Shannon E. Washburn

Maternal and Neonatal Plasma MicroRNA Biomarkers for Fetal Alcohol Exposure in an Ovine Model

BACKGROUND Plasma or circulating miRNAs (cir miRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol (EtOH) altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. METHODS Pregnant sheep were exposed to a binge model of EtOH consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a third-trimester-equivalent period from gestational day 4 (GD4) to GD132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte, and leukocytes obtained from nonpregnant ewes, and plasma from pregnant ewes 24 hours following the last binge EtOH episode, and from newborn lambs, at birth on ~GD147. RESULTS Pregnant ewe and newborn lamb cir miRNA profiles were similar to each other and different from nonpregnant female plasma, erythrocyte, or leukocyte miRNAs. Significant changes in cir miRNA profiles were observed in the EtOH-exposed ewe and, at birth, in the in utero, EtOH-exposed lamb. Cir miRNAs including miR-9, -15b, -19b, and -20a were sensitive and specific measures of EtOH exposure in both pregnant ewe and newborn lamb. Additionally, EtOH exposure altered guide-to-passenger strand cir miRNA ratios in the pregnant ewe, but not in the lamb. CONCLUSIONS Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. Cir miRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment.

Different patterns of regional Purkinje cell loss in the cerebellar vermis as a function of the timing of prenatal ethanol exposure in an ovine model

Studies in rat models of fetal alcohol spectrum disorders have indicated that the cerebellum is particularly vulnerable to ethanol-induced Purkinje cell loss during the third trimester-equivalent, with striking regional differences in vulnerability in which early-maturing regions in the vermis show significantly more loss than the late-maturing regions. The current study tested the hypothesis that the sheep model will show similar regional differences in fetal cerebellar Purkinje cell loss when prenatal binge ethanol exposure is restricted to the prenatal period of brain development equivalent to the third trimester and also compared the pattern of loss to that produced by exposure during the first trimester-equivalent. Pregnant Suffolk sheep were assigned to four groups: first trimester-equivalent saline control group, first trimester-equivalent ethanol group (1.75 g/kg/day), third trimester-equivalent saline control group, and third trimester-equivalent ethanol group (1.75 g/kg/day). Ethanol was administered as an intravenous infusion on 3 consecutive days followed by a 4-day ethanol-free interval, to mimic a weekend binge drinking pattern. Animals from all four groups were sacrificed and fetal brains were harvested on gestation day 133. Fetal cerebellar Purkinje cell counts were performed in an early-maturing region (lobules I-X) and a late-maturing region (lobules VIc-VII) from mid-sagittal sections of the cerebellar vermis. As predicted, the third trimester-equivalent ethanol exposure caused a significant reduction in the fetal cerebellar Purkinje cell volume density and Purkinje cell number in the early-maturing region, but not in the late-maturing region. In contrast, the first trimester-equivalent ethanol exposure resulted in significant reductions in both the early and late-maturing regions. These data confirmed that the previous findings in rat models that third trimester-equivalent prenatal ethanol exposure resulted in regionally-specific Purkinje cell loss in the early-maturing region of the vermis, and further demonstrated that first trimester ethanol exposure caused more generalized fetal cerebellar Purkinje cell loss, independent of the cerebellar vermal region. These findings support the idea that prenatal ethanol exposure in the first trimester interferes with the genesis of Purkinje cells in an unselective manner, whereas exposure during the third trimester selectively kills post-mitotic Purkinje cells in specific vermal regions during a vulnerable period of differentiation and synaptogenesis.

Formate metabolism in fetal and neonatal sheep.

By virtue of its role in nucleotide synthesis, as well as the provision of methyl groups for vital methylation reactions, one-carbon metabolism plays a crucial role in growth and development. Formate, a critical albeit neglected component of one-carbon metabolism, occurs extracellularly and may provide insights into cellular events. We examined formate metabolism in chronically cannulated fetal sheep (gestation days 119-121, equivalent to mid-third trimester in humans) and in their mothers as well as in normal full-term lambs. Plasma formate levels were much higher in fetal lamb plasma and in amniotic fluid (191 ± 62 and 296 ± 154 μM, respectively) than in maternal plasma (33 ± 13 μM). Measurements of folate, vitamin B12, and homocysteine showed that these high formate levels could not be due to vitamin deficiencies. Elevated formate levels were also found in newborn lambs and persisted to about 8 wk of age. Formate was also found in sheep milk. Potential precursors of one-carbon groups were also measured in fetal and maternal plasma and in amniotic fluid. There were very high concentrations of serine in the fetus (∼1.6 mM in plasma and 3.5 mM in the amniotic fluid) compared with maternal plasma (0.19 mM), suggesting increased production of formate; however, we cannot rule out decreased formate utilization. Dimethylglycine, a choline metabolite, was also 30 times higher in the fetus than in the mother.

The Role of Acidemia in Maternal Binge Alcohol-Induced Alterations in Fetal Bone Functional Properties

BACKGROUND Heavy alcohol consumption during pregnancy negatively impacts the physical growth of the fetus. Although the deleterious effects of alcohol exposure during late gestation on fetal brain development are well documented, little is known about the effect on fetal bone mechanical properties or the underlying mechanisms. The purpose of this study was to investigate the effects of late gestational chronic binge alcohol consumption and alcohol-induced acidemia, a critical regulator of bone health, on functional properties of the fetal skeletal system. METHODS Suffolk ewes were mated and received intravenous infusions of saline or alcohol (1.75 g/kg) over 1 hour on 3 consecutive days per week followed by 4 days without treatment beginning on gestational day (GD) 109 and concluding on GD 132 (term = 147 days). The acidemia group was exposed to increased inspired fractional concentrations of CO2 to closely mimic the alcohol-induced decreases in maternal arterial pH seen in the alcohol group. RESULTS Fetal femurs and tibias from the alcohol and acidemia groups were ~3 to 7% shorter in length compared with the control groups (p < 0.05). Three-point bending procedure demonstrated that fetal femoral ultimate strength (MPa) for the alcohol group was decreased (p < 0.05) by ~24 and 29%, while the acidemia group exhibited a similar decrease (p < 0.05) of ~32 and 37% compared with the normal control and saline control groups, respectively. Bone extrinsic and intrinsic mechanical properties including maximum breaking force (N) and normalized breaking force (N/kg) of fetal bones from the alcohol and acidemia groups were significantly decreased (p < 0.05) compared with both control groups. CONCLUSIONS We conclude that late gestational chronic binge alcohol exposure reduces growth and impairs functional properties of the fetal skeletal system and that the repeated episodes of alcohol-induced maternal acidemia may be at least partially responsible for these effects.

Interactive effects of in vitro binge-like alcohol and ATP on umbilical endothelial nitric oxide synthase post-translational modifications and redox modulation

Alcohol dysregulates the regulation of reproductive vascular adaptations. We herein investigated chronic in vitro binge-like alcohol effects on umbilical endothelial nitric oxide synthase (eNOS) multi-site phosphorylation and related redox switches under basal (unstimulated) and stimulated (with ATP) states. Alcohol decreased endothelial excitatory (Pser1177)eNOS (P<0.001), whereas excitatory (Pser635)eNOS exhibited a main effect of alcohol (↓P=0.016) and ATP (↑P<0.001). Alcohol decreased (Pthr495)eNOS (P=0.004) levels, whereas inhibitory (Pser116)eNOS exhibited an alcohol main effect in both basal and stimulated states (↑P=0.005). Total eNOS was reduced by alcohol (P=0.038). In presence of ATP, alcohol inhibited ERK activity (P=0.002), whereas AKT exhibited no alcohol effect. Alcohol main effect on S-nitroso-glutathione reductase (↓P=0.031) and glutathione-S-transferase (↓P=0.027) were noted. Increased protein glutathiolation was noted, whereas no alcohol effect on GSH, GSSG, NOX2 or SOD expression was noted. Thus, alcohol effects on multi-site post-translational modifications and redox switches related to vasodilatory eNOS underscore the necessity for investigating alcohol-induced gestational vascular dysfunction.

Effects of all three trimester moderate binge alcohol exposure on the foetal hippocampal formation and olfactory bulb

Abstract Objective: Pre-natal alcohol exposure results in injury to the hippocampus and olfactory bulb, but currently there is no consensus on the critical window of vulnerability. This study tested the hypothesis that pre-natal exposure to a moderate dose of alcohol during all three trimester-equivalents alters development of the hippocampal formation and olfactory bulb in an ovine model, where all brain development occurs pre-natally as it does in humans. Research design and methods: Pregnant sheep were divided into saline control and a binge drinking groups (alcohol dose 1.75 g kg−1; mean peak blood alcohol concentration 189 + 19 mg dl−1). Outcome and results: The density, volume and total cell number were not different between groups for the dentate gyrus, pyramidal cells in the CA1 and CA2/3 fields and mitral cells in the olfactory bulb. Conclusions: A moderate dose of alcohol administered in a binge pattern throughout gestation does not alter cell numbers in the hippocampus or olfactory bulb and exposure during the third trimester-equivalent is required for hippocampal injury, unless very high doses of alcohol are administered. This has important implications in establishing the sensitivity of imaging modalities such as MRI in which volumetric measures are being studied as biomarkers for pre-natal alcohol exposure.

Video-based data acquisition system for use in eye blink classical conditioning procedures in sheep

Pavlovian eye blink conditioning (EBC) has been extensively studied in humans and laboratory animals, providing one of the best-understood models of learning in neuroscience. EBC has been especially useful in translational studies of cerebellar and hippocampal function. We recently reported a novel extension of EBC procedures for use in sheep, and now describe new advances in a digital video-based system. The system delivers paired presentations of conditioned stimuli (CSs; a tone) and unconditioned stimuli (USs; an air puff to the eye), or CS-alone “unpaired” trials. This system tracks the linear distance between the eyelids to identify blinks occurring as either unconditioned (URs) or conditioned (CRs) responses, to a resolution of 5 ms. A separate software application (Eye Blink Reviewer) is used to review and autoscore the trial CRs and URs, on the basis of a set of predetermined rules, permitting an operator to confirm (or rescore, if needed) the autoscore results, thereby providing quality control for accuracy of scoring. Learning curves may then be quantified in terms of the frequencies of CRs over sessions, both on trials with paired CS–US presentations and on CS-alone trials. The latency to CR onset, latency to CR peak, and occurrence of URs are also obtained. As we demonstrated in two example cases, this video-based system provides efficient automated means to conduct EBC in sheep and can facilitate fully powered studies with multigroup designs that involve paired and unpaired training. This can help extend new studies in sheep, a species well suited for translational studies of neurodevelopmental disorders resulting from gestational exposure to drugs, toxins, or intrauterine distress.

Evaluation of performance and perceptions of electronic vs. paper multiple-choice exams

In the veterinary professional curriculum, methods of examination in many courses are transitioning from the traditional paper-based exams to electronic-based exams. Therefore, a controlled trial to evaluate the impact of testing methodology on examination performance in a veterinary physiology course was designed and implemented. Formalized surveys and focus group discussions were also used to determine student attitudes toward the examination formats. In total, 134 first-year veterinary students and 11 PhD/MS students were administered a total of 4 exams throughout 1 semester (2 on paper and 2 electronically) using a split-halves design. The paper (P) and electronic (E) exams contained 25 identical multiple-choice questions. Students were randomly assigned to two groups and were given exams in one of two sequences (E-P-E-P or P-E-P-E). Participants consented to and completed two anonymous surveys vis à vis their experience. Out of a maximum raw score of 25, the mean score for electronic examinations (20.8; 95% confidence interval, 20.3-21.2) was significantly (P = 0.01) greater than that for paper examinations (20.3; 95% confidence interval, 20.0-20.7). However, students expressed numerous concerns with the electronic examination format, and, at the completion of the study, 87% preferred to take their examination on paper rather than the electronic format. These data show that student attitudes concerning the examination format are not primarily determined by examination results, and that the additional anxiety related to the electronic examination format plays a large role in student attitudes.

Merging Clinical Cases, Client Communication, and Physiology to Enhance Student Engagement, Learning, and Skills

Understanding disease processes, making diagnoses, and guiding clinical therapy are predicated on an understanding of normal physiologic function. However, we have observed that many first-year students fail to appreciate the important role that a clear understanding of normal function plays in becoming well-prepared, practicing veterinarians. Students also struggle with application of basic knowledge to the diagnosis and treatment of disease, as evidenced by poor performance on exam questions requiring application. The purpose of this project was to help students link the physiologic concepts in the classroom with clinical application, as well as to improve their ability to explain those concepts to a client. We found that, as a result of this assignment, students developed a deeper understanding of physiologic processes and their clinical relevance and, subsequently, felt more confident conveying this knowledge to simulated clients. Implementation of this case project has been very well received by the students. Students improved their grasp of the material, and they indicated that the project contributed positively to their motivation to study and learn physiology.

Maternal and fetal effects of dexmedetomidine infusion in pregnant ewes anesthetized with sevoflurane

OBJECTIVE To characterize the maternal and fetal cardiopulmonary effects of a low-dose infusion of dexmedetomidine without a loading dose in pregnant ewes anesthetized with sevoflurane. ANIMALS 11 pregnant ewes. PROCEDURES Anesthesia was induced with propofol and maintained with sevoflurane. Ewes and fetuses were instrumented with arterial and venous catheters, and thermodilution-pulmonary arterial catheters were placed in the ewes. Baseline measurements were obtained at an end-tidal sevoflurane concentration of 3.4%, then dexmedetomidine (2 μg/kg/h, IV) was infused for 90 minutes without a loading dose. Cardiovascular and blood gas variables were measured at predetermined time points. RESULTS Dexmedetomidine infusion resulted in approximately 30% decreases in maternal systemic vascular resistance, blood pressure, and heart rate. Maternal cardiac index, oxygenation variables, and acid-base status remained unchanged, whereas pulmonary arterial pressure, pulmonary vascular resistance, and stroke volume increased, compared with baseline values. Uterine blood flow decreased by approximately 30% to 36%. Fetal heart rate and blood pressure remained unchanged, but significant increases in fetal plasma glucose and lactate concentrations were detected. CONCLUSIONS AND CLINICAL RELEVANCE Pregnant ewes receiving a combination of sevoflurane and an infusion of dexmedetomidine without a loading dose had cardiac index in acceptable ranges and maintained normoxia. This balanced anesthesia did not produce significant changes in fetal blood pressure or heart rate. However, the increase in fetal plasma lactate concentration and changes in maternal pulmonary vascular resistance and uterine blood flow require further investigation to better elucidate these effects.