Intramural ganglion structures in esophageal atresia: a morphologic and immunohistochemical study.

Introduction and Aim. Disorders of esophageal motility causing dysphagia and gastroesophageal reflux are frequent in survivors to esophageal atresia (EA) and distal tracheoesophageal fistula (TEF). The aim of the present study was to investigate the histologic and immunohistochemical features in both esophageal atretic segments to further understand the nature of the motor disorders observed in these patients. Material and Methods. Esophageal specimens from 12 newborns with EA/TEF and 5 newborns dead of unrelated causes were examined. The specimens were fixed in 5% buffered formalin, included in paraffin and cut in 5 micron sections that were stained with hematoxilin and eosin (H and E), and immunohistochemical stainings for Actin, S-100 protein, Neurofilament, Neuron-Specific-Enolase, Chromogranin A and Peripherin were evaluated under the microscope. Results. In controls, the distribution of the neural elements was rather homogenous at both levels of the esophagus. In contrast, the atretic segments showed quantitative and qualitative differences between them with sparser nervous tissue in the distal one in comparison with the proximal one and with controls. Conclusions. These results further support the assumption that histomorphological alterations of the muscular and nervous elements within the esophageal wall might contribute to esophageal dysmotility in patients surviving neonatal operations for EA/TEF.

The Modified Kimura's Technique for the Treatment of Duodenal Atresia.

Background/Purpose. Kimura's diamond-shaped-duodenoduodenostomy (DSD) is a known technique for the correction of congenital intrinsic duodenal obstruction. We present a modification of the technique and review the advantages of this new technique. Methods. From 1992 to 2006, 14 newborns were treated for duodenal atresia. We inverted the direction of the duodenal incisions: a longitudinal incision was made in the proximal duodenum while the distal was opened by transverse incision. Results. Our "inverted-diamond-shaped-duodenoduodenostomy" (i-DSD) allowed postoperative oral feeding to start on days 2 to 3, peripheral intravenous fluids discontinuity on days 3 to 8 (median values 3.6); time to achieve full oral feeds on days 8 to 12 (median values 9.4); the length of hospitalisation ranged from 10 and 14 days (median value 11.2). No complications related to the anastomosis, by Viz leakage, dehiscence, biliary stasis, or stenosis were observed. Conclusions. The i-DSD provides a safe procedure to protect the ampulla of Vater from injury and avoids any formation of a blind loop. The results show that patients who have i-DSD achieve full oral feeds in a very short time period and, consequently, the length of hospitalisation is also significantly reduced.

Mutations in INSL3 and RXFP2 genes in cryptorchid boys.

Mutations in the INSL3 and RXFP2 genes have been associated with human cryptorchidism but with contrasting data. We analyzed the frequency of mutations in these genes in 600 newborns with cryptorchidism (396 unilateral and 204 bilateral) and 300 noncryptorchid subjects. We found five RXFP2 mutations in five bilateral cryptorchid boys, one INSL3 mutation in a unilateral cryptorchid boy, and one INSL3 mutation in a boy with unilateral cryptorchidism at birth and spontaneous descent during the first month of life. Overall, the frequency of INSL3 and RXFP2 mutations was therefore 7/600 at birth (1.2%) and 7/303 (2.3%) in persistent cryptorchid boys, with a higher prevalence of bilateral forms (5/120, 4.2%). No mutations were found in controls. This study confirmed the association between INSL3 and RXFP2 gene mutations and human cryptorchidism.

Genetic alterations associated with cryptorchidism.

CONTEXT: Cryptorchidism is the most frequent congenital birth defect in male children and represents an important risk factor for infertility and testicular cancer. Major regulators of testicular descent are the hormones insulin-like factor 3 (INSL3) and testosterone, and disruption of these pathways might cause cryptorchidism. OBJECTIVE: To determine the frequency of genetic alterations in cryptorchidism. DESIGN AND SETTING: Case-control study in 2 departments of pediatric surgery in Italy between January 2003 and March 2005. PATIENTS: Six hundred male infants with cryptorchidism. Boys were followed up for 2 to 3 years (through January 2008) and orchidopexy was performed in those who were persistently cryptorchid. We analyzed 300 noncryptorchid male children aged 1 to 4 years as controls. MAIN OUTCOME MEASURES: Karyotype anomalies and INSL3, INSL3 receptor, and androgen receptor gene mutations. RESULTS: The frequency of genetic alterations in boys with cryptorchidism was low (17/600 [2.8%; 95% confidence interval {CI}, 1.7%-4.5%]) and was significantly higher in participants with persistent cryptorchidism (16/303 [5.3%; 95% CI, 3.0%-8.4%]; P = .001) and those with bilateral cryptorchidism (10/120 [8.3%; 95% CI, 4.1%-14.8%]; P = .001) than in controls (1/300 [0.3%; 95% CI, 0.1%-0.8%]). Boys with persistent cryptorchidism had a 17-fold greater odds of having a genetic alteration (odds ratio, 16.7; 95% CI, 2.2-126.5). The most common genetic findings in those with cryptorchidism were 8 cases of Klinefelter syndrome and 5 cases of mutations in the INSL3 receptor gene. Genetic alterations were not found in boys with low birth weight or low gestational age, who had frequent spontaneous descent of the testes. CONCLUSION: In a small percentage of the study population, there was a statistically significant association between bilateral and persistent cryptorchidism and genetic alterations, including Klinefelter syndrome and INSL3 receptor gene mutations.

Gastrointestinal sequelae in survivors of congenital diaphragmatic hernia.

BACKGROUND: Gastrointestinal sequelae have been sporadically reported in survivors of congenital diaphragmatic hernia (CDH). The aim of the present paper was to evaluate the gastrointestinal morbidity in infant, adolescent and adult patients who had undergone repair of CDH. METHODS: Thirty-one of 38 survivors after left-side CDH repair were followed up. They were subdivided in two groups. Group A consisted of 12 patients (39%) with a mean age of 4.5 years and group B, 19 patients (61%) with a mean age of 21.0 years. Patients underwent physical examination, barium meal study, gastroesophageal scintigraphy, esophageal pH monitoring and manometry of the esophagus and stomach. Upper intestinal endoscopy was performed in patients with confirmed gastroesophageal reflux (GER). RESULTS: All patients were within the normal range for height or weight. A total of 41.7% of group A and 15.8% of group B had typical symptoms suggesting GER. Barium meal study was pathological in 33.3% of group A and 21% of group B patients. In 58% of group A and 42% of group B, GER was documented on scintigraphy. In 41.7% of group A and 47% of group B the time taken for the stomach to empty half of its radioactive content (T(1/2)) was pathological. On 24 h pH monitoring pathological GER was documented in 54.5%, whereas in group B it was present in 33.3%. In 36.4% of group A and 46.7% of group B alteration of peristalsis of the stomach was recorded. Endoscopy showed esophageal pathology in 33%. CONCLUSIONS: Foregut dysmotility and GER are major sequelae in survivors of CDH repair. Long-term follow up of patients with CDH is recommended.

Intrinsic esophageal innervation in esophageal atresia without fistula.

Esophageal atresia and tracheo-esophageal fistula (EA + TEF) are often associated with malformations of neural crest origin. Esophageal innervation is also derived from the neural crest and it is abnormal in EA + TEF in which there is motor dysfunction. Our aim was to examine the intrinsic esophageal innervation in children with isolated EA in which different embryogenic mechanisms might be involved. Specimens from the proximal and distal esophageal segments of 6/35 patients who had esophageal replacement for isolated EA between 1965 and 2006 were suitable for the study. They were sectioned and immunostained with anti-neurofilament (NF) and anti-S-100 antibodies. The muscle and neural surfaces on each section were measured with the assistance of image processing software. The surface of the ganglia and the number of neurons per ganglion were determined at high power microscopy. The findings were compared with those of six autopsy specimens from newborns dead of other diseases by means of standard statistical tests and a significance threshold of P < 0.05. Unmatched age/size of babies in isolated EA and control groups precluded comparison of the relative surfaces occupied by neural elements. Patients with pure EA had denser fibrilar network and larger ganglia than controls. The number of neurons/ganglion were similar in both groups although the cells from EA patients were larger. The findings were similar at both esophageal levels studied. In spite of methodologic biases, it seems that intrinsic esophageal fibrilar network is denser and the intramural ganglia larger with larger cells in patients with pure EA than in controls on both esophageal ends of the organ. These neural anomalies are only in part reminiscent of those described in regular EA/TEF but may as well explain esophageal dysfunction in patients with repaired isolated EA.

Is a complete urological evaluation necessary in all newborns with asymptomatic renal ectopia?

AIM: To evaluate if a complete urological screening is justified by potential urological anomalies in newborns or infants with asymptomatic renal ectopia (RE). METHODS: The database records of 60 consecutive neonatal cases of RE diagnosed at the authors' hospital from 1990 to 2004 were retrospectively reviewed. RESULTS: At diagnosis, mean patient age was 1.6 months. In 58 of 60 patients, the suspected diagnosis of RE was made during prenatal or postnatal screening ultrasonography, including two newborns with anorectal malformation. There were 24 patients with crossed RE (C-RE) and 36 patients with simple RE (S-RE). A solitary RE was present in two patients. The most frequent associated urological abnormality was vesico-ureteral reflux (37.5% of C-RE and 16.6% of S-RE). Hydronephrosis was detected in seven RE and five contralateral kidneys. An obstructive megaureter was present in one patient with C-RE. In 40% of S-RE and 92.9% of C-RE, the (99m)Tc DMSA documented reduction of function of RE. The longitudinal diameter of the ectopic kidney was significantly smaller than the contralateral one. Other non-renal diseases were present in 15% of patients, of which the most frequent was cryptorchidism (6.6%). CONCLUSIONS: A complete urological evaluation is necessary in newborns with C-RE for the high incidence of associated urological anomalies, of which VUR is the most frequent. A complete urological evaluation is also reasonable in patients with S-RE who have a pelvic dilatation. No diagnosis or treatment of the associated urological diseases, in consideration of congenital decreased function of the ectopic kidney, might predispose these children to improvement of renal function impairment.

Altered serum inhibin b levels in adolescents with varicocele.

BACKGROUND/PURPOSE: Different studies have evaluated testicular hormonal dysfunction in adolescent varicocele but with variable results. Recently, inhibin B has been proposed as a marker for spermatogenesis and Sertoli cell function. The aim of the present study was to study in a homogeneous cohort of adolescents inhibin B and other hormones to detect whether untreated varicocele may be associated with any modifications of these factors. METHODS: Sixteen adolescents (mean age, 14.5 +/- 1.0 years), at Tanner stages 4 to 5 with grade II or III left-sided varicocele, underwent hormonal evaluation of inhibin B, basal testosterone, and both baseline and GnRH stimulated FSH and LH levels. Thirteen unaffected adolescents of age-matched and pubertal development were used as controls. RESULTS: Patients with varicocele showed a significant reduction in the testicular volume of the affected side (13.3 +/- 4.1 vs 15.8 +/- 4.8 mL; P = .002) and significant reduced levels of inhibin B compared with controls (271.9 +/- 70.2 vs 327.1 +/- 34.9 pg/mL; P = .042). Inhibin B levels were significantly correlated with testes volume (r = 0.62; P = .0097). Other hormonal parameters were similar in both varicocele and control groups. CONCLUSION: Inhibin B is reduced in adolescents with untreated varicocele, and it is positively correlated with testicular volume. This could be an early marker for Sertoli cell damage, with possible implications for spermatogenesis and could represent a new indication for varicocele repair.

Lipid peroxidation activates mitogen-activated protein kinases in testicular ischemia-reperfusion injury.

PURPOSE: Testicular damage after torsion has been attributed to many mechanisms, of which one is lipid peroxidation of the plasma membrane, which could cause the activation of the mitogen-activated protein kinase family. These proteins are of vital importance for signal transduction pathways and 2 of them, extracellular signal-regulated kinase and c-jun N-terminal kinase, participate in the pathogenesis of testicular ischemia. We investigated whether lipid peroxidation may trigger mitogen-activated protein kinase activation in testicular ischemia-reperfusion. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to 1-hour testicular ischemia, followed by 24 hours of reperfusion. Sham testicular ischemia-reperfusion rats served as controls. Animals were randomized to receive raxofelast, an inhibitor of lipid peroxidation (20 mg/kg intraperitoneally administered 15 minutes before detorsion and 15 minutes after detorsion) or vehicle (1 ml/kg 10% dimethyl sulfoxide/NaCl solution). A group of animals was sacrificed 0, 10, 15, 20, 25 and 30 minutes, and 1, 2 and 3 hours, respectively, after detorsion to evaluate testicular c-jun N-terminal kinase, extracellular signal-regulated kinase and tumor necrosis factor-alpha activation by Western blot analysis, and mRNA expression and conjugated dienes using a spectrophotometer technique. Another group was sacrificed 24 hours after detorsion to evaluate histological alterations. RESULTS: Testicular ischemia-reperfusion injury caused a significant increase in the conjugated diene levels, extracellular signal-regulated kinase c-jun N-terminal kinase activity and tumor necrosis factor-alpha expression in both testes. Furthermore, histological examination revealed marked damage. Raxofelast inhibited these parameters and decreased histological damage. CONCLUSIONS: These data suggest that lipid peroxidation triggers extracellular signal-regulated kinase and c-jun N-terminal kinase activation. Furthermore, mitogen-activated protein kinase blockade might represent a potential therapeutic approach to treatment in patients with unilateral testicular torsion.

Prenatal testicular torsion: ultrasonographic features, management and histopathological findings.

AIM: To highlight the ultrasonographic features of prenatal torsion of the testis in utero (IUTT) at presentation, the neonatal management and the histological findings postorchiectomy or biopsy. METHODS: Seven newborns underwent emergency exploration for IUTT. All patients underwent a sonography and real-time color Doppler ultrasound study of the scrotum before any surgical procedure. A histological examination was performed in the removed specimens. RESULTS: Sonography of the scrotum revealed enlarged, heterogeneous testes. In all cases the color and power Doppler did not reveal any flow signal on the affected side. Four newborn with unilateral testicular torsion underwent orchiectomy and contralateral orchidopexy. In one neonate after detorsion and with the absence of gangrenous changes and a reassuring biopsy, a twisted testis could be treated conservatively with orchidopexy. In another case, the parents, acknowledging the inviability of the affected testis, gave consent only for a biopsy of the testis. In the neonate with bilateral IUTT, bilateral testicular biopsies were performed. Histology of the removed testes variably showed interstitial red cell extravasion and coagulation or hemorrhagic necrosis. Light microscopy of the preserved testis highlighted surviving seminiferous tubules, with gonocytes, spermatogonia and fetal Sertoli cells. CONCLUSIONS: An early diagnosis and treatment in IUTT is essential. Surgical exploration should be always performed through the inguinal route. In bilateral IUTT testes should be left to try to assure, as long as possible, a residual Leydig cell function.

Scintigraphic evaluation of colonic motility in patients with anorectal malformations and constipation.

BACKGROUND AND PURPOSE: Constipation is one of the major sequelae in patients after correction of anorectal anomalies (ARAs). The aim of the present work has been to assess the colonic transit time, using radioisotope scintigraphy, in patients operated for ARA and experiencing constipation in the follow-up. The results were compared with transit time from children with true functional constipation. METHODS: Twelve or 32 patients operated for ARA during the period 1994-2003 experienced mild or severe constipation (6 with high or intermediate form of ARA and 6 with low type) at follow-up. The mean age of this group was 5.8 years. Eighteen patients, mean age 6.7 years, with true functional constipation were studied as well. Colonic transit times were investigated using radioisotope scintigraphy. Normal values for colonic transit time were derived from historical controls. Radioisotope diethylenetriamine pentaacetic acid labelled with indium 111 was administered orally to determine a segmental colonic transit. Images of the abdomen have been taken at 6, 24, 48, and again at 72 hours, if radioactivity was not cleared from the colon. To quantify colonic transit, we calculated the geometric centre (GC) dividing the colon into anatomic regions. RESULTS: According to normal controls, 2 different type of delayed transit can be observed: (a) slow-transit constipation if GC at 48 hours is less than 4.1; (b) functional rectosigmoid obstruction (FRSO) if GC at 48 hours is 4.1 or more but less than 6.1 at 72 hours. Patients with functional constipation were divided into 2 groups: (a) slow-transit constipation in 12 patients with a GC at 48 hours of 3.7 +/- 0.5; (b) FRSO in 6 patients with a GC of 4.7 +/- 0.04 and 5.02 at 48 and 72 hours, respectively. Patients operated for high ARA had values characteristic of FRSO with GC at 48 hours of 5.1 +/- 0.8 and 4.75 +/- 0.5 at 72 hours. In low ARA, the transit times were similar to the ones observed in patients with high ARA at 48 hours with a GC of 4.9 +/- 0.5. CONCLUSIONS: Patients with ARA frequently have functional sequelae in the postoperative period such as constipation. According to our results, constipation seems to be secondary to segmental motility disorders limited to the rectosigmoid area, similar to constipated children with FRSO. No evidence of more generalised motility disturbance, as previously postulated, could be recorded.

Mid- and long-term effects on pulmonary perfusion, anatomy and diaphragmatic motility in survivors of congenital diaphragmatic hernia.

The aim of the present study was to evaluate the pulmonary sequelae and diaphragmatic motility in infant, adolescent and adult patients (pts) who had undergone the repair of a congenital diaphragmatic hernia. Thirty-one (81.5%) out of 38 survivors after left side CDH repair, without using a patch, were followed-up. They were subdivided in two groups. Group A (mid-term follow-up): 12 pts (39%) (5 males, 7 females) with a mean age of 4.5 years; Group B (long-term follow-up): 19 pts (61%) (9 males, 10 females) with a mean age of 21.0 years. All pts underwent physical examination, chest X-ray, diaphragmatic ultrasonographic (US) examination, pulmonary perfusion scintigraphy. Patients of the group B were also submitted to spirometry. All pts had a normal life-style and no one complained of respiratory symptoms. The chest X-ray revealed pathologic findings in 12 pts (39%). 8 pts (26%) showed chest wall alterations. The profile of the left diaphragmatic dome appeared irregular in 9 pts (29%). In all pts M-mode sonography disclosed a reduced diaphragmatic motility on the treated side. The mean pulmonary perfusion scintigraphy value on the affected side was 39.2+/-0.7%. The spirometric study showed normal values. We noted that the lung perfusion significantly and rapidly improved after CDH repair even the apparently hypoplastic and small lungs, the diaphragm maintained a good contractility during forced respiration.

Evidence for a role of mitogen-activated protein kinase 3/mitogen-activated protein kinase in the development of testicular ischemia-reperfusion injury.

Mitogen-activated protein kinase (MAPK) 3/MAPK1 (also known as ERK1/ERK2) plays an important role in the signal transduction pathways. To our knowledge, however, its role in the development of testicular ischemia-reperfusion injury has not yet been investigated. Therefore, we studied the pattern of MAPK3/MAPK1 activation in a experimental model of testicular ischemia-reperfusion injury. We also investigated MAPK8 to understand whether an association exists between these two MAPKs. Adult male Sprague-Dawley rats were subjected to 1 h of testicular ischemia followed by 24 h of reperfusion or to a sham testicular ischemia-reperfusion. Animals were randomized to receive PD98059, which is an inhibitor of MAPK3/MAPK1 (10 mg/kg i.p. administered immediately after detorsion), or its vehicle. The time course of MAPK3/MAPK1, MAPK8, and tumor necrosis factor (TNF; also known as TNF alpha) expression and a histological examination in both the ischemic-reperfused testis and the contralateral one were performed. In both testes, MAPK3/MAPK1 and MAPK8 expression appeared following 10 min of reperfusion and reached their highest activation after 30 min. The MAPK levels slowly decreased, and no significant expression of either kinase was observed following 2 h of reperfusion. Expression of TNF was evident after 1 h of reperfusion and reached its maximum increase after 3 h. PD98059 blunted MAPK3/MAPK1 and MAPK8, reduced TNF expression, and improved the testicular damage caused by ischemia-reperfusion injury in both testes. These data emphasize that MAPK3/MAPK1 has a role in testicular damage and that its blockade might have a future therapeutic role for the management of patients with unilateral testicular torsion.

Raxofelast, a hydrophilic vitamin E-like antioxidant, reduces testicular ischemia-reperfusion injury.

Testis torsion is a surgical emergency that lead to permanent gonad damage. The damage has been ascribed to mechanisms of ischemia-reperfusion similar to other tissues. The mechanisms involved are different, but the lipid peroxidation of plasma membrane, caused by reactive oxygen species (ROS), generated particularly during reperfusion, is one of the most accredited. In the present study, we aimed to evaluate the effects of raxofelast, a vitamin E-like antioxidant with potent action and no systemic toxicity, on lipid peroxidation and histopathology in both testes after unilateral testicular torsion and detorsion. Adult male Wistar rats were subjected to total occlusion (3 h) of the left testis followed by 4 hours of reperfusion (TI/R). Sham testicular ischemia-reperfusion rats (SHAM TI/R) were used as controls. The animals were then randomized to receive either vehicle (1 ml/kg/i.p. of a dimetylsulphoxide/NaCl 0.9% 1:10 v/v solution, injected either 15 min before detorsion and 15 min after detorsion) or raxofelast (20 mg/kg i.p. 15 min before detorsion and 15 min after detorsion). Conjugated dienes (CD) levels, an index of lipid peroxidation, and testis histopathology were evaluated. Testicular ischemia reperfusion (TI/R) in untreated rats produced high testicular levels of CD (3.6+/-0.3 DeltaABS/g protein on the left side and 2.5+/-0.2 DeltaABS/g protein on the right side). Furthermore, histological examination revealed marked damage to the testis interstitium with severe haemorrhage and edema. The administration of raxofelast lowered CD levels (2.8+/-0.2 DeltaABS/g protein on the left side and 1.9+/-0.1 DeltaABS/g protein in the right side) and significantly reduced histological damage. These data suggest that the hydrophilic vitamin E-like antioxidants are good candidates for designing a novel therapeutic strategy to halt the oxidative stress that follows acute testis torsion.

Immunohistology of aquaporin-1 and stem cell factor-receptor in human undescended testes.

OBJECTIVES: Both aquaporin (AQP) 1 and the stem-cell factor/C-kit system seem to have a definite role in testis function, but very few studies have been reported in humans, especially in the paediatric age group. With the present study we wanted to investigate the expression of these proteins to better delineate their role in normal and pathologic testes. METHODS: Immunohistology using AQP 1 and C-kit antibodies was performed on paraffin sections of open-testicular biopsies from 32 undescended testes. The testes of cryptorchid patients, with ages ranging from 2 to 15 years, were biopsied during an orchidopexy operation, after obtaining informed consent. Control biopsies, from 8 patients of matched age, were obtained during operations for inguinal hernia or hydrocele, always after obtaining informed consent. Positive results were recorded as diffuse or focal patterns and scored as weak, moderate or strong immunostaining. RESULTS: AQP 1 antibody strongly depicted microvessel endothelial cells, but was unlabeled in endotubular and interstitial cell lines, in both control and undescended testes. The C-kit immunostaining in normal testes revealed a diffuse, strong staining in the cytoplasm of spermatogonia and primary spermatocytes. However, in the undescended testes a focal C-kit immunolabelling was weakly recognized in both spermatogonial and immature Sertoli cells. CONCLUSIONS: These results indicate a direct involvement of AQP 1 in the regulation of fluid transport across the endothelial cell membranes of testicular microvessels. A role of the C-kit receptor protein is also substantiated by its strong expression in the maturing spermatogonia of the normal testes, but was minimally or not recognizable in undescended prepubertal testes.