Temporal manipulation of transferrin-receptor-1-dependent iron uptake identifies a sensitive period in mouse hippocampal neurodevelopment.

Iron is a necessary substrate for neuronal function throughout the lifespan, but particularly during development. Early life iron deficiency (ID) in humans (late gestation through 2-3 yr) results in persistent cognitive and behavioral abnormalities despite iron repletion. Animal models of early life ID generated using maternal dietary iron restriction also demonstrate persistent learning and memory deficits, suggesting a critical requirement for iron during hippocampal development. Precise definition of the temporal window for this requirement has been elusive due to anemia and total body and brain ID inherent to previous dietary restriction models. To circumvent these confounds, we developed transgenic mice that express tetracycline transactivator regulated, dominant negative transferrin receptor (DNTfR1) in hippocampal neurons, disrupting TfR1 mediated iron uptake specifically in CA1 pyramidal neurons. Normal iron status was restored by doxycycline administration. We manipulated the duration of ID using this inducible model to examine long-term effects of early ID on Morris water maze learning, CA1 apical dendrite structure, and defining factors of critical periods including parvalbmin (PV) expression, perineuronal nets (PNN), and brain-derived neurotrophic factor (BDNF) expression. Ongoing ID impaired spatial memory and resulted in disorganized apical dendrite structure accompanied by altered PV and PNN expression and reduced BDNF levels. Iron repletion at P21, near the end of hippocampal dendritogenesis, restored spatial memory, dendrite structure, and critical period markers in adult mice. However, mice that remained hippocampally iron deficient until P42 continued to have spatial memory deficits, impaired CA1 apical dendrite structure, and persistent alterations in PV and PNN expression and reduced BDNF despite iron repletion. Together, these findings demonstrate that hippocampal iron availability is necessary between P21 and P42 for development of normal spatial learning and memory, and that these effects may reflect disruption of critical period closure by early life ID.

Identification and localization of divalent metal transporter-1 (DMT-1) in term human placenta.

The mechanism by which iron is transported from mother to fetus is incompletely understood. Whereas transferrin receptor (TfR) is responsible for iron uptake from maternal serum by the syncytiotrophoblast, the proteins responsible for intracytoplasmic transport and for delivery to the fetal serum remain unknown. The aim of this study was to determine whether the recently characterized endosomal membrane iron transporter, divalent metal ion transporter-1 (DMT-1), is expressed in human syncytiotrophoblast, and whether its cellular localization would support roles for cytoplasmic and placental-fetal iron transport. Six micron sections of frozen, term human placenta were assessed immunohistochemically using a polyclonal antibody to rat DMT-1 and a monoclonal antibody to human TfR. DMT-1 was found both in the cytoplasm and at the junction of the fetal (basal) membrane and fetal vessels, while TfR was localized predominantly to the maternal (apical) side of the syncytiotrophoblastic membrane. Double staining demonstrated no overlap between the two proteins on the apical membrane and minimal areas of overlap in the cytoplasm. We postulate that the syncytiotrophoblast takes up diferric transferrin from serum via TfR, subsequently incorporating the transferrin : TfR complex via endosomes. Subsequent transport of iron out of the endosome and across the basal membrane to the fetus may occur via DMT-1.

Perinatal iron deficiency decreases cytochrome c oxidase (CytOx) activity in selected regions of neonatal rat brain.

Intrauterine growth retardation and diabetes mellitus during human gestation result in significant losses of fetal and neonatal brain iron. Brain iron deficiency is associated with impaired cognitive processes including memory and attention. The regional distribution of iron staining and cytochrome c oxidase (CytOx) activity have not been mapped in the iron-sufficient or -deficient neonatal rat. CytOx is the iron-containing terminal enzyme in oxidative phosphorylation; its activity reflects neuronal metabolism. We hypothesized that neonatal brain iron deficiency differentially decreases iron and CytOx activity in brain regions, with more pronounced losses in structures involved in recognition memory. Pregnant Sprague Dawley rats were fed either an iron-deficient or -fortified diet from gestational d 1 until postnatal d 10. Iron staining and CytOx activity of 20 brain structures were mapped histochemically in 25 rats from each group. Brain iron staining was reduced from 75% to 100% and CytOx staining was decreased from 0% to 42% in the iron deficient group (p < 0.001). Areas with significantly reduced CytOx activity (p < 0.001) included all measured subareas of the hippocampus (CA1: 42%, CA3ab: 34%, CA3c: 33%, and dentate gyrus: 32%), the piriform cortex (17%), the medial dorsal thalamic nucleus (28%), and the cingulate cortex (41%). In contrast, the anterior thalamic nucleus, the lateral amygdaloid nucleus, and the medial habenula, areas not involved in higher cognitive functions, did not have significantly reduced CytOx activity (0%, 10%, and 16%, respectively). We conclude that perinatal iron deficiency differentially reduces neuronal metabolic activity, specifically targeting areas of the brain involved in memory processing.

Increased placental iron regulatory protein-1 expression in diabetic pregnancies complicated by fetal iron deficiency.

Placental transferrin receptor (TfR) protein expression is increased in diabetic pregnancies that are complicated by low fetal iron stores, suggesting regulation of placental iron transport by fetoplacental iron status. In cell culture, iron homeostasis is regulated by coordinate stabilization of TfR mRNA and translation inactivation of ferritin mRNA by iron regulatory proteins (IRP-1 and -2) which bind to iron-responsive elements (IREs) on the respective mRNAs. Concentrations of IRP-1, IRP-2 and TfR mRNA were measured in 10 placentae obtained from diabetic and non-diabetic human pregnancies with a wide range of fetoplacental iron status. IRP-1 activity was present in human placenta and correlated closely with TfR mRNA concentration (r=0.82; P=0.007). IRP-2 activity and protein were not detected. In a second experiment, placentae were collected from 12 diabetic pregnancies, six with low fetal cord serum ferritin and placental non-heme iron concentrations, and six with normal iron status. IRP-1 activity and TfR Bmax for diferric transferrin were greater in the iron-deficient group (P<0.05). IRP-1 activity correlated inversely with cord serum ferritin (r=0.75; P<0.01) and placental non-heme iron (r=0.61; P=0.05) concentration. Placental IRP-1 activity is directly related to TfR mRNA concentration and is more highly expressed in iron-deficient placentae. The study provides direct in vivo evidence for IRP regulation of TfR expression in the human placenta.

Perinatal brain iron deficiency increases the vulnerability of rat hippocampus to hypoxic ischemic insult.

Fetal brain iron deficiency occurs in human pregnancies complicated by diabetes mellitus or intrauterine growth retardation. Because neurocognitive deficits are more common in the offspring of these pregnancies, we tested the hypothesis that perinatal brain iron deficiency predisposes the neonatal hippocampus, a structure important for memory processing, to injury. Brain iron concentration was reduced by 45% in 45 neonatal rats by maternal dietary iron restriction during gestation. Right-sided neuronal injury in four hippocampal subareas was induced by hypoxic-ischemic insult (ipsilateral carotid artery ligation and subsequent hypoxia on postnatal d 7) and was quantified histochemically on d 8 by cytochrome c oxidase activity (n = 30), and on d 14 by Nissl staining (n = 15). Acute right-sided cytochrome c oxidase activity loss occurred in CA1 (P = 0.02), CA3c (P < 0.001) and dentate gyrus (P < 0.001) in the iron-deficient group, whereas only CA1 (P = 0. 003) was affected in the iron-sufficient group. Long-term right-sided Nissl substance loss occurred in CA1 (P = 0.001), CA3a,b (P < 0.001) and dentate gyrus (P = 0.008) in the iron-deficient group, but only in CA1 (P = 0.004) in the iron-sufficient group. No increase in right-sided free-iron staining was present in either group. Perinatal iron deficiency predisposes the neonatal hippocampus to a greater acute loss of neuronal metabolic activity after an hypoxic-ischemic event, suggesting compromised cellular energetics. The subsequently greater loss of hippocampal neuronal integrity suggests poorer recoverability after injury in the perinatal iron-deficient brain.

Increased N-glycosylation and reduced transferrin-binding capacity of transferrin receptor isolated from placentae of diabetic women.

Infants of diabetic mothers are frequently born iron deficient because their fetal iron demand exceeds placental iron transport capacity. Although transferrin receptor (TfR) expression is increased, binding to diferric transferrin is decreased proportionately to the severity of maternal disease. It is hypothesized that TfR isolated from diabetic placentae has altered N-glycosylation since proper glycosylation of N-linked oligosaccharides is important for normal TfR binding kinetics to diferric transferrin. TfR was obtained from syncytiotrophoblastic membranes of six diabetic and six non-diabetic human placentae. Competitive binding to 125I-transferrin demonstrated a higher Kd in the diabetic TfR (P = 0.04), directly correlated to cord serum C-peptide concentration (r = 0.81, P < 0.001). The molecular weight of the monomeric form of TfR prior to treatment with glycopeptidase F (PNG-F) was greater in the diabetic group (P < 0.001) was directly related to the Kd (r = 0.77, P = 0.002). Treatment with PNG-F eliminated the molecular weight difference between the two groups. Increased glycosylation of the N-linked oligosaccharides of TfR isolated from diabetic placentae may alter the three-dimensional structure or charge of the receptor, thus reducing its binding affinity for transferrin.

Liver and brain iron deficiency in newborn infants with bilateral renal agenesis (Potter's syndrome).

Significant changes in fetal iron status potentially occur in pregnancies in which reduced fetal nutrient delivery is severe enough to result in intrauterine growth retardation (IUGR), particularly if chronic fetal hypoxia is also present and increases fetal iron demand for hemoglobin synthesis. Neonates rarely die following IUGR secondary to maternal preeclampsia, but bilateral renal agenesis, which is also characterized by reduced maternal-fetal blood flow, late gestation placental failure, and IUGR, is uniformly fatal. We measured neonatal liver iron concentration, as an assessment of fetal storage iron status, and heart and brain iron concentrations, as assessments of nonheme tissue iron status, in 11 infants who died in the neonatal period of bilateral renal agenesis, and compared them with values for gestational age-matched control infants whose gestation was not complicated by fetal growth retardation or hypoxia. Stainable nonheme iron in the hepatocytes was significantly reduced in all and completely absent in 8 of the 11 cases of renal agenesis (P < .001 compared with control). The mean +/- SEM liver iron concentration of the bilateral renal agenesis group (999 +/- 218 micrograms/g dry tissue weight) was 26% of the control value (3894 +/- 548 micrograms/g dry tissue weight; P < .001). Brain iron concentration was also lower in the group with bilateral renal agenesis (109 +/- 17 vs. 161 +/- 19; P = .015) and was correlated with liver iron concentration (r = .47; P = .03). Heart iron concentrations were similar in the two groups. Nine of the subjects with bilateral renal agenesis had placental weights below the fifth percentile for gestational age. The bilateral renal agenesis group had a lower mean birth weight (P < .001) and had a higher prevalence of fetal growth retardation (55% vs. 0%; P < .001). We conclude that infants with bilateral renal agenesis are at risk for severe iron deficiency of storage and nonstorage tissues. Liveborn infants with nonfatal fetal conditions characterized by significant restriction of maternal-fetal blood flow may also be at significant risk for postnatal iron deficiency.

Reduced neonatal liver iron concentrations after uteroplacental insufficiency.

Neonatal liver (storage) but not heart (nonstorage) tissue iron concentrations were reduced by 60% at autopsy in 15 newborn infants who had gestations complicated by uteroplacental insufficiency because of maternal hypertension or Potter syndrome. The hepatic iron reductions in term and preterm infants, and with either antecedent condition, were similar.

Immunoreactive insulin in rat salivary glands and its dependence on age and serum insulin levels.

The major salivary glands of adult rodents contain immunoreactive insulin (IRI). To determine if the concentration of IRI in salivary glands is modulated by the level of serum insulin, insulin immunoreactivity in the parotid and submandibular glands of male rats at different ages (sucklings, pubescent, mature, and elderly) was assayed and compared with corresponding serum insulin concentrations. Salivary glands from suckling rats contained 94 ng/g (wet weight) insulin, which is 1.6 times higher than the level in pubescent rats, and about 10 times higher than levels in mature and elderly rats. No direct relationship between salivary gland content and serum IRI levels was indicated by the data. In an attempt to increase insulin levels in serum, insulin-secreting pancreatic islet adenomas were induced in young male rats by injecting streptozotocin (an islet tumor-inducing drug) with nicotinamide (which reduces the drug's beta-cell cytotoxicity). The mean insulin content of salivary glands from drug-treated rats that had not yet expressed tumors was no higher than controls. After the development of visible tumors of pancreatic islet tissue, however, salivary gland IRI was markedly elevated, reaching 40 times control levels, whereas serum insulin, and the immunoreactive insulin content of two insulin-sensitive tissues (vis. hepatic, adipose), were elevated only 2-fold. Examination of histologic sections of the parotid and submandibular glands from drug-treated animals revealed no evidence for the formation of salivary tumors. The data indicate that salivary gland insulin content (i) is age-related, being highest in neonates and declining thereafter, (ii) is generally identical in parotid and submandibular glands at a given age, and (iii) is not modulated solely by the animal's serum insulin concentration. These results are discussed in regard to the possible sources of insulin detected in the major salivary glands.

Placental transferrin receptor in diabetic pregnancies with increased fetal iron demand.

Augmented fetal hemoglobin synthesis during diabetic pregnancy increases fetal iron demand. To study the effect of increased fetal iron demand on placental transferrin receptor (TR), we utilized a monoclonal antibody to localize placental TR immunoreactivity and 125I-labeled transferrin to study TR binding characteristics in 10 placentas from poorly controlled diabetic mothers with increased fetal iron demand and 10 placentas from nondiabetic mothers. The infants born to the diabetics had higher cord serum C-peptide, erythropoietin, and hemoglobin concentrations, indicating fetal hyperinsulinemia and hypoxia, with augmented erythropoiesis and iron demand. TR immunoreactivity was localized to the syncytiotrophoblast in both groups, was greater in the diabetic group, and was inversely correlated with fetal storage iron (r = -0.75; P < 0.001). Scatchard analysis of 125I transferrin binding data confirmed greater receptor number (Bmax 17.9 +/- 2.2 vs. 12.6 +/- 1.3 pM/mg protein, P = 0.05), but reduced binding affinity [dissociation constant (Kd) 7.6 +/- 0.9 vs. 5.4 +/- 0.4 nM/l, P = 0.03] in the diabetic group. The TR staining intensity, Bmax, and Kd were each correlated with cord C-peptide, suggesting either a primary or secondary role for fetal hyperinsulinemia in TR expression. This study provides in vivo evidence that fetal factors, such as iron demand or hyperinsulinemia, influence regulation of placental TR in humans. The increase in placental syncytiotrophoblastic TR expression associated with reduced cord serum ferritin concentration suggests that the fetus utilizes both increased placental iron transport and mobilization of fetal iron stores to support augmented fetal erythropoiesis.

Pancreatic islet cell reaggregation systems: efficiency of cell reassociation and endocrine cell topography of rat islet-like aggregates.

Single cells isolated from rat islets of Langerhans were cultured under conditions that support reassociation into islet-like aggregates. Comparisons were made of enzymatic methods of islet dissociation, rotational or static culture conditions, and culture at basal or stimulatory glucose concentrations. Over a period of 4 days the aggregates progressed through three stages of organization: cell coalescence to cellular chains, rearrangement of chains into small spheroids, and growth of spheroids. The numerical yield of aggregates was optimum after islets were dissociated with dispase. Culture under rotation resulted in the production of more aggregates of significantly larger diameter than under static conditions. Medium glucose concentrations of 4 and 11 mM supported cell reassociation under rotator culture, but no aggregation occurred under static culture at the basal (4 mM) glucose level. Aggregates resulting from 4-day rotator culture exhibited endocrine cell distributions similar to intact islets. Islet aggregates released insulin in response to glucose, but nonaggregated cells, maintained in culture, did not. The present comparisons reveal significant variability in the cellular composition, rate of formation, and yield of aggregates, and suggest that the methodology for producing aggregates should be carefully considered in experimental design.

Iron deficiency of liver, heart, and brain in newborn infants of diabetic mothers.

Infants of diabetic mothers frequently have polycythemia, elevated serum erythropoietin concentrations, and decreased serum iron and ferritin concentrations, likely representing a redistribution of fetal iron into erythrocytes to support augmented fetal hemoglobin synthesis. We hypothesized that fetal liver, heart, and brain iron concentrations are also reduced in these infants. After obtaining autopsy tissue from infants who had died before 7 days of age, we measured liver, heart, and brain iron concentrations using atomic absorption spectrophotometry. Seven infants of diabetic mothers and seven gestational age-matched control infants were studied. All infants of diabetic mothers had pancreatic islet cell hyperplasia, indicating fetal hyperglycemia and hyperinsulinemia. Liver iron concentrations in the infants of diabetic mothers were 6.6% of control values (489.0 +/- 154.4 vs 7379.7 +/- 1473.8 micrograms/gm dry tissue weight (mean +/- SEM); p less than 0.001), heart iron concentrations were 43.9% of control values (124.7 +/- 20.5 vs 284.1 +/- 34.8 micrograms/gm dry tissue weight; p less than 0.002), and brain iron concentrations were 60.6% of control values (106.1 +/- 13.7 vs 175.2 +/- 10.7 micrograms/gm dry tissue weight; p less than 0.003). Heart and brain iron concentrations were directly correlated with liver iron concentrations (r = 0.80 for both; p less than 0.001) and indicated that hepatic iron was greater than 75% depleted before heart and brain iron reduction. We conclude that severely affected infants of diabetic mothers have reduced liver, heart, and brain iron concentrations. The role of tissue iron deficiency in the genesis of the abnormal clinical findings in these infants deserves further consideration.

Calcitonin and CGRP block bombesin- and substance P-induced increases in airway tone.

Calcitonin gene-related peptide (CGRP) and calcitonin (C) are two peptides that are cocontained and probably coreleased with the potent bronchocontrictors, bombesin (B) and substance P (SP), within the lung. Although CGRP and C have a wide intrapulmonary distribution, their actions have not been well defined. By the use of a computerized lung mechanics analyzer, changes in response to 10-min infusions of these agents were measured in spontaneously breathing, anesthetized guinea pigs. Infusion of 0.3 nmol.kg-1.min-1 CGRP and 2 nmol.kg-1.min-1 C caused little change in lung mechanics. Infusion of 0.06 nmol.kg-1.min-1 B and 0.3 nmol.kg-1.min-1 SP caused a marked increase in inspiratory, expiratory, and total pulmonary resistance (RT), from base-line values (P less than 0.02), with a maximal effect at 10 min postinfusion (PI) [RT = 326 +/- 20% (SE) (B), 490 +/- 73% (SP)]. Coinfusion of C or CGRP with B or SP at the above concentrations caused a marked reduction in SP - [RT = 189 +/- 28% (C), 142 +/- 16% (CGRP) at 10 min PI] and B - [RT = 157 +/- 18% (C), 158 +/- 10% (CGRP) at 10 min PI] induced changes in resistance (P less than 0.015). The mode of action of C and CGRP is unknown, but these peptides may antagonize the effects of B and SP via autonomic pathways by interfering with B- or SP-induced changes in intracellular calcium concentrations or by increasing intracellular cAMP levels by binding to specific cellular receptors linked to adenylate cyclase.

Glucose-stimulated hormone release in rats bearing streptozotocin/nicotinamide-induced islet adenomas: evidence for slow and fast responders.

Rats injected with streptozotocin and nicotinamide developed grossly visible islet cell tumors of the pancreas. During i.v. glucose tolerance tests, two populations of tumor-bearing rats were identified: fast responders exhibited significantly lower plasma glucose and markedly elevated plasma immunoreactive insulin (IRI) levels relative to those of the controls. In slow responders, the plasma glucose level was significantly elevated up to 2 h after glucose injection, and the plasma IRI level was lower than that of the controls. During in vitro perfusions with glucose at 300 mg/dl (16.7 mM), tumor-bearing pancreata of fast responders released elevated levels of IRI and immunoreactive somatostatin (IRS); after tumor removal, glucose-stimulated release of these hormones returned to control levels. However, during similar perfusions of pancreata from slow responders, the IRI and IRS release did not decrease after tumor removal, suggesting that the nontumorous pancreatic islets rather than the gross tumors of the slow-responder group were the source of the glucose-stimulated hormone release. These studies demonstrate that gross tumors in the two responder subgroups differ in their glucose-stimulated hormone release.

Changes in bombesin, calcitonin, and serotonin immunoreactive pulmonary neuroendocrine cells in cystic fibrosis and after prolonged mechanical ventilation.

Increases in bombesin, calcitonin, and serotonin immunoreactive pulmonary neuroendocrine cells have been documented in infants with bronchopulmonary dysplasia. As some of the secretory products of these postulated airway chemoreceptors are known to adversely affect pulmonary vasomotor and bronchomotor tone, the present study was undertaken to determine if similar changes occur in the lungs of older pediatric patients with chronic respiratory disease. Immunoreactive cells were identified using the antibody-peroxidase-antiperoxidase technique and expressed as immunoreactive bronchioles/cm2 of lung tissue. In subjects dying an accidental or noncardiopulmonary death (control group: n = 48, zero to 24 yr of age), the total number of bombesin, calcitonin, and serotonin immunoreactive bronchioles/cm2 was greatest at birth, then decreased rapidly to extremely low levels after the first year of life. In the cystic fibrosis (n = 55, 3 days to 29 yr of age) and prolonged ventilation (n = 24, 4 months to 18 yr of age) groups, there was a significant increase (p less than 0.035) in bombesin, calcitonin, and serotonin immunoreactive bronchioles/cm2 from 1 to 11 yr of age. In the cystic fibrosis group, there was a sixfold increase in the number of serotonin immunoreactive bronchioles/cm2 lung tissue (p less than 0.015) compared with that in the other 2 groups during the first decade of life, suggesting a response to specific factors present only in the lungs of patients with this disease. In all 3 groups, immunoreactive cells were infrequently identified after 11 yr, implying a fundamental change in neuroendocrine cell biology coincident with the termination of lung growth and/or the onset of puberty.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene-related peptide immunoreactivity in airway epithelial cells of the human fetus and infant.

Calcitonin gene-related peptide-immunoreactive cells were identified within the epithelium of distal conducting airways in the human fetus and infant. Several peptides and amines, including calcitonin, have been identified previously within a specific population of airway epithelial cells. These cells, referred to as pulmonary neuroendocrine cells, are postulated to be airway chemoreceptors responsible for changes in ventilation and perfusion in response to changes in airway gas composition. Calcitonin gene-related peptide immunoreactive cells could be identified throughout the period of development studies (20 weeks gestation to 3 months of age), but were present in only limited numbers in less than 50% of individuals (n = 23). In contrast, large numbers of calcitonin gene-related peptide immunoreactive cells were identified in 100% of infants (1-3 months, n = 5) with bronchopulmonary dysplasia. The differential processing of mRNA transcribed from the calcitonin gene in neural and non-neural tissue suggests that calcitonin, rather than calcitonin gene-related peptide, is the primary product of translation in pulmonary neuroendocrine cells. However, considering the potent vasodilatory and bronchoconstrictive effects of calcitonin gene-related peptide, its presence in pulmonary neuroendocrine cells, even in small amounts, may be important in controlling pulmonary vaso- and/or bronchomotor tone. The presence of large numbers of calcitonin gene-related peptide immunoreactive cells in infants with bronchopulmonary dysplasia suggests that calcitonin gene-related peptide may be one further agent contributing to the pulmonary pathophysiology seen in this disease.