Protein Ingredient Quality within Infant Formulas Impacts Plasma Amino Acid Concentrations in Neonatal Minipiglets.

BACKGROUND: Infant formulas (IFs), the only adequate substitute to human milk, are complex matrices that require numerous ingredients and processing steps that may impact protein digestion and subsequent amino acid (AA) absorption. OBJECTIVES: The objective was to understand the impact of the protein ingredient quality within IFs on postprandial plasma AA profiles. METHODS: Four isonitrogenous and isocaloric IFs were produced at a semi-industrial scale using whey proteins from different origins (cheese compared with ideal whey) and denaturation levels (IF-A, -B, -C), and caseins with different supramolecular organizations (IF-C, -D). Ten Yucatan minipiglets (12- to 27-d-old) were used as a human infant model and received each IF for 3 d according to a Williams Latin square followed by a 2-d wash-out period. Jugular plasma was regularly sampled from 10 min preprandial to 4 h postprandial on the third day to measure free AAs, urea, insulin, and glucose concentrations. Data were statistically analyzed using a mixed linear model with diet (IFs), time, and sex as fixed factors and piglet as random factor. RESULTS: IFs made with cheese whey (IF-A and -B) elicited significantly higher plasma total and essential AA concentrations than IFs made with ideal whey (IF-C and -D), regardless of the pre- and postprandial times. Most of the differences observed postprandially were explained by AA homeostasis modifications. IFs based on cheese whey induced an increased plasma concentration of Thr due to both a higher Thr content in these IFs and a Thr-limiting degrading capability in piglets. The use of a nonmicellar casein ingredient led to reduced plasma content of AA catabolism markers (IF-D compared with IF-C). CONCLUSIONS: Overall, our results highlight the importance of the protein ingredient quality (composition and structure) within IFs on neonatal plasma AA profiles, which may further impact infant protein metabolism.

Preclinical validation of NeoWarm, a low-cost infant warmer and carrier device, to ameliorate induced hypothermia in newborn piglets as models for human neonates.

Introduction: Approximately 1.5 million neonatal deaths occur among premature and small (low birthweight or small-for gestational age) neonates annually, with a disproportionate amount of this mortality occurring in low- and middle-income countries (LMICs). Hypothermia, the inability of newborns to regulate their body temperature, is common among prematurely born and small babies, and often underlies high rates of mortality in this population. In high-resource settings, incubators and radiant warmers are the gold standard for hypothermia, but this equipment is often scarce in LMICs. Kangaroo Mother Care/Skin-to-skin care (KMC/STS) is an evidence-based intervention that has been targeted for scale-up among premature and small neonates. However, KMC/STS requires hours of daily contact between a neonate and an able adult caregiver, leaving little time for the caregiver to care for themselves. To address this, we created a novel self-warming biomedical device, NeoWarm, to augment KMC/STS. The present study aimed to validate the safety and efficacy of NeoWarm. Methods: Sixteen, 0-to-5-day-old piglets were used as an animal model due to similarities in their thermoregulatory capabilities, circulatory systems, and approximate skin composition to human neonates. The piglets were placed in an engineered cooling box to drop their core temperature below 36.5°C, the World Health Organizations definition of hypothermia for human neonates. The piglets were then warmed in NeoWarm (n = 6) or placed in the ambient 17.8°C ± 0.6°C lab environment (n = 5) as a control to assess the efficacy of NeoWarm in regulating their core body temperature. Results: All 6 piglets placed in NeoWarm recovered from hypothermia, while none of the 5 piglets in the ambient environment recovered. The piglets warmed in NeoWarm reached a significantly higher core body temperature (39.2°C ± 0.4°C, n = 6) than the piglets that were warmed in the ambient environment (37.9°C ± 0.4°C, n = 5) (p < 0.001). No piglet in the NeoWarm group suffered signs of burns or skin abrasions. Discussion: Our results in this pilot study indicate that NeoWarm can safely and effectively warm hypothermic piglets to a normal core body temperature and, with additional validation, shows promise for potential use among human premature and small neonates.

White Matter Integrity and Motor Function Disruption Due to Traumatic Brain Injury in Piglets: Impacts on Motor-Related Brain Fibers.

Pediatric traumatic brain injury (TBI) often induces significant disability in patients, including long-term motor deficits. Early detection of injury severity is key in determining a prognosis and creating appropriate intervention and rehabilitation plans. However, conventional magnetic resonance imaging (MRI) scans, such as T2 Weighted (T2W) sequences, do not reliably assess the extent of microstructural white matter injury. Diffusion tensor imaging (DTI) tractography enables three-dimensional reconstruction of specific white matter tracts throughout the brain in order to detect white matter injury based on anisotropic diffusion. The objective of this study was to employ DTI tractography to detect acute changes to white matter integrity within the intersecting fibers of key motor-related brain regions following TBI. Piglets were assigned to either the sham craniectomy group (sham; n = 6) or the controlled cortical impact TBI group (TBI; n = 6). Gait and MRI were collected at seven days post-surgery (DPS). T2W sequences confirmed a localized injury predominately in the ipsilateral hemisphere in TBI animals. TBI animals, relative to sham animals, showed an increased apparent diffusion coefficient (ADC) and decreased fractional anisotropy (FA) in fiber bundles associated with key brain regions involved in motor function. TBI animals exhibited gait deficits, including stride and step length, compared to sham animals. Together these data demonstrate acute reductions in the white matter integrity, measured by DTI tractography, of fibers intersecting key brain regions that strongly corresponded with acute motor deficits in a pediatric piglet TBI model. These results provide the foundation for the further development of DTI-based biomarkers to evaluate motor outcomes following TBI.

Role of the GLP2-Wnt1 axis in silicon-rich alkaline mineral water maintaining intestinal epithelium regeneration in piglets under early-life stress.

Stress-induced intestinal epithelial injury (IEI) and a delay in repair in infancy are predisposing factors for refractory gut diseases in adulthood, such as irritable bowel syndrome (IBS). Hence, it is necessary to develop appropriate mitigation methods for mammals when experiencing early-life stress (ELS). Weaning, as we all know, is a vital procedure that all mammalian newborns, including humans, must go through. Maternal separation (MS) stress in infancy (regarded as weaning stress in animal science) is a commonly used ELS paradigm. Drinking silicon-rich alkaline mineral water (AMW) has a therapeutic effect on enteric disease, but the specific mechanisms involved have not been reported. Herein, we discover the molecular mechanism by which silicon-rich AMW repairs ELS-induced IEI by maintaining intestinal stem cell (ISC) proliferation and differentiation through the glucagon-like peptide (GLP)2-Wnt1 axis. Mechanistic study showed that silicon-rich AMW activates GLP2-dependent Wnt1/ß-catenin pathway, and drives ISC proliferation and differentiation by stimulating Lgr5+ ISC cell cycle passage through the G1-S-phase checkpoint, thereby maintaining intestinal epithelial regeneration and IEI repair. Using GLP2 antagonists (GLP23-33) and small interfering RNA (SiWnt1) in vitro, we found that the GLP2-Wnt1 axis is the target of silicon-rich AMW to promote intestinal epithelium regeneration. Therefore, silicon-rich AMW maintains intestinal epithelium regeneration through the GLP2-Wnt1 axis in piglets under ELS. Our research contributes to understanding the mechanism of silicon-rich AMW promoting gut epithelial regeneration and provides a new strategy for the alleviation of ELS-induced IEI.

Gut microbiota absence and transplantation affect diarrhea: an investigation in the germ-free piglet model.

This experiment was conducted to explore the effects of gut microbiota on neonatal diarrhea in a germ-free (GF) pig model. Twelve hysterectomy-derived GF piglets were housed in six sterile isolators. Among them, six piglets were treated as the GF group, and the other six piglets were orally introduced with healthy sow fecal suspension and regarded as the fecal microbiota transplantation (FMT) group. Another six piglets from natural birth were considered as the conventional (CV) group. The GF and FMT piglets were hand-fed with sterile milk powder for 21 days, and the CV piglets were suckled for the same days. Then, all piglets were fed with sterile feed for another 21 days. Results exhibited that the GF group's fecal score and moisture level were higher than those in the CV and FMT groups (p < 0.05). Meanwhile, the abundances of colonic AQP1 and AQP8 in the GF group were the greatest among these treatments (p < 0.05). However, FMT piglets had a lower fecal score in d 22-28 and d 29-35 than that in the CV piglets (p < 0.05). Collectively, the absence of gut microbiota may cause diarrhea in the piglet model, and transplantation of maternal fecal microbiota may reverse it.

Limosilactobacillus johnsoni and Limosilactobacillus mucosae and Their Extracellular Vesicles Alleviate Gut Inflammatory Injury by Mediating Macrophage Polarization in a Lipopolysaccharide-Challenged Piglet Model.

BACKGROUND: Limosilactobacillus johnsoni (L. j) and Limosilactobacillus mucosae (L. m) can alleviate the inflammatory response. OBJECTIVES: This study aimed to elucidate the underlying mechanisms by which L. j- and L. m-derived extracellular vesicles (EVs) mitigate lipopolysaccharide (LPS)-induced intestinal injury. METHODS: Piglets were assigned to 4 groups: oral phosphate-buffered saline inoculation for 2 wk prior to intraperitoneal injection of physiological saline or LPS, and oral L. j/L. m inoculation for 2 wk prior to intraperitoneal injection of LPS. The intestinal integrity, macrophage markers, cytokine levels, and microbiota were determined. The cytokine levels and macrophage phenotype were detected after L. j/L. m and their EVs were coincubated with macrophages. The levels of cytokines, tight junction proteins, and apoptosis were measured after intestinal epithelial cells were cocultured with macrophages. RESULTS: LPS challenge decreased jejunal villus length; expression levels of zonula occludens-1 (ZO-1), occludin, arginase-1 (Arg1), and interleukin (IL)-10; and number of CD163+ cells and increased the expression levels of inducible nitric oxide synthase (iNOS), IL-1ß, IL-6, and tumor necrosis factor (TNF)-α compared with that in the control. L. j and L. m pretreatment rescued the aforementioned indicators compared with LPS challenge. Pretreatment of L. j and L. m and their EVs reversed the levels of IL-1ß, IL-6, TNF-α, and IL-10 and the gene expression of iNOS and Arg1 in the LPS group in macrophages. Pretreatment with L. j and L. m-derived EVs increased ZO-1 and occludin mRNA expression and reduced IL-1ß, caspase-3, and bax gene expression in intestinal epithelial cells of the coculture system. Enzyme-treated EVs were less effective than native EVs. CONCLUSIONS: This study suggests that EVs secreted by L. j and L. m control inflammation by modulating macrophage polarization, thereby improving intestinal barrier function.

Long Non-Coding RNAs in Hypoxia and Oxidative Stress: Novel Insights Investigating a Piglet Model of Perinatal Asphyxia.

Birth asphyxia is the leading cause of death and disability in young children worldwide. Long non-coding RNAs (lncRNAs) may provide novel targets and intervention strategies due to their regulatory potential, as demonstrated in various diseases and conditions. We investigated cardinal lncRNAs involved in oxidative stress, hypoxia, apoptosis, and DNA damage using a piglet model of perinatal asphyxia. A total of 42 newborn piglets were randomized into 4 study arms: (1) hypoxia-normoxic reoxygenation, (2) hypoxia-3 min of hyperoxic reoxygenation, (3) hypoxia-30 min of hyperoxic reoxygenation, and (4) sham-operated controls. The expression of lncRNAs BDNF-AS, H19, MALAT1, ANRIL, TUG1, and PANDA, together with the related target genes VEGFA, BDNF, TP53, HIF1α, and TNFα, was assessed in the cortex, the hippocampus, the white matter, and the cerebellum using qPCR and Droplet Digital PCR. Exposure to hypoxia-reoxygenation significantly altered the transcription levels of BDNF-AS, H19, MALAT1, and ANRIL. BDNF-AS levels were significantly enhanced after both hypoxia and subsequent hyperoxic reoxygenation, 8% and 100% O2, respectively. Our observations suggest an emerging role for lncRNAs as part of the molecular response to hypoxia-induced damages during perinatal asphyxia. A better understanding of the regulatory properties of BDNF-AS and other lncRNAs may reveal novel targets and intervention strategies in the future.

Evaluation of a Plant-Based Infant Formula Containing Almonds and Buckwheat on Gut Microbiota Composition, Intestine Morphology, Metabolic and Immune Markers in a Neonatal Piglet Model.

A controlled-neonatal piglet trial was conducted to evaluate the impact of a plant-based infant formula containing buckwheat and almonds as the main source of protein compared to a commercially available dairy-based formula on the gut health parameters. Two day old piglets were fed either a plant-based or a dairy-based formula until day 21. Gut microbiome, cytokines, growth and metabolism related outcomes, and intestinal morphology were evaluated to determine the safety of the plant-based infant formula. This study reported that the plant-based formula-fed piglets had a similar intestinal microbiota composition relative to the dairy-based formula-fed group. However, differential abundance of specific microbiota species was detected within each diet group in the small and large intestinal regions and fecal samples. Lactobacillus delbrueckii, Lactobacillus crispatus, and Fusobacterium sp. had higher abundance in the small intestine of plant-based formula-fed piglets compared to the dairy-based group. Bacteroides nordii, Enterococcus sp., Lactobacillus crispatus, Prevotella sp., Ruminococcus lactaris, Bacteroides nordii, Eisenbergiella sp., Lactobacillus crispatus, Prevotella sp., and Akkermansia muciniphila had greater abundance in the large intestine of the plant based diet fed piglets relative to the dairy-based diet group. In the feces, Clostridiales, Bacteroides uniformis, Butyricimonasvirosa, Cloacibacillus porcorum, Clostridium clostridioforme, and Fusobacterium sp. were abundant in dairy-based group relative to the plant-based group. Lachnospiraceae, Clostridium scindens, Lactobacillus coleohominis, and Prevetolla sp. had greater abundance in the feces of the plant-based group in comparison to the dairy-based group. Gut morphology was similar between the plant and the dairy-based formula-fed piglets. Circulatory cytokines, magnesium, triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), vitamin D, vitamin K, and IgE levels were similar among all piglets independent of dietary group. Overall, the present study demonstrated that a plant-based formula with buckwheat and almonds as the primary source of protein can support similar gut microbiota growth and health outcomes compared to a dairy-based infant formula.

Evaluation of the Safety of a Plant-Based Infant Formula Containing Almonds and Buckwheat in a Neonatal Piglet Model.

A randomized neonatal piglet trial was conducted to evaluate the safety and the effects of a plant-based formula containing almonds and buckwheat as the main ingredients on growth and plasma parameters. From postnatal day (PND) 2 to 21, the piglets were fed a dairy-based milk formula (Similac Advance) or a plant-based formula (Else Nutrition) and all piglets were euthanized at day 21. No diarrhea was observed after PND 8 and all the piglets completed the trial. Body growth, kcal intake, the complete plasma count parameters and hematological parameters were within the reference range in both groups. Organ growth and development was similar between the two groups. Plasma glucose was higher in the dairy-based-fed piglets relative to the plant-based at 2 weeks of age. Liver function biomarkers levels were greater in the plasma of the plant-based compared to the dairy-based fed group. In addition, calcium levels were higher in the plant-based fed piglets at 1 week of age. Thus, the plant-based formula tested in this study was well tolerated by the piglets and supported similar growth compared to dairy-based milk formula. Therefore, the results support the safety of the tested plant-based infant formula during the neonatal period in comparison to the dairy-based formula fed group.

Impacts of sodium butyrate on intestinal mucosal barrier and intestinal microbial community in a weaned piglet model.

Objective: Butyrate is thought to enhance intestinal mucosal homeostasis, but the detailed mechanism remains unclear. Therefore, further investigation on the mechanism of butyrate regulation of intestinal mucosal homeostasis was performed. Materials and methods: This study used weaned piglets with similar intestinal metabolic function to humans as a research model. The dietary supplemented 0.2% sodium butyrate group (0.2% S) and negative control group (CON) were established to detect the effects of butyrate on growth performance, intestinal tissue morphology, mucosal barrier function, and intestinal microbial community structure in weaned piglets. Results: There was an increase in average daily gain (ADG) during three different experimental periods and a reduction in average daily feed intake (ADFI) and feed-to-gain ratio (F:G) during days 1-35 and days 15-35 in 0.2% S compared with CON (P > 0.05). Furthermore, villus height in the ileum and duodenum was increased, and crypt depths in the colon and jejunum were reduced in both groups (P < 0.05). Moreover, the ratio of villus height and crypt depth (V/C) in 0.2% S both in the ileum and jejunum was significantly increased (P < 0.05) compared with CON. The relative mRNA expression of PKC, MUC1, CLDN1, and ITGB1 was upregulated in the ileum of 0.2% S compared with CON (P < 0.05). The digesta samples of 0.2% S, both in the ileum (P < 0.05) and colon, contained greater intestinal bacterial abundance and diversity of probiotics, including Lactobacillus, Streptococcus, Megasphaera, and Blautia, which promoted amino acid metabolism and energy production and conversion in the colon and the synthesis of carbon-containing biomolecules in the ileum. Conclusion: In summary, dietary supplementation with 0.2% sodium butyrate was shown to have a tendency to improve the growth performance of weaned piglets and enhance intestinal mucosal barrier function via altering the gut microbiota.

Superior restoration of left ventricular performance after prolonged single-dose del Nido cardioplegia in conjunction with terminal warm blood cardioplegic reperfusion.

OBJECTIVES: An incomplete restoration of left ventricular contractility after del Nido cardioplegia was noted in our recent study. This study tested the hypothesis that terminal warm blood cardioplegia promotes a prompt restoration of left ventricular performance after a prolonged single-dose del Nido cardioplegia. METHODS: Fourteen piglets were subjected to 120 minutes of arrest by del Nido cardioplegia without terminal warm blood cardioplegia (del Nido cardioplegia group; n = 7) or with terminal warm blood cardioplegia before reperfusion (terminal warm blood cardioplegia group; n = 7). The other 7 piglets underwent total cardiopulmonary bypass without ischemia/reperfusion for 150 minutes (control group). Left ventricular function was assessed by percent recovery of end-systolic elastance as the contractility and percent end-diastolic pressure-volume relationship as the compliance using a conductance catheter. Troponin T and the mitochondrial score were also measured. RESULTS: Depressed percent recovery of end-systolic elastance was sustained in the del Nido cardioplegia group, and a prompt restoration of end-systolic elastance was achieved using terminal warm blood cardioplegia (57.9 ± 17.8 vs 94.7 ± 13.1, P < .028). Percent end-diastolic pressure-volume relationship at the early phase was better in the terminal warm blood cardioplegia compared with the del Nido group (88.5 ± 24.0 vs 101.4 ± 16.8, P = .050). Troponin T was higher in the terminal warm blood cardioplegia compared with the control group (0.80% ± 0.21% and 1.49% ± 0.31%, respectively, P = .002). The mitochondrial score was equivalent in all groups. Spontaneous restoration to sinus rhythm was more frequent in the terminal warm blood cardioplegia group than in the del Nido cardioplegia group (6/7 vs 1/7, P < .028). CONCLUSIONS: The supplementary use of terminal warm blood cardioplegia achieved prolongation of the safe ischemic time up to 120 minutes for a single-dose application.

Micafungin Population PK Analysis in Healthy and Septic Pigs: Can the Septic Porcine Model Predict the Micafungin PK in Septic Patients?

OBJECTIVES: To describe micafungin pharmacokinetic (PK) alterations of sepsis induced in piglets and to determine whether the porcine septic model is able to predict the PK of micafungin in septic patients at the plasma and peritoneal sites. METHODS: From healthy (n = 8) and septic piglet group (n = 16), total micafungin concentrations were subject to a population PK analysis using Monolix®. Data from 16 septic humans patients from others studies was used to compare micafungin PK between septic piglets and septic patients. RESULTS: Sepsis induced in piglets slightly alters the total clearance and the volume of distribution, while inter-compartment clearance is increased (from 3.88 to 5.74 L/h) as well as the penetration into peritoneal cavity (from 61 to 90%). In septic human patients, PK parameters are similar except for the Vd, which is corrected by an allometric factor based on the body weight of each species. Micafungin penetration into peritoneal cavity of humans is lower than in septic piglets (40 versus 90%). CONCLUSIONS: The sepsis induced in the porcine model alters the PK of micafungin comparable to that in humans. In addition, micafungin PK is similar between these two species at the plasma level taking into account the allometric relationship of the body weight of these species on the central volume of distribution. The porcine septic plasma model would be able to predict the micafungin PK in the septic patients. However, further studies on peritoneal penetration are necessary to characterize this inter-species difference.

Ventilation With or Without Endotracheal Tube Leak in Prolonged Neonatal Asphyxia.

Background Severe and prolonged asphyxia can result in either intrauterine fetal death and stillbirth or multiorgan failure in surviving neonates. Establishing effective ventilation is the primary aim of resuscitation in newborns with asphyxia. The objective of this study was to compare the outcome of resuscitation by applying an endotracheal tube (ETT) with less, an ETT with moderate, and an ETT with high leakage during mechanical ventilation in swine neonates after prolonged perinatal asphyxia. Materials and methods A prospective, randomized controlled laboratory study was performed. Thirty Landrace/large white pigs, aged one to four days and weighted 1.754±218 gr, were randomly allocated into three groups depending on the ETT size: Group C (less leak: ETT no 4.0, n=10); Group A (high leak: ETT no 3.0, n=10); and Group B (moderate leak: ETT no 3.5, n=10). Mechanical asphyxia was performed until their heart rate was less than 60 bpm or their mean arterial pressure was below 15 mmHg. All animals with return of spontaneous circulation (ROSC) were monitored for four hours for their hemodynamic parameters, arterial oxygen saturation, and lactate acid levels. Results We demonstrate that 70% of the surviving animals were ventilated with an ETT with a leak (no. 3.5 and 3). A statistically significant difference was noted in PO2 (p=0.032) between Group B (126.4±53.4 mmHg) compared to Group A (72.28±29.18 mmHg) and Group C (94.28±20.46 mmHg) as well as in the right atrial pressure (p<0.001) between Group C (4.5 mmHg) vs Groups A (2 mmHg) and B (2 mmHg) during ROSC time. Lactate levels were statistically significantly lower (p=0.035) in Group C (mean=0.92 ± 0.07mmol/L) as compared to Group A (mean=1.13 ± 0.1 mmol/L) and Group B (mean= 1.08 ± 0.07 mmol /L; p = 0.034) at 4h post-ROSC. Conclusion We provide preliminary evidence that ventilation with ETT with moderate leakage improves survival after 2h of ROSC, along with oxygenation and hemodynamic parameters, in a porcine model of neonatal asphyxia and resuscitation, compared to less leakage ETT.

Simulation of pelvic osteotomies applied for DDH treatment in pediatric patients using piglet models.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a common hip joint pathology seen in the pediatric orthopedist's practice. Pelvic osteotomies are the reliable surgical option for DDH treatment in walking patients, and 3 osteotomies (Salter, Dega and Pemberton) are widely used in patients under 6 years of age. Plastic changes in hinge points occur during iliac fragment movement, after the performed osteotomy. The locations of these points are described in the literature, but some debate still exists about their true positions. OBJECTIVES: To reveal hinge point locations during a simulation of pelvic osteotomies on biological models. MATERIAL AND METHODS: Eighteen piglet pelvis complexes were obtained and separated according to their age. Pelvic osteotomies were simulated, and bone changes were assessed on computed tomography (CT) scans after the performed surgeries. RESULTS: No bone changes were found after Salter osteotomy in younger piglets, while contralateral pubic bone metaphyseal fractures were found in older animals. After Pemberton osteotomy, greenstick fractures in iliac and pubic bones metaphyses in the triradiate cartilage area were revealed in younger and older piglets. After Dega osteotomy, a posterior medial cortical layer fracture of the uncut iliac bone in the greater sciatic notch was found in all piglets. In older piglets, an additional hinge point was detected in the ipsilateral pubic bone metaphysis. CONCLUSION: It was found that the age of the piglets has an impact on hinge point number and location, and this may be explained by an age-related decrease in pelvic bone and cartilage plasticity. The results of this study may help surgeons to decrease the number of preventable complications during pelvic osteotomies.

O-GlcNAc / Akt pathway regulates glucose metabolism and reduces apoptosis in liver of piglets with acute cold stress.

Cold stress is one of the serious factors restricting the development of animal husbandry in cold areas. Cold exposure can easily lead to cold stress, slow growth and even death of newborn animals. O-GlcNAcylation modification can act as type of "stress receptor" and"nutrition sensor" in a variety of stress responses, however, it is not clear how O-GlcNAcylation can regulate glucose metabolism in the liver of piglets under cold stress. In this study, piglets 21 days of age were exposed to 4 °C for 4 h or 8 h in a phytotron. Serum cortisol and other stress hormones were used to assess body status to establish a cold stress piglet model. The changes of glycogen in liver were detected by PAS. FDP and PA were also measured to study the glycolysis level of liver. To characterize potential mechanisms of O-GlcNAcylation on the livers of cold stress piglets, AKT, GSK3ß, GS, PFKFB2, AS160 and their corresponding phosphorylation were determined by Western blotting. Results show O-GlcNAcylation increased and apoptosis levels increased in the liver following cold exposure during excessive CORT or metabolic dysfunction. It is suggested that the acute cold exposure of piglets induced a sequential change in the level of O-GlcNAcylation, which may be one of the factors mediating liver cell apoptosis and glucose metabolism regulation by the O-GlcNAc/AKT pathway. These findings provide new insight into the mechanisms of the cold stress response, which can facilitate the development of new strategies to combat the effects of hypothermia.

Functional Correlates and Impact of Dietary Lactoferrin Intervention and its Concentration-dependence on Neurodevelopment and Cognition in Neonatal Piglets.

SCOPE: Lactoferrin (Lf), a sialylated milk glycoprotein, promotes early neurodevelopment and cognition. Functional concentrations of Lf, however, remain unknown. Our objective is to determine the concentration-dependency of Lf on genes associated with neurodevelopment and cognition in neonatal piglets. METHODS AND RESULTS: Piglets are given milk replacer with Lf at concentrations of 155 (low) or 285 mg kg-1 day-1 (high) from postnatal days 3 to 38. Gene expression associated with neurodevelopment, cognition, and cognate proteins were quantitated. This study found 1) The rate of learning and long-term memory was higher with 155 mg kg-1 day-1 assessed in an eight-arm radial maze; 2) Global gene transcription profiling showed this lower concentration upregulated genes and functions correlated with neurodevelopment and cognition, while the higher concentration regulated cellular processes for neuroprotection; 3) Expression of BDNF genes and proteins were higher with both concentrations, while genes regulating BDNF signaling, including SLC6A3, IGF-1 responded more to the lower concentration; 4) The lower concentration modulated genes in the five highest networks associated with cellularity and neurocognition, while the prevention of neurodevelopmental and neurological pathologies was associated with the higher concentration. CONCLUSION: The lower concentrations of Lf enhanced neurodevelopment and cognition, while higher concentrations are greater neuroprotective, findings of potential novel clinical relevance.

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.

Modified Del Nido Cardioplegia and Its Evaluation in a Piglet Model.

Del Nido cardioplegia (DN) is not available worldwide as the required solution is made from a commercial base (PlasmaLyte) that is not approved in all countries. We report our own modified DN solution and confirm its safety and effectiveness. Fourteen piglets were subjected to 90 minutes of global ischemia on cardiopulmonary bypass induced by original DN (n = 7) or NS (normal saline)-based DN (n = 7). Our DN solution begins with a base of NS (800 mL) and distilled water (200 mL), to which are added 15 mL KCl (2 mEq/mL), 17 mL NaHCO3 (1 mEq/mL), 10 mL MgSO4 (0.2 g/mL), 13 mL lidocaine 1%, and 13 mL mannitol 25%. LV function recovery was assessed in end-systolic elastance (EES) as systolic function and end-diastolic pressure-volume relationship (EDPVR) as diastolic function using a conductance catheter. Creatine kinase-MB (CK-MB) and mitochondrial score were also measured. Left ventricular (LV) contractility after ischemia (%EES ± SD) was not significantly different between the group induced by original DN (89.3 ± 20.6%) and the group induced by NS-based DN (99.3 ± 18.4%). LV compliance (%EDPVR ± SD) was likewise not significantly different between these groups (102.7 ± 28.2% vs 94.4 ± 22.8%, PL vs NS, respectively). CK-MB was equivalent between the groups. Mitochondrial scores were not significantly different between the groups, and this difference did not cause severe damage. NS-based DN preserves LV function recovery after prolonged global ischemia as effectively and as safely as original DN does. NS-based modified DN can be substituted for original DN.

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.

Iron Deficiency and Iron Excess Differently Affect Dendritic Architecture of Pyramidal Neurons in the Hippocampus of Piglets.

BACKGROUND: Both iron deficiency and overload may adversely affect neurodevelopment. OBJECTIVES: The study assessed how changes in early-life iron status affect iron homeostasis and cytoarchitecture of hippocampal neurons in a piglet model. METHODS: On postnatal day (PD) 1, 30 Hampshire × Yorkshire crossbreed piglets (n = 15/sex) were stratified by sex and litter and randomly assigned to experimental groups receiving low (L-Fe), adequate (A-Fe), or high (H-Fe) levels of iron supplement during the pre- (PD1-21) and postweaning periods (PD22-35). Pigs in the L-Fe, A-Fe, and H-Fe groups orally received 0, 1, and 30 mg Fe · kg weight-1 · d-1 preweaning and were fed a diet containing 30, 125, and 1000 mg Fe/kg postweaning, respectively. Heme indexes were analyzed weekly, and gene and protein expressions of iron regulatory proteins in duodenal mucosa, liver, and hippocampus were analyzed through qRT-PCR and western blot, respectively, on PD35. Hippocampal neurons stained using the Golgi-Cox method were traced and their dendritic arbors reconstructed in 3-D using Neurolucida. Dendritic complexity was quantified using Sholl and branch order analyses. RESULTS: Pigs in the L-Fe group developed iron deficiency anemia (hemoglobin = 8.2 g/dL, hematocrit = 20.1%) on PD35 and became stunted during week 5 with lower final body weight than H-Fe group pigs (6.6 compared with 9.6 kg, P < 0.05). In comparison with A-Fe, H-Fe increased hippocampal ferritin expression by 38% and L-Fe decreased its expression by 52% (P < 0.05), suggesting altered hippocampal iron stores. Pigs in the H-Fe group had greater dendritic complexity in CA1/3 pyramidal neurons than L-Fe group pigs as shown by more dendritic intersections with Sholl rings (P ≤ 0.04) and a greater number of dendrites (P ≤ 0.016). CONCLUSIONS: In piglets, the developing hippocampus is susceptible to perturbations by dietary iron, with deficiency and overload differentially affecting dendritic arborization.