Pediatric Surgery Firearm Injury Collaborative Symposium.

Injury from a firearm is now the leading cause of death of children and youth under age 19 in the United States (U.S.) [1] and the incidence of these deaths continues to increase each year [2]. For every death from firearm violence, there are several young people who have been injured by a bullet but not killed. As pediatric surgeons, we are on the front lines of treating these young patients. We have the unforgettable memories of delivering the horrible news to parents in "quiet rooms." [3]. As these injuries fall within our scope of practice, it is incumbent on us as professionals to work to prevent these injuries, apply best practices and work for the best pathways to recovery for our patients who do survive. There is a diverse community of pediatric surgeons tackling this public health problem in a variety of ways [4]. In a pre-meeting symposium at the APSA 2023 Annual meeting, we brought together a community of pediatric surgeons working on this critical area. The following summarizes the presentations of the symposium, with topics including Risk Factors, Injury Prevention, Treatment, Public Initiatives, and National Collaborative Efforts. TYPE OF STUDY: Review Article, Proceedings of a Symposium. LEVEL OF EVIDENCE: 1 through 4 all presented.

Neonatal peri-procedural extracorporeal membrane oxygenation in the management of tracheo-esophageal fistula/esophageal atresia and left pulmonary agenesis.

Congenital tracheo-esophageal fistula/esophageal atresia (TEF/EA) with concomitant pulmonary agenesis is exceedingly rare and has a high mortality rate. While there are several reported cases of successful repair, all but one patient had right-sided pulmonary agenesis. In the case of left-sided pulmonary agenesis, the patient had incomplete agenesis and underwent repair through a left thoracotomy. We present the first successful repair of TEF/EA with complete left-sided pulmonary agenesis. This patient also underwent elective pre-operative veno-venous extracorporeal membrane oxygenation (ECMO) and subsequent repair of the TEF/EA. We discuss the management, anesthesia risks, and role of periprocedural ECMO in pediatric patients who are high anesthetic risk.

In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease.

In utero base editing has the potential to correct disease-causing mutations before the onset of pathology. Mucopolysaccharidosis type I (MPS-IH, Hurler syndrome) is a lysosomal storage disease (LSD) affecting multiple organs, often leading to early postnatal cardiopulmonary demise. We assessed in utero adeno-associated virus serotype 9 (AAV9) delivery of an adenine base editor (ABE) targeting the Idua G→A (W392X) mutation in the MPS-IH mouse, corresponding to the common IDUA G→A (W402X) mutation in MPS-IH patients. Here we show efficient long-term W392X correction in hepatocytes and cardiomyocytes and low-level editing in the brain. In utero editing was associated with improved survival and amelioration of metabolic, musculoskeletal, and cardiac disease. This proof-of-concept study demonstrates the possibility of efficiently performing therapeutic base editing in multiple organs before birth via a clinically relevant delivery mechanism, highlighting the potential of this approach for MPS-IH and other genetic diseases.

Effect of Feeding Strategies on the Development of Fulminant Necrotizing Enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) causes significant neonatal morbidity. A subset of infants experience precipitous decline and death from fulminant-NEC (F-NEC). We sought to determine the effect of feeding practices on the development of this more virulent form of NEC. MATERIALS AND METHODS: Premature neonates developing Bell's stage II or III NEC between May 2011 and June 2017 were reviewed. Infants were stratified as having NEC or F-NEC, defined as NEC-totalis or NEC causing rapid decline and death within 72 hours. Risk factors extracted included demographics, gestational age, and weight at NEC diagnosis. Feeding data extracted included age at first feed, caloric density, type of feed (breast milk or formula), and whether full volume feeds were reached. Univariate analysis and multivariate analysis were performed. RESULTS: A total of 98 patients were identified, of which 80 were included. In total, 57 patients had NEC and 23 had F-NEC. Reaching full volume feeds was associated with F-NEC on both univariate and multivariate analysis (37.9 vs. 4.5%; odds ratio: 67, 95% confidence interval: 6.606-2041, p = 0.003). Infants developing F-NEC achieved full feeds earlier (22.5 vs. 19.8 days, p = 0.025) on univariate but not multivariate analysis. There was no difference in the rates of NEC and F-NEC among infants receiving breast milk (standard or fortified) or formula (standard or increased caloric density; p = 0.235). CONCLUSION: Among premature neonates with NEC, reaching full volume feedings was associated with a nearly 70-fold increased risk of F-NEC. Assuming it was possible to predict an infant's development of NEC, alternative feeding regimens might reduce the risk of F-NEC in this population.

A Rabbit Model for Optimization of Amniotic Fluid Components in the EXTrauterine Environment for Newborn Development (EXTEND) System.

In this model article, we present a protocol for continuous amniotic fluid exchange in rabbits using a novel system to test the effects of growth factor-deficient, artificial amniotic fluid on bowel development. BACKGROUND: Ideally, the EXTrauterine Environment for Neonatal Development (EXTEND) will provide physiologic support to the extreme premature infant. An important component of that environment is the amniotic fluid. Thus, we developed an animal model to study the growth factors found within amniotic fluid and inform design of a synthetic fluid to optimize fetal development. METHODS: We designed a model of amniotic fluid exchange within the pregnant rabbit, continuously removing the natural fluid from around 2 fetuses per doe and replacing it with a physiologic electrolyte solution during the final 100 h of gestation. Two fetuses from the contralateral uterine horn were used as sham-operated controls. Thirty-eight fetuses were analyzed, 19 in each group. We analyzed the fetal growth and bowel development. RESULTS: Ultrasound after 100 h of exchange showed equivalent fluid volumes, p = 0.63. Cultures were negative for bacterial colonization. Final fluid protein concentrations were 11.6% that of control fluid (mean 1,451 ± 224.2 vs. 12,491 ± 849.2 µg/mL). There was no significant difference in fetal growth, with experimental weights 91.4% of control weights, p = 0.07. Fetal bowel weights (90.1%, p = 0.16) and lengths (94.2%, p = 0.49) were also not significantly less compared to controls. There was no significant difference in villous height or crypt depth measurements between the groups, and absorptive capacity of the bowel was not different between groups, p = 0.44. CONCLUSION: This animal model allows for manipulation of the components of amniotic fluid. Marked reduction of natural amniotic fluid proteins during gestation does not appear to significantly impair fetal growth or bowel development. Further work with this model will assess the importance of amniotic fluid components for normal development to inform design of a synthetic fluid for use during EXTEND.

In utero gene editing for monogenic lung disease.

Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.

Premature Lambs Exhibit Normal Mitochondrial Respiration after Long-Term Extrauterine Support.

BACKGROUND: In an effort to mitigate the major morbidities and mortality associated with extreme prematurity, we have developed an EXTrauterine Environment for Neonatal Development (EXTEND) designed to provide physiologic support of extremely premature infants. OBJECTIVES: We have previously shown that long-term, physiologic support of premature fetal lambs is possible with EXTEND, but in this study, we sought to demonstrate bioenergetic equipoise at the tissue level. METHODS: Four premature fetal lambs were delivered by hysterotomy at gestational ages (GA) of 105-107 days (term ∼145 days), cannulated via the umbilical vessels, and transitioned to support on EXTEND for 3-4 weeks. Five control fetuses were age-matched to the GA of experimental fetuses at the time of study end (128-134 days GA) and immediately sacrificed after hysterotomy. Mitochondria were isolated from the heart, liver, kidney, and skeletal muscle of fetuses at the time of sacrifice, and oxygen consumption rates (OCRs) were measured. RESULTS: There were no differences in basal mitochondrial OCR between EXTEND and control fetuses for heart, kidney, or skeletal muscle. For liver, the basal OCR was higher in EXTEND fetuses compared to controls. There were no differences in physiologic maximal OCR or reserve capacity for any tissue analyzed. CONCLUSIONS: Fetal lambs supported by EXTEND demonstrate physiologic mitochondrial function as evidenced by adequate basal and physiologic maximal cellular respiration as well as preserved reserve capacity.

In utero CRISPR-mediated therapeutic editing of metabolic genes.

In utero gene editing has the potential to prenatally treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. We assessed the viral vector-mediated delivery of CRISPR-Cas9 or base editor 3 in utero, seeking therapeutic modification of Pcsk9 or Hpd in wild-type mice or the murine model of hereditary tyrosinemia type 1, respectively. We observed long-term postnatal persistence of edited cells in both models, with reduction of plasma PCSK9 and cholesterol levels following in utero Pcsk9 targeting and rescue of the lethal phenotype of hereditary tyrosinemia type 1 following in utero Hpd targeting. The results of this proof-of-concept work demonstrate the possibility of efficiently performing gene editing before birth, pointing to a potential new therapeutic approach for selected congenital genetic disorders.

Pre-Existing Maternal Antibodies Cause Rapid Prenatal Rejection of Allotransplants in the Mouse Model of In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative nonimmunosuppressive alternative to postnatal hematopoietic stem cell transplantation for the treatment of congenital hemoglobinopathies. Anti-HLA donor-specific Abs (DSA) are associated with a high incidence of graft rejection following postnatal hematopoietic stem cell transplantation. We determine if DSA present in the mother can similarly cause graft rejection in the fetus following IUHCT. Ten million C57BL/6 (B6, H2kb) bone marrow cells were transplanted in utero into gestational day 14 BALB/c (H2kd) fetuses. The pregnant BALB/c dams carrying these fetuses either had been previously sensitized to B6 Ag or were injected on gestational days 13-15 with serum from B6-sensitized BALB/c females. Maternal-fetal Ab transmission, Ab opsonization of donor cells, chimerism, and frequency of macrochimeric engraftment (chimerism >1%) were assessed by flow cytometry. Maternal IgG was transmitted to the fetus and rapidly opsonized donor cells following IUHCT. Donor cell rejection was observed as early as 4 h after IUHCT in B6-sensitized dams and 24 h after IUHCT in dams injected with B6-sensitized serum. Efficient opsonization was strongly correlated with decreased chimerism. No IUHCT recipients born to B6-sensitized dams or dams injected with B6-sensitized serum demonstrated macrochimeric engraftment at birth compared with 100% of IUHCT recipients born to naive dams or dams injected with naive serum (p < 0.001). In summary, maternal donor-specific IgG causes rapid, complete graft rejection in the fetus following IUHCT. When a third-party donor must be used for clinical IUHCT, the maternal serum should be screened for DSA to optimize the chance for successful engraftment.

Cessation of renal morphogenesis in mice.

The kidney develops by cycles of ureteric bud branching and nephron formation. The cycles begin and are sustained by reciprocal inductive interactions and feedback between ureteric bud tips and the surrounding mesenchyme. Understanding how the cycles end is important because it controls nephron number. During the period when nephrogenesis ends in mice, we examined the morphology, gene expression, and function of the domains that control branching and nephrogenesis. We found that the nephrogenic mesenchyme, which is required for continued branching, was gone by the third postnatal day. This was associated with an accelerated rate of new nephron formation in the absence of apoptosis. At the same time, the tips of the ureteric bud branches lost the typical appearance of an ampulla and lost Wnt11 expression, consistent with the absence of the capping mesenchyme. Surprisingly, expression of Wnt9b, a gene necessary for mesenchyme induction, continued. We then tested the postnatal day three bud branch tip and showed that it maintained its ability both to promote survival of metanephric mesenchyme and to induce nephrogenesis in culture. These results suggest that the sequence of events leading to disruption of the cycle of branching morphogenesis and nephrogenesis began with the loss of mesenchyme that resulted from its conversion into nephrons.

Laser capture-microarray analysis of Lim1 mutant kidney development.

The Lim1 gene has essential functions during several stages of kidney development. In particular, a tissue-specific knockout in the early metanephric mesenchyme results in the formation of the earliest nephron precursor, the renal vesicle, but failure of this structure to progress to the next stage, the comma-shaped body. To better understand the molecular nature of this developmental arrest, we used a laser capture microdissection-microarray strategy to examine the perturbed gene expression pattern of the mutant renal vesicles. Among the genes found differently expressed were Chrdl2, an inhibitor of BMP signaling, the proapoptotic factor Bmf, as well as myob5, an atypical myosin that modulates chemokine signaling, and pdgfrl, which is important in epithelial folding. Of particular interest, the microarray data indicated that the Dkk1 gene, which encodes an inhibitor of Wnt signaling, was downregulated ninefold in mutants. This was confirmed by in situ hybridizations. It is interesting to note that Lim1 and Dkk1 mutant mice have striking similarities in phenoytpe. These results suggest that the Dkk1 gene might be a key downstream effector of Lim1 function.

Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development.

BACKGROUND: The pygopus gene of Drosophila encodes an essential component of the Armadillo (beta-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions of Pygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs, Pygo1 and Pygo2. RESULTS: Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function. Pygo2 homozygous mutants, with rare exception, died shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate. Pygo1 homozygous mutants, however, were viable and fertile, with no detectable developmental defects. Double Pygo1/Pygo2 homozygous mutants showed no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression in Pygo1-/-/Pygo2-/- mutants, with tissue-specific variation in degree of diminution. The Pygo1 and Pygo2 genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud was altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there was an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeded normally. Microarray analysis showed changed expression of several genes, including Cxcl13, Slc5a2, Klk5, Ren2 and Timeless, which represent candidate Wnt targets in kidney development. CONCLUSION: The mammalian Pygopus genes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence of Pygopus function between mammals and Drosophila. In mammals, the Pygo1/Pygo2 genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity.

Comprehensive microarray analysis of Hoxa11/Hoxd11 mutant kidney development.

The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney.