Fetal hypoplastic lungs have multilineage inflammation that is reversed by amniotic fluid stem cell extracellular vesicle treatment.

Antenatal administration of extracellular vesicles from amniotic fluid stem cells (AFSC-EVs) reverses features of pulmonary hypoplasia in models of congenital diaphragmatic hernia (CDH). However, it remains unknown which lung cellular compartments and biological pathways are affected by AFSC-EV therapy. Herein, we conducted single-nucleus RNA sequencing (snRNA-seq) on rat fetal CDH lungs treated with vehicle or AFSC-EVs. We identified that intra-amniotically injected AFSC-EVs reach the fetal lung in rats with CDH, where they promote lung branching morphogenesis and epithelial cell differentiation. Moreover, snRNA-seq revealed that rat fetal CDH lungs have a multilineage inflammatory signature with macrophage enrichment, which is reversed by AFSC-EV treatment. Macrophage enrichment in CDH fetal rat lungs was confirmed by immunofluorescence, flow cytometry, and inhibition studies with GW2580. Moreover, we validated macrophage enrichment in human fetal CDH lung autopsy samples. Together, this study advances knowledge on the pathogenesis of pulmonary hypoplasia and further evidence on the value of an EV-based therapy for CDH fetuses.

Antenatal Administration of Extracellular Vesicles Derived From Amniotic Fluid Stem Cells Improves Lung Function in Neonatal Rats With Congenital Diaphragmatic Hernia.

BACKGROUND: The severity of pulmonary hypoplasia is a main determinant of outcome for babies with congenital diaphragmatic hernia (CDH). Antenatal administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) has been shown to rescue morphological features of lung development in the rat nitrofen model of CDH. Herein, we evaluated whether AFSC-EV administration to fetal rats with CDH is associated with neonatal improvement in lung function. METHODS: AFSC-EVs were isolated by ultracentrifugation and characterized by size, morphology, and canonical marker expression. At embryonic (E) day 9.5, dams were gavaged with olive oil (control) or nitrofen to induce CDH. At E18.5, fetuses received an intra-amniotic injection of either saline or AFSC-EVs. At E21.5, rats were delivered and subjected to a tracheostomy for mechanical ventilation (flexiVent system). Groups were compared for lung compliance, resistance, Newtonian resistance, tissue damping and elastance. Lungs were evaluated for branching morphogenesis and collagen quantification. RESULTS: Compared to healthy control, saline-treated pups with CDH had fewer airspaces, more collagen deposition, and functionally exhibited reduced compliance and increased airway resistance, elastance, and tissue damping. Conversely, AFSC-EV administration resulted in improvement of lung mechanics (compliance, resistance, tissue damping, elastance) as well as lung branching morphogenesis and collagen deposition. CONCLUSIONS: Our studies show that the rat nitrofen model reproduces lung function impairment similar to that of human babies with CDH. Antenatal administration of AFSC-EVs improves lung morphology and function in neonatal rats with CDH. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Anti-inflammatory effects of antenatal administration of stem cell derived extracellular vesicles in the brain of rat fetuses with congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) survivors may experience neurodevelopmental impairment, whose etiology remains elusive. Preclinical evidence indicates that amniotic fluid stem cell extracellular vesicle (AFSC-EV) administration promotes lung development but their effects on other organs are unknown. Herein, we investigated the brain of rat fetuses with CDH for signs of inflammation and response to AFSC-EVs. METHODS: CDH was induced by maternal nitrofen administration at E9.5. At E18.5, fetuses were injected intra-amniotically with saline or AFSC-EVs (isolated by ultracentrifugation, characterized as per MISEV guidelines). Fetuses from vehicle-gavaged dams served as controls. Groups were compared for: lung hypoplasia, TNFa and IL-1B brain expression, and activated microglia (Iba1) density in the subgranular zone (SGZ). RESULTS: CDH lungs had fewer airspaces compared to controls, whereas AFSC-EV-treated lungs had rescued branching morphogenesis. Fluorescently labeled AFSC-EVs injected intra-amniotically into CDH fetuses had fluorescent signal in the brain. Compared to controls, the brain of CDH fetuses had higher TNFa and IL-1B levels, and increased activated microglia density. Conversely, the brain of AFSC-EV treated fetuses had inflammatory marker expression levels and microglia density similar to controls. CONCLUSION: This study shows that the brain of rat fetuses with CDH has signs of inflammation that are abated by the intra-amniotic administration of AFSC-EVs.

Characterization of the congenital diaphragmatic hernia model in C57BL/6J fetal mice: a step toward lineage tracing experiments.

PURPOSE: Lineage tracing is key to study the fate of individual cells and their progeny especially in developmental biology. To conduct these studies, we aimed to establish a reproducible model of CDH in the most commonly used genetic background strain that is C57BL/6J mice. METHODS: CDH was induced in C57BL/6J dams by maternal administration of nitrofen + bisdiamine at E8.5. Fetuses from olive oil-gavaged mothers served as controls. Lungs from CDH and control fetuses were compared for (1) growth via radial airspace count (RAC), mean linear intercept (MLI) and gene expression for Fgf10, Nrp1, and Ctnnb1; (2) maturation (Pdpn, Spc, Ager, Abca3, Eln, Acta2, Pdgfra) via gene and protein expression; (3) vascularization via gene and protein expression (CD31, Vegfa, Vegfr1/2, Epas1, Enos). STATISTICS: unpaired t-test or Mann-Whitney test. RESULTS: Nitrofen + bisdiamine administration resulted in 36% left-sided CDH (31% mortality). CDH fetuses had hypoplastic lungs and impaired growth (lower RAC, higher MLI, lower Fgf10, Nrp1, Ctnnb1), maturation (decreased Pdpn, Ager, Eln gene expression), and vascularization (decreased Cd31, Vegfr1/2; Epas1 and Enos). Lower protein expression was confirmed for PDPN, ELN and CD31. CONCLUSION: Modeling CDH in C57BL/6J mouse fetuses is effective in reproducing the classical CDH hallmarks. This model will be critical for lineage tracing experiments.

Effects of early exercise on cardiac function and lipid metabolism pathway in heart failure.

We employed an early training exercise program, immediately after recovery from surgery, and before severe cardiac hypertrophy, to study the underlying mechanism involved with the amelioration of cardiac dysfunction in aortic stenosis (AS) rats. As ET induces angiogenesis and oxygen support, we aimed to verify the effect of exercise on myocardial lipid metabolism disturbance. Wistar rats were divided into Sham, trained Sham (ShamT), AS and trained AS (AST). The exercise consisted of 5-week sessions of treadmill running for 16 weeks. Statistical analysis was conducted by anova or Kruskal-Wallis test and Goodman test. A global correlation between variables was also performed using a two-tailed Pearson's correlation test. AST rats displayed a higher functional capacity and a lower cardiac remodelling and dysfunction when compared to AS, as well as the myocardial capillary rarefaction was prevented. Regarding metabolic properties, immunoblotting and enzymatic assay raised beneficial effects of exercise on fatty acid transport and oxidation pathways. The correlation assessment indicated a positive correlation between variables of angiogenesis and FA utilisation, as well as between metabolism and echocardiographic parameters. In conclusion, early exercise improves exercise tolerance and attenuates cardiac structural and functional remodelling. In parallel, exercise attenuated myocardial capillary and lipid metabolism derangement in rats with aortic stenosis-induced heart failure.

Anti-asthmatic activity of standardized hydro-ethanolic and aqueous extracts of Stachytarpheta cayennensis (Rich.) Vahl in a murine model.

ETHNOPHARMACOLOGICAL RELEVANCE: Stachytarpheta cayennensis (Verbenaceae) has been used in Brazilian traditional medicine to treat asthma and other respiratory diseases. AIMS OF THE STUDY: To investigate the effects of different doses of standardized hydro-ethanolic (SCH) and aqueous (SCA) extracts of aerial parts of S. cayennensis using a murine ovalbumin (OVA)-induced asthma model. MATERIALS AND METHODS: The major constituents of the plant extracts were identified and standardized by ultra-performance liquid chromatography coupled with mass spectrometry. Balb/c mice were challenged with OVA solution and treated concomitantly by intraperitoneal injection of standardized SCH or SCA extracts at 50, 100, and 200 mg/kg concentrations. OVA-challenged control animals were treated with either dexamethasone (OVA-DEX) or saline solution (OVA-SAL). After challenge, we assessed in vivo bronchial hyperresponsiveness, airway inflammation (number of cells), peribronchial inflammation (histological analysis) and production of OVA-specific IgE and interleukin (IL)-4, IL-5, and IL-13 (ELISA). RESULTS: Acteoside, isoacteoside, and ipolamiide were the major constituents of SCH and SCA. The respective concentrations of acteoside in SCH and SCA were 78 and 98 µg/mL, while those of ipolamiide were 30 and 19 µg/mL. Treatment with 200 mg/kg of SCH or SCA decreased IL-4, IL-5, and IL-13 in lung homogenates. These reductions were accompanied by a lower influx of inflammatory cells (eosinophils, lymphocytes, and macrophages) to the airways and lungs. In addition to the anti-inflammatory effects, administration of SCA, but not SCH, ameliorated the parameters of bronchial hyperresponsiveness and decreased levels of circulating OVA-specific IgE. CONCLUSION: The results presented herein demonstrate for the first time the anti-asthmatic activity of S. cayennensis extracts in a murine model, thereby supporting the ethnopharmacological uses of the plant.

Treatment with Amniotic Fluid Stem Cell Extracellular Vesicles Promotes Fetal Lung Branching and Cell Differentiation at Canalicular and Saccular Stages in Experimental Pulmonary Hypoplasia Secondary to Congenital Diaphragmatic Hernia.

Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired branching morphogenesis and differentiation. We have previously demonstrated that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues development of hypoplastic lungs at the pseudoglandular and alveolar stages in rodent models of CDH. Herein, we tested whether AFSC-EVs exert their regenerative effects at the canalicular and saccular stages, as these are translationally relevant for clinical intervention. To induce fetal pulmonary hypoplasia, we gavaged rat dams with nitrofen at embryonic day 9.5 and demonstrated that nitrofen-exposed lungs had impaired branching morphogenesis, dysregulated signaling pathways relevant to lung development (FGF10/FGFR2, ROBO/SLIT, Ephrin, Neuropilin 1, ß-catenin) and impaired epithelial and mesenchymal cell marker expression at both stages. AFSC-EVs administered to nitrofen-exposed lung explants rescued airspace density and increased the expression levels of key factors responsible for branching morphogenesis. Moreover, AFSC-EVs rescued the expression of alveolar type 1 and 2 cell markers at both canalicular and saccular stages and restored markers of club, ciliated epithelial, and pulmonary neuroendocrine cells at the saccular stage. AFSC-EV-treated lungs also had restored markers of lipofibroblasts and PDGFRA+ cells to control levels at both stages. EV tracking showed uptake of AFSC-EV RNA cargo throughout the fetal lung and an mRNA-miRNA network analysis identified that several miRNAs responsible for regulating lung development processes were contained in the AFSC-EV cargo. These findings suggest that AFSC-EV-based therapies hold potential for restoring fetal lung growth and maturation in babies with pulmonary hypoplasia secondary to CDH.

Autophagy Is Impaired in Fetal Hypoplastic Lungs and Rescued by Administration of Amniotic Fluid Stem Cell Extracellular Vesicles.

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC-conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. The expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17∼92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through microRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in congenital diaphragmatic hernia babies.

Fetal lung regeneration using stem cell-derived extracellular vesicles: A new frontier for pulmonary hypoplasia secondary to congenital diaphragmatic hernia.

The poor outcomes of babies with congenital diaphragmatic hernia (CDH) are directly related to pulmonary hypoplasia, a condition characterized by impaired lung development. Although the pathogenesis of pulmonary hypoplasia is not fully elucidated, there is now evidence that CDH patients have missing or dysregulated microRNAs (miRNAs) that regulate lung development. A prenatal therapy that supplements these missing/dysregulated miRNAs could be a strategy to rescue normal lung development. Extracellular vesicles (EVs), also known as exosomes when of small dimensions, are lipid-bound nanoparticles that can transfer their heterogeneous cargo (proteins, lipids, small RNAs) to target cells to induce biological responses. Herein, we review all studies that show evidence for stem cell-derived EVs as a regenerative therapy to rescue normal development in CDH fetal lungs. Particularly, we report studies showing that administration of EVs derived from amniotic fluid stem cells (AFSC-EVs) to models of pulmonary hypoplasia promotes fetal lung growth and maturation via transfer of miRNAs that are known to regulate lung developmental processes. We also describe that stem cell-derived EVs exert effects on vascular remodeling, thus possibly preventing postnatal pulmonary hypertension. Finally, we discuss future perspectives and challenges to translate this promising stem cell EV-based therapy to clinical practice.

Honeymoon Period in Newborn Rats With CDH Is Associated With Changes in the VEGF Signaling Pathway.

Background: Patients with congenital diaphragmatic hernia (CDH) have a short postnatal period of ventilatory stability called the honeymoon period, after which changes in pulmonary vascular reactivity result in pulmonary hypertension. However, the mechanisms involved are still unknown. The aim of this study was to evaluate mechanical ventilation's effect in the honeymoon period on VEGF, VEGFR-1/2 and eNOS expression on experimental CDH in rats. Materials and Methods: Neonates whose mothers were not exposed to nitrofen formed the control groups (C) and neonates with left-sided defects formed the CDH groups (CDH). Both were subdivided into non-ventilated and ventilated for 30, 60, and 90 min (n = 7 each). The left lungs (n = 4) were evaluated by immunohistochemistry of the pulmonary vasculature (media wall thickness), VEGF, VEGFR-1/2 and eNOS. Western blotting (n = 3) was performed to quantify the expression of VEGF, VEGFR-1/2 and eNOS. Results: CDH had lower biometric parameters than C. Regarding the pulmonary vasculature, C showed a reduction in media wall thickness with ventilation, while CDH presented reduction with 30 min and an increase with the progression of the ventilatory time (honeymoon period). CDH and C groups showed different patterns of VEGF, VEGFR-1/2 and eNOS expressions. The receptors and eNOS findings were significant by immunohistochemistry but not by western blotting, while VEGF was significant by western blotting but not by immunohistochemistry. Conclusion: VEGF, its receptors and eNOS were altered in CDH after mechanical ventilation. These results suggest that the VEGF-NO pathway plays an important role in the honeymoon period of experimental CDH.

The Role of Exosomes in the Treatment, Prevention, Diagnosis, and Pathogenesis of COVID-19.

The novel coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), continues to be a major health concern. In search for novel treatment strategies against COVID-19, exosomes have attracted the attention of scientists and pharmaceutical companies worldwide. Exosomes are small extracellular vesicles, secreted by all types of cells, and considered as key mediators of intercellular communication and stem-cell paracrine signaling. Herein, we reviewed the most recent literature about the role of exosomes as potential agents for treatment, prevention, diagnosis, and pathogenesis of COVID-19. Several studies and ongoing clinical trials have been investigating the anti-inflammatory, immunomodulatory, and reparative effects of exosomes derived from mesenchymal stem/stromal cells for COVID-19-related acute lung injury. Other studies reported that exosomes play a key role in convalescent plasma therapy for COVID-19, and that they could be of use for the treatment of COVID-19 Kawasaki's-like multisystem inflammatory syndrome and as drug delivery nanocarriers for antiviral therapy. Harnessing some advantageous aspects of exosome biology, such as their endogenous origin, capability of crossing biological barriers, high stability in circulation, and low toxicity and immunogenicity, several companies have been testing exosome-based vaccines against SARS-CoV-2. As they carry cargos that mimic the status of parent cells, exosomes can be isolated from a variety of sources, including plasma, and employed as biomarkers of COVID-19. Lastly, there is growing evidence supporting the role of exosomes in COVID-19 infection, spread, reactivation, and reinfection. The lessons learned using exosomes for COVID-19 will help determine their efficacy and applicability in other clinical conditions.

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, babies with CDH 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. Here, we describe a stem cell-based approach in rodents 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 promoted branching morphogenesis and alveolarization, rescued tissue homeostasis, and stimulated epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in in vitro models of lung injury using human material, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. MicroRNAs regulating the expression of genes involved in lung development, such as the miR17-92 cluster and its paralogs, were highly enriched in AFSC-EVs 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.

In situ Evidence of Collagen V and Interleukin-6/Interleukin-17 Activation in Vascular Remodeling of Experimental Pulmonary Hypertension.

Several studies have reported the pathophysiologic and molecular mechanisms responsible for pulmonary arterial hypertension (PAH). However, the in situ evidence of collagen V (Col V) and interleukin-17 (IL-17)/interleukin-6 (IL-6) activation in PAH has not been fully elucidated. We analyzed the effects of collagen I (Col I), Col V, IL-6, and IL-17 on vascular remodeling and hemodynamics and its possible mechanisms of action in monocrotaline (MCT)-induced PAH. Twenty male Wistar rats were randomly divided into two groups. In the PAH group, animals received MCT 60 mg/kg intraperitoneally, whereas the control group (CTRL) received saline. On day 21, the pulmonary blood pressure (PAP) and right ventricular systolic pressure (RVSP) were determined. Lung histology (smooth muscle cell proliferation [α-smooth muscle actin; α-SMA] and periadventitial fibrosis), immunofluorescence (Col I, Col V, and α-SMA), immunohistochemistry (IL-6, IL-17, and transforming growth factor-beta [TGF-ß]), and transmission electron microscopy to detect fibronexus were evaluated. The RVSP (40 ± 2 vs. 24 ± 1 mm Hg, respectively; p < 0.0001), right ventricle hypertrophy index (65 ± 9 and 25 ± 5%, respectively; p < 0.0001), vascular periadventitial Col I and Col V, smooth muscle cell α-SMA+, fibronexus, IL-6, IL-17, and TGF-ß were higher in the MCT group than in the CTRL group. In conclusion, our findings indicate in situ evidence of Col V and IL-6/IL-17 activation in vascular remodeling and suggest that increase of Col V may yield potential therapeutic targets for treating patients with PAH.

Systematic review of extracellular vesicle-based treatments for lung injury: are EVs a potential therapy for COVID-19?

Severe COVID-19 infection results in bilateral interstitial pneumonia, often leading to acute respiratory distress syndrome (ARDS) and pulmonary fibrosis in survivors. Most patients with severe COVID-19 infections who died had developed ARDS. Currently, ARDS is treated with supportive measures, but regenerative medicine approaches including extracellular vesicle (EV)-based therapies have shown promise. Herein, we aimed to analyse whether EV-based therapies could be effective in treating severe pulmonary conditions that affect COVID-19 patients and to understand their relevance for an eventual therapeutic application to human patients. Using a defined search strategy, we conducted a systematic review of the literature and found 39 articles (2014-2020) that reported effects of EVs, mainly derived from stem cells, in lung injury models (one large animal study, none in human). EV treatment resulted in: (1) attenuation of inflammation (reduction of pro-inflammatory cytokines and neutrophil infiltration, M2 macrophage polarization); (2) regeneration of alveolar epithelium (decreased apoptosis and stimulation of surfactant production); (3) repair of microvascular permeability (increased endothelial cell junction proteins); (4) prevention of fibrosis (reduced fibrin production). These effects were mediated by the release of EV cargo and identified factors including miRs-126, -30b-3p, -145, -27a-3p, syndecan-1, hepatocyte growth factor and angiopoietin-1. This review indicates that EV-based therapies hold great potential for COVID-19 related lung injuries as they target multiple pathways and enhance tissue regeneration. However, before translating EV therapies into human clinical trials, efforts should be directed at developing good manufacturing practice solutions for EVs and testing optimal dosage and administration route in large animal models.

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.

Gastroschisis: State of the Art in Translating Experimental Research to the Bedside.

The survival rate of newborns with gastroschisis (GS) has been increasing in the past decades; however, the morbidity continues to be high, mainly related to the length of hospitalization due to disturbances of motility, absorption, and risk of intestinal infections. The development of basic research with the creation of experimental models has provided enormous advances in the understanding of the pathophysiology of the disease. These models allowed the study of the target genes involved in the embryology of the defect, the influence of the amniotic fluid, and the use of drugs and fetal therapies in an attempt to reduce the intestinal damage and to provide a rapid return of intestinal motility. Our aim was to describe the main GS models and the translational, historical impact of these research advances on the disease.

Congenital Diaphragmatic Hernia: State of the Art in Translating Experimental Research to the Bedside.

Congenital diaphragmatic hernia (CDH) is a devastating disease that still carries a high mortality and morbidity rate. Poor outcomes for fetuses and infants with CDH are mainly related to pulmonary hypoplasia (PH) and pulmonary vascular remodeling that leads to pulmonary hypertension (PHTN). Over the last five decades, research efforts have focused on modeling CDH not only to study the pathophysiology of the diaphragmatic defect, pulmonary hypoplasia, and pulmonary hypertension, but also to identify therapies that would promote lung growth and maturation, and correct vascular remodeling. As CDH is a multifactorial condition whose etiology remains unknown, there is not a single model of CDH, rather several ones that replicate different aspects of this disease. While small animals like the mouse and the rat have mainly been used to uncover biological pathways underlying the diaphragmatic defect and poor lung growth, larger animals like the lamb and the rabbit models have been instrumental for pursuing medical and surgical interventions. Overall, the use of animal models has indeed advanced our knowledge on CDH and helped us test innovative therapeutic options. For example, the lamb model of CDH has been the paradigm for testing fetal surgical procedures, including tracheal occlusion, which has been translated to clinical use. In this review, we outline the induction protocols of CDH in animals with the use of chemicals, dietary changes, genetic alterations, and surgical maneuvers, and we describe the studies that have translated experimental results to the bedside.

Does Biliodigestive Anastomosis Have Any Effect on the Reversal of Hepatopulmonary Syndrome in a Biliary Cirrhosis Experimental Model?

BACKGROUND: Biliary cirrhosis is associated with hepatopulmonary syndrome (HPS), which is related to increased posttransplant morbidity and mortality. AIMS: This study aims to analyze the pathophysiology of biliary cirrhosis and the onset of HPS. METHODS: Twenty-one-day-old Wistar rats were subjected to common bile duct ligation and were allocated to two groups: group A (killed 2, 3, 4, 5, or 6 weeks after biliary obstruction) and group B (subjected to biliodigestive anastomosis 2, 3, 4, 5, or 6 weeks after the first procedure and killed 3 weeks later). At the killing, arterial blood was collected for the analyses, and samples from the liver and lungs were collected for histologic and molecular analyses. The gasometric parameters as well as the expression levels of ET-1, eNOS, and NOS genes in the lung tissue were evaluated. RESULTS: From a total of 42 blood samples, 15 showed hypoxemia (pO2 < 85 mmHg) and 17 showed an increased oxygen gradient [p (A-a) O2 > 18 mmHg]. The liver histology revealed increased ductular proliferation after common bile duct ligation, and reconstruction of bile flow promoted decreased ductular proliferation 5 and 6 weeks post-common bile duct ligation. Pulmonary alterations consisted of decreased parenchymal airspace and increased medial wall thickness. Biliary desobstruction promoted transitory improvements 5 weeks after biliary obstruction (increased parenchymal airspace and decreased MWT-p = 0.003 and p = 0.004, respectively) as well as increased endothelin expression levels (p = 0.009). CONCLUSIONS: The present model showed lung tissue alterations promoted by biliary obstruction. The biliodigestive anastomosis had no clear direct effects on these alterations.

Vascular and ventilatory mechanical responses in three different stages of pulmonary development in the rabbit model of congenital diaphragmatic hernia 1.

PURPOSE: To evaluate the vascular ventilatory response in different stages of lung development and to compare them to the neonates with congenital diaphragmatic hernia (CDH) in a rabbit model. METHODS: New Zealand rabbits were divided into 8 groups (n=5): E25, E27, E30, and CDH. All groups were ventilated on a FlexiVent (Scireq, Montreal, QC, Canada), compounding the other 4 groups. The CDH surgery was performed at E25 and the harvest at E30. Dynamic compliance (CRS), dynamic elastance (ERS) and dynamic resistance (RRS) were measured every 4 min/24 min. Median wall thickness (MWT) and airspace were measured. ANOVA Bonferroni tests were used to perform statistical analysis. Significance was considered when p<0.05. RESULTS: CRS was higher in E30 compared to all other groups (p<0.05). CRS and RRS of CDH and E27 were similar and were higher in E25 (p<0.05). MWT was decreased according to the gestational age, was increased in E27V and E30V (p<0.05) and decreased in CDHV (p<0.05), airspace was decreased in E25 and increased in all ventilated groups (p<0.05). CONCLUSIONS: The ventilation response of congenital diaphragmatic hernia is like the pseudoglandular stage of the lung development. These findings add information about the physiology of pulmonary ventilation in CDH.

Vascular and ventilatory mechanical responses in three different stages of pulmonary development in the rabbit model of congenital diaphragmatic hernia

Abstract Purpose: To evaluate the vascular ventilatory response in different stages of lung development and to compare them to the neonates with congenital diaphragmatic hernia (CDH) in a rabbit model. Methods: New Zealand rabbits were divided into 8 groups (n=5): E25, E27, E30, and CDH. All groups were ventilated on a FlexiVent (Scireq, Montreal, QC, Canada), compounding the other 4 groups. The CDH surgery was performed at E25 and the harvest at E30. Dynamic compliance (CRS), dynamic elastance (ERS) and dynamic resistance (RRS) were measured every 4 min/24 min. Median wall thickness (MWT) and airspace were measured. ANOVA Bonferroni tests were used to perform statistical analysis. Significance was considered when p<0.05. Results: CRS was higher in E30 compared to all other groups (p<0.05). CRS and RRS of CDH and E27 were similar and were higher in E25 (p<0.05). MWT was decreased according to the gestational age, was increased in E27V and E30V (p<0.05) and decreased in CDHV (p<0.05), airspace was decreased in E25 and increased in all ventilated groups (p<0.05). Conclusions: The ventilation response of congenital diaphragmatic hernia is like the pseudoglandular stage of the lung development. These findings add information about the physiology of pulmonary ventilation in CDH.