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  1. 1. 28th International Symposium on Pediatric Surgical Research ABSTRACT BOOK DUBLIN, IRELAND – 24-26 SEPTEMBER, 2015
  2. 2. 28th International Symposium on Pediatric Surgical Research 1 FRIDAY, 25TH SEPTEMBER, 2015 SCIENTIFIC SESSION I (PRIZE SESSION)
  3. 3. 28th International Symposium on Pediatric Surgical Research 2 THREE- AND FOUR-DIMENSIONAL ANALYSIS OF ALTERED BEHAVIOR OF ENTERIC NEURAL CREST DERIVED CELLS IN THE HIRSCHSPRUNG’S DISEASE MOUSE MODEL N Nakazawa-Tanaka1,2 , K Miyahara2 , N Fujiwara2 , M Urao1 , C Akazawa3 , G J. Lane2 , A Yamataka2 1 Department of Pediatric Surgery, Juntendo Nerima Hospital 2 Department of Pediatric Surgery, Juntendo University School of Medicine, 3 Department of Biochemistry and Biophysics, Graduate School of Health Care Science, Tokyo Medical and Dental University Tokyo, Japan Email address for correspondence: nana.nakazawa@gmail.com PURPOSE The behavior of enteric neural crest-derived cells (ENCC) during enteric nervous system (ENS) development is being gradually understood by the introduction of live-cell imaging technique. However, many of the analysis to date are two- dimensional and the precise multidirectional migration of ENCC has been challenging to interpret. Mice lacking the endothelin-B receptor gene, EDNRB(-/-) mice, are widely used as a model for Hirschsprung’s disease (HD). We have recently developed a SOX10 transgenic mouse to visualize ENCC with enhanced green fluorescent protein (VENUS). By breeding these two models, we have created a VENUS-positive, SOX10 transgenic mouse with a deletion of the EDNRB gene, SOX10-VENUS(+)/EDNRB(-/-) mouse, to investigate the ENS in HD. The aim of this study was to investigate the behavior of migrating ENCC in the hindgut of the SOX10-VENUS(+)/EDNRB(-/-) mouse by using three-dimensional and 4-dimensional (3D/4D) image analysis software. METHODS To compare the ENCC behavior when the wavefront of ENCC reaches the mid-hindgut between HD mouse and control, we harvested the fetal hindguts from SOX10-VENUS(+)/EDNRB(-/-) mice on D15.5 and SOX10-VENUS(+)/EDNRB(+/+) mice on D12.5, which was used as control. Dissected hindguts were cultured for 360 minutes and the time-lapse images were obtained using a confocal laser-scanning microscope. Each ENCC at the wavefront was tracked after adjusting the longitudinal axis of the gut to the Y axis and analyzed by using Imaris software (Bitplane, Zurich, Switzerland).
  4. 4. 28th International Symposium on Pediatric Surgical Research 3 RESULTS Track displacement (TD) of ENCC advancement in the rostral-caudal (vertical) plane was represented by the Y axis (TD-Y). Horizontal ENCC advancement and depth of ENCC advancement were represented by the X and Z axes as TD-X and TD-Z (Figure). Mean TD-Y was 34.56 µm in HD, but 63.48 µm in controls. Mean TD-X and TD- Z were similar in both groups. The mean track speeds were decreased in HD (72.87 µm/h) compared to controls (248.29 µm/h). CONCLUSIONS Our results showed that the track speed and ENCC advancement in a rostral-caudal direction were markedly decreased in the HD mice compared to controls. This technique provides added information by tracking ENCC with depth perception, which has potential for further elucidating the altered behavior of ENCC in HD. Figure Control Control
  5. 5. 28th International Symposium on Pediatric Surgical Research 4 CHARACTERIZATION OF VASCULOGENIC POTENTIAL OF HUMAN ADIPOSE-DERIVED ENDOTHELIAL CELLS IN A THREE-DIMENSIONAL VASCULARIZED SKIN SUBSTITUTE A. S. Klar1,2 , S. Güven3 , J. Zimoch1,2 , T. Biedermann1,2 , S. Böttcher-Haberzeth1,2,4 , C. Meuli-Simmen5 , Ivan Martin3 , A. Scherberich3 , E. Reichmann1,2 , M. Meuli2,4 (1) Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland (2) Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland (3) Department of Biomedicine, University Hospital of Basel, University of Basel, Basel, Switzerland (4) Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland (5) Department of Plastic, Reconstructive, Esthetical and Hand Surgery, Kantonsspital Aarau, Aarau, Switzerland Corresponding author: Martin.Meuli@kispi.uzh.ch Purpose: The clinical need for vascularized clinically applicable skin substitutes continues to grow. In this study, we explored the use of human endothelial cells derived from freshly isolated adipose stromal vascular fraction (SVF) in a 3D co-culture model of vascularized bio-engineered skin substitute. Methods: The SVF was isolated from human adipose tissue samples and keratinocytes from human skin biopsies. The SVF, in particular endothelial cells were characterized using flow cytometry and immuofluorescence analysis. Endothelial and mesenchymal progenitors from the SVF formed blood capillaries after seeding into a 3D collagen type I hydrogel in vitro. Subsequently, human keratinocytes were seeded on the top of those hydrogels to develop a vascularized dermo-epidermal skin substitute. Results: Flow cytometric analysis of surface markers of the freshly isolated SVF showed the expression of mesenchymal/stromal cell-associated markers (CD44, CD73, CD90, CD105), stem cell markers (CD133, CD49f), endothelial markers (CD31, CD34), and additionally hematopoietic markers (CD14, CD45). Further analysis of adipose-derived endothelial cells (ECs) revealed the co-expression of CD31, CD34, CD90, CD105, and partially CD146 on these cells. ECs were separated from adipose- stromal cells (ASCs) using FACS sorting. ASCs and ECs cultured separately in a 3D hydrogel for 3 weeks did not form any vascular structures. Only if co-cultured, both cell types aligned to develop a ramified vascular network in vitro with continuous endothelial lumen formation. Transplantation of those 3D-hydrogels onto immuno-incompetent rats displayed a rapid connection of human capillaries with the host vessels and formation of functional, blood-perfused mosaic human-rat vessels within only 3-4 days. Conclusions: Adipose tissue represents an attractive cell source due to the ease of isolation and abundance of endothelial and mesenchymal cell lineages. Adipose-derived SVF cells exhibit the ability to form microvascular structures in vitro and support the accelerated blood perfusion in skin substitutes when transplanted. Keywords: Adipose stem cells - Vascular network formation - Blood capillaries - Angiogenesis - Stromal vascular fraction - Mesenchymal cells - Endothelial cells - Skin tissue engineering
  6. 6. 28th International Symposium on Pediatric Surgical Research 5 Surfactant protein D attenuates lipopolysaccharide-induced inflammation in TLR-4-overexpressed human intestinal cells R Saka1) 2) 3) , T Wakimoto3) 4) , F Nishiumi3) , T Sasaki1) , S Nose1) , M Fukuzawa5) , T Oue1) , I Yanagihara3) , H Okuyama2) 1) Department of Pediatric Surgery, Hyogo College of Medicine 2) Department of Pediatric Surgery, Osaka University Graduate School of Medicine 3) Department of Developmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health 4) Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine 5) Department of Pediatric Surgery, Osaka Medical Center and Research Institute for Maternal and Child Health Address correspondence; Hiroomi Okuyama, MD, Ph. D Department of Pediatric Surgery, Osaka University Graduate School of Medicine Address: 2-2 Yamadaoka, Suita-shi, Osaka, Japan 5650871 E-mail: okuyama@pedsurg.med.osaka-u.ac.jp Abstract PURPOSE: Necrotizing enterocolitis (NEC) is a devastating disease specific to preterm infants. Recently, it has been reported that overexpressed toll-like receptor (TLR)-4 in the immature intestine is associated with the development of NEC. Surfactant protein (SP)-D is a member of the collectin family and plays an important role in innate immunity, particularly in the airways. Although SP-D also exists in the intestines, little is known about its function. The aim of this study was to investigate whether SP-D attenuates inflammation in TLR4 overexpressed intestinal cells. METHODS: All experimental procedures were performed using the human intestinal cell line INT407 originally derived from human embryonal intestines. Platelet activating factor (PAF), reported to be elevated in NEC patients, was used to overexpress TLR-4 in this cell line to mimic an immature intestine. Following stimulation with PAF, the TLR4 expression was measured using quantitative real- time PCR (normalized to GAPDH). The degree of inflammation induced by PAF and lipopolysaccharide (LPS) and the effects of SP-D were assessed with interleukin 8 (IL-8) measured by enzyme-linked immunosorbent assay (ELISA). The results of the quantitative RT-PCR and ELISA analyses are expressed as the mean ± SD. RESULTS: Following treatment with 5 µM of PAF, the expression levels of TLR4 mRNA were significantly upregulated (3.69+/-0.28) compared with that observed in the cells treated without PAF stimulation (1.00+/-0.10) (p<0.001). Stimulation with 5 µM of PAF and 100ng/ml of LPS resulted in significantly higher IL-8 production (1959.3+/-52.3 pg/ml) compared with control (141.2+/-12.4 pg/ml), LPS alone (167.3+/-65.8 pg/ml) and PAF alone (1527.2+/-129.4 pg/ml) (P<0.05) The IL-8 expression with the stimulation of PAF and LPS (1590.1+/-319.3 pg/ml) was attenuated by pretreatment with 20µg/ml of SP-D (1161.6+/-131.6 pg/ml) (p<0.05).
  7. 7. 28th International Symposium on Pediatric Surgical Research 6 CONCLUSION: SP-D attenuates LPS-induced IL-8 production in TLR4 overexpressed intestinal cells. These findings suggest that SP-D may have a protective effect in the development of NEC in preterm infants.
  8. 8. 28th International Symposium on Pediatric Surgical Research 7 NOTOCHORD MANIPULATION DOES NOT IMPACT OESOPHAGEAL AND TRACHEAL FORMATION FROM ISOLATED FOREGUT IN 3D EXPLANT CULTURE. Danielle Mc Laughlin1, 2 , Paula Murphy1 , Prem Puri2 (1) School of Natural Sciences, Trinity College Dublin, (2) National Children’s Research Centre, Our Lady’s Children’s Hospital Crumlin, DUBLIN, IRELAND, Background: Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of the notochord signaling on the adjacent foregut is not known. It was the purpose of this study to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. Methods: Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48 hours with native notochord intact (n=6), notochord removed (n=10) or additional notochord transplanted from stage matched controls (n=8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. Results: Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord (Fig A,B). In specimens with additional notochord transplants foregut morphology and molecular patterning was comparable to controls whether or
  9. 9. 28th International Symposium on Pediatric Surgical Research 8 not the native notochord was maintained (Fig C to G). In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. Conclusion: The relationship between the embryonic foregut and notochord is complex and ill- defined however the notochord does not contribute essentially to oesophagus and trachea formation beyond E9 in the mouse, and the transplantation of additional notochord does not disrupt foregut separation in 3D explant culture. Figure: Immunohistochemistry for Hnf3b and Sox2 on isolated foregut explant cryosections with (A) or without their native notochord (B) showing foregut (fg) division into oesophagus (oe) and trachea (tr). Transplant of additional notochord (D,E,G) does not impact foregut separation or molecular patterning compared to controls (C and F).
  10. 10. 28th International Symposium on Pediatric Surgical Research 9 IMPAIRED CYTOSKELETAL ARRANGEMENTS AND FAILURE OF VENTRAL BODY WALL CLOSURE IN CHICK EMBRYOS TREATED WITH ROCK INHIBITOR (Y-27632) Johannes W DUESS1,2 , Prem PURI1,2,3 , Jennifer THOMPSON1,2 (1) National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland (2) School of Medicine and Medical Science, University College Dublin, Ireland (3) Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland Purpose: Rho-associated kinase (ROCK) signaling regulates numerous fundamental developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. ROCK knockout mice exhibit a ventral body wall defect (VBWD) phenotype due to disorganization of actin filaments at the umbilical ring. However, the exact molecular mechanisms leading to VBWD still remain unclear. Improper somitogenesis has been hypothesized to contribute to failure of VBW closure. Adhesion molecules and connexins are involved in multiple morphological events, including somite formation, through cytoskeletal anchorage. Vinculin and microtubules are crucial for normal cell migration by binding to adhesion molecules and connexins. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) disrupts cytoskeletal arrangements in morphology during early chick embryogenesis, which may contribute to the development of VBWD. Methods: At 60h incubation, chick embryos were explanted into shell-less culture and treated with 50µL of vehicle for controls (n=33) or 50µL of 500µM of Y-27632 for the experimental group (Y-27, n=56). At 8h post-treatment, RT-PCR was performed to evaluate mRNA levels of N-cadherin, E- cadherin and connexin43. Immunofluorescence confocal microscopy was performed to analyze the expression and distribution of actin, vinculin and microtubules in the neural tube and somites. A further cohort of embryos was treated in ovo by dropping 50µL of vehicle or 50µL of 1000µM of Y- 27632 onto the embryo and allowing development to 14d for further assessment. Results: Gene expression levels of N-cadherin, E-cadherin and connexin43 were significantly decreased in treated embryos compared with controls (p<0.05) (Fig.1). Thickened actin filament bundles were recorded in the neural tube of Y-27 embryos. In somites, cells were disorientated with reduced actin distribution in affected embryos (Fig.2). Vinculin was over-expressed and clumped in the neural tube and somites (Fig.3), whereas reduced expression of tubulin was observed in Y-27 embryos compared with controls (Fig.4). At 14d of development, affected embryos presented with an enlarged umbilical ring and herniation of abdominal contents, associated with malformed lower limbs (Fig.5).
  11. 11. 28th International Symposium on Pediatric Surgical Research 10 Conclusion: ROCK inhibition impairs cytoskeletal arrangement during early chick embryogenesis, which may contribute to failure of anterior body wall closure causing VBWD at later stages of development.
  12. 12. 28th International Symposium on Pediatric Surgical Research 11 INTESTINAL MICROBIOTA OF INFANTS REQUIRING SURGERY ANALYSIS USING NEXT-GENERATION DNA SEQUENCING H. Murakami1 , Y. Shimomura2 , M. Matsumoto2 , GJ.Lane1 , A. Yamataka1 , M. Okawada1 1) Department of Pediatric General and Urogenital Surgery Juntendo University School of Medicine, Tokyo, Japan 2) Research Laboratories, Kyodo Milk Industry Co., Ltd, Tokyo, Japan E-mail address of corresponding author: manabu-o@juntendo.ac.jp PURPOSE: Development of the intestinal environment in infants is characterized by rapid, large changes in the abundance, diversity, and composition of microbiota under the influence of various factors that strongly influence both adult intestinal microbiota and development of the adult intestinal immune system. Probiotic therapy has been proposed as an effective way to enhance the intestinal environment during times of stress but there are few reports about probiotic use in children. The aim of this study was to conduct a comprehensive investigation of the intestinal microbiota of infants undergoing surgery using a next-generation DNA sequencing system and assess the value of probiotic administration. METHODS: We studied 8 infants requiring surgery. All were born at our institution. The probiotic preparation used was Bifidobacterium lactis LKM512 (LKM) 1.0 g/day administered orally. The 8 subjects were divided into two groups according to LKM use; LKM+ (n=4) and LKM- (n=4). As controls we included 2 infants who did not require surgery and who were not given LKM (C; n=2). Clinical examinations were performed according to our standard postoperative protocol and stool specimens (0.5g) were collected 5 times (after birth, and on days 3, 7, 10, and 14 after surgery). Extracted fecal DNA underwent PCR amplification for 16S rRNA amplicon library preparation and all stool specimens were sequenced with an Ion TorrentTM PGMTM Sequencer. RESULTS: Clinical status and all laboratory data were similar for LKM+ and LKM-. All stool specimens were successfully sequenced using only 20mg of stool. The microbiota in C stool was relatively varied, with Enterobacteriaceae, Bacteroidaceae, Bifidobacteriaceae, and Streptococcaceae identified in descending order of abundance. There were significantly more Enterococcaceae, Streptococcaceae, and Staphylococcaceae in both LKM+ and LKM- compared with controls. Unexpectedly, there were significantly more Bifidobacteriaceae in LKM- (39.8%) than LKM+ (12.8%) (p<0.005; Figure). CONCLUSION: The impact of surgical stress on intestinal microbiota in infants would appear to be considerable and warrants further investigation and the role of probiotic administration in infants requires clarification. New-generation DNA sequencing was particularly effective for identifying stool microbiota and will facilitate larger scale studies by reducing costs and labor.
  13. 13. 28th International Symposium on Pediatric Surgical Research 12
  14. 14. 28th International Symposium on Pediatric Surgical Research 13 Intraoperative acidosis and hypercapnia during thoracoscopic repair of esophageal atresia/tracheoesophageal fistula Augusto Zani, Ruben Lamas-Pinheiro, Irene Paraboschi, Giovanni Cobellis, Justyna Wolinska, Elke Zani-Ruttenstock, Agostino Pierro Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada PURPOSE: It has been reported that thoracoscopic repair of esophageal atresia/tracheoesophageal fistula (EA/TEF) is associated with severe intraoperative hypercapnia and acidosis. The aim of the present study was to confirm this phenomenon in a larger population from a North-American Institution. METHODS: Following ethical approval (REB1000046653), we retrospectively reviewed the medical charts of all neonates who underwent open or thoracoscopic EA/TEF repair between 2004 and 2014 at our Institution. Only patients with available intraoperative arterial gas values were included in this analysis. Data were compared using paired/unpaired t-tests and linear regression analysis and are reported as mean ± SD or median (range). RESULTS: During the study period, 205 infants underwent open (n=180) or thoracoscopic (n=25) EA/TEF repair. Intraoperative arterial gas values were recorded in 62 (34%) open and in 14 (56%) thoracoscopic operations. There were no differences in birth weight (p=0.2) or gestational age (p=0.06). Both groups had similar preoperative pH (p=0.3, Figure) and PaCO2 (p=0.2) but developed intraoperative acidosis (open: preop vs. intraop p<0.0003; thoracoscopic: preop vs. intraop p<0.0001) and hypercapnia (open: preop vs. intraop p=0.008; thoracoscopic: preop vs. intraop p=0.03). Intraoperatively, infants undergoing thoracoscopic repair had lower pH than those having open surgery (p= 0.01, Figure) and higher levels of hypercapnia (p=0.03). In none of the 5 (20%) patients who had a conversion to open surgery, this was due to ventilatory concerns. Postoperative ventilation was continued for 2 days (0-21) after open surgery and for 3 days (1-8) after thoracoscopy (p=0.9). There was no correlation between intraoperative acidosis and postoperative ventilation duration (r2 = 0.05). CONCLUSIONS: Neonates undergoing surgical repair of EA/TEF develop intraoperative acidosis and hypercapnia regardless the approach used. However, this phenomenon is more severe during thoracoscopic repair. The effects of acidosis and hypercapnia on brain development are unknown. Novel modalities to reduce intraoperative gas derangements particularly during thoracoscopic surgery need to be established.
  15. 15. 28th International Symposium on Pediatric Surgical Research 14 REDUCED EXPRESSION OF VOLTAGE-GATED KV11.1 (HERG) K+ CHANNELS IN AGANGLIONIC COLON IN HIRSCHSPRUNG’S DISEASE Tomuschat C, O’Donnell AM, Coyle D, Puri P National Childrens Research Centre, Ireland, Dublin Our Lady’s Children’s Hospital, Crumlin, Dublin prem.puri@ncrc.ie Purpose: The pathophysiology of Hirschsprung’s Disease (HD) is not fully understood. There is no clear explanation for the occurrence of the spastic or tonically contracted aganglionic segment of bowel. Kv11.1 (HERG) channels play a key role in the regulation of the resting membrane potential as well as affecting either the force or frequency of contraction of smooth muscles. We designed this study to investigate the expression and characterization of HERG channels in the normal colon and in the colon of patients with HD. Methods: We investigated Kv11.1 protein expression in both the ganglionic and aganglionic regions of HD patients (n=10) versus normal control colon (n=10). The distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression was quantified using quantitative real-time polymerase chain reaction, western blot analysis and densitometry. Results: Confocal microscopy of the normal colon revealed strong Kv11.1 channel expression in the enteric neurons, interstitial cells of Cajal (ICCs) and PDGFRα+ cells (Figure 2). HERG expression was markedly decreased in the aganglionic bowel. Colonic HERG gene expression levels were significantly decreased in HD specimen compared to controls (p < 0.003). Western blotting revealed decreased colonic HERG protein expression in aganglionic bowel compared to controls (Figure 1). Conclusions: We demonstrate for the first time the expression and distribution of HERG channels in enteric neurons, ICCs and PDGFRα+ The decreased expression of Kv11.1 (HERG) in the aganglionic colon may be responsible for the increased tone in the aganglionic narrow spastic segment of bowel. Figure 1
  16. 16. 28th International Symposium on Pediatric Surgical Research 15 Figure 2
  17. 17. 28th International Symposium on Pediatric Surgical Research 16 ENHANCED LIVER GENE EXPRESSION OF BILIARY ABC STEROL TRANSPORTERS IS LINKED WITH DEVELOPMENT OF GALLSTONES IN CHILDREN A Koivusalo, A Mutanen, MP Pakarinen Section of pediatric surgery, Pediatric Liver and Gut Research Group, Children´s Hospital, University of Helsinki, Finland mikko.pakarinen@hus.fi PURPOSE: To elucidate the pathogenesis of increasingly common pediatric cholelithiasis, we assessed liver RNA expression of canalicular bile transporters and their upstream nuclear receptor regulators in relation to gallstone sterol content and surrogate serum markers of cholesterol synthesis (precursors) and absorption (plant sterols). METHODS: After ethical approval, twenty children with gallstones (11 females), median age 11 (IQR 8.2) years and six with predisposing conditions underwent cholecystectomy, intraoperative liver biopsy and serum sampling. Liver RNA expression of biliary sterol (ABCG5, ABCG8), phospholipid (ABCB4), bile acid (ABCB11) and bilirubin (ABCC2) transporters and their upstream regulators (LXR and FXR) was measured with real time RT-PCR (ΔΔCt method). Serum and gallstone concentrations of cholesterol, cholesterol precursors (cholestenol and lathosterol) and plant sterols (stigmasterol, avenasterol, sitosterol, and campesterol) were measured using gas-liquid chromatography. Gallstones were classified as cholesterol gallstones (CGS, cholesterol > 35% of weight) or pigment stones (PS). Liver biopsies of six healthy donor livers (n = 6), and serum from 82 healthy children served as controls. RESULTS: As shown in the figure, RNA expression of canalicular sterol transporters (ABCG5 and ABCG 8) and their upstream regulator LXR were 1.5– 2.7 times higher in gallstone patients than in controls (p < 0.02– 0.0003), but were not related with stone or serum cholesterol content, predisposing conditions, age, sex or body mass index. Expression of biliary phospholipid, bile acid and bilirubin transporters were unaltered. Patients with CGS (n = 10) had 1.7 – 2.2 times higher serum cholestenol and lathosterol (p < 0.0001) and 1.3 – 2.0 lower stigmasterol and sitosterol (p < 0.03-0.0001), than patients with PS (n=10) or controls (n=82). CGS contained 4 – 24 times more cholestenol and lathosterol (ug /mg cholesterol) than PS (p = 0.0003 – 0.0005), whereas PS displayed 6.7 times higher concentration of stigmasterol (p = 0.0005). CONCLUSION: Enhanced liver gene expression of LXR and canalicular sterol transport proteins ABCG5 and ABCG8 suggest that increased biliary secretion of sterols, including cholesterol and plant sterols, is characteristic to pediatric gallstone disease. CGS developed in patients with increased cholesterol synthesis, whereas patients with normal cholesterol synthesis developed PS enriched by plant sterols. 0 ,5 1 1,5 2 2,5 3 3,5 Foldexpression(mean,95%CI) LXR ABCG5 ABCB8 control patient * * * * P < 0.04 vs controls
  18. 18. 28th International Symposium on Pediatric Surgical Research 17 BLADDER EXSTROPHY - A NATIONWIDE REGISTER STUDY ON MATERNAL AND FETAL FACTORS IN SWEDEN 1973-2011 G R Engberg1,2 , Ä Mantel3 , M Fossum1,2 , A Nordenskjöld1,2 1. Department of Women’s and Children’s Health and Center of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden 2. Department of Pediatric Surgery, Urology Section, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden 3. Clinical Epidemiology Unit, Department of Medicine, Solna (MedS), K2, Karolinska Institutet, Stockholm, Sweden gisela.reinfeldt.engberg@ki.se PURPOSE Bladder exstrophy is a rare congenital malformation where the underlying cause is largely unknown, but both environmental and genetic mechanisms are involved. The malformation often require multiple interventions by pediatric surgical teams from the newborn period into adolescence and sometimes adulthood. The aim of this study was to conduct a nationwide study related to maternal and fetal risk factors of bladder exstrophy in Sweden 1973-2011. METHODS 120 patients were identified in the Medical Birth Register with ICD codes; 753.50, 753F and Q64.1. Cases were matched with 5 controls per patient for calendar year of delivery and sex. A case-control study was performed by linkage of national registers. We studied additional malformations and environmental factors such as maternal age, BMI, smoking, parity, seasonal differences and assisted conception. We also studied delivery mode, birth weight, gestational week at birth, Apgar, survival rate and length of hospital stay. RESULTS The prevalence was 2.98:100 000 live births with a male-to-female ratio 1.14:1. Bladder exstrophy was an isolated malformation in 93% of the cases excluding inguinal hernia and cryptorchidism. 41% had received surgery for inguinal hernia, 63% of the male and 16% of the female cases. In addition, 11% of the boys had undergone surgery for non-descended testis but none of them were born premature or SGA. There was a tendency of high maternal age, extremes of BMI and smoking as risk factors for bladder exstrophy. Parity, seasonal differences and assisted conception showed no tendency of being risk factors. Delivery mode, birth weight, gestational age at birth, Apgar and survival rate corresponded to controls but patients stayed a few days longer at the hospital after birth. CONCLUSIONS This nationwide study demonstrates a prevalence of 3:100 000 live births with an almost equal sex ratio. Maternal high age, extremes of BMI and smoking appear to be associated with higher risk. The majority of the cases were isolated without major associated malformations. A high percentage of the affected children received surgery for inguinal hernia and non-descended testis. Bladder exstrophy did neither affect birth weight, gestational age at birth, Apgar nor survival rate.
  19. 19. 28th International Symposium on Pediatric Surgical Research 18 FRIDAY, 25TH SEPTEMBER, 2015 SCIENTIFIC SESSION II
  20. 20. 28th International Symposium on Pediatric Surgical Research 19 A NOVEL TREATMENT FOR HYPOPLASTIC LUNG ASSOCIATED WITH CONGENITAL DIAPHRAGMATIC HERNIA BY PRENATAL MATERNAL ADMINISTRATION OF SLOW-RELEASE SYNTHETIC PROSTACYCLIN AGONIST IN EXPERIMENTAL RAT MODEL S. Umeda1 , S. Miyagawa2 , S. Fukushima2 , A. Harada2 , A. Saito2 , Y. Sakai2 , Y. Sawa2 , H. Okuyama1 1 Department of Pediatric Surgery 2 Department of Cardiovascular Surgery Osaka University Graduate School of Medicine 2-2 Yamadaoka, Suita, Osaka, Japan E-mail address of corresponding author; sumeda@pedsurg.med.osaka-u.ac.jp PURPOSE: The major pathogenesis of congenital diaphragmatic hernia (CDH) is delayed lung development during fetal period, which leads to postnatal lethal lung hypoplasia and pulmonary hypertension (PH). It was recently reported that administration of sildenafil or vascular-endothelial growth factor (VEGF) enhanced development of fetal hypoplastic lungs in a rat model of CDH. On the other hand, ONO-1301, a synthetic prostacyclin receptor agonist, has been reported to upregulate proangiogenic and anti-inflammatory factors in a variety of pathologies including a rat model of PH. We therefore hypothesized that prenatal maternal administration of slow release form of ONO-1301 (ONO-1301SR), which is polylactic-co-glycolic acid copolymer (PLGA)-polymerized ONO-1301, would attenuates lung hypoplasia and pulmonary vascular remodeling associated with nitrofen-induced CDH. METHODS: Pregnant Sprague-Dawley rats were gavage fed with nitrofen to produce a CDH-relating lung hypoplasia model on embryonic day (E) 9.5, while control rats received vehicle only. At the same day, ONO-1301SR and placebo were injected subcutaneously. Rats were randomized into 3 groups: control (n=4), nitrofen treatment (n=7), nitrofen+ONO1301SR treatment (n=5). At term (E21.5), the fetal lungs were harvested for further pathobiological evaluation. In nitrofen and nitrofen+ONO1301SR group, only those which developed CDH fetuses were analyzed. RESULTS: The incidence of nitrofen-induced CDH was not influenced by prenatal ONO-1301SR administration. One-seventh of maternal plasma concentration of ONO-1301 was transferred to fetal plasma at E21.5, suggesting this molecule has an efficient placental permeability. Prostacyclin receptor expression was localized in pulmonary arterial smooth muscle cells in the fetus. Lung-to- body weight ratio (%) in the CDH+ONO group (1.88±0.07) was greater than that in the CDH group (1.60±0.04, p<0.01). Histologically, medial wall thickness in the CDH+ONO group was two-third thinner than that in the CDH group (p<0.01). In addition, mean linear intercept, an indicator of pulmonary airspace, were 1.5 times greater in the CDH+ONO group than that in the CDH group (p<0.05). These changes were associated with a greater expression of VEGF in the CDH+ONO group. CONCLUSION: Prenatal ONO-1301SR treatment attenuated lung hypoplasia and pulmonary vascular remodeling in experimental nitrofen-induced CDH, indicating potential of this treatment for pathologies having lung hypoplasia associated with CDH.
  21. 21. 28th International Symposium on Pediatric Surgical Research 20 Antenatal retinoic acid administration promotes lung growth by augmenting retinol levels in the nitrofen model of congenital diaphragmatic hernia Balazs Kutasy1 , Florian Friedmacher1 , Lara Pes2 , David Coyle1 , Takashi Doi1 , Francesca Paradisi2 , Prem Puri1 1:Our Lady's Children's Hospital, National Children's Research Centre, Dublin, Ireland, 2:Centre for Synthesis & Chemical Biology, School of Chemistry & Chemical Biology, University College Dublin, Dublin, Ireland Background: Low pulmonary retinol levels and disrupted retinoid signaling pathway (RSP) has been implicated in the pathogenesis of congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH). It has been demonstrated that nitrofen disturbs the retinol transport. Several studies have demonstrated that prenatal treatment with retinoic acid (RA) can reverse PH in the nitrofen-induced CDH model. We hypothesized that maternal administration of RA can increase fetal serum and pulmonary retinol levels and the gene expression of main components of the RSP in a nitrofen model of CDH. Methods: Pregnant rats were exposed to either olive oil or nitrofen on day9 of gestation (D9) and sacrificed on D21. RA was given intraperitoneally on D18,D19 and D20. Fetal left lungs were dissected on D21 and divided into four groups: control, control+RA, CDH and CDH+RA. Lungs and body weight were measured. Retinol levels were measured using HPLC. Expression levels of the primary RSP-genes (RBPI, RALDH3, RARa, RARb, RXRa) were determined using qRT-PCR and immunohistochemistry. Results: The incidence rate of diaphragmatic hernia was similar in both CDH and CDH+RA group ( 77/131, 58.7% vs 73/127, 57.4%). Both body and lungs weight were significantly higher in the CDH+RA group compared to CDH group (p<0.05)(Table). Serum and pulmonary retinol levels were significantly increased in control+RA compared to control (p<0.05)(Table) and in CDH+RA compared to CDH (p<0.05)(Table). The relative mRNA expression levels of RSP genes were significantly increased in control+RA compared to control (p<0.05) and in CDH+RA compared to CDH (p<0.05) (Table). Markedly increased immunoreactivity of RSP was observed in control+RA and CDH+RA lungs compared to control and CDH (Figure). Conclusions: Our data shows that prenatal treatment with RA elevates fetal retinol levels in lungs and serum. Enhanced retinol levels accompanied by the increased gene expression of main components of RSP suggest that retinol triggered RSP-activation may result in reversal of pulmonary hypoplasia in the nitrofen model.
  22. 22. 28th International Symposium on Pediatric Surgical Research 21 Weight (g) Control Control+RA CDH CDH+RA Left lungs 0.054±0.002 0.059±0.003 0.025 ±0.001*, ** 0.039 ±0.01 Right lungs 0.078 ±0.004 0.085 ±0.001 0.051±0.002*, ** 0.069±0.002 Body 5.1 ±0.08 6.2 ±0.1* 4.68±0.1*, ** 4.98±0.07 *: vs control, p<0.05; **: vs CDH+RA, p<0.05 Retinol level Control Control+RA CDH CDH+RA Serum 0.0872±0.001 0.181±0.001* 0.0151±0.003* 0.195±0.004*, ** Left lungs 0.0816 ± 0.01 0.121±0.01* 0.0717 ± 0.01* 0.151±0.01*, ** *: vs control, p<0.05; **: vs CDH+RA, p<0.05 RBP1 RALDH3 RARa RARb RXRa Control (n=8) 0.78 ± 0.09** 0.03 ± 0.01** 0.54 ± 0.04** 0.08 ± 0.01** 0.14 ± 0.01** Control+RA (n=8) 1.52 ± 0.07* 0.04 ± 0.01* 0.94 ± 0.04* 0.13 ± 0.01* 0.38 ± 0.01* CDH (n=8) 1.02 ± 0.40 0.07 ± 0.01 0.68 ± 0.03 0.09 ± 0.01 0.36 ± 0.03 CDH+RA (n=8) 1.71 ± 0.10** 0.10 ± 0.01** 1.22 ± 0.08** 1.99 ± 0.01** 0.48 ± 0.03** *: vs control, p<0.05; **: vs CDH, p<0.05;
  23. 23. 28th International Symposium on Pediatric Surgical Research 22
  24. 24. 28th International Symposium on Pediatric Surgical Research 23 MESENCHYMAL EXPRESSION OF THE FRAS1/FREM2 GENE UNIT IS DECREASED IN THE DEVELOPING DIAPHRAGM OF NITROFEN-INDUCED CONGENITAL DIAPHRAGMATIC HERNIA T. Takahashi1 , F. Friedmacher1 , J. Zimmer1 , P. Puri1 1 National Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland E-mail: prem.puri@ncrc.ie PURPOSE: Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1- related extracellular matrix2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH. METHODS: Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D13, D15 and D18. Dissected diaphragms (n=72) were divided into two groups: control and nitrofen-exposed samples (n=12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qPCR. Immunofluorescence-double-staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in developing fetal diaphragms. RESULTS: Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in pleuroperitoneal folds of nitrofen-exposed fetuses on D13 (1.76±0.86 vs. 3.09±1.15; p<0.05 and 0.47±0.26 vs. 0.82±0.36; p<0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45±0.80 vs. 2.63±0.84; p<0.05 and 0.41±0.16 vs. 1.02±0.49; p<0.05) and fully muscularized diaphragms of nitrofen-exposed fetuses on D18 (1.35±0.75 vs. 2.32±0.92; p<0.05 and 0.37±0.24 vs. 0.70±0.32; p<0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic FRAS1 and FREM2 immunofluorescence, which was associated with reduced proliferation of diaphragmatic mesenchymal cells in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls (Figure).
  25. 25. 28th International Symposium on Pediatric Surgical Research 24 CONCLUSION: Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.
  26. 26. 28th International Symposium on Pediatric Surgical Research 25 UPREGULATION OF S1P1 RECEPTOR IN THE PULMONARY VASCULATURE OF NITROFEN-INDUCED CONGENITAL DIAPHRAGMATIC HERNIA J. Zimmer1 , T. Takahashi1 , J. W. Duess1,2 , A.D. Hofmann1,3 , P. Puri1,2 1 National Children's Research Centre, Our Lady's Children's Hospital, Dublin,Ireland 2 School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland 3 Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany Purpose: The high morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH) is attributed to severe pulmonary hypoplasia and persistent pulmonary hypertension (PH). Sphingolipids have been demonstrated to play a crucial role in pulmonary development. The sphingosine kinase 1 (SphK1) is an oncogenic enzyme, which modulates the synthesis of sphingolipid sphingosine-1-phosphate (S1P). S1P is a key mediator regulating cell proliferation, migration and angiogenesis via different receptor subtypes, S1P1, S1P2 and S1P3. Recently, the SphK1/S1P pathway has been shown to be involved in adult human pulmonary arterial hypertension by promoting pulmonary artery smooth muscle cell (SMC) proliferation. The aim of this study was to investigate which S1P receptors are responsible for S1P induced abnormal pulmonary vascular remodelling in the nitrofen-induced CDH model. Methods: Pregnant Sprague Dawley rats were exposed to nitrofen or vehicle on D9 of gestation. Fetuses were sacrificed on D21 and divided into nitrofen and control group (n = 12). Pulmonary RNA was extracted and mRNA levels of SphK1, S1P1, S1P2 and S1P3 were determined by quantitative real-time PCR. Western blotting and confocal-immunofluorescence microscopy were performed to determine pulmonary protein expression as well as vascular localization of expressed SphK1, S1P1, S1P2 and S1P3. Results: Confocal-microscopy revealed an increased pulmonary vascular expression of S1P1 (Fig.1) and a decreased expression of S1P2 and S1P3 in lungs of nitrofen-exposed fetuses compared to controls. These results were confirmed by western blotting (Fig.2) and quantitative real-time PCR. SpHK1 expression was not found to be altered in treated rats compared to controls. Conclusion: We show for the first time the increased S1P1 receptor pulmonary vascular expression in the nitrofen induced CDH. These results suggest that S1P1 receptor is involved in the S1P induced pulmonary vascular remodeling resulting in PH in this model.
  27. 27. 28th International Symposium on Pediatric Surgical Research 26 Fig.1
  28. 28. 28th International Symposium on Pediatric Surgical Research 27 POSSIBLE ROLE OF INCREASED OXIDATIVE STRESS IN PULMONARY HYPERTENSION IN EXPERIMENTAL DIAPHRAGMATIC HERNIA R Aras-López1 , JA Tovar1,2 , L Martínez1,2 1 INGEMM and Idipaz Research Laboratory, Hospital Universitario La Paz, Madrid, Spain. 2 Department of Pediatric Surgery, Hospital Universitario La Paz, Madrid, Spain. rosa.aras@hotmail.com PURPOSE Congenital diaphragmatic hernia (CDH) is one of the causes of respiratory failure in newborns due to lung hypoplasia and pulmonary abnormalities leading to pulmonary hypertension (PH). NAD(P)H oxidase (Nox), is a family of isoenzymes, composed of seven members Nox1-5, and the dual oxidases (Duox) Duox1 and Duox2, that generate reactive oxygen species (ROS) such as anion superoxide (•O2 − ) and hydrogen peroxide (H2O2) in pulmonary vascular cells which can contribute to PH-induced vascular dysfunction. Our aim is to examine whether PH-associated to CDH is due to a dysregulation of ROS production in lungs from CDH fetuses. METHODS Pregnant rats received either 100 mg nitrofen or vehicle on E9.5. Fetuses were sacrificed on E21 and divided into control and CDH groups. Fetal lungs were recovered to determine (1) Nox activity by a lucigenin-enhanced chemiluminescence assay, (2) H2O2 production by Amplex Red assay and (3) mRNA levels of Nox1, Nox2 and Nox4 isoenzymes by real-time polymerase chain reaction (RT-PCR). RESULTS Nox activity and Nox1 and Nox2 mRNA levels were increased in the lungs of fetuses with CDH. However, there were no changes in H2O2 production and Nox4 mRNA levels. CONCLUSION These preliminary data highlight the complexity of Nox function in lungs from CDH fetuses, emphasizing that more than one Nox isoenzyme may be involved in Nox-mediated •O2 − production. Moreover, an altered H2O2 production regulated by Nox4 may suggest a malfunction of the detoxification processes in the lungs. The raised oxidative stress seems to be a potential mechanism involved on PH-associated with CDH.
  29. 29. 28th International Symposium on Pediatric Surgical Research 28 MiRacles for babies with abnormal lungs: the story of miR-10a and lung development R. Visser1, C. Fraser2, D. Mulhall2, F. Zhu2, C. Day2 , B. Iwasiow2, T. Mahood2,3, R. Keijzer1,2 1Department of General Surgery, University of Manitoba 2 Department of Pediatric Surgery and Child Health and Children’s Hospital Research Institute of Manitoba, University of Manitoba; Winnipeg, MB, Canada 3 Department of Physiology and Pathophysiology, University of Manitoba; Winnipeg, MB, Canada Corresponding author: richardkeijzer@gmail.com Purpose Worldwide, 150 babies are born every day with congenital diaphragmatic hernia (CDH). One third of these infants will die from respiratory failure due to pulmonary hypoplasia. MicroRNAs are essential epigenetic factors for lung development. We identified microRNA miR-10a as a key regulator in CDH. We aimed to define the role of miR-10a in both normal and abnormal lung development. Methods Using the nitrofen rat model to induce CDH, we employed real-time quantitative polymerase chain reaction (RT-qPCR) and fluorescent in situ hybridization to study miR-10a expression during development. Control- and nitrofen-treated fetal rat lungs were extracted for explant cultures and treated with miR-10a inhibitors and mimics. We are investigating miR-10a’s interaction with the retinoic acid signalling pathway using RT-qPCR and will confirm its participation in vitro using a retinoic acid response element (RARE) dual luciferase assay. Results We found that miR-10a expression is localized in the mesenchyme of fetal lungs. Nitrofen treatment reduces miR-10a’s expression, particularly early in gestation. Using nitrofen-treated fetal rat lungs as explant cultures, we could reverse the hypoplastic phenotype by treating lungs early in development with a miR-10a mimic. We are currently using RT-qPCR and the RARE dual luciferase assay to confirm that miR-10a affects lung development through the retinoic acid signalling pathway. Conclusion In CDH, reduced expression of miR-10a early in development contributes to pulmonary hypoplasia. Treating developing lungs with miR-10a mimics can reverse the hypoplastic phenotype. We believe this microRNA can help to develop a prenatal treatment to improve the outcomes of CDH babies.
  30. 30. 28th International Symposium on Pediatric Surgical Research 29 MiRacles for babies with abnormal lungs: the role of miR-200b in lung development R. Visser1, N. Khoshgoo2, Vinaya Kumar Siragam2,3 , Barbara Iwasiow2,3 , Fuqin Zhu2,3 , Arzu Öztürk4,5 , Sujata Basu3 , Molly Pind4,5 , Agnes Fresnosa4,5 , Gerald Stelmack3 , Geoff Hicks4,5 , Andrew Halayko3 , Richard Keijzer1,2,3 1Department of General Surgery, University of Manitoba; Winnipeg, MB, Canada 2 Department of Pediatric Surgery and Child Health and Children’s Hospital Research Institute of Manitoba, University of Manitoba; Winnipeg, MB, Canada 3Department of Physiology and Pathophysiology, University of Manitoba; Winnipeg, MB, Canada 4 Manitoba Institute of Cell Biology University of Manitoba, Winnipeg, Canada, 5 Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Canada Corresponding author: richardkeijzer@gmail.com Purpose Respiratory failure is the leading cause of neonatal death in infants with congenital diaphragmatic hernia (CDH). Reduced airway branching and a thickened layer of connective tissue characterize the lungs of CDH babies. One explanation for the pathogenesis of CDH is defective epithelial-to- mesenchymal interactions (EMT) during development. We have identified that miR200b is abnormally expressed in CDH lungs. This microRNA is well known as an epigenetic moderator of EMT in oncology. We aimed to define the effects of miR-200b on EMT during normal and abnormal lung organogenesis. Methods We developed a miR-200b knockout mouse that allowed us to localized miR-200b expression with a LacZ reporter gene. We then characterized both lung branching and pulmonary function in fetal and newborn mice. By crossing our miR-200b knockout mice with CFP-ECadherin mice, we were able to study the pulmonary epithelial phenotype in the presence and absence of miR-200b expression. Results LacZ staining revealed high miR-200b expression in fetal lungs. Heterozygous knockout lungs showed reduced branching. Homozygous mice had decreased pulmonary function after birth. CFP-ECadherin miR200b knockout lung explants indicated increased epithelial expression, consistent with altered EMT signaling. Conclusion Epithelial-to-mesenchymal signaling is essential to proper lung development. Our findings support that miR-200b is a key regulator of the crosstalk between the multiple pathways that participate in this capacity. By altering the expression of miR-200b early in development, we believe this microRNA could be the key to developing a prenatal treatment to improve the outcome in CDH babies.
  31. 31. 28th International Symposium on Pediatric Surgical Research 30 DOWNREGULATION OF HEPATIC ADIPOSE DIFFERENTIATION-RELATED PROTEIN (ADRP) IN NITROFEN INDUCED CONGENITAL DIAPHRAGMATIC HERNIA MODEL. Takahashi H.1 , Friedmacher F.1 , Takahashi T.1 , Puri P. 1 National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland, 2 Conway Institue of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland Background: Vitamin A plays an essential role in biologic processes during embryogenesis. Disruption of vitamin A homeostasis is a part of pathogenesis of congenital diaphragmatic hernia accompanied by lung hypoplasia in mammalian and human cases. Since Vitamin A is not be synthesized by the body, mammals absorb all Vitamin A from diet. After absorption, Vitamin A transported to the lipid droplet in hepatic stellate cells for storage. Adipose differentiation-related protein (ADRP) stabilize lipid droplet interface, which is a reliable and sensitive marker for lipid droplets. The teratogen induced nitrofen model has been widely used to investigate the CDH-associated pathology in rodents. It has been reported that nitrofen disturbs the mobilization of retinoid from placenta to fetus. However, retinoid storage in the hepatic lipid droplet in the CDH nitrofen model remains unclear. We designed this study to test the hypothesis that the hepatic ADRP expression is downregulated in the nitrofen-induced CDH model. Methods: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D21, and livers were divided into two groups: controls and nitrofen (n = 24 at each time-point). ADRP gene expression level was analyzed by qRT-PCR. Immunohistochemistry were performed to investigate ADRP protein expression. Results: ADRP gene expression was significantly reduced in liver (ADRP: 1.72±0.19 vs. 0.99±0.10; p=0.00721) and protein expression was markedly decreased in liver. Conclusion: Our findings of downregulation of hepatic ADRP gene expression, suggest that the retinoid storage in hepatic lipid droplets may be impaired in the nitrofen-induced CDH model.
  32. 32. 28th International Symposium on Pediatric Surgical Research 31 Minimal invasive lung support via umbilical vein with a double-lumen cannula in a neonatal lamb model: A proof of principle F.Schmidt1 , J.Kuebler2 , M.Ganter3 , T.Jack1 , L.Meschenmoser4 , M.Sasse1 , M.Boehne1 , H.Bertram1 , P.Beerbaum1 , H.Koeditz1 1 Department of Pediatric Cardiology and Intensive Care Medicine, Medical School Hannover, Carl- Neuberg-Strasse 1, 30625, Hannover, Germany 2 Department of Pediatric Surgery, Medical School Hannover, Germany 3 University of Veterinary Medicine Hannover Foundation, Germany 4 Department of Cardiothoracic Surgery, Transplantation and Vascular Surgery, Medical School Hannover, Germany Corresponding author: Schmidt.florian@mh-hannover.de Purpose: Acute respiratory distress syndrome, with the need for invasive mechanical ventilation (MV), remains a major cause of neonatal mortality and morbidity. Although veno-venous extracorporeal lung support (VV-ECLS) has become a standard of care procedure in neonatal patients with acute pulmonary failure there are no reports regarding the use of a double-lumen cannula for extracorporeal minimal invasive lung support via the umbilical vein. Methods: A near-term neonatal lamb model was used (n=3). Umbilical vein was cannulated with a double-lumen catheter allowing veno-venous extracorporeal gas exchange. Cannula was positioned with its tip in the right atrium. VV-ECLS was started and ventilation was stopped. Providing oxygenation and CO2 removal solely through VV-ECLS hemodynamics, blood gases were measured. Results: Total VV-ECLS without MV was applied to all three neonatal lambs. Time on veno-venous ECLS was 60, 120 and 120min. Initial pCO2 was 60, 55 and 65 mmHg compared to 31, 32 and 32 mmHg at the end of VV-ECLS. Initial pO2 was 30, 27 and 26 mmHg compared to 22, 19 and 23 mmHg. Initial lactate was 5, 10 and 4 mmol/l compared to 13, 13, 11 mmol/l at the end of VV-ECLS. MAP at baseline was 51, 55 and 65 mmHg compared to 36,38 and 41 mmHg at the end of VV-ECLS. In all three lambs inotropes were admitted to maintain MAD >35mmHg.
  33. 33. 28th International Symposium on Pediatric Surgical Research 32 Conclusion: VV-ECLS using a double lumen catheter via the umbilical vein can sufficiently remove pCO2 without MV in a neonatal lamb model for a period of at least 120min. pO2 were measured 22, 19 and 23mmHg respectively at the end of VV-ECLS. At least partially caused by recirculation phenomena, which could possibly be improved by different cannula design. Inotropic support was necessary during VV-ECLS to achieve targeted MAD > 35mmHg.
  34. 34. 28th International Symposium on Pediatric Surgical Research 33 Outcome of esophageal atresia/tracheoesophageal fistula in extremely-low-birth-weight neonates (<1000 grams) Augusto Zani, Giovanni Cobellis, Justyna Wolinska, Priscilla Chiu, Agostino Pierro Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada PURPOSE: Recent advances in intensive care have resulted in a high survival of extremely low birth weight (ELBW, <1000 g) neonates. The management of ELBW infants born with esophageal atresia/tracheoesophageal fistula (EA/TEF) remains challenging and controversial. In this study, we reviewed our experience with the surgical management of these neonates at our institution over a fifteen year period. METHODS: Ethical approval was obtained (REB1000046653). Medical records of ELBW infants with EA/TEF treated at our institution between 2000 and 2014 were reviewed. Demographics, associated anomalies, operative approach and postoperative complications were reported. Data are reported as median (range). RESULTS: Of the 268 infants with EA/TEF managed at our institution during the study period, 8 (3%, 5 females) infants had a birth weight of 930 grams (540-995) and a gestational age of 28 weeks (23- 32). Seven infants had type-C EA/TEF and 1 type B. Patient details and outcome are reported in the Table. CONCLUSION: This is the largest series of <1000 gram EA/TEF neonates. The incidence of ELBW infants with EA/TEF is rare (3%). The association of ELBW and EA/TEF results in high morbidity and mortality. In our series, the cause of death is mainly related to complications not associated with EA/TEF repair. Fistula ligation followed by delayed anastomosis seems to achieve a better outcome in ELBW infants. Ass. anomalies Surgery (DOL= day of life) Complications Outcome 1 PDA DOL 1: Trans-pleural fistula ligation and Stamm gastrostomy DOL 120: Delayed anastomosis Anastomotic leak + stricture (dilated x1) PO feeds FU of 5yrs 2 VSD, PDA, ARM, annular pancreas , omphalo- mesenteric duct DOL 1: Extra-pleural fistula ligation, small bowel resection, stoma, Stamm gastrostomy Gastric leak and peritonitis, bilateral IVH Death 3 Truncus arteriosus DOL 5: Extra-pleural fistula ligation DOL 16: truncus arteriosus repair Intraop. liver hemorrhage due to peritoneal drain insertion Death 4 Butterfly vertebra, tethered cord, VSD, PDA, PFO, fused kidney DOL 2: Primary anastomosis + sigmoidostomy loop colostomy DOL 125: PSARP DOL 208: stoma closure Occlusive saphenous thrombus PO feeds FU of 3yrs 5 Fused vertebrae, tethered cord, PDA, PFO, ASD, Talipes DOL: 1 Trans-pleural fistula division DOL 5: Re-thoracotomy for ?air leak, primary anastomosis RDS, persistent pneumothorax multiple cerebellar and brain hemorrhages - palliated Death 6 Trisomy 18 No surgery - palliated - Death 7 ASD, tethered cord DOL 1: : Extra-pleural fistula Thoracic duct injury,
  35. 35. 28th International Symposium on Pediatric Surgical Research 34 ligation DOL 60: Stamm gastrostomy DOL 178: Delayed anastomosis DOL 416: Lap Nissen fundoplication Pseudomonas pneumonia, pulmonary hypertension, anastomotic strictures, GERD G-tube feeds 8 PDA, RDS, disseminated fungemia DOL 61: Stamm gastrostomy DOL 126: TEF ligation and delayed anastomosis DOL 217: Aortopexy (open) Complications of severe prematurity, anastomotic strictures, GERD PO feeds FU of 3yrs
  36. 36. 28th International Symposium on Pediatric Surgical Research 35 TITLE: MODELLING THE INTERACTION OF COMORBIDITIES PREDICTING RISK OF FUNDOPLICATION AUTHOR(S): Dr E.W. Macharia-Coates AFFILIATION: Institute of Child Health, University College London, WC1N 1EH, United Kingdom Email: eve.macharia-coates@ucl.ac.uk PURPOSE Prior studies of fundoplication risk focus on individual effects of comorbid risk factors. However, in vivo, children with gastro-oesophageal reflux disease (GORD) may have multiple and interacting pathologies, whose risk can be estimated. Our aim is to model fundoplication risk incorporating a search for these interacting comorbidities. METHODS Data mining of an electronic clinical documents database was conducted. A retrospective 13-year cohort of children (0-18 years) on prescription anti-reflux medications reviewed at a single institution was identified. Putative risk factors identified i.e. demographics, comorbidities and investigations. Exposure of interest was first fundoplication. Data partitioned into training and testing datasets. Multivariable logistic regression with a search for 2-way interactions used to model patient-specific fundoplication risk. RESULTS 1080 patients (7.7%) of the cohort (n=13902) underwent fundoplication between January 2000 – December 2012. There were 29 associated comorbidities. Table 1: Variables predicting risk of fundoplication. Beta- coefficients for significant comorbidities (p<0.05) are re- expressed as odds ratios (OR) and confidence intervals (CI). Statistical inference: In multivariate regression, 10 variables were significantly associated with an increase in fundoplication risk. Additionally, we identified 6 interaction terms. Notably, neurological impairment (NI) with prematurity decreases fundoplication risk. Model performance: The model exhibited a sensitivity of 73% and specificity of 80%. The receiver operating characteristic (ROC) analysis demonstrated an area under the curve of 0.82. The model is a “good” predictor of fundoplication risk.
  37. 37. 28th International Symposium on Pediatric Surgical Research 36 Figure 1: ROC analysis for multivariate logistic regression model incorporating 2-way interaction terms Independent variables OR 2.5%CI 97.5%CI ni 10.05 6.79 15.09 cdh 7.69 4.32 13.50 achalasia 7.18 2.28 19.88 renal 5.93 2.38 14.53 oatof 5.83 3.67 9.13 skeletal 5.66 2.44 13.43 prem 4.51 2.93 6.97 dental 3.76 0.99 13.31 cld 2.80 1.92 4.06 metabolic 1.81 1.04 3.07 Interaction terms ni:cleft 108.51 9.53 4242.43 ni:swallow 12.80 3.00 91.39 ni:tracheal 10.79 3.16 45.72 cld:metabolic 7.38 1.15 47.16 ni:cardiac 3.36 1.99 5.69 ni:prem 0.40 0.24 0.68 CONCLUSION We observe comorbidities with no individual effects that, in concert with NI, dramatically increase fundoplication risk. Modelling to include a search for interactions reveals hidden risk factors. This analysis demonstrates the importance of modelling with in vivo assumptions i.e. interacting comorbidities.
  38. 38. 28th International Symposium on Pediatric Surgical Research 37 Morphological changes and altered vascular measurements in the chick embryo model following exposure to cadmium. A Kaskova Gheorghescu, J. Thompson School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland Abstract Purpose: Chick embryos exposed to teratogenic doses of cadmium acetate (CdAc) at H-H stage 16-17 are known to develop abnormal body axes, with exaggerated lordosis in the lumbosacral region and subsequent ventral body wall defect. It has been noted previously that cadmium causes abnormal angiogenesis. The purpose of this study was to describe and quantify anti-angiogenic effect of Cd utilizing the chick embryo model. Methods: After 60 hours incubation, chicks were explanted according to Dugan’s method and treated with 5, 25, 50, 75 and 100 µl of 50 µmol CdAc. Equimolar sodium acetate 50 µl was given to controls. Embryos were returned to the incubator and re-examined at 4, 8, 24 and 48 hours later. The dose at which Cd produces an anti-angiogenic effect was established. Morphological abnormalities were observed. Vascular and embryo measurements (length of the sinus terminalis, the size of the area vasculosa, vessel density and crown-rump length) were performed. Cadmium uptake within the chick embryo was quantified. Quantitative real-time RT-PCR was used to determine the relative expression of Ang-2 and VE-cadherin. Results: 50 μL of 50 μM CdAc produced an anti-angiogenic effect. In Cd explants, extra-embryonic vasculature was present in all treated explants, but a disturbed pattern was seen, omphalomesenteric vessels appeared retarded with reduced branching. The sinus terminalis, appeared tortuous and vascular development in the AV appeared suppressed generally. Complete extra-embryonic avascularity wasn’t observed. The length of the sinus terminalis, the size of the area vasculosa, vessel density, and CRL of the embryos were significantly reduced in the Cd group at all time points when compared with controls. Treatment with 50 μL of 50 μM of CdAc resulted in a mean embryo (n=14) Cd concentration of 4.1 ± 1.2 μM and a mean concentration of 7.6 ± 1.0 μM in the extra-embryonic membranes (n=14) at 8 hours. The relative mRNA expression levels of Ang-2 were increased in the Cd group at 4 hours. VE-cadherin expression was significantly decreased in the Cd group compared to controls at 1 hour and 4 Conclusion: Vascular endothelium is a target of Cd toxicity. Cadmium exposure impairs vascular development, as evidenced by reduction in vascular and embryo measurements. Association between abnormal vasculature and abnormal embryos was very strong. Results clearly indicate that abnormal extra-embryonic vasculature is linearly associated with the growth retardation of the embryo.
  39. 39. 28th International Symposium on Pediatric Surgical Research 38 Table 1 Effects of cadmium dose on embryo survival and gross vascular abnormality after treatment at 60 hours (Hamburger-Hamilton developmental stages 16-17). Treatment (50 μL) N Dead at 60+24h Vascular abnormality 60+24h (% of survivors) 5 μM CdAc 10 3 (30%) 0 (0%) 25 μM CdAc 10 4 (40%) 0 (0%) 50 μM CdAc 10 3 (30%) 4 (57.1%)* 75 μM CdAc 10 7 (70%) 3 (100%) 100 μM CdAc 10 7 (70%) 3 (100%) NaAc 10 2 (20%) 0 (0%) Table 2 The effects of Cd on vascular development in the chick area vasculosa. TREATMENT N Dead Disturbed pattern (% of survivors) 4 hours Control (NaAc) Cd (CdAc) 140 170 6 (4.2%) 16 (%) 0 (0%) 73 (47.4%)*** 8 hours Control (NaAc) Cd (CdAc) 134 154 3 (2.2%) 14 (%) 0 (0%) 71 (50.7%)*** 24 hours Control (NaAc) Cd (CdAc) 131 140 6 (4.5%) 27 (%) 0 (0%) 78 (77.9%)*** 48 hours Control (NaAc) Cd (CdAc) 125 113 5 (4.0%) 26 (%) 0 (0%) 69 (79.3%)***
  40. 40. 28th International Symposium on Pediatric Surgical Research 39 Table 3 Gross abnormalities in embryos 48 hours after treatment at 60 hours. ABNORMALITY N Limb abnormalities, including hypoplasia and malposition 7 (28%) Longitudinal body axis malformation with dorsal kink in the caudal end of the embryo 7 (28%) Eye abnormalities, including microphthalmia and unilateral anopthalmia 0 (0%) Defects of the cranial part of the neural tube 0 (0%) Facial abnormalities, including midfacial cleft and hypoplasia 0 (0%) Figure 1 The length of the sinus terminalis.
  41. 41. 28th International Symposium on Pediatric Surgical Research 40 Figure 2 The size of the area vasculosa. Figure 3 Vessel density. Figure 4 Crown-rump length in control and Cd-treated embryos.
  42. 42. 28th International Symposium on Pediatric Surgical Research 41 Figure 5 Relative mRNA expression levels of Ang-2 at 60 hours. Figure 6 Relative mRNA expression levels of VE-cadherin at 60 hours.
  43. 43. 28th International Symposium on Pediatric Surgical Research 42 FRIDAY, 25TH SEPTEMBER, 2015 SCIENTIFIC SESSION III
  44. 44. 28th International Symposium on Pediatric Surgical Research 43 Non-Invasive Ablation of Fetal Rabbit Tissue In Utero Using Magnetic Resonance Guided High Intensity Focused Ultrasound K. Piorkowska1 , A.C. Waspe1 , C. Mougenot2 , T. Wang3 , J.T. Gerstle1,4 and J. Drake,1,5 . 1 Centre for Image Guided Innovation and Therapeutic Intervention, Hospital for Sick Children, Toronto, Ontario, Canada. 2 Philips Healthcare, Toronto, Ontario, Canada. 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 4 Department of General and Thoracic Surgery, Hospital for Sick Children, Toronto, Ontario, Canada. 5 Department of Neurosurgery, Hospital for Sick Children, Toronto, Ontario, Canada. Email of Corresponding author: Karolina.Piorkowska@sickkids.ca PURPOSE: Magnetic resonance guided high intensity focused ultrasound (MRgHIFU) is a potential non-invasive therapy for fetal conditions including lung malformations and other solid organ lesions requiring invasive surgical interventions. This acute study assessed feasibility and safety parameters of in utero MRgHIFU delivery and determined accuracy and tissue response to treatment. METHODS: High resolution 3T MR images were acquired in late gestation rabbits (~30 days, term = 32; n=5) to identify clinically relevant fetal targets. HIFU sonications were applied continuously for ~20 seconds to a specific target organ at various powers to achieve areas of necrosis guided by MR and thermal imaging. HIFU exposures are presented as average acoustic power in Watts (Wac) ± standard deviation reaching maximum temperatures, averaged over all exposures, in degrees Celsius. Tissues were collected for histological analysis. RESULTS: Lungs (n=8) were targeted with 85 ± 15 Wac reaching 74°C and livers (n=6) with 80 ± 15 Wac reaching 75°C. Histological changes for both tissue types showed distinct necrotic areas with circumferential hemorrhage which transitioned to healthy tissue. In one fetus, a “banding pattern” occurred due to rib interference. Kidneys (n=5) were treated with 100 Wac and reached 67°C: one successful treatment resulted in a focal area of necrosis and surrounding vasodilation of arteries; other treatments were less effective due to fetal movement and high perfusion of the kidney. Necropsy revealed mild maternal skin burns (n=2) that was rectified by improved coupling and increased skin cooling time; also, mild architectural changes in the smooth muscle of the external uterine wall (n=3) not spanning the breadth of tissue layers to the internal surface. Examination of the fetuses revealed focal epidermal vasodilation on the chest wall of fetus (n=8) associated with lung targeting. Collateral organ damage ranged from mild vasodilation to diminutive necrosis and was due to fetal size and overlapping orientation of organs. There was no apparent affect to adjacent untreated fetuses. CONCLUSION: MRgHIFU therapy can effectively target and thermally treat specific in utero organs in this acute fetal rabbit model. Clinical MRgHIFU therapy for specific organ anomalies may improve overall fetal outcome over traditional invasive surgical procedures.
  45. 45. 28th International Symposium on Pediatric Surgical Research 44 HUMAN ADIPOSE MESENCHYMAL CELLS SUPPRESS MELANOCYTE FUNCTION AND SKIN PIGMENTATION BY SECRETING TRANSFORMING GROWTH FACTOR β-1 A. S. Klar1,2 *, T. Biedermann1,2 *, K. Michalak1,2 , T. Michalczyk1,2 , C. Meuli-Simmen3 , M. Meuli2,4 , E. Reichmann1,2 (1) Tissue Biology Research Unit, Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland (2) Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland (3) Department of Plastic, Reconstructive, Esthetical and Hand Surgery, Kantonsspital Aarau, Aarau, Switzerland (4) Department of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland *authors contributed equally Correspondence should be addressed to: Ernst.Reichmann@kispi.uzh.ch Purpose: We have previously shown that skin pigmentation is determined by mesenchymal- epithelial interactions via the interplay of transcription factors and growth factors influencing skin melanocytes. In this experimental study, we investigated the effect of adipose-derived stromal cells (ASCs) on the melanocyte functions such as differentiation, proliferation, melanogenesis, and dendritogenesis in dermo-epidemal skin substitutes. Methods: Human epidermal melanocytes, keratinocytes, and fibroblasts were isolated from dark- pigmented skin biopsies, whereas ASCs were isolated from human adipose tissue biopsies. After in vitro cell expansion, bovine collagen hydrogels containing ASCs or fibroblasts were prepared, and melanocytes and keratinocytes were seeded in a 1:5 ratio onto those hydrogels. The dermo- epidermal skin substitutes were transplanted onto full-thickness wounds of immuno-deficient rats and analyzed after 5 weeks. Results: ASCs significantly suppressed the growth and pigmentation of melanocytes in dermo- epidermal skin substitutes as compared with skin-fibroblasts. ELISA and western blot analyses showed higher levels of TGFβ-1 in ASCs as compared to skin-fibroblasts. We showed that melanocytes respond to TGFβ-1 in vitro and in vivo by suppressing cell proliferation and the expression of melanosomal markers, such as tyrosinase and tyrosinase-related protein 1, which are essential for maturation of melanosomes and melanin synthesis. Furthermore, the distribution of melanin in the epidermis was also suppressed as demonstrated by Fontana Masson staining. Conclusions: Our data suggest that mesenchymal cells of different tissue origin - adipose and skin - used to construct pigmented dermo-epidermal skin substitutes, have distinct influences on the melanocyte functions and skin pigmentation. Adipose-ASCs significantly decreased melanocyte function that resulted in generation of light-pigmented skin substitutes, which did not correspond to the dark-pigmented donor skin color. Keywords: Melanocytes - Melanogenesis - Adipose-derived stromal cells - TGFβ-1 - Skin tissue engineering - Pigmented skin substitutes - Rat model
  46. 46. 28th International Symposium on Pediatric Surgical Research 45 Colonic epithelia disruption after maternal separation is rescued by corticotropin releasing hormone Bo Li1 , Tali Filler1 , Carol Lee1 , Augusto Zani1 , Elke Zani-Ruttenstock1 , Wan Ip2 , Tanja Gonska2 , Agostino Pierro1 1 Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, ON, Canada 2 Department of Pediatrics and Gastroenterology, The Hospital for Sick Children, Toronto, ON, Canada PURPOSE: Early maternal separation (MS) induced colonic disruption of morphology and permeability, enhancing the risk of early intestinal disorders. This effect could be associated with brain-gut interactions through the release of corticotropin releasing hormone (CRH) by hypothalamus. The aim of the present study was to determine whether intestinal epithelial damage could be restored by modulation of the CRH pathway. METHODS: Following approval (license 32238), C57BL/6 neonatal mice underwent MS for 3 hours daily between postnatal day 5 and 9. Mice were randomly assigned to receive an intraperitoneal injection of: i) DMSO (MS+DMSO group, n=10), or ii) Antalarmin, a CRF antagonist (10 mg/kg/day, MS+Antalarmin group, n=10). MS was performed after treatment with DMSO or Antalarmin. Untreated pups served as control. Proximal colon (most injured gut area) was harvested and analyzed for goblet cell density per crypt (alcian blue), crypt length (H&E) and tissue permeability. Ussing chamber was used to test tissue viability (trans-epithelial resistance) and trans- cellular flux. Data was compared using one-way ANOVA with Bonferroni post-test; p<0.05 was considered significant. RESULTS: Morphology - Compared to controls, the number of goblet cells per crypt was reduced in MS+DMSO mice (p<0.001), but increased by Antalarmin (p<0.01 to MS+DMSO; Figure 1a). Similarly, colonic crypt length was decreased in MS+DMSO mice (p<0.01 to control), but returned to normal levels after Antalarmin administration (p<0.05; Figure 1b). Permeability - Trans-epithelial resistance remained unchanged across the groups confirming tissue viability of all samples. MS+DMSO increased trans-cellular permeability compared to control (p<0.001) but MS+Antalarmin reduced it to level observed in control (p<0.001) (Figure 1c). CONCLUSION: Colonic mucosal damage and increased trans-cellular permeability induced by maternal separation are rescued by Antalarmin, a CRH antagonist. These findings suggest that maternal separation induced bowel damage could be due to a disruption of the gut-brain axis. This provides insights on the development of new drugs that could be used for the treatments of neonatal intestinal diseases, such as necrotizing enterocolitis.
  47. 47. 28th International Symposium on Pediatric Surgical Research 46 AUTOPHAGY AS A CHARACTERISTIC FEATURE OF NEONATAL TISSUE MACROPHAGES Authors: T. Winterberg1 , Y. Yu1 , S. Groos2 , J.K. Park1 , G. Vieten1 , B. Ure1 , JF. Kuebler1 Institutional affiliations 1 Department of Pediatric Surgery 2 Institute of Cell Biology in the Center of Anatomy Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover E-mail address Kuebler.Joachim@mh-hannover.de Purpose: Macrophage autophagy has been the focus of recent interest and a growing body of evidence indicates that it is a key component of the innate immune response, involved in many aspects of macrophage development and polarization. In former studies we observed distinct phenotypic and functional features of neonatal tissue macrophages. The aim of this study was to investigate the occurrence of autophagosomes in neonatal macrophages. Methods: Peritoneal macrophages were gained by injury-free lavages of neonatal and adult C57/BL6 mice. Pelleted cells were investigated by electron microscopy. FACSorted, purified macrophages were stained using standard May-Grünwald-Giemsa staining or further cultured and stimulated for 6h with TLR agonists. Total mRNA was extracted and detected using Agilent Micro Array Detection System. After data normalization specific software was used to investigate primarily homeostatic, pro- and anti inflammatory processes. Results: Naive neonatal macrophages displayed a high amount of cytoplasmic vesicular structures visible after staining (Fig.1). Electron microscopy of macrophages from neonatal mice regularly showed autophagosomes (Fig. 2B). Microarray gene network analysis revealed that the increase in autophagy was associated with a signature of increased homeostatic activity as well as a decrease in genes responsible for resolving inflammation (Alox15, Tgfb2, Ikbkg). Conclusion: Neonatal macrophages appear to display an increased autophagic activity in combination with a tissue and developmental homeostatic “priming”. Although this seems to be well adapted to the major changes in the tissue microenvironment after birth, these changes could have adverse effects on the immunological balance by down regulation of pro-resolving and anti- inflammatory mediators. Fig.1 Fig.2
  48. 48. 28th International Symposium on Pediatric Surgical Research 47 Fig.1 Naive neonatal (A) and adult (B) peritoneal macrophages (May-Grünwald-Giemsa staining; one representative experiment of >8 experiments; bars: 10 µm. Fig.2 Thin sections of naive neonatal (A, B) and adult (C, D) peritoneal macrophages; (AP) autophagosome, asterisks: Endoplasmic Reticulum, (G) Golgi stack, (M) Mitochondrium, (N) nucleus; bars A, C: 2 µm; B, D: 1 µm.
  49. 49. 28th International Symposium on Pediatric Surgical Research 48 Modified epidermal self renewal of engineered pigmented human skin after UVB exposure in an in vivo assay Teresa Michalczyk, Thomas Biedermann, Sophie Böttcher- Haberzeth, Agnes Klar, Ernst Reichmann, Martin Meuli T. Michalczyk, T. Biedermann, S. Böttcher- Haberzeth, A. Klar, E. Reichmann Tissue Biology Research Unit, University Children’s Hospital Zurich, Zurich, Switzerland S. Böttcher- Haberzeth, M. Meuli Departement of Surgery, University Children’s Hospital Zurich, Zurich, Switzerland T. Michalczyk, T. Biedermann, S. Böttcher- Haberzeth, A. Klar, E. Reichmann, M.Meuli Children’s Research Center, University Children’s Hospital Zurich, Zurich, Switzerland e-mail: Teresa.Michalczyk@kispi.uzh.ch Abstract Purpose The basal compartment of the human epidermis is constantly renewing, but still little is known about the epidermal mechanism of maintaining and adapting its homeostasis, in particular when exposed to external stress factors like UV irradiation. UVB light provokes tanning in human skin. Also, it can lead to inflammation and can induce mutagenic events in the genome of skin cells. We recently demonstrated successful engineering of pigmented dermo-epidermal skin substitutes, with restoration of the original donor skin color in long-term in vivo experiments. In view of future clinical application, we now studied the behavior of these tissue-engineered substitutes after exposure to UVB irradiation. Methods Human dermo-epidermal pigmented skin was engineered with keratinocytes, melanocytes and fibroblasts originating of the same donor biopsy. Then, keratinocytes and melanocytes were seeded on collagen type I hydrogels, previously populated with fibroblasts. Skin substitutes were transplanted onto full-thickness wounds of immuno-incompetent rats and irradiated with a single dose of 250mJ/cm2 UVB irradiation 4 weeks after transplantation. Punch biopsies were taken from the grafts before and after irradiation for immunohistochemical staining. Results After UVB irradiation transplanted skin substitutes showed physiological tanning properties. Temporarily, Cytokeratin 16 and Desmoglein 3 were expressed in keratinocytes, indicating a wound healing response. In engineered skin without UVB exposure, as well as in foreskin, few proliferating keratinocytes were mostly located in the suprabasal layer, directly adjacent to the basal layer. After UVB exposure proliferation of keratinocytes, but not melanocytes or fibroblasts, massively increased notably in the basal layer and normalized 4 weeks after irradiation. The expression of the Wnt- pathway inhibitors Dickkopf 3 and Wif 1 was lost in basal keratinocytes after UVB irradiation, authorizing proliferation of keratinocytes. Interestingly, melanocytes continued to express Wnt- Inhibitors after UVB treatment.
  50. 50. 28th International Symposium on Pediatric Surgical Research 49 Conclusion These findings suggest that our engineered human skin is capable to maintain epidermal homeostasis after external stress, via modified controlled proliferation of keratinocytes. This is crucial regarding a future clinical application of tissue-engineered pigmented autologous skin substitutes on patients. Furthermore, there is evidence that the basal compartment itself is balancing inhibitors of the Wnt-pathway to initiate epidermal self-renewal.
  51. 51. 28th International Symposium on Pediatric Surgical Research 50 Comparison of different methods of preservation to store decellularised oesophagi for tissue engineering purposes L. Urbani1 , P. Maghsoudlou1 , A. Milan1,2 , S. Eaton1 , P. De Coppi1 . 1 UCL Institute of Child Health and Great Ormond Street Hospital, UCL, London, UK 2 University of Padua, Padua, Italy Corresponding author: l.urbani@ucl.ac.uk Purpose Several conditions may require esophageal tissue replacement. Tissue engineering of the oesophagus (TEO) has been proposed as a therapeutic alternative to oesophageal substitution. Acellular matrices are ideal for TEO because they are made up by tissue-specific extracellular matrix (ECM). However, the appropriate preservation of the ECM components may significantly affect the behavior of scaffolds in vivo. Thus far, there is no consensus on the most appropriate storage methodology for decellularised scaffolds. Here we aim at establishing the best way to storage oesophageal scaffolds from a large animal model and preserve the structure of their ECM. Methods Rabbit oesophagi were decellularized using detergent-enzymatic treatment (DET) and evaluated at 2 weeks, 1, 3 and 6 months of storage by means of histology, immunofluorescence, biomechanical testing, ECM component quantification and angiogenic assay. Four different storage methods were compared: A) phosphate-buffered saline solution at 4°C, B) at -20°C after a freeze-drying step, C) at - 80°C after snap-freezing in liquid nitrogen or D) in liquid nitrogen after cooling in medium with 10% DMSO at -1°C/min then stored in liquid nitrogen. Results Efficient decellularization was achieved after 2 cycles of DET as evidenced by histology and DNA quantification, with preservation of collagen, elastin and glycosaminoglycans levels. As a storage methodology the PBS/4°C option (method A) led to the most structural destruction of the tissue, whereas gradual freezing of the tissue (method D) was best at preserving the tissue for long term. Scaffolds stored with method D had a preserved structure and orientation throughout all the time points, showing intact layers and collagen and elastin fibers. Oesophagi stored at -80°C post snap- freezing (method C) were substantially different compared to the rest, displaying irreversible collapse and breaking up of the tissue. Conclusion Developing of an acellular scaffold for oesophageal tissue engineering that preserves the ECM components and architecture of the original tissue is essential for therapy. Efficient storage allows their use as an ‘off-the-shelf’ product. This study demonstrated that, similarly to cellular cryopreservation, slow cooling in a DMSO/medium solution and subsequent storage in liquid nitrogen allows long term storage of decellularized scaffolds.
  52. 52. 28th International Symposium on Pediatric Surgical Research 51 Development of an Extracellular Matrix Hydrogel for use in Intestinal Tissue Engineering. C.Crowley1 , L.Urbani1 , V.Gaillet1 , F.Onofri1 , L.Maughan1 , S.Eaton1 ,P.Coppi1 . 1 UCL Institute of Child Health and Great Ormond Street Hospital, UCL, London, UK Corresponding author: claire.crowley.09@ucl.ac.uk Abstract: Purpose: Irreversible intestinal failure (IF) is a condition that can lead to significant morbidity and mortality and occurs as a result of anatomical or functional loss. Perenteral nutrition has been shown to improve the condition substantially, however, there are many associated complications with this approach. As a result, many patients eventually require an intestinal transplant, which is unfortunately limited by the availability of donor organs and the need for aggressive immunosuppression. Recent years have brought many advances to this field particularly regarding isolation and culture of intestinal stem cells. However, their expansion for translational application remains limited, so the discovery of alternative culturing methods is crucial. Here we investigate the ability to create a hydrogel from the extracellular matrix (ECM) of intestinal tissue. ECM hydrogels from other tissues have shown great potential for many different applications. This study aims to develop and characterise an intestinal ECM hydrogel for eventual use in intestinal organoid culture and as a hybrid scaffold for tissue engineered intestinal replacement. Methods: A decellularisation protocol was developed specifically for newborn porcine intestinal tissue. This method was assessed and characterised using SEM, histology and quantification of collagen, elastin, GAG and DNA content after each cycle. The decellularised tissue was then lyophilised, milled into a powder, pepsin digested and brought to a physiological pH and temperature to produce a soluble ECM gel. The gel was characterised for its rheological properties and gelation potential using spectrometry and oscillatory tests. Whole intestine containing both the muscle and mucosa and the mucosa alone were compared, as this method can be highly tissue specific. Results: One cycle of the decellularisation protocol was sufficient to successfully remove the nuclei and reduce the DNA content of the intestinal tissue. Collagen, GAG and Elastin remained intact after decellularisation. An ECM hydrogel was successfully produced and characterised. Optimal conditions (pH, concentration, timing) for gel formation were established to obtain a hydrogel suitable for cell culture and in vivo application. Conclusion: In this study we established a tissue specific ECM-derived hydrogel for intestinal tissue engineering. The applications for these gels is extensive, both for clinical and research purposes.
  53. 53. 28th International Symposium on Pediatric Surgical Research 52 Quantitation of residual detergent in decellularised organs for tissue engineering with gas chromatography-mass spectrometry E. Maughan1 , C. Crowley1 , R. Bhondi1 , F. Tommasini1 , L. Urbani1 , C. Butler1 , M. Birchall1 , S. Eaton1 , P. De Coppi1 . 1 Institute of Child Health and Great Ormond Street Hospital, UCL, London, UK Corresponding author: claire.crowley.09@ucl.ac.uk Purpose Sodium deoxycholate (SDC) is a cytotoxic detergent widely used in organ decellularisation for tissue engineering for the removal of cell membrane components. It has been reported that residual SDC can compromise cell viability during subsequent recellularisation, 3D culture and implantation. However, quantification of residual SDC in the tissue has not been optimized. This study was designed to evaluate the use of Gas Chromatography-Mass Spectrometry (GC-MS) in determining the efficacy of residual SDC removal following post-decellularisation washing. Methods Rat tracheae (n=16) were decellularised using a detergent-enzyme technique containing SDC over three cycles. Following decellularisation, a washing step using double-distilled water (MilliQ) was performed with agitation for 24 hours, 48 or 72 hours. Wash solutions were changed every 24 hours. Samples were acidified, cholic acid added as an internal standard, extracted with diethyl ether, derivatised to the pentaflurobenzyl, trimethylsilyl derivative, and analyzed by GC-MS. Results GC-MS proved a highly sensitive method for detection of very small residual concentrations of SDC (as low as 2nmol). The concentration of residual SDC was cumulative with increasing cycle number (1.64 vs 3.91 vs 9.56 nmol for cycles 1, 2 and 3 respectively). Increasing washing times resulted in a demonstrable decrease in SDC concentration (e.g. 9.56 to 3.56 nmol from 24- to 72-hour timepoints). Conclusion The novel use of GC-MS as a highly sensitive method for detecting and quantifying residual SDC within decellularised tissue could enable the comparison of washing protocol efficacy. Further validation of this analytical tool could enable its development as a GMP release criteria for decellularised tissue in clinical trials.
  54. 54. 28th International Symposium on Pediatric Surgical Research 53 CYTOREDUCTIVE SURGERY (CRS) AND HYPERTHERMIC INTRAPERITONEAL CHEMOTHERAPY (HIPEC) IN PEDIATRIC OVARIAN TUMORS: A NOVEL TREATMENT APPROACH A Hayes-Jordan 1 , CLopez2 , HL Green1 , LC Xiao3 , W Huh MD4 , C Herzog 4 ahjordan@mdanderson.org 1. University of Texas MD Anderson Cancer Center, Department of Surgical Oncology/Pediatric Surgical Oncology, Houston, Texas, USA 2. University of Texas Houston Health Sciences Center 3. University of Texas MD Anderson Cancer Center, Department of Biostatistics 4. University of Texas MD Anderson Cancer Center, Division of Pediatrics Purpose: CRS and HIPEC have been used in adults with ovarian carcinoma proving overall survival benefit in randomized trials, measured in months. Diffuse peritoneal disease from pediatric type ovarian tumors is rare. We applied this approach to a select group of pediatric girls with diffuse peritoneal disease. These patients were all included as part of a phase 1 or phase 2 clinical trial for CRS and HIPEC in children. Methods: In all patients complete cytoreduction followed by HIPEC using 100mg/M2 of Cisplatin for 90 minutes in a closed technique, was utilized. All patients were treated with the same strict peri-- operative management, as part of an investigator initiated clinical trial. All received neoadjuvant chemotherapy. Patients with disease outside of the abdominal cavity were excluded. Results: Of 101 pediatric CRS and HIPEC operations, 8 had ovarian primary tumors and multifocal peritoneal disease. There were 3 yolk sac tumors( germ cell, mixed teratoma), one Sertoli-Leydig, one PNET of the ovary, one choriocarcinoma, one juvenile granulosa cell tumor and one adenocarcinoma. Age at diagnosis ranged from 4 to 18 years. Two of the 7 (28%) recurred and died. The remaining 70% are disease free 2 to 8 years post HIPEC. Overall survival and relapse free survival in this cohort was 64% and 62% respectively. [CI 0.64 (0.34,1 ); 0.62 ( 0.37, 1 )] Complications included 2 wound infections, and 1 urinary tract infection and 1 enterocutaneous fistula. Conclusions: This is the first report of CRS and HIPEC in pediatric ovarian tumors. HIPEC is a safe approach to diffuse peritoneal disease secondary to pediatric-type ovarian tumors. More treated patients are required to determine efficacy of this approach.
  55. 55. 28th International Symposium on Pediatric Surgical Research 54 FRIDAY, 25TH SEPTEMBER, 2015 SCIENTIFIC SESSION IV
  56. 56. 28th International Symposium on Pediatric Surgical Research 55 PLATELET-LEUKOCYTE FIBRIN MEMBRANES AS POTENTIAL PLATFORMS FOR WOUND HEALING * F. Grandi1 , E. Stocco2,5 , S. Barbon2,5 , S. Capelli3 , A. Borean3 , A. Porzionato4 , V. Macchi4 , G. Albertin4 , R. De Caro4 , PG. Gamba1 , P.P. Parnigotto5 , C. Grandi2 1 Pediatric Surgery, Department of Woman and Child Health, University of Padua, Padua, Italy 2 Department of Pharmaceutical an Pharmacological Sciences, University of Padua, Padua, Italy 3 Department of Immunohematology and Transfusion-Medicine of Belluno Hospital, Belluno, Italy 4 Section of Human Anatomy, Department of Molecular Medicine, University of Padua, Padua, Italy 5 Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling (TES) ONLUS, Caselle di Selvazzano Dentro, Padua, Italy * Corresponding and presenting author: Dr. F. Grandi francesca.grandi@studenti.unipd.it PURPOSE Translational medicine has emerged as a new trend in medical practice; one of the main topics deals with those therapies focused on enhancing tissue repair and regeneration. In particular, the use of autologous haemocomponents as biomolecules delivering systems is gaining wide attention. Recently, we developed a new method to collect haemocomponents for regenerative use, obtaining gels rich in platelets, monocytes/macrophages, fibrinogen and CD34+ cells. Even though these preparations are available today to stimulate tissue healing, they do not fully satisfy surgeons’ requests, looking for easily applicable and manipulable membranes. Hence, our aim was to manufacture a novel Platelet-Leukocyte Fibrin membrane (PLFm) and to study its morpho- mechanical properties as well as its attitude to act as a scaffold. METHODS PLFms were prepared from a Platelet-Leukocyte concentrate/Plasma MIX as we previously described(Caloprisco et al., Transfus Apher Sci. 2010;42:117-24) after activation with calcium gluconate. The resulting PLFms were characterized haemocytometrically for cell concentration in comparison with the MIX. To predict the in vivo behaviour of PLFms, supports were incubated in PBS at 37 °C up to day 21, and their morphology/histoarchitecture were investigated by histology, immunohistochemistry and SEM, at different end-points. In addition, contingent variations in mechanical properties of PLFms were also assessed by tensile tests. Lastly, PLFms were used as scaffolds to verify their ability in sustaining cell adhesion/proliferation. RESULTS From a MIX volume of 11±2 ml were obtained membranes of 2.8±0.6 ml in volume with an area of 8.9±1.4 cm2 and 0.31±0.06 cm in thickness. A significant increase in cell concentration was observed for PLFms compared to the MIX. Histology and SEM showed that samples morphology changed along with time, as the fibrin scaffold, previously masked by erythrocytes, became progressively visible. The presence of cellular elements of interest was also assessed by immunohystochemistry. Moreover, any significant difference in the maximum percentage of deformation was observed within 10 days of incubation. Cell culture on PLFms showed the attitude of these haemocomponents in sustaining cell adhesion/proliferation. CONCLUSIONS Autologous PLFms represent a platform with structural and biological properties that promote tissue healing and with a possible routinary application in surgery.
  57. 57. 28th International Symposium on Pediatric Surgical Research 56 SPHINGOSINE-1-PHOSPHATE CONTROLS CELL MOTILITY OF PLACENTA MESENCHYMAL STEM CELLS Giulio Innamorati#* , Emanuela Fontana, Federica Steccanella, Giovanni Ridolfi, Kushal Gandhi, Luca Giacomello* Paediatric Surgical Research Laboratories, Department of Surgery, University of Verona, Italy *Corresponding authors: giulio.innamorati@univr.it, luca.giacomello@univr.it PURPOSE A major concerns in using stem cells for therapeutic approaches is the very limited percent of engraftment and, as a consequence, the large number of cells to be administered. Sphingosine 1 phosphate (S1P) is emerging as a crucial regulator of cell motility and chemotaxis acting in concert with CXCL12 to regulate the egression immature progenitors. We explored the functional implications of S1P signaling in placenta derived mesenchymal stem cells (PDMSC), a promising opportunity for a number of diseases. METHODS Primary cultures of fetal cells were obtained from chorion of human term placenta. Mesenchymal properties were confirmed by osteocytic and adypocytic differentiation. S1P receptors (S1PRs) gene expression was assessed by RT-PCR utilizing specific primers for each of the 5 existing subtypes. ERK1/2 and PKD1 activation was measured by western blot. Cell motility was monitored in DMEM supplemented with 0.3% fetal calf serum, migration was digitally quantified after fixation and crystal violet staining. RESULTS RT-PCR revealed mRNA expression of S1P receptors subtypes 1, 3, 4. Subtypes 2 and 5 were absent. Consistently, SEW2871 and CYM50179 (selective agonists for S1P1R and S1P4R respectively) and FTY-P (active on S1PR 1,3,5,4) induced ERK1/2 activation (A). 0%# 20%# 40%# 60%# 80%# 100%# 120%# 0# 60# 120# 180# 240# 300# 360# 420# 480# 540# 600# 660# 720# 0%# 20%# 40%# 60%# 80%# 100%# 120%# SEW2871# CYM# FTY720# S1P# C YM 50179 S1P ** ** ** FTY720PSEW2871 A ERK1/2activation(%S1Pstimulated-basal) FTY720P SEW 2871 S1P CYM 50179 B ERK1/2activation (%maximalstimulation-basal) 0%# 10%# 20%# 30%# 40%# 50%# 60%# 70%# 1.E-08# 1.E-07# 1.E-06# 1.E-05# Woundclosure(%) C [S1P] (log M) -7 -6 -5Time (hours) 1 2 3 4 5 18 24
  58. 58. 28th International Symposium on Pediatric Surgical Research 57 Addition of S1P promoted the transient activation of ERK1/2 (B). An analogous effect was observed with PKD1. Wound healing assays revealed that increasing concentrations of S1P significantly and progressively reduced PDMSC motility (C). CONCLUSION We demonstrated that PDMSC express more than one G protein coupled receptor specific for S1P. S1P signaling inhibits cell migration and could be exploited to design novel approaches aimed to promote PDMSC engraftment in the target tissues.
  59. 59. 28th International Symposium on Pediatric Surgical Research 58 Effects of curcumin on a pediatric hepatocellular carcinoma model in vivo and curcuminoid concentrations after oral application in mice V. Ellerkamp1 , N. Bortl1 , J. Frank2 , C. Schiborr2 , S. Armeanu-Ebinger1 , E. Schmid1 , B. Kirchner1 , S.W. Warmann1 , J. Fuchs1 University Children`s Hospital Tuebingen, Department for Pediatric Surgery and pediatric Urology, Tuebingen, Germany University Hohenheim, Institute of Biological Chemistry and Nutrition, Hohenheim, Germany Abstract In children with hepatocellular carcinoma (pHCC) the 5-year overall survival rate is poor. In adult HCC several antitumor properties are described in in vitro models for the use of curcumin. Methods: Orthotopic growth of the pediatric hepatocellular carcinoma cell line HC-AWF1 in NOD/LtSz-scid/IL-2Rgamma(null)mice was induced. By the increase serum alpha fetoprotein AFP >5 U/mL mice were randomly assigned to one of four groups: control (no treatment); micellar curcumin; cisplatin, and micellar curcumin + cisplatin. Curcuminoid levels in serum and organ lysates as well as AFP serum levels were investigated. Results: Serum curcumin decreased from 3513.89 ± 2791.84 nmol/L two hours after administration to 769.74 ± 448.61 nmol/L after five hours. Curcumin concentrations significantly differed between organs (p=0.000), highest concentrations were observed in the lungs 11.33 ± 9.17 nmol/Kg, lowest in the brain 0.16 ± 0.24 nmol/Kg. The concentrations in the tumor tissue (2.57 ± 1.49 nmol/Kg) were higher than in the liver (1.77 ± 1.50 nmol/Kg). Combination therapy (micellar curcumin + cisplatin) significantly reduced AFP concentrations compared to control group (week 3: 1.04 ± 0.67 vs. 2.73 ± 0.64, p = 0.004; week 4: 2.05 ± 1.01 vs. 3.35 ± 0.43, respectively, p = 0.02). Conclusion: These data prove the potential of micellar curcumin as a complementary agent in pediatric oncology to enhance the overall survival of patients with pediatric liver tumors.