Chronic Binge Alcohol Exposure During Pregnancy Alters mTOR System in Rat Fetal Hippocampus.

BACKGROUND: Gestational alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), an array of cognitive, behavioral, and physical developmental impairments. Mammalian target of rapamycin (mTOR) plays a key role in regulating protein synthesis in response to neuronal activity, thereby modulating synaptic plasticity and long-term memory formation in the brain. Based on our previous quantitative mass spectrometry proteomic studies, we hypothesized that gestational chronic binge alcohol exposure alters mTOR signaling and downstream pathways in the fetal hippocampus. METHODS: Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol (peak BAC, 216 mg/dl) from GD 5-10 and progressed to 6 g/kg alcohol (peak BAC, 289 mg/dl) from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin. RESULTS: In the Alcohol group, following this exposure paradigm, fetal body weight and crown-rump length were decreased. The phosphorylation level of mTOR (P-mTOR) in the fetal hippocampus was decreased in the Alcohol group compared with controls. Alcohol exposure resulted in dysregulation of fetal hippocampal mTORC1 signaling, as evidenced by an increase in total 4E-BP1 expression. Phosphorylation levels of 4E-BP1 and p70 S6K were also increased following alcohol exposure. P-mTOR and P-4E-BP1 were exclusively detected in the dentate gyrus and oriens layer of the fetal hippocampus, respectively. DEPTOR and RICTOR expression levels in the fetal hippocampus were increased; however, RAPTOR was not altered by chronic binge alcohol exposure. CONCLUSION: We conclude that chronic binge alcohol exposure during pregnancy alters mTORC1 signaling pathway in the fetal hippocampus. We conjecture that this dysregulation of mTOR protein expression, its activity, and downstream proteins may play a critical role in FASD neurobiological phenotypes.

Impact of e-cigarette vaping aerosol exposure in pregnancy on mTOR signaling in rat fetal hippocampus.

Electronic cigarette (e-cig) use during pregnancy has become a major health concern in recent years and many view them as less harmful and may help quit or reduce combustible cigarettes. Implementing a state-of-the-art engineered vaping system, comprising an atomizer similar to those sold in vape shops, we aimed to utilize a translational e-cig inhalation delivery method to provide crucial information on the impact of prenatal e-cig aerosols on the developing brain hippocampal mTOR system in a rat model system. Gestational e-cig vaping significantly increased P-mTOR levels (p < 0.05) in the rat fetal hippocampi in the nicotine group (comprising of VG/PG + nicotine) compared to the control and the juice (comprising of VG/PG) groups. Total mTOR expression was not different among groups. Immunofluorescence imaging demonstrated P-mTOR was detected exclusively in the granule cells of the dentate gyrus of the fetal hippocampus. E-cig did not alter DEPTOR, but RAPTOR and RICTOR were higher (p < 0.05) in the Nicotine group. Gestational e-cig vaping with nicotine increased (p < 0.05) the activity and expression of 4EBP1, p70S6K, but decreased (p < 0.05) P-PKCα in the fetal hippocampi. In summary, dysregulation of mTORC1 and the related mTORC2, their activity, and downstream proteins together may play a critical role in e-cig-vaping-induced neurobiological phenotypes during development.

A Forehead Wearable Sensor for the Objective Measurement of Chronic Pain.

Chronic pain impacts one in five Americans and is difficult to manage, costing ~USD 600 billion annually. The subjective experience of pain is a complex processing of central nervous system input. Recent advances in magnetic resonance imaging revealed the prefrontal cortex as vital to the perception of pain and that changes in the cerebral hemodynamics can be used to detect painful sensations. Current pain monitoring is dependent on the subjective rating provided by patients and is limited to a single time point. We have developed a biomarker for the objective, real-time and continuous chronic pain assessment using proprietary algorithms termed ROPA and cerebral optical spectrometry. Using a forehead sensor, the cerebral optical spectrometry data were collected in two clinical sites from 41 patients (19 and 22, respectively, from sites 1 and 2), who elected to receive an epidural steroid injection for the treatment of chronic pain. Patients rated their pain on a numeric rating scale, ranging from 0-10, which were used to validate the ROPA objective pain scoring. Multiple time points, including pre- and post-procedure were recorded. The steroid injection was performed per standard medical practice. There was a significant correlation between the patient's reported numeric rating scale and ROPA, for both clinical sites (Overall ~0.81). Holding the subjective pain ratings on a numeric rating scale as ground truth, we determined that the area under the receiver operator curves for both sites revealed at least good (AUC: 64%) to excellent (AUC > 98%) distinctions between clinically meaningful pain severity differentiations (no/mild/moderate/severe). The objective measure of chronic pain (ROPA) determined using cerebral optical spectrometry significantly correlated with the subjective pain scores reported by the subjects. This technology may provide a useful method of detection for the objective and continuous monitoring and treatment of patients with chronic pain, particularly in clinical circumstances where direct assessment is not available, or to complement the patient-reported pain scores.

Interaction of alcohol & phosphatidic acid in maternal rat uterine artery function.

Alcohol has been demonstrated to impair maternal uterine arterial adaptations in Fetal Alcohol Spectrum Disorder (FASD) animal models. However, the exact mechanism remains inconclusive. We hypothesized that phosphatidic acid (PA), a direct target of alcohol metabolism, would alleviate alcohol-induced vascular dysfunction of the maternal uterine artery. Mean fetal weight, and crown-rump length of the alcohol administered rats were ~9 % and 7.6 % lower than the pair-fed control pups, respectively. Acetylcholine (Ach)-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats (P < 0.05). Supplementation of 10-5 M PA reversed alcohol-induced vasodilatory deficit; no difference was detected after PA treatment between pair-fed control and alcohol groups (P = 0.37). There was a significant interaction between PA concentrations and alcohol exposure (PA X Alcohol effect, P < 0.0001). Pair-wise comparisons showed a concentration-dependent vasodilatory effect on uterine arteries of the alcohol-administered rats, with % relaxation significantly improved at PA concentrations > 10-7 M (P < 0.05). Alcohol significantly reduced vasodilatory P-Ser1177 endothelial nitric oxide synthase (eNOS) levels in the uterine artery (↓90.7 %; P = 0.0029). PA treatment significantly reversed P-Ser1177 eNOS level in alcohol uterine arteries (153.7 %↑; P = 0.005); following ex vivo PA, there was no difference in P-Ser1177 eNOS levels between Control and Alcohol. Neither alcohol treatment nor PA affected total eNOS levels. Our data provide the first evidence of the interaction of alcohol and PA in rat maternal uterine artery vascular function and demonstrates PA's relationship with the eNOS system. Overall, the current study demonstrates that PA may be a promising therapeutic molecule of interest in alcohol-related gestational vascular dysfunction.

Impact of E-cig aerosol vaping on fetal and neonatal respiratory development and function.

Electronic cigarette (e-cig) use has increased over the past decade, and exposure to e-cig aerosols during pregnancy raises concern for maternal and fetal health. The developing fetal lung is known to be sensitive to prenatal tobacco product exposure. Utilizing a 3-pronged approach, we examined the effects of prenatal e-cig aerosols with, and without nicotine on respiratory development in a murine model. RNAseq analysis of fetal lungs revealed extensive dysregulation in gene expression. Morphologic assessment of distal airspaces in neonatal lungs display an emphysematic phenotype. Respiratory mechanics of neonates display signs of increased respiratory workload, with increased resistance and decreased compliance. These data are novel and provide evidence that prenatal e-cig exposure may result in altered lung function or development of disease.

Morphological alteration in rat hippocampal neuronal dendrites following chronic binge prenatal alcohol exposure.

Prenatal alcohol exposure (PAE) may result in Fetal Alcohol Spectrum Disorders (FASD). The hippocampus has been recognized as a vulnerable target to alcohol-induced developmental damage. However, the effect of prenatal exposure to alcohol on dendritic morphological adaptations throughout the hippocampal fields in the developing brain still remains largely unknown in the context of FASD. We hypothesized that chronic binge alcohol exposure during pregnancy alters dendrite arborization throughout the developing rat hippocampus. Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol from gestational day (GD) 5-10 and progressed to 6 g/kg alcohol from GD 11-21. Pair-fed dams similarly received isocaloric maltose dextrin. After parturition, all dams received an ad libitum diet and nursed their offspring until postnatal day (PND) 10 when the pup brains were collected for morphological analysis. PAE increased dendritic arborization and complexities of CA1, CA2/3, and DG neurons in the PND 10 rat hippocampus. The number of primary dendrites, total dendritic length, and number of dendritic branches were significantly increased following PAE, and Sholl analysis revealed significantly more intersections of the dendritic processes in PND 10 offspring following PAE compared with those in the PF-Cont group. We conclude that chronic binge PAE significantly alters hippocampal dendritic morphology in the developing hippocampus. We conjecture that this morphological alteration in postnatal rat hippocampal dendrites following chronic binge prenatal alcohol exposure may play a critical role in FASD neurobiological phenotypes.

Impact of gestational electronic cigarette vaping on amino acid signature profile in the pregnant mother and the fetus.

BACKGROUND: Electronic cigarettes (e-cigs) are a form of tobacco product that has become increasingly popular over the past decade. Despite the known health consequences of tobacco product exposure during pregnancy, a substantial number of daily smokers will continue to smoke during pregnancy. Our current knowledge on the effects of e-cig aerosol exposure during pregnancy is limited to a small number of animal studies, which have identified several e-cig aerosol-induced disruptions to the physiology of normal development. METHODS: To further assess the impact of prenatal e-cig aerosol exposure on maternal and fetal health, we examined the amino acid signature profiles in maternal and fetal plasma, as well as in the fetal lungs, a sensitive target organ for prenatal tobacco product exposure. Pregnant Sprague Dawley rats were randomly assigned to one of three groups and were exposed to either e-cig aerosols containing nicotine, e-cig aerosols without nicotine, or room air. Dams were exposed utilizing a state-of-the-art custom engineered e-cig vaping system that is compatible with commercially available e-cig atomizers and enables a translational inhalation delivery method comparable to human vaping. RESULTS: We determined that gestational exposure to e-cig aerosols results in significant alterations to the amino acid profile in the maternal and fetal compartments, including the fetal lungs. The data shows a targeted disruption to the nitric oxide pathway, branched-chain amino acid metabolism, fetal protein synthesis, and urea cycle. CONCLUSION: The data presented herein provides additional support that gestational e-cig aerosol exposure can impact crucial biological processes and exemplifies the need for extensive research on exposure to e-cig aerosols.

Impact of Electronic Cigarette Aerosols on Pregnancy and Early Development.

As the use of electronic cigarettes (e-cigs) continues to increase, especially among youth and pregnant women, so does the need for investigations into the effects of e-cig aerosols on prenatal development and early life. Herein, the most recent findings on the effects of e-cig aerosols during pregnancy and early life are reviewed. The results of these studies support the need for immediate action to further understand the potential harm that e-cigs may cause to pregnant women and their children. The effects of e-cigs is completely unknown in regards to human development. This review provides evidence that e-cigs may be harmful to early life development and that the use of e-cigs should be avoided during pregnancy.

Hippocampal transcriptome reveals novel targets of FASD pathogenesis.

INTRODUCTION: Prenatal alcohol exposure can contribute to fetal alcohol spectrum disorders (FASD), characterized by a myriad of developmental impairments affecting behavior and cognition. Studies show that many of these functional impairments are associated with the hippocampus, a structure exhibiting exquisite vulnerability to developmental alcohol exposure and critically implicated in learning and memory; however, mechanisms underlying alcohol-induced hippocampal deficits remain poorly understood. By utilizing a high-throughput RNA-sequencing (RNA-seq) approach to address the neurobiological and molecular basis of prenatal alcohol-induced hippocampal functional deficits, we hypothesized that chronic binge prenatal alcohol exposure alters gene expression and global molecular pathways in the fetal hippocampus. METHODS: Timed-pregnant Sprague-Dawley rats were randomly assigned to a pair-fed control (PF) or binge alcohol (ALC) treatment group on gestational day (GD) 4. ALC dams acclimatized from GDs 5-10 with a daily treatment of 4.5 g/kg alcohol and subsequently received 6 g/kg on GDs 11-20. PF dams received a once daily maltose dextrin gavage on GDs 5-20, isocalorically matching ALC counterparts. On GD 21, bilateral hippocampi were dissected, flash frozen, and stored at -80° C. Total RNA was then isolated from homogenized tissues. Samples were normalized to ~4nM and pooled equally. Sequencing was performed by Illumina NextSeq 500 on a 75 cycle, single-end sequencing run. RESULTS: RNA-seq identified 13,388 genes, of these, 76 genes showed a significant difference (p < 0.05, log2 fold change ≥2) in expression between the PF and ALC groups. Forty-nine genes showed sex-dependent dysregulation; IPA analysis showed among female offspring, dysregulated pathways included proline and citrulline biosynthesis, whereas in males, xenobiotic metabolism signaling and alaninine biosynthesis etc. were altered. CONCLUSION: We conclude that chronic binge alcohol exposure during pregnancy dysregulates fetal hippocampal gene expression in a sex-specific manner. Identification of subtle, transcriptome-level dysregulation in hippocampal molecular pathways offers potential mechanistic insights underlying FASD pathogenesis.

Chronic exposure to e-cig aerosols during early development causes vascular dysfunction and offspring growth deficits.

Increasing popularity of electronic cigarettes (e-cigs), including among women of reproductive age, is attributed to its perceived safety compared to conventional tobacco. However, there is a major knowledge gap surrounding the effects of e-cig aerosols on pregnancy and fetal development. We aimed to evaluate the effects of vaping e-cigs during gestation on offspring growth and to asses if growth deficits are accompanied by altered maternal and fetal vascular hemodynamics. Sprague-Dawley dams were assigned to Pair-Fed Control, Pair-Fed Juice, or Juice+Nicotine groups, and then underwent either a prenatal or prenatal+postnatal exposure paradigm in a custom-engineered vaping system. Mass spectrometry identified major aerosolized constituents from e-cig vaping. The Juice+Nicotine group exhibited significantly decreased fetal weight and crown-rump length (↓46.56%, and ↓23.83%, respectively). Pre- and postnatal exposure to Juice+Nicotine resulted in decreased pup weight at postnatal day (PND) 4-10. Crown-rump length was decreased by 24.71% on PND 10. Blood flow in the Juice+Nicotine group was decreased in the maternal uterine and fetal umbilical circuits by 49.50% and 65.33%, respectively. We conclude that chronic exposure to e-cig aerosols containing nicotine during early development can have deleterious health effects on the exposed offspring. Vaping e-cigs containing nicotine during pregnancy lead to a reduction in offspring weight and crown-rump length, associated with a marked decrease in blood flow in both the maternal uterine and fetal umbilical circulation (a strong indicator of growth restriction). Thus, chronic exposure to e-cig aerosols containing nicotine can lead to potentially harmful developmental effects in early life.

Gestational binge alcohol-induced alterations in maternal uterine artery transcriptome.

Binge alcohol exposure during pregnancy results in diminished vessel function and altered proteome in the maternal uterine artery. We aimed to utilize high throughput RNA-seq deep-sequencing to characterize specific effects of binge alcohol exposure during pregnancy on the uterine artery transcriptome, and gain insight into mechanisms underlying alcohol-mediated uterine artery dysfunction. Pregnant Sprague-Dawley rats assigned to Pair-Fed Control or Alcohol groups, received a once-daily orogastric gavage in a binge paradigm. RNA-sequencing using Illumina NextSeq 500, identified 13,941 genes; 40 significantly altered genes were altered by log2(fold change) > 2; 27 genes were upregulated and 13 were downregulated in the Alcohol group. Transcripts altered included those which encode for aldehyde dehydrogenases, matrix metalloproteases, and molecules vital for vasodilation and vascular remodeling. Biological pathways that were disproportionally altered by alcohol were proline and citrulline biosynthesis/metabolism. Disruption of these pathways suggests candidate mechanism(s) for alcohol-mediated impairments to the proteome and vascular function.

Chronic binge alcohol consumption during pregnancy alters rat maternal uterine artery pressure response.

We aimed to investigate pressure-dependent maternal uterine artery responses and vessel remodeling following gestational binge alcohol exposure. Two groups of pregnant rats were used: the alcohol group (28.5% wt/v, 6.0 g/kg, once-daily orogastric gavage in a binge paradigm between gestational day (GD) 5-19) and pair-fed controls (isocalorically matched). On GD20, excised, pressurized primary uterine arteries were studied following equilibration (60 mm Hg) using dual chamber arteriograph. The uterine artery diameter stabilized at 20 mm Hg, showed passive distension at 40 mm Hg, and redeveloped tone at 60 mm Hg. An alcohol effect (P = 0.0025) was observed on the percent constriction of vessel diameter with greater pressure-dependent myogenic constriction. Similar alcohol effect was noted with lumen diameter response (P = 0.0020). The percent change in media:lumen ratio was higher in the alcohol group (P < 0.0001). Thus, gestational alcohol affects pressure-induced uterine artery reactivity, inward-hypotrophic remodeling, and adaptations critical for nutrient delivery to the fetus.