Naho Fujiwara

Rectal mucosal dissection during transanal pull-through for Hirschsprung disease: the anorectal or the dentate line?

PURPOSE Both the dentate line (DL) and anorectal line (ARL) are anatomic landmarks for rectal mucosal dissection during transanal pull-through for Hirschsprung disease. We compared outcome after rectal mucosal dissection commencing above the DL (DL group; n = 8) with outcome after rectal mucosal dissection commencing on the ARL (ARL group; n = 6) with normal babies (Cont group; n = 10). METHODS Rectal mucosal dissection commenced on the ARL in the ARL group and at various levels (0-10 mm) above the DL in the DL group. Outcome was assessed prospectively for 6 months using a standard structured questionnaire. RESULTS Subject demographics were not significantly different. Differences in frequency of motions between the ARL and Cont groups were not statistically significant after 3 months of age, but the DL group had significantly more motions than the other 2 groups after 4 months of age (P < .01). At 6 months of age, fecal staining was 17% in the ARL group, 63% in the DL group, and 0% in the Cont group. Anal shape was normal in the ARL and Cont groups, but 50% of the DL group had visible anal mucosa. CONCLUSION Bowel function in the ARL group is similar to normal, and because the ARL is easily identifiable without the need for subjective interpretation, we recommend using the ARL as a landmark for rectal mucosal dissection during transanal pull-through for Hirschsprung disease.

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.

Prenatal retinoic acid increases lipofibroblast expression in hypoplastic rat lungs with experimental congenital diaphragmatic hernia.

BACKGROUND/PURPOSE Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH. METHODS Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo, and Nitrofen+ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining. RESULTS Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo (0.31±0.02 vs. 0.08±0.01; P<0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen+ATRA compared to Nitrofen+Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen+ATRA compared to Nitrofen+Placebo. CONCLUSIONS Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development.

Expression of Eya1 and Six1 is decreased in distal airways of rats with experimental pulmonary hypoplasia.

BACKGROUND/PURPOSE Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. Eyes absent 1 (Eya1) and sine oculis homebox 1 (Six1) have been identified as essential components of the gene network that regulates foetal lung development. Eya1 and Six1 are expressed in distal epithelial tips of branching airways as well as in surrounding mesenchymal cells, highlighting their important role during branching morphogenesis. Lungs of Eya1(-/-) and Six1(-/-) knockouts display PH with reduced epithelial branching, appearing to be arrested in the pseudoglandular stage. We hypothesized that Eya1 and Six1 expression is decreased in branching airways of nitrofen-induced PH. METHODS Time-mated rats received either nitrofen or vehicle on E9.5. Foetal lungs were dissected on E15.5 and divided into control and nitrofen groups, whereas lungs harvested on E18.5 were divided into controls, PH without CDH [PH(-)], and PH with CDH [PH(+)]. Pulmonary gene expression levels of Eya1 and Six1 were analyzed by quantitative real-time PCR. Immunofluorescence staining was performed to investigate Eya1 and Six1 protein expression and localization by confocal laser scanning microscopy (CLSM). RESULTS Relative mRNA expression of Eya1 and Six1 was significantly decreased in PH(-) and PH(+) on E18.5 compared to controls. CLSM confirmed markedly diminished immunofluorescence of Eya1 and Six1 in distal airway epithelium as well as in surrounding mesenchymal cells of nitrofen-induced PH on E18.5 compared to controls. CONCLUSIONS Downregulation of Eya1 and Six1 gene expression in nitrofen-induced PH suggests that decreased Eya1 and Six1 expression during the late pseudoglandular stage may interfere with epithelial branching and distal airway maturation, thus resulting in PH.

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

PurposeLaminin, 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.MethodsWe 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.ResultsThe 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.ConclusionsAltered 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.

Spatiotemporal alterations in Sprouty-2 expression and tyrosine phosphorylation in nitrofen-induced pulmonary hypoplasia

BACKGROUND/PURPOSE Pulmonary hypoplasia (PH) is a life-threatening condition of newborns presenting with congenital diaphragmatic hernia (CDH). Sprouty-2 functions as a key regulator of fibroblast growth factor receptor (FGFR) signalling in developing foetal lungs. It has been reported that FGFR-mediated alveolarization is disrupted in nitrofen-induced PH. Sprouty-2 knockouts show severe defects in lung morphogenesis similar to nitrofen-induced PH. Upon FGFR stimulation, Sprouty-2 is tyrosine-phosphorylated, which is essential for its physiological function during foetal lung development. We hypothesized that Sprouty-2 expression and tyrosine phosphorylation are altered in nitrofen-induced PH. METHODS Time-pregnant rats received either nitrofen or vehicle on gestation day 9 (D9). Foetal lungs were dissected on D18 and D21. Pulmonary Sprouty-2 gene and protein expression levels were analyzed by qRT-PCR, Western blotting and immunohistochemical staining. RESULTS Relative mRNA expression of Sprouty-2 was significantly decreased in hypoplastic lungs without CDH (0.1050±0.01 vs. 0.3125±0.01; P<.0001) and with CDH (0.1671±0.01 vs. 0.3125±0.01; P<.0001) compared to controls on D18. Protein levels of Sprouty-2 were markedly decreased in hypoplastic lungs on D18 with decreased tyrosine phosphorylation levels on D18 and D21 detected at the molecular weight of Sprouty-2 consistent with Sprouty-2 tyrosine phosphorylation. Sprouty-2 immunoreactivity was markedly decreased in hypoplastic lungs on D18 and D21. CONCLUSION Spatiotemporal alterations in pulmonary Sprouty-2 expression and tyrosine phosphorylation during the late stages of foetal lung development may interfere with FGFR-mediated alveolarization in nitrofen-induced PH.

Increased expression of Semaphorin 3A in the endothelin receptor-B null mouse model of Hirschsprung disease

PURPOSE Semaphorins are guidance cues for developing neurons, implicated in the determination of the migratory pathway of neural crest-derived neural precursors during enteric nervous system development. Recently, it has been reported that Semaphorin 3A (SEMA3A) expression is up-regulated in the aganglionic colon in Hirschsprung disease (HD) patients, suggesting that increased SEMA3A expression may be a risk factor for HD. Thus, the aim of our study was to determine the expression of SEMA3A using Sox10-Venus mice gut. METHODS We harvested the gut on postnatal day 2 (P2). SOX10-Venus+/EDNRB-/- mice were compared with SOX10-Venus+/EDNRB+/+ mice as controls. QRT-PCR was performed to determine gene expression of SEMA3A (n=8). Fluorescent immunohistochemistry was performed to assess protein distribution. RESULTS On P2, gene expression levels of SEMA3A were significantly increased in the HD group compared to controls in the proximal and distal colon (p<0.05). Laser scanning microscopy revealed SEMA3A expression was localized within the submucosa and muscle layer of the gut in both HD and controls. In HD, SEMA3A was highly expressed in the proximal and distal colon. CONCLUSIONS In the present study, we demonstrated that SEMA3A expression is increased in the EDNRB-/- HD model on P2, suggesting that SEMA3A may interfere with ENCC migration, resulting in an absence of enteric neurons.

Pulmonary FGF9 Gene Expression is Downregulated during the Pseudoglandular Stage in Nitrofen-Induced Hypoplastic Lungs

INTRODUCTION The pathogenesis of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains unclear. Fibroblast growth factor 9 (FGF9) is an essential component of the gene network that regulates lung development. FGF9 knockouts exhibit disrupted mesenchymal proliferation and reduced airway branching. The authors hypothesized that pulmonary FGF9 gene expression is downregulated during the pseudoglandular stage in nitrofen-induced hypoplastic lungs. MATERIAL AND METHODS Pregnant rats received either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were dissected on D15 and D18, and were divided into controls, hypoplastic lungs with CDH (CDH+) and hypoplastic lungs without CDH (CDH-). Pulmonary FGF9 gene expression levels were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to investigate FGF9 protein expression/distribution. RESULTS Relative messenger RNA levels of FGF9 were significantly decreased on D15 in hypoplastic lungs compared with controls (p < 0.01), and on D18 in CDH+ and CDH- compared with controls (p< 0.05, respectively). Immunoreactivity of FGF9 was markedly diminished in mesothelium and distal airway epithelium on D15 and decreased in overall intensity on D18 in hypoplastic lungs compared with controls. CONCLUSIONS Downregulation of FGF9 gene expression during the pseudoglandular stage may cause pulmonary hypoplasia in the nitrofen model by decreasing distal airway epithelial and mesenchymal proliferation throughout the branching morphogenesis.