Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis

Amniotic fluid; Neonatal mortality; ExencephalyLíquido amniótico; Mortalidad neonatal; ExencefaliaLíquid amniòtic; Mortalitat neonatal; ExencefàliaExencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.This work was supported by Prof. Jose L. Peiro internal Cincinnati Children's Hospital funding

Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord

Robotic Assisted Surgery in Pediatric Gynecology: Promising Innovation in Mini Invasive Surgical Procedures

Study Objective: Robotic assisted surgery is not yet widely applied in the pediatric field. We report our initial experience regarding the feasibility, safety, benefits, and limitations of robot-assisted surgery in pediatric gynecological patients. Design: Descriptive, retrospective report of experience with pediatric gynecological patients over a period of 12 months. Setting: Department of Pediatric Surgery, IRCCS Policlinico San Matteo Foundation. Participants: Children and adolescents, with a surgical diagnosis of ovarian and/or tubal lesions. Main Outcome Measures: Robot assembly time and operative time, days of hospitalization, time to cessation of pain medication, complication rate, conversion rate to laparoscopic procedure and trocar insertion strategy. Results: Six children and adolescents (2.4-15 yrs), weighing 12-55 kg, underwent robotic assisted surgery for adnexal pathologies: 2 for ovarian cystectomy, 2 for oophorectomy, 1 for right oophorectomy and left salpingo-oophorectomy for gonadal disgenesis, 1 for exploration for suspected pelvic malformation. Mean operative time was 117.5 \ub1 34.9 minutes. Conversion to laparatomy was not necessary in any of the cases. No intra- or postoperative complications occurred. Conclusion: Initial results indicate that robotic assisted surgery is safely applicable in the pediatric gynecological population, although it is still premature to conclude that it provides better clinical outcomes than traditional laparoscopic surgery. Randomized, prospective, comparative studies will help characterize the advantages and disadvantages of this new technology in pediatric patients

Combined antenatal therapy with retinoic acid and tracheal occlusion in a rat model of congenital diaphragmatic hernia

To investigate the effect of combined prenatal treatment with retinoic acid (RA) and tracheal occlusion (TO) on the pulmonary vascular morphology and expression of vascular endothelial growth factors (VEGF) and its receptors in a rat model of congenital diaphragmatic hernia (CDH). Rats were given nitrofen at 9 days of gestation followed by no treatment (CDH), RA (CDH + RA), TO (CDH + TO), or both (CDH + RA + TO) (n = 16). We measured the median wall thickness of the pulmonary arterioles (MWT) and analyzed the expression of VEGF and its receptors (VEGFR1 and VEGFR2). Compared to control animals, CDH had increased MWT (44 +/- 15 vs. 58 +/- 7; p < 0.05) and decreased expression of VEGF, VEGFR1, and VEGFR2 (p < 0.05). Treatment with RA or TO alone, and RA + TO reduced the MWT (46 +/- 9, 42 +/- 11, 46 +/- 8, respectively) and improved the expression of VEGF, VEGFR1, and VEGFR2 compared to CDH (p < 0.05). However, the combination of RA + TO did not confer additional benefit in the reduction of the MWT or in increasing the VEGF and its receptors compared to either treatment alone. Antenatal treatment with either RA or TO improved the MWT and expression of VEGF and its receptors in a CDH rat model. However, combined treatment with RA + TO was not superior to either treatment alone

A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats

BACKGROUND: Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. METHODS: Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. RESULTS: DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. CONCLUSIONS: This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. Impact There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future

Isolated colon ischemia with norovirus infection in preterm babies: a case series

Introduction. Norovirus infection with necrotizing enterocolitis has so far been reported as a specific tropism of the small bowel in premature newborns. Case presentation. Three cases of premature newborns presenting with extensive isolated colonic ischemia due to norovirus infection are reported.Patient 1 was a Caucasian girl with a gestational age of 29+2 weeks. She had sudden onset of abdominal distension on the 30th day of life. Radiological signs of colonic pneumatosis were present 48 hours before perforation and stool analysis was positive for norovirus. On the 34th day, free air was detected on plain abdominal X-ray. At laparotomy, stenosis, necrosis and perforations involved the whole colon. The patient underwent ileostomy. A large colon resection and ileosigmoid anastomosis were done 3 months later.Patient 2 was a Caucasian boy with a gestational age of 28+3 weeks. On the 19th day, bloody stools with abdominal distension appeared. Stool analysis resulted positive for norovirus. A plain abdominal X-ray showed distended bowel loops. Antibiotic treatment was started. On the 32nd day due to the progressive deterioration of clinical conditions and the appearance of colic pneumatosis, a laparotomy was performed. Severe damage of the transverse colon and multiple areas of necrosis were found. Terminal ileostomy was performed. Six months later surgery consisted of mid-transverse colon resection as far as the splenic flexure, colocolic anastomosis and closure of ileostomy.Patient 3 was a Caucasian boy with a gestational age of 30 weeks. On the 44th day bloody-mucous stools appeared and stool analysis was positive for norovirus infection. Even with institution of antibiotic therapy clinical abdominal radiologic signs of colonic pneumatosis of the upper right quadrant were found. At the 70th day an explorative laparotomy showed dilated bowel loops and stenotic right colon and ileostomy was mandatory. Partial colectomy was later necessary and ileocolic anastomosis was performed. Conclusion: We hypothesize that norovirus infection may be responsible for severe, distinctive colonic lesions, even in premature newborn infants

Excessive Reversal of Epidermal Growth Factor Receptor and Ephrin Signaling Following Tracheal Occlusion in Rabbit Model of congenital Diaphragmatic Hernia

Abstract Congenital diaphragmatic hernia (CDH) causes severe pulmonary hypoplasia from herniation of abdominal contents into the thorax. Tracheal occlusion (TO) for human CDH improves survival, but morbidity and mortality remain high, and we do not fully understand the cellular pathways and processes most severely impacted by CDH and TO. We created a left diaphragmatic hernia (DH) in rabbit fetuses with subsequent TO and collected left lung sections for NextGen mRNA sequencing. DH, TO and DHTO fetuses had comparable body and organ growth to control except for lower lung weights in DH (p < 0.05). Of 13,687 expressed genes, DHTO had 687 differentially expressed genes compared with DH, but no other group-group comparison had more than 10. Considering genes in combination, many of the genes reduced in DH were more highly expressed in DHTO than in control. Benchmarking fetal rabbit lung gene expression to published lung development data, both DH and DHTO lungs were more highly correlated with the gene expression of immature lung. DNA synthesis was upregulated in DHTO compared with DH and ribosome and protein synthesis pathways were downregulated. DH reduced total and epithelial cell proliferation by half and two-thirds respectively, and DHTO increased proliferation by 2.5 and 3.4-fold respectively. Signaling pathways downregulated by DH and upregulated in DHTO were epidermal growth factor receptor signaling, ephrin signaling and cell migration; however, levels of ephrin and EGFR signaling in DHTO exceeded that of control. Identification and inhibition of the ligands responsible for this dysregulated signaling could improve lung development in CDH

La sorveglianza dello stato di salute della popolazione del Comune di Parona (PV) (II Fase)

Nell'ambito di un intervento di sorveglianza epidemiologica di una popolazione residente in una zona che si è trasformata da prevalentemente agricola ad industriale, il nostro gruppo di lavoro ha svolto nel 2007 la seconda fase dell'indagine, nell'ottica di uno studio prospettico iniziato nel 2000, per monitorare lo stato di salute della popolazione residente. Scopo di questa indagine epidemiologica è stato la rilevazione di eventuali modificazioni nello stato di salute in relazione alle patologie respiratorie associate all'inquinamento atmosferico. La rilevazione dei dati è stata effettuata mediante somministrazione di questionari e di un esame spirometrico. Allo studio hanno partecipato 399 soggetti dai 15 ai 79 anni, appartenenti ad una popolazione di 1484 persone, convocate mediante lettera individuale. I rispondenti sono stati complessivamente il 27% dei convocati. L'età media della popolazione rispondente è stata di 48,43 (±15,75) anni. Solo 149 soggetti (37,2% del totale degli attuali rispondenti) avevano partecipato alla precedente indagine. I dati anamnestici relativi alla sintomatologia associata a patologie respiratorie non hanno dimostrato modificazioni significative nel periodo considerato. 386 soggetti sono stati sottoposti a spirometria: i reperti patologici sono risultati essere 36, corrispondenti al 9,3% delle persone monitorate. Rispetto al 2000 si registra un modico aumento della prevalenza di reperti spirometrici patologici da ostruzione (5,9% vs 2,6%) nelle femmine mentre la prevalenza delle spirometrie patologiche da restrizione è rimasta simile sia tra i maschi (1,7% vs 2,4%) che tra le femmine (2% vs 1,5%). Tra i soggetti che hanno partecipato ad entrambe le indagini (149 soggetti, 65 maschi e 84 femmine) sono state rilevate nel tempo differenze verosimilmente associate all'invecchiamento in quanto le spirometri patologiche sono moderatamente aumentate sia per quel che riguarda i reperti spirometrici da "ostruzione" (9% vs 6,8%) che per quelli da "restrizione" (2,2% vs 1,4%). Sia nella prima che nella seconda indagine, la stessa metodologia operativa è stata applicata ad una popolazione di controllo, omogenea per caratteristiche con quella indagata, eccetto che per la residenza nei pressi dell'inceneritore e di industrie potenzialmente inquinanti. Il confronto dei risultati ottenuti dalle indagini eseguite a Parona e nella località scelta come controllo (Rovescala) per quanto riguarda la funzionalità respiratoria, non ha evidenziato differenze significative nella prevalenza di reperti patologici da ostruzione, anche se tra i maschi la prevalenza è risultata maggiore a Parona (9,2% vs 6,9%). La partecipazione della popolazione all'indagine nei due Comuni è risultata percentualmente inferiore rispetto al 2000, nonostante i ripetuti tentativi di sensibilizzazione. Ciò non ha consentito una valutazione soddisfacente delle condizioni di salute degli abitanti di Parona