Adverse neurological outcome is a major cause of long-term morbidity in ex-preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non-infectious model of preterm birth by administering RU486. Pro-inflammatory cytokines (IL-10, IL-1β, IL-6 and TNF-α) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro-inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammationinduced preterm birth, but not non-infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubuleassociated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation-induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology. Keywords mouse model of preterm birth; neuroinflammation; neuronal injuryIn the United States, approximately 12% of all live births are delivered preterm (Green et al., 2005). Preterm birth (PTB) is the leading cause of neonatal mortality and morbidity in the NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptUnited States. Specifically, PTB is a risk factor for adverse neurological outcome for expreterm children (Anderson and Doyle, 2003;Hack et al., 2005).It has long been believed that cerebral palsy is the primary neurological outcome of clinical interest. However, it is now known that ex-preterm children also are at a significant risk for a spectrum of cognitive and neurobehavioral disorders Wood et al., 2005;Costeloe, 2006;Limperopoulos et al., 2007;Lindstrom et al., 2009) including autism spectrum disorders (Brimacombe et al., 2007;Limperopoulos et al., 2008;Schendel and Bhasin, 2008). Current understanding of the pathogenesis of fetal brain injury in a PTB focuses mainly on specific structural findings of white matter damage (WMD) (Cai et al., 2000;Paintlia et al., 2004;Rousset et al., 2006). This current paradigm may be insufficient to explain the increasing prevalence of adverse cognitive and neurobehavioral outcomes in ex-preterm infants.While adverse neurological outcomes are increasingly prevalent in ex-preterm children, it remains unknown whether th...

Objective-To elucidate possible mechanisms of fetal neuronal injury in inflammation-induced preterm birth.Study design-Utilizing mouse model of preterm birth, primary cultures were prepared from fetal brains: 1) control neurons (CN); 2) LPS-exposed neurons (LN); 3) control co-culture (CCC), consisting of neurons and glia; 4) LPS-exposed co-culture (LCC), consisting of LPS-exposed neurons and glia. CN and LN were treated with culture media from CN, LN, CCC and LCC after 24 hours in vitro. Immunocytochemistry was performed for culture characterization and neuronal morphology. Quantitative PCR was performed for neuronal differentiation marker, MAP2, and for cell death mediators, Caspases 1, 3 and 9.Results-LPS exposure in vivo did not influence neuronal or glial content in co-cultures but decreased expression of MAP2 in LN. Media from LN and LCC induced morphological changes in control neurons comparable to LN. The neuronal damage caused by in vivo exposure (LN) could not be reversed by media from control groups.Conclusion-LPS-induced preterm birth may be responsible for irreversible neuronal injury.

Premature cervical ripening is believed to contribute to preterm birth (PTB). Preterm cervical ripening may be due to an aberrant regulation in timing of the same processes that occur at term, or may result from unique molecular mechanisms. Using mouse models of PTB, this study sought to investigate if the molecular mechanisms that govern cervical ripening were similar between preterm and term. Lipopolysaccharide (LPS) is infused into the uterine horn to create a mouse model of inflammation-induced PTB. For a noninfectious model of PTB, RU486 was administered. Both models result in delivery of pups in 8-24 h. Cervical tissues were collected from these models, as well as throughout gestation. Cervical tissues from E15 (preterm), E15 LPS (preterm inflammation), and E18.5 (term) were used for microarray analysis (n = 18). Additional experiments using gestational time course specimens were performed to confirm microarray results. Specific gene pathways were differentially expressed between the groups. Genes involved in immunity and inflammation were increased in the cervix in inflammation-induced PTB; term labor was not associated with differential expression of immune pathways. Cytokine expression was not increased in cervices during term labor, but was increased in the pospartum period. Epithelial cell differentiation pathway was significantly altered in term, but not preterm, labor. Activation of immune pathways may be sufficient for cervical ripening, but does not appear necessary. Differential expression of the epithelial cell differentiation pathway appears necessary in the process of cervical repair. Our results indicate that the molecular mechanisms governing preterm and term cervical ripening are distinctly different.

OBJECTIVE-To investigate whether magnesium sulfate (MgSO 4 ) prevents fetal brain injury in inflammation-associated preterm birth (PTB).STUDY DESIGN-Utilising a mouse model of PTB, LPS or normal saline (NS)-exposed mice via intrauterine injection, were randomized to intraperitoneal treatment with MgSO 4 or NS. From the 4 treatment groups, 1)NS+NS; 2)LPS+NS; 3)LPS+MgSO 4 ; and 4)NS+MgSO 4 , fetal brains were collected for QPCR studies and primary neuronal cultures. mRNA expression of cytokines, cell death, and markers of neuronal and glial differentiation were assessed.Immunocytochemistry and confocal microscopy were performed. RESULTS-There was no difference between LPS+NS and LPS+MgSO 4 groups in expression ofpro-inflammatory cytokines, cell death markers as well markers of pro-oligodendrocyte and astrocyte development (P>0.05 for all). Neuronal cultures from LPS+NS demonstrated morphological changes and this neuronal injury was prevented by MgSO 4 (P<0.001).CONCLUSION-Amelioration of neuronal injury in inflammation-associated PTB may be a key mechanism by which MgSO 4 prevents cerebral palsy.

Taste buds are chemosensory structures widely distributed on the surface of the oral cavity and larynx. Taste cells, exposed to the oral environment, face great challenges in defense against potential pathogens. While immune cells, such as T-cells and macrophages, are rarely found in taste buds, high levels of expression of some immune-response-associated molecules are observed in taste buds. Yet, the cellular origins of these immune molecules such as cytokines in taste buds remain to be determined. Here, we show that a specific subset of taste cells selectively expresses high levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α). Based on immuno-colocalization experiments using taste-cell-type markers, the TNF-α-producing cells are predominantly type II taste cells expressing the taste receptor T1R3. These cells can rapidly increase TNF-α production and secretion upon inflammatory challenges, both in vivo and in vitro. The lipopolysaccharide (LPS)-induced TNF-α expression in taste cells was completely eliminated in TLR2−/−/TLR4−/− double-gene-knockout mice, which confirms that the induction of TNF-α in taste buds by LPS is mediated through TLR signaling pathways. The taste-cell-produced TNF-α may contribute to local immune surveillance, as well as regulate taste sensation under normal and pathological conditions.

Inflammatory cytokines are important regulators of metabolism and food intake. Over production of inflammatory cytokines during bacterial and viral infections leads to anorexia and reduced food intake. However, it remains unclear whether any inflammatory cytokines are involved in the regulation of taste reception, the sensory mechanism governing food intake. Previously, we showed that tumor necrosis factor (TNF), a potent proinflammatory cytokine, is preferentially expressed in a subset of taste bud cells. The level of TNF in taste cells can be further induced by inflammatory stimuli. To investigate whether TNF plays a role in regulating taste responses, in this study, we performed taste behavioral tests and gustatory nerve recordings in TNF knockout mice. Behavioral tests showed that TNF-deficient mice are significantly less sensitive to the bitter compound quinine than wild-type mice, while their responses to sweet, umami, salty, and sour compounds are comparable to those of wild-type controls. Furthermore, nerve recording experiments showed that the chorda tympani nerve in TNF knockout mice is much less responsive to bitter compounds than that in wild-type mice. Chorda tympani nerve responses to sweet, umami, salty, and sour compounds are similar between TNF knockout and wild-type mice, consistent with the results from behavioral tests. We further showed that taste bud cells express the two known TNF receptors TNFR1 and TNFR2 and, therefore, are potential targets of TNF. Together, our results suggest that TNF signaling preferentially modulates bitter taste responses. This mechanism may contribute to taste dysfunction, particularly taste distortion, associated with infections and some chronic inflammatory diseases.

Although inflammatory responses are a critical component in defense against pathogens, too much inflammation is harmful. Mechanisms have evolved to regulate inflammation, including modulation by the anti-inflammatory cytokine interleukin-10 (IL-10). Previously we have shown that taste buds express various molecules involved in innate immune responses, including the proinflammatory cytokine tumor necrosis factor (TNF). Here, using a reporter mouse strain, we show that taste cells also express the anti-inflammatory cytokine IL-10. Remarkably, IL-10 is produced by only a specific subset of taste cells, which are different from the TNF-producing cells in mouse circumvallate and foliate taste buds: IL-10 expression was found exclusively in the G-protein gustducin-expressing bitter receptor cells, while TNF was found in sweet and umami receptor cells as reported previously. In contrast, IL-10R1, the ligand-binding subunit of the IL-10 receptor, is predominantly expressed by TNF-producing cells, suggesting a novel cellular hierarchy for regulating TNF production and effects in taste buds. In response to inflammatory challenges, taste cells can increase IL-10 expression both in vivo and in vitro. These findings suggest that taste buds use separate populations of taste receptor cells that coincide with sweet/umami and bitter taste reception to modulate local inflammatory responses, a phenomenon that has not been previously reported. Furthermore, IL-10 deficiency in mice leads to significant reductions in the number and size of taste buds, as well as in the number of taste receptor cells per taste bud, suggesting that IL-10 plays critical roles in maintaining structural integrity of the peripheral gustatory system.

While our understanding of the molecular and cellular aspects of taste reception and signaling continues to improve, the aberrations in these processes that lead to taste dysfunction remain largely unexplored. Abnormalities in taste can develop in a variety of diseases, including infections and autoimmune disorders. In this study, we used a mouse model of autoimmune disease to investigate the underlying mechanisms of taste disorders. MRL/MpJ-Faslpr/J (MRL/lpr) mice develop a systemic autoimmunity with phenotypic similarities to human systemic lupus erythematosus and Sjögren's syndrome. Our results show that the taste tissues of MRL/lpr mice exhibit characteristics of inflammation, including infiltration of T lymphocytes and elevated levels of some inflammatory cytokines. Histological studies reveal that the taste buds of MRL/lpr mice are smaller than those of wild-type congenic control (MRL/+/+) mice. 5-Bromo-2′-deoxyuridine (BrdU) pulse-chase experiments show that fewer BrdU-labeled cells enter the taste buds of MRL/lpr mice, suggesting an inhibition of taste cell renewal. Real-time RT-PCR analyses show that mRNA levels of several type II taste cell markers are lower in MRL/lpr mice. Immunohistochemical analyses confirm a significant reduction in the number of gustducin-positive taste receptor cells in the taste buds of MRL/lpr mice. Furthermore, MRL/lpr mice exhibit reduced gustatory nerve responses to the bitter compound quinine and the sweet compound saccharin and reduced behavioral responses to bitter, sweet, and umami taste substances compared with controls. In contrast, their responses to salty and sour compounds are comparable to those of control mice in both nerve recording and behavioral experiments. Together, our results suggest that type II taste receptor cells, which are essential for bitter, sweet, and umami taste reception and signaling, are selectively affected in MRL/lpr mice, a model for autoimmune disease with chronic inflammation.