Development of cortical interneurons continues until the end of human pregnancy. Premature birth deprives the newborns from the supply of maternal estrogen and a secure intrauterine environment. Indeed, preterm infants suffer from neurobehavioral disorders. This can result from both preterm birth and associated postnatal complications, which might disrupt recruitment and maturation of cortical interneurons. We hypothesized that interneuron subtypes, including parvalbumin-positive (PV), somatostatin-positive (SST), calretinin-positive (CalR), and neuropeptide Y-positive (NPY) interneurons, were recruited in the upper and lower cortical layers in a distinct manner with advancing gestational age. In addition, preterm birth would disrupt the heterogeneity of cortical interneurons, which might be reversed by estrogen treatment. These hypotheses were tested by analyzing autopsy samples from premature infants and evaluating the effect of estrogen supplementation in prematurely delivered rabbits. The PV and CalR neurons were abundant, whereas SST and NPY neurons were few in cortical layers of preterm human infants. Premature birth of infants reduced the density of PV or GAD67 neurons and increased SST interneurons in the upper cortical layers. Importantly, 17 β-estradiol treatment in preterm rabbits increased the number of PV neurons in the upper cortical layers relative to controls at postnatal day 14 (P14) and P21 and transiently reduced SST population at P14. Moreover, protein and mRNA levels of Arx, a key regulator of cortical interneuron maturation and migration, were higher in estrogen-treated rabbits relative to controls. Therefore, deficits in PV and excess of SST neurons in premature newborns are ameliorated by estrogen replacement, which can be attributed to elevated Arx levels. Estrogen replacement might enhance neurodevelopmental outcomes in extremely preterm infants. Premature birth often leads to neurodevelopmental delays and behavioral disorders, which may be ascribed to disturbances in the development and maturation of cortical interneurons. Here, we show that preterm birth in humans is associated with reduced population of parvalbumin-positive (PV) neurons and an excess of somatostatin-expressing interneurons in the cerebral cortex. More importantly, 17 β-estradiol treatment increased the number of PV neurons in preterm-born rabbits, which appears to be mediated by an elevation in the expression of Arx transcription factor. Hence the present study highlights prematurity-induced reduction in PV neurons in human infants and reversal in their population by estrogen replacement in preterm rabbits. Because preterm birth drops plasma estrogen level 100-fold, estrogen replacement in extremely preterm infants might improve their developmental outcome and minimize neurobehavioral disorders.

Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic environment and supply of maternal hormones. As the production of interneurons continues until the end of pregnancy, we hypothesized that premature birth would disrupt interneuron production and that restoration of the hypoxic milieu or estrogen treatment might reverse interneuron generation. To test these hypotheses, we compared interneuronal progenitors in the medial ganglionic eminences (MGEs), lateral ganglionic eminences (LGEs), and caudal ganglionic eminences (CGEs) between preterm-born [born on embryonic day (E) 29; examined on postnatal day (D) 3 and D7] and term-born (born on E32; examined on D0 and D4) rabbits at equivalent postconceptional ages. We found that both total and cycling Nkx2.1, Dlx2, and Sox2 cells were more abundant in the MGEs of preterm rabbits at D3 compared with term rabbits at D0, but not in D7 preterm relative to D4 term pups. Total Nkx2.1 progenitors were also more numerous in the LGEs of preterm pups at D3 compared with term rabbits at D0. Dlx2 cells in CGEs were comparable between preterm and term pups. Simulation of hypoxia by dimethyloxalylglycine treatment did not affect the number of interneuronal progenitors. However, estrogen treatment reduced the density of total and proliferating Nkx2.1 and Dlx2 cells in the MGEs and enhanced Ascl1 transcription factor. Estrogen treatment also reduced Ki67, c-Myc, and phosphorylation of retinoblastoma protein, suggesting inhibition of the G1-to-S phase transition. Hence, preterm birth disrupts interneuron neurogenesis in the MGE and estrogen treatment reverses interneuron neurogenesis in preterm newborns by cell-cycle inhibition and elevation of Ascl1. We speculate that estrogen replacement might partially restore neurogenesis in human premature infants. Prematurity results in developmental delays and neurobehavioral disorders, which might be ascribed to disturbances in the development of cortical interneurons. Here, we show that preterm birth disrupts interneuron neurogenesis in the medial ganglionic eminence (MGE) and, more importantly, that estrogen treatment reverses this perturbation in the population of interneuron progenitors in the MGE. The estrogen seems to restore neurogenesis by inhibiting the cell cycle and elevating Ascl1 expression. As preterm birth causes plasma estrogen level to drop 100-fold, the estrogen replacement in preterm infants is physiological. We speculate that estrogen replacement might ameliorate disruption in production of interneurons in human premature infants.

Intraventricular hemorrhage (IVH) leads to reduced myelination and astrogliosis of the white matter in premature infants. No therapeutic strategy exists to minimize white matter injury in survivors with IVH. Epidermal growth factor (EGF) enhances myelination, astrogliosis, and neurologic recovery in animal models of white matter injury. Here, we hypothesized that recombinant human (rh) EGF treatment would enhance oligodendrocyte precursor cell (OPC) maturation, myelination, and neurological recovery in preterm rabbits with IVH. In addition, rhEGF would promote astrogliosis by inducing astroglial progenitor proliferation and GFAP transcription. We tested these hypotheses in a preterm rabbit model of IVH and evaluated autopsy samples from human preterm infants. We found that EGF and EGFR expression were more abundant in the ganglionic eminence relative to the cortical plate and white matter of human infants and that the development of IVH reduced EGF levels, but not EGFR expression. Accordingly, rhEGF treatment promoted proliferation and maturation of OPCs, preserved myelin in the white matter, and enhanced neurological recovery in rabbits with IVH. rhEGF treatment inhibited Notch signaling, which conceivably contributed to OPC maturation. rhEGF treatment contributed to astrogliosis by increasing astroglial proliferation and upregulating GFAP as well as Sox9 expression. Hence, IVH results in a decline in EGF expression; and rhEGF treatment preserves myelin, restores neurological recovery, and exacerbates astrogliosis by inducing proliferation of astrocytes and enhancing transcription of GFAP and Sox9 in pups with IVH. rhEGF treatment might improve the neurological outcome of premature infants with IVH.