Jeffrey J. Dehmer

Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation.

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.

Competence and confidence with basic procedural skills: the experience and opinions of fourth-year medical students at a single institution.

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.

Massive transfusion and blood product use in the pediatric trauma patient.

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.

Expansion of Intestinal Epithelial Stem Cells during Murine Development

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.

IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations

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‐EGFPLow) and reserve/facultative ISCs (Sox9‐EGFPHigh) 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‐EGFPLow ISCs and percentage of these cells in M‐phase. IGF1 increased percentages of Sox9‐EGFPHigh ISCs inS‐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‐EGFPHigh facultative ISCs but not Sox9‐EGFPLow 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.—Van Landeghem, L., Santoro, M. A., Mah, A. T., Krebs, A. E., Dehmer, J. J., McNaughton, K. K., Helmrath, M.A., Magness, S.T., Lund, P. K. IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations. FASEB J. 29, 2828‐2842 (2015). www.fasebj.org

Who teaches basic procedural skills: student experience versus faculty opinion.

INTRODUCTION Learning procedural skills as a medical student has evolved, as task trainers and simulators are now ubiquitous. It is yet unclear whether they have supplanted bedside teaching or are adjuncts to it, and whether faculty or residents are responsible for student skills education in this era. In this study we sought to characterize the experience and opinions of both medical students and faculty on procedural skills training. METHODS Surveys were sent to clinical medical students and faculty at UNC Chapel Hill. Opinions on the ideal learning environment for basic procedural skills, as well as who serves as primary teacher, were gathered using a 4-point Likert scale. Responses were compared via Fisher exact test. RESULTS A total of 237 students and 279 faculty responded. Third-year students were more likely to report simulation as the primary method of education (64%), compared to either fourth-year students (35%; P < 0.0001) or faculty (43%; P = 0.0018). Third- and fourth-year students were also more likely to report interns as a primary teacher (15% and 10%, respectively) as opposed to faculty (2%), and less likely to suggest faculty were the primary teacher (30% and 21%, respectively, versus 35%), P < 0.0001. Residents were the primary teachers for all three groups (55%, 70%, and 63% respectively). CONCLUSIONS Our data suggest that both medical students and faculty recognize the utility of simulation in procedural skills training, but vary in the degree to which they think simulation is or should be the primary instructional tool. Both groups suggest residents are the primary teacher of these skills.

Management of Pediatric Intestinal Failure

Patients and families suffering from intestinal failure are exposed daily to physical, emotional, and fiscal burdens that result in an immeasurable amount of distress [1]. Intestinal failure occurs in these patients because of an inability of their bowel to meet fluid and/or nutritional needs required to sustain normal physiology and growth without parenteral nutritional support. In children, short-bowel syndrome (SBS) is the major cause of intestinal failure and results from both congenital disorders and extensive surgical resection. The common causes of SBS include intestinal atresia, abdominal wall defects (primarily gastroschisis), intestinal volvulus, long-segment Hirschsprung disease, complicated meconium ileus, and necrotizing enterocolitis (30% of cases and the most common cause). Of all pediatric SBS, 80% occurs during the neonatal period. Data on the incidence and mortality related to SBS are sparse. A recent cohort of very low and extremely low birth weight neonates at 16 tertiary centers in the United States demonstrated incidences of SBS at 0.7% and 1.1%, respectively, although this excluded cases in term infants [2]. Given the increase in the overall number and survival of these at-risk patients, it is logical to assume that the overall number of pediatric SBS will continue to increase as well. Mortality in this population of patients occurs in a bimodal fashion, with the first peak corresponding to infants who undergo massive initial bowel resections and a later peak corresponding to complications of SBS, namely central venous catheter sepsis and intestinal failure–associated liver disease (IFALD). Survival rates for children with SBS have been quoted as 73% to 89%, with lower rates in patients requiring chronic total parenteral nutrition (TPN) [3–6]. Recently, the development of formal multidisciplinary programs has greatly improved the care provided to these patients by reducing both the morbidity and mortality associated with intestinal failure [7]. In general, these programs

Localized Intestinal Radiation and Liquid Diet Enhance Survival and Permit Evaluation of Long-Term Intestinal Responses to High Dose Radiation in Mice

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 14Gy-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.