Mucosal Monosaccharide Transporter Expression in Newborns With Jejunoileal Atresia and Along the Adult Intestine.

OBJECTIVES: In newborn rodents, intestinal maturation involves delayed fructose transporter GLUT5 expression until weaning. In jejunoileal atresia (JIA), distal intestinal segments lack exposure to amniotic fluid-containing carbohydrates. We assessed in human newborns, the impact of intestinal maturation and obstruction on mucosal monosaccharide transporter expression. METHODS: Samples were obtained from 10 newborns operated for small intestinal atresia and from 17 adults undergoing gastroduodenoscopy and/or ileocolonoscopy. mRNA expression of the transporters SGLT1, GLUT1, GLUT2, GLUT5, and GLUT7 was measured in neonate samples proximal and distal of the atresia as well as in adult duodenum, ileum, and colon. Protein expression and localization was assessed using immunofluorescence. RESULTS: Although mRNA expression of monosaccharide transporters did not significantly differ between newborn and adult samples, luminal fructose transporter GLUT5 protein was absent in 0- to 4-day-old neonates, but expressed in adults. The mRNA expression of the 5 tested monosaccharide transporters was unchanged distal from the JIA relative to proximal. Similarly, luminal sodium-dependent glucose transporter SGLT1 and basolateral GLUT2 were expressed proximal and distal to JIA as visualized by immunofluorescence staining. With the exception of glucose transporter GLUT1 that showed highest expression levels in colon, all investigated hexose transporters showed strongest expression in duodenum, lower levels in ileum and lowest in colon. CONCLUSIONS: Human newborns lack small intestinal fructose transporter GLUT5 protein expression and small intestinal atresia does not affect the expression of hexose transporters.

Umbilical Cord Ulcer and Intrauterine Death in Fetal Intestinal Atresia.

OBJECTIVE: To examine the incidence of umbilical cord ulcer (UCU) that causes intrauterine fetal death (IUFD) in fetal duodenal or jejunoileal atresia and the association between UCU and bile acid concentrations in amniotic fluid. METHODS: Perinatal outcomes were evaluated in cases of fetal intestinal atresia between 2003 and 2017. A pathological examination of the umbilical cord was performed, and bile acid concentrations in the amniotic fluid were measured. RESULTS: Among the 46 cases included in this study, there were 27 with duodenal atresia and 19 with jejunoileal atresia. There were 4 cases (8.7%) of IUFD and 1 (2.2%) neonatal death with multiple structural anomalies. UCUs were found in 37.5% (15/40) of cases, and severe UCUs with exposed vessels were significantly more common in IUFD (3/4) than in livebirth (0/42) cases (p < 0.01). The incidences of chromosomal abnormality and structural anomalies were not markedly different between livebirth (9/30 and 11/42, respectively) and IUFD (1/3 and 1/4, respectively) cases. Bile acid concentrations in amniotic fluid were significantly higher in cases of UCUs than in those without (p < 0.01). CONCLUSION: UCUs were not rare in fetal intestinal atresia and were associated with high bile acid concentrations in amniotic fluid. UCUs with exposed vessels were associated with IUFD in intestinal atresia.

LncRNA-MEG3 protects against ganglion cell dysplasia in congenital intestinal atresia through directly regulating miR-211-5p/GDNF axis.

Background LncRNAs are known to take part in normal brain functions and nervous system diseases. Little evidence has pointed to the dysregulation of lncRNAs in congenital intestinal atresia. We aimed to investigate the underlying molecular mechanism of congenital intestinal atresia that involves in lncRNA-MEG3. Materials and methods The expressions of LncRNA-MEG3, miR-211-5p and GDNF were determined by the qRT-PCR and Western blot assay when appropriate. The results were verified in intestinal atresia Wistar rat model and bone marrow derived stem cell (BMSCs)-derived into intestinal ganglion cells. RNA immunoprecipitation and RNA pull-down assays were performed to analyze the regulatory mechanism between MEG3 and miR-211-5p. The effects of MEG3 on the cell proliferation and apoptosis of isolated intestinal ganglion cells were detected with an MTT assay and flow cytometry, respectively. Results The expression of MEG3 was detected to be declined in congenital intestinal atresia tissues at clinic and animal levels. MEG3 promoted the differentiation of BMSCs into intestinal ganglion cells and regulated GDNF expression in retinal ganglion cells (RGC-5 cells) via targeting miR-211-5p. Hypoxia induced the apoptosis of intestinal ganglion cells via MEG3/miR-211-5p/GDNF axis. Conclusion MEG3 promoted the differentiation of BMSCs into intestinal ganglion cells and inhibited the apoptosis of intestinal ganglion cells under the exposure of hypoxia to protect against CIA injury via directly regulating miR-211-5p/GDNF axis.

Characterization and origin of heme precursors in amniotic fluid: lessons from normal and pathological pregnancies.

BACKGROUND: Heme is the prosthetic group of numerous proteins involved in vital processes such as oxygen transport, oxidative stress, and energetic mitochondrial metabolism. Free heme also plays a significant role at early stages of development and in cell differentiation processes. The metabolism of heme by the fetal placenta unit is not well-established in humans. METHODS: In a retrospective study, we measured heme precursors in the amniotic fluid (AF) of 51 healthy women, and 10 AF samples from pregnancies with either upper or lower intestinal atresia or ileus were also analyzed. RESULTS: We showed that the porphyrin precursors aminolevulinic acid, porphobilinogen, and protoporphyrin IX are present at the limit of detection in the AF. Total porphyrin levels decreased progressively from week 13 to week 33 (p < 0.01). Interestingly, uroporphyrin, initially detected as traces, increased with maturation, in contrast to coproporphyrin. Uro- and coproporphyrins were type I immature isomers (>90%), suggesting a lack of maturity in the fetal compartment of the heme pathway. Finally, the differential analysis of AF from normal and pathological pregnancies demonstrated the predominant hepatic origin of fetal porphyrins excreted in the AF. CONCLUSION: This study gives the first insight into heme metabolism in the AF during normal and pathological pregnancies.

MicroRNA-211 causes ganglion cell dysplasia in congenital intestinal atresia via down-regulation of glial-derived neurotrophic factor.

BACKGROUND: MicroRNAs (miRNAs) are known to be involved in normal brain functions and nervous system diseases. Some evidence have pointed to the dysregulation of miRNAs in congenital intestinal atresia. In this study, we investigated the differential expression of miRNAs and the posttranscriptional regulation of glial-derived neurotrophic factor (GDNF) by endogenous miRNA in congenital intestinal atresia. METHODS: Quantitative real-time PCR and a Western blot were performed to determine the regulation of miRNA and GDNF in patients with congenital intestinal atresia. The results were verified in rat model of intestinal atresia and bone marrow derived stem cell BMSCs-derived into intestinal ganglion cells. The effects of miRNA and GDNF on the cell proliferation and apoptosis of isolated intestinal ganglion cells were detected with an 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. KEY RESULTS: Only miR-211 was greatly up-regulated in the patients with congenital intestinal atresia. The other miRNAs examined showed no change. Overexpression of miR-211 suppressed the differentiation of BMSCs into intestinal ganglion cells. In retinal ganglion cells (RGC-5 cells), miR-211 regulated the expression of GDNF. The MTT and TUNEL assays revealed that miR-211 overexpression suppressed the cell proliferation of isolated intestinal ganglion cells and that GDNF overexpression reversed the effect of pre-miR-211 on cell proliferation and apoptosis. CONCLUSIONS & INFERENCES: Our results indicate that overexpression of miR-211 suppresses the differentiation of BMSCs into intestinal ganglion cells by directly down-regulating the expression of GDNF. The findings elucidate the role of miRNA in congenital intestinal atresia.

The clinical significance of pathological studies of congenital intestinal atresia.

OBJECTIVE: The purpose of this study was to explore the mechanisms of postoperative intestinal motility disorders in intestinal atresia patients by investigating the expression profiles of proteins, including calretinin (CR), glial-derived neurotrophic factor (GDNF), bone morphogenetic protein 2 (BMP-2), c-kit, α-smooth muscle actin (α-SMA), and S-100 protein; to decipher the correlation between the area of the pathological segment and the alteration of the above 6 proteins; and thereby to provide a clinical specific reference values to determine the removal length for intestinal tract resection. METHODS: Immunohistochemistry technique was applied to detect the CR, c-kit, GDNF, BMP-2, α-SMA, and S-100 protein in specimens of atretic, proximal, and distal intestine from 25 cases of intestinal atresia and samples of intestinal walls from 10 non-atresia control specimens. The alteration of the enteric nervous system, nerve growth and its regulatory factors, the interstitial cells of Cajal (ICCs), and the enteric muscle system were examined, with particular attention being paid to pathological changes and the lesion area. RESULTS: The expression of all of the abovementioned 6 proteins in the proximal side of the atresia was significantly lower than in control group. The expression of the abovementioned proteins tended to be higher farther away from the atresia site. The expressions of both GDNF and BMP-2 had returned to normal level at 10 cm proximal to the atresia site, whereas the expressions of CR, c-kit, α-SMA, and S-100 protein only returned to normal at 15 cm proximal to the atresia site. On the distal side, the expression of all 6 markers at 3 cm distal to the atresia site was normal. CONCLUSION: Pathological deterioration of the myenteric ganglia, nerve growth factor, and ICCs are the causes of intestinal motility disorders after the surgical repair of intestinal atresia. Our data support resecting an intestinal segment extending from 15 cm proximal to 3 cm distal to the atretic segment. In proximal jejunal atresia, when it is not possible to resect 15 cm, we suggest resecting as much of the hypertrophic proximal intestine as possible. Based on our data, we believe this surgical practice could improve postoperative dysmotility in these patients.

Haploinsufficiency of retinaldehyde dehydrogenase 2 decreases the severity and incidence of duodenal atresia in the fibroblast growth factor receptor 2IIIb-/- mouse model.

BACKGROUND: Homozygous null mutation of the fibroblast growth factor receptor 2IIIb (Fgfr2IIIb) gene in mice results in 42% of embryos developing duodenal atresias. Retinaldehyde dehydrogenase 2 (Raldh2, a gene critical for the generation of retinoic acid) is expressed in the mouse duodenum during the temporal window when duodenal atresias form. Raldh2 is critical for the normal development of the pancreatoduodenal region; therefore, we were interested in the effect of a Raldh2 mutation on duodenal atresia formation. To test this, we rendered Fgfr2IIIb(-/-) embryos haploinsufficient for the Raldh2 and examined these embryos for the incidence and severity of duodenal atresia. METHODS: Control embryos, Fgfr2IIIb(-/-) mutants, and Fgfr2IIIb(-/-); Raldh2(+/-) mutants were harvested at embryonic day 18.5, genotyped, and fixed overnight. Intestinal tracts were isolated. The type and severity of duodenal atresia was documented. RESULTS: A total of 97 Fgfr2IIIb(-/-) embryos were studied; 44 had duodenal atresias, and 41 of these presented as type III. In the 70 Fgfr2IIIb(-/-); Raldh2(+/-) embryos studied, a lesser incidence of duodenal atresia was seen (15 of 70; P = .0017; Fisher exact test). Atresia severity was also decreased; there were 12 embryos with type I atresias, 3 with type II atresias, and 0 with type III atresias (P < 2.81E-013; Fisher exact test). CONCLUSION: Haploinsufficiency of Raldh2 decreases the incidence and severity of duodenal atresia in the Fgfr2IIIb(-/-) model. The ability to alter defect severity through manipulation of a single gene in a specific genetic background has potentially important implications for understanding the mechanisms by which intestinal atresias arise.

Significance of abnormalities in systems proximal and distal to the obstructed site of duodenal atresia.

BACKGROUND: Duodenal atresia (DA) is a well-known neonatal intestinal disease. Even after surgery, the proximal segment can continue to be severely dilated with hypoperistalsis, resulting in intestinal dysmotility problems in later life. No data have been published regarding the morphologic differences between the proximal and distal regions of obstructed sites of the intramural components in DA. METHODS: Operative duodenal samples (N = 12) from cases with DA (age 1-3 days) were used. Age-matched controls (N = 2) were used. All of the specimens were immunohistochemically stained with antibodies to S-100 protein, α-smooth muscle actin, and c-kit protein. RESULTS: At the proximal segments of the obstructed site in DA, the number of neuronal cells decreased in size and number. The circular musculature was moderately to severely hypertrophic. Unusual ectopic smooth muscle bundles were also identified. The innermost layer of the circular musculature was thinner. Interstitial cells of Cajal are decreased, even around the myenteric plexus. All of the staining in the distal segments in DA was similar to the control tissues. CONCLUSIONS: Proximal and distal segments in DA differ in the neural cells, musculature, and distributions of the interstitial cells of Cajal. Based on the present study, these morphologic changes may contribute to the onset of postoperative duodenal dysmotility.

Does small intestinal atresia affect epithelial protein expression in human newborns?

OBJECTIVES: Bowel segments distal to a congenital intestinal obstruction have been suggested to be immature. In other words, luminal components such as amniotic fluid (before birth) and/or enteral nutrition (after birth) may be required to activate intestinal epithelial protein expression, thereby influencing epithelial differentiation. We investigated cell-type-specific protein expression proximal and distal to jejunal and ileal atresias in human newborns. PATIENTS AND METHODS: We immunohistochemically studied intestinal tissue specimens of 16 newborns who had undergone surgery for jejunal or ileal atresia. Sections were taken from both the proximal and distal sides of the atresias. RESULTS: For all patients, the enterocyte-specific markers lactase, sucrase-isomaltase, sodium glucose cotransporter 1, glucose transporters 2 and 5, intestinal fatty acid-binding protein and alkaline phosphatase were expressed at a mean 3 +/- 1 days after birth, both proximal and distal to jejunal and ileal atresias. Expression of goblet cell-specific markers mucin 2 and trefoil factor 3 and that of the Paneth cell marker lysozyme was maintained at either side of the atretic segment. CONCLUSIONS: With respect to the markers used, the human small intestinal epithelium is already differentiated shortly after birth. The absence of intestinal continuity in case of a jejunal or ileal atresia does not affect epithelial protein expression. This would seem to indicate that the developing small intestinal epithelium matures independently of luminal components.

A genetic mechanism for cecal atresia: the role of the Fgf10 signaling pathway.

BACKGROUND: Intestinal atresia represents a significant surgically correctable cause of intestinal obstruction in neonates. Intestinal development proceeds as a tube-like structure with differentiation along its axis. As the intestine differentiates, the cecum develops at the transition from small to large intestine. Fgf10 is known to serve a key role in budding morphogenesis; however, little is known about its role in the development of this transitional structure. Here we evaluate the effect of Fgf10/Fgfr2b invalidation on the developing cecum. MATERIALS AND METHODS: Wild-type C57Bl/6, Fgf10(-/-), and Fgfr2b(-/-) embryos harvested from timed pregnant mothers were analyzed for cecal phenotype, Fgf10 expression, and differentiation of smooth muscle actin. RESULTS: Wt cecal development is first evident at E11.5. FGF10 is discreetly expressed in the area of the developing cecum at early stages of development. One hundred percent of Fgf10(-/-) and Fgfr2b(-/-) mutant embryos demonstrate cecal atresia with absence of epithelial and muscular layers. The development of neighboring anatomical structures such as the ileocecal valve is not affected by Fgf10/Fgfr2b invalidation. CONCLUSIONS: FGF10 expression is localized to the cecum early in the normal development of the cecum. Fgf10(-/-) and Fgfr2b(-/-) mutant embryos demonstrate cecal atresia with complete penetrance. Epithelial and muscular layers of the cecum are not present in the atretic cecum. The Fgf10(-/-) and Fgfr2b(-/-) mutants represent a genetically reproducible animal model of autosomal recessive intestinal atresia.

The occurrence of unusual smooth muscle bundles expressing alpha-smooth muscle actin in human intestinal atresia.

BACKGROUND/PURPOSE: Intestinal dysmotility is an important problem in the postoperative management of patients with intestinal atresia (IA). Changes in the intramural components have so far been histochemically and immunohistochemically examined in both the proximal and distal segments of IA, but no detailed analysis of the muscular elements has been performed. The aim of this study was to carefully examine any alterations in the muscular elements in the intestines from patients with IA. METHODS: Resected intestines were obtained from 6 patients with ileal atresia, 4 patients with jejunal atresia, and 3 controls without gastrointestinal diseases obtained by autopsy (congenital diaphragmatic hernia). All specimens were immunochemically stained with a monoclonal antibody to alpha-smooth muscle actin (alpha-SMA) as a smooth muscle marker. RESULTS: In the normal small intestine, almost all the enteric smooth musculature were positive for alpha-SMA antiserum, except for the bulk of the circular musculature. In the proximal segments of all cases with IA, a reduced staining intensity for alpha-SMA was observed mainly in the severely hypertrophic muscle layers. In addition, some bundles of smooth muscle also were located in the submucosal connective tissue near the border of the innermost layer of the circular musculature, in which large amounts of smooth muscle fibers extended occasionally from the innermost layer of the circular musculature to the muscularis mucosae in the proximal segments of 4 cases. In the distal segments of IA, the distribution of alpha-SMA-positive smooth muscle fibers was similar to that in the control intestines, excluding mild to moderate hypertrophy of the muscular layers. CONCLUSIONS: Both severe hypertrophy and a reduced immunoreactivity for alpha-SMA were observed in the circular muscle layer of the proximal segments. In addition, the occurrence of alpha-SMA-positive abnormal smooth muscle fibers was recognized in the submucosal layers of the proximal segments, thus, suggesting a delay in the intestinal muscular formation or a regressive reaction secondary to dilatation. These muscular alterations in the proximal segments might be considered to contribute to the postoperative intestinal dysmotility in IA cases.

Abnormalities of enteric neurons, intestinal pacemaker cells, and smooth muscle in human intestinal atresia.

BACKGROUND/PURPOSE: Intestinal dysmotility, which usually has been encountered in the severely dilated proximal segment, is an important problem in postoperative management of patients with intestinal atresia (IA). Changes of enteric nerves had been histochemically examined in both the proximal and distal segments of IA, but a systemic immunohistochemical analysis is still lacking. The aim of this study was to examine precisely alterations of neuronal and muscular elements and pacemaker cells in intestines from patients with IA. METHODS: Resected intestines were obtained from 5 patients with ileal atresia, 3 patients with jejunal atresia, and 3 controls without gastrointestinal diseases (congenital diaphragmatic hernia). All specimens were immunochemically stained with a monoclonal antibody to alpha-smooth muscle actin (SMA) as a smooth muscle marker, polyclonal antibodies to protein gene product (PGP) 9.5 as a general neuronal marker, and to c-kit protein as a maker of intestinal pacemaker cells. In addition, all specimens also were stained by NADPH-diaphorase (NADPH-d) to know the distribution of inhibitory nitrergic nerves. RESULTS: A hypoplasia of the myenteric ganglia and a marked reduction of intramuscular nerve fibers, including nitrergic neurons, were observed in the dilated proximal segment of IA. C-kit-positive cells were localized around the myenteric plexus, but rarely found within the muscularis propria in the proximal segment. The distribution of nerves and c-kit-positive cells in the distal segment was comparable with that seen in controls. A reduced staining intensity for alpha-SMA was mainly observed in the hypertrophic circular muscle layer of the proximal segment. CONCLUSIONS: A hypoplasia of intramural nerves and pacemaker cells was seen predominantly in the proximal segments of IA. Hypertrophy and reduced immunoreactivity for alpha-SMA also were observed in the circular muscle layer of the proximal segment. These alterations of the proximal segment may thus contribute to the postoperative intestinal dysmotility in IA cases.

[Fetal nutrition in intestinal atresia. Studies on the chick embryo]. / Nutrición fetal en la atresia intestinal. Estudios en el embrión de pollo.

This paper examines the effects of experimental prenatal intestinal obstruction on the growth and blood composition of chick embryos. Intestinal atresia (IA) was produced by bipolar bowel electrocoagulation in fertile eggs on the 14th day of incubation. The chicks sacrificed on the 19th day were measured, weighed and blood-sampled. Twenty-three control, 10 sham-operated and 11 IA chicks were studied. Animals with IA were severely undernourished by weight (43.4 +/- 4.7 vs 70.3 +/- 7.6% of egg weight, p < 0.001) and length (15.3 +/- 1.1 vs 18.1 +/- 9 mm. tibial length, p < 0.001) in comparison with sham-operated ones. Their haematocrit was slightly lower, and total protein increased. Pre-albumin was absent in their sera and albumin, alpha and beta globulins were significantly decreased whereas gamma-globulin was greatly increased. Sodium, potassium chloride, urea and glucose remained within normal limits. The lack of placenta in the avian embryo precludes any supply of nutrients by this route and the ingestion of amniotic fluid, which is protein-rich after the 13th day of incubation, when the opening of the sero-amniotic connection allows albumen to be mixed with it, becomes the main source of nutrients until hatching. Obstruction of the main incoming avenue by IA induces severe malnutrition in this model which relies on this route to a greater extent than the human foetus. In spite of the obvious biological differences between the avian embryo and the human foetus, the present evidence supports the hypothesis that prenatal interruption of the amniotic fluid transit contributes to foetal undergrowth in IA.

Respiratory gas exchange in response to fat-free parenteral nutrition: a comparison after thoracic or abdominal surgery in newborn infants.

Thoracic surgery is known to cause a postoperative respiratory failure because of the mechanical problems following chest wall disruption and/or diaphragmatic dysfunction. This study was to verify whether the fat-free intravenous nutritional support of neonates who underwent thoracic surgery could lead to a CO2 production exceeding the patients' respiratory reserves. Respiratory gas exchange and alveolar ventilation were obtained by indirect calorimetry and continuous recordings of transcutaneous PO2 and PCO2. These noninvasive measurements were compared at the same age of 7 +/- 1 days between a group of 7 newborn infants (mean +/- SEM: 3.09 +/- 0.14 kg, 39 +/- 1 weeks) after thoracic surgery versus a group of 8 newborn infants (2.88 +/- 0.17 kg, 37 +/- 1 weeks) after abdominal surgery. The intravenous macronutrient support was the same between both groups: 14 g/kg/d of glucose, 2 g/kg/d of amino acids, 250 kJ/kg/d of energy. One week after surgery, the global metabolic rate (195 kJ/kg/d) was not increased, and comparable between both groups. We documented that early after thoracic surgery, the ventilatory compensation required to handle the CO2 production (6.7 +/- 0.2 mL/kg/min) associated with a positive energy balance (45 +/- 8 kJ/kg/d) was effective.

Composition of amniotic fluid with experimental jejuno-ileal atresia.

The possibility that the composition of amniotic fluid is significantly altered with fetal intestinal obstruction was examined in 15 normal, 15 sham operated and 15 ewes in which jejuno-ileal atresia was created by devascularizing a 15 cm intestinal segment in the fetus at 90--100 days gestation. Samples of amniotic fluid for osmolality, urea N, creatinine, electrolytes, amylase, lipase, bilirubin, and proteins were obtained at 100 days gestation and at C-section performed 5 days prior to the anticipated date of delivery. One sham operated and four operated fetuses aborted prior to term for an 83% overall fetal survival rate. All surviving operated animals had total intestinal obstruction with the classical appearance of jejuno-ileal atresia. The volume of amniotic fluid at term varied from gelatinous material to as much as 1.0 liter of clear yellow to dark orange fluid. Amniotic fluid composition in term animals was not significantly different from those at 100 days gestation when the creatinine was below 15 mg%. However, sodium and chloride were significantly elevated in all groups at term when the creatinine was above 15 mg%. The osmolality and urea N in the latter group was not significantly different from animals at 100 days gestation. The amylase, lipase, total proteins, albumin, total and direct, and bilirubin in control animals at term was not significantly different from sham operated and experimental subjects. This study suggests that routine amniocentesis in the "high risk" patient would not be diagnostic of fetal intestinal obstruction.