Hippocampal neurogenesis and memory in adolescence following intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is associated with hippocampal alterations that can increase the risk of short-term memory impairments later in life. Despite the role of hippocampal neurogenesis in learning and memory, research into the long-lasting impact of IUGR on these processes is limited. We aimed to determine the effects of IUGR on neuronal proliferation, differentiation and morphology, and on memory function at adolescent equivalent age. At embryonic day (E) 18 (term ∼E22), placental insufficiency was induced in pregnant Wistar rats via bilateral uterine vessel ligation to generate IUGR offspring (n = 10); control offspring (n = 11) were generated via sham surgery. From postnatal day (P) 36-44, spontaneous location recognition (SLR), novel object location and recognition (NOL, NOR), and open field tests were performed. Brains were collected at P45 to assess neurogenesis (immunohistochemistry), dendritic morphology (Golgi staining), and brain-derived neurotrophic factor expression (BDNF; Western blot analysis). In IUGR versus control rats there was no difference in object preference in the NOL or NOR, the similar and dissimilar condition of the SLR task, or in locomotion and anxiety-like behavior in the open field. There was a significant increase in the linear density of immature neurons (DCX+) in the subgranular zone (SGZ) of the dentate gyrus (DG), but no difference in the linear density of proliferating cells (Ki67+) in the SGZ, nor in areal density of mature neurons (NeuN+) or microglia (Iba-1+) in the DG in IUGR rats compared to controls. Dendritic morphology of dentate granule cells did not differ between groups. Protein expression of the BDNF precursor (pro-BDNF), but not mature BDNF, was increased in the hippocampus of IUGR compared with control rats. These findings highlight that while the long-lasting prenatal hypoxic environment may impact brain development, it may not impact hippocampal-dependent learning and memory in adolescence.

Dose-dependent exacerbation of ventilation-induced lung injury by erythropoietin in preterm newborn lambs.

Erythropoietin (EPO) is being trialled in preterm infants to reduce brain injury, but high doses increase lung injury in ventilated preterm lambs. We aimed to determine whether early administration of lower doses of EPO could reduce ventilation-induced lung injury and systemic inflammation in preterm lambs. Ventilation was initiated in anaesthetized preterm lambs [125 ± 1 (SD) days gestation] using an injurious strategy for the first 15 min. Lambs were subsequently ventilated with a protective strategy for a total of 2 h. Lambs were randomized to receive either intravenous saline (Vent; n = 7) or intravenous 300 ( n = 5), 1,000 (EPO1000; n = 5), or 3,000 (EPO3000; n = 5) IU/kg of human recombinant EPO via an umbilical vein. Lung tissue was collected for molecular and histological assessment of inflammation and injury and compared with unventilated control lambs (UVC; n = 8). All ventilated groups had similar blood gas and ventilation parameters, but EPO1000 lambs had a lower fraction of inspired oxygen requirement and lower alveolar-arterial difference in oxygen. Vent and EPO lambs had increased lung interleukin (IL)-1ß, IL-6, and IL-8 mRNA, early lung injury genes connective tissue growth factor, early growth response protein 1, and cysteine-rich 61, and liver serum amyloid A3 mRNA compared with UVCs; no difference was observed between Vent and EPO groups. Histological lung injury was increased in Vent and EPO groups compared with UVCs, but EPO3000 lambs had increased lung injury scores compared with VENT only. Early low-doses of EPO do not exacerbate ventilation-induced lung inflammation and injury and do not provide any short-term respiratory benefit. High doses (≥3,000 IU/kg) likely exacerbate lung inflammation and injury in ventilated preterm lambs. NEW & NOTEWORTHY Trials are ongoing to assess the efficacy of erythropoietin (EPO) to provide neuroprotection for preterm infants. However, high doses of EPO increase ventilation-induced lung injury (VILI) in preterm lambs. We investigated whether early lower doses of EPO may reduce VILI. We found that lower doses did not reduce, but did not increase, VILI, while high doses (≥3,000 IU/kg) increase VILI. Therefore, lower doses of EPO should be used in preterm infants, particularly those receiving respiratory support.

Intrauterine Growth Restriction Alters the Postnatal Development of the Rat Cerebellum.

Intrauterine growth restriction (IUGR) is a major cause of antenatal brain injury. We aimed to characterize cerebellar deficits following IUGR and to investigate the potential underlying cellular and molecular mechanisms. At embryonic day 18, pregnant rats underwent either sham surgery (controls; n = 23) or bilateral uterine vessel ligation to restrict blood flow to fetuses (IUGR; n = 20). Offspring were collected at postnatal day 2 (P2), P7, and P35. Body weights were reduced at P2, P7, and P35 in IUGR offspring (p < 0.05) compared with controls. At P7, the width of the external granule layer (EGL) was 30% greater in IUGR than control rats (p < 0.05); there was no difference in the width of the proliferative zone or in the density of Ki67-positive cells in the EGL. Bergmann glia were disorganized at P7 and P35 in IUGR pups, and by P35, there was a 10% decrease in Bergmann glial fiber density (p < 0.05) compared with controls. At P7, trophoblast antigen-2 (Trop2) mRNA and protein levels in the cerebellum were decreased by 88 and 40%, respectively, and astrotactin 1 mRNA levels were increased by 20% in the IUGR rats (p < 0.05) compared with controls; there was no difference in ASTN1 protein. The expressions of other factors known to regulate cerebellar development (astrotactin 2, brain-derived neurotrophic factor, erb-b2 receptor tyrosine kinase 4, neuregulin 1, sonic hedgehog and somatostatin) were not different between IUGR and control rats at P7 or P35. These data suggest that damage to the migratory scaffold (Bergmann glial fibers) and alterations in the genes that influence migration (Trop2 and Astn1) may underlie the deficits in postnatal cerebellar development following IUGR.

Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury.

BACKGROUND: Human amnion epithelial cells (hAECs) are clonogenic and have been proposed to reduce inflammatory-induced tissue injury. Perturbation of the immune response is implicated in the pathogenesis of perinatal brain injury; modulating this response could thus be a novel therapy for treating or preventing such injury. The immunomodulatory properties of hAECs have been shown in other animal models, but a detailed investigation of the effects on brain immune cells following injury has not been undertaken. Here, we investigate the effects of hAECs on microglia, the first immune responders to injury within the brain. METHODS: We generated a mouse model combining neonatal inflammation and perinatal hyperoxia, both of which are risk factors associated with perinatal brain injury. On embryonic day 16 we administered lipopolysaccharide (LPS), or saline (control), intra-amniotically to C57Bl/6 J mouse pups. On postnatal day (P)0, LPS pups were placed in hyperoxia (65% oxygen) and control pups in normoxia for 14 days. Pups were given either hAECs or saline intravenously on P4. RESULTS: At P14, relative to controls, LPS and hyperoxia pups had reduced body weight, increased density of apoptotic cells (TUNEL) in the cortex, striatum and white matter, astrocytes (GFAP) in the white matter and activated microglia (CD68) in the cortex and striatum, but no change in total microglia density (Iba1). hAEC administration rescued the decreased body weight and reduced apoptosis and astrocyte areal coverage in the white matter, but increased the density of total and activated microglia. We then stimulated primary microglia (CD45lowCD11b+) with LPS for 24 h, followed by co-culture with hAEC conditioned medium for 48 h. hAEC conditioned medium increased microglial phagocytic activity, decreased microglia apoptosis and decreased M1 activation markers (CD86). Stimulating hAECs for 24 h with LPS did not alter release of cytokines known to modulate microglia activity. CONCLUSIONS: These data demonstrate that hAECs can directly immunomodulate brain microglia, probably via release of trophic factors. This observation offers promise that hAECs may afford therapeutic utility in the management of perinatal brain injury.

Caffeine for apnea of prematurity: Effects on the developing brain.

Caffeine is a methylxanthine that is widely used to treat apnea of prematurity (AOP). In preterm infants, caffeine reduces the duration of respiratory support, improves survival rates and lowers the incidence of cerebral palsy and cognitive delay. There is, however, little evidence relating to the immediate and long-term effects of caffeine on brain development, especially at the cellular and molecular levels. Experimental data are conflicting, with studies showing that caffeine can have either adverse or benefical effects in the developing brain. The aim of this article is to review current understanding of how caffeine ameliorates AOP, the cellular and molecular mechanisms by which caffeine exerts its effects and the effects of caffeine on brain development. A better knowledge of the effects of caffeine on the developing brain at the cellular and/or molecular level is essential in order to understand the basis for the impact of caffeine on postnatal outcome. The studies reviewed here suggest that while caffeine has respiratory benefits for preterm infants, it may have adverse molecular and cellular effects on the developing brain; indeed a majority of experimental studies suggest that regardless of dose or duration of administration, caffeine leads to detrimental changes within the developing brain. Thus there is an urgent need to assess the impact of caffeine, at a range of doses, on the structure and function of the developing brain in preclinical studies, particularly using clinically relevant animal models. Future studies should focus on determining the maximal dose of caffeine that is safe for the preterm brain.

Human Amnion Epithelial Cells Modulate Ventilation-Induced White Matter Pathology in Preterm Lambs.

BACKGROUND: Preterm infants can be inadvertently exposed to high tidal volumes (VT) during resuscitation in the delivery room due to limitations of available equipment. High VT ventilation of preterm lambs produces cerebral white matter (WM) pathology similar to that observed in preterm infants who develop cerebral palsy. We hypothesized that human amnion epithelial cells (hAECs), which have anti-inflammatory and regenerative properties, would reduce ventilation-induced WM pathology in neonatal late preterm lamb brains. METHODS: Two groups of lambs (0.85 gestation) were used, as follows: (1) ventilated lambs (Vent; n = 8) were ventilated using a protocol that induces injury (VT targeting 15 ml/kg for 15 min, with no positive end-expiratory pressure) and were then maintained for another 105 min, and (2) ventilated + hAECs lambs (Vent+hAECs; n = 7) were similarly ventilated but received intravenous and intratracheal administration of 9 × 10(7) hAECs (18 × 10(7) hAECs total) at birth. Oxygenation and ventilation parameters were monitored in real time; cerebral oxygenation was measured using near-infrared spectroscopy. qPCR (quantitative real-time PCR) and immunohistochemistry were used to assess inflammation, vascular leakage and astrogliosis in both the periventricular and subcortical WM of the frontal and parietal lobes. An unventilated control group (UVC; n = 5) was also used for qPCR analysis of gene expression. Two-way repeated measures ANOVA was used to compare physiological data. Student's t test and one-way ANOVA were used for immunohistological and qPCR data comparisons, respectively. RESULTS: Respiratory parameters were not different between groups. Interleukin (IL)-6 mRNA levels in subcortical WM were lower in the Vent+hAECs group than the Vent group (p = 0.028). IL-1ß and IL-6 mRNA levels in periventricular WM were higher in the Vent+hAECs group than the Vent group (p = 0.007 and p = 0.001, respectively). The density of Iba-1-positive microglia was lower in the subcortical WM of the parietal lobes (p = 0.010) in the Vent+hAECs group but not in the periventricular WM. The number of vessels in the WM of the parietal lobe exhibiting protein extravasation was lower (p = 0.046) in the Vent+hAECs group. Claudin-1 mRNA levels were higher in the periventricular WM (p = 0.005). The density of GFAP-positive astrocytes was not different between groups. CONCLUSIONS: Administration of hAECs at the time of birth alters the effects of injurious ventilation on the preterm neonatal brain. Further studies are required to understand the regional differences in the effects of hAECs on ventilation-induced WM pathology and their net effect on the developing brain.

Trop2: from development to disease.

BACKGROUND: Trop2 was first discovered as a biomarker of invasive trophoblast cells. Since then most research has focused on its role in tumourigenesis because it is highly expressed in the vast majority of human tumours and animal models of cancer. It is also highly expressed in stem cells and in many organs during development. RESULTS: We review the multifaceted role of Trop2 during development and tumourigenesis, including its role in regulating cell proliferation and migration, self-renewal, and maintenance of basement membrane integrity. We discuss the evolution of Trop2 and its related protein Epcam (Trop1), including their distinct roles. Mutation of Trop2 leads to gelatinous drop-like corneal dystrophy, whereas over-expression of Trop2 in human tumours promotes tumour aggressiveness and increases mortality. Although Trop2 expression is sufficient to promote tumour growth, the surprising discovery that Trop2-null mice have an increased risk of tumour development has highlighted the complexity of Trop2 signaling. Recently, studies have begun to identify the mechanisms underlying TROP2's functions, including regulated intramembrane proteolysis or specific interactions with integrin b1 and claudin proteins. CONCLUSIONS: Understanding the mechanisms underlying TROP2 signaling will clarify its role during development, aid in the development of better cancer treatments and unlock a promising new direction in regenerative medicine.

Protective ventilation of preterm lambs exposed to acute chorioamnionitis does not reduce ventilation-induced lung or brain injury.

BACKGROUND: The onset of mechanical ventilation is a critical time for the initiation of cerebral white matter (WM) injury in preterm neonates, particularly if they are inadvertently exposed to high tidal volumes (VT) in the delivery room. Protective ventilation strategies at birth reduce ventilation-induced lung and brain inflammation and injury, however its efficacy in a compromised newborn is not known. Chorioamnionitis is a common antecedent of preterm birth, and increases the risk and severity of WM injury. We investigated the effects of high VT ventilation, after chorioamnionitis, on preterm lung and WM inflammation and injury, and whether a protective ventilation strategy could mitigate the response. METHODS: Pregnant ewes (n = 18) received intra-amniotic lipopolysaccharide (LPS) 2 days before delivery, instrumentation and ventilation at 127±1 days gestation. Lambs were either immediately euthanased and used as unventilated controls (LPSUVC; n = 6), or were ventilated using an injurious high VT strategy (LPSINJ; n = 5) or a protective ventilation strategy (LPSPROT; n = 7) for a total of 90 min. Mean arterial pressure, heart rate and cerebral haemodynamics and oxygenation were measured continuously. Lungs and brains underwent molecular and histological assessment of inflammation and injury. RESULTS: LPSINJ lambs had poorer oxygenation than LPSPROT lambs. Ventilation requirements and cardiopulmonary and systemic haemodynamics were not different between ventilation strategies. Compared to unventilated lambs, LPSINJ and LPSPROT lambs had increases in pro-inflammatory cytokine expression within the lungs and brain, and increased astrogliosis (p<0.02) and cell death (p<0.05) in the WM, which were equivalent in magnitude between groups. CONCLUSIONS: Ventilation after acute chorioamnionitis, irrespective of strategy used, increases haemodynamic instability and lung and cerebral inflammation and injury. Mechanical ventilation is a potential contributor to WM injury in infants exposed to chorioamnionitis.

Prophylactic erythropoietin exacerbates ventilation-induced lung inflammation and injury in preterm lambs.

Ventilation-induced lung injury (VILI) of preterm neonates probably contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Erythropoietin (EPO) has been suggested as a therapy for BPD. The aim of this study was to determine whether prophylactic administration of EPO reduces VILI in preterm newborn lambs. Lambs at 126 days of gestation (term is 147 days) were delivered and ventilated with a high tidal volume strategy for 15 min to cause lung injury, then received gentle ventilation until 2 h of age. Lambs were randomized to receive intravenous EPO (5000 IU kg(-1): Vent+EPO; n = 6) or phosphate-buffered saline (Vent; n = 7) soon after birth: unventilated controls (UVC; n = 8) did not receive ventilation or any treatment. Physiological parameters were recorded throughout the experimental procedure. Samples of lung were collected for histological and molecular assessment of inflammation and injury. Samples of liver were collected to assess the systemic acute phase response. Vent+EPO lambs received higher F IO 2, P aO 2 and oxygenation during the first 10 min than Vent lambs. There were no differences in physiological indices beyond this time. Total lung injury score, airway wall thickness, inflammation and haemorrhage were higher in Vent+EPO lambs than in Vent lambs. Lung inflammation and early markers of lung and systemic injury were elevated in ventilated lambs relative to unventilated lambs; EPO administration further increased lung inflammation and markers of lung and systemic injury. Prophylactic EPO exacerbates VILI, which may increase the incidence and severity of long-term respiratory disease. More studies are required before EPO can be used for lung protection in preterm infants.

The anti-inflammatory agent N-acetyl cysteine exacerbates endotoxin-induced hypoxemia and hypotension and induces polycythemia in the ovine fetus.

BACKGROUND: Lipopolysaccharide (LPS) delivered acutely to the ovine fetus induces cerebral white matter injury and brain inflammation. N-acetyl cysteine (NAC) is potentially neuroprotective as it blocks the production of inflammatory cytokines and increases glutathione levels; however, it is unknown whether NAC affects the physiological status of the fetus already exposed to an inflammatory environment. OBJECTIVES: Our objective was to determine whether NAC influences the physiological effects of LPS exposure in the ovine fetus. METHODS: Catheterized fetal sheep underwent one of four treatments (saline, n = 6; LPS, n = 6; LPS + NAC, n = 6; NAC, n = 3) on 5 consecutive days from 95 days of gestation (term approximately 147 days). Fetal arterial pressure and heart rate were recorded and blood samples collected. RESULTS: LPS administration resulted in fetal hypoxemia and hypotension; simultaneous treatment with NAC exacerbated these effects and induced polycythemia. NAC treatment alone had no effect on the fetus. CONCLUSION: In the presence of LPS, NAC compromises fetal physiological status, suggesting that it may not be a suitable antenatal treatment for a fetus with evidence of inflammation.

Investigating structural and biochemical correlates of ganglion cell dysfunction in streptozotocin-induced diabetic rats.

The aim of this study was to determine whether inner retinal dysfunction in diabetic rats is correlated with structural and/or biochemical changes in the retina and optic nerve. Using the electroretinogram (ERG; -5.83 to 1.28 log cd.s.m(-2)) retinal function (photoreceptor, bipolar, amacrine and ganglion cell components) was measured in control (n=13; citrate buffer) and diabetic (n=13; streptozotocin, STZ, 50 mg kg(-1)) rats, 12 weeks following treatment. Retinae and optic nerves were analyzed for structural changes and retinae were assessed for alterations in growth factor/cytokine expression using quantitative real-time PCR. We found that phototransduction efficiency was reduced 12 weeks after STZ-induced diabetes (-30%), leading to reduced amplitude of ON-bipolar (-18%) and amacrine cell (-29%) dominated responses; ganglion cell dysfunction (-84%) was more profound. In the optic nerve, nerve fascicle area and myelin sheath thickness were reduced (p<0.05), whereas the ratio of blood vessels and connective tissue to total nerve cross-sectional area was increased (p<0.05) in diabetic compared to control rats. In the retina, connective tissue growth factor (CTGF), transforming growth factor beta, type 2 receptor (TGFbeta-r2) mRNA and platelet-derived growth factor B (PDGF-B) mRNA were increased (p<0.035). Reduced ganglion cell function was correlated with increased CTGF and TGFbeta-r2, but not PDGF-B mRNA. In summary, the ganglion cell component exhibited the greatest level of dysfunction within the ERG components examined after 12 weeks of STZ-induced diabetes; the level correlated with increased CTGF and TGFbeta-r2 mRNA, but not with gross morphological changes in the retina or optic nerve.

HGF promotes survival and growth of maturing sympathetic neurons by PI-3 kinase- and MAP kinase-dependent mechanisms.

Hepatocyte growth factor (HGF) is a pleiotrophic factor whose many functions include promoting neuronal survival and growth. Hitherto, these effects have been observed in the presence of other neurotrophic factors like NGF and CNTF, and this requirement for an accessory factor has made it difficult to elucidate the signaling pathways that mediate its survival and growth-enhancing effects. Here, we show that HGF promotes the survival of mature sympathetic neurons of the superior cervical ganglion (SCG) grown at low density in defined medium lacking other neurotrophic factors. This effect was first clearly observed in cultures established from postnatal day 20 (P20) mice and became maximal by P40. HGF also enhanced the growth of neurite arbors from neurons throughout postnatal development and in the adult. HGF treatment resulted in phosphorylation of Akt and ERK1/ERK2. Preventing Akt activation with the phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 blocked the HGF survival response, and inhibition of ERK activation with the MEK inhibitors PD98059 or U0126 reduced the HGF survival response and the neurite growth-promoting effects of HGF. These results indicate that HGF promotes the survival and growth of maturing sympathetic neurons by both PI-3 kinase- and MAP kinase-dependent mechanisms.

HGF regulates the development of cortical pyramidal dendrites.

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.

The fetal brainstem is relatively spared from injury following intrauterine hypoxemia.

Our aim was to test the hypothesis that the fetal brainstem is relatively spared, compared to other brain regions, from hypoxia-induced damage. We have used established experimental models of acute and chronic intrauterine compromise in sheep to mimic conditions that can arise in human pregnancy. The acute insult was 12 h of placental insufficiency induced by restricted utero-placental blood flow at 90 days of gestation (term approximately 147 days). Five weeks after this insult (n=7 fetuses) there was no overt damage to the brainstem nor were there alterations to the blood vessel morphology, volume of the medulla or of medullary nuclei compared to controls (n=8). This regimen is known to have significant effects on the forebrain and cerebellum. The chronic insult was induced in five fetuses via embolisation of the umbilico-placental circulation from 120 to 140 days of gestation. An additional three fetuses were found to be spontaneously hypoxemic (SH) immediately after surgery. At 140 days, in brainstems of all chronically hypoxemic fetuses compared to controls (n=8), there was an increase (P<0.05) in the percentage of neuropil occupied by blood vessels and abnormal myelin in the most severely SH fetus but no other morphological or neurochemical alterations. This regimen is known to cause marked damage to the cerebral hemispheres and to a lesser extent to the cerebellum. We suggest that the absence of marked structural or neurochemical alterations in the brainstem is most likely due to the maintenance of oxygen delivery to the brainstem during fetal hypoxemia.