Surgeons_and_Clinics RSS : Gourt

Disruption of calreticulin-mediated cellular adhesion signaling in the cadmium-induced omphalocele in the chick model

Abstract Purpose Administration of cadmium (Cd) causes omphalocele in the chick embryo. The earliest histological changes in the chick Cd model are the breakdown of adherens junctions (AJs). Calreticulin (CRT) plays a key role in Ca2+ signaling and cell adhesion. Ca2+ signaling in the Cd chick model is known to be altered. The calcium-dependent adhesion molecule, E-cadherin, and its associate, β-catenin, are key components of AJs regulated by CRT. CRT knockouts display omphalocele. We hypothesized that CRT, E-cadherin and β-catenin are downregulated during early embryogenesis in the Cd chick model. Methods After 60 h (H) incubation, chicks were harvested 1H, 4H, and 8H post treatment with saline or Cd and divided into controls and Cd. RT-PCR was performed to evaluate mRNA levels of CRT, E-cadherin and β-catenin in the Cd chick model. Results The mRNA levels of CRT were significantly decreased in the Cd group at 1H compared to controls (p < 0.05). The mRNA levels of E-cadherin and β-catenin were significantly decreased at 4H in the Cd group compared to controls (p < 0.05). There were no significant differences at 8H. Conclusion Downregulation of CRT, E-cadherin and β-catenin genes may cause omphalocele in the Cd chick model by disrupting CRT-mediated Ca2+ signaling and AJs. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2505-9Authors Takashi Doi, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 IrelandPrem Puri, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 IrelandJohn Bannigan, University College Dublin School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical Research Dublin IrelandJennifer Thompson, University College Dublin School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical Research Dublin Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Transglutaminases, involucrin, and loricrin as markers of epidermal differentiation in skin substitutes derived from human sweat gland cells

Abstract Background/Purpose In a multi-project research line, we are currently testing whether a morphologically and functionally near normal epidermis can be cultured from human sweat gland (SG) cells and be used as a skin substitute. The present study focuses on the stratum corneum of the epidermis that assumes a vital barrier function for the skin. The main process in the formation of the cornified cell envelope in human epidermis, i.e. crosslinking of proteins and lipids, is catalyzed by several transglutaminases (TG). Therefore, we compared the expression patterns of various TG and their substrates in SG-derived versus keratinocyte-derived epidermal substitutes. Methods Sweat gland cells, keratinocytes, and fibroblasts were isolated from human skin samples and cultivated separately to generate epidermal substitutes. These were transplanted onto the back of athymic rats. After 2 weeks, the transplants were excised and analyzed histologically as well as by indirect immunofluorescence. We looked at the expression of TG1, 3, 5, and their substrates involucrin and loricrin (=markers of epidermal differentiation) in SG-derived and keratinocyte-derived skin substitutes as well as in normal skin. Results The SG cell-derived epidermis was near normal anatomically, formed a cornified cell envelope and demonstrated TG1, 3, and 5 as well as involucrin and loricrin expression patterns similar to those found in keratinocyte-derived epidermis and normal control skin. Conclusion These findings support the thesis that SG cells have the potential to form a near normal stratified epidermal analog that might be used as a skin substitute. The expression of TG1 and 3, not normally expressed in human SG, suggests the presence of re-programmed SG cells and/or stem cells capable of both de novo generating and maintaining an epidermis. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2517-5Authors Sasha Tharakan, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich SwitzerlandLuca Pontiggia, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich SwitzerlandThomas Biedermann, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich SwitzerlandSophie Böttcher-Haberzeth, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich SwitzerlandClemens Schiestl, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich SwitzerlandErnst Reichmann, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich SwitzerlandMartin Meuli, University Children’s Hospital Tissue Biology Research Unit, Department of Surgery Zurich Switzerland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Microscopic magnetic resonance in congenital diaphragmatic hernia and associated malformations in rats

Abstract Background/aim The research on congenital diaphragmatic hernia (CDH) is often carried out on the nitrofen fetal rat model in which most investigations involve microdissections and fastidious assessment of serial sections of different anatomic areas. Current microscopic magnetic resonance (MMR) equipment allows detailed anatomic studies of alive, fresh or fixed fetuses. The purpose of the present study was to demonstrate that CDH itself and most of the associated malformations are adequately imaged and measured by MMR. Materials and methods Fetuses from pregnant rats treated with either i.g. vehicle (control, n = 10) or 100 mg nitrofen (only those with CDH, n = 18) on E9.5 were recovered on E21 (term = E22) and total body was scanned by MMR under sedation in a 7 T MRI system (Bruker Medical, Ettlingen, Germany). CDH was detected with a coronal multislice fast spin echo sequence with a long repetition time and short effective echo time. Oblique MPR and 3D reconstructions were used. All studies were processed with attention to the hernia and its contents and the structure of the tracheobronchial tree and the lung, the heart and great vessels, the thymus and cervico-thoracic vertebrae. The findings in both groups were compared. Results Congenital diaphragmatic hernia, lung hypoplasia and parenchymal features were clearly depicted. Tracheal ring anomalies were also demonstrated. The thymus was significantly smaller in CDH pups (2.9 × 1 × 2.4 mm) than in controls (4 × 1.3 × 2.8 mm) (p < 0.01). MRI was particularly performant for imaging cardiovascular anomalies: 4 double aortic arches, 3 Fallots, 3 right aortic arches, 3 ventricular septal defects and 1 aberrant subclavian artery. Conclusions Microscopic magnetic resonance involves refined and expensive equipment but it provides a powerful research tool for the study of CDH and other malformations in rat fetuses. Further work on this area is warranted. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2518-4Authors Montserrat Bret, Hospital Universitario La Paz Department of Radiology Madrid SpainAna Lourdes Luis, Hospital Universitario La Paz Department of Pediatric Surgery Paseo de la Castellana 261 28046 Madrid SpainEmilio Cuesta, Hospital Universitario La Paz Department of Radiology Madrid SpainFederica Pederiva, Hospital Universitario La Paz Department of Pediatric Surgery Paseo de la Castellana 261 28046 Madrid SpainRosa Aras, Hospital Universitario La Paz Department of Pediatric Surgery Paseo de la Castellana 261 28046 Madrid SpainLeopoldo Martinez, Hospital Universitario La Paz Department of Pediatric Surgery Paseo de la Castellana 261 28046 Madrid SpainJuan A. Tovar, Hospital Universitario La Paz Department of Pediatric Surgery Paseo de la Castellana 261 28046 Madrid Spain Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Effect of simvastatin on intestinal recovery following gut ischemia–reperfusion injury in a rat

Abstract Background Pleiotropic (lipid lowering-independent) effects of statins are attributed to their antiinflammatory, antioxidant, and/or vascular actions. Extensive studies in various experimental models have established that pretreatment with simvastatin significantly protects heart and kidney injured by ischemia–reperfusion (IR). The purpose of the present study was to examine the effect of simvastatin on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. Methods Male Sprague–Dawley rats were divided into three experimental groups: (1) sham rats underwent laparotomy, (2) IR-rats underwent occlusion of both superior mesenteric artery and portal vein for 30 min followed by 48 h of reperfusion, and (3) IR-SIM rats underwent IR and were treated with oral simvastatin (10 mg/kg) given by gavage immediately before and 24 h after operation. Intestinal structural changes, Park’s injury score, enterocyte proliferation and enterocyte apoptosis were determined 24 h following IR. A non-parametric Kruskal–Wallis ANOVA test was used for statistical analysis with P less than 0.05 considered statistically significant. Results Treatment with simvastatin resulted in a significant increase in bowel and mucosal weight in ileum, villus height and crypt depth in jejunum and ileum compared to IR animals. IR-SIM rats had also a significantly lower intestinal injury score as well as lower apoptotic index in jejunum and ileum compared to IR animals. Conclusions Treatment with simvastatin prevents gut mucosal damage and inhibits programmed cell death following intestinal IR in a rat. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2508-6Authors Nadav Slijper, Technion-Israel Institute of Technology Laboratory of Intestinal Adaptation and Recovery, The Bruce Rappaport Faculty of Medicine Haifa IsraelIgor Sukhotnik, Technion-Israel Institute of Technology Laboratory of Intestinal Adaptation and Recovery, The Bruce Rappaport Faculty of Medicine Haifa IsraelElena Chemodanov, Bnai Zion Medical Center Department of Pathology Haifa IsraelYulia Bashenko, Technion-Israel Institute of Technology Laboratory of Intestinal Adaptation and Recovery, The Bruce Rappaport Faculty of Medicine Haifa IsraelRon Shaoul, Rambam Medical Center Pediatric Gastroenterology and Nutrition Unit, Meyer Children’s Hospital Haifa IsraelArnold G. Coran, University of Michigan Medical School Section of Pediatric Surgery, C.S. Mott Children’s Hospital Ann Arbor MI USAJorge Mogilner, Bnai Zion Medical Center Department of Pediatric Surgery B 47 Golomb St. P.O.B. 4940 31048 Haifa Israel Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Altered expression of c-kit-positive cells in the ureterovesical junction after surgically created vesicoureteral reflux

Abstract Purpose Peristaltic contractions propel urine unidirectionally from the renal pelvis trough the ureter and into the bladder. A morphologically and functionally competent ureterovesical junction prevents vesicoureteral reflux (VUR). According to current knowledge, pyeloureteral peristalsis is driven by atypical muscle cells within the upper urinary tract. Another likely relevant cell population, which is c-kit-positive, has been shown to be present within the whole urinary tract. Morphological changes of c-kit-positive cells have been described in VUR and pyeloureteral junction (PUJ) obstruction. Nevertheless, the functional importance of c-kit-positive interstitial cells has not yet been elucidated fully. Therefore, we investigated the influence of experimentally created VUR on the expression of c-kit-positive cells. Materials and methods We created left-sided unilateral VUR in eight Vietnamese pigs using an open surgical technique. The VUR was shown to be grade II–III by voiding cystourethrogram (VCUG), 1 and 6 months after the procedure. The animals were killed after 6 months. The vesicoureteral junction and the distal ureters were excised and fixed in 4% PFA. Paraffin sections were stained using c-kit immunohistochemistry. A quantitative evaluation was performed by two independent investigators. The unaffected, contralateral, nonrefluxing ureters served as controls. Results We identified two types of c-kit-immunoreactive cells within the ureterovesical junction and distal ureter. The first group was comprised of round-shaped cells with substantial intracellular granulas, which resembled mast cells. Mast cells were found in the subepithelial region as well as between the muscle bundles. The number of mast cells was slightly increased in the VUR group. The second group consisted of spindle-shaped, bipolar ICC-like cells, which were seen mainly in the submucosal and muscular layers. The number of spindle-shaped c-kit-immunoreactive cells was markedly decreased in the refluxing ureter compared to the controls. Conclusions Surgically created VUR leads to altered expression of c-kit-positive cells. The altered distribution of c-kit-positive ICC-like cells might further contribute to the impairment of coordinated pyeloureteral peristalsis. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2487-7Authors Zsolt Oberritter, University of Pecs Department of Paediatric Surgery Pecs HungaryUdo Rolle, Johann Wolfgang-Goethe University Department of Paediatric Surgery Frankfurt GermanyZsolt Juhasz, University of Pecs Department of Paediatric Surgery Pecs HungaryTamas Cserni, University College of Dublin Children’s Research Centre Dublin IrelandPrem Puri, University College of Dublin Children’s Research Centre Dublin Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Is C-reactive protein a reliable test for suspected appendicitis in extremely obese children?

Abstract Purpose The diagnosis of acute appendicitis by physical examination can sometimes be difficult in extremely obese children. C-reactive protein (CRP) is commonly used to support the clinical diagnosis of appendicitis. However, obesity has been widely recognized as a chronic inflammatory condition and associated with elevated inflammatory indicators including CRP. The aim of this study was to examine the association between obesity and CRP levels in extremely obese children presenting with suspected appendicitis. Materials The hospital records of 947 consecutive patients, who underwent appendectomy for acute appendicitis between 2002 and 2008 were retrospectively analyzed. 164 children (17.3%) were extremely obese. Extreme obesity was defined, as greater than two standard deviations above the standardized mean weight for age. The diagnostic value of CRP level was compared between extremely obese and non-obese children. Results The incidence of histologically normal appendix was significantly higher in extremely obese children [42 out of 164 (25.6%)] compared to non-obese children [85 out of 783 (10.8%) (P < 0.001)]. The mean CRP levels were significantly higher in extremely obese children with histologically normal appendix compared to non-obese children with normal appendix (P < 0.001). The specificity and the positive predictive value were significantly lower in the extremely obese children group than in the non-obese group (P < 0.001). Conclusion CRP is not a reliable marker of inflammation in extremely obese children presenting with suspected appendicitis. Our data highlight the importance of obesity when interpreting the significance of an elevated CRP level in children with suspected diagnosis of appendicitis. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2511-yAuthors Balazs Kutasy, The National Children’s Hospital, Dublin Dublin 24 IrelandGanapathy Laxamanadass, The National Children’s Hospital, Dublin Dublin 24 IrelandPrem Puri, The National Children’s Hospital, Dublin Dublin 24 Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Loss of intestine during stoma closure: an experimental model comparing laparoscopic and conventional techniques

Abstract Purpose This study compared laparoscopy-assisted stoma closure (Lap) with conventional closure (Co) to assess loss of intestine. Methods Ileostomies (loop L; single S) were performed 5 cm proximal to the ileocecal junction through a right lower quadrant incision in forty 11-week-old Lewis rats (L = 20, S = 20). Stoma closure was performed 60 days later using laparoscopy (Lap) or conventional closure (Co) in 10 rats each, to give 4 groups, Lap-L, Lap-S, Co-L, and Co-S. End-to-end anastomosis was performed through the stoma site in all rats. Bowel resected from the skin to the anastomosis was termed resected unusable bowel (RUB) and measured blindly. Laparotomy was performed 30 days later to assess the status of the anastomosis and complications. Results Average RUB with Lap was significantly shorter; Lap-L (17.8 mm) versus Co-L (23.8 mm), P = 0.002, and Lap-S (10.6 mm) versus Co-S (13.8 mm), P = 0.001. During Co, accidental full-thickness injury to underlying bowel during stoma take-down occurred in 3 Co-L and 2 Co-S rats. All Lap rats were uncomplicated. Average times taken until end of stoma take-down were 6.1 min for Lap-L (3.2 min for trocar insertion, 2.8 min for stoma take-down), 5.6 min for Lap-S (2.8 and 2.7 min), 6.3 min for Co-L (from first incision to stoma take-down), and 5.1 min for Co-S (P = NS). At laparotomy there was no evidence of complications such as wound infection, incisional hernia or anastomotic stenosis in any rat. Conclusions Our results suggest that laparoscopy-assisted stoma closure is safe and quick, and results in less loss of intestine during stoma closure. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2513-9Authors Go Miyano, Juntendo University School of Medicine Department of Pediatric General and Urogenital Surgery 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421 JapanSatoko Ichikawa, Juntendo University School of Medicine Department of Pediatric General and Urogenital Surgery 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421 JapanGeoffrey J. Lane, Juntendo University School of Medicine Department of Pediatric General and Urogenital Surgery 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421 JapanYoshifumi Kato, Juntendo University School of Medicine Department of Pediatric General and Urogenital Surgery 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421 JapanTadaharu Okazaki, Juntendo University School of Medicine Department of Pediatric General and Urogenital Surgery 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421 JapanAtsuyuki Yamataka, Juntendo University School of Medicine Department of Pediatric General and Urogenital Surgery 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421 Japan Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Amniotic fluid stem cell migration after intraperitoneal injection in pup rats: implication for therapy

Abstract Purpose Despite being commonly used in clinical practice, the intraperitoneal (i.p.) route has been rarely used for cell delivery. We evaluated the capacity of amniotic fluid stem (AFS) cells, administered i.p., to diffuse systemically and to integrate into tissues of healthy newborn rats. Methods AFS cells were obtained from pregnant GFP + Sprague–Dawley rats by c-kit selection. Wild-type Sprague–Dawley newborn rats were divided into two groups receiving i.p.: (1) 2 × 106 AFS cells (n = 12); (2) of phosphate buffer saline (PBS) (n = 2) at 24 and 48 h after birth. Animals were either killed at 96 h of life, and organs collected for gfp amplification, or at 3 weeks of life and tissues isolated for green fluorescence protein (GFP) immunofluorescence. Results No adverse effects were observed after i.p. injection of PBS or AFS cells. Gfp was amplified in at least one organ in all rats injected with AFS cells except one (11/12). The intestine was the organ found most frequently positive (67%) followed by liver (25%), spleen (16%), heart (16%), lungs (16%), femur (8%) and brain (0%). Immunohistochemistry confirmed PCR results. Conclusion In the short term, the i.p. administration of AFS cells, is a safe procedure and allows their migration, homing and integration into various organs of healthy newborn rats. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2504-xAuthors Marco Ghionzoli, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKMara Cananzi, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKAugusto Zani, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKCarlo Alberto Rossi, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKFrancesco Fascetti Leon, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKAgostino Pierro, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKSimon Eaton, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UKPaolo De Coppi, Institute of Child Health Department of Surgery 30 Guilford Street London WC1N 1EH UK Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Fgf10 gene expression is delayed in the embryonic lung mesenchyme in the adriamycin mouse model

Abstract Background The adriamycin mouse model is a well-established teratogenic model of esophageal atresia/tracheoesophageal fistula. Fibroblast growth factor 10 (Fgf10) plays a key role in branching of the lung buds during lung morphogenesis. Fgf10 knockout mice exhibit the absence of the lungs. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Fgf10 during the critical period of separation of the trachea and esophagus in normal and adriamycin-treated embryos using OPT. Methods Time-mated CBA/Ca mice received intraperitoneal injections of adriamycin (6 mg/kg) or saline on days 7 and 8 of gestation. Embryos were harvested on days 10–13, stained after whole mount in situ hybridization with labeled RNA probes to detect Fgf10 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with endoderm marker Hnf3β was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains. Results Computer reconstructed specimens allowed precise staging of developing embryos according to Theiler Staging (TS) criteria. OPT elegantly displayed Fgf10 gene expression in the pulmonary mesenchyme around the tip of the lung buds in both controls and treated embryos in the same spatial territory. Fgf10 gene expression was first detected in the control embryos at TS17. However, Fgf10 gene expression in adriamycin-treated embryos was first only observed at TS18 in 67% of the specimens. Conclusion Delayed Fgf10 gene expression during the critical period of separation of the trachea and esophagus may affect lung bud formation in the adriamycin model leading to tracheoesophageal malformations. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2519-3Authors Piotr Hajduk, Our Lady’s Children’s Hospital Children’s Research Centre Crumlin Dublin 12 IrelandPaula Murphy, Trinity College, University of Dublin Department of Zoology Dublin 2 IrelandPrem Puri, Our Lady’s Children’s Hospital Children’s Research Centre Crumlin Dublin 12 Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Does intestinal permeability lead to organ failure in experimental necrotizing enterocolitis?

Abstract Purpose The rat gavage model is used to explore the pathogenesis and treatment of necrotizing enterocolitis (NEC). Although intestinal histological damage is seen in this model, intestinal perforation is rarely observed. Whether organ failure occurs in this model is largely unknown. We hypothesised that increased intestinal permeability leads to organ failure in experimental NEC. Methods NEC was induced in neonatal rats by gavage feeding of hypertonic formula plus exposure to hypoxia plus oral lipopolysaccharide (4 mg/kg per day daily). Breast-fed rats were used for comparison. At 92 h, lactulose (3 mg) and mannitol (2 mg) were administered orally in 0.1 ml water. Four hours later, rats were killed and blood samples collected. Lactulose and mannitol were measured by gas chromatography–mass spectrometry and lactulose/mannitol ratio calculated as index of intestinal permeability. Plasma cardiac troponin-I was measured by ELISA as a marker of cardiac damage and plasma creatinine measured spectrophotometrically as a marker of renal failure. Results Experimental NEC induced an increase in intestinal permeability (P = 0.0002). This was associated with cardiac damage (P < 0.0001), and renal failure (P = 0.004). Conclusion Intestinal permeability is increased in experimental NEC in association with increased cardiac damage. Rat mortality may be due to cardiac failure secondary to an inflammatory response caused by increased intestinal permeability. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2507-7Authors Augusto Zani, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKMarco Ghionzoli, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKGiuseppe Lauriti, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKMara Cananzi, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKVirpi V. Smith, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKAgostino Pierro, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKPaolo De Coppi, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UKSimon Eaton, Institute of Child Health Surgery Unit, Department of Surgery 30 Guilford Street London WC1N 1EH UK Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Fluorescence-activated cell sorting of PCK-26 antigen-positive cells enables selection of ovine esophageal epithelial cells with improved viability on scaffolds for esophagus tissue engineering

Abstract Objective For esophagus tissue engineering, isolation and proliferation of esophageal epithelial cells (EEC) is a pre-requisite for scaffold seeding to create constructs. The aim of this study was to sort EEC expressing cytokeratin markers and their proliferative subpopulations; also, to investigate the viability of differentiated EEC subpopulations on collagen scaffolds. Methods Ovine esophageal epithelial cells (OEECs) from sheep esophagus were analyzed using flow cytometry for pan cytokeratin (PCK-26) and proliferation cell nuclear antigen (PCNA). Using fluorescent-activated cell sorting, OEEC were separated and analyzed for PCNA expression. The OEEC subpopulations were seeded on collagen scaffolds for a week in vitro culture. Results Proliferation cell nuclear antigen was expressed in >45% of OEEC isolated. In flow cytometry, 30% OEEC were PCK-26 positive which exhibited a high-proliferative capacity of 80%. PCK-26-negative OECC exhibited a low-proliferative capability of 13%. Scanning electron microscopy demonstrated organized attachment and uniform scaffold coverage in PCK-26-positive cells. Conclusion Ovine esophageal epithelial cells can be divided into PCK-26-positive and negative subpopulations. PCK-26-positive OEEC constitute one-third of the isolated cells with high-proliferative capability. Seeding of PCK-26-positive OEEC on collagen scaffolds leads to uniform distribution of cells in vitro. In esophagus, tissue engineering PCK-26-positive OEEC subpopulation is important for optimal construct generation. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2512-xAuthors Kristina Kofler, Medical University of Graz Department of Pediatric and Adolescent Surgery Auenbruggerplatz 34 8036 Graz AustriaHerwig Ainoedhofer, Medical University of Graz Department of Pediatric and Adolescent Surgery Auenbruggerplatz 34 8036 Graz AustriaMichael E. Höllwarth, Medical University of Graz Department of Pediatric and Adolescent Surgery Auenbruggerplatz 34 8036 Graz AustriaAmulya K. Saxena, Medical University of Graz Department of Pediatric and Adolescent Surgery Auenbruggerplatz 34 8036 Graz Austria Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Disturbance of parathyroid hormone-related protein signaling in the nitrofen-induced hypoplastic lung

Abstract Purpose Despite remarkable progress in resuscitation and intensive care, the morbidity and mortality rates in congenital diaphragmatic hernia (CDH) remain high due to severe pulmonary hypoplasia. The pathogenesis of pulmonary hypoplasia associated with CDH is still not clearly understood. Pulmonary parathyroid hormone-related protein (PTHrP) is expressed in the type II epithelial cells and stimulates surfactant production by a paracrine feedback loop regulated by PTHrP receptor (PTHrP-R), which is expressed in the mesenchyme, during terminal airway differentiation. It has been reported that PTHrP knockout and PTHrP-R null mice both exhibit pulmonary hypoplasia, disrupting alveolar maturation before birth. We designed this study to test the hypothesis that gene expression of PTHrP and PTHrP-R is downregulated in the late stages of lung morphogenesis in the nitrofen-induced hypoplastic lung. Methods Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, 18, and 21 and divided into three groups: control, nitrofen without CDH [CDH(−)], and nitrofen with CDH [CDH(+)] (n = 8 at each time point for each group, respectively). Total mRNA was extracted from fetal lungs and mRNA expression of PTHrP and PTHrP-R was analyzed by real-time RT-PCR and the significant differences between the groups were accepted at P < 0.05 by statistical analysis. Immunohistochemical studies were also performed to evaluate PTHrP and PTHrP-R protein expression at each time point. Results Pulmonary mRNA expression of PTHrP-R was significantly decreased in both nitrofen groups [CDH(−) and CDH(+)] compared to controls at D18 and 21. The mRNA level of PTHrP was significantly decreased at D21 in both nitrofen groups compared to controls. Immunoreactivity of PTHrP and PTHrP-R at D18 and 21 was diminished in the distal epithelium and in the mesenchyme, respectively, in the nitrofen-induced hypoplastic lung compared to control lungs. There were no significant differences in both gene/protein expression of PTHrP and PTHrP-R on D15. Conclusion Downregulation of PTHrP and PTHrP-R gene expression during late lung morphogenesis may cause pulmonary hypoplasia in the nitrofen CDH model, disrupting alveolar maturation and surfactant production by interfering with mesenchymal–epithelial interactions. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2506-8Authors Takashi Doi, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 IrelandAušra Lukošiūtė, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 IrelandElke Ruttenstock, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 IrelandJens Dingemann, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 IrelandPrem Puri, Our Lady’s Children’s Hospital The Children’s Research Centre Dublin 12 Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Upregulation of Endothelin Receptors A and B in the nitrofen induced hypoplastic lung occurs early in gestation

Abstract Purpose Pulmonary hypoplasia and persistent pulmonary hypertension (PPH) aggravate clinical courses in congenital diaphragmatic hernia (CDH). Endothelin 1 enhances PPH by vasoconstriction and proliferation of vessel walls. Up-regulation of pulmonary Endothelin Receptors A and B (EDNRA, EDNRB) has been reported in human CDH and animal models, but the onset of those alterations during lung development remains unclear. We hypothesized that pulmonary expression of EDNRA and EDNRB is up-regulated at early gestational stages in the nitrofen model. Methods Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Embryos were sacrificed on D15, D18 and D21 and divided into nitrofen- and control group. Pulmonary RNA was extracted and mRNA levels of EDNRA and EDNRB were determined by real-time PCR. Immunohistochemistry for protein expression of both receptors was performed. Results mRNA levels of EDNRA and EDNRB were significantly increased in the nitrofen group on D15, D18 and D21. Immunohistochemistry revealed increased pulmonary vascular expression of EDNRA and EDNRB compared to controls. Conclusion Altered expression of EDNRA and EDNRB is an early event in lung morphogenesis in the nitrofen model. We speculate that pulmonary arteries in CDH become excessively muscularised in early fetal life, becoming unable to adapt normally at birth. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2514-8Authors Jens Dingemann, The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 IrelandTakashi Doi, The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 IrelandElke Ruttenstock, The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 IrelandPrem Puri, The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

The 22nd international symposium on paediatric surgical research

The 22nd international symposium on paediatric surgical research Content Type Journal ArticleCategory EditorialDOI 10.1007/s00383-009-2510-zAuthors Prem Puri, Our Lady’s Children’s Hospital Children’s Research Centre Dublin Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Oral insulin stimulates intestinal epithelial cell turnover in correlation with insulin-receptor expression along the villus–crypt axis in a rat model of short bowel syndrome

Abstract Purpose It has been reported that oral insulin (OI) has a trophic effect on intestinal mucosa. In the present study, we evaluated the effect of OI on enterocyte turnover and correlated it with insulin-receptor expression along the villus–crypt axis in a rat model of short bowel syndrome (SBS). Methods Male rats were divided into three groups: Sham rats underwent bowel transection, SBS rats underwent a 75% bowel resection, and SBS–OI rats underwent bowel resection and were treated with OI given in drinking water (1 U/ml) from the fourth postoperative day. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined on day 15. Real-time PCR was used to determine the level of insulin receptor-beta (IRB) mRNA. Insulin-receptor expression along the villus–crypt axis (villus tips, lateral villi and crypts) was assessed by immunohistochemistry. The effect of OI on cell turnover for each compartment was evaluated in correlation with the receptor expression. Statistical analysis was performed using the one-way ANOVA test, with P < 0.05 considered statistically significant. Results Treatment with OI resulted in a significant increase in all parameters of intestinal adaptation. Insulin-receptor expression in crypts significantly increased in SBS rats (vs. Sham rats) and was accompanied by a significant increase in enterocyte proliferation following OI administration. A significant increase in insulin-receptor expression at the tip of the villous and in the lateral villous in SBS rats (vs. Sham) was accompanied by decreased cell apoptosis in these compartments following treatment with OI. Conclusions In a rat model of SBS, OI enhances enterocyte turnover and stimulates intestinal adaptation. The stimulating effect of insulin on enterocyte turnover correlates with insulin-receptor expression along the villus–crypt axis. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2520-xAuthors Shani Ben Lulu, The Ruth & Bruce Rappaport Faculty of Medicine Laboratory of Intestinal Adaptation and Recovery, Technion-Israel Institute of Technology Haifa IsraelArnold G. Coran, University of Michigan Medical School Section of Pediatric Surgery, C.S. Mott Children’s Hospital Ann Arbor MI USAJorge G. Mogilner, Bnai Zion Medical Center Department of Pediatric Surgery B 47 Golomb St. P.O.B. 4940 31048 Haifa IsraelRon Shaoul, Rambam Medical Center Department of Pediatric Gastroenterology and Nutrition, Meyer Children’s Hospital Haifa IsraelRaanan Shamir, Tel-Aviv University Institute of Gastroenterology, Nutrition and Liver Diseases, Schneider Children’s Medical Center, Sackler School of Medicine Tel Aviv IsraelNaim Shehadeh, Rambam Medical Center Department of Pediatrics, Meyer Children’s Hospital Haifa IsraelIgor Sukhotnik, Bnai Zion Medical Center Department of Pediatric Surgery B 47 Golomb St. P.O.B. 4940 31048 Haifa Israel Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Carbon dioxide modifies the morphology and function of mesothelial cells and facilitates transepithelial neuroblastoma cell migration

Abstract Background The response of mesothelial cells to surgical trauma and bacterial contamination is poorly defined. We have recently shown that CO2 pneumoperitoneum increases systemic metastasis of neuroblastoma cells in a murine model. Thus, we hypothesized that CO2 alters the morphology and function of mesothelial cells and facilitates transmesothelial tumor cell migration. Materials and methods Murine mesothelial cells were exposed to 100% CO2 and 5% CO2 as control. Scanning electron microscopy (SEM) investigations, as well as LPS-induced granulocyte-colony stimulating factor (G-CSF) production and mitochondrial activity (MTT assay) were measured. Transmesothelial migration of neuroblastoma cells (Neuro2a) was determined using a transwell chamber system. Results CO2 incubation was associated with a significant destruction of the microvillar formation in SEM. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was identified after 100% CO2 exposure (P < 0.05). Although the conversion of MTT as an indicator of mitochondrial activity was only slightly and not significantly reduced after CO2 incubation, the release of G-CSF induced by LPS was completely blocked during the incubation with 100% CO2 (P < 0.05). The capacity of G-CSF release recovered after the incubation. Conclusion We observed that peritoneal mesothelial cells lose their typical cell morphology by CO2 incubation, which is accompanied by facilitated migration of neuroblastoma cells. Moreover, the synthesis of immunological factors is blocked, but this effect is not long lasting. These mechanisms may explain an increased metastasis rate of neuroblastoma cells after CO2 pneumoperitoneum, which was recently observed in a murine model. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2503-yAuthors Yi Yu, Hannover Medical School Department of Pediatric Surgery Carl-Neuberg-Str.1 30625 Hannover GermanyJoachim Kuebler, Hannover Medical School Department of Pediatric Surgery Carl-Neuberg-Str.1 30625 Hannover GermanyStephanie Groos, Hannover Medical School Department of Cell Biology Carl-Neuberg-Str.1 30625 Hannover GermanyMartin Metzelder, Hannover Medical School Department of Pediatric Surgery Carl-Neuberg-Str.1 30625 Hannover GermanySilvia Kurpanik, Hannover Medical School Department of Pediatric Surgery Carl-Neuberg-Str.1 30625 Hannover GermanyBenno Manfred Ure, Hannover Medical School Department of Pediatric Surgery Carl-Neuberg-Str.1 30625 Hannover GermanyGertrud Vieten, Hannover Medical School Department of Pediatric Surgery Carl-Neuberg-Str.1 30625 Hannover Germany Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Management of acute appendicitis: an imaging strategy in children

Abstract The pre-operative diagnosis of acute appendicitis (AA) has markedly changed during the last couple of decades due to the advent of modern imaging technology. Nowadays, the use of imaging has dramatically changed the way we approach children admitted to emergency room for abdominal pain with suspected AA. This change is mainly manifested in our diagnostic strategy. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2493-9Authors David Neufeld, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Surgery 59 Tchernichovsky St. 44281 Kfar Sava IsraelMichael Vainrib, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Urology Kfar Sava IsraelGenady Buklan, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Pediatric Surgery Kfar Sava IsraelMichael Gutermacher, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Pediatric Surgery Kfar Sava IsraelHaim Paran, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Surgery 59 Tchernichovsky St. 44281 Kfar Sava IsraelMyriam Werner, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Diagnostic Imaging Kfar Sava IsraelValeria Rathause, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Diagnostic Imaging Kfar Sava IsraelRivka Zissin, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Diagnostic Imaging Kfar Sava IsraelLudwig Lazar, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Pediatric Surgery Kfar Sava IsraelIlan Erez, Meir Medical Center (affiliated to the Sackler School of Medicine, Tel Aviv University) Department of Pediatric Surgery Kfar Sava Israel Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Downregulation of insulin-like growth factor binding protein 3 and 5 in nitrofen-induced pulmonary hypoplasia

Abstract Purpose The high mortality in congenital diaphragmatic hernia (CDH) is mainly attributed to pulmonary hypoplasia. Recent studies suggest that retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung. The insulin-like growth factor (IGF) system plays a crucial role in fetal lung development by interaction of IGFBP-3 and IGFBP-5 with RSP. We hypothesized that pulmonary IGFBP-3 and IGFBP-5 gene expression levels are downregulated in the nitrofen-induced pulmonary hypoplasia. Methods Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 (D9.5) of gestation. Fetal lungs were harvested on D18 and D21 and divided into control and nitrofen groups. IGFBP-3 and IGFBP-5 pulmonary gene and protein expression were determined using real-time RT–PCR and immunohistochemistry. Results Relative levels of IGFBP-3 mRNA were significantly decreased in the nitrofen group (8.00 ± 14.44) in D21 compared to controls (14.81 ± 16.11; p < 0.05). Expression levels of IGFBP-5 mRNA were also significantly decreased in nitrofen group (10.66 ± 4.83) on D18 compared to controls (17.92 ± 4.77). Immunohistochemistry showed decreased IGFBP-3 expression on D21 and decreased IGFBP-5 immunoreactivity on D18 in hypoplastic lungs compared to controls. Conclusion Downregulation of IGFBP-3 and IGFBP-5 gene expression may cause pulmonary hypoplasia in the nitrofen-induced CDH model by interfering with retinoid signaling pathway. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2509-5Authors Elke Ruttenstock, University College Dublin The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 IrelandTakashi Doi, University College Dublin The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 IrelandJens Dingemann, University College Dublin The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 IrelandPrem Puri, University College Dublin The Children’s Research Centre, Our Lady’s Children’s Hospital Dublin 12 Ireland Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Neonatal pneumoperitoneum: a critical appraisal of its causes and subsequent management from a developing country

Abstract Background The diagnosis and management of neonatal pneumoperitoneum revolves around necrotizing enterocolitis (NEC) in most of the published literature. Although NEC remains the major cause of pneumoperitoneum in a neonate, there are several other causes leading to free air in the peritoneal cavity. A number of case reports have appeared describing pneumoperitoneum in a newborn due to rupture of one particular organ, but there have been only few collective reviews on the subject. The present study shares the experience with neonates admitted with a diagnosis of pneumoperitoneum in a pediatric surgical center of a developing country. The various causes of pneumoperitoneum in a newborn, their management and subsequent outcome are described. Materials and methods The study was conducted in the Department of Pediatric Surgery, CSMMU (upgraded King Georges Medical College), Lucknow, India. All the neonates admitted with a diagnosis of pneumoperitoneum during the period of last 3 years (2005–2008) were retrospectively analyzed. Other neonatal admissions were also retrieved for the same period. Free air was confirmed by erect abdominal X-ray or lateral decubitus films in certain cases. The data sheets were analyzed regarding age of presentation, cause of bowel perforation, management offered and subsequent outcome achieved. All patients of NEC without evidence of perforation were not included in the study (n = 21). Results Out of total 537 neonatal admissions, 89 (16.5%) neonates were admitted with a diagnosis of pneumoperitoneum. There were 79 (88.7%) males and only 10 (11.6%) female neonates admitted during the study period. All of them had evidence of pneumoperitoneum at the time of admission. The age at presentation ranged from 4 to 32 days. NEC remained the single major cause of pneumoperitoneum in the newborn; however, in 44 (49.4%) patients the cause was not related to NEC. Perforated pouch colon, isolated colonic perforations, caecal perforations, gastric and duodenal perforations were the main causes of pneumoperitoneum not related to NEC. There were seven patients in whom no cause of pneumoperitoneum could be ascertained. The treatment was individualized according to the presentation. Most of the NEC-related perforations were managed by peritoneal drains. Laparotomy was done in rest of the patients. Three patients were managed conservatively. Overall, 19 (21.6%) patients expired. Most of those expired were of low birth weight with NEC and congenital pouch colon with perforation. Conclusion Neonatal pneumoperitoneum remains a surgical emergency and outcome can be lethal if the problem is not addressed early. NEC remains the major cause; however, there are several other important causes of isolated gastrointestinal perforations leading to neonatal pneumoperitoneum. The management should be individualized in these patients and the outcome largely depends on the early recognition of the condition. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2488-6Authors Tanvir Roshan Khan, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow IndiaJile Dar Rawat, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow IndiaIntezar Ahmed, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow IndiaKumar A. Rashid, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow IndiaMadhukar Maletha, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow IndiaAshish Wakhlu, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow IndiaShiv Narain Kureel, CSM Medical University (Upgraded KGMC) Department of Pediatric Surgery Lucknow India Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358

Pediatric tracheostomy: complications and role of home care in a developing country

Abstract Introduction Tracheotomy in its earlier days was most commonly performed for acute airway infection in children. Its indications are now changing; it is now most commonly performed for congenital malformations (McMurray and Prescott in Practical pediatric otolaryngology. W.B. Saunders Company, Philadelphia, pp 575–592, 1996). This shift in indication has increased the rate of survival of such patients, and therefore the number of children going home after tracheostomy has also increased. Objective This study was conducted (1) to observe the pattern of indication and complications for tracheostomy, in our part of the world, (2) the rate at which tracheostomy can help wean patients off the ventilator, and (3) the feasibility of sending these children home with tracheostomy. Materials and methods A retrospective study was done on 127 patients. The indications, final outcome and the complications encountered in and outside the hospital were studied through review of charts. Results Based on the main indications, patients were grouped into: prolonged ventilation group (PV) 61%, followed by mechanical obstruction group (MO) 22%, and the last being adjunct to surgery group (AS) 17%. The in-hospital complication rate was 30% and that at home was 18.11%. The most common complications included upper respiratory tract infections, and blockage or displacement of tubes. The late complication rate was 4%. Hundred (78.8%) patients on the ventilator could be successfully weaned off, with a p value of 0.001; 81 were sent home with the tracheostomy tube (TT). Forty of these were successfully decannulated and the overall decannulation rate was 48.8%. Conclusion A large number of tracheostomies have been performed in the PV group to reduce the intensive care unit (ICU) stay and to prevent nosocomial infections. The need arises from the high cost of prolonged stay in an ICU setup, which is a cause of major economic burden, and lack of financial assistance for these patients worsens the scenario. Home care of the tracheostomy tubes remains a good option for patients requiring long periods of time to overcome their primary pathology. Content Type Journal ArticleCategory Original ArticleDOI 10.1007/s00383-009-2494-8Authors Sadaf Zia, Aga Khan University Hospital Department of Otolaryngology Head and Neck Surgery Karachi PakistanMuhammad Arshad, Aga Khan University Hospital Department of Pediatric Surgery Karachi PakistanZafar Nazir, Aga Khan University Hospital Department of Pediatric Surgery Karachi PakistanSohail Awan, Aga Khan University Hospital Department of Otolaryngology Head and Neck Surgery Karachi Pakistan Journal Pediatric Surgery InternationalOnline ISSN 1437-9813Print ISSN 0179-0358