The effect on the intestines of continuous release of methylene blue from a drug delivery system: an experimental study in a chick embryo gastroschisis model

Increased small bowel nitric oxide synthase (NOS) activity has been suspected as a cause of postnatal intestinal dysmotility in gastroschisis. The effect of continuous delivery of methylene blue loaded polymer (MBLP) hydroxy-propyl methyl cellulose-ethyl cellulose (HPEC—MC) and daily injection of methylene blue (MB) on the intestinal damage (ID) was evaluated using a chick embryo gastroschisis model. Fourteen-day-old fertilized chick eggs were divided into five groups. In the control (C) group, no intervention was performed. In the sham (S) group, the allantoic and amniotic membranes were opened to create a common cavity that resembles the amniotic cavity in human. In the gastroschisis only (GO) group, a defect in the abdominal wall of the embryo was made, and intestinal loops were exteriorized following connection of amniotic and allantoic cavities. In the gastroschisis plus methylene blue (G+MB) group, gastroschisis was created and MB administered into the amnioallantoic cavity (AAC) by daily injections for 5 days. In the gastroschisis plus methylene blue loaded polymer (G+MBLP) group, MBLP was placed into AAC after gastroschisis was created. At the end of the 19th day of incubation, intestinal morphological changes were investigated macroscopically and microscopically. Although the survival rates were decreased in the chick embryos with creation of gastroschisis compared with C and S groups (p<0.001), the survival rates were increased in G+MBLP group (76.92%) when compared with the GO group (41%) (p<0.001). Because of multiple intervention of embryos, higher mortality was observed in the G-MB group (75.61%). Macroscopic and microscopic scores of ID and mean intestinal wall thickness were significantly higher in the GO group when compared with C, S, G+MB, and G+MBLP groups (p<0.001). The mean score of intestinal ganglia morphology was significantly increased and the total number of ganglion cells was significantly decreased in the GO group when compared with C, S, G+MB, and G+MBLP groups (p<0.001). It is possible to decrease intrauterine intestinal morphological changes in gastroschisis by inhibiting NOS. As a first preliminary study, we believe that use of MBLP may be an alternative for fetal treatment by eliminating the harmful effects of multiple interventions or amniotic fluid exchanges.     

Correlation of sonographic findings and outcome in necrotizing enterocolitis

Background There is little in the literature regarding the use of gray-scale and Doppler sonography of the bowel in necrotizing enterocolitis (NEC) and how findings depicted by this modality might assist in predicting outcome and influence management. Objective To correlate sonographic findings with outcome in NEC. Materials and methods This was a retrospective analysis of clinical and abdominal ultrasonography (AUS) findings in NEC from January 2003 to December 2005. AUS findings were evaluated for portal venous gas, free gas, peritoneal fluid, bowel wall thickness, echogenicity, perfusion and intramural gas. Patients were categorized into two groups, according to their outcome. Results A total of 40 infants were identified who had AUS for NEC prior to any surgical intervention. Group A comprised 18 neonates treated medically and recovered fully, and group B comprised 22 neonates who required surgery or died. Free gas (six patients) and focal fluid collections (three patients) were only found in group B. Increased bowel wall echogenicity, absent bowel perfusion, portal venous gas, bowel wall thinning, bowel wall thickening, free fluid with echoes and intramural gas were seen in both groups, but more frequently in group B. Anechoic free fluid was seen more frequently in group A. Increased bowel perfusion was seen equally in both groups. Conclusion An adverse outcome was associated with the sonographic findings of free gas, focal fluid collections or three or more of the following: increased bowel wall echogenicity, absent bowel perfusion, portal venous gas, bowel wall thinning, bowel wall thickening, free fluid with echoes and intramural gas. Sonographic findings are useful in predicting outcome and therefore might help guide management. This material was presented at the 5th International Pediatric Radiology Meeting in Montreal, Canada, May 2006.     

大鼠腹裂模型肠管损伤的研究

目的研究先天性腹裂的肠管受损害情况，探讨该病术后并发症的原因。方法利用大鼠腹裂模型，运用组织学、生化学和免疫组织化学方法，分析腹裂胎鼠肠管的组织结构，DNA和蛋白质，细胞增生和凋亡等方面的改变。结果共获得腹裂胎鼠16只，对照胎鼠21只。与对照组相比，腹裂鼠肠管变短、充血水肿、粘连，肠壁表面纤维覆盖，壁内胶原沉积，DNA总量下降，蛋白质总量基本不变，细胞增生率下降，凋亡率上升。结论腹裂的肠管损伤是多方面的，是术后肠管运动和吸收功能异常的原因，大鼠的腹裂模型是对先天性腹裂的病因、病理等方面研究的合适工具。     

Radiographic predictors determined with an objective assessment tool for neonatal patients with necrotizing enterocolitis

ObjectiveThe objective of this study was to develop and validate a computational tool to assist radiological decisions on necrotizing enterocolitis.MethodologyPatients that exhibited clinical signs and radiographic evidence of Bell's stage 2 or higher were included in the study, resulting in 64 exams. The tool was used to classify localized bowel wall thickening and intestinal pneumatosis using full-width at half-maximum measurements and texture analyses based on wavelet energy decomposition. Radiological findings of suspicious bowel wall thickening and intestinal pneumatosis loops were confirmed by both patient surgery and histopathological analysis. Two experienced radiologists selected an involved bowel and a normal bowel in the same radiography. The full-width at half-maximum and wavelet-based texture feature were then calculated and compared using the Mann–Whitney U test. Specificity, sensibility, positive and negative predictive values were calculated.ResultsThe full-width at half-maximum results were significantly different between normal and distended loops (median of 10.30 and 15.13, respectively). Horizontal, vertical, and diagonal wavelet energy measurements were evaluated at eight levels of decomposition. Levels 7 and 8 in the horizontal direction presented significant differences. For level 7, median was 0.034 and 0.088 for normal and intestinal pneumatosis groups, respectively, and for level 8 median was 0.19 and 0.34, respectively.ConclusionsThe developed tool could detect differences in radiographic findings of bowel wall thickening and IP that are difficult to diagnose, demonstrating the its potential in clinical routine. The tool that was developed in the present study may help physicians to investigate suspicious bowel loops, thereby considerably improving diagnosis and clinical decisions.     

Transperitoneal exchanges of water and solutes in the fetus with gastroschisis. Experimental study in the chick embryo.

Aiming at testing the hypothesis that, in the foetus with gastroschisis (GX) peritoneal dialysis during antenatal bowel exposure to the amniotic fluid may induce some modifications of the internal environment, we have studied the composition of blood, amniotic fluid and allantoic fluid in a chick embryo model of GX. Intestinal loops were surgically eviscerated in 184 embryos on the 14th day of incubation and exposed to the mixture of both embryonal fluids until the 19th day (hatching expected at the 21th). Typical lesions of GX were present in half of the 94 survivors (n = 42). For comparison we used a control group of non-manipulated animals (n = 19) and a "sham" group of animals in which the operation was carried out only until umbilical manipulation but without bowel exposure (n = 22). Chicks with GX were smaller and slightly dehydrated as attested by clinical signs and had moderately increased hematocrit. They had significant hyperkaliaemia and slight decreases in serum Na and Cl. Urea, total protein and protein fractions were not modified. Taking into account the gradients for such substances existing between the dialysate (amino-allantoic mixture) and the embryonal blood, the changes observed, albeit slight, suggest that there was some evidence of dialysis through the peritoneum: lower Na and Cl concentrations in the dialysate than in blood, induced slight falls in serum Na and Cl whereas higher K concentration resulted in severe hyperkaliemia. The high levels of albumin in the chick amniotic fluid did not allow protein depletion and therefore total protein and its fractions remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

先天性肠闭锁肠神经节细胞NT-3蛋白表达研究

目的了解神经节细胞在先天性小肠闭锁中的分布情况,并探讨其临床意义。方法应用免疫组化技术检测15例先天性小肠闭锁患儿肠壁内NT-3的表达情况,并以正常小肠标本作为对照。结果肠闭锁组肠壁肌间神经丛中神经节细胞NT-3蛋白表达明显低于对照组(t'=9.90,P<0.05)。结论小肠闭锁近端肠壁内神经节细胞减少和神经干的变细可以影响闭锁区域肠道的功能,是病人术后肠道功能不良的原因所在。     

鸡胚神经管缺陷脊髓病理与组织化学研究

目的 鸡胚内注射甲氨喋呤（ＭＴＸ）,制作鸡神经管缺陷动物模型。利用该模型,进一步研究观察病变的病理变化及组织化学改变。方法 选用上海农科院“莱杭”鸡受蛋６６校,在孵化期第４、５天,注射ＭＴＸ,计量为：０．０１ｍｇ／ｋｇ及０．０２ｍｇ／ｋｇ。孵化３周后,选取病变鸡,采用ＮＡＤＰＨ－黄麦组织化学染色,测定变脊髓神经元ＮＯＳ含量;电镜观察病变脊髓神经元内线粒体结构变化。结果 ＮＡＤＰＨ－黄递酶组织化学染     

Differentiation of food protein-induced enterocolitis syndrome and necrotizing enterocolitis in neonates by abdominal sonography

ObjectivesTo summarize and differentiate abdominal ultrasound findings of necrotizing enterocolitis and food protein-induced enterocolitis syndrome.MethodsFrom January 2017 to December 2018, the abdominal ultrasound results of 304 cases diagnosed necrotizing enterocolitis or food protein-induced enterocolitis syndrome were retrospectively analyzed. The presence of pneumatosis intestinalis, portal venous gas, bowel wall thickening, intestinal motility, focal fluid collections and hypoechoic change of gallbladder wall were calculated, and the results were compared and analyzed.ResultsPneumatosis intestinalis, portal venous gas, bowel wall thickening, intestinal motility weakened/absent, focal fluid collections and hypoechoic change of gallbladder wall can be found in both necrotizing enterocolitis and food protein-induced enterocolitis syndrome infants. However, in infants with necrotizing enterocolitis, intestinal motility was weakened/absent in whole abdomen, and in food protein-induced enterocolitis syndrome, it only involved isolated segment of bowel. The positive rates of above signs in necrotizing enterocolitis infants were significantly higher than those in food protein-induced enterocolitis syndrome (p < 0.01). Moreover, it was observed that the rate of weakened intestinal motility besides the lesion segment of bowel in necrotizing enterocolitis infants was 100%, and in food protein-induced enterocolitis syndrome infants, it was 0%, which is supposed to be a main sign for identification.ConclusionIn the early stage, abdominal ultrasound can be used to differentiate necrotizing enterocolitis and food protein-induced enterocolitis syndrome.     

Antenatal diagnosis of intestinal malrotation on fetal MRI

We report a case of intestinal malrotation without any associated GI tract complications diagnosed antenatally by fetal MRI. Antenatal US revealed a midline stomach. Subsequent fetal MRI confirmed the midline stomach and, in addition, revealed all loops of small bowel to the right of the midline and all large bowel to the left. All these features were consistent with intestinal malrotation. There was no abnormal bowel wall thickening, bowel dilatation, ascites or polyhydramnios. To our knowledge, this is a unique case of intestinal malrotation without associated GI tract complications diagnosed antenatally on fetal MRI.     

Elective preterm section for improved primary repair of gastroschisis

For the first time in some 36 years of experience with gastroschisis. the author has encountered in short order both his first and second cases of gastroschisis without any thickening of the eviscerated intestine. Both infants, whose gastroschisis had been detected by maternal alphafetoprotein and ultrasound studies, were delivered by elective preterm cesarean section and were repaired easily with closure of the abdomen in layers. It is suggested that the bowel thickening and rigidity in gastroschisis may result from the trauma of labor and delivery and that elective preterm section, as soon as lung maturity can be demonstrated, may substantially improve the ease of primary repair in gastroschisis.     

Bowel wall thickening in infants with food allergy

BACKGROUND: Food allergy may be partly due to impairment of the intestinal barrier and is frequently associated with atopic dermatitis (AD) in early life. OBJECTIVE: To determine whether bowel wall thickening, as demonstrated by US, is useful for the identification of food allergy. MATERIALS AND METHODS: We used US to study eight infants with food allergy and ten non-allergic controls to clarify whether specific intestinal findings in the jejunum, ileum and colon are present in infants with AD who have food allergy. RESULTS: Wall thickening was observed in all eight patients with food allergy, but only in the jejunum; it disappeared after elimination of suspected dietary allergens. Bowel wall thickening returned in the two allergic patients who received a food challenge test. CONCLUSIONS: Wall thickening of more than 2 mm on US in the jejunum in infants with AD may be a useful marker to evaluate the presence of food allergy and therapeutic effects.     

Extent of intestinal damage in the developing chick embryo after repetitive hypoxia under normoxic or hyperoxic conditions.

BACKGROUND: Episodes of hypoxia and reperfusion play an important role in the development of intestinal damage during perinatal development. The aim of this study was to investigate the histopathology of the intestine in the developing chick embryo after exposure to repetitive hypoxia and recovery under two different conditions: normoxic and hyperoxic (60% O2). METHODS: Chick embryos were exposed to 5 minutes of hypoxia. This was repeated six times with a recovery period of 15 minutes under normoxic conditions (21% O2) for chick embryos in test group 1 (TG1) and under hyperoxic conditions (100% O2) for chick embryos in test group 2 (TG2), from day 11 until day 20. Chick embryos that recovered under hyperoxic conditions (100% O2) were previously incubated under hyperoxic conditions (60% O2 for 24 hours). Histologic evaluation of the ileum was performed at different times after the interventions (2, 4, 8, 16, and 24 hours). RESULTS: In both test groups, only chick embryos older than 19 days showed intestinal damage. Intestinal histology on day 19 showed vasodilation of villus capillaries (10% in TG1 and 15% in TG2), necrosis in the top of the villi (29% in TG1 and 30% in TG2), and necrosis with preservation of base of the crypts (2% in TG1) and transmucosal necrosis (2% in TG2). CONCLUSIONS: Significant histologic changes, compared with the control group, were only found in chick embryos that were studied 2 hours after the interventions. Furthermore, recovery under hyperoxic conditions did not cause more intestinal damage compared with recovery under normoxic conditions.     

Vagal neural crest provides inhibitory neurotransmission to the chick embryo cloaca

Introduction  The intrinsic innervation of the developing chick cloaca originates in the vagal and sacral regions of the neural tube. Its major inhibitory neurotransmitters are nitric oxide (NO) and vasoactive intestinal peptide (VIP). It has previously been shown that the majority of neurons in the chick embryo cloaca are derived from vagal neural crest cells. This study aimed to identify the phenotype of these vagal-derived neurons using quail-chick chimeras. Materials and methods  Chicken embryos were incubated until the 10–12 somite stage. The vagal neural tube was then microsurgically ablated in ovo and replaced with the vagal neural tube from age-matched quail embryos. Quail-chick chimera embryos were harvested at E12, and E14, and fixed and embedded in paraffin wax, and serially sectioned. Immunohistochemistry was performed using human natural killer-1 (HNK-1), quail-cell-specific perinuclear (QCPN), NOS and VIP antibodies. Expression of NOS and VIP neurons in the developing chick embryo cloaca was also further analysed using immunohistochemistry. Results  HNK-1 labelled all ganglia in the myenteric and submucosal plexuses of the cloaca, whilst the quail-specific QCPN antibody labelled all ganglia derived from the transplanted quail vagal neural tube. NOS- and VIP-immunoreactive neurons appeared to make up a large proportion of the quail-derived vagal neural crest cells. Both NOS and VIP expression was seen to increase throughout development. Conclusion  This data suggests for the first time that the inhibitory neurons in the chick cloaca primarily originate in the vagal neural crest, thus providing new insights into the developmental origin of the intrinsic innervation of the developing cloaca.     

Cardiopulmonary effects of chronic administration of the NO synthase inhibitor L-NAME in the chick embryo

BACKGROUND: Experimental observations in mammalian models suggest that endothelial nitric oxide (NO) synthase (NOS) content and activity are decreased in persistent pulmonary hypertension of the newborn. OBJECTIVES: To test the hypothesis that disruption of NO signaling in the developing chick embryo lung may contribute to pulmonary hypertension. METHODS: We analyzed pulmonary arterial reactivity and structure and heart morphology of 19-day chick embryos (incubation time 21 days) that received a daily injection of the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 20 mug per gram egg) or vehicle from day 12 until day 18. RESULTS: Exposure to L-NAME did not affect embryonic survival or body mass of the embryos. The contractile responses to KCl, endothelin-1, the thromboxane A2 mimetic U46619, noradrenaline, and electrical-field stimulation were not affected by exposure to L-NAME. In contrast, in ovo L-NAME exposure reduced the sensitivity of pulmonary arteries to acetylcholine (pD2: 6.53 +/- 0.14 vs. 6.96 +/- 0.13; p < 0.05) and this effect was reversed by the NOS substrate L-arginine. Relaxations induced by sodium nitroprusside or forskolin were not altered by chronic L-NAME. Pulmonary vessel density was not different, but the percentage medial wall area of small pulmonary arteries (external diameter 10-50 microm) was slightly but significantly increased in the embryos exposed to L-NAME. In addition, hearts of L-NAME-exposed embryos showed an increase in right and left ventricular wall area. CONCLUSIONS: Chronic inhibition of NOS produced, in the chick embryo, impairment of endothelium-dependent relaxation, structural remodeling of the pulmonary vascular bed and biventricular cardiac enlargement.     

Value of echography in the diagnosis of hematomas of the digestive tract wall in rheumatoid purpura

Three cases of Henoch-Sch?nlein purpura with intramural hematoma of the duodenum, jejunum or colon are reported. In the first 2 cases, there was complete agreement between the X-ray and ultrasonographic data: multiple thumb print defects and irregular narrowing of the digestive lumen, and thickening of the intestinal wall, respectively. Ultrasonography was the only investigation performed in the third case. Finally, in 2 of 3 cases an exudative enteropathy of unclear mechanism was discovered.     

A comparative investigation of the bowel wall in gastroschisis and omphalocele: relation to postoperative complications

In gastroschisis the protruding bowel is generally covered by a fibrous coating as a result of prolonged exposure to amniotic fluid but no such coating is found on the exposed bowel loops in a ruptured omphalocele. In patients with gastroschisis, the postoperative course is often complicated by periods of hypoperistalsis. Based on studies of animal models of gastroschisis, various authors have suggested that the postoperative complications might be due to structural defects of the bowel wall. In our own experimental investigation of gastroschisis using chick embryos, we found no evidence of a structural defect of the bowel wall. With a view to clinical substantiation of our experimental findings, we conducted a comparative investigation of patients material, involving 10 cases of gastroschisis, 5 cases of ruptured omphalocele, and 9 cases of intact omphalocele. The investigation revealed an entirely normal structure of the bowel wall in all 10 cases of gastroschisis. On the serosa we noted an inflammation infiltrate rich in collagen fibers, frequently containing squamous cells (vernix) and lanugo. In the cases of ruptured omphalocele, the bowel wall specimens revealed a subacute inflammatory reaction. No major abnormalities were found in the bowel wall of patients with an intact omphalocele. Normal development of the autonomic nervous system was found in all patient material. Severe ischemic changes of the bowel wall were found in 4 of the gastroschisis cases and these were the 4 patients who suffered from postoperative hypoperistalsis. Thus the complications occurring during the postoperative phase of gastroschisis are most probably due to ischemic bowel damage.     

ROCK inhibitor (Y-27632) disrupts somitogenesis in chick embryos

Aim

In chick embryos, administration of cadmium (Cd) induces ventral body wall defects (VBWD) similar to human omphalocele. It has been shown that failure of proper VBW formation may be due to disruption of somite development during early embryogenesis. In the VBWD chick model, Cd causes abnormal cell death in the somitic region resulting in improperly developed somites and tortuosity of the neural tube. However, the exact molecular mechanisms leading to VBWD still remain unclear. Wnt signaling is crucial during embryogenesis and plays a key role in normal somite formation. The Rho-associated coiled-coil containing protein kinase (ROCK) is involved in the non-canonical Wnt pathway which controls actin cytoskeleton assembly and cell contractility, and contributes to several developmental processes including somitogenesis. ROCK gene expression levels have recently been reported to be significantly decreased in the Cd-induced VBWD chick model. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) in the absence of Cd disrupts somitogenesis and could contribute to the development of VBWD during early embryogenesis.

Methods

After 60 h of incubation chick embryos were transferred from eggs to culture dishes containing 20 μM of Y-27632 for experimental group (Y-27, n = 22) or chick saline for controls (n = 14). Following 24 h in the incubator they were assessed for stage development and gross abnormalities in morphology using the dissecting microscope. Western blot was performed to confirm Y-27632 inhibition of ROCK downstream signaling using an antibody against phosphorylated cofilin-2.

Results

20 (90.9 %) embryos from Y-27 group and all controls were alive at examination. Morphological abnormalities were detected in 14 (70 %) Y-27 embryos. Somites appeared improperly developed, flattened in the cranio-caudal direction, and elongated in transverse direction in relation to controls. Chick embryos in Y-27 also presented with tortuosity of the neural tube in the lumbosacral region. Western blot analysis showed inhibition of cofilin-2 phosphorylation in affected embryos in comparison to controls.

Conclusion

Our study provides evidence that ROCK inhibitor results in the disruption of normal somitogenesis in chick embryos which may contribute to the failure of fusion of the anterior abdominal wall causing VBWD.     

Intestinal ultrasonographic findings in Schönlein-Henoch syndrome

Thirteen children with Schönlein-Henoch syndrome were evaluated for ultrasonographic changes of the gastro-intestinal tract. One patient was examined twice because of a relapse 1 year later. Out of 14 sonographic scans 10 showed abnormal findings, consisting of circumscribed hypo-echoic asymmetrical enlargement of the intestinal wall, the sonographic result of intramural effusions. One patient also had ileocoecal intussusception. Our results suggest that sonography of the intestine is useful to detect changes of the intestinal wall in Schönlein-Henoch syndrome.     

Severe hypoalbuminaemia in a systemic lupus erythematosus-like patient

Diffuse oedema due to hypoalbuminaemia is a common manifestation in patients affected with systemic lupus erythematosus. Hypoalbuminaemia is usually secondary to an ongoing glomerulonephritis leading to proteinuria or, more rarely, to decreased protein synthesis or deficient protein intake. Here, we report the case of a 6-year-old girl with a previous history of Evans' syndrome and mesangial glomerulonephritis who subsequently developed severe anasarca without apparent proteinuria. Faecal oal-antitrypsin showed a persistent severe intestinal protein loss (ranging from 1500 to 6.500 yl/g humid faeces, normal value < 200), consistent with the diagnosis of protein-losing enteropathy. An echographic examination of the abdomen revealed a diffuse thickening of the intestinal wall, particularly at the level of the small bowel, with an almost exclusive involvement of the submucosal layer. Colonoscopy revealed the presence of diffuse purpuric lesions at the level of the submucosa. Aggressive immunosuppressive treatment completely resolved the clinical picture. CONCLUSION: protein-losing enteropathy is an uncommon cause of hypoalbuminaemia during the course of systemic lupus erythrematosus. It may be considered as a clinical syndrome related to many pathological conditions leading to an excessive intestinal protein loss. Some conditions are related with an altered mucosal permeability, others with primary or secondary intestinal lymphangiectasia. A review of the possible causes of systemic lupus erythrematosus-associated protein-losing enteropathy reported in the paediatric literature is given.