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.

Lower NPAS3 expression during the later stages of abnormal lung development in rat congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is characterized by a developmental defect in the diaphragm, pulmonary hypoplasia and pulmonary hypertension. NPAS3 is a PAS domain transcription factor regulating Drosophila tracheogenesis. NPAS3 null mice develop pulmonary hypoplasia in utero and die after birth due to respiratory failure. We aimed to evaluate NPAS3 expression during normal and abnormal lung development due to CDH. METHODS: CDH was induced by administering 100 mg/ml nitrofen to time-pregnant dams on embryonic day (E) 9 of gestation. Lungs were isolated on E15, E18 and E21 and NPAS3 localization was determined by immunohistochemistry and quantified using Western blotting. RESULTS: We found that only E21 hypoplastic CDH lungs have reduced expression of NPAS3 in the terminal saccules. Western blotting confirmed the down-regulation of NPAS3 protein in the nitrofen-induced hypoplastic lungs. CONCLUSIONS: We demonstrate for the first time that nitrofen-induced hypoplastic CDH lungs have reduced NPAS3 expression in the terminal saccules during the later stages of abnormal lung development. Our findings suggest that NPAS3 is associated with pulmonary hypoplasia in CDH.

Unique Tracheal Fluid MicroRNA Signature Predicts Response to FETO in Patients With Congenital Diaphragmatic Hernia.

OBJECTIVE AND BACKGROUND: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. METHODS: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-ß2 in postnatal lung sections. We investigated miR-200b effects on TGF-ß signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. RESULTS: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-ß2 expression was lower in CDH lungs. miR-200b inhibited TGF-ß-induced SMAD signaling in cultures of human bronchial epithelial cells. CONCLUSIONS: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-ß/SMAD signaling.

MicroRNAs and lung development.

MicroRNAs (miRNAs) constitute a large group of small (∼22 nucleotides), non-coding RNA sequences that are highly conserved among animals, plants and microorganisms, suggesting that microRNAs represent a highly conserved and important regulatory mechanism. They have been demonstrated to play an important role in gene regulation. Recently, miRNAs have become a major focus of interest for research in organ development. Research focusing on the potential role of microRNAs during lung development is slowly starting to emerge. A number of miRNAs have been demonstrated to play important roles during early and late lung development. Several studies have begun to profile miRNA expression at various stages of lung development and this article provides an overview of the various miRNAs that have been implicated in lung organogenesis.

Attending surgeon burnout and satisfaction with the establishment of a regional acute care surgical service.

BACKGROUND: Establishment of the Acute Care Surgical Service (ACSS) has dramatically changed the management of acute, nontrauma surgical patients in Winnipeg, Manitoba. Its formation was partially driven by increasing strain on surgeons and surgical services. We sought to determine surgeon level of burnout and satisfaction with the ACSS. METHODS: All Winnipeg ACSS surgeons were mailed surveys. Burnout was established using the Maslach Burnout Inventory Human Services Survey. Satisfaction was ascertained with a series of questions. RESULTS: We attained a response rate of 76%. Most surgeons were married men with children. A burnout level of 61% was determined. Although most surgeons felt the ACSS was a positive change in their careers, they felt that operating room accessibility and teaching opportunities were lacking. CONCLUSION: Although a high level of burnout exists among ACSS surgeons, most are satisfied with its establishment. Factors such as operating room accessibility and teaching opportunities must be addressed.

Pulmonary development considerations in the surgical management of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia remains a clinical challenge for both neonatologists and pediatric surgeons. Advancements in mechanical ventilation strategies and neonatal intensive care have improved survival and transformed treatment of congenital diaphragmatic hernia from emergent surgery to early stabilization of the newborn followed by delayed repair of the diaphragmatic defect. Surgical technique has evolved and minimally invasive surgical approaches to close the diaphragmatic defect in these babies will likely improve with increasing experience. Finally, as more patients are diagnosed prenatally, attempts have been made to close the diaphragmatic defect prenatally. Unfortunately, this approach did not change the outcome of affected babies. Recently, progress has been made with prenatal tracheal plugging to improve prenatal lung development. In the near future experimental studies will start to explore new ways of treating affected babies prior to birth. This article reviews the evolution of the current treatment strategies in congenital diaphragmatic hernia and its future directions.

Evaluation of a regional acute care surgery service by residents in general surgery.

BACKGROUND: Acute care surgery (ACS) services dedicated to care of acute general surgery patients have been established in many tertiary centers across Canada. Little is known about the impact of this trend on postgraduate education. In this study we aimed to evaluate ACS through a cross-sectional survey of general surgery residents in Winnipeg, Manitoba. METHODS: General surgery residents at the University of Manitoba were asked to complete an anonymous survey. Basic demographic data were obtained. The educational value of ACS was assessed using 10 statements derived from the CanMEDS framework for training physicians. Resident burnout was measured using the Maslach Burnout Inventory, on emotional exhaustion, depersonalization, and personal accomplishment. RESULTS: The response rate was 70% (14/20). ACS was evaluated positively based on the CanMEDS roles by the following proportions of responders: surgical skills (79%), clinical knowledge (100%), communicator (100%), collaborator (100%), manager (86%), health advocate (100%), scholar (64%), and professional (93%). Fifty percent of responders had a high score on emotional exhaustion, 43% on depersonalization, and 0% on low sense of personal accomplishment. The overall burnout was 64%. CONCLUSIONS: ACS provides a comprehensive clinical experience based on the CanMEDS competencies. Despite an increased sense of personal accomplishment, residents experienced a high incidence of burnout, as demonstrated by high scores on emotional exhaustion and depersonalization of patients.

Multicenter study of compliance and drop administration in glaucoma.

BACKGROUND: Poor compliance with medication is a major concern in the management of glaucoma. Improper administration technique can lead to contamination and inaccurate dosing. This study estimates the prevalence and predictors of noncompliance and improper administration technique among Canadian glaucoma patients. METHODS: Data were collected using a standardized questionnaire. Noncompliance was defined as missing at least 1 drop of medication per week and (or) the inability to accurately describe the medication regimen. Patients were asked to indicate the most common reason for missing medication. Study personnel assessed drop administration technique as patients were applying eye drops. Physicians provided information, including measures of disease stability, regarding the patient's glaucoma. Predictors were assessed using odds ratios from a logistic regression model. RESULTS: 500 patients from 10 centers across Canada participated in the study. Of these, 25.6% reported missing at least 1 drop of medication per week, and 4.2% were unable to accurately describe their medication regimen. The overall proportion of noncompliance was 27.9%. With regard to drop administration, 6.8% missed their eye and 28.8% contaminated the bottle tip; overall, 33.8% demonstrated improper technique. The most common reasons given for missing eye drops were "forgetfulness" and "being away from drops." Formal education limited to elementary school and treatment duration of <5 years increased patient-reported noncompliance. Factors associated with improper administration technique were age 60 years and older and formal education limited to elementary school. INTERPRETATION: Over 50% of the patients surveyed were either noncompliant or demonstrated improper administration technique. Glaucoma patients should be educated on the importance of compliance and instructed on proper drop administration.

Risk factors for bile duct injury during laparoscopic cholecystectomy: a case-control study.

Common bile duct injury is a serious but uncommon complication of laparoscopic cholecystectomy. A case-control epidemiologic study of patients who had undergone cholecystectomy in Ontario, Canada, between 1991 and 1997 was performed. Four patients who had undergone a laparoscopic cholecystectomy at the same hospital 2 months prior to a case were selected as controls. The risk of bile duct injury associated with various exposures was estimated by unconditional logistic regression. There were 28 cases and 88 controls. Emergency operation (adjusted odds ratio = 5.0; 95% confidence interval, 1.4-17.8) and failure to identify the cystic duct (adjusted odds ratio = 13.7; 95% confidence interval, 2.5-76.3) were statistically significant risk factors for operative bile duct injury. No other characteristics were independent risk factors for bile duct injury. Failure to identify the cystic duct and the emergency surgery are independent risk factors for bile duct injury.