Stereological Lung Parameters After Fetoscopic Abdominal Decompression of Congenital Diaphragmatic Hernia in an Ovine Model: A Pilot Study.

Background: Fetoscopic endoluminal tracheal occlusion (FETO) improves lung maturation in severe cases of congenital diaphragmatic hernia (CDH) but it does not ameliorate lung compression by herniated abdominal organs. Surgically opening the fetal abdomen (abdominal decompression [AD]) reduces the intrathoracic pressure by diverting the abdominal organs into the amniotic cavity-a probable causal therapy for lung hypoplasia and pulmonary hypertension in CDH. Open surgical abdominal decompression has been reported: we describe a minimally invasive approach in an ovine model of CDH as a probable fetoscopic intervention. Materials and Methods: Eight fetuses were included, 2 were kept as HEALTHY controls. A CDH (left side, liver down) was created by open fetal surgery at midgestation in 6 fetuses, 2 were taken as CDH controls. Fetoscopic abdominal decompression (fAD) was performed 21 days later in 4 animals. The fetuses were retrieved at the end of gestation and evaluated by lung stereology. Results: fAD led to a near total evacuation of the thoracic cavity in 2 of the 4 animals. Fetuses with CDH had a lower total volume and fraction of alveolar air space, a lower volume fraction of the parenchyma, and an increase of the volume fraction of the alveolar septa and atelectasis, as well as an increased mean thickness of alveolar septa compared with HEALTHY fetuses. Fetuses treated with abdominal decompression showed an improvement of stereological parameters. Conclusions: In spite of relevant limitations (pilot study, small groups, spontaneous closure of the abdominal incision) we were able to demonstrate that abdominal decompression for CDH can be performed by fetoscopy. Our results support the hypothesis of causally improving lung development by abdominal decompression, thus implying increased survival in extreme cases of CDH. A refinement of the fetoscopic techniques and direct comparison to FETO appears warranted.

Fetoscopic techniques for prenatal covering of gastroschisis in an ovine model are technically demanding and do not lead to permanent anchoring on the fetus until the end of gestation.

INTRODUCTION: This is the cumulative technical report on the operative procedures and limitations of fetoscopic bag insertion, intestinal bag placement, and bag fixation to the fetus in a series of pilot studies in an ovine model for prenatal treatment of gastroschisis. MATERIAL AND METHODS: In 24 German blackhead sheep, a surgically created gastroschisis was managed by fetoscopic placement of the extruded intestines into a bag. The bag was then fastened onto the fetal abdominal wall. Different materials (sterile gloves, latex condoms, laparosopic retrieval bags) and different fixation techniques (laparoscopic staplers, interrupted and continuous sutures) have been examined. The fetuses were retrieved and evaluated at the end of gestation. RESULTS: Uterine bag insertion was successful in 15 of 24 (62.5%) and intestinal bag placement in 10 of 15 available fetuses (66.6%). The main factor limiting fetoscopic procedures was chorioamniotic separation (CAS). Sterilized condoms provided the most appropriate type of bags and the V-Loc™ running suture, the most expedient type of fixation, which was achieved in 9 of the 10 fetuses (complete = 2, partially = 7) by using a three port access (5 mm and 2 × 3 mm). All bags were encountered completely or partially dislocated from the fetus at the end of gestation. CONCLUSIONS: Fetoscopic intestinal bag placement and fixation in gastroschisis technically demanding. None of the evaluated techniques led to permanent anchorage of the bag to the fetus. The development of specially designed instruments, bags and fixation methods is required to optimize this approach.

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.

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.

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.

Reintroduction of the Rat for Experimental Subarachnoid Hemorrhage with Accelerated Clot Formation: A Low Mortality Model with Persistent Clots as a Precondition for Studies in Vasospasm.

BACKGROUND: Cerebral vasospasm as a delayed, possibly treatable sequel of subarachnoid hemorrhage (SAH) is a focus of experimental animal research. For this purpose, the rat is not a good model because of the difficulty creating a stable subarachnoid clot that persists > 1 to 2 days and could induce vasospasm. Only in rat models with a high mortality of ∼ 50% or more can SAH and its effects be investigated. Therefore, other animals than rodents are used for investigating the delayed effects of SAH. Only animal studies addressing the acute effects of SAH use rats. OBJECTIVE: We designed a model that allows intensive clot formation combined with low mortality to facilitate studies on the delayed effects of experimental SAH, for example, delayed vasospasm or other alterations of vessels. METHODS: After in vitro acceleration of the clotting process in the rats' blood by tissue factor and preliminary in vivo testing, we induced a SAH by injecting blood together with tissue factor in 22 rats. We analyzed clot expansion, length of clot persistence, chronic alterations, and histologic changes. RESULTS: The injection of blood supplemented by tissue factor led to persistent voluminous blood clots in the subarachnoid space close to the large arteries. Despite the pronounced SAH, all animals survived, allowing investigation of delayed SAH effects. All animals killed within the first 7 days after surgery had extensive clots; in some animals, the clots remained until postoperative day 12. During further clot degradation connective tissue appeared, possibly as a precursor of SAH-related late hydrocephalus. CONCLUSION: The injection of blood together with tissue factor significantly improves SAH induction in the rat model. This rat model allows studying delayed SAH effects as found in humans.

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.

Modulation of Thrombosis Significantly Reduces Testicular Damage after Testicular Torsion in Rats: Anti-Thrombotic Treatment and Testicular Torsion.

OBJECTIVE: To evaluate the effects of thrombolysis and/or anticoagulation on testicular viability after testicular tortion (TT) was the aim of this study. It has been suggested that alterations of circulation during TT result in thrombus formation that might prevent sufficient perfusion after detorsion. Due to the narrow safety margin of testicular perfusion, even moderate disturbances in blood supply can cause major testicular damage. METHODS: In 112 rats, the right testicle was torsed for 3 or 6 hours. After detorsion and randomization, they received either enoxaparin, alteplase, both, or placebo, according to their subgroup. Thrombus formation was accessed via D-dimers, pDNA, oxidative testicular damage was evaluated via glutathione peroxidase and malondialdehyde, and cellular damage via inhibin B, testosterone, histological analysis (Johnsen score, Cosetino grading), and TUNEL assay. RESULTS: One hundred and twelve rats were included in the study. The treatment with alteplase or enoxaparin showed significantly less testicular damage and significantly improved Sertoli cell function. Enoxaparin significantly reduced oxidative impairment. CONCLUSION: The results of the study indicate that TT induces thrombus formation and demonstrate that modulation of thrombosis significantly ameliorates testicular damage in rats. Hence, this treatment option after TT ought to be evaluated in humans.

Gut inflammation and expression of ICC in a fetal lamb model of fetoscopic intervention for gastroschisis.

BACKGROUND: The pathogenesis of intestinal dysmotility in gastroschisis is not completely understood. Peel formation and disorganization of interstitial Cajal cells (ICC) have been proposed in humans. The aim of this study was to evaluate the impact of prenatal coverage of gastroschisis on gut inflammation and expression of ICC in a fetal lamb model. METHODS: Twenty-one German blackhead sheep with an abdominal wall defect that was created fetoscopically on day 77 of 145 days gestation were used in this study. Intrauterine surgery with the aim to cover the defect was performed 3 weeks later; two fetuses were covered completely, 5 partially and 11 remained uncovered. Three fetuses without gastroschisis were used as controls. All fetuses were retrieved by cesarean section at day 135. Samples of the small intestine were stained with hematoxylin and eosin for histologic analysis of peel formation and serosal and muscular thickness. For ICC detection, immunohistochemistry using anti-CD117 (c-Kit) antibody was used. RESULTS: In all samples with exposure to amniotic fluid, peel formation and significantly decreased ICC were found. Complete coverage reduced peel formation and disorganization of ICC compared to uncovered animals almost to the level of controls. CONCLUSIONS: Peel formation and ICC derangement were significantly reduced by prenatal coverage of gastroschisis. Moreover, this animal model mimics the histopathological bowel changes as seen in human gastroschisis and may, therefore, be used for further research on the pathophysiology and fetal therapy of this malformation.

Mucosal loss with increased expression of IL-6, IL-8, and COX-2 in a formula-feeding only neonatal rat model of necrotizing enterocolitis.

INTRODUCTION: The aim of our study is to establish a reliable neonatal rat model by formula feeding only for evaluation of early surgical intervention on the course of experimental necrotizing enterocolitis (NEC). MATERIAL AND METHODS: Newborn Sprague-Dawley rats were divided into 50 breast-fed (group 1) and 38 formula fed (Similac/Esbilac, group 2) animals. The pups were sacrificed on the 4th, 5th, and 6th day of life and the terminal intestine examined for macroscopic and histologic changes as well as cytokine expression. RESULTS: The histological mucosal damage was significantly higher of group 2 compared to group 1. The area of the vital mucosa of group 2 was significantly (58.57%, p<0.001) lower compared to group 1 (75.12%). The mRNA expression of the inflammatory cytokines IL-6, IL-8 and COX-2 was significantly 2-, 5- and 10-fold increased in group 2 compared to group 1. DISCUSSION: Formula fed newborn rats displayed an inflammatory enterocolitis similar to human NEC. Our study demonstrates a significant loss of mucosa in animals with NEC having increased expression levels of IL-6, IL-8 and COX-2. Mucosal loss appears to be a distinct feature of experimental NEC and has to be correlated with the human disease.

Abdominal wall incision with or without exteriorization of bowel: results from a fetal lamb model for the embryogenesis of gastroschisis.

INTRODUCTION: The embryogenesis of gastroschisis is not completely understood. The aim of our study was to evaluate the impact of a simple abdominal wall defect versus a defect including eviscerated intestine or omentum for the development of gastroschisis in a fetal lamb model. MATERIAL AND METHODS: At mid-gestation (day 77) an abdominal wall defect was fetoscopically created with three different approaches in 19 German blackhead sheep. The intestine was eviscerated in 7 fetuses (group 1). The peritoneum was incised and a patch of the omentum pulled through the incision in 5 fetuses (group 2). In 7 fetuses (group 3) the skin and rectus muscle were incised until the peritoneum was visible. In this group, no abdominal contents were exteriorized and the peritoneum was left intact. A second fetoscopic procedure was performed 21 days later, assessing the condition and extension of eviscerated bowel. The fetus was retrieved by Cesarean section on day 132 and evaluated. RESULTS: The second fetoscopy acting as a control for the creation of gastroschisis demonstrated eviscerated and inflamed intestine in all 3 groups. The amount of eviscerated intestine did not appear to depend on the size of the defect nor on its duration. DISCUSSION: It appears that a simple incision of the abdominal wall with intact peritoneum is sufficient for the development of gastroschisis in a fetal sheep model. This finding may improve the understanding of the etiology of gastroschisis.

Fetoscopic management of gastroschisis in a lamb model.

BACKGROUND: Gastroschisis is a malformation consisting of an abdominal wall defect with eviscerated bowel. Its standard treatment is postnatal repositioning or temporary prosthetic bag placement. The aim of our study is to evaluate the feasibility of its fetoscopic management in a lamb model. METHODS: At mid-gestation (day 75), gastroschisis was created in eight German blackhead sheep. A second fetoscopic procedure was performed on day 105, assessing the viability, extension, and potential for repositioning of eviscerated bowel. The fetus was retrieved by Cesarean section on day 132 and evaluated. RESULTS: In six fetuses gastroschisis could be successfully created and assessed with fetoscopy. Two fetuses were lost due to technical complications. Primary repair by repositioning the intestine into the abdomen was not possible because it grew into an inflammatory conglomerate too large for the small fetal abdominal cavity. CONCLUSIONS: Although technically demanding, we were able to produce and reassess six cases of gastroschisis by fetoscopy. As primary repositioning appears unfavorable, fetoscopic prosthetic bag placement may become an alternative.

Long-term subarachnoid catheter placement in the middle cranial fossa of the rat.

Research using rats sometimes requires long-term placement of catheters in the subarachnoid space, the cavity between the arachnoid mater and the pia mater in the brain. These catheters can be used to experimentally induce subarachnoid bleeding by injecting blood or to locally administer drugs or other substances. To date, published techniques for penetrating the subarachnoid space of small experimental animals require the use of inflexible or relatively inflexible catheters. These catheters typically consist of metal or stiff plastic and are used to access the occipital or frontal cranial cavity or to directly access the cisterna magna via the atlantooccipital membrane. However, inflexible catheters are not ideal for long-term placement in the subarachnoid space. In this paper, the authors describe a reliable procedure for long-term catheterization of the subarachnoid cavity of the rat. For this method, personnel insert the catheter and keep it in place in the rat's middle cranial cavity, in the vicinity of the cerebral arterial circle. This new approach allows personnel to repeatedly use the catheter for a period of at least 2 weeks. The catheter, which is well-tolerated by rats, can be used for administering saline solutions and for injecting blood that has not been treated with heparin into the subarachnoid space.

Transfer of human leukocytes into double-knockout Pfp-/-Rag2-/- mice grafted with human skin: increased accumulation of neutrophils in human dermal microvessels.

Severe combined immunodeficient mice reconstituted with human leukocytes have been useful to model parts of the human immune system, including some of its diseases (e.g., AIDS). Because no human polymorphonuclear leukocytes (huPMN) develop in these xenograft models, diseases such as several forms of vasculitis cannot be modeled using this approach. To provide such a model for vasculitis, human skin patches were grafted onto double-knockout Pfp(-/-)Rag2(-/-) mice, which not only lack functional T and B cells but which are also devoid of natural killer cells. After intravenous injection, a high proportion of huPMNs survived within the circulation and accumulated in the human blood vessels. The accumulation increased considerably after the endothelium of the skin patches had been stimulated by tumor necrosis factor-alpha. Alpha mild perivascular neutrophilic infiltration and vascular necrosis was observed in the microvessels of the skin patches. Thus, a xenograft model of vasculitis with predominant huPMNs infiltration has been established for the first time.