Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord

Fetal lung underdevelopment is rescued by administration of amniotic fluid stem cell extracellular vesicles in rodents

Fetal lung underdevelopment, also known as pulmonary hypoplasia, is characterized by decreased lung growth and maturation. The most common birth defect found in babies with pulmonary hypoplasia is congenital diaphragmatic hernia (CDH). Despite research and clinical advances, CDH babies still have high morbidity and mortality rates, which are directly related to the severity of lung underdevelopment. To date, there is no effective treatment that promotes fetal lung growth and maturation. Herein, we describe a stem cell-based approach that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). Using fetal rodent models of pulmonary hypoplasia (primary epithelial cells, organoids, explants, and in vivo), we demonstrated that AFSC-EV administration promotes branching morphogenesis and alveolarization, rescues tissue homeostasis, and stimulates epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in human models of lung injury, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action by tracking AFSC-EV cargo transfer, profiling EV protein and RNA content, and sequencing target lung epithelial cells, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. Particularly, miRNAs that regulate the expression of genes involved in lung development, such as the miR17~92 cluster and its paralogues, were highly enriched in AFSCEVs and were increased in AFSC-EV-treated primary lung epithelial cells compared to untreated cells. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application in patients with pulmonary hypoplasia

Evaluation of different screening models for optimal pulmonary transduction with lentiviral vectors

status: publishe

The female condom as a temporary silo: A simple and inexpensive tool in the initial management of the newborn with gastroschisis

Objective. the aim of this study is to report the use of a female condom as a non-surgical silon pouch in the early management of newborns with gastroschisis with large visceroabdominal disproportion. Methods. Pre-washed, sterile female condoms without spermicide were used as an early approach to treat gastroschisis in 20 newborns with large defects and in whom staged correction was anticipated. the condom was placed in the neonatal intensive care unit using sterile technique, with no anesthesia, and it was removed only at the time of the surgical procedure for gastroschisis correction. Results. There were no complications associated with the use of a female condom as a temporary silo for gastroschisis. It protected the exposed organs and also allowed a careful evaluation of the bowel and a better pre-operative planning without the need for emergency procedures. Conclusion. the use of a female condom as a silon pouch is a low-cost and simple alternative in the initial management of newborns with gastroschisis in whom primary correction is considered non-feasible.Univ Estadual Campinas, UNICAMP, Div Pediat Surg, Dept Surg,Sch Med Sci, Campinas, SP, BrazilUniv Estadual Campinas, UNICAMP, Div Neonatol, Dept Pediat,Sch Med Sci, Campinas, SP, BrazilWeb of Scienc

Prenatal retinoic acid improves lung vascularization and VEGF expression in CDH rat

OBJECTIVE: We sought to investigate the effects of antenatal retinoic acid on the pulmonary vasculature and vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) expression in a nitrofen-induced congenital diaphragmatic hernia (CDH) model. STUDY DESIGN: Rat fetuses were exposed to nitrofen at gestational day 9.5 and/or all-trans retinoic acid (ATRA) at gestational days 18.5-20.5. We assessed lung growth, airway, and vascular morphometry. VEGF, VEGFR1, and VEGFR2 expression was analyzed by Western blotting and immunohistochemistry. Continuous data were analyzed by analysis of variance and Kruskal-Wallis test. RESULTS: CDH decreased lung to body weight ratio, increased mean linear intercept and mean transection length/airspace, and decreased mean airspace cord length. ATRA did not affect lung growth or morphometry. CDH increased proportional medial wall thickness of arterioles while ATRA reduced it. ATRA recovered expression of VEGF and receptors, which were reduced in CDH. CONCLUSION: Retinoic acid and VEGF may provide pathways for preventing pulmonary hypertension in CDH

Treatment of bowel in experimental gastroschisis with a nitric oxide donor

To reduce the harmful effect of bowel exposure to amniotic fluid in gastroschisis, we used the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) in an animal model of gastroschisis and assessed the ideal concentration for treatment of changes in bowel. Gastroschisis was surgically induced in rat fetuses on day 18.5 of gestation. The fetuses were divided into 5 groups (n = 12 animals/group): control (C), gastroschisis (G), gastroschisis + GSNO 5 mu mol/L (GNO1), gastroschisis + GSNO 0.5 mu mol/L (GNO2), and gastroschisis + GSNO 0.05 mu mol/L (GNO3). On day 21.5 of gestation, fetuses were collected by cesarean delivery. Body and intestinal weight were measured and the bowels were either fixed for histometric and immunohistochemical study or frozen for Western blotting. We analyzed bowel morphometry on histological sections and expression of the NO synthase (NOS) enzymes by Western blotting and immunohistochemistry. Data were analyzed by analysis of variance or Kruskal-Wallis test when appropriate. Morphological and histometric measurements of weight, diameter, and thickness of the layers of the intestinal wall decreased with GSNO treatment, especially in the GNO3 group, when compared with the G group (P .05). Fetal treatment with 0.05 mmol/L GSNO resulted in significant improvement of bowel morphology in gastroschisis2123FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP08/51487-9; 11/00794-1; 08/52772-9; 11/12587-0Higher Education Consortia Program, Brazilian Ministry of Educatio

Hydrogel protection: a novel approach to reduce bowel inflammation in experimental gastroschisis

Objective: In gastroschisis there is herniation of the fetal bowel into the amniotic cavity that results in severe intestinal dysfunction. In order to reduce bowel exposure to amniotic fluid we used a hydrogel of N-isopropylacrylamide copolymerized with acrylic acid (P(NIPAAm-co-AAc)) to coat the herniated bowel through the use of a fibrin adhesive (Beriplast(R)). Study design: Gastroschisis was created in fetuses of 31 pregnant Sprague-Dawley rats by evisceration of the bowel through a right paramedian incision in the abdominal wall on day 18.5 of pregnancy. The fetuses were separated in four groups of 12 fetuses: control (C), gastroschisis (G), gastroschisis + fibrin adhesive (GA) and gastroschisis + fibrin adhesive + dry hydrogel (GAH). Animals were harvested at day 21.5 of pregnancy and the hydrogel was removed. Fetuses and bowels were weighed and morphometric analysis was performed. Isoelectric focusing of the amniotic fluid determined its electrical charge. We evaluated the hydrogel swelling ratio (Q) in the amniotic fluid. Histological analysis and scanning electronic microscopy (SEM) of the bowel and hydrogel were performed. Our primary outcome was bowel intactness after hydrogel removal and our secondary outcome was the effectiveness of the hydrogel in protecting the bowel against amniotic fluid and its components. Differences among the groups were tested by the ANOVA and Tukey-Kramer post-test method and the statistical significance accepted was for p values <0.05. Results: The mass of swollen hydrogel was 34 times the mass of dry hydrogel. Isoelectric focusing of the amniotic fluid showed that most of its proteins are negatively charged as the hydrogel. SEM showed that removal of the hydrogel did not damage bowel serosa. Bowel weight, diameter and wall thickness were similar between groups C and GAH but bowel diameter and wall thickness was significantly reduced in C and GAH compared to G and GA (p < 0.001). Conclusion: The P(NIPAAm-co-AAc) hydrogel does not harm the bowel and provides a safe effective protection with reduction of bowel damage in gastroschisis. (C) 2009 Elsevier Ireland Ltd. All rights reserved

The Ideal Timing for Experimental Cleft Lip Creation

Cleft lip and palate (CLP) is the most common congenital defect of the face. Many animal models have been utilized to study embryogenesis and pathogenesis of CLP, including the development of secondary anomalies and consequent deformities. However, the ideal gestational age for surgical creation of lip or palate defects in rat models has never been determined. The aim of the present study is to improve the experimental model utilizing rat fetuses, defining the most appropriate timing for creation of the lip defect model. The study was composed of three groups of fetuses undergoing surgical creation of a lip defect at the left side of the superior lip at 17.5, 18.5, and 19.5 days of gestation. Fetuses were harvested at 21.5 days of gestation (term = 22 days) and underwent macroscopic and microscopic analyses. We found that the most appropriate moment for lip defect creation was at 19.5 days, given the presence of lip depression at the site of the defect and asymmetry and retraction associated with interruption of the lip and complete reepithelialization of the borders of the defect.Sao Paulo Research Foundation - FAPESP[08/50347-9

Prenatal retinoic acid improves lung vascularization and VEGF expression in CDH rat

OBJECTIVE: We sought to investigate the effects of antenatal retinoic acid on the pulmonary vasculature and vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) expression in a nitrofen-induced congenital diaphragmatic hernia (CDH) model. STUDY DESIGN: Rat fetuses were exposed to nitrofen at gestational day 9.5 and/or all-trans retinoic acid (ATRA) at gestational days 18.5-20.5. We assessed lung growth, airway, and vascular morphometry. VEGF, VEGFR1, and VEGFR2 expression was analyzed by Western blotting and immunohistochemistry. Continuous data were analyzed by analysis of variance and Kruskal-Wallis test. RESULTS: CDH decreased lung to body weight ratio, increased mean linear intercept and mean transection length/airspace, and decreased mean airspace cord length. ATRA did not affect lung growth or morphometry. CDH increased proportional medial wall thickness of arterioles while ATRA reduced it. ATRA recovered expression of VEGF and receptors, which were reduced in CDH. CONCLUSION: Retinoic acid and VEGF may provide pathways for preventing pulmonary hypertension in CDH.Sao Paulo Research FoundationSao Paulo Research Foundation [08/50347-9, 11/00794-1, 08/52772-9, 11/12587-0