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PURPOSE: The use of materials to facilitate dural closure during spina bifida (SB) repair has been a highly studied aspect of the surgical procedure. The overall objective of this review is to present key findings pertaining to the success of the materials used in clinical and pre-clinical studies. Additionally, this review aims to aid fetal surgeons as they prepare for open or fetoscopic prenatal SB repairs. METHODS: Relevant publications centered on dural substitutes used during SB repair were identified. Important information from each article was extracted including year of publication, material class and sub-class, animal model used in pre-clinical studies, whether the repair was conducted pre-or postnatally, the bioactive agent delivered, and key findings from the study. RESULTS: Out of 1,121 publications, 71 were selected for full review. We identified the investigation of 33 different patches where 20 and 63 publications studied synthetic and natural materials, respectively. From this library, 43.6% focused on clinical results, 36.6% focused on pre-clinical results, and 19.8% focused on tissue engineering approaches. Overall, the use of patches, irrespective of material, have shown to successfully protect the spinal cord and most have shown promising survival and neurological outcomes. CONCLUSION: While most have shown significant promise as a therapeutic strategy in both clinical and pre-clinical studies, none of the patches developed so far are deemed perfect for SB repair. Therefore, there is an opportunity to develop new materials and strategies that aim to overcome these challenges and further improve the outcomes of SB patients.

To date, fetal liver implication is not a well-understood phenomenon in congenital diaphragmatic hernia (CDH). We evaluated the fetal morphologic changes on liver growth after surgical procedure in CDH experimental model. A diaphragmatic defect at gestational day E25 and tracheal occlusion (TO) at E27 were surgically created in rabbit fetuses. Five experimental groups were assessed: control group, left CDH, right CDH, CDH + TO, and TO alone. Body and organ growth were measured. For histological evaluation of the CDH effect, liver sections were collected. Left-CDH group had livers with increased leukocyte infiltration in comparison with controls (p = 0.02). Increased capillary sinusoid congestion and hepatocyte vacuolation were greater in left-CDH compared with the right-CDH group (p = 0.05). Capillary sinusoid congestion and interstitial edema were more evident in the left-CDH compared with CDH + TO group (p = 0.05). Increases in sinusoid congestion, hepatocyte vacuolation, and interstitial edema were also greater in the CDH + TO compared with controls (p ≤ 0.02). Intrathoracic liver weight was higher in right-CDH compared with left-CDH group (p 0.001). Total lung weights (TLW) were significantly lower in both left-CDH compared with controls (p 0.001), CDH + TO (p = 0.01), and TO (p 0.01) and in right-CDH compared with CDH + TO (p 0.01) and TO (p 0.01). Decreased kidney and heart weights were also recorded. Hemodynamics and structural fetal liver changes in laterality were noted in CDH model. Regulation of intrathoracic liver weights seems to be disturbed by the absence of diaphragmic contact. Pulmonary injury is supported by the effect of a first hit, while the growth of internal organs suggests a multisystemic remodeling related to the fetal adaptation.

Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p

During embryonic spinal cord development, neural progenitor cells (NPCs) generate three major cell lines: neurons, oligodendrocytes, and astrocytes at precise times and locations within the spinal cord. Recent studies demonstrate early astrogenesis in animal models of spina bifida, which may play a role in neuronal dysfunction associated with this condition. However, to date, the pathophysiological mechanisms related to this early astrocytic response in spina bifida are poorly understood. This study aimed to characterize the development of early astrogliosis over time from Pax6+, Olig2+, or Nkx2.2+ NPCs using a retinoic acid-induced spina bifida rat model. At three gestational ages (E15, E17, and E20), spinal cords from fetuses with retinoic acid-induced spina bifida, their healthy sibling controls, or fetuses treated with the vehicle control were analyzed. Results indicated that premature astrogliosis and astrocytic activation were associated with an altered presence of Pax6+, Olig2+, and Nkx2.2+ NPCs in the lesion compared to the controls. Finally, this response correlated with an elevation in genes involved in the Notch-BMP signaling pathway. Taken together, changes in NPC patterning factor expression with Notch-BMP signaling upregulation may be responsible for the altered astrogenesis patterns observed in the spinal cord in a retinoic acid-induced spina bifida model.

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 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 tracheal occlusion (TO) with transcriptomic signatures implicating basal cells. To test this hypothesis, we deconvolved mRNA sequencing (mRNA-seq) data and used quantitative image analysis in fetal rabbit lung TO, which had 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 subpleural airways. 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 tracheal occlusion had clusters of subpleural basal cells that were not present in the control. TO increases lung epithelial cell nuclear Yap, leading to basal cell expansion.

Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development.Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed.DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT.This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH.There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

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.

Open spina bifida or myelomeningocele (MMC) is a devastating neurologic congenital defect characterized by primary failure of neural tube closure of the spinal column during the embryologic period. Cerebrospinal fluid leak caused by the MMC spinal defect in the developing fetus can result in a constellation of encephalic anomalies that include hindbrain herniation and hydrocephalus. The exposure of extruded spinal cord to amniotic fluid also poses a significant risk for inducing partial or complete paralysis of the body parts beneath the spinal aperture by progressive spinal cord damage in-utero. A randomized trial demonstrated that prenatal repair by fetal surgery, sometimes using patches, to cover the exposed spinal cord with a watertight barrier is effective in reducing the postnatal neurologic morbidity as evidenced by decreased incidence and severity of postnatal hydrocephalus and the reduced need for ventricular-peritoneal shunting. Currently, the use of inert or collagen-based patches are associated with high costs and inadequate structural properties. Specifically, the inert patches do not degrade after implantation, causing the need for a post-natal removal surgery associated with trauma for the newborn. Our present study is aimed towards in-vitro degradation studies of a newly designed patch, which potentially can serve as a superior alternative to existing patches for MMC repair. This novel patch was fabricated by blending poly(l-lactic acid) and poly(ε-caprolactone). The 16-week degradation study in amniotic fluid was focused on tracking changes in crystallinity and mechanical properties. An additional set of designed patches was exposed to phosphate-buffered saline (PBS), as a time-paired control. Crystallinity studies indicate the progress of hydrolytic degradation of the patch in both media, with a preference to bulk erosion in phosphate buffered saline and surface erosion in amniotic fluid. Mechanical testing results establish that patch integrity is not compromised up to 16 weeks of exposure either to body fluids analog (PBS) or to amniotic fluid.

Introduction: Thoracoscopic repair of congenital diaphragmatic hernia (CDH) has become a popular approach and several benefits have been published. Patch closure requires demanding thoracoscopic sk...

Congenital hydrocephalus (HCP) is a developmental brain disorder characterized by the abnormal accumulation of cerebrospinal fluid within the ventricles. It is caused by genetic and acquired factors that start during early embryogenesis with disruption of the neurogerminal areas. As might be expected, early-onset hydrocephalus alters the process of brain development leading to irreparable neurological deficit. A primary alteration of the ependyma/neural stem cells (affecting vesicle trafficking and abnormal cell junctions) leads to its loss or denudation and translocation of neural progenitor cells (NPCs) and neural stem cells (NSCs) into the cerebrospinal fluid (CSF). Under these abnormal conditions, morphological and functional processes, underlying the concept of astroglial reaction, are initiated in an attempt to recover homeostasis in the periventricular zone. This astroglial reaction includes astrocyte hypertrophy, hyperplasia, and development of a new layer with reorganized functional features that resemble the ependyma. Despite decades of research, there is a lack of information concerning the biological basis of the brain abnormalities that are associated with HCP. The present review of current literature discusses the neuropathological changes during gestation following the onset of congenital hydrocephalus and the unanswered questions into the pathophysiology of the disease. A better understanding of those missing points might help create novel therapeutic strategies that can reverse or even prevent the ultimate neurological impairment that affects this population and improve long-term clinical outcome.

Our study focuses on the development and characterization of a self-expanding, watertight and biodegradable patch for fetoscopic myelomeningocele (MMC) prenatal repair. We fabricated poly(l-lactic acid) (PLA) and poly(ɛ-caprolactone) (PCL) blend films by solution casting. Formulation c with average glass transition temperature of 37.6 ± 1.2°C was chosen for temporospatial recovery. Favorable results from surface studies reflected homogeneous dispersion of polymers in the blend. The cytotoxicity was studied in human foreskin fibroblasts. The blend film was cytocompatible, evidenced by matching percentage of live cells in exposed and control solutions. Subsequently, liquid water permeability experiments confirmed watertight nature of films. Finally, in vitro degradation was investigated in phosphate buffered saline (PBS) and amniotic fluid (AF) separately for 16 weeks. Similar weight loss (n = 6, p = 0.912) and significantly different (n = 3, p = 0.025) surface roughness was observed in PBS and AF, respectively, at 16 weeks. Functional group analysis displayed increasing carbonyl and hydroxyl bonds in PBS and AF, respectively, over time, indicating progression of hydrolytic degradation. Favorable characterization results provide strong evidence to employ PLA-PCL blend films as surgical patches in fetoscopic MMC repair. Designed patch serves as standalone system to successfully tackle impending hurdles of MMC repair and proves to be a superior alternative compared to existing patches. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 295-305, 2019.

A better understanding of the transcriptomic modifications that occur in spina bifida may lead to identify mechanisms involved in the progression of spina bifida in utero and the development of new therapeutic strategies that aid in spinal cord regeneration after surgical interventions. In this study, RNA-sequencing was used to identify differentially expressed genes in fetal spinal cords from rats with retinoic acid-induced spina bifida at E15, E17, and E20. Gene ontology, KEGG, and protein–protein interaction analysis were conducted to predict pathways involved in the evolution of the disease. Approximately 3000, 1000 and 300 genes were differentially expressed compared to the control groups at E15, E17 and E20, respectively. Overall, the results suggest common alterations in certain pathways between gestational time points, such as upregulation in p53 and sonic hedgehog signaling at E15 and E17 and downregulation in the myelin sheath at E17 and E20. However, there were other modifications specific to gestational time points, including skeletal muscle development at E15, downregulated glucose metabolism at E17, and upregulated inflammation at E20. In conclusion, this work provides evidence that gestational age during spina bifida repair may be a significant variable to consider during the development of new regenerative therapeutics approaches.

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.

The lipopolysaccharide (LPS)– toll-like receptor-4 (TLR4) pathway plays an important role in liver failure. Recombinant alkaline phosphatase (recAP) deactivates LPS. The aim of this study was to determine whether recAP prevents the progression of acute and acute-on-chronic liver failure (ACLF). Eight groups of rats were studied 4-weeks after sham surgery or bile duct ligation and were injected with saline or LPS to mimic ACLF. Acute liver failure was induced with Galactosamine-LPS and in both models animals were treated with recAP prior to LPS administration. In the ACLF model, the severity of liver dysfunction and brain edema was attenuated by recAP, associated with reduction in cytokines, chemokines, liver cell death, and brain water. The activity of LPS was reduced by recAP. The treatment was not effective in acute liver failure. Hepatic TLR4 expression was reduced by recAP in ACLF but not acute liver failure. Increased sensitivity to endotoxins in cirrhosis is associated with upregulation of hepatic TLR4, which explains susceptibility to development of ACLF whereas acute liver failure is likely due to direct hepatoxicity. RecAP prevents multiple organ injury by reducing receptor expression and is a potential novel treatment option for prevention of ACLF but not acute liver failure.

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.

OBJECTIVEThe authors hypothesized that new agents such as BioGlue would be as efficacious as kaolin in the induction of hydrocephalus in fetal sheep.METHODSThis study was performed in 34 fetal lambs randomly divided into 2 studies. In the first study, fetuses received kaolin, BioGlue (2.0 mL), or Onyx injected into the cisterna magna, or no injection (control group) between E85 and E90. In the second study, fetuses received 2.0-mL or 2.5-mL injections of BioGlue into the cisterna magna between E85 and E90. Fetuses were monitored using ultrasound to assess lateral ventricle size and progression of hydrocephalus. The fetuses were delivered (E120–E125) and euthanized for histological analysis. Selected brain sections were stained for ionized calcium binding adaptor 1 (Iba1) and glial fibrillary acidic protein (GFAP) to assess the presence and activation of microglia and astroglia, respectively. Statistical comparisons were performed with Student’s t-test for 2 determinations and ANOVA 1-way and 2-way repeated measures for multiple determinations.RESULTSAt 30 days after injection, the lateral ventricles were larger in all 3 groups that had undergone injection than in controls (mean diameter in controls 3.76 ± 0.05 mm, n = 5). However, dilatation was greater in the fetuses injected with 2 mL of BioGlue (11.34 ± 4.76 mm, n = 11) than in those injected with kaolin (6.4 ± 0.98 mm, n = 7) or Onyx (5.7 ± 0.31 mm, n = 6) (ANOVA, *p ≤ 0.0001). Fetuses injected with 2.0 mL or 2.5 mL of BioGlue showed the same ventricle dilatation but it appeared earlier (at 10 days postinjection) in those injected with 2.5 mL. The critical threshold of ventricle dilatation was 0.1 for all the groups, and only the BioGlue 2.0 mL and BioGlue 2.5 mL groups exceeded this critical value (at 30 days and 18 days after injection, respectively) (ANOVA, *p ≤ 0.0001). Moderate to severe hydrocephalus with corpus callosum disruption was observed in all experimental groups. All experimental groups showed ventriculomegaly with significant microgliosis and astrogliosis in the subventricular zone around the lateral ventricles. Only kaolin resulted in significant microgliosis in the fourth ventricle area (ANOVA, *p ≤ 0.005).CONCLUSIONSThe results of these studies demonstrate that BioGlue is more effective than Onyx or kaolin for inducing hydrocephalus in the fetal lamb and results in a volume-related response by obstructive space-occupancy without local neuroinflammatory reaction. This novel use of BioGlue generates a model with potential for new insights into hydrocephalus pathology and the development of therapeutics in obstructive hydrocephalus. In addition, this model allows for the study of acute and chronic obstructive hydrocephalus by using different BioGlue volumes for intracisternal injection.

Comparative, functional, developmental, and some morphological studies on animal anatomy require accurate visualization of three-dimensional structures. Nowadays, several widely applicable methods exist for non-destructive whole-mount imaging of animal tissues. The purpose of this study was to optimize specimen preparation and develop a method for quantitative analysis of the total pulmonary vasculature in fetal rats. Tissues were harvested at E21 and fetuses fixed overnight in 4% paraformaldehyde/phosphate buffered saline. They were treated with 25% Lugol solution for 72 hours to ensure perfusion. Four different methods were used for fetal specimen preparation; isolated lung, upper torso, direct right ventricle contrast injection, and whole body with partial thoracic skin excision. The microCT scan was performed, and pulmonary vasculature was segmented. Vessels were analyzed for diameter, length, and branching. Of the four preparation methods, only whole body with partial thoracic skin excision resulted in adequate reconstruction of the pulmonary vasculature. In silico generated 3D images gathered by micro CT showed pulmonary vasculature distributed throughout the lung, which was representative of the shape and structure of the lungs. The mean number of vessels segmented in the pulmonary tree was 900 +/- 24 with a mean diameter of 134.13 mu m (range 40.72-265.69 mu m). While up to the 30th generation of vessels could be segmented, both for arteries and veins, the majority of branching was between the 21st and 30th generations. Passive diffusion of contrast material enables quantitative analysis of the fetal pulmonary vasculature. This technique is a useful tool to analyze the characteristics and quantify the fetal pulmonary vasculature., NA

The authors wish to make the following corrections to this paper [...]

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.

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.

Kaolin (aluminum silicate) has been used to generate hydrocephalus by direct cisterna magna injection in animal models. The aim of the present study is to compare which method of Kaolin injection into fetal cisterna magna is feasible, safer, and more effective to induce hydrocephalus in fetal lambs. Twenty-five well-dated pregnant ewes at gestational 85–90 days (E85-90) were used to compare three different kaolin injection puncture techniques into the fetal cisterna magna. Group 1, ultrasound guidance in a maternal percutaneous transabdominal (TA); group 2, without opening the uterus in a transuterine (TU) technique; group 3, by occipital direct access after exteriorizing fetal head (EFH); and group 4, control group, was normal fetal lambs without injection. The fetal lambs were assessed using lateral ventricle diameter ultrasonographic measurements prior the kaolin injection and on the subsequent days. We analyzed the effectivity, mortality, and fetal losses to determine the best technique to create hydrocephalus in fetal lamb. After fetal intracisternal kaolin (2%, 1mL) injection, lateral ventricle diameters increased progressively in the three different interventional groups compared with the normal values of the control group (p ≤ 0.05). We observed that the transabdominal method had a 60% of fetal losses, considering failure of injection and mortality, compared with the 12.5% in the open group (EFH), and 0% for the transuterine group. Based on our study, we believe that both, open uterine (EFH) and transuterine approaches are more effective and safer than the transabdominal ultrasound-guided method to induce hydrocephalus.

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

Background & Aims: Liver failure is characterized by endothelial dysfunction, which results in hemodynamic disturbances leading to renal failure. Albumin infusion improves hemodynamics and prevents renal dysfunction in advance liver failure. These effects are only partly explained by the oncotic properties of albumin. This study was designed to test the hypothesis that albumin exerts its beneficial effects by stabilising endothelial function. Methods: In vivo: systemic hemodynamics, renal function, markers of endothelial dysfunction (ADMA) and inflammation were studied in analbuminaemic and Sprague–Dawley rats, 6-weeks after sham/bile duct ligation surgery. In vitro: human umbilical vein endothelial cells were stimulated with LPS with or without albumin. We studied protein expression and gene expression of adhesion molecules, intracellular reactive oxygen species, and cell stress markers. Results: Compared to controls, analbuminaemic rats had significantly greater hemodynamic deterioration after bile duct ligation, resulting in worse renal function and shorter survival. This was associated with significantly greater plasma renin activity, worse endothelial function, and disturbed inflammatory response. In vitro studies showed that albumin was actively taken up by endothelial cells. Incubation of albumin pre-treated endothelial cells with LPS was associated with significantly less activation compared with untreated cells, decreased intracellular reactive oxygen species, and markers of cell stress. Conclusions: These results show, for the first time, that absence of albumin is characterised by worse systemic hemodynamics, renal function and higher mortality in a rodent model of chronic liver failure and illustrates the important non-oncotic properties of albumin in protecting against endothelial dysfunction. 2015 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver.

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.

Our study focuses on the development and characterization of a self-expanding, watertight and biodegradable patch for fetoscopic myelomeningocele (MMC) prenatal repair. We fabricated poly(l-lactic acid) (PLA) and poly(ɛ-caprolactone) (PCL) blend films by solution casting. Formulation c with average glass transition temperature of 37.6 ± 1.2°C was chosen for temporospatial recovery. Favorable results from surface studies reflected homogeneous dispersion of polymers in the blend. The cytotoxicity was studied in human foreskin fibroblasts. The blend film was cytocompatible, evidenced by matching percentage of live cells in exposed and control solutions. Subsequently, liquid water permeability experiments confirmed watertight nature of films. Finally, in vitro degradation was investigated in phosphate buffered saline (PBS) and amniotic fluid (AF) separately for 16 weeks. Similar weight loss (n = 6, p = 0.912) and significantly different (n = 3, p = 0.025) surface roughness was observed in PBS and AF, respectively, at 16 weeks. Functional group analysis displayed increasing carbonyl and hydroxyl bonds in PBS and AF, respectively, over time, indicating progression of hydrolytic degradation. Favorable characterization results provide strong evidence to employ PLA-PCL blend films as surgical patches in fetoscopic MMC repair. Designed patch serves as standalone system to successfully tackle impending hurdles of MMC repair and proves to be a superior alternative compared to existing patches. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 295-305, 2019.

Experimental models of hepatic encephalopathy (HE) are limited by difficulties in objectively monitoring neuronal function. There are few models that examine a well-defined neuronal pathway and lack the confounding effects of anesthetics. Motor-evoked potentials (MEPs) assess the function of the motor tract, which has been shown to be impaired in patients with cirrhosis. MEPs were elicited by cranial stimulation (central) and compound motor action potential by sciatic nerve stimulation (peripheral) in several models of HE in the rat. The experiments were performed using subcutaneous electrodes without anesthetics. Brain water content was assessed by gravimetry, brain metabolites were measured by magnetic resonance spectroscopy, and amino acids in microdialysates from the frontal cortex were analyzed by high-performance liquid chromatography. Abnormalities of MEP were observed in acute liver failure (ALF) induced by hepatic devascularization in relation to the progression of neurological manifestations. Similar disturbances were seen in rats with portocaval anastomosis after the administration of blood or lipopolysaccharide, but were absent in rats with biliary duct ligation. Hypothermia (≤35°C) and mannitol prevented the development of brain edema in acute liver failure, but only hypothermia avoided the decrease in the amplitude of MEP. Disturbances of MEP caused by the administration of blood into the gastrointestinal tract in rats with portocaval anastomosis were associated with an increase in ammonia, glutamine, and glutamate in brain microdialysate. Conclusion: Assessment of MEP in awake rats is a valid method to monitor HE in models of ALF and precipitated HE. This method shows the lack of efficacy of mannitol, a therapy that decreases brain edema, and relates disturbances of the function of the motor tract to ammonia and its metabolites. (HEPATOLOGY 2010)

Background/Aims Splanchnic vasodilation initiates the hyperdynamic syndrome in portal hypertension. We aimed to explore molecular mechanisms involved in the development of mesenteric vasodilation in portal hypertension. Methods Superior mesenteric artery (SMA) samples from portal vein ligated (PVL) and sham rats were compared in a time course experiment using DNA microarrays. Selected genes were quantified by qRT-PCR in PVL and cirrhotic rats. Inmunohistochemistry of tyrosine hydroxylase (Th) and norepinephrine was assessed in SMA sections of PVL and sham rats. Western blot analysis of Th, dopamine β-hydroxylase (Dbh) and synaptosome-associated protein (Snap-25) was performed in SMA and jejunum samples from the animal models. Results Fifty differentially expressed genes implicated in neurotransmission, especially adrenergic, were detected in SMA samples from PVL rats. Sequential analysis showed a profound down-regulation at 14 days in PVL rats. These down-regulated genes were confirmed by RT-PCR in SMA from PVL and cirrhotic rats. Th and NE detection by immunohistochemistry was reduced in PVL compared to sham. Th, Dbh and Snap-25 expression was lower in SMA from 14-day PVL and cirrhotic rats compared to sham and control rats, respectively. Conclusions Genetic down-regulation of genes related to the adrenergic system might have a role in splanchnic vasodilation of portal hypertension.

The goal of this investigation was to develop a method to study the neurophysiological integrity of the central motor tract using motor evoked potentials in the awake rat and assess the effects of different anesthetics in this model. Rats were implanted with six subcutaneous electrodes (pediatric myocardial pacing leads) and one cranial screw. Motor evoked potentials of the hind limb were elicited after cranial and sciatic nerve stimulation. Experiments were repeated on different days during three weeks studying the effect of three different anesthetics (propofol, ketamine/xylazine, pentobarbital) at three different doses. Stimulation of motor evoked potentials in the awake rat was well tolerated with no effects on behavior. The electrodes could be kept chronically in place without signs of infection. The repeated recordings on different days showed high reproducibility after the fourth day following implantation of the electrodes. All three anesthetics induced an increase in the latency and a decrease in the amplitude of the motor evoked potentials which were dose dependent. Propofol (up to 1 mg/kg x min(1)) affected motor evoked potentials to a lesser extent than the other anesthetics. Based upon these findings, we believe that our approach provides a new method of chronically implanting electrodes in the rat to assess the neurophysiological function of the motor tract without the need of anesthetics. This model may prove useful in the investigation of various diseases that affect the motor pathways without the confounding effects of anesthesia.

Spinal cord injury (SCI) results in neural loss and consequently motor and sensory impairment below the injury. There are currently no effective therapies for the treatment of traumatic SCI in humans. Various animal models have been developed to mimic human SCI. Widely used animal models of SCI are complete or partial transection or experimental contusion and compression, with both bearing controversy as to which one more appropriately reproduces the human SCI functional consequences. Here we present in details the widely used procedure of complete spinal cord transection as a faithful animal model to investigate neural and functional repair of the damaged tissue by exogenous human transplanted cells. This injury model offers the advantage of complete damage to a spinal cord at a defined place and time, is relatively simple to standardize and is highly reproducible.

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.

International audience; This paper synthesizes the technical requirements made by a group of French scientists and defence users expert in hyperspectral imagery to design a new space borne imaging spectrometer. This project called HYPXIM is currently in phase 0 mission study and two French aerospace companies have proposed solutions that are analysed by the CNES. These technical requirements are converted into at-sensor radiance specifications for each scientific application and the final radiance set used for the instrument design is defined.

Ornithine phenylacetate (OP) is a new drug that has been proposed for the treatment of hepatic encephalopathy (HE) because it decreases plasma ammonia. We performed a study to assess if OP would impact on neuronal function.Motor-evoked potentials (MEP), a surrogate of hepatic encephalopathy, were assessed (without anesthesia) in rats with portacaval anastomosis (PCA) that received gastrointestinal blood (GIB). Rats were pre-treated with OP prior to GIB. Ammonia and related metabolites (plasma, urine, and brain microdialysis) were assessed by HPLC and mass spectroscopy.OP (one dose or 3 days) prevented disturbances in MEP induced by GIB in PCA rats. In rats treated with OP for 3 days, the amplitude and latency of MEP remained stable (-1% and +1%), while in the control group the amplitude decreased -21% and the latency increased +12% (p0.01). OP attenuated the rise of ammonia in plasma by 45%, ammonia in brain microdialysate by 48%, induced a faster glutamine rise and the appearance of phenylacetylglutamine in plasma and urine. In addition, OP was associated with a lower concentration of ammonia and glutamate in brain microdialysate (approx. 50%).OP prevents abnormalities in MEP precipitated by GIB in a model of HE. This is probably due to the enhancement of glutamine synthesis and metabolism, which results in a lower rise of plasma ammonia and the prevention of changes in glutamate in microdialysate. Thus, OP may be a good drug to prevent HE precipitated by gastrointestinal bleeding.

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.

Damage of neural elements (spinal cord and encephalus) in myelomeningocele (MMC) seems to be progressive during gestation because of amniotic fluid chemical contact and continuous leakage of CSF. We studied the effect of preterm delivery and steroid treatment in a model of MMC in the rabbit foetus. Twelve New Zealand White rabbits underwent laparotomy and hysterotomy at 23 days of gestation. Fifty-nine out of 107 foetuses underwent lumbar laminectomy (three to four levels). Dura was opened to expose the neural elements to the amniotic fluid. Six rabbits underwent caesarean section on gestational day 31 for fetal harvest; three of them had no treatment (group T) and three received corticosteroid treatment (group TC). The other six rabbits underwent caesarean section on gestational day 29 for fetal harvest (preterm delivery); three of them had no treatment (group P) and three received corticosteroid treatment (group PC). Alive newborns were clinically, neurophysiologically and histologically analysed. None of mothers died during the procedure. After birth, animals in group preterm showed statistically significant less deformity than animals in group at term. Lower kyphosis was observed in group PC (preterm and steroids). Pain related and spontaneous mobility of lower extremities was higher in groups treated with corticosteroids (TC and PC). Only newborns at term (T and TC groups) showed response to evoked potentials (CMEPs). The response was earlier and higher in group treated with steroids (TC). Histologically, we observed progressive lesion of the spinal cord. Groups treated with steroids (TC and PC) show less inflammatory response. Arnold-Chiari malformation was present in all groups. Animals in group preterm with steroids show statistically significant less herniation than those group at term. Preterm delivery and prenatal steroid therapy seem to be an effective treatment to get less neural injury (spinal cord and encephalus) in myelomeningocele foetuses.

Introduction: Hepatic encephalopathy is a neurologic syndrome secondary to liver failure that causes cognitive and motor abnormalities. Impairment in the function of the first neuron of the motor tract (corticospinal tract) has been demonstrated in patients with cirrhosis and minimal hepatic encephalopathy. Aim: Investigate the function of the first neuron of the motor tract in experimental models of minimal hepatic encephalopathy. Material and methods: Rats with portocaval anastomosis (n=8) and rats with carbon tetrachloride induced cirrhosis (n=11) underwent neurophysiological recording under light anesthesia with propofol. Motor evoked potentials were elicited applying a transcranial electric pulse and were recorded in the tibialis anterior muscle. The effect of the dose of anesthesia was assessed in a group of normal rats (n=10). Results: Rats with portocaval anastomosis exhibited a decrease in motor evoked potentials amplitude following surgery (67±11 to 41±16%, P < 0.001). Cirrhotic rats exhibited an increase in motor evoked potentials latency after the appearance of ascites (4.65±0.43 to 5.15±0.67 ms., P=0.04). Increasing doses of propofol produced a decrease in the amplitude and an increase in the latency of motor evoked potentials. Conclusion: It is possible to reproduce functional abnormalities of the central motor tract in rats with portocaval anastomosis and carbon tetrachloride induced cirrhosis. The development of motor abnormalities in experimental models of minimal hepatic encephalopathy offers the possibility to investigate the mechanisms involved in the pathogenesis of hepatic encephalopathy and test therapeutic strategies.