Abdominal Wall Movements Predict Intra-Abdominal Pressure Changes in Rats: A Novel Non-Invasive Intra-Abdominal Pressure Detection Method.

(1) Background: As increases in intra-abdominal pressure (IAP) result in irreversible tissue damage, monitoring IAP in critically ill patients using the common urinary bladder catheter method is essential. However, this method can result in complications and is not suitable for very low birth weight neonates. The aim of this study was to establish a non-invasive and accurate method to detect IAP changes using an animal model. (2) Methods: IAP changes via intra-abdominal air application (up to 20 mmHg) were measured in 19 Wistar rats via an intra-abdominally placed intracranial pressure probe. Concurrently, abdominal surface tension was measured using a Graseby capsule (GC). (3) Results: A high correlation between abdominal wall distension and IAP (r = 0.9264, CI 0.9249-0.9279) was found for all subjects. (4) Conclusions: IAP changes in rats can be detected non-invasively using a GC. However, further studies are necessary to assess whether IAP changes can be measured using a GC in the neonatal population.

Clinical outcome, quality of life, and mental health in long-gap esophageal atresia: comparison of gastric sleeve pull-up and delayed primary anastomosis.

INTRODUCTION: Pediatric surgeons have yet to reach a consensus whether a gastric sleeve pull-up or delayed primary anastomosis for the treatment of esophageal atresia (EA), especially of the long-gap type (LGEA) should be performed. Thus, the aim of this study was to evaluate clinical outcome, quality of life (QoL), and mental health of patients with EA and their parents. METHODS: Clinical outcomes of all children treated with EA from 2007 to 2021 were collected and parents of affected children were asked to participate in questionnaires regarding their Quality of Life (QoL) and their child's Health-Related Quality of Life (HRQoL), as well as mental health. RESULTS: A total of 98 EA patients were included in the study. For analysis, the cohort was divided into two groups: (1) primary versus (2) secondary anastomosis, while the secondary anastomosis group was subdivided into (a) delayed primary anastomosis and (b) gastric sleeve pull-up and compared with each other. When comparing the secondary anastomosis group, significant differences were found between the delayed primary anastomosis and gastric sleeve pull-up group; the duration of anesthesia during anastomosis surgery (478.54 vs 328.82 min, p < 0.001), endoscopic dilatation rate (100% vs 69%, p = 0.03), cumulative time spent in intensive care (42.31 vs 94.75 days, p = 0.03) and the mortality rate (0% vs 31%, p = 0.03). HRQoL and mental health did not differ between any of the groups. CONCLUSION: Delayed primary anastomosis or gastric sleeve pull-up appear to be similar in patients with long-gap esophageal atresia in many key aspects like leakage rate, strictures, re-fistula, tracheomalacia, recurrent infections, thrive or reflux. Moreover, HrQoL was comparable in patients with (a) gastric sleeve pull-up and (b) delayed primary anastomosis. Future studies should focus on the long-term results of either preservation or replacement of the esophagus in children.

Surgical Experience Affects the Outcome of Central Venous Access Catheter Implantation in Children: A Retrospective Cohort Study.

INTRODUCTION: Surgical complications occur in up to third of children, limiting the benefits of tunneled central venous catheters (tCVCs) in children. We aimed to identify risk factors for complications related to catheter implantation. METHODS: All children and adolescents undergoing tCVC implantation at a single center over a period of 9 years were analyzed. Infection, thrombosis, dislocation, and catheter dysfunction were defined as complications. Both patient-related (ie, age, sex, vessel characteristics, revision surgery) and surgical factors (ie, sex of surgeon, surgical experience) were analyzed for their association with complications. RESULTS: A total of 1024 catheters were inserted, 887 ports and 137 broviac catheters. In terms of patient-related factors, Broviac catheters, and nononcological patients had a higher complication rate. The use of the internal jugular vein and revision surgery was associated with significantly increased complications in patients with port catheters. Experience of the surgeon correlated with various outcome parameters. Implantation performed by an attending were associated with lower complication rates in comparison to those performed by residents. Within the resident group, insertions performed by experienced residents had more complications compared with those performed by residents during their first years. CONCLUSION: The study suggests that the outcome of tCVCs insertion is affected by the type of catheter used, the utilized vessel and above all by surgical experience. Residents had significantly increased complication rates in comparison to board-certified surgeons and amongst resident's outcome got worse with increasing experience of the residents. The presence of an experienced attending did not compensate for this effect. To improve the outcome of tCVCs, strategies like direct feedback after every procedure to achieve proficiency should be implanted in residency programs.

Religious and cultural aspects of organ donation: Narrowing the gap through understanding different religious beliefs.

BACKGROUND: The supply of viable organ donations falls significantly below the demand. Discrepancies concerning the availability of transplants cannot be explained solely by the various consent models used in different countries. There is evidence that religious beliefs of patients, potential donors, and healthcare professionals also play an important role in the decision-making process. However, to date, very little research has been conducted on the consequences of specific religious beliefs on transplantation rates. The aim of this review was to outline the religious views of Christians, Muslims, Jews, Hindus, and Buddhists on organ donation. Additionally, different approaches to address this topic throughout the world are presented and can function as a helpful background for medical professionals. METHODS: The umbrella organizations of the five largest religious movements were asked for written statements concerning the subject of organ transplantation, and a literature review was performed. RESULTS: All German religious umbrella organizations have a positive view on organ donation, if certain rules are respected. Particularly, deceased donations are supported as a sign of altruism, love, and respect for another human being. Different aspects of the transplant process (e.g., consent, brain death, and respect for the dead body) are particularly stressed in some religions. CONCLUSIONS: In general, the religious groups addressed here are in favor of organ donations. Nevertheless, there are important particularities to be considered. Hopefully, understanding the different religious views on organ donations will help in reducing religious concerns about transplantation and narrowing the gap between the need and the availability of organ donations.

The Spaced Learning Concept in Combination With Halsted and Peyton - A Randomized Controlled Study.

BACKGROUND: Several motor learning models have been used to teach highly complex procedural skills in medical education. Two approaches are often employed amongst health care professionals: Halsted's "See one - do one - teach one" concept and Peyton's Four-step approach. Peyton's deconstruction of the learning process into 4 sub-steps was reported to be preferable for learning/acquiring/teaching complex clinical skills. However, a new increasingly popular technique is known as the spaced learning method. In a previous study, we were able to confirm that the spaced learning concept is superior for laparoscopic suturing and knot tying compared to conventional training curricula, this current study aimed to assess the influence of spaced learning in combination with Halsted's and Peyton's tutoring approaches on laparoscopic knot tying of medical students. METHODS: After randomization, 20 medical students were either assigned to Halsted's or Peyton's teaching approach and trained one-on-one (teacher-student). Additionally, all subjects were trained according to the spaced learning concept, containing 40 minutes of content-blocks, followed by a 20-minute break involving coordinated, standardized physical activity. This was repeated three times. Primary endpoints were time, knot quality, precision, knot strength, as well as overall laparoscopic knotting performance and competency. To evaluate the motivation of the subjects, an 18-item questionnaire was utilized to measure four motivational factors (anxiety, probability of success, interest, and challenge). RESULTS: All trainees significantly improved after training in all knot attributes. Trainees assigned to Halsted's method were able to significantly outperform the Peyton group in knot quantity within 30 minutes (p = 0.013), time/knot (p = 0.033), performance score (p = 0.009), and precision (p = 0.032). No significant difference between Halsted and Peyton was found for knot strength and quality. Furthermore, no significant difference was identified comparing motivation pre- and post-training. However, subjects in the Peyton appeared to be significantly more anxious after training. CONCLUSION: Combining spaced learning technique with Halsted's "see one - do one - teach one" appears to be superior to Peyton's Four-step approach in conjunction with spaced learning in surgical naïve students. We recommend further studies evaluating the combination of spaced learning with Halsted and Peyton's instructional methods.

Are We Hitting the Target?: The Effectiveness of Distinct Antibiotic Regimens in Pediatric Appendectomies.

BACKGROUND: Currently, a range of different antibiotics are administered perioperatively during appendectomy surgery in the pediatric population. One reason for the lack of a uniform treatment regimen is the paucity of large studies on microbiologic data and susceptibility patterns. METHODS: A retrospective, monocentric study on antibiotic susceptibility, including 1258 pediatric patients undergoing appendectomy from 2012 to 2020 was performed. Predictors for prolonged length of stay (LOS) and complications were determined using regression analysis, and objective criteria for broad-spectrum antibiotic regimens were identified. Simulations of the effectiveness of different antibiotic regimens against identified bacterial susceptibility results were conducted. RESULTS: The most important predictors for prolonged LOS and an increased incidence of infectious complications are elevated preoperative C-reactive protein levels, high intraoperative severity gradings and insufficient primary antibiotic treatment. Based on our results, a simple antibiotic treatment, consisting of cefuroxime-metronidazole (C-M) or ampicillin-sulbactam (AS), is sufficient in around 85% of all patients; both options being equally efficient. In severe appendicitis, the likelihood of resistance is 31% and 32% for C-M and AS, respectively. For piperacillin-tazobactam and C-M-tobramycin, the incidence of bacterial resistance decreases to 12%. Resistance is reduced to 8% for AS-tobramycin treatment. The patient populations benefiting most from an extended antibiotic regimen are those with a high intraoperative severity grading. CONCLUSIONS: Evidence suggests that an insufficient antibiotic regimen is associated with a prolonged LOS and a higher rate of infectious complications, justifying the expansion of a simple antibiotic regimen in cases of severe intraabdominal findings. In all other cases, a simple antibiotic treatment with AS is recommended.

Telementoring of Open and Laparoscopic Knot Tying and Suturing Is Possible: A Randomized Controlled Study.

AIM: Several motor learning models have been used to teach highly complex procedural skills in medical education. The aim of this randomized controlled trial was to assess the efficiency of telementoring of open and laparoscopic suturing of medical students compared to conventional in-person teaching and training. METHODS: After randomization, 23 medical students were assigned to either the telementoring or the in-person training group. Both groups were taught by surgically trained residence with a student-teacher ratio of 1:1 (teacher-student). Open suturing was assessed in a model of congenital diaphragmatic hernia and laparoscopic suturing in a model of bowel anastomosis. All subjects were trained according to the spaced learning concept for 3 hours. Primary end points were time, knot quality, precision, knot strength, and overall knotting performance/competency. Furthermore, we utilized the Surgery Task Load Index to evaluate the cognitive load of both teaching techniques. Students' subjective progress regarding skill acquisitions and acceptance of telementoring was assessed using a nine-item questionnaire. RESULTS: All 23 trainees significantly improved after training in all knot attributes. More than 90% of all subjects reached proficiency in both groups. In-person training and telementoring were similarly practical, and no significant differences regarding speed, knot quality, precision, knot stability, and procedure performance/competency were found. Students perceived no difference in acquisition of factual or applicational knowledge between the two groups. General acceptance of telementoring was moderate in both groups before training, but increased during training in students actually assigned to this group, in comparison to students assigned to conventional teaching. CONCLUSION: The current study shows that telementoring of open and laparoscopic suturing is an ideal answer to the current coronavirus disease 2019 pandemic, ensuring continuous training. On-site training and telementoring are similarly effective, leading to substantial improvement in proficiency in intracorporeal suturing and knot tying. Likewise, students' subjective progress regarding skill acquisitions and cognitive load does not differ between teaching methods. Skepticism toward telementoring decreases after exposure to this learning method. Given our results, telementoring should be considered a highly effective and resource-saving educational approach even after the current pandemic.

Therapeutic Targeting of Neutrophil Extracellular Traps Improves Primary and Secondary Intention Wound Healing in Mice.

Background: Neutrophils are the first responders in wound healing after injury that mediate pro- and anti-inflammatory activities i.a. through the formation of extracellular traps (NETs). However, excessive NETs presence in wound tissue can cause local hyperinflammation and -coagulation resulting in delayed wound healing. To improve wound healing, we aimed to examine the role of NETs and DNase1 on primary and secondary wound healing. Methods: The study included 93 C57BL/6 mice, with 3 different genotypes: wildtype, Pad4-, and DNase1-Knockout (KO). Pad4-KO mice show limited NETs formation, while DNase1-KO mice cannot disintegrate them. All 3 genotypes were included in (1) a laparotomy group and (2) a thermal injury group. Animals in both groups either received DNase1 or a vehicle i.p. post wound induction and wound assessment and euthanasia were conducted. Laparotomy and burn scars were assessed using the stony brook scar evaluation scale and modified Yeong scale respectively. Tissue was analyzed histologically using H&E staining. Ly6g, Collagen I and III, SMA, and Fibrinogen were visualized and neutrophils activation (NE, MPO) and NETs (H3cit) formation assessed. Results: All animals survived with no complications. DNase1 treatment led to a significantly improved scar appearance in both groups, which was also seen in Pad4-KO mice. In the laparotomy group DNase1 improved collagen deposition and fibrin concentration was significantly reduced by DNase1 treatment. Markers of neutrophil activation were significantly reduced in the treatment and Pad4-KO group. In the thermal injury group wound closure time was significantly reduced after DNase1 treatment and in the Pad4-KO group. Even though inflammation remained high in the thermal injury model over time, neutrophil activation and NETs formation were significantly reduced by DNase1 treatment compared to controls. Discussion: Primary and secondary intention wound healing is improved by targeting NETs through DNase1 treatment or genetic KO, as assessed by wound closure time and scar appearances. Additionally, wound stability was not affected by DNASE treatment. The results suggest that overall wound healing is accelerated and DNase1 appears to be a promising option to reduce scar formation; which should be evaluated in humans.

Hirschsprung Disease - Clinical Relevance of RET Mutations.

Introduction To date, several genes involved in the pathogenesis of HD have been recognized. Out of these, the RET gene (chromosomal locus 10q11), one of the first genes identified in combination with HD, is still considered the basis for HD development. However, even with over a hundred RET gen coding sequence mutations identified, the mutations do not fully explain the observed sex bias of HD and the elevated risk of developing HD among siblings. Thus, our aim was to evaluate the clinical relevance of an as yet undescribed genotype in a family with HD to improve genetic counseling for families with RET mutation-associated HD.Patients This case report provides an overview of a family with a history of HD with a novel, unreported autosomal dominant RET mutation.Results/Summary The family examined in this study clearly demonstrates that (1) the genotype to phenotype correlation of patients with RET mutation-associated HD is not directly related, and (2) genetic mechanisms underlying the different HD phenotypes, as well as the model of inheritance of HD, are complex and not yet fully understood. As such, a multifactorial genesis of HD appears more likely and should be the center of genetic counseling for concerned families. Having identified another RET mutation with a possible correlation of severity of HD and gender will aid in filling the gaps of the incomplete picture of the pathogenesis of HD.Beim Morbus Hirschsprung handelt es sich um eine angeborene Aganglionose des Darms mit multifaktorieller Vererbung. Das RET-Protoonkogen (chromosomaler Locus 10q11), eines der ersten Gene, die in Kombination mit Morbus Hirschsprung identifiziert wurden, spielt dabei eine zentrale Rolle. Es wurden bereits über hundert Mutationen der RET-Gen-Codierungssequenz beschrieben. Vollständig verstanden ist jedoch weder das Wiederholungsrisiko noch die geschlechtsspezifische Ausprägung des Phänotyps. Vorgestellt wird eine Familie mit einer familiären Form eines Morbus Hirschsprung. Alle betroffenen Familienmitglieder weisen eine heterozygote Deletion c.1384_1410del (p.Ser462_THr470del) einer bisher in der Literatur noch nicht beschriebenen autosomal dominanten RET-Mutation auf. Die männlichen Betroffenen zeigen phänotypisch einen langstreckigen Morbus Hirschsprung, wohingegen bei den weiblichen betroffenen Familienmitgliedern lediglich das Rektosigmoid von der Aganglionose betroffen ist. Die untersuchte Familie ist ein erneuter Beweis für (1) die geringe Genotyp-Phänotyp-Korrelation von Patienten mit RET-assoziiertem Morbus Hirschsprung und (2) die komplexen genetischen Mechanismen, die dem Ausprägungsgrad von Morbus Hirschsprung zugrunde liegen.

Lithocholic bile acid induces apoptosis in human nephroblastoma cells: a non-selective treatment option.

Lithocholic bile acid (LCA) has been reported to selectively kill cancer cells within many tumor cell lines including neuroblastoma or glioblastoma. Wilms' tumor shares similarities with neuro- and glioblastoma. Hence, the aim of the study was to evaluate the effects of LCA on nephroblastoma. To test the effects of LCA, nephroblastoma cell line WT CLS1 was used. SK NEP1 was tested as well. It was originally classified as a nephroblastoma cell line but was meanwhile reclassified as an ewing sarcoma cell line. As control cell lines HEK 293 from embryonic kidney and RC 124 from adult kidney tissue as well as podocytes were used. The effects were evaluated using proliferation assay, caspase activity assay, FACS and Western blot. LCA showed a dose and time-dependent selective effect inducing apoptosis in nephroblastoma cells. However, these effects were not limited to the nephroblastoma cell line but also affected control kidney cell lines and the sarcoma cells; only podocytes are significantly less affected by LCA (at dosages < 200 µm). There were no significant differences regarding the TGR5 receptor expression. The study showed that LCA has a strong, yet unselective effect on all used in vitro cell-lines, sparing the highly differentiated podocytes in lower concentrations. Further studies are needed to verify our results before dismissing LCA as an anti-cancer drug.

Ultrasound-guided ilioinguinal-iliohypogastric block (ILIHB) or perifocal wound infiltration (PWI) in children: a prospective randomized comparison of analgesia quality, a pilot study.

BACKGROUND: Ilioinguinal-iliohypogastric block (ILIHB) is a well-established procedure for postoperative analgesia after open inguinal surgery in children. This procedure is effective and safe, especially when ultrasound is used. Data availability for comparing ultrasound-guided blocks versus wound infiltration is still weak. The study was designed to determine the efficacy of ultrasound-guided ILIHB (US-ILIHB) on postoperative pain control in pediatric patients following a inguinal daycase surgery, compared with perifocal wound infiltration (PWI) by the surgeon. METHODS: This randomized, double-blinded trail was conducted in pediatric patients aged from 6 months to 4 years. The total number of children included in the study was 103. Patients were allocated at random in two groups by sealed envelopes. The ILIHB group recieved 0,2% ropivacain for US-ILIHB after anesthesia induction. The PWI group recieved 0,2% ropivacain for PWI performed by a surgeon before wound closure. Parameters recorded included the postoperative pain score, pain frequency, time to first analgesics and consumption of analgesics. RESULTS: US-ILIHB significantly reduced the occurrence of pain within the first 24 h after surgery (7.7%, p = 0.01). Moreover, the pain-free interval until administration of the first dose of opioids was 21 min longer, on average (p = 0.003), following US-ILIHB compared to perifocal wound infiltration. 72% of children who received US-ILIHB did not require additional opioids, as compared to 56% of those who received PWI. CONCLUSION: Thus our study demonstrates that US-ILIHB ensures better postoperative analgesia in children and should be prioritized over postoperative PWI. TRAIL REGISTRATION: UIHBOPWIIC, DRKS00020987 . Registered 20 March 2020 - Retrospectivley registered.

The Impact of Oral Sodium Chloride Supplementation on Thrive and the Intestinal Microbiome in Neonates With Small Bowel Ostomies: A Prospective Cohort Study.

Background: Infants with ileostomies often suffer from sodium depletion, ultimately leading to a failure to thrive. Moreover, early-infantile microbial dysbiosis may potentially aggravate weight faltering. Given that sodium supplementation has been used to restore weight gain and feeding practices largely determine infantile microbiota, the current study investigated the effect of sodium chloride (NaCl) on weight gain and intestinal microbiome in infants with jejuno- and ileostomies. Methods: A prospective cohort study including 24 neonates with enterostomies compared 19 subjects receiving oral NaCl (5.85%) to five subjects without supplementation with respect to postoperative changes in thrive and the intestinal microbiome. Results: Infants receiving NaCl after enterostomy-surgery showed vastly improved weight gain and an increased abundance of Lactobacillus in fecal samples, as compared to subjects without oral supplement who displayed decreasing percentiles for weight and did not reveal a higher abundance of probiotic strains within the ostomy effluent. Contrarily, Klebsiella was equally enriched in supplemented infants, reflecting a higher susceptibility for infections in preterm neonates. Discussion: Our findings support oral NaCl supplementation as a mainstay of postoperative treatment in infants with small bowel ostomies who are predisposed to suffer from a sodium depletion-associated failure to thrive. Not only does NaCl promote weight gain by increasing glucose resorption, but it also appears to induce microbial restoration by enhancing the abundance of health-promoting probiotic bacteria. This finding has an even greater significance when facing an elevated Klebsiella/Bifidobacteria (K/B) ratio, believed to represent an early-life microbial biomarker for development of allergic disease.

Development of an improved murine model of necrotizing enterocolitis shows the importance of neutrophils in NEC pathogenesis.

Various research models to induce necrotizing enterocolitis (NEC) in animals exist, yet significant differences in NEC severity between murine animal models and human patients persist. One possible explanation for the difference in severity may be the variance in neutrophil concentration among newborn humans (50-70%) in comparison to neonatal mice (10-25%). However, neutrophil activity has yet to be evaluated in NEC pathogenesis. Thus, the aim of the study was to evaluate the effects of altered neutrophil concentrations in neonatal mice while simultaneously undergoing a NEC induction. A total of 44 neonatal mice were included in this study and 40 were subjected to an established NEC induction paradigm and 4 were assigned a sham group. Of the 40 mice, 30 received granulocyte-colony stimulating factor (G-CSF) on a daily basis, while 10 were used as controls (receiving inactivated G-CSF). Mice undergoing G-CSF treatment were further divided into two subgroups: (1) wildtype and (2) ELANE-knockout (KO). ELANE - KO mice are incapable of producing neutrophil elastase (NE) and were used to evaluate the role of neutrophils in NEC. For each of the groups, the following metrics were evaluated: survival, NEC severity, tissue damage, neutrophil count and activation, and NETs formation. An improved murine model of NEC was developed using (1) Lipopolysaccharides and Neocate gavage feeding, (2) hypoxia, and (3) G-CSF administration. The results suggest that the addition of G-CSF resulted in significantly elevated NEC manifestation rates with consequent tissue damage and intestinal inflammation, without affecting overall mortality. Animals without functioning NE (ELANE-KO) appeared to have been protected from NEC development. This study supports the importance of neutrophils in NEC pathogenesis. The optimized NEC induction paradigm, using G-CSF administration, resulted in elevated neutrophil counts, resembling those of neonatal humans. Elevation of neutrophil levels significantly improved NEC disease manifestation by modeling human physiology more accurately than current NEC models. Thus, in the future, murine NEC experiments should include the elevation of neutrophil levels to improve the transition of research findings from mice to humans.

Cardiac and Inflammatory Necrotizing Enterocolitis in Newborns Are Not the Same Entity.

Background: Necrotizing enterocolitis (NEC) is an often-fatal neonatal disease involving intestinal hyperinflammation leading to necrosis. Despite ongoing research, (1) conflicting results and (2) comorbidities of NEC patients make early NEC detection challenging and may complicate therapy development. Most research suggests that NEC pathogenesis is multifactorial, involving a combination of (1) gut prematurity; (2) abnormal bacterial colonization; and (3) ischemia-reperfusion (I/R) injury. As neutrophil extracellular traps (NETs) partially mediate I/R injury and drive inflammation in NEC, we hypothesized that NETs contribute to NEC development; particularly in cardiac patients. Methods: A retrospective analysis of baseline characteristics, clinical signs, laboratory parameters, and imaging was conducted for surgically verified NEC cases over 10 years. Patients were stratified into two groups: (1) prior medically or surgically treated cardiac disease (cardiac NEC) and (2) no cardiac comorbidities (inflammatory NEC). Additionally, histology was reassessed for neutrophil activation and NETs formation. Results: A total of 110 patients (cNEC 43/110 vs. iNEC 67/110) were included in the study, with cNEC neonates being significantly older than iNEC neonates (p = 0.005). While no significant differences were found regarding clinical signs and imaging, laboratory parameters revealed that cNEC patients have significantly increased leucocyte (p = 0.024) and neutrophil (p < 0.001) counts. Both groups also differed in pH value (p = 0.011). Regarding histology: a non-significant increase in staining of myeloperoxidase within the cNEC group could be found in comparison to iNEC samples. Neutrophil elastase (p = 0.012) and citrullinated histone H3 stained (p = 0.041) slides showed a significant markup for neonates diagnosed with cNEC in comparison to neonates with iNEC. Conclusion: The study shows that many standardized methods for diagnosing NEC are rather unspecific. However, differing leucocyte and neutrophil concentrations for iNEC and cNEC may indicate a different pathogenesis and may aid in diagnosis. As we propose that iNEC is grounded rather in sepsis and neutropenia, while cNEC primarily involves I/R injuries, which involves neutrophilia and NETs formation, it is plausible that I/R injury due to interventions for cardiac comorbidities results in pronounced neutrophil activation followed by a hyperinflammation reaction and NEC. However, prospective studies are necessary to validate these findings and to determine the accuracy of the potential diagnostic parameters.

Degradation of Extracellular DNA Significantly Ameliorates Necrotizing Enterocolitis Severity in Mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is one of the most devastating diseases in neonates and is characterized by high morbidity and mortality. It has been suggested that neutrophils play a crucial role in NEC pathogenesis and contribute to the hyperinflammatory reaction after bacterial colonization, which ultimately induces NEC. The aim of this study was to investigate whether dissolution of neutrophil extracellular traps (NETs) by systemic DNase1 therapy reduces NEC manifestation and morbidity. METHODS: NEC was induced in neonatal mice by gavage feeding of lipopolysaccharide mixed in Neocate, followed by hypoxia q12 h for 5d. Inactivated DNase1 and DNase1 were administered intraperitoneally twice daily in the control and treatment groups, respectively, starting on day 5 for 72 h. Survival, NEC score, intestinal damage (Chiu score, malondialdehyde [MDA], glutathione peroxidase [GPx]), inflammation (neutrophil elastase [NE], myeloperoxidase [MPO], toll-like receptor 4 [TLR4]), and NETs markers (SYTOX orange, cell-free DNA [cfDNA], DNase, citrullinated Histone 3 [H3cit]) were then assessed. RESULTS: In total, 44 neonatal mice were used in the experiment. Mice in the treatment group demonstrated significantly reduced NEC rates (44 versus 86%, P = 0.029) and improved survival in comparison to controls (65 versus 35%, P = 0.01). Furthermore, mice treated with DNase1 showed significantly less tissue damage (cfDNA, Chiu score), oxidative stress (MDA, GPx), and inflammation (NE, MPO, H3cit, TLR4), which ultimately lead to a significant reduction in mortality. CONCLUSIONS: The results of the study indicate that systemic DNase1 treatment leads to a significant reduction in tissue damage, NEC severity, and mortality. Therefore, after validation of our findings in human subjects, DNase1 treatment should be considered as a therapeutic option in neonates diagnosed with NEC.

Markers of NETosis Do Not Predict Neonatal Early Onset Sepsis: A Pilot Study.

Introduction: Early-onset sepsis in neonates potentially results in substantial morbidity and mortality. A key player in sepsis a neutrophil extracellular traps (NETs) to limit dissemination of pathogens. Aim of this study was to evaluate markers of NET formation in umbilical cord blood as a predictor of neonatal sepsis. Methods: Prospective study including term and preterm neonates. Umbilical cord blood samples were obtained immediately after birth and following markers of inflammation and NET formation were assessed: complete blood count, C-reactive protein (CRP), interleukin 6 (IL-6), levels of cell-free DNA (cfDNA), neutrophil elastase (NE), and myeloperoxidase (MPO). The study population included neonates with confirmed early-onset sepsis and propensity score matched controls. Results: Umbilical cord blood samples of 491 neonates were obtained, of whom 17 neonates (n = 17) presented clinical and laboratory signs of infection within the first 72 h postpartum. Seventeen neonates without infection were matched as controls. IL-6 differed significantly between both groups, whereas other infection parameters such as CRP and neutrophil levels, and in particular the NET surrogate markers (cfDNA, NE, MPO), did not show any significant differences. Conclusion: NET markers in umbilical cord blood appear to not predict the onset of neonatal sepsis. These findings probably result from the neonates' inability or delayed ability to form NETs, which is suspected to be a main reason for the increased risk of severe infections in neonates, but is also assumed to prevent negative NET-mediated consequences during perinatal adaptation.

NEC is likely a NETs dependent process and markers of NETosis are predictive of NEC in mice and humans.

Necrotizing enterocolitis (NEC) is one of the most devastating diseases affecting premature and mature infants. It is hypothesized that NEC is the result of neutrophils' active role in hyperinflammation after bacterial gut colonization, through their nuclear DNA release and formation of neutrophil extracellular traps (NETs) to combat pathogens. The aim of this study was to evaluate the importance of NETs in NEC pathogenesis, as well as to identify and validate markers of NETosis to predict NEC. NEC was induced in mice by gavage feeding of Neocate and lipopolysaccharide, followed by ten minutes of hypoxia (5% O2) q12h for five days, starting on day four postpartum (p.p.). The interrelation of NEC and neutrophils, including NETs, was assessed macroscopically (i.e. NEC score, SYTOX Orange), microscopically (i.e. Chiu score, citrullinated histone H3, neutrophil elastase), and in blood samples (i.e. cell-free DNA (cfDNA), DNase). In order to determine the exact role of NETs in NEC pathogenesis, a protein arginine deiminase (PAD) inhibition model was established (preventing NETs formation in mice) by injecting BB-Cl-amidine once daily, starting on day one p.p. Additionally, human intestinal samples of diagnostically verified NEC were analyzed. In total, 76 mice were analyzed in the experiment. Serum cfDNA correlated positively with NEC manifestation, as measured by macroscopic NEC score (r = 0.53, p = 0.001), and microscopic evaluation with Chiu score (r = 0.56, p < 0.001). Markers of neutrophil activation and NETosis were significantly increased in animals with NEC and in human samples as compared to controls. Further, prevention of NETosis by protein arginine deiminase (PAD) inhibition in mice significantly reduced mortality, tissue damage, and inflammation in mice induced with NEC. Our results suggest that the hyperinflammation observed in NEC is a NETs-dependent process, as NEC severity was significantly reduced in mice incapable of forming NETs (PAD inhibition) and markers for NEC and NETs correlated positively during the time course of NEC induction. Further, serum surrogate markers of NETosis (such as cfDNA and DNase) appear to predict NEC in neonatal mice. As findings of the mouse NEC model correlate positively with human NEC samples immunohistochemically, the hyperinflammation reaction observed in mice could potentially be applied to human NEC pathogenesis.

Therapeutic targeting of extracellular DNA improves the outcome of intestinal ischemic reperfusion injury in neonatal rats.

Thrombosis and inflammation cooperate in the development of intestinal infarction. Recent studies suggest that extracellular DNA released by damaged cells or neutrophils in form of extracellular traps (NETs) contributes to organ damage in experimental models of ischemia-reperfusion injury. Here we compared the therapeutic effects of targeting fibrin or extracellular DNA in intestinal infarction after midgut volvulus in rats. Following iatrogenic midgut volvulus induction for 3 hours, we treated animals with a combination of tissue plasminogen activator (tPA) and low molecular weight heparin (LMWH) to target fibrin or with DNase1 to degrade extracellular DNA. The therapeutic effects of tPA/LMWH and DNase1 were analyzed after 7 days. We observed that both therapeutic interventions ameliorated tissue injury, apoptosis, and oxidative stress in the intestine. DNase1, but not tPA/LMWH, reduced intestinal neutrophil infiltration and histone-myeloperoxidase-complexes, a surrogate marker of NETs, in circulation. Importantly, tPA/LMWH, but not DNase1, interfered with hemostasis as evidenced by a prolonged tail bleeding time. In conclusion, our data suggest that the therapeutic targeting of fibrin and extracellular DNA improves the outcome of midgut volvulus in rats. DNase1 therapy reduces the inflammatory response including NETs without increasing the risk of bleeding. Thus, targeting of extracellular DNA may provide a safe therapy for patients with intestinal infarction in future.

Degradation of Extracellular DNA by DNase1 Significantly Reduces Testicular Damage After Testicular Torsion in Rats.

OBJECTIVE: To examine the effects of DNase1 treatment on testicular damage after testicular torsion (TT). It has been demonstrated that TT induces thrombus formation and that anticoagulation significantly reduces testicular damage after TT. It was hypothesized that these thrombi are dependent on neutrophil extracellular traps (NETs) and thus NETs disintegration would reduce testicular cell damage. METHODS: A sham operation was performed in 10 rats. Thirty-four rats underwent induction of iatrogenic TT for 3 hours. After de-torsion and randomization, 24 rats received DNase1 or inactivated DNase1. The following parameters were assessed: testicular damage via Cosentino grading; spermatogenesis via Johnsen score; stem cell factor and c-Kit, apoptosis via Bax, Bcl2, Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling assay, and cleaved caspase3 staining; oxidative stress via superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde; neutrophil recruitment via myeloperoxidase and neutrophil elastase staining; and NET formation via cell-free DNA. RESULTS: Forty-three rats were included in the study. Subjects treated with DNase1 showed significantly less cellular damage, oxidative stress, and apoptosis. Further, DNase1-treated rats demonstrated a significant improvement of spermatogenesis, compared with the controls. CONCLUSION: The results of the study indicate that thrombus formation during TT is quite likely NET associated, and that dissolution of cell-free DNA (including NETs) significantly improves testicular damage in rats. As treatment with DNase1 reduced apoptosis, oxidative stress, and inflammation, without adversely affecting coagulation, it might be a suitable treatment for (neonatal) TT and ought to be evaluated in humans.