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Fetal endoscopic tracheal occlusion (FETO) is an emerging surgical therapy for congenital diaphragmatic hernia (CDH). Ovine and rabbit data suggested altered lung epithelial cell populations after TO with transcriptomic signatures implicating basal cells. To test this hypothesis, we deconvolved mRNA-seq data and used quantitative image analysis in fetal rabbit lungs to showed increased basal cells and reduced ciliated cells after TO. In a fetal mouse TO model, flow cytometry showed increased basal cells, and immunohistochemistry demonstrated basal cell extension to the subpleura. Nuclear yap, a known regulator of basal cell fate, was increased in TO lung, and Yap ablation on the lung epithelium abrogated TO-mediated basal cell expansion. mRNA-seq of TO lung showed increased activity of downstream Yap genes. Human lung specimens with congenital and fetal endoscopic tracheal occlusion had clusters of subpleural basal cell that were not present in control. TO increases lung epithelial cell nuclear Yap leading to basal cell expansion.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Despite benefits of prenatal in utero repair of myelomeningocele, a severe type of spina bifida aperta, many of these patients will still suffer mild to severe impairment. One potential source of stem cells for new regenerative medicine-based therapeutic approaches for spinal cord injury repair is neural progenitor cells (NPCs) in cerebrospinal fluid (CSF). To this aim, we extracted CSF from the cyst surrounding the exposed neural placode during the surgical repair of myelomeningocele in 6 fetuses (20 to 26 weeks of gestation). In primary cultured CSF-derived cells, neurogenic properties were confirmed by in vitro differentiation into various neural lineage cell types, and NPC markers expression (TBR2, CD15, SOX2) were detected by immunofluorescence and RT-PCR analysis. Differentiation into three neural lineages was corroborated by arbitrary differentiation (depletion of growths factors) or explicit differentiation as neuronal, astrocyte, or oligodendrocyte cell types using specific induction mediums. Differentiated cells showed the specific expression of neural differentiation markers (βIII-tubulin, GFAP, CNPase, oligo-O1). In myelomeningocele patients, CSF-derived cells could become a potential source of NPCs with neurogenic capacity. Our findings support the development of innovative stem-cell-based therapeutics by autologous transplantation of CSF-derived NPCs in damaged spinal cords, such as myelomeningocele, thus promoting neural tissue regeneration in fetuses.

Background Fetal tracheal occlusion (TO) is an experimental therapeutic approach to stimulate lung growth in the most severe congenital diaphragmatic hernia (CDH) cases. We have previously demonstrated a heterogeneous response of normal fetal rabbit lungs after TO with the appearance of at least two distinct zones. The aim of this study was to examine the fetal lung response after TO in a left CDH fetal rabbit model. Methods Fetal rabbits at 25 d gestation underwent surgical creation of CDH followed by TO at 27 d and harvest on day 30. Morphometric analysis, global metabolomics, and fluorescence lifetime imaging microscopy (FLIM) were performed to evaluate structural and metabolic changes in control, CDH, and CDH + TO lungs. Results Right and left lungs were different at the baseline and had a heterogeneous pulmonary growth response in CDH and after TO. The relative percent growth of the right lungs in CDH + TO was higher than the left lungs. Morphometric analyses revealed heterogeneous tissue-to-airspace ratios, in addition to size and number of airspaces within and between the lungs in the different groups. Global metabolomics demonstrated a slower rate of metabolism in the CDH group with the left lungs being less metabolically active. TO stimulated metabolic activity in both lungs to different degrees. FLIM analysis demonstrated local heterogeneity in glycolysis, oxidative phosphorylation (OXPHOS), and FLIM “lipid-surfactant” signal within and between the right and left lungs in all groups. Conclusions We demonstrate that TO leads to a heterogeneous morphologic and metabolic response within and between the right and left lungs in a left CDH rabbit model.

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Development of brain edema in acute liver failure can increase intracranial pressure, which is a severe complication of the disease. However, brain edema is neither entirely cytotoxic nor vasogenic and the specific action of the brain microvasculature is still unknown. We aimed to analyze gene expression of brain cortex microvessels in two rat models of acute liver failure. In order to identify global gene expression changes we performed a broad transcriptomic approach in isolated brain cortex microvessels from portacaval shunted rats after hepatic artery ligation (HAL), hepatectomy (HEP), or sham by array hybridization and confirmed changes in selected genes by RT-PCR. We found 157 and 270 up-regulated genes and 143 and 149 down-regulated genes in HAL and HEP rats respectively. Western blot and immunohistochemical assays were performed in cortex and ELISA assays to quantify prostaglandin E metabolites were performed in blood of the sagittal superior sinus. We Identified clusters of differentially expressed genes involving inflammatory response, transporters-channels, and homeostasis. Up-regulated genes at the transcriptional level were associated with vasodilation (prostaglandin-E synthetase, prostaglandin-E receptor, adrenomedullin, bradykinin receptor, adenosine transporter), oxidative stress (hemoxygenase, superoxide dismutase), energy metabolism (lactate transporter) and inflammation (haptoglobin). The only down-regulated tight junction protein was occludin but slightly. Prostaglandins levels were increased in cerebral blood with progression of liver failure. In conclusion, in acute liver failure, up-regulation of several genes at the level of microvessels might suggest an involvement of energy metabolism accompanied by cerebral vasodilation in the cerebral edema at early stages.

Congenital diaphragmatic hernia (CDH) causes severe pulmonary hypoplasia from herniation of abdominal contents into the thorax. Tracheal occlusion (TO) for human CDH improves survival, but morbidity and mortality remain high, and we do not fully understand the cellular pathways and processes most severely impacted by CDH and TO. We created a left diaphragmatic hernia (DH) in rabbit fetuses with subsequent TO and collected left lung sections for NextGen mRNA sequencing. DH, TO and DHTO fetuses had comparable body and organ growth to control except for lower lung weights in DH (p < 0.05). Of 13,687 expressed genes, DHTO had 687 differentially expressed genes compared with DH, but no other group-group comparison had more than 10. Considering genes in combination, many of the genes reduced in DH were more highly expressed in DHTO than in control. Benchmarking fetal rabbit lung gene expression to published lung development data, both DH and DHTO lungs were more highly correlated with the gene expression of immature lung. DNA synthesis was upregulated in DHTO compared with DH and ribosome and protein synthesis pathways were downregulated. DH reduced total and epithelial cell proliferation by half and two-thirds respectively, and DHTO increased proliferation by 2.5 and 3.4-fold respectively. Signaling pathways downregulated by DH and upregulated in DHTO were epidermal growth factor receptor signaling, ephrin signaling and cell migration; however, levels of ephrin and EGFR signaling in DHTO exceeded that of control. Identification and inhibition of the ligands responsible for this dysregulated signaling could improve lung development in CDH.

Amniotic fluid; Neonatal mortality; Exencephaly Líquido amniótico; Mortalidad neonatal; Exencefalia Líquid amniòtic; Mortalitat neonatal; Exencefàlia 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. This work was supported by Prof. Jose L. Peiro internal Cincinnati Children's Hospital funding.

Congenital diaphragmatic hernia (CDH) occurs in similar to 1:2,000 pregnancies and is associated with substantial morbidity and mortality. Fetal tracheal occlusion (TO) is an emerging therapy that improves lung growth and reduces mortality, although substantial respiratory compromise persists in survivors. In this study, we used tracheal fluid in a fetal sheep model of CDH with TO for proteomic analysis with subsequent validation of findings in sheep lung tissue. We found that the proteomic profiles of CDH tracheal fluid was most similar to control lung and CDH/TO lung most similar to TO lung. Among 118 proteins altered in CDH, only 11 were reciprocally regulated in CDH/TO. The most significantly altered pathways and processes were cell proliferation, phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling, inflammation, and microtubule dynamics. CDH suppressed and TO promoted cell proliferation and AKT-related signaling cascades. By Western blot analysis and immunohistochemistry, epithelial PCNA and phosphorylated AKT were decreased in CDH and increased in TO and CDH/TO lungs. The Wnt target Axin2 was decreased threefold in CDH lung compared with control without a significant increase in CDH/TO lung. Cilia-related pathways were among the most dysregulated with CDH lung having a nearly twofold increase in acetylated alpha-tubulin and a relative increase in the number of ciliated cells. While TO improves lung growth and patient survival in CDH, the procedure substantially alters many processes important in lung development and cell differentiation. Further elucidation of these changes will be critical to improving lung health in infants with CDH treated with TO., National Heart, Lung, and Blood Institute; Spanish Health Institute Carlos III Economy and Development Ministry; Parker B. Francis Fellowship Award; Cincinnati Children’s Hospital Research Foundation Procter Award

Spina bifida aperta is a congenital malformation characterized by the failure of neural tube closure resulting in an unprotected fetal spinal cord. The spinal cord then undergoes progressive damage, likely due to chemical and mechanical factors related to exposure to the intrauterine environment. Astrogliosis in exposed spinal cords has been described in animal models of spina bifida during embryonic life but its relationship with neuroinflammatory processes are completely unknown. Using a retinoic acid-induced rat model of spina bifida we demonstrated that, when exposed to amniotic fluid, fetal spinal cords showed progressive astrogliosis with neuronal loss at mid-gestation (E15) compared to unexposed spinal cords. The number of microglial cells with a reactive phenotype and activation marker expression increased during gestation and exhibited progressive disruption in the inhibitory immune ligand-receptor system. Specifically we demonstrate down-regulation of CD200 expression and up-regulation of CD200R. Exposed spinal cords demonstrated neuroinflammation with increased tissue water content and cytokine production by the end of gestation (E20), which correlated with active Caspase3 expression in the exposed layers. Our findings provide new evidence that microglia activation, including the disruption of the endogenous inhibitory system (CD200-CD200R), may participate in the pathogenesis of spina bifida through late gestation.

Background Fetal tracheal occlusion (TO) is an emerging surgical therapy in congenital diaphragmatic hernia that improves the fetal lung growth. Different animal models of congenital diaphragmatic hernia and TO present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and available genetic tools. We developed a minimally invasive murine transuterine TO model, which will be useful in defining how TO impacts lung molecular biology, cellular processes, and overall lung physiology. Materials and methods Time-mated C57BL/6 mice underwent laparotomy at embryonic day 16.5 (E16.5) with transuterine TO performed on two fetuses in each uterine horn. At E18.5, dams were sacrificed and fetuses harvested. The lungs of the TO fetuses were compared with the nonmanipulated counterparts by morphometric and histologic analysis. Results Successful TO was confirmed in 16 of 20 TO fetuses. Twelve of them survived to E18.5 (75%). Fetal weights were comparable, but lung weights were significantly greater in TO (28.41 ± 5.87 versus 23.38 ± 3.09, P = 0.043). Lung to body weight ratio was also greater (0.26 ± 0.003 versus 0.22 ± 0.002, P = 0.006). E18.5 TO lungs demonstrated dilated central and distal airspaces with increased cellularity. DNA/protein and DNA/lung weight ratios were elevated while protein/lung weight ratio was lower in TO compared to control. Conclusions Mice fetal transuterine TO is feasible with comparable outcomes to other current animal models. The increase in the lung weight, lung to body weight ratio and the DNA/protein ratio indicate organized lung growth rather than edema or cell hypertrophy.

Hepatic encephalopathy has traditionally been considered a reversible disorder. However, recent studies suggested that repeated episodes of hepatic encephalopathy cause persistent impairment leading to neuronal loss. The aims of our study were the development of a new animal model that reproduces the course of episodic hepatic encephalopathy and the identification of neurodegeneration evidences. Rats with portacaval anastomosis underwent simulated episodes of hepatic encephalopathy, triggered by the regular administration of ammonium acetate, and/or lipopolysaccharide. The neurological status was assessed and neuronal loss stereologically quantified in motor areas. During the simulated episodes, ammonia induced reversible motor impairment in portacaval anastomosis rats. In cerebellum, stereology showed a reduction in Purkinje cell population in portacaval anastomosis and PCA+NH3 groups and morphological changes. An increase in astrocyte size in PCA+NH3 group and activated microglia in groups treated with ammonium acetate and/or lipopolysaccharide was observed. A modulation of neurodegeneration-related genes and the presence of apoptosis in Bergmann glia were observed. This new animal model reproduces the clinical course of episodic hepatic encephalopathy when ammonia is the precipitant factor and demonstrates the existence of neuronal loss in cerebellum. The persistence of over-activated microglia and reactive astrocytes could participate in the apoptosis of Bergmann glia and therefore Purkinje cell degeneration.

9 páginas, 4 figuras.-- et al., Human embryonic stem cells (hESC) hold great promise for the treatment of patients with many neurodegenerative diseases particularly those arising from cell loss or neural dysfunction including spinal cord injury. This study evaluates the therapeutic effects of transplanted hESC-derived oligodendrocyte progenitors (OPC) and/or motoneuron progenitors (MP) on axonal remyelination and functional recovery of adult rats after complete spinal cord transection. OPC and/or MP were grafted into the site of injury in the acute phase. Based on Basso-Beattie-Bresnahan scores recovery of locomotor function was significantly enhanced in rats treated with OPC and/or MP when compared with control animals. When transplanted into the spinal cord immediately after complete transection, OPC and MP survived, migrated, and differentiated into mature oligodendrocytes and neurons showing in vivo electrophysiological activity. Taken together, these results indicate that OPC and MP derived from hESC could be a useful therapeutic strategy to repair injured spinal cord., This work was supported by funds for research in the field of Regenerative Medicine from the Regional Government Health Department (Generalitat Valenciana), the Instituto Carlos III belonging to the Spanish Ministry of Health and Consumer Affairs, and through the grants from the Spanish Ministry of Science and Innovation (SAF2007-63193) and La Marato (TV3 070,330).

Background & Aim Acute-on-chronic liver failure is the term that refers to sustained liver injury with acute decompensation, usually induced by a precipitating factor. A common link between ensuing failures of various organs is impairment of the vascular supply, which may also induce vasogenic oedema in the brain. The aim of this study was to perform magnetic resonance (MR) study of the brain in a rat model combining bile duct ligation (BDL) and lipopolysaccharide (LPS) administration to investigate brain oedema in liver failure. Methods Bile duct-ligated rats underwent in vivo brain MR imaging at 4, 5 and 6 weeks, and after superimposed administration of LPS. The MR techniques applied enabled assessment of brain metabolites, and intra- or extracellular water distribution. Brain water content was assessed by gravimetry. Results MR spectroscopy showed an increase in brain glutamine and a decrease in myo-inositol and choline in relation to progression of liver disease. BDL rats showed a slight, progressive increase in the amount of cortical brain water that was significant after LPS injection. These changes did not modify the apparent diffusion coefficient, supporting a mixed origin of brain oedema (vasogenic and cytotoxic). Conclusions The mechanisms leading to the development of brain oedema in an experimental liver disease model were related to the time course of liver failure and to pro-inflammatory stimuli. MR findings support the presence of cytotoxic and vasogenic mechanisms in induced brain oedema in BDL rats exposed to LPS.

Background & Aims Brain edema is a severe complication of acute liver failure (ALF) that has been related to ammonia concentrations. Two mechanisms have been proposed in the pathogenesis: vasogenic edema that is secondary to the breakdown of the blood–brain barrier and cytotoxic edema caused by ammonia metabolites in astrocytes. Methods We applied magnetic resonance techniques to assess the intracellular or extracellular distribution of brain water and metabolites in a rat model of devascularized ALF. The brain water content was assessed by gravimetry and blood–brain barrier permeability was determined from the transfer constant of 14C-labeled sucrose. Results Rats with ALF had a progressive decrease in the apparent diffusion coefficient (ADC) in all brain regions. The average decrease in ADC was significant in precoma (−14%) and coma stages (−20%). These changes, which indicate an increase of the intracellular water compartment, were followed by a significant increase in total brain water (coma 82.4% ± 0.3% vs sham 81.6% ± 0.3%; P = .0001). Brain concentrations of glutamine (6 hours, 540%; precoma, 851%; coma, 1086%) and lactate (6 hours, 166%; precoma, 998%; coma, 3293%) showed a marked increase in ALF that paralleled the decrease in ADC and neurologic outcome. In contrast, the transfer constant of 14C-sucrose was unaltered. Conclusions The pathogenesis of brain edema in an experimental model of ALF involves a cytotoxic mechanism: the metabolism of ammonia in astrocytes induces an increase of glutamine and lactate that appears to mediate cellular swelling. Therapeutic measures should focus on removing ammonia and improving brain energy metabolism.

Liver Failure Group, UCL Institute for Liver and Digestive Health, The Royal Free Hospital, Pond Street, London NW3 2PF, United KingdomSee Article, pages 554–560Pathogenesis of intracranial hypertension in acute liver failure(ALF) has been a subject of intense investigation and debate overa number of years. Hyperammonemia, systemic and braininflammation, and alterations in cerebral blood flow have all beensuggested as interrelated pathogenic mechanisms [1]. Theprevailing hypothesis is that increased brain ammonia resultsin cytotoxic brain edema due to an increase in astrocytic gluta-mine, which acts as an osmolyte producing accumulation ofwater in the cell [2]. Cerebral hyperlactatemia has been sug-gested to be associated with increased ICP in patients with ALF.In fact, close correlation between increased brain lactate andincreased ICP was shown in humans with ALF [3]. The pathophys-iological mechanism of this increase in brain lactate is not fullyunderstood but has been suggested to occur due to relative cere-bral hypoperfusion, reduced cerebral oxygenation and an ammo-nia induced inhibition of a key enzyme in the tricarboxylic acidcycle, alpha-ketoglutarate dehydrogenase driving anaerobicmetabolism [4,5]. Arguments countering this contention are theobservations that cerebral blood flow is indeed increased in latestages of ALF, which correlates with the severity of intracranialhypertension, a maintained lactate/pyruvate ratio, undisturbedcerebral alpha-ketoglutarate dehydrogenase in ALF animals andno significant differences in brain mitochondrial complex activi-ties [6,7]. Taken together, the studies favour the hypothesis thatthe increase in brain lactate is a consequence of pathophysiologicmechanisms rather than its cause and therefore a marker ratherthan a therapeutic target. The study by Bosoi et al. questions thishypothesis [8]. Data about brain lactate metabolism in chronicliver disease is scarce and therefore the paper in the present issueof the Journal adds further to the literature.The authors of the study interpret their data to suggest thatbrain lactate and not brain glutamine acts as a pivotal osmolytein the pathogenesis of brain edema in a chronic liver failureanimal model and therefore is a potential target for therapy.The study was performed in 6-weeks bile duct ligated rats(BDL) rats. BDL rats demonstrated a significant increase in brainlactate (p