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Sustaining impactful research within the field of perinatal biology requires training and retention of the next generations of physician-scientists and basic-scientists. Professional societies such as the Perinatal Research Society (PRS) have a unique role to play in training and retention of perinatal biologists. Here we report outcomes for an innovative Young Investigator Training Workshop created for the PRS. The PRS Workshop uses immersive, active-writing, and active-oral presentation design, with one-on-one feedback from NIH-funded faculty-mentors drawn from the PRS membership. Young Investigator data were collected by anonymous surveys of Young Investigators, NIH RePORTER, and individual Young Investigator follow-up. Ninety-seven Young Investigators attended the Workshops over the period 2013-2018. Young Investigators were physician- (73%) and PhD- (27%) scientists at the rank of clinical fellow/postdoctoral fellow (27%) or Instructor/Assistant Professor (73%). Participation by underrepresented minority (URM) Young Investigators was 14%. Young Investigators received NIH and non-NIH funding, with 80% of Young Investigators receiving new funding since the Workshop that they attended. NIH funding was received by 31% of Young Investigators in the form of K-series awards, R01 equivalents, and other NIH awards. In conclusion, our PRS Young Investigator Workshop serves as a model to facilitate training of emerging physician- and basic-scientists by scientific societies.

Background: Nucleated red blood cells (NRBC) are very uncommon in the blood of children and adults, but small numbers are not rare in neonates on the day of birth. Elevated NRBC counts in neonates can be seen following erythropoietin dosing. Limited studies in human neonates suggest the time-interval between erythropoietin dosing and the first appearance of NRBC in the blood (the “NRBC emergence-time”) is in excess of 24 hours. Methods: We made serial blood counts (Sysmex veterinary analyzer) on ten newborn lambs; five were dosed with darbepoetin (10 μg/kg), and five were dosed with a vehicle-control to assess the NRBC emergence time under relatively controlled laboratory conditions. Results: The first appearance of NRBC was at 24 h (2757 ± 3210 NRBC/μL vs. 0/μL in controls). Peak was 48-72 h (16,758 ± 8434/μL vs. 0/μL in controls), followed by fewer at 96 hours (7823 ± 7114/μL vs. 0/μL in controls). Similarly, reticulocytes peaked at 48-72 h (113,094 ± 3210/μL vs. 10,790 ± 5449/μL in controls), with no changes in platelets or leukocytes. Conclusions: The NRBC emergence time in newborn lambs is similar to reports from newborn hu-mans. By extrapolation, if a neonate has a high NRBC at birth, the erythropoietic stimulus likely occurred within the interval 24 to perhaps 96+ hours prior to birth.

Background: Adults born preterm (< 37 weeks’ gestation) exhibit altered cardiac growth and are susceptible to cardiac dysfunction. Sheep studies have shown that moderate preterm birth results in maladaptive structural remodelling of the cardiac ventricles. The aim of this study was to examine ventricular structure in lambs born at a greater severity of preterm birth and ventilated postnatally. Methods: Former-preterm lambs delivered at 128 days’ gestation, and mechanically ventilated for a week after birth, were compared with unventilated lambs born at term (150 days’ gestation), at 2 months (term: n = 10, former-preterm: n = 8), and 5 months (term: n = 9, former-preterm: n = 8) term-equivalent age. The right ventricle and left ventricle plus septum were analysed using immunohistochemistry, histology, and stereology. Results: Cardiomyocyte number, cross-sectional area, proliferation, and apoptosis were not affected by preterm birth or age. Left ventricle plus septum interstitial collagen levels increased with age (P = 0.0015) and were exacerbated by preterm birth (P = 0.0006; 2 months term: 0.57% ± 0.07%, former-preterm: 1.44% ± 0.18%; 5 months term: 1.37% ± 0.25%, former-preterm: 2.15% ± 0.31%). Right ventricle interstitial collagen levels increased with age (P = 0.012) but were not affected by preterm birth. Conclusion: This study is the first to explore the effect of preterm birth combined with modern neonatal interventions on the ventricular myocardium in lambs. There was no adverse impact on cardiomyocyte growth in early postnatal life. Of concern, however, there was increased collagen deposition in the preterm hearts, which has the potential to induce cardiac dysfunction, especially if it becomes exaggerated with ageing. Résumé: Introduction: Les adultes nés avant terme (< 37 semaines de grossesse) montrent une altération de la croissance cardiaque et sont exposés à une dysfonction cardiaque. Les études sur les moutons ont montré que la prématurité modérée entraîne un remodelage structurel inadapté des ventricules du cœur. L’objectif de la présente étude était d’examiner la structure ventriculaire des agneaux grands prématurés et oxygénés après la naissance. Méthodes: Les agneaux anciens prématurés nés après 128 jours de gestation et sous respirateur durant une semaine ont été comparés aux agneaux nés à terme qui n’avaient pas été sous respirateur (150 jours de gestation) à l’âge du terme, soit deux mois (à terme : n = 10, anciens prématurés : n = 8) et cinq mois (à terme : n = 9, anciens prématurés : n = 8). Le ventricule droit et le ventricule gauche plus le septum ont été analysés par immunohistochimie, histologie et stéréologie. Résultats: Le nombre de cardiomyocytes, la surface en coupe transversale, la prolifération et l’apoptose n’étaient pas affectés par la naissance prématurée ou l’âge. Les concentrations interstitielles en collagène du ventricule gauche plus le septum augmentaient avec l’âge (P = 0,0015) et étaient exacerbées par la naissance prématurée (P = 0,0006; âge du terme, deux mois : [à terme : 0,57 % ± 0,07 %, anciens prématurés : 1,44 % ± 0,18 % ]; âge du terme, cinq mois : [à terme : 1,37 % ± 0,25 %, anciens prématurés : 2,15 % ± 0,31 %]). Les concentrations interstitielles en collagène du ventricule droit augmentaient avec l’âge (P = 0,012), mais n’étaient pas affectées par la naissance avant terme. Conclusion: Il s’agit de la première étude qui porte sur la combinaison des effets de la naissance avant terme aux interventions néonatales modernes sur le myocarde ventriculaire des agneaux. Aucune conséquence sur la croissance des cardiomyocytes dans la phase précoce de la vie postnatale n’a été observée. Toutefois, le dépôt accru de collagène dans le cœur des prématurés est préoccupant puisqu’il a le potentiel d’induire une dysfonction cardiaque, particulièrement s’il s’exacerbe avec le vieillissement.

Background The brain of chronically ventilated preterm human infants is vulnerable to collateral damage during invasive mechanical ventilation (IMV). Damage is manifest, in part, by learning and memory impairments, which are hippocampal functions. A molecular regulator of hippocampal development is insulin-like growth factor 1 (IGF1). A gentler ventilation strategy is noninvasive respiratory support (NRS). We tested the hypotheses that NRS leads to greater levels of IGF1 messenger RNA (mRNA) variants and distinct epigenetic profile along the IGF1 gene locus in the hippocampus compared to IMV. Methods Preterm lambs were managed by NRS or IMV for 3 or 21 days. Isolated hippocampi were analyzed for IGF1 mRNA levels and splice variants for promoter 1 (P1), P2, and IGF1A and 1B, DNA methylation in P1 region, and histone covalent modifications along the gene locus. Results NRS had significantly greater levels of IGF1 P1 (predominant transcript), and 1A and 1B mRNA variants compared to IMV at 3 or 21 days. NRS also led to more DNA methylation and greater occupancy of activating mark H3K4 trimethylation (H3K4me3), repressive mark H3K27me3, and elongation mark H3K36me3 compared to IMV. Conclusions NRS leads to distinct IGF1 mRNA variant levels and epigenetic profile in the hippocampus compared to IMV. Impact Our study shows that 3 or 21 days of NRS of preterm lambs leads to distinct IGF1 mRNA variant levels and epigenetic profile in the hippocampus compared to IMV. Preterm infant studies suggest that NRS leads to better neurodevelopmental outcomes later in life versus IMV. Also, duration of IMV is directly related to hippocampal damage; however, molecular players remain unknown. NRS, as a gentler mode of respiratory management of preterm neonates, may reduce damage to the immature hippocampus through an epigenetic mechanism.

A group of original papers is assembled for this thematic-papers issue on traditional Chinese medicine (TCM). The idea for a thematic-papers issue came from Associate Editor and Guest Editor, Jicheng Li. Jicheng's idea was to introduce concepts and studies of TCM to the editorial board of The Anatomical Record and the Journal's readership. Thus, nine papers are published that deal with biomarkers, animal models, or treatments of TCM syndromes. Uniquely, Jicheng's effort culminated in acupuncture treatment of the editorial board members by a licensed physician of TCM. We learned, first-hand by Dr. Weixue Wang, about this ancient and vibrant holistic philosophy and medical practice. Our pleasure is to share the topic and our experience with you.

Low levels of insulin-like growth factor-1 (IGF-1) protein in preterm human infants are associated with bronchopulmonary dysplasia (BPD). We used our preterm lamb model of BPD to determine (1) dosage of recombinant human (rh) IGF-1 bound to binding protein-3 (IGFBP-3) to reach infant physiologic plasma levels; and (2) whether repletion of plasma IGF-1 improves pulmonary and cardiovascular outcomes.Group 1: normal, unventilated lambs from 128 days gestation through postnatal age 5 months defined normal plasma levels of IGF-1. Group 2: continuous infusion of rhIGF-1/rhIGFBP-3 (0.5, 1.5, or 4.5 mg/kg/day; n = 2) for 3 days in mechanically ventilated (MV) preterm lambs determined that 1.5 mg/kg/day dosage attained physiologic plasma IGF-1 concentration of ~125 ng/mL, which was infused in four more MV preterm lambs.Group 1: plasma IGF-1 protein increased from ~75 ng/mL at 128 days gestation to ~220 ng/L at 5 months. Group 2: pilot study of the optimal dosage (1.5 mg/kg/day rhIGF-1/rhIGFBP-3) in six MV preterm lambs significantly improved some pulmonary and cardiovascular outcomes (p 0.1) compared to six MV preterm controls. RhIGF-1/rhIGFBP-3 was not toxic to the liver, kidneys, or lungs.Three days of continuous iv infusion of rhIGF-1/rhIGFBP-3 at 1.5 mg/kg/day improved some pulmonary and cardiovascular outcomes without toxicity.Preterm birth is associated with rapid decreases in serum or plasma IGF-1 protein level. This decline adversely impacts the growth and development of the lung and cardiovascular system. For this pilot study, continuous infusion of optimal dosage of rhIGF-1/rhIGFBP-3 (1.5 mg/kg/day) to maintain physiologic plasma IGF-1 level of ~125 ng/mL during mechanical ventilation for 3 days statistically improved some structural and biochemical outcomes related to the alveolar formation that would favor improved gas exchange compared to vehicle-control. We conclude that 3 days of continuous iv infusion of rhIGF-1/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs.

Research within the anatomical sciences often relies on human cadaveric tissues. Without the good will of these donors who allow us to use their bodies to push forward our anatomical knowledge, most human anatomical research would come to a standstill. However, many research papers omit an acknowledgement to the donor cadavers or, as no current standardized versions exist, use language that is extremely varied. To remedy this problem, 20 editors-in-chiefs from 17 anatomical journals joined together to put together official recommendations that can be used by authors when acknowledging the donor cadavers used in their studies. The goal of these recommendations is to standardize the writing approach by which donors are acknowledged in anatomical studies that use human cadaveric tissues. Such sections in anatomical papers will not only rightfully thank those who made the donation but might also encourage, motivate, and inspire future individuals to make such gifts for the betterment of the anatomical sciences and patient care.

Recent clinical studies have shown that traumatic brain injury is a significant risk factor for stroke. Motivated to better understand possible mechanisms of this association, we studied subfailure disruption of the intima in overstretched sheep cerebral arteries, as this has been implicated in the increased risk of stroke following blunt cerebrovascular injury. Middle cerebral arteries from four age groups (ranging from fetal to adult) were stretched axially to failure, and intimal disruption was captured with a video camera. All vessels demonstrated intimal disruption prior to catastrophic failure, with nearly all incurring disruption at stretch values well below those at ultimate stress (means of 1.56 and 1.73, respectively); the lowest stretch associated with intimal disruption was 1.29. The threshold of intimal failure was independent of age. Additional analysis showed that disruption included failure of both the endothelium and internal elastic lamina. Although our experiments were conducted at quasi-static rates, the results likely have important implications for vessel function following trauma. Future work should seek to identify subfailure disruption of the cerebrovasculature in head trauma.

Pulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that NF-κB promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF-κB activation in the pulmonary vasculature are unknown. To identify mechanisms that activate proangiogenic NF-κB signaling in the developing pulmonary vasculature, proteomic analysis of the lung secretome was performed using two-dimensional difference gel electrophoresis. NF-κB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PECs) and TGFBI (transforming growth factor-β-induced protein)-regulated genes identified using RNA sequencing. Alveolarization and pulmonary angiogenesis was assessed in wild-type and

RationalePulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that nuclear factor kappa-B (NFκB) promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NFκB activation in the pulmonary vasculature are unknown.ObjectivesTo identify mechanisms that activate pro-angiogenic NFκB signaling in the developing pulmonary vasculature.MethodsProteomic analysis of the lung secretome was performed using 2D-DIGE. NFκB activation and angiogenic function was assessed in primary pulmonary endothelial cells (PEC) and TGFBI-regulated genes identified using RNA-sequencing. Alveolarization and pulmonary angiogenesis was assessed in WT and TGFBI null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support.Measurements and Main ResultsSecreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PEC. Proteomic analysis identified transforming growth factor beta-induced protein (TGFBI) as a protein highly expressed by myofibroblasts in the early alveolar lung that promoted PEC migration by activating NFκB via αvβ3 integrins. RNA-sequencing identifiedCsf3as a TGFBI-regulated gene that enhances nitric oxide production in PEC. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization.ConclusionsOur studies identify TGFBI as a developmentally-regulated protein that promotes NFκB-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

Objectives To assess the National Institute of Child Health and Human Development's Pediatric Critical Care Trauma Scientist Development Program national K12 program. Design Mixed-methods study. Setting Pediatric Critical Care Trauma Scientist Development Program participants from 2005 to 2018. Subjects Past participants in the Pediatric Critical Care Trauma Scientist Development Program, including those who received funding (scholars), those who did not receive funding (applicants), and those who participated as diversity fellows. Interventions None. Measurements and main results Thirty-four past scholars, participants, and fellows in the Pediatric Critical Care Trauma Scientist Development were interviewed, including 19 women (56%) and 15 men (44%) via Skype. Interviews were audio recorded and transcribed, with permission. Codes were developed, using qualitative methods, that included the following: Community Building and Mentorship, Career and Research Development, and Tensions and Growth Opportunities. Quantitative data about physician-scholar grant success were retrieved from the National Institutes of Health system to search for funded grants, RePORT, physician-scholar curriculum vitae, and university websites. Since inception of the program, 46 scholars have been appointed. Scholars are equally split between women and men. Four members of the total cohort (9%) are from under-represented minority groups in medicine. Among the total past 46 participants, 72% of those who completed the K12 achieved an National Institutes of Health K-award and 36% of those not on K-level funding achieved at least one Research Program Grant-level award. All scholars, except one, remain academically active, as noted by recent publications in the peer reviewed literature; scholars from 2005 to 2013 are progressing in their careers, with 60% promoted to associate or full professor. Conclusions The Pediatric Critical Care Trauma Scientist Development Program is reaching its programmatic goals of buildin g a community of scientists in pediatric critical care and trauma surgery as shown by the qualitative analysis. Key challenges include increasing the diversity of applicants, encouraging applicants who are not funded, increasing the rate of K- to R-conversion, and preserving National Institute of Child Health and Human Development Program priorities for national K12 programs and individual K-awards.

Background Dysregulated nitric oxide (NO) signaling contributes to chronic hypoxia (CH)-induced pulmonary hypertension (PH). NO signaling is improved and pulmonary vascular resistance (PVR) is reduced in CH piglets treated with the l-arginine-NO precursor, l-citrulline. We hypothesized that l-citrulline might cause structural changes in the pulmonary circulation that would contribute to the reduction in PVR and that the l-citrulline-induced structural changes would be accompanied by alterations in vascular endothelial growth factor (VEGF) signaling. Methods We evaluated small pulmonary arterial (PA) wall thickness, lung capillary density, and protein abundances of VEGF, VEGFR2, and phospho (p)-VEGFR2 in PA and peripheral lung samples of piglets raised in the lab in CH (10%-12% O2 ) from the day of life (DOL) 2 until DOL 11 to 12 or raised in room air (normoxia) by the vendor and studied on arrival to the lab on DOL 11 to 12. Some CH piglets were treated with oral l-citrulline (1-1.5 g/kg/d) starting on the third day of hypoxia. Results PA wall thickness was 32% less and lung capillary formation was nearly doubled in l-citrulline treated than untreated CH piglets. Both of these l-citrulline-induced structural changes in the pulmonary circulation were accompanied by altered amounts of VEGF protein but not by altered amounts of either VEGFR2 or p-VEGFR2 proteins. Conclusions Alterations in the structure of the pulmonary circulation in CH piglets by l-citrulline are unlikely to be mediated by overall VEGF signaling. Nonetheless, l-citrulline- induced structural changes should reduce PVR and thereby contribute to the amelioration of CH-induced PH.

Diffusion tensor imaging (DTI) is an MRI technique that can be used to map cardiomyocyte tracts and estimate local cardiomyocyte and sheetlet orientation within the heart. DTI measures diffusion distances of water molecules within the myocardium, where water diffusion generally occurs more freely along the long axis of cardiomyocytes and within the extracellular matrix, but is restricted by cell membranes such that transverse diffusion is limited. DTI can be undertaken in fixed hearts and it allows the three-dimensional mapping of the cardiac microarchitecture, including cardiomyocyte organization, within the whole heart. The objective of this study was to use DTI to compare the cardiac microarchitecture and cardiomyocyte organization in archived fixed left ventricles of lambs that were born either preterm (n = 5) or at term (n = 7), at a postnatal timepoint equivalent to about 6 years of age in children. Although the findings support the feasibility of retrospective DTI scanning of fixed hearts, several hearts were excluded from DTI analysis because of poor scan quality, such as ghosting artifacts. The preliminary findings from viable DTI scans (n = 3/group) suggest that the extracellular compartment is altered and that there is an immature microstructural phenotype early in postnatal life in the LV of lambs born preterm. Our findings support a potential time-efficient imaging role for DTI in detecting abnormal changes in the microstructure of fixed hearts of former-preterm neonates, although further investigation into factors that affect scan quality is required.

Among Cetartiodactyla, cetaceans are the only obligate aquatic dwellers. Given morphological similarities between cetacean relatives such as Indohyus (the best represented Eocene raoellid artiodactyl) with other, later artiodactyls, any crown artiodactyl that engages in aquatic behaviors is of interest as an evolutionary model for the adaptations that accompanied the origins of cetaceans. The American moose (Alces alces) is the only non-cetacean artiodactyl to engage in aquatic foraging and, other than Hippopotamus, is distinctive in its diving behaviors. This study surveyed the soft and hard tissue nasal morphology of Alces alces to assess phylogenetic polarity and the presence of adaptations for diving and feeding in fresh water habitats. A fresh dissection of the facial musculature and nasal cavity was performed on one subadult male individual and osteological analyses were also performed on dry crania. This species was analyzed alongside fossil crania of Cervalces (its presumed ancestor), other cervids (e.g., Odocoileus virginianus, the white tail deer; Dama dama, the fallow deer), a bovid (Bos taurus, domestic cattle), and a carnivoran (Ursus americanus, the American black bear). A fresh dissection of the facial musculature and nasal anatomy of one fallow deer specimen was also performed for comparison with the moose. Results indicate that Alces alces exhibited a primitive configuration of maxillolabial muscles and, like Dama, exhibited a series of subcutaneous fibrous tissues connecting these muscles to skin. Alces and Dama, however, both exhibited autapomorphies in the soft tissue anatomy of the external nares. The former possessed a series of muscles that act to constrict the anterior nares, likely during diving. Extremely large fibrofatty pads that were perforated by muscle tendon supported their alar fold. Internally, a double-scrolled maxilloturbinal occupied nearly the entire volume of the anterior nasal cavity and protruded beyond the rim of the piriform aperture in dry crania. Dama had long, thin muscles taking origin on their nasal conchae and inserting onto the alar fold. Yet, despite these anterior nasal autapomorphies, the ethmoturbinal patterns of all observed cervids and the one bovid all appeared primitive with a posteroinferiorly oriented array of ethmoturbinals in close contact with a relatively straight cribriform plate, a macrosmatic condition. These differed from the curved cribriform plate of Ursus whose posterior nasal anatomy appeared hyper-macrosmatic. Indohyus exhibits no skeletal sign of a fleshy proboscis such as an enlarged piriform aperture or shortened nasal bones. Thus, there is little evidence that the early ancestors of cetaceans engaged in prolonged bouts of diving for aquatic foods but more probably were surface swimmers traveling between terrestrial food sources or fleeing predators. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:667-692, 2019. © 2018 Wiley Periodicals, Inc.

Preterm birth and mechanical ventilation (MV) frequently lead to bronchopulmonary dysplasia, the histopathological hallmark of which is alveolar simplification. How developmental immaturity and ongoing injury, repair, and remodeling impact completion of alveolar formation later in life is not known, in part because of lack of suitable animal models. We report a new model, using former-preterm lambs, to test the hypothesis that they will have persistent alveolar simplification later in life. Moderately preterm lambs (~85% gestation) were supported by MV for ~6 days before being transitioned from all respiratory support to become former-preterm lambs. Results are compared with term control lambs that were not ventilated, and between males (M) and females (F). Alveolar simplification was quantified morphometrically and stereologically at 2 mo (4 M, 4 F) or 5 mo (4 M, 6 F) corrected postnatal age (cPNA) compared with unventilated, age-matched term control lambs (4 M, 4 F per control group). These postnatal ages in sheep are equivalent to human postnatal ages of 1–2 yr and ~6 yr, respectively. Multivariable linear regression results showed that former-preterm lambs at 2 or 5 mo cPNA had significantly thicker distal airspace walls ( P < 0.001 and P < 0.009, respectively), lower volume density of secondary septa ( P < 0.007 and P < 0.001, respectively), and lower radial alveolar count ( P < 0.003 and P < 0.020, respectively) compared with term control lambs. Sex-specific differences were not detected. We conclude that moderate preterm birth and MV for ~6 days impedes completion of alveolarization in former-preterm lambs. This new model provides the opportunity to identify underlying pathogenic mechanisms that may reveal treatment approaches.

Integrity in the pursuit of discovery through application of the scientific method and reporting the results is an obligation for each of us as scientists. We cannot let the value of science be diminished because discovering knowledge is vital to understand ourselves and our impacts on the earth. We support the value of science by our stewardship of integrity in the conduct, training, reporting, and proposing of scientific investigation. The players who have these responsibilities are authors, reviewers, editors, and readers. Each role has to be played with vigilance for ethical behavior, including compliance with regulations for protections of study subjects, use of select agents and biohazards, regulations of use of stem cells, resource sharing, posting datasets to public repositories, and so on. The positive take-home message is that the scientific community is taking steps in behavior to protect the integrity of science. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 301:1481-1487, 2018. © 2018 Wiley Periodicals, Inc.

OBJECTIVE: To provide a non-invasive approach to monitoring lung function in spontaneously breathing lambs, from birth to five months of life, by the forced oscillation technique (FOT). This report describes the experimental set-up, data processing, and identification of normal predicted values of resistance (Rrs) and reactance (Xrs) of the respiratory system, along with normal bronchodilator response for bronchial reversibility testing. APPROACH: Rrs and Xrs at 5, 11, and 19 Hz were measured monthly for five months in 20 normal term lambs that breathed spontaneously. In seven lambs, repeated measurements also were made within the first month of life (at 3, 7, 14, and 21 d of life). We determined the repeatability and reproducibility of the measurements and characterized the relationship between lung mechanics and age, sex, and body dimensions, using regression analysis, and measured changes in lung mechanics in response to inhaled bronchodilator. MAIN RESULTS: The measurements provided repeatable and reproducible data. Rrs decreased, whereas Xrs increased, with growth from birth through the first two months of life, after which no statistically significant differences were detected. We identified normal value equations for Rrs and Xrs and for each of the measured anthropometric variables. Respiratory system mechanics were not affected by the bronchodilator. SIGNIFICANCE: The FOT provides reliable non-invasive measurement of respiratory system mechanics in spontaneously breathing term lambs from birth to five months of age. The methods and normal reference values defined in this study will facilitate testing of the pathophysiological consequences of preterm birth and prolonged respiratory support on respiratory system mechanics.

Diffusion tensor imaging (DTI) has emerged as a promising method for noninvasive quantification of myocardial microstructure. However, the origin and behavior of DTI measurements during myocardial normal development and remodeling remain poorly understood. In this work, conventional and bicompartmental DTI in addition to three-dimensional histological correlation were performed in a sheep model of myocardial development from third trimester to postnatal 5 months of age. Comparing the earliest time points in the third trimester with the postnatal 5 month group, the scalar transverse diffusivities preferentially increased in both left ventricle (LV) and right ventricle (RV): secondary eigenvalues D2 increased by 54% (LV) and 36% (RV), whereas tertiary eigenvalues D3 increased by 85% (LV) and 67% (RV). The longitudinal diffusivity D1 changes were small, which led to a decrease in fractional anisotropy by 41% (LV) and 33% (RV) in 5 month versus fetal hearts. Histological analysis suggested that myocardial development is associated with hyperplasia in the early stages of the third trimester followed by myocyte growth in the later stages up to 5 months of age (increased average myocyte width by 198%, myocyte length by 128%, and decreased nucleus density by 70% between preterm and postnatal 5 month hearts.) In a few histological samples (N = 6), correlations were observed between DTI longitudinal diffusivity and myocyte length (r = 0.86, P

Background: Ventilated preterm infants may have respiratory morbidities early in life and later at school age and adulthood (Clemm HH, 2014). Modeling with large animals is needed to identify mechanisms Aim: Use a new former preterm (FPT) lamb model to evaluate lung mechanics, airway reactivity, and airway smooth muscle (ASM) thickness. Methods: 6 preterm lambs (128d; term ~150d; ~28w gestation in humans) were resuscitated and supported by mechanical ventilation for 3-6d before weaning from respiratory support (FPT lambs). They lived for ~6m (~5m corrected postnatal age; ≈6y in humans). Control lambs (n=20), born at term, lived 5m. We measured respiratory mechanics monthly by forced oscillation technique (FOT; 11Hz) and airway reactivity by changes in FOT variables after increased doses of nebulized methacholine (MCh; 0.06-4.00 mg; ATS guideline, 1999). We used quantitative histology to measure ASM thickness in terminal bronchioles. Results: FPT lambs had increased airway reactivity during the first 2m of life (figure). ASM thickness was significantly greater in FPT lambs (6.7±1.1vs 3.1±0.5 mm control lambs). Conclusions: The new FPT lamb model recapitulates altered lung mechanics and airway reactivity in FPT humans. Interestingly, airway muscularization persists at 5m when lung mechanics and airway reactivity are no longer impaired. This model provides opportunity to identify pathogenic mechanisms.

Background: SLI at birth has been shown to promote lung aeration in intubated preterm lambs (Tingay et al PedRes 2014). The effectiveness of applying SLI by full-face mask might be impaired by leaks, glottis closure, and accumulation of air in the stomach. Aim: To compare timing and magnitude of lung aeration at birth in preterm lambs receiving non-invasive resuscitation with and without the application of SLI. Methods: Preterm lambs (128d; ~28w in humans) were randomly assigned to receive SLI at birth (SLI group, n=7) or not (noSLI group, n=6). SLI group received two SLIs of 35s at 30cmH2O separated by 55s of nIPPV. All lambs received surfactant and were maintained with nIPPV (PEEP=8cmH2O, RR=60). PIP and FiO2 were adjusted to maintain PaCO2 at 35-55 mmHg and PaO2 at 60-80 mmHg up to 3h of life in both groups. Lung aeration was assessed as changes in lung reactance (Xrs) at 5Hz (Dellaca’ et al, ICM2011) measured by the forced oscillation technique. Data reported as median (IQR). Results: In both groups, Xrs increased with time (Figure). Xrs was significantly greater in SLI group at the end of each SLI and more variable in noSLI group. At 3h, Xrs stabilized at similar values for both groups. Conclusions: SLI applied non-invasively improved lung aeration and a more homogenous response in preterm lambs at birth. However, the improvements compared to CPAP disappear within few minutes.

Objective To (1) compare physiologic changes during rigid bronchoscopy during spontaneous and mechanical ventilation and (2) evaluate the efficacy of a helium-oxygen (heliox) gas mixture as compared with room air during rigid bronchoscopy. Study Design Crossover animal study evaluating physiologic parameters during rigid bronchoscopy. Outcomes were compared with predicted computational fluid analysis. Setting Simulated ventilation via computational fluid dynamics analysis and term lambs undergoing rigid bronchoscopy. Methods Respiratory and physiologic outcomes were analyzed in a lamb model simulating bronchoscopy during foreign body aspiration to compare heliox with room air. The main outcome measures were blood oxygen saturation, heart rate, blood pressure, partial pressure of oxygen, and partial pressure of carbon dioxide. Computational fluid dynamics analysis was performed with SOLIDWORKS within a rigid pediatric bronchoscope during simulated ventilation comparing heliox with room air. Results For room air, lambs desaturated within 3 minutes during mechanical ventilation versus normal oxygen saturation during spontaneous ventilation ( P = .01). No improvement in respiratory outcomes was seen between heliox and room air during mechanical ventilation. Computational fluid dynamics analysis demonstrates increased turbulence within size 3.5 bronchoscopes when comparing heliox and room air. Meaningful comparisons could not be made due to the intolerance of the lambs to heliox in vivo. Conclusion During mechanical ventilation on room air, lambs desaturate more quickly during rigid bronchoscopy on settings that should be adequate. Heliox does not improve ventilation during rigid bronchoscopy.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature human infants, which may persist through adulthood. Airway inflammation has been firmly established in the pathogenesis of BPD. Previous studies to reduce airway inflammation with high-dose dexamethasone demonstrated adverse neurological outcomes, despite lower incidences of BPD. Instillation of budesonide and surfactant can facilitate early extubation and reduce the incidence of BPD and death among very low birth weight infants. However, the pharmacokinetics of budesonide and its distribution into the lung and brain are unknown. Therefore, 5 premature lambs were administered 0.25 mg/kg budesonide, with surfactant as the vehicle. Plasma and tissue samples were taken from the lambs for measurement of budesonide, 16α-hydroxy prednisolone, and budesonide palmitate using LC/MS/MS. Peak plasma budesonide concentrations were inversely correlated with the oxygenation index (correlation coefficient of −0.75). Plasma budesonide concentrations were extremely low (~10% of expected) for two lambs that had high oxygenation indices and were excluded from further analyses. For the remaining 5 premature lambs, a non-compartmental analysis demonstrated an AUCinf of 148.77 ± 28.16 h*μg/L, half-life of 4.76 ± 1.79 h, and Cmax of 46.17 ± 17.71 μg/L. Using population pharmacokinetic methods, a one-compartment model with exponential residual error and first-order absorption adequately described the data. The apparent clearance and apparent volume of distribution of budesonide were estimated at 6.29 L/h (1.99 L/h/kg) and 29.1 L (9.2 L/kg), respectively. Budesonide and budesonide palmitate, but not 16α-hydroxy prednisolone, were detected in lung tissue. In this study, budesonide and its metabolites were not detected in the brain, which suggests that intratracheal instillation suggests that after local pulmonary deposition, there is no evidence of budesonide accumulation in the central nervous system. Overall, these results show that peak plasma budesonide concentrations are inversely correlated with the oxygenation index and that lung-specific delivery of budesonide avoids accumulation of budesonide in the brain.