrent hypoinsulinemic hyperglycemia of 2 h duration twice daily from postnatal (P) day 3-P12 and killed on P13. mRNA and protein expression of PARP-1/NF-κB and their downstream effectors were determined in the cerebral cortex. Microgliosis was determined using CD11 immunohistochemistry. RESULTS: Recurrent hyperglycemia increased PARP-1 expression confined to the nucleus and without causing PARP-1 overactivation and cell death. NF-κB mRNA expression was increased, while IκB mRNA expression was decreased. inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) mRNA expressions were decreased. Hyperglycemia significantly increased the number of microglia. CONCLUSION: Recurrent hyperglycemia in neonatal rats is associated with upregulation of PARP-1 and NF-κB expression and subsequent microgliosis but not neuronal cell death in the cerebral cortex.

Acute hypoglycemia results in reduced cortical neuronal injury in the developing IUGR rat.

BACKGROUND: Hypoglycemia (HG) is common in intrauterine growth restricted (IUGR) neonates. In normally grown (NG) neonatal rats, acute HG causes neuronal injury in the brain; the cerebral cortex is more vulnerable than the hippocampus (HPC). We hypothesized that the IUGR brain is less vulnerable to HG-induced injury while preserving regional variation in vulnerability. METHODS: We induced IUGR via bilateral uterine artery ligation on gestational day 19 (term 22 d) rats. On postnatal day 14, insulin-induced HG of equivalent severity and duration (blood glucose < 40 mg/dl for 240 min) was produced in IUGR and NG (IUGR/HG and NG/HG). Neuronal injury in the cortex and HPC was quantified 6-72 h later using Fluoro-Jade B (FJB) histochemistry. The mRNA expression of monocarboxylate transporters, MCT1 and MCT2, and glucose transporters, GLUT1 and GLUT3, was determined using quantitative PCR. RESULTS: There were fewer FJB-positive (FJB+) cells in the cortex of IUGR/HG; no difference was observed in FJB+ cells in HPC. Core body temperature was lower in IUGR/HG compared with NG/HG. MCT2 expression was increased in the IUGR cortex. CONCLUSION: HG-induced neuronal injury is decreased in the cortex of the developing IUGR brain. Adaptations including systemic hypothermia and enhanced delivery of alternative substrates via MCT2 might protect against HG-induced neuronal injury in IUGR.

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.

Hyperglycemia accentuates and ketonemia attenuates hypoglycemia-induced neuronal injury in the developing rat brain.

BACKGROUND: Prolonged hypoglycemia leads to brain injury, despite treatment with 10% dextrose. Whether induction of hyperglycemia or ketonemia achieves better neuroprotection is unknown. Hyperglycemia is neuroprotective in other brain injuries during development; however, it worsens hypoglycemia-induced injury in the adult brain via poly(ADP-ribose)polymerase-1 (PARP-1) overactivation. METHODS: Three-week-old rats were subjected to insulin-induced hypoglycemia and treated with 10% dextrose or 50% dextrose. Neuronal injury, PARP-1, and brain-derived neurotrophic factor (BDNF) III/TrkB/p75(NTR) expressions were determined. In the second experiment, ketonemia was induced by administering ß-hydroxybutyrate during hypoglycemia and its effect on neuronal injury was compared with those conventionally treated using 10% dextrose. RESULTS: Both 10 and 50% dextrose administration led to hyperglycemia (50% dextrose > 10% dextrose). Compared with the 10% dextrose group, neuronal injury was greater in the 50% dextrose group and was accompanied by PARP-1 overactivation. BDNF III and p75(NTR), but not TrkBFL, mRNA expressions were upregulated. Neuronal injury was less severe in the rats subjected to ketonemia, compared with those conventionally treated using 10% dextrose. CONCLUSION: Hyperglycemia accentuated hypoglycemia-induced neuronal injury, likely via PARP-1 overactivation. Although BDNF was upregulated, it was not neuroprotective and potentially exaggerated injury by binding to p75(NTR) receptor. Conversely, ketonemia during hypoglycemia attenuated neuronal injury.

Metabolomic analysis of cerebrospinal fluid indicates iron deficiency compromises cerebral energy metabolism in the infant monkey.

Iron deficiency anemia affects many pregnant women and young infants worldwide. The health impact is significant, given iron's known role in many body functions, including oxidative and lipid metabolism, protein synthesis and brain neurochemistry. The following research determined if (1)H NMR spectroscopy-based metabolomic analysis of cerebrospinal fluid (CSF) could detect the adverse influence of early life iron deficiency on the central nervous system. Using a controlled dietary model in 43 infant primates, distinct differences were found in spectra acquired at 600 MHz from the CSF of anemic monkeys. Three metabolite ratios, citrate/pyruvate, citrate/lactate and pyruvate/glutamine ratios, differed significantly in the iron deficient infant and then normalized following the consumption of dietary iron and improvement of clinical indices of anemia in the heme compartment. This distinctive metabolomic profile associated with anemia in the young infant indicates that CSF can be employed to track the neurological effects of iron deficiency and benefits of iron supplementation.

Iron supplementation dose for perinatal iron deficiency differentially alters the neurochemistry of the frontal cortex and hippocampus in adult rats.

BACKGROUND: Long-term prefrontal cortex (PFC)- and hippocampus-based cognitive deficits are the sequelae of perinatal iron deficiency, despite iron supplementation starting in the newborn period. Whether high-dose iron supplementation prevents these deficits is yet to be determined. METHODS: Perinatal iron deficiency was induced in rat pups using a low-iron (3 mg/kg diet) diet during gestation until postnatal day (P)8. Iron was supplemented using a standard (40 mg/kg diet) or a 10-fold higher (400 mg/kg diet) iron-containing diet until P21. PFC and hippocampal neurochemistry was determined using in vivo (1)H nuclear magnetic resonance (NMR) spectroscopy at 9.4 Tesla on P90. RESULTS: Both standard and 10-fold higher iron supplementation doses corrected anemia and brain iron deficiency by P21. The neurochemical profile of the PFC in both supplementation groups was comparable with the control group. In the hippocampus, standard-dose iron supplementation resulted in lower concentrations of N-acetylaspartate (NAA) and phosphoethanolamine (PE) and higher concentrations of N-acetylaspartylglutamate (NAAG) and glycerophosphocholine + phosphocholine (GPC + PC). High-dose iron supplementation resulted in lower PE and higher GPC + PC concentrations. CONCLUSION: The iron supplementation dose for perinatal iron deficiency differentially alters the neurochemical profile of the PFC and hippocampus in adults. The neurochemical changes suggest altered glutamatergic neurotransmission, hypomyelination, and abnormal phospholipid metabolism in the formerly iron-deficient (FID) hippocampus.

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.

Determination of oxidative glucose metabolism in vivo in the young rat brain using localized direct-detected ¹³C NMR spectroscopy.

Determination of oxidative metabolism in the brain using in vivo ¹³C NMR spectroscopy (¹³C MRS) typically requires repeated blood sampling throughout the study to measure blood glucose concentration and fractional enrichment (input function). However, drawing blood from small animals, such as young rats, placed deep inside the magnet is technically difficult due to their small total blood volume. In the present study, a custom-built animal holder enabled temporary removal of the animal from the magnet for blood collection, followed by accurate repositioning in the exact presampling position without degradation of B0 shimming. ¹³C label incorporation into glutamate C4 and C3 positions during a 120 min [1,6-¹³C2] glucose infusion was determined in 28-day-old rats (n = 4) under α-chloralose sedation using localized, direct-detected in vivo ¹³C MRS at 9.4T. The tricarboxylic acid cycle activity rate (V(TCA)) determined using a one-compartment metabolic modeling was 0.67 ± 0.13 µmol/g/min, a value comparable to previous ex vivo studies. This methodology opens the avenue for in vivo measurements of brain metabolic rates using ¹³C MRS in small animals.