Effect of Oral Chondroitin Sulfate Supplementation on Acute Brain Injury in a Murine Necrotizing Enterocolitis Model.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating condition where inflammatory changes and necrosis in the gut results in activation of brain microglia and subsequent neurodevelopmental impairment. Chondroitin sulfate (CS) is a glycosaminoglycan in human breast milk that is absent in conventional formulas. We hypothesized that oral formula supplementation with CS during a murine model of experimental NEC would not only attenuate intestinal injury, but also brain injury. STUDY DESIGN: NEC was induced in mouse pups on postnatal days (PNDs) 5 to 8. Three conditions were studied: (1) breastfed controls, (2) NEC, and (3) NEC+enteral CS (formula+200 mg/kg/d of CS). Pups were euthanized on PND 9 or reunited with dams by the evening of PND 8. Intestinal segments were H&E stained, and immunohistochemistry was performed on brain tissue for Iba-1 to assess for microglial morphology and cortical changes. Neurodevelopmental assays were performed on mice reunited with foster dams on PND 9. Single-cell RNA-sequencing analysis was performed on human intestinal epithelial cells exposed to (1) nothing, (2) hydrogen peroxide (H 2 O 2 ) alone, or (3) H 2 O 2 + CS to look at the differential gene expression between groups. Groups were compared with ANOVA or Kruskal-Wallis tests as appropriate with p < 0.05 considered significant. RESULTS: Compared with NEC, mice treated with oral CS showed improved clinical outcomes, decreased intestinal injury, and attenuated microglial activation and deleterious cortical change. Mice with CS performed better on early neurodevelopmental assays when compared with NEC alone. Single-cell analysis of HIEC-6 cells demonstrated that CS treatment down regulated several inflammatory pathways including nuclear factor κB-suggesting an explanation for the improved Th17 intestinal cytokine profile. CONCLUSIONS: Oral CS supplementation improved both physiological, clinical, and developmental outcomes. These data suggest that CS is a safe compound for formula supplementation for the prevention of NEC.

Hydrogen Sulfide Improves Outcomes in a Murine Model of Necrotizing Enterocolitis via the Cys440 Residue on Endothelial Nitric Oxide Synthase.

BACKGROUND: Hydrogen sulfide (H2S) has been shown to improve outcomes in a murine model of necrotizing enterocolitis (NEC). There is evidence in humans that H2S relies on endothelial nitric oxide synthase (eNOS) to exert its protective effects, potentially through the persulfidation of eNOS at the Cysteine 443 residue. We obtained a novel mouse strain with a mutation at this residue (eNOSC440G) and hypothesized that this locus would be critical for GYY4137 (an H2S donor) to exert its protective effects. METHODS: Necrotizing enterocolitis was induced in 5-day old wild type (WT) and eNOSC440G mice using intermittent exposure to hypoxia and hypothermia in addition to gavage formula feeds. On postnatal day 9, mice were humanely euthanized. Data collected included daily weights, clinical sickness scores, histologic lung injury, intestinal injury (macroscopically and histologically), and intestinal perfusion. During the NEC model, pups received daily intraperitoneal injections of either GYY4137 (50 mg/kg) or PBS (vehicle). Data were tested for normality and compared using t-test or Mann-Whitney, and a p-value <0.05 was considered significant. RESULTS: In WT mice, the administration of GYY4137 significantly improved clinical sickness scores, attenuated intestinal and lung injury, and improved mesenteric perfusion compared to vehicle (p < 0.05). In eNOSC440G mice, the treatment and vehicle groups had similar clinical sickness scores, intestinal and lung injury scores, and intestinal perfusion. CONCLUSIONS: GYY4137 administration improves clinical outcomes, attenuates intestinal and lung injury, and improves perfusion in a murine model of necrotizing enterocolitis. The beneficial effects of GYY4137 are dependent on the Cys440 residue of eNOS.

Chondroitin sulfate supplementation improves clinical outcomes in a murine model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) continues to be a devastating disease in preterm neonates and has a paucity of medical management options. Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) in human breast milk (HM) and has been shown to reduce inflammation. We hypothesized that supplementation with CS in an experimental NEC model would alter microbial diversity, favorably alter the cytokine profile, and (like other sulfur compounds) improve outcomes in experimental NEC via the eNOS pathway. NEC was induced in 5-day-old pups. Six groups were studied (n = 9-15/group): (1) WT breastfed and (2) Formula fed controls, (3) WT NEC, (4) WT NEC + CS, (5) eNOS KO (knockout) NEC, and (6) eNOS KO NEC + CS. Pups were monitored for clinical sickness score and weights. On postnatal day 9, the pups were killed. Stool was collected from rectum and microbiome analysis was done with 16 s rRNA sequencing. Intestinal segments were examined histologically using a well-established injury scoring system and segments were homogenized and analyzed for cytokine profile. Data were analyzed using GraphPad Prism with p < 0.05 considered significant. CS supplementation in formula improved experimental NEC outcomes when compared to NEC alone. CS supplementation resulted in similar improvement in NEC in both the WT and eNOS KO mice. CS supplementation did not result in microbial changes when compared to NEC alone. Our data suggest that although CS supplementation improved outcomes in NEC, this protection is not conferred via the eNOS pathway or alteration of microbial diversity. CS therapy in NEC does improve the intestinal cytokine profile and further experiments will explore the mechanistic role of CS in altering immune pathways in this disease.

Volatile Organic Compound Assessment as a Screening Tool for Early Detection of Gastrointestinal Diseases.

Gastrointestinal (GI) diseases have a high prevalence throughout the United States. Screening and diagnostic modalities are often expensive and invasive, and therefore, people do not utilize them effectively. Lack of proper screening and diagnostic assessment may lead to delays in diagnosis, more advanced disease at the time of diagnosis, and higher morbidity and mortality rates. Research on the intestinal microbiome has demonstrated that dysbiosis, or unfavorable alteration of organismal composition, precedes the onset of clinical symptoms for various GI diseases. GI disease diagnostic research has led to a shift towards non-invasive methods for GI screening, including chemical-detection tests that measure changes in volatile organic compounds (VOCs), which are the byproducts of bacterial metabolism that result in the distinct smell of stool. Many of these tools are expensive, immobile benchtop instruments that require highly trained individuals to interpret the results. These attributes make them difficult to implement in clinical settings. Alternatively, electronic noses (E-noses) are relatively cheaper, handheld devices that utilize multi-sensor arrays and pattern recognition technology to analyze VOCs. The purpose of this review is to (1) highlight how dysbiosis impacts intestinal diseases and how VOC metabolites can be utilized to detect alterations in the microbiome, (2) summarize the available VOC analytical platforms that can be used to detect aberrancies in intestinal health, (3) define the current technological advancements and limitations of E-nose technology, and finally, (4) review the literature surrounding several intestinal diseases in which headspace VOCs can be used to detect or predict disease.

Stem cells as a therapeutic avenue for active and long-term complications of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal disease associated with significant morbidity and mortality. Although decades of research have been dedicated to understanding the pathogenesis of NEC and developing therapies, it remains the leading cause of death among neonatal gastrointestinal diseases. Mesenchymal stem cells (MSCs) have garnered significant interest recently as potential therapeutic agents for the treatment of NEC. They have been shown to rescue intestinal injury and reduce the incidence and severity of NEC in various preclinical animal studies. MSCs and MSC-derived organoids and tissue engineered small intestine (TESI) have shown potential for the treatment of long-term sequela of NEC such as short bowel syndrome, neurodevelopmental delay, and chronic lung disease. Although the advances made in the use of MSCs are promising, further research is needed prior to the widespread use of these cells for the treatment of NEC.

Gut-Brain cross talk: The pathogenesis of neurodevelopmental impairment in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating condition of multi-factorial origin that affects the intestine of premature infants and results in high morbidity and mortality. Infants that survive contend with several long-term sequelae including neurodevelopmental impairment (NDI)-which encompasses cognitive and psychosocial deficits as well as motor, vision, and hearing impairment. Alterations in the gut-brain axis (GBA) homeostasis have been implicated in the pathogenesis of NEC and the development of NDI. The crosstalk along the GBA suggests that microbial dysbiosis and subsequent bowel injury can initiate systemic inflammation which is followed by pathogenic signaling cascades with multiple pathways that ultimately lead to the brain. These signals reach the brain and activate an inflammatory cascade in the brain resulting in white matter injury, impaired myelination, delayed head growth, and eventual downstream NDI. The purpose of this review is to summarize the NDI seen in NEC, discuss what is known about the GBA, explore the relationship between the GBA and perinatal brain injury in the setting of NEC, and finally, highlight the existing research into possible therapies to help prevent these deleterious outcomes.

Stem cell derived therapies to preserve and repair the developing intestine.

Stem cell research and the use of stem cells in therapy have seen tremendous growth in the last two decades. Neonatal intestinal disorders such as necrotizing enterocolitis, Hirschsprung disease, and gastroschisis have high morbidity and mortality and limited treatment options with varying success rates. Stem cells have been used in several pre-clinical studies to address various neonatal disorders with promising results. Stem cell and patient population selection, timing of therapy, as well as safety and quality control are some of the challenges that must be addressed prior to the widespread clinical application of stem cells. Further research and technological advances such as the use of cell delivery technology can address these challenges and allow for continued progress towards clinical translation.

Age disparities in intestinal stem cell quantities: a possible explanation for preterm infant susceptibility to necrotizing enterocolitis.

PURPOSE: Preterm infants are more susceptible to necrotizing enterocolitis (NEC) than term Queryinfants. This may be due to a relative paucity of Lgr5+ or Bmi1+-expressing intestinal stem cells (ISCs) which are responsible for promoting intestinal recovery after injury. We hypothesized that the cellular markers of Lgr5+ and Bmi1+, which represent the two distinct ISC populations, would be lower in younger mice compared to older mice. In addition, we hypothesized that experimental NEC would result in a greater loss of Lgr5+ expression compared to Bmi1+ expression. METHODS: Transgenic mice with EGFP-labeled Lgr5 underwent euthanasia at 10 different time points from E15 to P56 (n = 8-11/group). Lgr5+-expressing ISCs were quantified by GFP ELISA and Bmi1+ was assessed by qPCR. In addition, Lgr5EGFP mice underwent experimental NEC via formula feeding and hypoxic and hypothermic stress. Additional portions of the intestine underwent immunostaining with anti-GFP or anti-Bmi1+ antibodies to confirm ELISA and PCR results. For statistical analysis, p < 0.05 was significant. RESULTS: Lgr5+ and Bmi1+expression was lowest in embryonal and early postnatal mice and increased with age in all segments of the intestine. Experimental NEC was associated with loss of Lgr5+-expressing ISCs but no significant change in Bmi1+ expression. CONCLUSION: Lgr5+ and Bmi1+ expression increase with age. Lgr5+-expressing ISCs are lower following experimental necrotizing enterocolitis while Bmi1+ expression remains relatively unchanged. Developing a targeted medical therapy to protect the low population of ISCs in preterm infants may promote tissue recovery and regeneration after injury from NEC.

Recent Development of the Molecular and Cellular Mechanisms of Hydrogen Sulfide Gasotransmitter.

Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H2S in mammals. H2S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H2S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H2S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H2S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H2S is involved in many important cellular and physiological processes. Compounds that donate H2S to biological systems can be developed as therapeutics for different diseases.

Sildenafil attenuates intestinal injury in necrotizing enterocolitis independently of endothelial nitric oxide synthase.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease that impacts the intestine of premature infants. Sildenafil has shown benefit in colitis and ischemia/reperfusion models but has not been adequately studied in NEC. Sildenafil's best studied mechanism involves augmenting nitric oxide induced vasodilation. We hypothesized that sildenafil would improve outcomes during experimental NEC in an eNOS dependent manner. MATERIALS: NEC was induced in five-day old mouse pups with gavage formula feeds plus intermittent hypoxia and hypothermia. Using wild type (WT) mice, the route of sildenafil administration was studied in the following groups: (1) breastfed controls, (2) NEC + oral (PO) sildenafil, (3) NEC + PO vehicle, (4) NEC + intraperitoneal (IP) sildenafil, (5) NEC + IP vehicle. The eNOS KO groups studied included: (1) breastfed controls, (2) NEC + PO sildenafil, (3) NEC + PO vehicle. Data were tested for normality and compared using t-tests or Mann-Whitney with a p-value <0.05 considered significant. RESULTS: In WT mice, oral and IP sildenafil resulted in improved clinical outcomes compared to their respective vehicle group. Only orally administered sildenafil significantly improved perfusion to the intestine and protected it from macroscopic and histologic injury. When repeated in eNOS KO mice, oral sildenafil improved clinical scores and attenuated intestinal injury scores, despite no effect on intestinal perfusion. CONCLUSIONS: Sildenafil, when administered orally, improves clinical outcomes and protects the intestine in a murine model of experimental necrotizing enterocolitis. While sildenafil requires eNOS to impact mesenteric perfusion, it does not appear to be dependent on eNOS to attenuate intestinal injury.