Karyl K. Buddington

Maternal-to-Infant Transmission of Probiotics: Concept Validation in Mice, Rats, and Pigs

Background: Postnatal introduction of probiotics results in a low incidence of colonization, whereas maternal fecal and vaginal bacteria colonize the gastrointestinal tract (GIT) of vaginally delivered infants. Objective: We tested if probiotic bacteria, fed to three pregnant animal models, would colonize the GIT of offspring delivered vaginally. Methods: Probiotic strains of Lactobacillus acidophilus and Bifidobacterium lactis were fed to pregnant mice, rats, and sows for at least 7 days prior to vaginal delivery. Cultural approaches and genotyping were used to determine if the probiotic bacteria colonized the GIT after birth. Results: The probiotic bacteria were detected in the feces and vagina of maternal mice, rats, and sows after, but not before, administration. L. acidophilus was detected at postnatal day 14 in 22, 33, and 75% of the mice, rats, and pigs, respectively, and after weaning in 35% of the mice and 1 of 5 pigs. B. lactis was present at postnatal day 14 in 30 and 80% of the mice and pigs. Bacterial assemblages in the GIT of the colonized young differed from those in which the probiotics were not detected. Conclusions: Probiotic bacteria administered to mothers during late gestation are transferred to infants born vaginally and influence the assemblages of GIT bacteria. However, colonization of the neonatal GIT and persistence past weaning does not occur in all offspring and varies among probiotics and animal models.

A preterm pig model of lung immaturity and spontaneous infant respiratory distress syndrome

Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia remain the leading causes of preterm infant morbidity, mortality, and lifelong disability. Research to improve outcomes requires translational large animal models for RDS. Preterm pigs delivered by caesarian section at gestation days (GD) 98, 100, 102, and 104 were provided 24 h of neonatal intensive care, monitoring (pulse oximetry, blood gases, serum biomarkers, radiography), and nutritional support, with or without intubation and mechanical ventilation (MV; pressure control ventilation with volume guarantee). Spontaneous development of RDS and mortality without MV are inversely related with GD at delivery and correspond with inadequacy of tidal volume and gas exchange. GD 98 and 100 pigs have consolidated lungs, immature alveolar architecture, and minimal surfactant protein-B expression, and MV is essential at GD 98. Although GD 102 pigs had some alveoli lined by pneumocytes and surfactant was released in response to MV, blood gases and radiography revealed limited recruitment 1-2 h after delivery, and mortality at 24 h was 66% (35/53) with supplemental oxygen provided by a mask and 69% (9/13) with bubble continuous positive airway pressure (8-9 cmH2O). The lungs at GD 104 had higher densities of thin-walled alveoli that secreted surfactant, and MV was not essential. Between GD 98 and 102, preterm pigs have ventilation inadequacies and risks of RDS that mimic those of preterm infants born during the saccular phase of lung development, are compatible with standards of neonatal intensive care, and are alternative to fetal nonhuman primates and lambs.

The Weaned Pig as a Model for Doxorubicin-Induced Mucositis

Background: Chemotherapy-induced mucositis (CIM) complicates cancer therapy and limits maximum tolerated doses and efficacy. Rodent models do not reproducibly mimic clinical CIM, so alternative models are needed. Methods: CIM severity was assessed after weaned pigs were treated with doxorubicin (5 and 3.75 mg/kg) using clinical observations, laboratory parameters and gastrointestinal structure and functions. Bovine colostrum was provided as an experimental intervention to the pigs treated receiving the 3.75 mg/kg dose. Results: Doxorubin at 3.75 mg/kg decreased food intake and weight gain (p < 0.05) and caused diarrhea and vomiting that coincided with damage to the small intestine mucosa based on histological scoring (p < 0.05). It resulted in higher serum TNF-α concentrations, increased chloride secretion and reduced brush border membrane disaccharidase activities and carrier-mediated glucose uptake (all p < 0.05). The gastrointestinal damage and dysfunction resemble the clinical and laboratory features of CIM in humans; these can be partially prevented by providing cow colostrum. Conclusion: The weaned pig is a relevant large animal for studying CIM and evaluating existing and experimental interventions for mucositis.

Intestinal adaptations to a combination of different diets with and without endurance exercise

BackgroundEndurance athletes search for diet regimens that will improve performance and decrease gastrointestinal disturbances during training and events. Although the intestine can adapt to changes in the amount and composition of dietary inputs, the responses to the combination of endurance exercise and diet are poorly understood.MethodsWe evaluated small intestinal dimensions and mucosal architecture and calculated the capacities of the entire small intestine to digest maltose and maltodextrin and absorb glucose in response to two different diet types; a western human diet and the Daniel Fast, a vegan style diet, and with moderate intensity endurance training or a no-exercise sedentary lifestyle for a 13xa0week period (nu2009=u20097 per group). The influences of diet and exercise, alone and in combination, were analyzed by analysis of variation.ResultsRats fed the western diet gained more weight (Pu2009<u20090.05) due to more fat mass (Pu2009<u20090.05), with a similar response for the sedentary compared with the exercised rats in each diet group (Pu2009<u20090.05). The Daniel Fast rats had longer and heavier intestines with deeper crypts with villi that were wider (Pu2009<u20090.05), but not taller. Despite increased energetic demands, the exercised rats had shorter and lighter intestines with shorter villi (Pu2009<u20090.05). Yet, the percentage of mucosa did not differ among groups. Total small intestinal activities for maltase and α-glucoamylase, and capacities for glucose absorption were similar regardless of diet or exercise.ConclusionsThese findings indicate the structural responses of the small intestine to a vegan style diet are modified by exercise, but without altering the capacities of the brush border membrane to digest and absorb carbohydrates.

A Phosphatidylserine Source of Docosahexanoic Acid Improves Neurodevelopment and Survival of Preterm Pigs

The amount, composition, and sources of nutrition support provided to preterm infants is critical for normal growth and development, and particularly for structural and functional neurodevelopment. Although omega-3 long chain polyunsaturated fatty acids (LC-PUFA), and particularly docosahexanoic acid (DHA), are considered of particular importance, results from clinical trials with preterm infants have been inconclusive because of ethical limitations and confounding variables. A translational large animal model is needed to understand the structural and functional responses to DHA. Neurodevelopment of preterm pigs was evaluated in response to feeding formulas to term-equivalent age supplemented with DHA attached to phosphatidylserine (PS-DHA) or sunflower oil as the placebo. Newborn term pigs were used as a control for normal in utero neurodevelopment. Supplementing formula with PS-DHA increased weight of the brain, and particularly the cerebellum, at term-equivalent age compared with placebo preterm pigs (P’s < 0.10 and 0.05 respectively), with a higher degree of myelination in all regions of the brain examined (all p < 0.06). Brains of pigs provided PS-DHA were similar in weight to newborn term pigs. Event-related brain potentials and performance in a novel object recognition test indicated the PS-DHA supplement accelerated development of sensory pathways and recognition memory compared with placebo preterm pigs. The PS-DHA did not increase weight gain, but was associated with higher survival. The benefits of PS-DHA include improving neurodevelopment and possibly improvement of survival, and justify further studies to define dose-response relations, compare benefits associated with other sources of DHA, and understand the mechanisms underlying the benefits and influences on the development of other tissues and organ systems.

Growth Responses of Preterm Pigs Fed Formulas with Different Protein Levels and Supplemented with Leucine or β-Hydroxyl β-Methylbutyrate

Growth after preterm birth is an important determinant of long-term outcomes. Yet, many preterm infants suffer ex utero growth retardation. We evaluated effects of leucine and the metabolite, β-hydroxy β-methylbutyrate (HMB) on growth of preterm pigs, a previously-validated translational model for preterm infants. After 48 h of parenteral nutrition preterm pigs were fed for 6 to 7 days isocaloric formulas with different levels of protein (50 or 100 g/L) with leucine (10 g/L, 76 mM) or HMB (at 1.1 g/L, 4 mM) added to stimulate protein synthesis or with alanine (6.8 g/L; 76 mM) as the control. Rates of growth of pigs fed the low protein formula with alanine (3.4 ± 0.2% gain per day) or leucine (3.7 ± 0.2) exceeded that of pigs fed the high protein formula (2.8 ± 0.2, p = 0.02 for comparison with both low protein formulas; p = 0.01 compared with low protein + leucine). Supplementing the high protein formula with leucine or HMB did not increase growth relative to alanine (2.72 ± 0.20, 2.74 ± 0.27, and 2.52 ± 0.20, respectively). Small pigs (<700 g birth weight) grew slower during parenteral nutrition and had a more pronounced response to leucine. Females fed the high protein formulas grew faster than males, and particularly for small pigs (p < 0.05). Blood urea nitrogen values were lower for pigs fed the low versus the high protein formulas (p < 0.05). Leucine and HMB improved growth of preterm pigs fed low, but not high protein formulas, even after controlling for birth weight and sex, which independently correlated with growth rates. They offer an option to improve growth without increasing the amino acid load, with its attendant metabolic disadvantages.

Localized delivery of Cl-amidine from electrospun polydioxanone templates to regulate acute neutrophil NETosis: A preliminary evaluation of the PAD4 inhibitor for tissue engineering

Upon interaction, neutrophils can potentially release neutrophil extracellular traps (NETs) on the surface of an implanted electrospun template, which may be a significant preconditioning event for implantable biomaterials of yet unknown consequences. In this study, we investigated the potential of polydioxanone templates as a delivery vehicle for Cl-amidine, an inhibitor of peptidyl arginase deiminase 4 (PAD4), and if drug elution could attenuate PAD4-mediated NETosis in the vicinity of implanted templates. Electrospun polydioxanone templates were fabricated with distinct architectures, small diameter (0.4 μm) or large diameter (1.8 μm) fibers, and incorporated with 0–5 mg/mL Cl-amidine to examine dose-dependent effects. Acute neutrophil-template interactions were evaluated in vitro with freshly isolated human neutrophils and in vivo with a rat subcutaneous implant model. The in vitro results suggest large diameter templates with 0 mg/mL Cl-amidine significantly attenuate NETosis compared to small diameter templates. As the drug concentration increased, NETosis was significantly decreased on small diameter templates in a dose-dependent manner. The opposite was observed for large diameter templates, indicating multiple mechanisms of NETosis may be regulating neutrophil template preconditioning. Similar results were observed in vivo, verifying local NETosis inhibition by Cl-amidine eluting templates in a physiological environment. Importantly, large diameter templates with Cl-amidine enhanced neutrophil invasion and survival, supporting the potential for long-term modulation of tissue integration and regeneration. This preliminary study demonstrates a novel delivery vehicle for Cl-amidine that can be used to regulate acute NETosis as the potential critical link between the innate immune response, inflammation, and template-guided tissue regeneration.

Maturation of the Coordination Between Respiration and Deglutition with and Without Recurrent Laryngeal Nerve Lesion in an Animal Model

The timing of the occurrence of a swallow in a respiratory cycle is critical for safe swallowing, and changes with infant development. Infants with damage to the recurrent laryngeal nerve, which receives sensory information from the larynx and supplies the intrinsic muscles of the larynx, experience a significant incidence of dysphagia. Using our validated infant pig model, we determined the interaction between this nerve damage and the coordination between respiration and swallowing during postnatal development. We recorded 23 infant pigs at two ages (neonatal and older, pre-weaning) feeding on milk with barium using simultaneous high-speed videofluoroscopy and measurements of thoracic movement. With a complete linear model, we tested for changes with maturation, and whether these changes are the same in control and lesioned individuals. We found (1) the timing of swallowing and respiration coordination changes with maturation; (2) no overall effect of RLN lesion on the timing of coordination, but (3) a greater magnitude of maturational change occurs with RLN injury. We also determined that animals with no surgical intervention did not differ from animals that had surgery for marker placement and a sham procedure for nerve lesion. The coordination between respiration and swallowing changes in normal, intact individuals to provide increased airway protection prior to weaning. Further, in animals with an RLN lesion, the maturation process has a larger effect. Finally, these results suggest a high level of brainstem sensorimotor interactions with respect to these two functions.

Responses of Preterm Pigs to an Oral Fluid Supplement During Parenteral Nutrition

BACKGROUNDnNutrients and electrolytes in amniotic fluid swallowed by fetuses are important for growth and development. Yet, preterm infants requiring parenteral nutrition (PN) receive minimal or no oral inputs. With the limited availability of amniotic fluid, we evaluated the responses of preterm pigs receiving PN to an oral fluid supplement (OFS) based on the electrolyte and nutrient composition of amniotic fluid.nnnMATERIALS AND METHODSnPreterm pigs (92% of term) received a combination of PN (6 mL/kg-h) and 4 mL/kg-h of supplemental fluid as an experimental OFS (n = 9), lactated Ringers either enterally (n = 10) or intravenously (n = 8). Outcome measures after 96 hours were weight gain, blood chemistry, organ weights, and small intestine mass and brush-border membrane carbohydrases.nnnRESULTSnThe OFS did not improve weight gain compared with providing lactated Ringers orally or intravenously, or increase serum urea nitrogen values, but resulted in higher serum total and low-density lipoprotein cholesterol, as well as improved glucoregulation and heavier intestines, livers, kidneys, and brains and lighter lungs.nnnCONCLUSIONSnProviding supplemental fluid and electrolytes during PN either intravenously or orally increases weight gain after preterm birth. An oral fluid supplement based on amniotic fluid may accelerate development and maturation of organs critical for extrauterine life after preterm birth and may enhance neurodevelopment.

PO-0769 The Preterm Pig As A Model For Acute Lung Disease

Background and aims Despite advances in ventilation support, acute lung disease (ALD) remains the leading cause of morbidity, mortality, and disability after preterm birth. There is a need for a spontaneous translational model of ALD after preterm birth. Methods Preterm pigs delivered at gestation days (GD) 98, 100, 102, and 104 days were provided ventilation support using supplemental oxygen (NC), bubble Continuous Positive Airway Pressure (bCPAP; 7–8 cm H2O), or mechanical ventilation (MV; Pressure Control Ventilation with Volume Guarantee; 5 ml/kg; PEEP 5 cm H2O). Monitoring included pulse oximetry, arterial blood gases, and radiography. Lungs were harvested after 24 h or after premature death for histology and measurements of surfactant protein B, phosphatidylcholine, and cytokines. Results All pigs breathed spontaneously. Lungs at GD 98 and 100 were consolidated with immature alveolar architecture, minimal surfactant protein B expression, and MV was essential for 24 h survival. GD 102 pigs had alveoli lined by pneumocytes and surfactant was released in response to MV. Blood gases and radiography for NC and bCPAP pigs 1–2 h after delivery revealed limited recruitment and mortality at 24 h was 66% (35/53) and 69% (9/13), respectively. GD 104 pigs had higher densities of thin walled alveoli that secreted surfactant and MV was not essential. Conclusions Preterm pigs have developmental changes in ventilation inadequacies that mimic those of preterm infants and represent a spontaneous model of ALD that is clinically relevant, compatible with standards of chronic neonatal intensive care, and is an alternative for nonhuman primates and lambs.