Ultrasound-guided intrauterine injection of lipopolysaccharide as a novel model of preterm birth in the mouse.

Mouse models are used to study mechanisms that link intrauterine infection and preterm birth (PTB). To mimic intrauterine infection, lipopolysaccharide (LPS) is commonly injected into the uterus via minilaparotomy, which is invasive, and can cause PTB in control animals. We hypothesized that less-invasive ultrasound-guided intrauterine LPS injection or intravaginal LPS administration could induce PTB by stimulating an inflammatory response of the uteroplacental tissues, while minimizing PTB in control animals. On day 17 of gestation mice received LPS intravaginally (10 to 240 µg; n = 3 to 8) or into the uterus (20 µg) under ultrasound guidance (n = 7) or via laparotomy (n = 7). Control animals received phosphate-buffered saline (PBS; n = 5 to 7). Intrauterine administration of LPS, both under ultrasound guidance and via laparotomy, induced delivery earlier than in PBS control groups (P < 0.01). Intravaginal LPS administration did not stimulate PTB. Quantitative real-time PCR and immunohistochemistry of tissues harvested 6 hours after treatment confirmed that ultrasound-guided LPS administration induced a localized inflammatory response. Ultrasound-guided intrauterine LPS injection reliably induces PTB in the mouse and mimics the local inflammatory and immune responses observed in the more-invasive laparotomy model of inflammation-induced PTB. Ultrasound-guided intrauterine LPS injection is a useful novel model of PTB for future studies and concords with the principles of reduction, replacement, and refinement.

Decidual neutrophil infiltration is not required for preterm birth in a mouse model of infection-induced preterm labor.

Parturition is associated with a leukocyte influx into the intrauterine tissues; however, the exact role these leukocytes play in the onset of labor remains unclear. Neutrophil infiltration of the uteroplacental tissues has been particularly associated with infection-associated preterm labor (PTL) in both women and mouse models. In this study, we investigated the role of neutrophils in a mouse model of infection-induced PTL. Intrauterine administration of LPS on day 17 of gestation resulted in a 7-fold increase in the number of decidual neutrophils compared with control mice receiving PBS (p < 0.01; n = 8-11). We hypothesized that neutrophil influx is necessary for PTL and that neutrophil depletion would abolish preterm birth. To test this hypothesis, mice were depleted of neutrophils by treatment with anti-Gr-1, anti-Ly-6G, or the appropriate IgG control Ab on day 16 of gestation prior to LPS on day 17 (n = 6-7). Successful neutrophil depletion was confirmed by flow cytometry and immunohistochemistry. Neutrophil depletion with Gr-1 resulted in reduced uterine and placental Il-1ß expression (p < 0.05). Neutrophil depletion with Ly-6G reduced uterine Il-1ß and Tnf-α expression (p < 0.05). However, neutrophil depletion with either Ab did not delay LPS-induced preterm birth. Collectively, these data show that decidual neutrophil infiltration is not essential for the induction of infection-induced PTL in the mouse, but that neutrophils contribute to the LPS-induced inflammatory response of the uteroplacental tissues.

Complement inhibition and statins prevent fetal brain cortical abnormalities in a mouse model of preterm birth.

Premature babies are particularly vulnerable to brain injury. In this study we focus on cortical brain damage associated with long-term cognitive, behavioral, attentional or socialization deficits in children born preterm. Using a mouse model of preterm birth (PTB), we demonstrated that complement component C5a contributes to fetal cortical brain injury. Disruption of cortical dendritic and axonal cytoarchitecture was observed in PTB-mice. Fetuses deficient in C5aR (-/-) did not show cortical brain damage. Treatment with antibody anti-C5, that prevents generation of C5a, also prevented cortical fetal brain injury in PTB-mice. C5a also showed a detrimental effect on fetal cortical neuron development and survival in vitro. Increased glutamate release was observed in cortical neurons in culture exposed to C5a. Blockade of C5aR prevented glutamate increase and restored neurons dendritic and axonal growth and survival. Similarly, increased glutamate levels - measured by (1)HMRS - were observed in vivo in PTB-fetuses compared to age-matched controls. The blockade of glutamate receptors prevented C5a-induced abnormal growth and increased cell death in isolated fetal cortical neurons. Simvastatin and pravastatin prevented cortical fetal brain developmental and metabolic abnormalities -in vivo and in vitro. Neuroprotective effects of statins were mediated by Akt/PKB signaling pathways. This study shows that complement activation plays a crucial role in cortical fetal brain injury in PTL and suggests that complement inhibitors and statins might be good therapeutic options to improve neonatal outcomes in preterm birth.

Oxygen flux reduces Cux1 positive neurons and cortical growth in a gestational rodent model of growth restriction.

BACKGROUND: The mammalian cerebral cortex forms in an inside-out manner, establishing deep cortical layers before superficial layers and is regulated by transcription factors which influence cell differentiation. Preterm birth interrupts the trajectory of normal neurodevelopment and adverse perinatal exposures have been implicated in cortical injury. We hypothesise that growth restriction (GR) and fluctuating hyperoxia (ΔO2) impair cortical laminar development. METHODS: Sprague-Dawley rats received 18% (non-restricted, NR) or 9% (growth restricted, GR) protein diet from E15-P7. Litters were reared in air or fluctuating hyperoxia (circa 10kPa) from P0 to P7. Cortical laminae were stained and measured. Neuronal subtypes were quantified using immunofluorescence for subtype-specific transcription factors (Satb2, Cux1, Ctip2, Tbr1). RESULTS: ΔO2 did not affect brain weight at P7 but reduced cortical thickness in both NR (p<0.05) and GR groups (p<0.001). ΔO2 resulted in superficial cortical thinning in both groups and in the deep layers of GR pups (p<0.001). Cell density was preserved. ΔO2 did not affect proportions of callosal, corticothalamic and corticospinal neurons but resulted in a reduction of neurons expressing Cux1 (p<0.01) implicated in dendritic branching and synapse formation. CONCLUSION: Postnatal ΔO2, a modifiable factor in neonatal care, impairs cortical development in a rodent model with preferential disadvantage to superficial neurons.

Chlamydia trachomatis infection of human endometrial stromal cells induces defective decidualisation and chemokine release.

Miscarriage affects ~20% of pregnancies and maternal infections account for ~15% of early miscarriages. Chlamydia trachomatis (Ct) has been associated with miscarriage but the underlying mechanisms are unknown. Successful implantation requires endometrial stromal cell (ESC) decidualisation. Maintenance of pregnancy requires angiogenesis, establishment of the correct cellular milieu and trophoblast invasion, all of which involve the action of chemokines. Our objective was to determine whether Ct infection impacts upon ESC decidualisation and chemokine secretion. Human primary ESC were decidualised in-vitro, infected with Ct serovar E, and changes in expression of genes of interest were measured using RT-PCR, proteomic array and ELISA. We demonstrate for the first time that Ct can infect and proliferate in ESC. Expression of the decidualisation marker prolactin was decreased in Ct-infected ESC at both mRNA and protein levels. Ct infection altered the chemokine profile of decidualised ESC as shown by proteomic array. Chemokines CXCL12 and CXCL16, important for trophoblast invasion, were analysed further and expression was reduced in infected decidualised cells at mRNA and protein levels. Our data indicate that Ct infection of ESC impairs decidualisation and alters chemokine release. These findings at least partially explain how Ct infection could result in adverse pregnancy outcomes.

Myelin expression is altered in the brains of neonatal rats reared in a fluctuating oxygen atmosphere.

BACKGROUND: Preterm infants receiving supplemental oxygen therapy experience frequent fluctuations in their blood oxygen levels, the magnitude of which has been associated with the incidence and severity of retinopathy of prematurity in such infants. OBJECTIVE: Our objective was to investigate in a relevant animal model whether the immature brain with its poorly vascularised white matter might also be susceptible to injury when exposed to such fluctuations in blood oxygen. METHODS: Newborn rats were reared in an atmosphere in which a computer reproduced the oxygen fluctuations derived from the transcutaneous oxygen levels of a 24-week preterm infant who had developed severe retinopathy. Following 14 days of exposure, we measured the expression of active caspase-3, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in the brains comparing with rat pups raised in room air. RESULTS: Compared to room air controls, at day 14, the expression of active caspase-3 was increased by up to 162% (significant increase in 7 of 9 regions), MBP decreased by up to 70% (significant in the hypothalamus only) and GFAP increased by up to 103% (significant in 6 of 7 regions. On day 21, following 7 days of reparative recovery, GFAP levels in most areas of oxygen-exposed brains had returned to near control levels. There were no longer significant differences in caspase-3 levels apart from the cerebral cortex, cerebellum and striatum. In contrast, MBP expression was now much higher in most regions of the treated brains compared to controls. CONCLUSION: We conclude that fluctuations in blood oxygen, observed in preterm survivors, may constitute a source of injury to the white matter and corpus striatum of the developing brain and contribute to the neurological sequelae in extremely premature infants.

Hypoxic oxygen fluctuations produce less severe retinopathy than hyperoxic fluctuations in a rat model of retinopathy of prematurity.

The aim of this study was to investigate whether the mean around which arterial oxygen fluctuations take place was important in a unique animal model of oxygen-induced retinopathy. Retinopathy of prematurity (ROP) is associated with fluctuating arterial oxygen. A recent retrospective study suggested that management of high-risk preterm infants at lower oxygen saturations was associated with less severe ROP. Rat pups were raised in a variable oxygen environment around a high (24%), normal (21%) or low (17%) mean inspired oxygen for 14 d. Rat pups raised in the high (24%) mean variable oxygen environment had more retarded retinal vascular development than did rats raised in an environment that fluctuated around 21% mean oxygen. In contrast, rats raised in a lower mean (17%) but still variable oxygen environment had no discernible retinal differences from controls raised in constant room air. Rats raised in a relatively hypoxic but variable oxygen environment develop less severe retinal vascular abnormalities than those raised in variable oxygen around higher oxygen means.