Preterm Pups Research Articles

Ganaxolone Therapy After Preterm Birth Restores Cerebellar Oligodendrocyte Maturation and Myelination in Guinea Pigs.

The postnatal environment is challenging for the preterm neonate with exposure to hypoxic and excitotoxic events, amplified by premature loss of placentally derived neurosteroids. Between preterm birth and term equivalent age (TEA), cerebellar development continues despite these challenges. We hypothesize that neurosteroid replacement therapy during this time will support optimal cerebellar development. Guinea pig sows delivered at term (∼69 days gestation) or were induced to deliver preterm (∼62 days), with preterm pups receiving ganaxolone or vehicle until TEA. Postnatal assessments comprised salivary cortisol (corrected postnatal age [CPA] 0, 7, 38), behavioral analysis (CPA7, 38), and tissue collection (CPA0 and CPA40). Neurodevelopmental markers (MBP, Olig2, and NeuN) were assessed in the cerebellum by immunohistochemistry, whereas RT-PCR was utilized to investigate key inhibitory/excitatory pathways and oligodendrocyte lineage markers. Following preterm birth, there was evidence of a hyperactive phenotype, increased salivary cortisol concentrations, and impaired myelination and oligodendrocyte maturation at the protein level. mRNA expressions of key inhibitory/excitatory pathways and myelin stability were also altered following preterm birth. Importantly, we showed that neurosteroid replacement therapy returns cerebellar development and behavior toward a term-like phenotype. Therefore, ganaxolone may reduce the vulnerability of the cerebellum to postnatal challenges arising from preterm birth.

Neuroprotective Effects of Delayed TGF-β1 Receptor Antagonist Administration on Perinatal Hypoxic-Ischemic Brain Injury

Hypoxic-ischemic (HI) brain injury in neonatal encephalopathy triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the primary damage. The cellular processes mediating this prolonged neurodegeneration in HI injury are not sufficiently understood. Consequently, current therapies are not fully protective. In a recent study, we found significant improvements in neurologic outcomes when a small molecule antagonist for activin-like kinase 5 (ALK5), a transforming growth factor beta (TGF-β) receptor was used as a therapeutic in a rat model of moderate term HI. Here, we have extended those studies to a mouse preterm pup model of HI. For these studies, postnatal day 7 CD1 mice of both sexes were exposed to 35–40 min of HI. Beginning 3 days later, SB505124, the ALK5 receptor antagonist, was administered systemically through intraperitoneal injections performed every 12 h for 5 days. When evaluated 23 days later, SB505124-treated mice had ∼2.5-fold more hippocampal area and ∼2-fold more thalamic tissue. Approximately 90% of the ipsilateral hemisphere (ILH) was preserved in the SB505124-treated HI mice compared to the vehicle-treated HI mice, where the ILH was ∼60% of its normal size. SB505124 also preserved the subcortical white matter. SB505124 treatment preserved levels of aquaporin-4 and n-cadherin, key proteins associated with blood-brain barrier function. Importantly, SB505124 administration improved sensorimotor function as assessed by a battery of behavioral tests. Altogether, these data lend additional support to the conclusion that SB505124 is a candidate neuroprotective molecule that could be an effective treatment for HI-related encephalopathy in moderately injured preterm infants.

Severe intraventricular hemorrhage causes long-lasting structural damage in a preterm rabbit pup model

BackgroundIntraventricular hemorrhage causes significant lifelong mortality and morbidity, especially in preterm born infants. Progress in finding an effective therapy is stymied by a lack of preterm animal models with long-term follow-up. This study addresses this unmet need, using an established model of preterm rabbit IVH and analyzing outcomes out to 1 month of age.MethodsRabbit pups were delivered preterm and administered intraperitoneal injection of glycerol at 3 h of life and approximately 58% developed IVH. Neurobehavioral assessment was performed at 1 month of age followed by immunohistochemical labeling of epitopes for neurons, synapses, myelination, and interneurons, analyzed by means of digital quantitation and assessed via two-way ANOVA or Student’s t test.ResultsIVH pups had globally reduced myelin content, an aberrant cortical myelination microstructure, and thinner upper cortical layers (I–III). We also observed a lower number of parvalbumin (PV)-positive interneurons in deeper cortical layers (IV–VI) in IVH animals and reduced numbers of neurons, synapses, and microglia. However, there were no discernable changes in behaviors.ConclusionsWe have established in this preterm pup model that long-term changes after IVH include significant wide-ranging alterations to cortical organization and microstructure. Further work to improve the sensitivity of neurocognitive testing in this species at this age may be required.ImpactThis study uses an established animal model of preterm birth, in which the rabbit pups are truly born preterm, with reduced organ maturation and deprivation of maternally supplied trophic factors.This is the first study in preterm rabbits that explores the impacts of severe intraventricular hemorrhage beyond 14 days, out to 1 month of age.Our finding of persisting but subtle global changes including brain white and gray matter will have impact on our understanding of the best path for therapy design and interventions.

Examining Neurosteroid-Analogue Therapy in the Preterm Neonate For Promoting Hippocampal Neurodevelopment.

Background: Preterm birth can lead to brain injury and currently there are no targeted therapies to promote postnatal brain development and protect these vulnerable neonates. We have previously shown that the neurosteroid-analogue ganaxolone promotes white matter development and improves behavioural outcomes in male juvenile guinea pigs born preterm. Adverse side effects in this previous study necessitated this current follow-up dosing study, where a focus was placed upon physical wellbeing during the treatment administration and markers of neurodevelopment at the completion of the treatment period. Methods: Time-mated guinea pigs delivered preterm (d62) by induction of labour or spontaneously at term (d69). Preterm pups were randomized to receive no treatment (Prem-CON) or ganaxolone at one of three doses [0.5 mg/kg ganaxolone (low dose; LOW-GNX), 1.0 mg/kg ganaxolone (mid dose; MID-GNX), or 2.5 mg/kg ganaxolone (high dose; HIGH-GNX) in vehicle (45% β-cyclodextrin)] daily until term equivalence age. Physical parameters including weight gain, ponderal index, supplemental feeding, and wellbeing (a score based on respiration, activity, and posture) were recorded throughout the preterm period. At term equivalence, brain tissue was collected, and analysis of hippocampal neurodevelopment was undertaken by immunohistochemistry and RT-PCR. Results: Low and mid dose ganaxolone had some impacts on early weight gain, supplemental feeding, and wellbeing, whereas high dose ganaxolone significantly affected all physical parameters for multiple days during the postnatal period when compared to the preterm control neonates. Deficits in the preterm hippocampus were identified using neurodevelopmental markers including mRNA expression of oligodendrocyte lineage cells (CSPG4, MBP), neuronal growth (INA, VEGFA), and the GABAergic/glutamatergic system (SLC32A1, SLC1A2, GRIN1, GRIN2C, DLG4). These deficits were not affected by ganaxolone at the doses used at the equivalent of normal term. Conclusion: This is the first study to investigate the effects of a range of doses of ganaxolone to improve preterm brain development. We found that of the three doses, only the highest dose of ganaxolone (2.5 mg/kg) impaired key indicators of physical health and wellbeing over extended periods of time. Whilst it may be too early to see improvements in markers of neurodevelopment, further long-term study utilising the lower doses are warranted to assess functional outcomes at ages when preterm birth associated behavioural disorders are observed.

Antenatal Betamethasone Induces Increased Surfactant Proteins and Decreased Foxm1 Expressions in Fetal Rabbit Pups.

Introduction: Antenatal steroid improves respiratory distress syndrome in preterm infants. The molecular mechanism of the process is not well established. The aim of this study is to investigate the possible association between antenatal steroid and fetal Forkhead box M1(Foxm1) expression.Materials and methods: An animal study using mated pregnant New Zealand white rabbits and their fetuses was designed. Fourteen mother rabbits were assigned to four groups to undergo a cesarean section. In groups 1, 2, and 3, preterm pups were harvested on day 27 of gestation. In group 4, term pups were harvested on day 31. Antenatal maternal intramuscular injection was performed in groups 2 (normal saline) and 3 (betamethasone). Using qRT-PCR and Western blot, mRNA transcription and protein expression of surfactant protein (SP) A, B, C, and Foxm1 were compared between the pups of those four groups.Results: Sixty two fetal rabbits were harvested. One-way ANOVA test showed higher mRNA transcription of SPs in groups 3 and 4 than groups 1 and 2. Significantly lower Foxm1 mRNA transcription and protein expression were observed in group 3 or 4 compared with group 1 or 2.Conclusion: Decreased Foxm1 expression was associated in an antenatal betamethasone animal model.

Iatrogenic Prelabor Premature Rupture of Membrane and Preterm Birth: an Experimental Mouse Study

Objective: We aimed to investigate whether mouse mothers born prematurely give birth to preterm pups. Methods: Sexually mature female C57BL/6 mice were used. To create the pPROM model, the fetal membranes of the right uterus were pierced using sterile 21- or 26-gauge needles on gestational day 15.5. Results: The mean gestational duration of the first-generation pPROM model was 17.5 days. The mean gestational duration for the second-generation female mice born prematurely from the pPROM model was 19.8 days. The gestational duration between the first and second generations was significantly different (P<0.01). The average pup weight was greater in the third-generation mice than in the second-generation mice. Conclusion: Mice born to pPROM model mice did not give birth to preterm pups. It appears that mother born prematurely through iatrogenic pPROM does not have preterm birth.

Preterm birth impairs postnatal lung development in the neonatal rabbit model

BackgroundBronchopulmonary dysplasia continues to cause important respiratory morbidity throughout life, and new therapies are needed. The common denominator of all BPD cases is preterm birth, however most preclinical research in this area focusses on the effect of hyperoxia or mechanical ventilation. In this study we investigated if and how prematurity affects lung structure and function in neonatal rabbits.MethodsPups were delivered on either day 28 or day 31. For each gestational age a group of pups was harvested immediately after birth for lung morphometry and surfactant protein B and C quantification. All other pups were hand raised and harvested on day 4 for the term pups and day 7 for the preterm pups (same corrected age) for lung morphometry, lung function testing and qPCR. A subset of pups underwent microCT and dark field imaging on day 0, 2 and 4 for terms and on day 0, 3, 5 and 7 for preterms.ResultsPreterm pups assessed at birth depicted a more rudimentary lung structure (larger alveoli and thicker septations) and a lower expression of surfactant proteins in comparison to term pups. MicroCT and dark field imaging revealed delayed lung aeration in preterm pups, in comparison to term pups. Preterm birth led to smaller pups, with smaller lungs with a lower alveolar surface area on day 7/day 4. Furthermore, preterm birth affected lung function with increased tissue damping, tissue elastance and resistance and decreased dynamic compliance. Expression of vascular endothelial growth factor (VEGFA) was significantly decreased in preterm pups, however in the absence of structural vascular differences.ConclusionsPreterm birth affects lung structure and function at birth, but also has persistent effects on the developing lung. This supports the use of a preterm animal model, such as the preterm rabbit, for preclinical research on BPD. Future research that focuses on the identification of pathways that are involved in in-utero lung development and disrupted by pre-term birth, could lead to novel therapeutic strategies for BPD.

The effect of salt supplements on thyroid hormones and quality of pregnancy in female hypothyroid rats.

The use of nutrient supplements along with medication to optimize the treatment of diseases yields desirable outcomes. Hypothyroidism causes abnormalities in cells, and organs, and induces gene expression changes. The use of salt supplements and vitamins considerably helps to treat hypothyroidism. To evaluate the effect of a food supplement containing iron, iodine, and folic acid on thyroid hormones changes as well as the quality and quantity of hypothyroid female rat's offspring. In the current experimental study, 40 female rats were divided into six experimental and two control groups. The study was conducted in three phases. In the first phase, the role of a combinatory supplement along with levothyroxine to treat hypothyroidism by assessing T3, T4, and TSH hormones was investigated. In the second phase, the dose-depended effects of a combinatory supplement were investigated. Additionally, in the third phase, the quality and quantity of the next generation were measured in the hypothyroid female rats receiving the salt supplement. The plasma level of T3, T4 and TSH in hypothyroid rats receiving nutrient supplements indicated that the use of combinatory supplements along with levothyroxine could have desirable effects on the treatment of hypothyroidism to such an extent that the level of T3 and T4 hormones in the intervention group was significantly higher than that of the control group (P≤0.01). The second phase demonstrated that the desired effects of combinatory supplements on the serum levels of T3, T4, and TSH hormones were dose-dependent so that by increasing the dosage of supplementation, a significant decrease in the TSH level was observed (P <0.05), while T3 and T4 levels increased (P <0.01).The results of the third phase demonstrated that salt supplements could be effective in reducing the number of dead or preterm pups, and the use of mineral salts along with levothyroxine could promote a healthy birth. Salt supplements have considerable effects on the health status of the offspring of hypothyroid rats, resulting in the birth of more healthy pups and reducing the rate of abortion or preterm births.

Long-term neurological effects of neonatal caffeine treatment in a rabbit model of preterm birth.

Neonatal caffeine treatment might affect brain development. Long-term studies show conflicting results on brain-related outcomes. Herein we aimed to investigate the long-term effects of neonatal caffeine administration in a rabbit model of preterm birth. Preterm (born day 29) and term (day 32) pups were raised by wet nurses and allocated to treatment with saline or caffeine for 7 or 17 days. At pre-puberty, neurobehavioral tests were performed and brains were harvested for immunostaining of neurons, synapses, myelin, and astrocytes. Survival was lower in preterm saline pups than in controls, whereas caffeine-treated preterm pups did not differ from term control pups. Preterm saline pups covered less distance compared to controls and were more likely to stay in the peripheral zone of the open field. Corresponding differences were not seen in preterm caffeine pups. Preterm animals had lower neuron density compared to controls, which was not influenced by caffeine treatment. Synaptic density, astrocytes, and myelin were not different between groups. Caffeine appeared to be safe. All preterm rabbits had lower neuron density but anxious behavior seen in preterm saline rabbits was not seen in caffeine-treated preterm pups.

Neurosteroid replacement therapy using the allopregnanolone-analogue ganaxolone following preterm birth in male guinea pigs.

Children born preterm, especially boys, are at increased risk of developing attention deficit hyperactivity disorder (ADHD) and learning difficulties. We propose that neurosteroid-replacement therapy with ganaxolone (GNX) following preterm birth may mitigate preterm-associated neurodevelopmental impairment. Time-mated sows were delivered preterm (d62) or at term (d69). Male preterm pups were randomized to ganaxolone (Prem-GNX; 2.5 mg/kg subcutaneously twice daily until term equivalence), or preterm control (Prem-CON). Surviving male juvenile pups underwent behavioural testing at d25-corrected postnatal age (CPNA). Brain tissue was collected at CPNA28 and mature myelinating oligodendrocytes of the hippocampus and subcortical white matter were quantified by immunostaining of myelin basic protein (MBP). Ganaxolone treatment returned the hyperactive behavioural phenotype of preterm-born juvenile males to a term-born phenotype. Deficits in MBP immunostaining of the preterm hippocampus and subcortical white matter were also ameliorated in animals receiving ganaxolone. However, during the treatment period weight gain was poor, and pups were sedated, ultimately increasing the neonatal mortality rate. Ganaxolone improved neurobehavioural outcomes in males suggesting that neonatal treatment may be an option for reducing preterm-associated neurodevelopmental impairment. However, dosing studies are required to reduce the burden of unwanted side effects.

Disruption of Interneuron Neurogenesis in Premature Newborns and Reversal with Estrogen Treatment.

Many Preterm-born children suffer from neurobehavioral disorders. Premature birth terminates the hypoxic in utero 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.SIGNIFICANCE STATEMENT 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.

Different concentrations of docosahexanoic acid supplement during lactation result in different outcomes in preterm Sprague-Dawley rats

ProposeIn this study, we evaluated the effects of different concentrations of docosahexanoic acid (DHA) supplement on preterm Sprague-Dawley rat pups, and in parallel, measured the phosphorylation activity of the mTOR pathway in the hippocampal CA1 area. MethodsPreterm Sprague-Dawley rat pups were randomly assigned to experimental groups which included; a sufficient DHA group (100 mg/kg/day); an enriched DHA group (300 mg/kg/day); an excess DHA group (800 mg/kg/day); and a deficient DHA group (normal saline gavage 0.1 ml/10 g). Body weight (g) was measured at days 1/7/14/21/28/42, respectively. Spatial learning and memory were also tested using the Morris water maze at week 6 (day 42). Finally, activation of the mTOR signaling pathway in hippocampal CA1 area were evaluated by western blotting. ResultsPostnatal sufficient/enriched docosahexanoic acid supplement ameliorated body weight restriction, spatial learning and memory restriction, and decreased phosphorylation of AKT, mTOR, P70S6K1, and 4EBP1 in hippocampal CA1 area. Furthermore, excess docosahexanoic acid supplement impeded weight gain and spatial learning and memory, perturbed serum unsaturated fatty acid, and downregulated phosphorylation of AKT, mTOR, P70S6K1, and 4EBP1 in hippocampal CA1 area. ConclusionPostnatal sufficient/enriched DHA supplement ameliorated growth and spatial learning and memory impairment and upregulated the mTOR pathway in preterm pups, although excessive DHA supplement did not have any beneficial effects.

OP 24 Nrf2 deficiency induces placental TNF- α expression and influences the fetal growth in mice

Introduction and objectives Increased oxidative stress and intrauterine inflammation are etiological factors for intrauterine growth restriction (IUGR). The transcription of many antioxidative genes is mediated mainly through the transcription factor Nrf2. A link between Nrf2 and the vascular haemeostasis in preeclampsia has been discussed. The influence of Nrf2 deficiency on fetal growth has not been well described. Here, we studied the effect of Nrf2-deletion on fetal and placental development in the knockout mice model. Materials and methods We paired Nrf2 heterozygote mice to conditionally induce gene deletion in the coming embryos. Placentas from Nrf2 −/− and Nrf2 +/+ fetuses were collected on days 13.5, 15.5 and 18.5 post coitum . One fixed half was processed into paraffin wax, exhaustively sectioned and then stained with Periodic Acid Schiff (PAS).The total volume of each placenta was estimated using the Cavalieri estimator of volume. The other half was used for molecular biology analyses. Expression of the pro-inflammatory cytokines IL-6, IL-1, TNF- α , the anti-inflammatory enzyme Heme oxygenase-1 and VEGF were analyzed. Results The Nrf2-deficient pups presented reduced birth weight (0.7 ± 0.06 g) when compared to the wild type pups (0.95 ± 0.03 g). In addition the placental volume of Nrf2 deleted embryos was significantly reduced in comparison to the wild type placentas (Nrf2 +/+ 121 ± 4 vs. Nrf2 −/− 92 ± 5 mm 3 ). Placentas from preterm Nrf2-deleted pups showed increased levels of the pro-inflammatory cytokines (IL-6 and TNF- α ), and decreased expression of HO-1 and VEGF. Conclusion Our results show that a disturbed Nrf2 signaling leads to a significant reduction in the growth of the fetuses. The increased expression of the pro-inflammatory cytokines IL-6 and TNF- α as well as the reduced expression of the anti-inflammatory enzyme HO-1 and VEGF in Nrf2 deleted placentas suggest that intrauterine inflammation may be a cause for the IUGR in this model.

Transcriptome Analysis of the Preterm Rabbit Lung after Seven Days of Hyperoxic Exposure.

The neonatal management of preterm born infants often results in damage to the developing lung and subsequent morbidity, referred to as bronchopulmonary dysplasia (BPD). Animal models may help in understanding the molecular processes involved in this condition and define therapeutic targets. Our goal was to identify molecular pathways using the earlier described preterm rabbit model of hyperoxia induced lung-injury. Transcriptome analysis by mRNA-sequencing was performed on lungs from preterm rabbit pups born at day 28 of gestation (term: 31 days) and kept in hyperoxia (95% O2) for 7 days. Controls were preterm pups kept in normoxia. Transcriptomic data were analyzed using Array Studio and Ingenuity Pathway Analysis (IPA), in order to identify the central molecules responsible for the observed transcriptional changes. We detected 2217 significantly dysregulated transcripts following hyperoxia, of which 90% could be identified. Major pathophysiological dysregulations were found in inflammation, lung development, vascular development and reactive oxygen species (ROS) metabolism. To conclude, amongst the many dysregulated transcripts, major changes were found in the inflammatory, oxidative stress and lung developmental pathways. This information may be used for the generation of new treatment hypotheses for hyperoxia-induced lung injury and BPD.

Premature guinea pigs: a new paradigm to investigate the late-effects of preterm birth.

Preterm birth is common and the associated short-term morbidity well described. The adult-onset consequences of preterm birth are less clear, but cardiovascular and metabolic health may be adversely affected. Although large animal models of preterm birth addressing important short-term issues exist, long-term studies are hampered by significant logistical constraints. Current small animal models of prematurity require terminal caesarean section of the mother; both caesarean birth and early maternal care modify offspring adult cardio-metabolic function. We describe a novel method for inducing preterm labour in guinea pigs. With support comparable to that received by moderately preterm human infants, preterm pups are viable. Growth trajectories between preterm and term-born pups differ significantly; between term equivalent age and weaning ex-preterm animals demonstrate increased weight and ponderal index. We believe this novel paradigm will significantly improve our ability to investigate the cardio-metabolic sequelae of preterm birth throughout the life course and into the second generation.

Functional assessment of hyperoxia-induced lung injury after preterm birth in the rabbit

The objective of this study was to document early neonatal (7 days) pulmonary outcome in the rabbit model for preterm birth and hyperoxia-induced lung injury. Preterm pups were delivered at 28 days (term = 31 days; early saccular phase of lung development) by cesarean section, housed in an incubator, and gavage fed for 7 days. Pups were divided into the following groups: 1) normoxia (21% O2; normoxia group) and 2) and hyperoxia (>95% O2; hyperoxia group). Controls were pups born at term who were housed in normoxic conditions (control group). Outcome measures were survival, pulmonary function tests using the whole body plethysmograph and forced oscillation technique, and lung morphometry. There was a significant difference in survival of preterm pups whether they were exposed to normoxia (83.3%) or hyperoxia (55.9%). Hyperoxic exposure was associated with increased tissue damping and elasticity and decreased static compliance compared with normoxic controls (P < 0.01). Morphometry revealed an increased linear intercept and increased mean wall transection length, which translates to larger alveoli with septal thickening in hyperoxia compared with normoxia (P < 0.01). In conclusion, the current experimental hyperoxic conditions to which preterm pups are exposed induce the typical clinical features of bronchopulmonary dysplasia. This model will be used to study novel preventive or therapeutic interventions.

Effect(s) of Preterm Birth on Normal Retinal Vascular Development and Oxygen-Induced Retinopathy in the Neonatal Rat

ABSTRACTPurpose: Maturity is a critical factor in the pathogenesis of retinopathy of prematurity (ROP). One widely used method for studying this condition is that of oxygen-induced retinopathy (OIR). The general conditions of an OIR term animal, both at the time of birth and following birth, differ from those of the preterm infant. This, to simulate preterm conditions and to provide a basis for further studies on ROP, we investigated the effect(s) of preterm birth on retinal vascularization using the neonatal rat.Materials and methods: Sprague-Dawley (SD) rats were delivered preterm by caesarean section on the day 19 of gestation. Term pups were used as controls. On the day of birth, preterm and term pups were housed under conditions of room air or cyclic oxygen. Retinas of pups housed in room air on days 4, 7, 10, 14, 18 and 22, as well as pups housed in oxygen on days 14, 18, and 22 were whole-mounted and stained with isolectin-B4. On day 18, cross-sections of the retina were cut and stained with hematoxylin and eosin for the identification of preretinal neovascular tufts. Images of avascular and neovascular areas were compared using light and fluorescence microscopy.Results: Preterm pups had significantly larger avascular retinal areas than term rats on the various postnatal days. After exposure to cyclic oxygen, preterm pups demonstrated significantly larger avascular (days 14 and 18) and neovascular areas (day 18) compared with term rats. On day 22, residual retinopathy of preterm pups was greater than that of term pups.Conclusions: Preterm birth of rats, which are comparable in their physiology to humans, had negative effects on retinal vascularization. The impaired retinal vascular development and subsequent vasoproliferation resulting from hyperoxia in preterm pups is more severe and enduring.

Preliminary study of retinal pathological features in preterm birth pups exposed to an animal model of oxygen-induced retinopathy in mice

The main risk factors of retinopathy of prematurity (ROP) are low gestational age and low birth weight, which are mainly caused by preterm birth. Currently, the animal model of oxygen-induced retinopathy (OIR) in mice is the most widely used model in ROP-associated studies. However, the experimental mice are normal-term pups, and may not mimic the pathogenic status of human ROP patients. In this study, we investigated the retinal pathological features in preterm birth pups exposed to an animal model of oxygen-induced retinopathy in mice. Preterm-birth mice were obtained from pregnant C57BL/6J mice that were induced by an intraperitoneal injection of lipopolysaccharide (LPS). The preterm and control mice were treated with high oxygen (75%) from postnatal day 7 (P7) to P12. The mice were perfused with high-molecular-weight FITC-dextran on P12, P15 and P17, and the retinas were whole-mounted and imaged. Vascular endothelial growth factor (VEGF) mRNA was also detected. Cross-sections of the retina were stained with hematoxylin and eosin (H&E) to identify preretinal neovascular tufts. For general observation, whole retinal images were also obtained using a microscope. Leakage of the retinal blood vessels was aggravated in the preterm mice, particularly on P12 and P15. The non-perfused areas of the retina (pixel value, 183,673 ± 28,148 vs 132,110 ± 23,732, P = 0.009) and the number of preretinal endothelial cell nuclei were smaller (30.17 ± 8.33 vs 22.17 ± 6.74, P < 0.0001) on P17. The VEGF mRNA levels in the retinas were higher on P12 and P15 but lower on P17, compared with the control mice. Retinal hemorrhage was observed in the preterm mouse group (five out of six examined eyes). Preterm-birth mice that were subject to OIR exhibited several pathological features, such as retinal hemorrhage, severe retinal leakage and moderate retinal neovascularization, which were similar to the clinical manifestations in ROP patients.

Neurogenesis Continues in the Third Trimester of Pregnancy and Is Suppressed by Premature Birth

Premature infants exhibit neurodevelopmental delay and reduced growth of the cerebral cortex. However, the underlying mechanisms have remained elusive. Therefore, we hypothesized that neurogenesis in the ventricular and subventricular zones of the cerebral cortex would continue in the third trimester of pregnancy and that preterm birth would suppress neurogenesis. To test our hypotheses, we evaluated autopsy materials from human fetuses and preterm infants of 16-35 gestational weeks (gw). We noted that both cycling and noncycling Sox2(+) radial glial cells and Tbr2(+) intermediate progenitors were abundant in human preterm infants until 28 gw. However, their densities consistently decreased from 16 through 28 gw. To determine the effect of premature birth on neurogenesis, we used a rabbit model and compared preterm [embryonic day 29 (E29), 3 d old] and term (E32, <2 h old) pups at an equivalent postconceptional age. Glutamatergic neurogenesis was suppressed in preterm rabbits, as indicated by the reduced number of Tbr2(+) intermediate progenitors and the increased number of Sox2(+) radial glia. Additionally, hypoxia-inducible factor-1α, vascular endothelial growth factor, and erythropoietin were higher in term than preterm pups, reflecting the hypoxic intrauterine environment of just-born term pups. Proneural genes, including Pax6 and Neurogenin-1 and -2, were higher in preterm rabbit pups compared with term pups. Importantly, neurogenesis and associated factors were restored in preterm pups by treatment with dimethyloxallyl glycine, a hypoxia mimetic agent. Hence, glutamatergic neurogenesis continues in the premature infants, preterm birth suppresses neurogenesis, and hypoxia-mimetic agents might restore neurogenesis, enhance cortical growth, and improve neurodevelopmental outcome of premature infants.

Enteral intestinal alkaline phosphatase administration in newborns decreases iNOS expression in a neonatal necrotizing enterocolitis rat model

PurposeTo determine if intestinal alkaline phosphatase (IAP) decreases intestinal injury resulting from experimentally induced necrotizing enterocolitis (NEC). We hypothesized that IAP administration prevents the initial development of NEC related intestinal inflammation. MethodsPre- and full-term newborn Sprague–Dawley rat pups were sacrificed on day 1 of life. Pre-term pups were exposed to intermittent hypoxia and formula containing LPS to induce NEC. Select NEC pups were given 40, 4 or 0.4units/kg of bovine IAP (NEC+IAP40u, IAP4u or IAP0.4u) enterally, once daily. Ileal sections were evaluated by real-time PCR (qRT-PCR) for IAP, iNOS, IL-1β, IL-6, and TNF-α mRNA and immunofluorescence for 3-nitrotyrosine (3-NT). ResultsExperimentally induced NEC decreased IAP mRNA expression by 66% (p≤0.001). IAP supplementation increased IAP mRNA expression to control. Supplemental enteral IAP decreased nitrosative stress as measured by iNOS mRNA expression and 3-NT staining in the NEC stressed pups (p≤0.01), as well as decreased intestinal TNF-α mRNA expression. In addition, IAP decreased LSP translocation into the serum in the treated pups. ConclusionsWe conclude that enterally administered IAP prevents NEC-related intestinal injury and inflammation. Enteral IAP may prove a useful strategy in the prevention of NEC in preterm neonates.