Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Intraluminal chloride regulates lung branching morphogenesis: involvement of PIEZO1/PIEZO2.

BACKGROUND: Clinical and experimental evidence shows lung fluid volume as a modulator of fetal lung growth with important value in treating fetal lung hypoplasia. Thus, understanding the mechanisms underlying these morphological dynamics has been the topic of multiple investigations with, however, limited results, partially due to the difficulty of capturing or recapitulating these movements in the lab. In this sense, this study aims to establish an ex vivo model allowing the study of lung fluid function in branching morphogenesis and identify the subsequent molecular/ cellular mechanisms. METHODS: Ex vivo lung explant culture was selected as a model to study branching morphogenesis, and intraluminal injections were performed to change the composition of lung fluid. Distinct chloride (Cl-) concentrations (5.8, 29, 143, and 715 mM) or Cl- channels inhibitors [antracene-9-carboxylic acid (A9C), cystic fibrosis transmembrane conductance regulator inhibitor172 (CFTRinh), and calcium-dependent Cl- channel inhibitorA01 (CaCCinh)] were injected into lung lumen at two timepoints, day0 (D0) and D2. At D4, morphological and molecular analyses were performed in terms of branching morphogenesis, spatial distribution (immunofluorescence), and protein quantification (western blot) of mechanoreceptors (PIEZO1 and PIEZO2), neuroendocrine (bombesin, ghrelin, and PGP9.5) and smooth muscle [alpha-smooth muscle actin (α-SMA) and myosin light chain 2 (MLC2)] markers. RESULTS: For the first time, we described effective intraluminal injections at D0 and D2 and demonstrated intraluminal movements at D4 in ex vivo lung explant cultures. Through immunofluorescence assay in in vivo and ex vivo branching morphogenesis, we show that PGP9.5 colocalizes with PIEZO1 and PIEZO2 receptors. Fetal lung growth is increased at higher [Cl-], 715 mM Cl-, through the overexpression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. In contrast, intraluminal injection of CFTRinh or CaCCinh decreases fetal lung growth and the expression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. Finally, the inhibition of PIEZO1/PIEZO2 by GsMTx4 decreases branching morphogenesis and ghrelin, bombesin, MLC2, and α-SMA expression in an intraluminal injection-independent manner. CONCLUSIONS: Our results identify PIEZO1/PIEZO2 expressed in neuroendocrine cells as a regulator of fetal lung growth induced by lung fluid.

Predicting Insulin Resistance in a Pediatric Population With Obesity.

OBJECTIVES: Insulin resistance (IR) affects children and adolescents with obesity and early diagnosis is crucial to prevent long-term consequences. Our aim was to identify predictors of IR and develop a multivariate model to accurately predict IR. METHODS: We conducted a cross-sectional analysis of demographical, clinical, and biochemical data from a cohort of patients attending a specialized Paediatric Nutrition Unit in Portugal over a 20-year period. We developed multivariate regression models to predict IR. The participants were randomly divided into 2 groups: a model group for developing the predictive models and a validation group for cross-validation of the study. RESULTS: Our study included 1423 participants, aged 3-17 years old, randomly divided in the model (n = 879) and validation groups (n = 544). The predictive models, including uniquely demographic and clinical variables, demonstrated good discriminative ability [area under the curve (AUC): 0.834-0.868; sensitivity: 77.0%-83.7%; specificity: 77.0%-78.7%] and high negative predictive values (88.9%-91.6%). While the diagnostic ability of adding fasting glucose or triglycerides/high density lipoprotein cholesterol index to the models based on clinical parameters did not show significant improvement, fasting insulin appeared to enhance the discriminative power of the model (AUC: 0.996). During the validation, the model considering demographic and clinical variables along with insulin showed excellent IR discrimination (AUC: 0.978) and maintained high negative predictive values (90%-96.3%) for all models. CONCLUSION: Models based on demographic and clinical variables can be advantageously used to identify children and adolescents at moderate/high risk of IR, who would benefit from fasting insulin evaluation.

Objective structured assessment ultrasound skill scale for hyomental distance competence - psychometric study.

BACKGROUND: Ultrasound assessment of the airway recently integrates the point-of-care approach to patient evaluation since ultrasound measurements can predict a difficult laryngoscopy and tracheal intubation. Because ultrasonography is performer-dependent, a proper training and assessment tool is needed to increase diagnostic accuracy. An objective, structured assessment ultrasound skill (OSAUS) scale was recently developed to guide training and assess competence. This work aims to study the psychometric properties of OSAUS Scale when used to evaluate competence in ultrasound hyomental distance (HMD) measurement. METHODS: Prospective and experimental study. Volunteers were recruited and enrolled in groups with different expertise. Each participant performed three ultrasonographic HMD evaluation. The performance was videorecorded and anonymized. Five assessors blindly rated participants' performance using OSAUS scale and a Global Rating Scale (GRS). A psychometric study of OSAUS scale as assessment tool for ultrasound HMD competence was done. RESULTS: Fifteen voluntaries participated on the study. Psychometric analysis of OSAUS showed strong internal consistency (Cronbach's alpha 0.916) and inter-rater reliability (ICC 0.720; p < 0.001). The novice group scored 15.4±0.18 (mean±SD), the intermediate 14.3±0.75 and expert 13.6±0.1.25, with a significant difference between novice and expert groups (p = 0.036). The time in seconds to complete the task was evaluated: novice (90±34) (mean±SD), intermediate (84±23) and experts (83±15), with no significant differences between groups. A strong correlation was observed between OSAUS and global rating scale (r = 0.970, p < 0.001). CONCLUSION: The study demonstrated evidence of validity and reliability. Further studies are needed to implement OSAUS scale in the clinical setting for training and assessment of airway ultrasound competence.

Diaphragmatic hernia repair porcine model to compare the performance of biodegradable membranes against Gore-Tex®.

BACKGROUND: Patch repair of congenital diaphragmatic hernia (CDH) using Gore-Tex® is associated with infection, adhesions, hernia recurrence, long-term musculoskeletal sequels and poor tissue regeneration. To overcome these limitations, the performance of two novel biodegradable membranes was tested to repair CDH in a growing pig model. METHODS: Twelve male pigs were randomly assigned to 3 different groups of 4 animals each, determined by the type of patch used during thoracoscopic diaphragmatic hernia repair (Gore-Tex®, polycaprolactone electrospun membrane-PCLem, and decellularized human chorion membrane-dHCM). After 7 weeks, all animals were euthanized, followed by necropsy for diaphragmatic evaluation and histological analysis. RESULTS: Thoracoscopic defect creation and diaphragmatic repair were performed without any technical difficulty in all groups. However, hernia recurrence rate was 0% in Gore-Tex®, 50% in PCLem and 100% in dHCM groups. At euthanasia, Gore-Tex® patches appeared virtually unchanged and covered with a fibrotic capsule, while PCLem and dHCM patches were replaced by either floppy connective tissue or vascularized and floppy regenerated membranous tissue, respectively. CONCLUSION: Gore-Tex® was associated with a higher survival rate and lower recurrence. Nevertheless, the proposed biodegradable membranes were associated with better tissue integration when compared with Gore-Tex®.

Glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3D culture.

Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.

Characterization of technical skill progress in a standardized rabbit model for training in laparoscopic duodenal atresia repair.

BACKGROUND: Laboratory skills training is an essential step before conducting minimally invasive surgery in clinical practice. Our main aim was to develop an animal model for training in clinically highly challenging laparoscopic duodenal atresia repair that could be useful in establishing a minimum number of repetitions to indicate safe performance of similar interventions on humans. MATERIALS AND METHODS: A rabbit model of laparoscopic duodenum atresia surgery involving a diamond-shaped duodeno-duodenostomy was designed. This approach was tested in two groups of surgeons: in a beginner group without any previous clinical laparoscopic experience (but having undergone previous standardized dry-lab training, n = 8) and in an advanced group comprising pediatric surgery fellows with previous clinical experience of laparoscopy (n = 7). Each participant performed eight interventions. Surgical time, expert assessment using the Global Operative Assessment of Laparoscopic Skills (GOALS) score, anastomosis quality (leakage) and results from participant feedback questionnaires were analyzed. RESULTS: Participants in both groups successfully completed all eight surgeries. The surgical time gradually improved in both groups, but it was typically shorter in the advanced group than in the beginner group. The leakage rate was significantly lower in the advanced group in the first two interventions, and it reached its optimal level after five operations in both groups. The GOALS and participant feedback scores showed gradual increases, evident even after the fifth surgery. CONCLUSIONS: Our data confirm the feasibility of this advanced pediatric laparoscopic model. Surgical time, anastomosis quality, GOALS score and self-assessment parameters adequately quantify technical improvement among the participants. Anastomosis quality reaches its optimal value after the fifth operation even in novice, but uniformly trained surgeons. A minimum number of wet-lab operations can be determined before surgery can be safely conducted in a clinical setting, where the development of further non-technical skills is also required.

ASSOCIATION OF SERUM VASOGENIC AND PROINFLAMMATORY FACTORS WITH CLINICAL RESPONSE TO ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR FOR DIABETIC MACULAR EDEMA.

PURPOSE: To study the role of serum biomarkers as prognostic factors for qualitative and quantitative response to anti-vascular endothelial growth factor injections for diabetic macular edema (DME). METHODS: Sixty-seven eyes with DME were treated with intravitreal bevacizumab during a 12-month follow-up period. All cases underwent a baseline workup consisting of 12 inflammatory, metabolic and prothrombotic factors. The following outcomes were evaluated at 3-month intervals until 1 year of follow-up: visual acuity, central subfield thickness (CST), macular volume (MV), % of change from baseline in CST, occurrence of a CST change < 10%, a CST change >20%, and a CST <330 µm, achieving an improvement ≥2 lines of visual acuity, achieving visual acuity ≥20/40. RESULTS: A significant improvement in CST and visual acuity was seen from third month onwards. Twenty-eight (48.1%) cases were classified as "early responders," 24 (35.8%) as "late responders", and 15 (22.4%) as "poor responders." Serum vascular endothelial growth factor-A levels were significantly lower in "poor responders" (P = 0.006). C-reactive protein (hsCRP) was associated with a limited anatomic response (<10% CST change) (P = 0.002, OR = 1.845, cutoff value of hsCRP = 1.84 mg/L). hsCRP was also negatively associated with obtaining a final CST <330 µm (P = 0.04, r2 = 0.112, OR = 0.643). Baseline visual acuity was significantly associated with 12th month visual acuity (P < 0.001, r2 = 0.602) and also with an improvement ≥2 visual acuity lines (P = 0.009, OR = 20.54). CONCLUSION: Increased high-sensitivity C-reactive protein was associated with limited anatomic response to anti-vascular endothelial growth factor treatment and persistent DME. Poor responders had significantly lower values of serum vascular endothelial growth factor-A, suggesting an alternative pathogenic pathway for persisting DME.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. METHODS: The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and ß-Catenin were quantified by Western blot. RESULTS: Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho ß-Catenin, and BMP4. CONCLUSIONS: These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia.

OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.

STATs in Lung Development: Distinct Early and Late Expression, Growth Modulation and Signaling Dysregulation in Congenital Diaphragmatic Hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a life-threatening developmental anomaly, intrinsically combining severe pulmonary hypoplasia and hypertension. During development, signal transducers and activators of transcription (STAT) are utilized to elicit cell growth, differentiation, and survival. METHODS: We used the nitrofen-induced CDH rat model. At selected gestational time points, lungs were divided into two experimental groups, i.e., control or CDH. We performed immunohistochemistry and western blotting analysis to investigate the developmental expression profile of the complete family of STATs (STAT1-6), plus specific STATs activation (p-STAT3, p-STAT6) and regulation by SOCS (SOCS3) in normal lungs against those of diseased lungs. The normal fetal lung explants were treated with piceatannol (STAT3 inhibitor) in vitro followed by morphometrical analysis. RESULTS: Molecular profiling of STATs during the lung development revealed distinct early and late expression signatures. Experimental CDH altered the STATs expression, activation, and regulation in the fetal lungs. In particular, STAT3 and STAT6 were persistently over-expressed and early over-activated. Piceatannol treatment dose-dependently stimulated the fetal lung growth. CONCLUSION: These findings suggest that STATs play an important role during normal fetal lung development and CDH pathogenesis. Moreover, functionally targeting STAT signaling modulates fetal lung growth, which highlights that STAT3 and STAT6 signaling might be promising therapeutic targets in reducing or preventing pulmonary hypoplasia in CDH.

Neurodevelopment impact of CO2-pneumoperitoneum in neonates: experimental study in a rat model.

BACKGROUND: Laparoscopy is becoming more common in neonates. However, concerns remain about the impact of the carbon-dioxide (CO2)-insufflation over the neonatal brain. We aim to evaluate the peripheral (serum) and central (cerebrospinal fluid [CSF]) cytokine response after neonatal CO2-pneumoperitoneum and its impact over neurodevelopmental milestones acquisition and long-term behavioral outcomes. MATERIALS AND METHODS: Rats were subjected to a systematic assessment of neurodevelopmental milestones between postnatal day 1 (PND 1) and PND 21. At PND 10, neonatal rats were anesthetized, mechanically ventilated, and exposed to different pressures and times of abdominal CO2-insufflation. Immediately after pneumoperitoneum, corticosterone was analyzed in serum. Twenty-four hours after intervention, serum and CSF were collected to assess inflammatory response (interleukin [IL]-10, IL-1ß, tumor necrosis factor [TNF]-α, and interferon [IFN]-γ). In adulthood, animals from each group were submitted to several tests to assess different behavioral domains (locomotion, anxiety, mood, and cognition). RESULTS: The antiinflammatory cytokine IL-10 was significantly increased in CSF in CO2-insufflated groups, with no other significant changes in the other biomarkers. Acquisition of neurodevelopmental milestones was maintained in all studied groups. No significant differences were observed in adult behavior in the different CO2-insufflation conditions. CONCLUSIONS: Neonatal CO2-pneumoperitoneum does not seem to have any negative impact on neurodevelopment or induce behavioral alterations in adulthood. Minimally invasive surgery results in a central antiinflammatory profile, and further studies on the functional consequences of these phenomena are needed.

Retinoic acid regulates avian lung branching through a molecular network.

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarß) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfß2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarß, meis2, hoxb5, tgfß2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfß2, and id2 spatial distribution; to increase rarß, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.

Thoracoscopic repair of congenital diaphragmatic hernia: review of the results.

Congenital diaphragmatic hernia (CDH) remains one of the major challenges in neonatal surgery. Survival rate has increased in the last decades mainly due to perinatal care and surgical technique improvements. Classically, a laparotomy has been performed after cardiovascular and respiratory stabilization. Introduction of thoracoscopy in the repair of CDH brought the known advantages of reduced postoperative pain and better cosmesis. However, its safety and effectiveness have been questioned in the last few years. Although there is lack of high evidence data, it seems consensual that thoracoscopy is associated with: 1) longer operative time on account of the learning curve; 2) increased acidosis during surgery whose effects have not been clarified but there is evidence suggesting that neurodevelopment is not affected; 3) reduced morbidity, namely postoperative ileum and adhesions, pain and scar formation; 4) similar mortality rate; 5) higher number of recurrences. While the majority of outcomes are comparable between open and thoracoscopic repair, reduced postoperative morbidity and better cosmesis are advantages to be considered. Technique improvements are still required to reduce recurrence rate. Division of the pleura from the peritoneum is a major step of the procedure; suture type and quality must simulate those in the open repair. The authors believe that careful and meticulous execution of the principles of open surgery will improve outcomes regarding recurrence.

Mauritian cynomolgus macaques with M3M4 MHC genotype control SIVmac251 infection.

BACKGROUND: Understanding natural HIV control may lead to new preventative or therapeutic strategies. Several protective major histocompatibility complex (MHC) genotypes were found in humans and rhesus macaques. Here, we report a simian immunodeficiency virus (SIV) controller MHC genotype in Mauritian cynomolgus macaques (MCMs). METHODS: Twelve MHC-genotyped MCMs were infected with SIVmac251 and monitored for viral loads and CD4+ T-cell counts. RESULTS: Two macaques with M3M4 genotype exhibited the lowest peak viral loads (log plasma SIV RNA copies/mL), nearly 3 logs lower than those in most macaques with other MHC haplotype combinations, and set point viral loads below the level of detection limit by RT-qPCR (<2 log RNA copies/mL). They maintained healthy CD4+ T-cell counts of >500 cells/µL blood, while CD4 counts in the vast majority of other macaques were below this level. CONCLUSIONS: The M3M4 MHC genotype may confer enhanced control of SIV replication in MCMs.

Expression analysis of Shh signaling members in early stages of chick lung development.

Lung organogenesis is guided by epithelial-mesenchymal interactions that coordinate cellular events responsible for the formation of the respiratory system. Several signaling pathways have been implicated in this process; among them, sonic hedgehog (Shh) signaling has emerged as a crucial regulator of branching morphogenesis in the mammalian lung. Canonical Shh signaling requires the presence of patched (Ptch) and smoothened (Smo) transmembrane receptors in order to induce the activation of glioblastoma (Gli) zinc finger transcription factors that are the true effectors of the pathway. Signal transduction is finely regulated by Ptch1, Gli, and Hhip (hedgehog-interacting protein). The present work characterizes, for the first time, the expression pattern of shh, ptch1, smo, gli1, and hhip in early stages of the embryonic chick lung. In situ hybridization studies revealed that these genes are expressed in the same cellular compartments as their mammalian counterparts, although their proximo-distal distribution is slightly changed. Moreover, the molecular interactions between fibroblast growth factor (FGF) and Shh signaling pathway were assessed, in vitro, by grafting beads soaked in SU5402 (an FGF receptor inhibitor). In the chick lung, Shh signaling seems to have some features that are species specific since shh is not a downstream target of FGF signaling. Nonetheless and despite the observed differences, these findings suggest a role for Shh signaling in the epithelial-mesenchymal interactions that control chick lung morphogenesis.

Resection of Sentinel Lymph Nodes by an Extraperitoneal Minilaparoscopic Approach Using Indocyanine Green for Uterine Malignancies: A Preclinical Comparative Study.

Background The sentinel lymph node (SLN) concept might minimize surgical aggressiveness in cervical and endometrial malignancies. The aim of the study was to test the feasibility and reliability of minilaparoscopic extraperitoneal SLN excision after indocyanine green (ICG) cervical injection using a high-definition near infrared (NIR) imaging system in an in vivo porcine model. The same procedure was performed using conventional laparoscopic instruments and both outcomes were compared. Methods Twenty-four animals were equally and randomly divided into a minilaparoscopic group (group A) and a 5-mm conventional laparoscopic group (group B). A high-definition NIR imaging system and a 30° ICG endoscope were used. First, ICG (0.5 mL) was injected in the paracervical region. The SLN coloring time was recorded. An extraperitoneal approach to the SLN was executed with the same CO2 retropneumoperitoneum pressures (10 mm Hg). In both groups, the times for SLN localization and excision, as well as complications, were registered. Finally, a laparotomy was then done to evaluate whether any stained SLN still remained. The same surgical team performed all experiments. Results SLNs were identified and extraperitoneally excised in all animals without major complications. The SLN localization varied between animals from external iliac to preaortic regions. The surgical times were shorter with minilaparoscopy (39.3 ± 13 minutes) than with conventional 5-mm instruments (51.3 ± 14.17 minutes; P = .042). In group B, one stained SLN remained and was only detected by laparotomy. Conclusions We confirmed the feasibility and reliability of extraperitoneal minilaparoscopic approach for identification, dissection, and excision of SLN using an NIR imaging system and ICG.

Assessment of Laparoscopic Skills Performance: 2D Versus 3D Vision and Classic Instrument Versus New Hand-Held Robotic Device for Laparoscopy.

INTRODUCTION AND OBJECTIVES: Laparoscopic surgery has undeniable advantages, such as reduced postoperative pain, smaller incisions, and faster recovery. However, to improve surgeons' performance, ergonomic adaptations of the laparoscopic instruments and introduction of robotic technology are needed. The aim of this study was to ascertain the influence of a new hand-held robotic device for laparoscopy (HHRDL) and 3D vision on laparoscopic skills performance of 2 different groups, naïve and expert. MATERIALS AND METHODS: Each participant performed 3 laparoscopic tasks-Peg transfer, Wire chaser, Knot-in 4 different ways. With random sequencing we assigned the execution order of the tasks based on the first type of visualization and laparoscopic instrument. Time to complete each laparoscopic task was recorded and analyzed with one-way analysis of variance. RESULTS: Eleven experts and 15 naïve participants were included. Three-dimensional video helps the naïve group to get better performance in Peg transfer, Wire chaser 2 hands, and Knot; the new device improved the execution of all laparoscopic tasks (P < .05). For expert group, the 3D video system benefited them in Peg transfer and Wire chaser 1 hand, and the robotic device in Peg transfer, Wire chaser 1 hand, and Wire chaser 2 hands (P < .05). CONCLUSION: The HHRDL helps the execution of difficult laparoscopic tasks, such as Knot, in the naïve group. Three-dimensional vision makes the laparoscopic performance of the participants without laparoscopic experience easier, unlike those with experience in laparoscopic procedures.

Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development.

KEY POINTS: Retinoic acid (RA) and ghrelin levels are altered in human hypoplastic lungs when compared to healthy lungs. Although considerable data have been obtained about RA, ghrelin and bombesin in the congenital diaphragmatic hernia (CDH) rat model, neuroendocrine factors have never been associated with the RA signalling pathway in this animal model. In this study, the interaction between neuroendocrine factors and RA was explored in the CDH rat model. The authors found that normal fetal lung explants treated with RA, bombesin and ghrelin showed an increase in lung growth. Hypoplastic lungs presented higher expression levels of the RA receptors α and γ. Moreover bombesin and ghrelin supplementation, in vitro, to normal lungs increased RA receptor α/γ expression whereas administration of bombesin and ghrelin antagonists to normal and hypoplastic lungs decreased it. These data reveal for the first time that there is a link between neuroendocrine factors and RA, and that neuroendocrine factors sensitise the lung to the RA action through RA receptor modulation. ABSTRACT: Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, ß and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ.