RESUMO
Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.
Assuntos
Células-Tronco Adultas/classificação , Fator de Crescimento Insulin-Like I/fisiologia , Intestino Delgado/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Animais , Ciclo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiologia , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/classificação , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/tratamento farmacológico , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/fisiopatologia , Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
BACKGROUND: Pediatric electrical and chemical burns are rare injuries, and the care of these patients varies significantly. We reviewed our experience in management of electrical and chemical burns to analyze the clinical course, management, and outcomes. METHODS: A retrospective review was conducted on children with chemical and electrical burns presenting to two large regional pediatric burn centers over a 10-y period (2002-2012). Clinical data including patient demographics, nature of burns, management, and outcomes were collected and analyzed. RESULTS: There were 50 cases, 25 chemical and electrical burns each. Overall, the mean±standard deviation age was 6.2±5.6 y, and the mean total body surface area burn was 4.3±3.2%. Chemical burns were larger, had less depth, and shorter length of stay, whereas electrical burns were smaller, deeper, and had a longer length of stay. Two chemical burns and six electrical burns required grafting. Twelve percent of electrical burns required rehabilitation, and 20% required compression garments for hypertrophic scars. Six percent required late surgeries. CONCLUSIONS: Pediatric electric and chemical burns are rare and require specialized care. Graft rates are not high but are mostly noted in electrical burns.
Assuntos
Queimaduras Químicas/cirurgia , Queimaduras por Corrente Elétrica/cirurgia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Tempo de Internação , Masculino , Estudos RetrospectivosRESUMO
Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.
Assuntos
Proteínas de Fluorescência Verde/biossíntese , Jejuno/efeitos da radiação , Fatores de Transcrição SOX9/biossíntese , Células-Tronco/efeitos da radiação , Animais , Proliferação de Células/efeitos da radiação , Células Cultivadas , Expressão Gênica/efeitos da radiação , Proteínas de Homeodomínio/biossíntese , Jejuno/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/biossíntese , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Regeneração/efeitos da radiação , Proteínas Repressoras/biossínteseRESUMO
Pediatric post-operative abdominal pain can present a unique diagnostic challenge. The case presented here describes a 9-year-old female who presented with fever and worsening abdominal pain 4 days after laparoscopic resection of a benign ovarian teratoma. Computed tomography failed to provide adequate diagnostic imaging. Ultrasound was subsequently used to rule-out a major post-operative complication and ultimately led to a successful non-operative approach while avoiding repeat radiation exposure. Thin body habitus, increased radiosensitivity of pediatric organs, and increased lifetime risk of cancer complicate the use of computed tomography in the pediatric population. Ultrasound, when correlated to clinical findings, has unique advantages over CT such as detailed delineation of soft tissue structures and dynamic assessment of anatomy that make it advantageous in the pediatric post-operative setting.
RESUMO
INTRODUCTION: Traditionally open resection with hepaticojejunostomy (HJ) reconstruction has been the surgical treatment for cases of choledochal cyst. Our center has recently transitioned from open to laparoscopic and HJ to hepaticoduodenostomy (HD) as our preferred method of excision and biliary reconstruction. Our initial experience is presented here. MATERIALS AND METHODS: A single-center retrospective chart review was performed from 2005 to 2014. All patients undergoing surgical treatment for choledochal disease were considered. RESULTS: During the study period 18 patients had surgical treatment for choledochal cyst disease. The average age of all patients was 4.7 years (range, 2 months-15.5 years). Eleven of these patients had laparoscopic excision and reconstruction. Of these 11 patients, 7 had an HD anastomosis. Comparing the laparoscopic with the open group and the HD with the HJ group, there was no significant difference in operative time, estimated blood loss, time to regular diet, length of stay, or complication rate. Mean follow-up of 3.1 years revealed no documented cases of bile reflux or cholangitis. A recent adaptation in technique may improve ease of HD anastomosis. In this method, two strands of temporary monofilament suture cut to 8-10 cm each are tied extracorporeally. This knot is then placed on the outside of the medial corner. The anastomosis is then completed in a running fashion with the two strands and then secured intracorporeally at the lateral corner. CONCLUSIONS: Laparoscopic choledochal cyst resection with both HJ and HD reconstruction appears safe and has equivalent outcomes to open procedures in our series.
Assuntos
Cisto do Colédoco/cirurgia , Duodeno/cirurgia , Jejuno/cirurgia , Laparoscopia , Fígado/cirurgia , Adolescente , Anastomose Cirúrgica/métodos , Criança , Pré-Escolar , Colangite/cirurgia , Feminino , Seguimentos , Humanos , Lactente , Masculino , Duração da Cirurgia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
PURPOSE: Data indicate that students are unprepared to perform basic medical procedures on graduation. The authors' aim was to characterize graduating students' experience with and opinions about these skills. METHOD: In 2011, an online survey queried 156 fourth-year medical students about their experience with, and actual and desired levels of competence for, nine procedural skills (Foley catheter insertion, nasogastric tube insertion, venipuncture, intravenous catheter insertion, arterial puncture, basic suturing, endotracheal intubation, lumbar puncture, and thoracentesis). Students self-reported competence on a four-point Likert scale (4=independently performs skill; 1=unable to perform skill). Data were analyzed by analysis of variance and Student t test. A five-point Likert scale was used to assess student confidence. RESULTS: One hundred thirty-four (86%) students responded. Two skills were performed more than two times by over 50% of students: Foley catheter insertion and suturing. Mean level of competence ranged from 3.13±0.75 (Foley catheter insertion) to 1.7±0.7 (thoracentesis). A gap in desired versus actual level of competence existed for all procedures (P<.0001). There was a correlation between the number of times a procedure had been performed and self-reported competence for all skills except arterial puncture and suturing. CONCLUSIONS: Participants had performed most skills infrequently and rated themselves as being unable to perform them without assistance. Strategies to improve student experience and competence of procedural skills must evolve to improve the technical competency of graduating students because their current competency varies widely.
Assuntos
Competência Clínica/estatística & dados numéricos , Educação de Graduação em Medicina , Autoeficácia , Estudantes de Medicina/psicologia , Adulto , Análise de Variância , Competência Clínica/normas , Educação de Graduação em Medicina/métodos , Educação de Graduação em Medicina/normas , Educação de Graduação em Medicina/estatística & dados numéricos , Feminino , Humanos , Masculino , North Carolina , AutorrelatoRESUMO
BACKGROUND: In vivo studies of high dose radiation-induced crypt and intestinal stem cell (ISC) loss and subsequent regeneration are typically restricted to 5-8 days after radiation due to high mortality and immune failure. This study aimed to develop murine radiation models of complete crypt loss that permit longer-term studies of ISC and crypt regeneration, repair and normalization of the intestinal epithelium. METHODS: In C57Bl/6J mice, a predetermined small intestinal segment was exteriorized and exposed to 14 Gy-radiation, while a lead shield protected the rest of the body from radiation. Sham controls had segment exteriorization but no radiation. Results were compared to C57Bl/6J mice given 14 Gy-abdominal radiation. Effects of elemental liquid diet feeding from the day prior to radiation until day 7 post-radiation were assessed in both models. Body weight and a custom-developed health score was assessed every day until day 21 post-radiation. Intestine was assessed histologically. RESULTS: At day 3 after segment radiation, complete loss of crypts occurred in the targeted segment, while adjacent and remaining intestine in segment-radiated mice, and entire intestine of sham controls, showed no detectable epithelial damage. Liquid diet feeding was required for survival of mice after segment radiation. Liquid diet significantly improved survival, body weight recovery and normalization of intestinal epithelium after abdominal radiation. Mice given segment radiation combined with liquid diet feeding showed minimal body weight loss, increased food intake and enhanced health score. CONCLUSIONS: The segment radiation method provides a useful model to study ISC/crypt loss and long-term crypt regeneration and epithelial repair, and may be valuable for future application to ISC transplantation or to genetic mutants that would not otherwise survive radiation doses that lead to complete crypt loss. Liquid diet is a simple intervention that improves survival and facilitates long-term studies of intestine in mice after high dose abdominal or segment radiation.
Assuntos
Alimentos Formulados , Intestino Delgado/fisiologia , Intestino Delgado/efeitos da radiação , Modelos Animais , Regeneração/fisiologia , Células-Tronco/efeitos da radiação , Análise de Variância , Animais , Mucosa Intestinal/efeitos da radiação , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Estatísticas não ParamétricasRESUMO
Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.
Assuntos
Biomarcadores/metabolismo , Linhagem da Célula , Células Epiteliais/citologia , Intestinos/citologia , Células-Tronco/citologia , Animais , Células Epiteliais/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismoRESUMO
Hemorrhagic shock in the pediatric trauma patient is an uncommon but fundamental problem for the treating clinician. Current management of hemorrhagic shock involves initial resuscitation with crystalloid fluids followed by infusion of blood components as necessary. In management of the adult trauma patient, many institutions have implemented massive transfusion protocols to guide transfusion in situations requiring or anticipating the use of greater than 10 U of packed red blood cells. In the pediatric population, guidelines for massive transfusion are vague or nonexistent. Adult trauma transfusion protocols can be applied to children until a pediatric protocol is validated. Here, we attempt to identify certain principles of transfusion therapy specific to pediatric trauma and outline a sample pediatric massive transfusion protocol that may be used to guide resuscitation. Also, adjuncts to transfusion, such as colloid fluids, other plasma expanders or hemoglobin substitutes, and recombinant activated factor VII, are discussed.