Association between 4%-tetrasodium EDTA and sepsis in neonatal piglets: A retrospective cohort study.

BACKGROUND: Central line-associated bloodstream infections are a major concern for children with intestinal failure and in animal research using parenteral nutrition (PN). In neonatal piglets receiving PN, we compared sepsis, line occlusions, line replacements, mortality, and costs with and without the use of a 4%-tetrasodium ethylenediaminetetraacetic acid (T-EDTA) locking solution. METHODS: We performed a retrospective review of piglets with a central venous jugular catheter enrolled in 14-day exclusive PN (TPN) trials or in 7-day short bowel syndrome (SBS) trials, before and after initiation of T-EDTA. Lines were locked with a 1-ml solution for 2 h daily (T-EDTATPN, n = 17; T-EDTASBS, n = 48) and compared with our prior standard of care using 1.5-ml heparin flushes twice daily (CONTPN, n = 34; CONSBS, n = 48). Line patency and signs of sepsis were checked twice daily. Jugular catheters were replaced for occlusions whenever possible. Humane end points were used for sepsis not responding to antibiotic treatment or unresolved catheter occlusions. RESULTS: Compared with CON, sepsis was reduced using T-EDTA, significantly for TPN (P = 0.006) and with a trend for SBS piglets (P = 0.059). Line occlusions necessitating line changes were reduced 15% in TPN studies (P = 0.16), and no line occlusions occurred for T-EDTA SBS piglets. CONCLUSION: In our neonatal piglet research, use of T-EDTA locking solution decreased sepsis and, although not statistically significant, reduced occlusions requiring line replacements. Given the expense of animal research, adding a locking solution must be cost-effective, and we were able to show that T-EDTA significantly reduced total research costs and improved animal welfare.

In parenteral nutrition-fed piglets, fatty acids vary by lipid emulsion and tissue sampled.

BACKGROUND: Children with intestinal failure without liver disease may be given soy-based lipid emulsion (SLE) or mixed lipid emulsion (MLE; containing soy, medium-chain triglyceride, olive, and/or fish oils). Both differ in essential fatty acid content: MLE has added arachidonic acid (AA) and docosahexaenoic acid (DHA). The aim of this study, in neonatal piglets, was to compare serum and tissue fatty acid composition when the emulsions were given at unrestricted doses. METHODS: We compared SLE (n = 15) and MLE (n = 15) at doses of 10-15 g/kg/day in parenteral nutrition (PN). On day 14 we collected serum and tissues. Using gas-liquid chromatography, percentage fatty acids were measured in serum, brain, and liver phospholipid. Comparisons were made to reference values from litter-matched controls (n = 8). RESULTS: Comparing median values, linoleic acid (LA) was lower for MLE vs SLE in serum (-27%), liver (-45%), and brain (-33%) (P < 0.001). AA was lower for MLE in serum (-25%), liver (-40%), and brain (-10%). DHA was higher for MLE in serum (+50%), liver (+200%), and brain (+10%). AA levels were lower for MLE vs control piglets in serum (-81%), liver (-63%), and brain (-9%). DHA levels were higher in serum (+41%), liver (+38%), and brain (+19%). CONCLUSION: This study in piglets has shown that, at unrestricted doses, MLE treatment is associated with low serum and tissue AA compared with SLE and healthy litter-matched controls. Although not yet proven, low tissue AA levels may have functional consequences, and these data support current practice avoiding MLE dose restriction.

Probiotic treatment vs empiric oral antibiotics for managing dysbiosis in short bowel syndrome: Impact on the mucosal and stool microbiota, short-chain fatty acids, and adaptation.

BACKGROUND: Infants and children with short bowel syndrome (SBS) are presumed to be at risk of gut microbial dysbiosis with potential sequelae of bacterial overgrowth that include sepsis, d-lactic acidosis, mucosal inflammation, and malabsorption. In neonatal piglets with SBS, we compared intestinal microbial composition, short-chain fatty acids (SCFAs), and adaptation given probiotic (PRO) treatment (Lactobacillus and Bifidobacterium spp) vs oral metronidazole (MET). METHODS: Following 75% distal small intestinal resection, piglets were allocated to PRO (500 mg twice a day, n = 7), MET (15 mg/kg twice a day, n = 8), and placebo (PLA) (500 mg twice a day, n = 8). After 10 days of parenteral and enteral nutrition, 16S ribosomal RNA gene amplicon sequencing (colon tissue and stool) was undertaken and SCFA analysis (stool and colon effluent) was performed using gas chromatography. RESULTS: In colon, Shannon diversity was higher for PRO compared with MET and PLA (P = 0.002). PRO and PLA increased abundance of Bacteroidetes species (eg, Bacteroides fragilis) compared with MET (P < 0.001). PRO, compared with PLA, increased abundance of Firmicutes species (eg, Lactobacillus fermentum) (P < 0.001). MET increased abundance of Proteobacteria members, predominately Enterobacteriaceae, compared with PRO (P = 0.004). In stool, microbial findings were similar and SCFA (butyrate) concentrations were highest for PRO (P = 0.003) compared with MET. CONCLUSION: In pediatric SBS, the empiric use of oral antibiotics, such as MET, is common for presumed clinical consequences of microbial dysbiosis. In this study of SBS piglets, that approach was associated with decreased microbial diversity and increased abundance of potentially inflammatory Proteobacteria. In contrast, a PRO treatment using Lactobacillus and Bifidobacterium spp increased both diversity and SCFAs.

The effect of fecal microbial transplant on intestinal microbial composition in short-bowel neonatal piglets.

BACKGROUND: Short-bowel syndrome (SBS) in neonates is associated with microbial dysbiosis due to intestinal surgery, prolonged hospitalization, enteral nutrition, and repeated antibiotic exposure. Sepsis and liver disease, leading causes of morbidity and mortality in SBS, may relate to such intestinal dysbiosis. We investigated the safety and feasibility of fecal microbial transplant (FMT) to alter intestinal microbial composition in SBS piglets. METHODS: Following a 75% distal small-intestinal resection, piglets were fed parenteral nutrition with an elemental diet and randomized to saline (SAL; n = 12) or FMT (n = 12) treatments delivered by gastric tube on day 2 (d2). The FMT donor was a healthy adult pig. Comparisons were also made to healthy sow-fed littermate controls (SOW; n = 6). Stool samples were collected daily, and tissue samples were collected at baseline and termination. Microbial DNA was extracted from stool and analyzed using 16S ribosomal RNA sequencing. RESULTS: All piglets survived to the end point. On d2-d4, FMT piglets had some differences in microbiota composition compared with SAL, SOW, and donor counterparts. Between base and term, there were transitory changes to alpha and beta diversity in FMT and SAL. CONCLUSION: FMT treatment in postsurgical neonatal piglets with SBS appears safe, with no increase in sepsis and no mortality. In SBS piglets, FMT induced transient changes to the intestinal microbiota. However, these changes did not persist long-term.

Comparing the Intestinotrophic Effects of 2 Glucagon-Like Peptide-2 Analogues in the Treatment of Short-Bowel Syndrome in Neonatal Piglets.

BACKGROUND: In treating short-bowel syndrome (SBS), autonomy from parenteral nutrition (PN) relies upon intestinal adaptation, which can be augmented by glucagon-like peptide-2 (GLP-2) analogues. In neonatal piglets with SBS, we compared intestinal adaptation following treatment with 2 GLP-2 analogues: teduglutide (TED) and apraglutide (APRA) METHODS: Following 75% distal small-intestinal resection, piglets were allocated to 4 treatment groups: saline (CON: n = 8), twice weekly APRA (5 mg/kg/dose; n = 8), and TED once daily (TED, 0.05 mg/kg/dose; n = 8) or twice daily (TEDBID, 0.05 mg/kg/dose; n = 7). Pharmacokinetic (PK) studies were undertaken, and on day 7, small-intestinal length and weight were measured and jejunal tissue collected for histology. RESULTS: PK profiles were different between the 2 analogues. To achieve a comparable exposure to APRA, TED requires twice daily injection (TEDBID). Compared with CON, APRA and TEDBID increased small-bowel length (cm) (CON: 141, APRA: 166, TED: 153, TEDBID: 165; P = .004), whereas APRA increased small-bowel weight (g) (CON: 26, APRA: 33, TED: 28, TEDBID: 31; P = .007) and villus height (mm) (CON: 0.59, APRA: 0.90, TED: 0.58, TEDBID: 0.74; P < .001). CONCLUSION: APRA injected only twice during the 7 consecutive days demonstrated a superior intestinotrophic effect compared with TED injected once daily. Even at more comparable drug exposure, when TED was injected twice a day, APRA showed superior trophic activity at the mucosal level. This is highly relevant for the treatment of pediatric SBS, given the markedly lower dose frequency by subcutaneous injection of APRA.

Intestinal resection affects whole-body arginine synthesis in neonatal piglets.

BACKGROUND: Previous studies in piglets show a direct relationship between intestinal mass and arginine (Arg) synthesis. We aimed to study the effects of 75% intestinal resection on whole-body Arg synthesis. METHODS: Piglets were allocated to sham or jejunocolic (JC) surgery and to enteral nutrition (EN) at 20% [sham (n = 8), JC (n = 10)], or 40% [sham (n = 4), JC (n = 5)]. A gastric tube was placed for EN and a venous catheter for parenteral nutrition and blood sampling. On day 6, a primed bolus and constant infusion of Arg m + 2 label and proline m + 1 label was delivered. In addition, 40% EN piglets received a citrulline (Cit) m + 3 tracer. Blood sampling was undertaken and whole-body Arg synthesis was calculated. On day 7, intestinal length was measured, and samples were collected for gene expression (PCR quantification) and histopathology. RESULTS: On Day 7, sham piglets showed intestinal lengthening compared to JC (p = 0.02). Whole-body Arg synthesis was similar between groups (p = 0.50). Adjusting for absolute small intestinal length, JC piglets had greater Arg synthesis (p = 0.01). Expression of arginosuccinase was upregulated in the jejunum of JC compared to sham on 20% EN (p = 0.03). CONCLUSION: This demonstrates for the first-time adaptive changes in intestinal Arg synthesis following intestinal resection. IMPACT: The intestine makes a critical contribution to whole-body arginine synthesis, particularly in neonates, a human population at risk for short bowel syndrome. Therefore, we studied intestinal arginine synthesis in a neonatal piglet model of short bowel syndrome and demonstrated adaptive changes in the intestine that may preserve whole-body arginine synthesis, despite loss of intestinal mass. This research adds new information to our understanding of the effects a massive intestinal resection has on amino acid metabolism during neonatal development.

Durability of Linear Small-Intestinal Growth Following Treatment Discontinuation of Long-Acting Glucagon-Like Peptide 2 (GLP-2) Analogues.

BACKGROUND: Short-bowel syndrome is the leading cause of pediatric intestinal failure, resulting in dependency on long-term parenteral nutrition (PN). To promote enteral autonomy in neonates, a key outcome may be intestinal growth in length. The purpose of this study was to determine if intestinal lengthening persists following discontinuation of treatment with 1 of 2 GLP-2 analogues with different pharmacokinetic profiles. METHODS: Neonatal short-bowel piglets were assigned to saline control (S), 7-day treatment with teduglutide (T) (0.05 mg/kg twice daily), or 7-day treatment with apraglutide (A) (5 mg/kg twice weekly). Comparisons were made between day 7 and day 14 endpoints using analysis of variance. Data included small-intestine length, weight, histology, and quantitative polymerase chain reaction analysis of mucosal transcripts for peptide growth factors and their receptors, nutrient transporters, and tight-junction proteins. RESULTS: Compared with control, 7 days of GLP-2 analogue treatment induced mucosal adaptation based on villus hyperplasia (P = .003), which was not durable 7 days after treatment cessation (day 14; P = .081). Treatment increased intestinal growth in length by day 7 (P = .005), which was maintained (by T) or further increased (by A) at day 14 (P < .001). No significant differences in mucosal transcripts were detected. CONCLUSION: Unlike mucosal adaptation, intestinal growth appears to be a lasting outcome of treatment with long-acting GLP-2 analogues in a neonatal piglet short-bowel model. This has significant clinical implications for neonates, given their potential for intestinal growth. Intestinal lengthening varies between analogues with different half-lives; however, molecular mechanisms require further elucidation.

In the Short-term, Milk Fat Globule Epidermal Growth Factor-8 Causes Site-specific Intestinal Growth in Resected Piglets.

OBJECTIVES: Short bowel syndrome (SBS) remains the leading cause of neonatal intestinal failure. Milk fat globule epidermal growth factor-8 (MFG-E8), present in human milk, has homology with epidermal growth factor (EGF), known to enhance adaptation in SBS. In this pilot study, the role of oral MFG-E8 treatment in SBS was explored in neonatal piglets. METHODS: Neonatal piglets underwent 75% intestinal resection, either distal (jejunal-colonic [JC] anastomosis) or mid-intestinal (jejunal-ileal [JI] anastomosis). Piglets were randomized to intragastric treatment with MFG-E8  (5 mg/kg per day) or saline and were maintained on parenteral nutrition and enteral nutrition for 7 days. Adaptation was assessed by intestinal length and weight, histopathology, fecal fat analysis and RT-qPCR analysis of mucosal transcripts, including growth factors. RESULTS: JI piglets demonstrated intestinal lengthening (P < 0.001), 2-fold greater in ileum than jejunum (P = 0.02), where lengthening was increased by MFG-E8 treatment (P = 0.02). JC piglets did not exhibit jejunal lengthening, regardless of treatment. Fat absorption was greater for JI piglets (P = 0.02), unaffected by treatment. In JI piglets, expression of Egf was increased in the ileum (P < 0.01) and MFG-E8 treatment increased Egfr (receptor) expression (P = 0.02). CONCLUSIONS: MF-EG8 demonstrated site-specific trophic effects, only with JI anatomy. This may limit the utility of this treatment for SBS, except for rare patients with retained ileum. The mechanisms of these site-specific effects, however, and the role of MFG-E8 in neonatal gut growth and in diseases, such as necrotizing enterocolitis that commonly target ileum, warrant further exploration.

Plasma citrulline is not a biomarker for intestinal adaptation in short bowel syndrome, studied in piglets: a model for human neonates.

BACKGROUND: There are no in vivo methods to measure adaptation in neonatal short bowel syndrome (SBS). We evaluated citrulline (Cit) levels in neonatal piglet surgical models of SBS. METHODS: Piglets underwent 75% mid-intestinal resection with jejunoileal anastomosis (JI), 75% distal resection of ileum with jejunocolic anastomosis (JC) or sham surgery. Jugular and gastric catheters were inserted for parenteral and enteral nutrition. On D7, small intestine length and weight were measured, jejunum collected for histopathology and Cit level determined. RESULTS: JI (n = 5) compared to JC (n = 5) had increased small intestinal length (JC - 17.5 cm; JI +22.0 cm; p = 0.02) and mass (JC 43.1 mg/cm/kg; JI 51.3 mg/cm/kg; p = 0.02), while Cit did not differ (JI 801.0 µM; JC 677.7 µM; p = 0.90). Including non-resected shams (n = 4), Cit correlated with length (R2 = 0.48; p = 0.006), but not for SBS alone (R2 = 0.11; p = 0.4), mass (R2 = 0.05; p = 0.5). A second experiment compared change in Cit levels from baseline to D7. Levels declined in sham (n = 8) and JC (n = 10) (sham - 110.1 µM; JC - 56.6 µM; p = 0.17), regardless of intestinal lengthening (sham 29.9 cm; JC - 10.4 cm; p = 0.002). CONCLUSION: Citrulline levels predict large differences in intestinal length and 'identify' SBS. However, citrulline cannot discriminate between adaptation in JI and JC, nor predict intestinal lengthening.

Mixed Lipid, Fish Oil, and Soybean Oil Parenteral Lipids Impact Cholestasis, Hepatic Phytosterol, and Lipid Composition.

OBJECTIVES: In parenteral nutrition-dependent infants and children, intestinal failure (IF)-associated liver disease (IFALD) remains an important problem. A comparative study was undertaken of parenteral mixed lipid (ML), ω-3 predominant fish oil (FO), and ω-6 predominant soybean oil (SO) emulsions in regards to hepatic phytosterol, neutral lipid, fatty acid (FA) content, and the relationship to cholestasis in piglets. METHODS: Neonatal piglets received parenteral nutrition, varying in lipid dose (5 or 10 g·â€Škg ·â€Šday) and formulation: SO5 (n = 5), SO10 (n = 5), FO5 (n = 5), and ML10 (n = 5). On day 14, liver chemistry, bile flow, histology and neutral lipid staining were assessed. Hepatic triglyceride FA content was determined using thin layer and gas chromatography, and phytosterol content was assessed using gas chromatography-mass spectrometry. RESULTS: SO groups had higher prevalence of biochemical cholestasis (P < 0.04) and lower bile flow (P < 0.0001). Hepatic campesterol, stigmasterol, and ß-sitosterol were highest in SO10 (P < 0.0001). Hepatic FA (P < 0.03) and ω-6/ω-3 FA ratio (P < 0.0001) were higher in the SO groups. Neutral lipid accumulation (P = 0.3) and liver histology (P = 0.16) were not different between groups. Univariate predictors of bile flow were: campesterol (r = -0.77, P = 0.001), ß-sitosterol (r = -0.74, P = 0.002), stigmasterol (r = -0.74, P = 0.002), ω-6 FA (r = -0.72, P = 0.002), and ω-3 FA (r = 0.59, P = 0.02). Only campesterol independently predicted bile flow. CONCLUSIONS: ML and FO lipid emulsions reduce cholestasis in association with lowered hepatic phytosterol and lipid content. Lower hepatic phytosterol and ω-6 FA content, and higher ω-3 FA content are hepatoprotective. Multivariate analysis suggests reduced phytosterol accumulation may best explain the hepatoprotective effect of fish oil-containing lipids.

Novel Long-Acting GLP-2 Analogue, FE 203799 (Apraglutide), Enhances Adaptation and Linear Intestinal Growth in a Neonatal Piglet Model of Short Bowel Syndrome with Total Resection of the Ileum.

BACKGROUND: Glucagon-like peptide-2 (GLP-2) is an intestinotrophic factor released from L-cells in the ileum, a segment commonly resected or atretic in neonatal short bowel syndrome (SBS). In piglets, ileal resection decreases intestinal adaptation and endogenous GLP-2 production, whereas exogenous replacement promotes adaptation. In this study, we determined the effect of a novel long-acting GLP-2 analogue, FE 203799 (FE; apraglutide), upon intestinal growth, adaptation, and function in neonatal SBS piglets without ileum. METHODS: Neonatal piglets were randomized to saline (n = 10) vs FE treatment (n = 8). All piglets underwent 75% intestinal resection with jejunocolic anastomosis and were pair-fed parenteral and enteral nutrition. Saline and FE (5 mg/kg) treatments were administered subcutaneously on days 0 and 4. On day 6, 24-hour fecal samples were collected for subsequent nutrient analysis. On day 7, small-intestinal length and weight were measured and tissue collected for analyses. RESULTS: On day 7, saline and FE-treated piglets were healthy and gained equivalent weight (P = 0.12). Compared with saline piglets, FE-treated piglets had lower fecal fat (P = 0.043) and energy (P = 0.043) losses and exhibited intestinal lengthening (P = 0.001), greater small-intestinal weight (P = 0.004), longer villus height (P = 0.027), and greater crypt depth (P = 0.054). CONCLUSIONS: The subcutaneous GLP-2 analogue, FE, enhanced intestinal adaptation in a neonatal model of SBS without ileum. The observed intestinal lengthening with FE treatment was unique compared with our prior experience with native GLP-2 in this same model and has important clinical implications for treating neonatal SBS. At this developmental stage, growth in the intestine, if augmented, could accelerate weaning from parenteral nutrition.

Impact of Clinical Use of Parenteral Lipid Emulsions on Bile Acid Metabolism and Composition in Neonatal Piglets.

BACKGROUND: Neonates with intestinal failure dependent on parenteral nutrition (PN) are at risk of intestinal failure-associated liver disease (IFALD). PN lipid composition relates to the risk of IFALD, but the mechanisms are poorly understood. We investigated the effects of soybean oil (SO), a mixed-lipid (ML) emulsion containing fish oil (FO), and a pure FO. We hypothesized FO-containing PN lipids would result in increased gene expression of canalicular bile acid transporters and a larger, more hydrophilic bile acid pool, predictive of increased bile flow. METHODS: Neonatal piglets were allocated to receive 1 of SO, ML, or FO throughout 14 days of PN feeding. Relative expression of genes involved in bile acid synthesis and transport were determined through quantitative polymerase chain reaction. Bile secreted from the liver was collected and measured. Bile acid composition was determined using tandem mass spectrometry. Regression analysis was used to determine predictors of bile flow. RESULTS: PN reduced bile acid secretion (P < .001). FO-containing PN lipids were associated with greater expression of bile acid and organic solute transport genes (P < .05) and greater secretion of hydrophobic bile acids (P < .001). Farnesoid X receptor (P = .01), bile salt export pump (P < .01), multidrug resistant protein 2 (P < .01), and unconjugated hyocholic acid (P < .001) independently predicted bile flow. CONCLUSIONS: PN lipid modulation altered bile acid metabolism and composition. These alterations may explain the hepatoprotective effects of FO-containing PN lipids and support their use in the prevention and treatment of IFALD.

Parenteral Lipid Dose Restriction With Soy Oil, Not Fish Oil, Preserves Retinal Function in Neonatal Piglets.

BACKGROUND: A dietary supply of docosahexaenoic acid (DHA) and arachidonic acid (AA) is critical for neonatal retinal development. Both are absent/minimal in parenteral nutrition (PN) using soy-oil emulsions ([SO] Intralipid®) traditionally used for neonatal intestinal failure. In contrast, fish-oil emulsions ([FO] Omegaven®) are enriched in DHA/AA. The aim of this study was to compare retinal function and fatty acid content in neonatal piglets fed PN with SO or FO. METHODS: Two-5-day-old piglets were randomly allocated to SO (n = 4) or FO (n = 4), provided at equivalent doses (5g/kg/d). After 14 days of PN, retinal function was assessed by electroretinography and retinas were harvested for fatty acid content analysis. Sow-fed piglets served as a reference (REF). RESULTS: Light flash-elicited stoppage of cone and rod dark-currents (a-waves) and the ensuing postsynaptic activation of cone and rod ON bipolar cells (b-waves) were comparable between SO and REF. Responses recorded from FO were subnormal (P <0.001) when compared with both SO and REF. Retinal DHA content was similar in both groups (FO, 14.59% vs SO, 12.22%; P = 0.32); while AA was lower in FO (FO, 6.01% vs SO, 8.21%; P = .001). CONCLUSION: Paradoxically, FO containing more DHA and AA did not preserve retinal function when compared with the same low dose of SO. This may be due to the reduced AA enrichment in the retina with FO treatment. Further investigation into the ideal amounts of DHA and AA for optimal neonatal retinal function is required.

Surgical Anatomy Does Not Affect the Progression of Intestinal Failure-Associated Liver Disease in Neonatal Piglets.

BACKGROUND: Intestinal failure-associated liver disease (IFALD) causes significant morbidity in neonates with short bowel syndrome (SBS) dependent on parenteral nutrition (PN). Resected ileum, with loss of the ileocecal valve (ICV), is the most common anatomy in SBS, yet its impact on IFALD has not been adequately studied. METHODS: Neonatal piglets were randomized to 75% intestinal resection with jejunocolic anastomosis (JC, n = 12), 75% resection with jejunoileal anastomosis and intact ICV (JI, n = 13), PN-fed sham (sham, n = 14), or sow-fed control (SF, n = 8). Surgical and sham piglets received 100% PN for 14 days before bile flow was measured and blood chemistry, liver pathology, jejunal permeability, and bacterial translocation were assessed. RESULTS: Bile flow was lower for PN-fed compared with SF (P = .002) but not different between the PN-fed groups. Total bilirubin (P = .03) and liver pathology (P < .001) were greater in PN-fed than SF groups but not different between PN-fed groups. Serum bile acids were increased in sham (P = .01) but not different between SBS groups. PN-fed piglets with sepsis had lower bile flow (P = .001) and increased bilirubin (P = .04). Neither jejunal permeability nor bacterial translocation were different between JC, JI, or sham groups. CONCLUSION: Contrary to our hypothesis, the remnant anatomy does not appear to worsen the progression of IFALD. However, the role of sepsis in IFALD should be further explored, in addition to other mechanisms, including PN factors, host immune responses, and intestinal bacterial dysbiosis.

Synergy of glucagon-like peptide-2 and epidermal growth factor coadministration on intestinal adaptation in neonatal piglets with short bowel syndrome.

Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) treatment enhance intestinal adaptation. To determine whether these growth factors exert synergistic effects on intestinal growth and function, GLP-2 and EGF-containing media (EGF-cm) were administered, alone and in combination, in neonatal piglet models of short bowel syndrome (SBS). Neonatal Landrace-Large White piglets were block randomized to 75% midintestinal [jejunoileal (JI) group] or distal intestinal [jejunocolic (JC) group] resection or sham control, with 7-day infusion of saline (control), intravenous human GLP-2 (11 nmol·kg-1·day-1) alone, enteral EGF-cm (80 µg·kg-1·day-1) alone, or GLP-2 and EGF-cm in combination. Adaptation was assessed by intestinal length, histopathology, Üssing chamber analysis, and real-time quantitative PCR of intestinal growth factors. Combined EGF-cm and GLP-2 treatment increased intestinal length in all three surgical models (P < 0.01). EGF-cm alone selectively increased bowel weight per length and jejunal villus height in the JI group only. The JC group demonstrated increased intestinal weight and villus height (P < 0.01) when given either GLP-2 alone or in combination with EGF-cm, with no effect of EGF-cm alone. Jejunal permeability of mannitol and polyethylene glycol decreased with combination therapy in both SBS groups (P < 0.05). No difference was observed in fat absorption or body weight gain. IGF-1 mRNA was differentially expressed in JI vs. JC piglets with treatment. Combined treatment with GLP-2 and EGF-cm induced intestinal lengthening and decreased permeability, in addition to the trophic effects of GLP-2 alone. Our findings demonstrate the benefits of novel combination GLP-2 and EGF treatment for neonatal SBS, especially in the JC model representing most human infants with SBS.NEW & NOTEWORTHY Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) are intestinotrophic, with demonstrated benefit in both animal models and human studies of short bowel syndrome (SBS). The current research shows that over and above known trophic effects, the combination of GLP-2 and EGF synergistically lengthens the bowel in neonatal piglet models of SBS. Most notable benefit occurred with resection of the terminal ileum, the common clinical anatomy seen in neonatal SBS and associated with least de novo lengthening postsurgery.

Supplemental Parenteral Vitamin E Into Conventional Soybean Lipid Emulsion Does Not Prevent Parenteral Nutrition-Associated Liver Disease in Full-Term Neonatal Piglets.

BACKGROUND: Parenteral nutrition-associated liver disease (PNALD) continues to cause morbidity and mortality for neonates with intestinal failure. Lipid peroxidation is one potential etiological factor. This study was designed to test if supplementing vitamin E into conventional soy-based lipid would reduce the risk of PNALD. METHODS: Sixteen piglets, aged 2-5 days and weighing 1.8-2.5 kg, were randomized to parenteral nutrition (PN) with soy lipid (SO, n = 8) or the same lipid plus α-tocopherol, the most bioactive form of vitamin E (SO+E, n = 8). After 17 days, bile flow, liver chemistry, gene expression associated with bile acid metabolism, and bile acid composition were assessed. C-reactive protein (CRP) and oxidative stress markers, including plasma 8-isoprostane, were measured. All results were compared with a sow-reared control group (CON). RESULTS: Comparing PN-treated groups, SO vs SO+E mean bile flow (5.91 vs 5.54 µL/g liver; P = .83), serum bile acid concentration (39.2 vs 26.6 µmol/L; P = .12), and total bilirubin (35.2 vs 26.9 µmol/L; P = .56) were not different. Gene expression related to bile acid metabolism and bile composition was not different between PN groups. There was no difference in CRP (41.8 vs 36.8 µg/mL; P = .22) or in plasma 8-isoprostane (27.9 vs 26.1 pg/mL; P = .77). CONCLUSIONS: In term neonatal piglets, supplemental vitamin E did not prevent cholestasis. Additional vitamin E was not associated with reduced inflammation or oxidative stress. The benefit of supplementing vitamin E into conventional lipid, vs adding fish oil, to prevent early onset of PNALD requires further clarification.

Lipid Emulsion Formulation of Parenteral Nutrition Affects Intestinal Microbiota and Host Responses in Neonatal Piglets.

BACKGROUND: Total parenteral nutrition (TPN) is a cause of intestinal microbial dysbiosis and impaired gut barrier function. This may contribute to life-threatening parenteral nutrition-associated liver disease and sepsis in infants. We compared the effects of a lipid emulsion containing long-chain ω-3 polyunsaturated fatty acids (PUFAs; SMOFlipid) and a predominantly ω-6 PUFA emulsion (Intralipid) on microbial composition and host response at the mucosal surface. MATERIALS AND METHODS: Neonatal piglets were provided isocaloric, isonitrogenous TPN for 14 days versus sow-fed (SF) controls. Equivalent lipid doses (10 g/kg/d) were given of either SMOFlipid (ML; n = 10) or Intralipid (SO; n = 9). Ileal segments and mucosal scrapings were used to characterize microbial composition by 16S rRNA gene sequencing and quantitative gene expression of tight junction proteins, mucins, antimicrobial peptides, and inflammatory cytokines. RESULTS: The microbial composition of TPN piglets differed from SF, while ML and SO differed from each other (analysis of molecular variance; P < .05); ML piglets were more similar to SF, as indicated by UniFrac distance ( P < .05). SO piglets showed a specific and dramatic increase in Parabacteroides ( P < .05), while ML showed an increase in Enterobacteriaceae ( P < .05). Gene expression of mucin, claudin 1, ß-defensin 2, and interleukin 8 were higher in TPN; overall increases were significantly less in ML versus SO ( P < .05). CONCLUSION: The formulation of parenteral lipid is associated with differences in the gut microbiota and host response of TPN-fed neonatal piglets. Inclusion of ω-3 long-chain PUFAs appears to improve host-microbial interactions at the mucosal surface, although mechanisms are yet to be defined.

In a Neonatal Piglet Model of Intestinal Failure, Administration of Antibiotics and Lack of Enteral Nutrition Have a Greater Impact on Intestinal Microflora Than Surgical Resection Alone.

BACKGROUND: Data are limited on how short bowel syndrome (SBS) affects the healthy developing intestinal microbiome, with even less assessing different SBS anatomical surgical models. This study was conducted to describe the "ileal" and "colonic" microflora in 2 surgical models of SBS. MATERIALS AND METHODS: Neonatal piglets (2-5 days old) underwent intestinal resection, leaving the ileum (JI anatomy, n = 6) or removing the ileum and ileocecal valve (JC anatomy, n = 5), or sham surgery (sham; n = 4). JI, JC, and sham piglets commenced parenteral nutrition on day 0 and received ampicillin and trimethoprim-sulfadoxine on days 0-4 for prevention of line sepsis. At day 7, ileal and colonic digesta were collected, and they were also collected from age-matched sow-fed piglets (n = 6). DNA extraction, sequencing, and annotation followed standard procedures. RESULTS: Colonic and ileal bacterial genus diversity and relative bacterial abundance were greater ( P < .05) in sow-fed compared with JI, JC, and sham piglets; however, minor differences were observed in either location between sham, JI, and JC piglets and within the surgical model. In the colon, sow-fed piglets had higher ( P < .05) abundance of Lactobacillus (26%) and tended to have lower ( P = .06) abundance of Enterococcus (<.1%) than JI, JC, or sham piglets, in which Lactobacillus and Enterococcus abundance averaged <.1% and 9%, respectively. CONCLUSIONS: Intestinal resection reduces bacterial diversity in the large bowel, and the difference is associated with the presence/absence of the ileum and ileocecal valve. The lack of enteral nutrition and antibiotic administration (ie, sow-fed vs surgery) had a greater influence on the observed shift in diversity and relative abundance than intestinal resection.

Differential Effects on Intestinal Adaptation Following Exogenous Glucagon-Like Peptide 2 Therapy With and Without Enteral Nutrition in Neonatal Short Bowel Syndrome [Formula: see text].

BACKGROUND: We aim to study the efficacy of exogenously administered glucagon-like peptide 2 (GLP-2) on intestinal adaptation in 2 preclinical models of neonatal short bowel syndrome (SBS) according to remnant intestinal anatomy, with and without ileum. Furthermore, we aim to determine if this adaptive effect was potentiated with enteral nutrition (EN). METHODS: Neonatal piglets were block-randomized to 75% mid-intestinal (JI group, retains ileum) or distal-intestinal (JC group, has no ileum) resection or no resection (sham control) and GLP-2 treatment (11 nmol/kg/d) or saline control for 7 days. Piglets received nutrition support, either 100% parenteral nutrition (PN; 0% EN, n = 32 in total) or 80% PN + 40% EN (n = 28 in total). Adaptation was assessed by morphological and histological changes, as well as RT quantitative polymerase chain reaction of nutrient transporters and tight junctional proteins and fat absorption. Data are analyzed by 3-way analysis of variance (ANOVA) and 2-way ANOVA per EN level. RESULTS: GLP-2 treatment lengthened villi, deepened crypts, and improved intestinal weight in the remnant intestine of JC piglets. EN was a more potent adaptive stimulus for JI piglets. Small intestinal lengthening occurred only in the JI group, when given EN. There was no difference in total fat absorption and messenger RNA expression of nutrient transporters and tight junctional proteins. CONCLUSIONS: GLP-2 administration augmented structural adaptation in JC piglets with distal intestinal resection. Given JI anatomy, further stimulation by GLP-2 treatment over innate adaptation and stimulation by EN was modest and restricted to ileum. The differential effect of GLP-2 in neonatal SBS, depending on remnant anatomy, has important implications for clinical translation and planning of clinical trials.

A Third-Generation Lipid Emulsion that Contains n-3 Long-Chain PUFAs Preserves Retinal Function in Parenterally Fed Neonatal Piglets.

BACKGROUND: Preterm neonates and those with intestinal failure require prolonged parenteral nutrition (PN) during a critical time of early central nervous system maturation. Conventional lipid emulsions fed to preterm neonates lack n-3 (ω-3) long-chain polyunsaturated fatty acids (LC-PUFAs; >20 carbon chain in length). Recently, fish oil lipid emulsions have been developed that provide both n-6 (ω-6) and n-3 LC-PUFAs, precursors of very long-chain PUFAs (VLC-PUFAs; >24 carbon chain in length). OBJECTIVE: Our objective was to determine the effect of fish oil lipid on retinal function in neonatal piglets fed total PN with the use of the lipid emulsions available in clinical practice. We hypothesized that fish oil-containing parenteral lipid would preserve retinal function more than conventional parenteral lipid. METHODS: Male neonatal piglets (2-5 d of age) were fed isonitrogenous (16 g · kg-1 · d-1), isocaloric (1.1 MJ · kg-1 · d-1) PN that varied only in the lipid emulsion: Intralipid or SMOFlipid at 10 g · kg-1 · d-1 (n = 8/group). Retinal function was assessed after 14 d of treatment by recording electroretinograms under various light intensity conditions. Retinas were then harvested for histology and to determine fatty acid composition. RESULTS: Electroretinogram intensity response curves showed greater photoreceptor a-wave amplitude in piglets fed SMOFlipid than in those fed Intralipid (percentage), for postsynaptic depolarizing bipolar cell b-waves (percentage) and for flicker electroretinogram amplitudes (percentage) (P < 0.05). Compared with those fed Intralipid, SMOFlipid-fed piglets had greater retinal total n-3 LC-PUFAs (15.7% compared with 18.4%; P = 0.04) and n-3 VLC-PUFAs (0.9% compared with 1.5%; P = 0.02), whereas Intralipid-fed piglets had greater total n-6 LC-PUFAs (13.1% compared with 10.5%; P < 0.01) and n-6 VLC-PUFAs (0.7% compared with 0.5%; P = 0.01). Histologically, retinas were indistinguishable between groups. CONCLUSIONS: In a neonatal piglet model of PN feeding, the inclusion of fish oil-based n-3 LC-PUFAs in the lipid emulsion leads to their accretion and endogenous elongation to VLC-PUFAs in the retina, which is associated with better retinal function.