Role of neurotrophic factors in enhancing linear axonal growth of ganglionic sensory neurons in vitro.

Neurotrophins play a major role in the regulation of neuronal growth such as neurite sprouting or regeneration in response to nerve injuries. The role of nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor in maintaining the survival of peripheral neurons remains poorly understood. In regenerative medicine, different modalities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. This study was to investigate the influence of nerve growth factor, neurotrophin-3 and brain-derived neurotrophic factor on the growth of neurites using two in vitro models of dorsal root ganglia explants and dorsal root ganglia-derived primary cell dissociated cultures. Quantitative data showed that the total neurite length and tortuosity were differently influenced by trophic factors. Nerve growth factor and, indirectly, brain-derived neurotrophic factor stimulate the tortuous growth of sensory fibers and the formation of cell clusters. Neurotrophin-3, however, enhances neurite growth in terms of length and linearity allowing for a more organized and directed axonal elongation towards a peripheral target compared to the other growth factors. These findings could be of considerable importance for any clinical application of neurotrophic factors in peripheral nerve regeneration. Ethical approval was obtained from the Regione Piemonte Animal Ethics Committee ASLTO1 (file # 864/2016-PR) on September 14, 2016.

Characterization of glial cell models and in vitro manipulation of the neuregulin1/ErbB system.

The neuregulin1/ErbB system plays an important role in Schwann cell behavior both in normal and pathological conditions. Upon investigation of the expression of the neuregulin1/ErbB system in vitro, we explored the possibility to manipulate the system in order to increase the migration of Schwann cells, that play a fundamental role in the peripheral nerve regeneration. Comparison of primary cells and stable cell lines shows that both primary olfactory bulb ensheathing cells and a corresponding cell line express ErbB1-ErbB2 and neuregulin1, and that both primary Schwann cells and a corresponding cell line express ErbB2-ErbB3, while only primary Schwann cells express neuregulin1. To interfere with the neuregulin1/ErbB system, the soluble extracellular domain of the neuregulin1 receptor ErbB4 (ecto-ErbB4) was expressed in vitro in the neuregulin1 expressing cell line, and an unexpected increase in cell motility was observed. In vitro experiments suggest that the back signaling mediated by the transmembrane neuregulin1 plays a role in the migratory activity induced by ecto-ErbB4. These results indicate that ecto-ErbB4 could be used in vivo as a tool to manipulate the neuregulin1/ErbB system.

Urocortin increases IL-4 and IL-10 secretion and reverses LPS-induced TNF-alpha release from human trophoblast primary cells.

PROBLEM: As urocortin (Ucn) is a placental peptide belonging to the corticotrophin-releasing hormone (CRH) family that modulates immune function in other biological models, this study evaluated Ucn effects on cytokines secretion from cultured human trophoblast cells. METHOD OF STUDY: Placentas were collected from normal term pregnancies after elective caesarean section, and primary trophoblast culture was prepared followed by the treatment of Ucn and/or CRH selective antagonists, antalarmin and astressin 2b. The anti-inflammatory cytokines IL-4 and IL-10 and the pro-inflammatory cytokine TNF-alpha were measured by ELISA. RESULTS: Urocortin treatment induced a significant and dose-dependent increase of IL-4 and IL-10, whereas it did not affect TNF-alpha secretion. When incubated in the presence of LPS, Ucn reversed LPS-induced TNF-alpha release from cultured trophoblast cells, an effect that was blocked by the CRH-R2 selective antagonist, astressin 2b. CONCLUSION: Urocortin stimulates IL-4 and IL-10 secretion and reverses LPS-induced TNF-alpha release from trophoblast cells through action on CRH-R2 receptors, suggesting that this peptide may play a possible role as an anti-inflammatory agent.

Amniotic fluid urocortin, CRF, oestriol, dehydroepiandrosterone sulfate and cortisol concentrations at mid-trimester: putative relationship with preterm delivery.

OBJECTIVE: Stress-related peptide and steroid hormones are involved in the pathogenesis of preterm delivery, even though their clinical usefulness as predictive markers of preterm delivery remains unclear. The present study evaluated whether mid-trimester amniotic fluid concentrations of stress-related peptides, that is corticothophin-releasing factor (CRF) and urocortin (Ucn) and feto-placental steroids (oestriol, DHEA-S and cortisol) correlated with preterm delivery. STUDY DESIGN: It is a retrospective case-control study. Healthy women (n=130) undergoing amniocentesis at mid-gestation for genetic indications, of whom 15 had a preterm delivery (cases) and 115 delivered at term (controls). CRF, urocortin, cortisol, DHEA-S and oestriol concentrations were measured by specific and sensitive immunoenzymatic assays. RESULTS: Amniotic fluid urocortin concentrations in the cases (0.50+/-0.07 ng/ml) (M+/-SD) were significantly lower (P<0.0001) than in the control group (0.90+/-0.26 ng/ml), while CRF concentrations did not differ between the cases (1.52+/-0.39 ng/ml) and control group (1.64+/-0.68 ng/ml). Amniotic fluid cortisol (17.71+/-3.72 ng/ml vs. 17.32+/-3.17 ng/ml), DHEA-S (0.16+/-0.06 ng/ml vs. 0.17+/-0.09 ng/ml) and oestriol (4.68+/-1.95 ng/ml vs. 4.79+/-1.84 ng/ml) concentrations were similar in the two groups. CONCLUSIONS: The low amniotic fluid concentrations of urocortin at mid-trimester may be a signal of predisposition to preterm delivery, while the unchanged CRF and steroid hormones concentrations in women delivering preterm suggest that this mechanisms are not yet activated at mid-trimester.

Labor (term and preterm) is associated with changes in the placental mRNA expression of corticotrophin-releasing factor.

Because maternal plasma corticotrophin-releasing factor (CRF) levels increase during the last weeks of pregnancy and at parturition, the present study evaluated whether placental mRNA expression of CRF and CRF-binding protein (CRF-BP) are modified in preterm delivery. A group of 30 women with singleton pregnancies were enrolled in the study. A placental tissue specimen was collected from pregnant women (1) at term after cesarean delivery (39.3 +/- 0.1 gestational weeks; n = 10), (2) at term after spontaneous vaginal delivery (40.1 +/- 0.2 gestational weeks; n = 10), or (3) at preterm delivery (32.4 +/- 0.4 gestational weeks; n = 10). Changes of placental mRNA expression were evaluated by real-time quantitative reverse transcriptase polymerase chain reaction analysis. Placental CRF mRNA expression at term (P < .001) and preterm delivery (P < .001) was significantly higher than in cesarean delivery and highest in preterm placentas. With respect to CRF-BP, no significant difference in placental mRNA expression was observed among samples collected after term or preterm delivery and cesarean delivery. The present study showed for the first time that both term and preterm labor are associated with increased expression of placental CRF but not CRF-BP mRNA.

High maternal and fetal plasma urocortin levels in pregnancies complicated by hypertension.

OBJECTIVE: We evaluated maternal and fetal plasma levels and placental mRNA expression of urocortin, a placental vasoactive neuropeptide, in singleton pregnancies (n = 70) complicated by hypertensive disorders classified as gestational hypertension (n = 36), pre-eclampsia (n = 19), and pre-eclampsia complicated by intrauterine growth restriction (PE/IUGR, n = 15), and in 70 healthy normotensive singleton pregnancies. METHODS: Plasma levels were assayed by radioimmunoassay, fetal biometry by ultrasound scans, utero-placental and fetal perfusion by Doppler velocimetry, and placental urocortin mRNA expression by quantitative real time reverse transcriptase-polymerase chain reaction. The main outcome measures were the correlation of urocortin concentrations with patterns of the utero-placental and fetal circulation, and the early prediction of a poor neonatal outcome such as the occurrence of perinatal death and intraventricular hemorrhage. RESULTS: Maternal and fetal urocortin levels were significantly (both P < 0.001) higher in gestational hypertension, pre-eclampsia and PE/IUGR women than in controls, and correlated with Doppler velocimetry patterns. Fetal concentrations were significantly (P < 0.0001) higher than and significantly (P < 0.0001) correlated to maternal levels. Placental mRNA expression did not change. Ten out of 140 newborns had a poor neonatal outcome, with an overall prevalence of 7.14% (pretest probability). Using the receiver operator characteristics curve analysis cut-off values, the probability of a poor neonatal outcome was 66.7% when urocortin was used, and was 0% if levels were unaltered. CONCLUSIONS: Maternal and fetal urocortin levels are increased in hypertensive disorders of pregnancy. Since urocortin has vasoactive properties, the evidence of increased urocortin levels in hypertensive disorders may represent an adaptive fetal response.

Maternal plasma corticotrophin-releasing factor and urocortin levels in post-term pregnancies.

OBJECTIVE: Corticotrophin-releasing factor (CRF) and urocortin are two placental neuropeptides that are involved in the mechanisms of labour by modulating myometrial activity. Maternal plasma levels of both CRF and urocortin are increased at term and preterm labour, whilst those of CRF are reduced in women who are destined to experience post-term delivery. The present study evaluated maternal plasma levels in term and post-term pregnancies out of labour. DESIGN: A group of healthy pregnant women was enrolled and subdivided as follows: (i) at term out of labour (n = 19; 276 +/- 0.7 days of gestation; samples collected at the time of elective caesarean section due to previous uterine surgery); (ii) post-term (n = 19; 291 +/- 1.4 days of gestation), from whom samples were collected before induction of labour. METHODS: Urocortin and CRF measurements by radioimmunoassay; digital palpatory cervical examination and Bishop score computation; cervical length and funnelling presence assessment by transvaginal ultrasonography. RESULTS: Maternal plasma CRF concentrations were significantly (P < 0.05) lower whilst those of urocortin were unchanged in post-term compared with term pregnancy. However, CRF and urocortin levels were both significantly (P < 0.05 and P < 0.001 respectively) higher in pregnancies delivered within 12 h of labour induction than in those that remained undelivered, and were significantly correlated with the induction-delivery interval (CRF: r = -0.676, P = 0.0015; urocortin: r = -0.783, P < 0.0001). CONCLUSIONS: CRF and urocortin levels are decreased and unchanged, respectively, in post-term pregnancy when compared with term pregnancy. Both CRF and urocortin correlate with the time of labour onset after induction. Since CRF derives from the placenta, and urocortin from the fetus, the concerted expression of these neuropeptides appears to be relevant in determining the length of human gestation.