The impact of the recipient intestinal site on the differentiation of transplanted enteric neural crest cells.

PURPOSE: It has long been established that the failure of enteric neural crest cells (ENCCs) to colonize the entire gut results in aganglionosis at the distal colon in Hirschsprung disease (HD). However, it is still unclear how the intestinal microenvironment of the distal aganglionic gut differs from that of the proximal ganglionic gut in HD versus normal gut. We have recently succeeded in transplanting ENCC into aganglionic gut in endothelin receptor B (Ednrb) knockout (KO) mice. to advance the development of cell therapy for HD, it is essential to determine if the transplanted ENCCs differentiate normally in aganglionic gut. Therefore, we designed this study to investigate the impact of the environment of the recipient intestinal tract, at various sites of aganglionic gut, on the differentiation of transplanted ENCCs. METHODS: ENCCs were isolated from Sox10 Venus transgenic (Tg) mouse gut on embryonic day 18.5 (E18.5) and neurospheres (NS) were generated. Then, NS were transplanted into aganglionic KO and wildtype (WT) gut that had been transected just distal to the ENCC wavefront (KO-wf: n = 6, WT: n = 7), and into distal KO gut transected at a site equivalent to that of the WT (KO-d: n = 6) on E12.5. ENCC differentiation was evaluated using whole-mount immunohistochemistry with Tuj-1 (neuronal marker) and GFAP (glial marker) antibodies. RESULTS: The transplanted ENCCs migrated to form the myenteric and submucosal plexus in all groups. The ratio of the area of Tuj-1-positive cells/GFAP-positive cells in migrated cells in the recipient gut was found to be significantly lower in KO-d compared to KO-wf and WT, while there was no significant difference between KO-wf and WT groups. This suggests that neuronal/glial differentiation was decreased in KO-d compared to that in KO-wf and WT groups. CONCLUSION: Our study highlights the differences in ENCC differentiation depending on the site of transplantation. To further develop cell therapy for HD, it is important to consider the impact of the recipient intestinal environment on transplanted ENCCs.

A novel mouse model of intestinal neuronal dysplasia: visualization of the enteric nervous system.

PURPOSE: Intestinal neuronal dysplasia (IND) is a congenital anomaly affecting gastrointestinal neural innervation, but the pathogenesis remains unclear. The homozygous Ncx/Hox11L.1 knockout (Ncx-/-) mice exhibit megacolon and enteric ganglia anomalies, resembling IND phenotypes. Sox10-Venus transgenic mouse were used to visualize enteric neural crest cells in real time. This study aims to establish a novel mouse model of Sox10-Venus+/Ncx-/- mouse to study the pathogenesis of IND. METHODS: Sox10-Venus+/Ncx-/- (Ncx-/-) (n = 8) mice and Sox10-Venus+/Ncx+/+ controls (control) (n = 8) were euthanized at 4-5 weeks old, and excised intestines were examined with fluorescence microscopy. Immunohistochemistry was performed on tissue sections with neural marker Tuj1. RESULTS: Ncx-/- mice exhibited dilated cecum and small intestine. Body weight of Ncx-/- mice was lower with higher ratio of small intestine length relative to body weight. The neural network (Sox10-Venus) was observed along the intestine wall in Ncx-/- and control mice without staining. Ectopic and increased expression of Tuj1 was observed in both small intestine and proximal colon of Ncx-/- mice. CONCLUSION: This study has established a reliable animal model that exhibits characteristics similar to patients with IND. This novel mouse model can allow the easy visualization of ENS in a time- and cost-effective way to study the pathogenesis of IND.

Comparison of robotic assistance and laparoscopy for pediatric choledochal cyst: advantages of robotic assistance.

AIM: Surgery for pediatric choledochal cyst (CC), complete excision (CE), and Roux-en-Y hepaticojejunostomy anastomosis (HJA) can be performed using laparoscopy (Lap), robotic-assistance (Rob; da Vinci Xi/Si), or both (Lap/Rob). METHODS: Lap was used exclusively between 2009 and 2021 (n = 31) and Rob was introduced in 2017 (n = 23). All subjects were matched for age, weight, BMI, and episodes of preoperative pancreatitis. For Rob, the first 15/23 were Lap-CE/Rob-HJA and the last 8/23 were Rob-CE/Rob-HJA. RESULTS: Total anastomotic time (TAT), TAT per suture during HJA, and time taken for dissection during CE were significantly shorter with less variance for Rob, although overall operative times were similar. Serum amylase on postoperative days 3, 5, and 7 were significantly higher for Lap. Times taken to ambulate, for return of bowel sounds, and discharge home were all significantly shorter for Rob. All postoperative complications occurred after Lap; HJA leak (n = 1; 3.2%), HJA stricture (n = 1; 3.2%), both treated by open re-HJA; and pancreatic fistula (n = 6; 19%), all treated conservatively. CONCLUSION: Dissection and recovery were faster with Rob while overcoming Lap-associated shortcomings to prevent complications associated with suturing. Both CE and HJA were safer and more reliable with Rob, a reflection of Rob's superiority.

In vitro investigation of the differentiation of enteric neural crest-derived cells following transplantation of aganglionic gut in a mouse model.

PURPOSE: Cell therapy is a promising approach to treat enteric neuropathies such as Hirschsprung disease (HD). Recent studies have reported that enteric neurons derived from stem cells (ENCCs) can be grafted into the HD colon. Thus, we investigated the migration and generation of enteric neurospheres from SOX10-VENUS+ mice after transplantation into control or Ednrb KO mice, which are a model of HD. METHODS: Single-cell suspensions were isolated from the fetal guts of SOX10-VENUS+ mice E13.5 and dissociated. These cells were cultured for 7 days under non-adherent conditions to generate neurospheres, which were co-cultured with dissociated control or SOX10-VENUS- Ednrb KO mouse gut on E13.5. 4 days later, these cells were fixed and the expression of the neuronal marker, Tuj1, was evaluated. RESULTS: Transplanted neurospheres had undergone abundant neuronal migration and differentiation of ENCCs in the control gut compared with the HD gut. The average length and intersections were significantly decreased in HD colon compared with controls (p < 0.05), and a similar pattern was observed in the HD small intestine (p < 0.05). CONCLUSIONS: We demonstrated that transplanted ENCCs did not differentiate properly in HD gut. These results highlight the importance of the neuronal environment in the recipient gut for enteric nervous system development.

Increased enteric neural crest cell differentiation after transplantation into aganglionic mouse gut.

PURPOSE: In recent years, many studies have made considerable progress in the development of stem cell-based therapies for Hirschsprung's disease (HD). However, the question of whether enteric neural crest-derived cells (ENCCs) that are transplanted into the aganglionic gut can migrate, proliferate, and differentiate in a normal manner remains unanswered. Thus, we designed this study to compare the behavior of ENCCs transplanted into the aganglionic gut of endothelin receptor B knockout (Ednrb-KO) mice versus wild-type (WT) mice. METHODS: ENCCs were isolated from the fetal guts of Sox10 transgenic mice, in which ENCCs were labeled with an enhanced green fluorescent protein, Venus, on an embryonic day 18.5 (E18.5). Neurospheres were generated and transplanted into the aganglionic region of either Ednrb-KO mice gut, or WT mice gut that had not yet been colonized, on E12.5. Time-lapse imaging of the transplanted ENCCs was performed after 24, 48, and 72 h of culture. Neuronal differentiation was evaluated using whole-mount immunohistochemistry. RESULTS: Sox10-positive ENCCs were seen to successfully migrate into the myenteric region of the aganglionic gut following transplantation in both the Ednrb-KO and WT mice. The ratio of Tuj1-positive/Sox10-positive cells was significantly increased after 72 h of culture compared to 24 h in the Ednrb-KO mice, which suggests that the transplanted ENCCs differentiated over time. In addition, at the 72 h timepoint, neuronal differentiation of transplanted ENCC in the aganglionic gut of Ednrb-KO mice was significantly increased compared to that of WT mice. CONCLUSIONS: The results of our study demonstrated that transplanted ENCCs migrated into the myenteric region of the aganglionic recipient gut in mice. The increased neuronal differentiation of transplanted ENCC in Endrb-KO mice gut suggests that the microenvironment of this region affects ENCC behavior following transplantation. Further research to explore the characteristics of this microenvironment will improve the potential of developing cell therapy to treat HD patients.

Differentiation of enteric neural crest cells transplanted from SOX10-Venus mouse embryonic stem cells into the gut of the endothelin receptor B null mouse model.

PURPOSE: Failure of enteric neural crest-derived cells (ENCCs) to correctly colonize the embryonic gut results in Hirschsprung's disease (HD). Embryonic stem cells (ESCs) have the potential to differentiate into all tissue-specific cells and lineages, including ENCCs. We investigated the cellular differentiation of ESCs from Sox10-Venus + mice into both control and endothelin receptor-B knockout (Ednrb KO) mouse gut to assess each region. METHODS: We established ESCs from Sox10-Venus + mice. These cells were cultured for 2 days, then selected and co-cultured with either a dissociated control or Sox10-Venus - Ednrb KO mouse gut (both small intestine and colon) on embryonic day (E) 13.5. Four days later, cells were immunolabeled for Tuj1 and visualized using confocal microscopy. RESULTS: Confocal microscopy revealed that transplanted Sox10-Venu + cells from ESCs migrated extensively within the host gut. Moreover, Tuj1-positive neurites were detected in the transplanted ESCs. Tuj1 expression was significantly decreased in aganglionic HD colon compared to controls (p < 0.05) and the HD small intestine (p < 0.05). CONCLUSIONS: This study demonstrated that an appropriate host environment is crucial for normal and complete colonization of the gut. Further investigations are required to confirm whether modifying this environment can improve the results of this model.

High intestinal vascular permeability in a murine model for Hirschsprung's disease: implications for postoperative Hirschsprung-associated enterocolitis.

PURPOSE: Intestinal vascular permeability (VP) in a murine model for Hirschsprung's disease (HD) and postoperative Hirschsprung-associated enterocolitis (HAEC) were investigated. METHODS: Intestinal VP was determined using a Miles assay using 1% Evans blue injected into a superficial temporal vein of newborn endothelin receptor-B KO HD model (KO) and syngeneic wild-type (WT) mice (n = 5, respectively). Extravasated Evans blue in normoganglionic ileum (Ng-I), normoganglionic proximal colon (Ng-PC) and aganglionic distal colon (Ag-DC) was quantified by absorbance at 620 nm. Quantitative polymerase chain reaction (qPCR) for Vascular Endothelial Growth Factor A (VEGF-A), VEGF-B, CDH5, SELE and CD31, and immunofluorescence for CD31 were performed. RESULTS: VP was significantly higher in Ng-I, Ng-PC, and Ag-DC from KO than WT (p < 0.01, p < 0.05, and p < 0.05, respectively). qPCR demonstrated upregulated VEGF-A in Ng-I and Ag-DC, VEGF-B in Ng-I, and SELE in Ng-I and Ng-PC (p < 0.05, p < 0.05, p < 0.05, p < 0.01 and p < 0.05, respectively), and downregulated CDH5 in Ng-I and Ng-PC from KO (p < 0.05, respectively). Expression of CD31 mRNA in Ng-I and Ag-DC from KO was significantly higher on qPCR (p < 0.05) but differences on immunofluorescence were not significant. CONCLUSIONS: VP may be etiologic for postoperative HAEC throughout the intestinal tract even after excision of aganglionic bowel.

DPP4 inhibitor reinforces cell junction proteins in mouse model of short bowel syndrome.

PURPOSE: Bacterial overgrowth commonly occurs and favors bacterial translocation in short bowel syndrome (SBS). Glucagon-like peptide-2 (GLP-2) is effective for treating SBS, but is rapidly inactivated by dipeptidyl peptidase IV (DPP4). DPP4 inhibitor (DPP4I) is known to be effective for treating SBS. Here, we investigated cell junction protein function following DPP4I administration in a mouse model of SBS. METHODS: Mice were divided into four groups: naïve (n = 5), naïve + DPP4I (n = 6), control (n = 6), and DPP4I (n = 5). All control and DPP4I mice had 50% of their proximal small bowel resected. DPP4I or normal saline was administered orally twice daily from days 1-7 postoperatively. The functions of cell junction proteins were assessed by RT-PCR and immunohistochemistry. Body weights and blood glucose levels were recorded. RESULTS: E-Cadherin was significantly higher in the DPP4I group than in the control group. E-Cadherin, occludin, and claudin-4 were significantly higher in the naïve group than in the control group. Positive staining for E-cadherin and occludin varied widely between the control and DPP4I groups. CONCLUSION: Up-regulation of E-cadherin and occludin by DPP4I may be correlated with the anti-inflammatory action of DPP4I. Therefore, DPP4I may reduce bacterial translocation in SBS.

Decreased expression of ß1 integrin in enteric neural crest cells of the endothelin receptor B null mouse model.

BACKGROUND: Interactions between enteric neural crest-derived cells (ENCC) and the surrounding intestinal microenvironment, such as the extracellular matrix (ECM), are critical for regulating enteric nervous system (ENS) development. Integrins are the major receptors for ECM molecules, such as laminin, which have been reported to be involved in the pathogenesis of Hirschsprung's disease. In this study, we examined the expression of ß1 integrin in the endothelin receptor B (Ednrb) knock out (KO) mouse gut, which presents with an aganglionic colon. METHODS: A Sox10-Venus-positive Ednrb KO mouse, where ENCC is labeled with fluorescent protein, 'Venus', was created. Sox10-Venus-positive Ednrb wild type (WT) were used as controls. Small intestine, proximal colon and distal colon were dissected on E13.5 and E15.5 and ß1 integrin expression of the gut tissue was examined by immunohistochemistry and real time RT-PCR. The cells of the gut dissected on E11.5 were isolated and cultured for 2 days. Venus-positive ENCC were immunostained with ß1 integrin and Tuj-1, which is a marker for neurons. RESULTS: The expression of ß1 integrin was not significantly different between KO and WT in all parts of the gut examined. However, the ß1 integrin expression in the isolated ENCC was significantly decreased in KO compared to WT. The average threshold area was 42.98 ± 17.47% in KO and 73.53 ± 13.77 in WT (p < 0.001). CONCLUSIONS: We demonstrated that ß1 integrin expression was specifically decreased in ENCC in Ednrb KO mice. Our results suggest that impaired interaction between integrin and its ligands may disturb normal ENS development, resulting in an aganglionic colon.

The learning curve of laparoscopic percutaneous extraperitoneal closure (LPEC) for inguinal hernia: protocoled training in a single center for six pediatric surgical trainees.

BACKGROUND: Laparoscopic percutaneous extraperitoneal closure (LPEC) has become a common procedure for repairing inguinal hernia. As a laparoscopic approach, pediatric surgical trainees require more training to learn LPEC than a traditional open approach. This study aimed to clarify the experience needed to acquire the skill to perform LPEC adequately. METHODS: This descriptive single-center study used clinical data from patients who underwent LPEC between May 2009 and May 2016. The mean operative time for ten consecutive unilateral repairs was used as an index of proficiency with the procedure. The number of repairs performed before the mean operative time became less than 20 min was evaluated for each trainee. RESULTS: During the study period, six pediatric surgical trainees participated in the training independently. The number of the patients was 987. The total number of repairs was 1436, including 538 unilateral repairs and 449 concurrent bilateral repairs. Overall, the mean operative time was 21.8 ± 8.1 min for unilateral repair and 31.4 ± 9.7 min for concurrent bilateral repairs. The mean number of repairs performed before the acquisition of skill for dexterous LPEC was 125.1 ± 29.5. CONCLUSIONS: Although there were individual differences, all trainees acquired the skill to perform LPEC adequately within one year. With appropriate guidance, LPEC can become a standard technique for pediatric surgical trainees, along with traditional open surgery. These results provide valuable information for planning LPEC training.

Bone mineral density is increased in the cadmium-induced omphalocele chick model by using three-dimensional micro-computed tomography.

PURPOSE: The cadmium (Cd) chick model has been described as a reliable model of omphalocele. Skeletal anomalies, including lumber lordosis, can be seen in the Cd chick model, as well as in the human omphalocele. Bone deformations, such as lordosis, are associated with high bone mineral density (BMD). Recently, three-dimensional microcomputed tomography (3DMCT) has been used to investigate skeletal development in small animal embryos. We used 3DMCT to test the hypothesis that the BMD is increased in the Cd-induced omphalocele chick model. METHODS: After a 60-h incubation, chicks were exposed to either chick saline or Cd in ovo. Chick embryos were harvested at embryonic day 16.5 (E16.5) and were divided into control (n = 8) and Cd (n = 9). Chicks were then scanned by 3DMCT. The body volume, bone volume, bone/body volume ratio, bone mineral quantity and BMD were analysed statistically (significance was accepted at p < 0.05). RESULTS: Bone mineral density (mg/cm3) was significantly increased in the Cd group compared to control group (235.3 ± 11.7 vs 223.4 ± 4.6, p < 0.05), whereas there was no significant difference in the bone/body volume ratio between the Cd group and the control group (0.7 ± 0.1 vs 0.6 ± 0.0). The body volume (cm3) (0.3 ± 0.2 vs 0.3 ± 0.1), bone volume (cm3) (0.2 ± 0.2 vs 0.2 ± 0.1), and bone mineral quantity (mg) (51.3 ± 41.6 vs 41.5 ± 16.5) were not significantly different between the two groups. CONCLUSIONS: Increased BMD may be associated with lordosis of the vertebral column in the Cd-induced omphalocele chick model, stimulating osteogenesis by activating the canonical Wnt signalling pathway.

Endothelin receptor B affects the perfusion of newborn intestine: possible mechanism of necrotizing enterocolitis development.

BACKGROUND: Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. Hypoxia is known as one of the major risk factors for the development of NEC. Endothelin, known to regulate vasoconstriction, has two receptors (A and B). However, the role of endothelin receptor B (EDNRB) in neonatal intestinal injury remains unclear. We aimed to investigate whether EDNRB is involved in NEC pathophysiology. METHODS: Following ethical approval (#44032), EDNRB hetero knockout mice pups (EDNRB±) and their wild-type (WT) littermates were studied. NEC was induced from postnatal day 5-9 (P5-P9) by hypoxia, gavage feeding of formula and administration of lipopolysaccharide. On P9, the ileum was harvested. RESULTS: NEC induction in WT mice was associated with mucosal injury. However, EDNRB± NEC mice had reduced mucosal injury. Similarly, EDNRB± mice had significantly lower expression of IL-6 mRNA compared to WT NEC mice. Pimonidazole immunostaining was also significantly lower in EDNRB± compared to WT NEC, suggesting reduced tissue hypoxia. CONCLUSIONS: Partial knockout of EDNRB results in reduced NEC severity and reduced tissue hypoxia. Intestinal perfusion and hypoxia are important elements of NEC pathogenesis. These findings are relevant to the understanding of NEC pathophysiology and to the development of novel preventive strategies for NEC.

Altered expression of laminin alpha1 in aganglionic colon of endothelin receptor-B null mouse model of Hirschsprung's disease.

PURPOSE: Laminin, an extracellular matrix molecule, is essential for normal development of the nervous system. The alpha1 subunit of laminin-1 (LAMA1) has been reported to promote neurites and outgrowth and is expressed only during embryogenesis. Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize Enteric neural crest-derived cell (ENCC)s with a green fluorescent protein, Venus. We designed this study to investigate the expression of LAMA1 using Sox10-VENUS mice gut. METHODS: We harvested the gut on days 13.5 (E13.5) and 15.5 (E15.5) of gestation. Sox10-VENUS+/Ednrb -/- mice (n = 8) were compared with Sox10-VENUS+/Ednrb +/+ mice (n = 8) as controls. Gene expression of LAMA1 was analysed by real-time RT-PCR. Fluorescent immunohistochemistry was performed to assess protein distribution. RESULTS: The relative mRNA expression levels of LAMA1 were significantly increased in HD in the proximal and distal colon on E15.5 compared to controls (p < 0.05), whereas there were no significant differences on E13.5. LAMA1 was expressed in the serosa, submucosa and basal lamina in the gut, and was markedly increased in the proximal and distal colon of HD on E15.5. CONCLUSIONS: Altered LAMA1 expression in the aganglionic region may contribute to impaired ENCC migration, resulting in HD. These data could help in understanding the pathophysiologic interactions between LAMA1 and ENCC migration.

Altered differentiation of enteric neural crest-derived cells from endothelin receptor-B null mouse model of Hirschsprung's disease.

PURPOSE: Hirschsprung's disease (HD) is caused by a failure of enteric neural crest-derived cells (ENCC) to colonize the bowel, resulting in an absence of the enteric nervous system (ENS). Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize ENCC with the green fluorescent protein, Venus. The aim of this study was to isolate Sox10-Venus+ cells, which are differentiated neurons and glial cells in the ENS, and analyze these cells using Sox10-Venus mice gut. METHODS: The mid-and hindgut of Sox10-Venus+/Ednrb +/+ and Sox10-Venus+/Ednrb -/- at E13.5 and E15.5 were dissected and cells were dissociated. Sox10-Venus+ cells were then isolated. Expression of PGP9.5 and GFAP were evaluated neurospheres using laser scanning microscopy. RESULTS: 7 days after incubation, Sox10-Venus+ cells colonized the neurosphere. There were no significant differences in PGP9.5 expressions on E13.5 and E15.5. GFAP was significantly increased in HD compared to controls on E15.5 (P < 0.05). CONCLUSIONS: Our results suggest increased glial differentiation causes an imbalance in ENCC lineages, leading to a disruption of normal ENS development in this HD model. Isolation of ENCC provides an opportunity to investigate the ENS with purity and might be a useful tool for modeling cell therapy approaches to HD.

Three- and four-dimensional analysis of altered behavior of enteric neural crest derived cells in the Hirschsprung's disease mouse model.

BACKGROUND/AIM: The behavior of enteric neural crest-derived cells (ENCC) during enteric nervous system (ENS) development is being gradually understood with the introduction of live-cell imaging. However, many of the analyses 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 (Tg) mouse to visualize ENCC with enhanced green fluorescent protein (Venus). By breeding these two models, we have created a Venus-positive, Sox10 Tg 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 using three-dimensional and four-dimensional 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 of Sox10-Venus(+)/Ednrb (-/-) mice on embryonic day 15.5 (E15.5) and Sox10-Venus(+)/Ednrb (+/+) mice on E12.5, which was used as control. Dissected hindguts were cultured for 360 min 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 using Imaris software. RESULTS: Track displacement (TD)-Y indicates ENCC advancement in a rostral-caudal direction. TD-X and TD-Z indicate ENCC advancement perpendicular to the rostral-caudal axis. Mean TD-Y was 34.56 µm in HD, but 63.48 µm in controls. TD-Y/TD-XZ was not significantly different in both groups. However, the mean track speeds were significantly decreased in HD (72.87 µm/h) compared to controls (248.29 µm/h). CONCLUSIONS: Our results showed that the track speed of ENCC advancement was 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.

Downregulation of p300 gene expression in airway mesenchyme of nitrofen-induced hypoplastic lungs.

PURPOSE: Congenital diaphragmatic hernia (CDH) is a relatively common developmental abnormality causing life-threatening respiratory distress at birth. The nitrofen model has been widely used to investigate the pathogenesis of hypoplastic lungs associated with CDH. Embryos lacking p300 and CBP genes are significantly smaller in lung formation. We hypothesized that pulmonary gene expression of p300 and CBP is downregulated during late gestation in the nitrofen-induced CDH model. METHODS: Time-pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 (n = 8 at each time point). Pulmonary gene expression of p300 and CBP was analyzed by quantitative real-time PCR. Immunohistochemistry was performed to investigate expression and localization of pulmonary p300 and CBP proteins. RESULTS: Relative mRNA expression levels of p300 were significantly decreased in nitrofen-induced hypoplastic lungs on D18 compared to controls (3.00 ± 0.20 vs. 3.76 ± 0.14; p = 0.0039), while CBP levels were not altered. p300 immunoreactivity was markedly diminished in surrounding mesenchymal compartments and nuclei of proximal and distal airway cells, while CBP expression was not altered. CONCLUSION: Downregulation of p300 gene expression during the early canalicular stage may disrupt epithelial-mesenchymal signaling interactions, contributing to the development of hypoplastic lungs in the nitrofen-induced CDH model.

Evidence for decreased lipofibroblast expression in hypoplastic rat lungs with congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypoplasia (PH) is a serious condition in newborns with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) play an essential role in fetal lung maturation by stimulating alveolarization and lipid homeostasis. In rodents, LIFs are first evident during the canalicular phase of lung development with a significant increase over the last 4 days of gestation. Adipocyte differentiation-related protein (ADRP), a functional lipogenic molecular marker characterizing LIFs, is highly expressed in fetal lungs during this critical time period. We hypothesized that LIF expression in hypoplastic rat lungs is decreased in the nitrofen-induced CDH model, which is accompanied by reduced alveolar ADRP expression and lipid content. METHODS: On embryonic day 9.5 (E9.5), time-mated rats received either nitrofen or vehicle. Fetuses were sacrificed on selected time points E18.5 and E21.5, and dissected lungs were divided into controls and CDH-associated PH. Pulmonary gene expression levels of ADRP were determined by quantitative real-time polymerase chain reaction. ADRP immunohistochemistry and oil red O staining were used to assess pulmonary protein expression and lipid content. Immunofluorescence double staining for alpha smooth muscle actin, which is known to be absent in LIFs, and lipid droplets was performed to evaluate the pulmonary expression of this specific subset of fibroblasts. RESULTS: Relative mRNA expression of ADRP was significantly reduced in lungs of CDH-associated PH on E18.5 and E21.5 compared to controls. ADRP immunoreactivity and lipid staining were markedly diminished in alveolar mesenchymal cells of CDH-associated PH on E18.5 and E21.5 compared to controls. Confocal laser scanning microscopy demonstrated markedly decreased LIF expression in alveolar interstitium of CDH-associated PH on E18.5 and E21.5 compared to controls. CONCLUSION: Decreased pulmonary LIF expression during late gestation suggests impaired LIF functioning in the nitrofen-induced CDH model, which may cause disruption in fetal alveolarization and lipid homeostasis, and thus contribute to the development of PH.

Optical Trocar Access for Retroperitoneal Robotic-Assisted Pyeloplasty in Children with Ureteropelvic Junction Obstruction.

Purpose: Retroperitoneal robotic-assisted pyeloplasty (ret-RAP) for ureteropelvic junction obstruction (UPJO) requires a larger retroperitoneal space (RS) to maintain specified distances between robotic (da Vinci) trocars and between trocars and the region of interest. A modified closed technique (MOT) and conventional closed technique (COT) were compared for creating an adequate RS with optical trocars. Methods: RS access in children with UPJO who underwent ret-RAP (n = 30) was MOT (n = 15) and COT (n = 15). All patients were positioned laterally. For MOT, a 5 mm optical trocar was inserted at the angle formed between the 12th rib and the erector spinae muscles. As the trocar was advanced under direct vision, it pierced the superficial subcutaneous layer, Scarpa's fascia, lumbar fascia, internal/external oblique and transversus abdominalis muscles, and the posterior renal fascia. Once in the RS, the tip of the scope was used for blunt dissection of perirenal fat, the tip was withdrawn until it was outside the perirenal fascia, and used to dissect toward the anterior abdomen in the pararenal fat layer. Results: Ages and weights at ret-RAP were similar (MOT: 5.6 ± 1.8 years versus COT: 7.8 ± 4.6 years; MOT: 20.6 ± 10.1 kg versus COT: 27.6 ± 13.9 kg). Times for RS access were similar (MOT: 1.6 ± 0.5 minutes versus COT: 1.9 ± 0.7 minutes), but RS expansion was significantly quicker in MOT (32.3 ± 8.7 minutes versus 52.0 ± 15.1 minutes; P < .001). Peritoneal injury caused carbon dioxide leakage in 4 of 15 COT cases and 0 of 15 MOT cases. Conclusion: RS expansion with MOT was safer because there were no peritoneal injuries and MOT was quicker than COT.

Autologous concentrated bone marrow injection for precollapse osteonecrosis of the femoral head concurrent with contralateral total hip arthroplasty: protocol for a clinical trial.

INTRODUCTION: The femoral head contralateral to the collapsed femoral head requiring total hip arthroplasty (THA) often manifests in the precollapse stage of osteonecrosis of the femoral head (ONFH). It is not yet demonstrated how autologous concentrated bone marrow injection may prevent collapse of the femoral head concurrent with contralateral THA. The primary objective is to evaluate the efficacy of autologous concentrated bone marrow injection for the contralateral, non-collapsed, femoral head in patients with bilateral ONFH, with the ipsilateral collapsed femoral head undergoing THA. METHODS AND ANALYSIS: This is a multicentre, prospective, non-randomised, historical-data controlled study. We will recruit patients with ONFH who are scheduled for THA and possess a non-collapsed contralateral femoral head. Autologous bone marrow will be collected using a point-of-care device. After concentration, the bone marrow will be injected into the non-collapsed femoral head following the completion of THA in the contralateral hip. The primary outcome is the percentage of femoral head collapse evaluated by an independent data monitoring committee using plain X-rays in two directions 2 years after autologous concentrated bone marrow injection. Postinjection safety, adverse events, pain and hip function will also be assessed. The patients will be evaluated preoperatively, and at 6 months, 1 year and 2 years postoperatively. ETHICS AND DISSEMINATION: This protocol has been approved by the Certified Committee for Regenerative Medicine of Tokyo Medical and Dental University and Japan's Ministry of Healthy, Labour and Welfare and will be performed as a class III regenerative medicine protocol, in accordance with Japan's Act on the Safety of Regenerative Medicine. The results of this study will be submitted to a peer-review journal for publication. The results of this study are expected to provide evidence to support the inclusion of autologous concentrated bone marrow injections in the non-collapsed femoral head in Japan's national insurance coverage. TRIAL REGISTRATION NUMBER: jRCTc032200229.

Disruption of the bone morphogenetic protein receptor 2 pathway in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Congenital diaphragmatic hernia (CDH) remains a major therapeutic challenge despite advances in neonatal resuscitation and intensive care. The high mortality and morbidity in CDH has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). Bone morphogenetic protein receptor 2 (BMPR2) plays a key role in pulmonary vasculogenesis during the late stages of fetal lung development. BMPR2 is essential for control of endothelial and smooth muscle cell proliferation. Dysfunction of BMPR2 and downstream signaling have been shown to disturb the crucial balance of proliferation of smooth muscle cells contributing to the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that BMPR2 signaling is disrupted in nitrofen-induced CDH. METHODS: Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of BMPR2 and related proteins. RESULTS: Pulmonary Bmpr2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy revealed decreased pulmonary BMPR2 protein expression and increased activation of p38(MAPK) in CDH compared to controls. CONCLUSION: The observed disruption of the BMPR2 signaling pathway may lead to extensive vascular remodeling and contribute to PH in the nitrofen-induced CDH model. BMPR2 may therefore represent a potential target for the treatment of PH in CDH.