bluuebunny/crossref_metadata_2025 huggingface dataset:
Temperature effect on cardiorespiratory responses and autonomic modulation of heart rate in premetamorphic bullfrog tadpoles, Lithobates catesbeianus
Authors: Longhini Leonardo S , Zena Lucas A , da Silva Glauber SF , Bícego Kênia C , Gargaglioni Luciane H
Published: November 2023

American bullfrog tadpoles exhibit an inhibitory vagal tone in the heart, while in adults; vagal tone becomes considerably reduced and virtually independent of temperature. Temperature effects on cardiac autonomic tonus in amphibian's larval stages have never been investigated. Therefore, we evaluated the effect of different temperatures (15, 25, and 30°C) on the cardiorespiratory responses and cardiac autonomic tonus of premetamorphic bullfrog tadpoles, Lithobates castebeianus. A non‐invasive method for measuring ECG and breathing frequency (fR) was developed. For pharmacological study, intraperitoneal injections of ringer solution (control), atropine (cholinergic muscarinic antagonist), and sotalol (β‐adrenergic antagonist) were performed. Ringer injections did not affect heart rate (fH) and fR across temperatures. Cardiorespiratory parameters were significantly augmented by temperature [fH (beats.min−1): 15°C: 24.5±1.0; 25°C: 54.5±2.0; 30°C: 75.8±2.8 and fR (breaths.min−1): 15°C: 30.3±1.1; 25°C: 73.1±4.0; 30°C: 100.6±3.7]. A predominant vagal tone was observed at 15 (32.0±3.2%) and 25°C (27.2±6.7%) relative to the adrenergic tone. At 30°C, adrenergic tone increases relative to the lower temperature. This study provides a suitable non‐invasive method for studying cardiorespiratory function (ECG and electromyography of the buccal floor) in tadpoles. Furthermore, cholinergic and adrenergic tone seems to be independent of temperature for the thermal interval between 15 and 25°C, while animals' exposure to 30°C is followed by a significant increase in adrenergic tone that may reflect cardiovascular adjustment in order to match oxygen delivery to demand.

Support or Funding Information

FAPESP and CNPq
Black Adults Display Reduced Sympathetic Reactivity to Mental Stress Compared to Non‐Hispanic White Adults
Authors: Fonkoue Ida T. , Schwartz Christopher E. , Gervais Brett M. , Carter Jason R.
Published: November 2023

Black adults have a higher risk of developing essential hypertension compared to non‐Hispanic white (NHW) adults, but the physiological mechanisms underlying this predisposition are not clearly elucidated. The purpose of this study was to compare muscle sympathetic nerve activity (MSNA) responses to mental stress in a group of young black and NHW participants. We hypothesized that the sympathoexcitation associated with mental stress would be greater in black adults compared to NHW. Thirty‐five male adults (19 black, 23±1 years; 16 NHW, 22±1 years) were examined during a 5 min supine baseline and 5 min of mental stress (via mental arithmetic). Neural and cardiovascular measurements included blood pressure (sphygmomanometer and beat‐to‐beat finger plethysomography), heart rate (HR; electrocardiogram), and MSNA (microneurography). Baseline mean arterial pressure (MAP; 72±2 vs. 77±2 mmHg), HR (68±4 vs. 66±4 beats/min), MSNA (13±1 vs. 15±3 bursts/min), and spontaneous sympathetic baroreflex sensitivity (−1.1 ± 0.4 vs −1.5 ± 0.3 bursts/100hb/mmHg) were not significantly different between NHW and black adults (p>0.05), respectively. Systolic arterial pressure (SAP) responsiveness to mental stress was significantly higher in NHW compared to black adults (time × race, p=0.028). Likewise, MSNA reactivity to mental stress was significantly higher in NHW compared to black adults (time × race, p=0.017), with a particularly divergent responsiveness during the first minute of mental stress in NHW (Δ4±1 burst/min) and black (Δ‐4±2 burst/min; p = 0.007). HR reactivity to mental stress was similar between the two groups (time × race, p=0.721). In summary, and contrary to our initial hypothesis, NHW participants demonstrated a higher MSNA responsiveness to mental stress compared to black adults. These findings suggest that although black adults have a higher prevalence of essential hypertension, they do not appear to have higher neural and cardiovascular responsiveness to mental stress when compared to NHW.
Reciprocal Inhibitory Integration and Respiratory Modulation of Arterial Chemoreceptor and Baroreceptor Inputs to Nucleus Tractus Solitarii
Authors: Moraes Davi J.A. , Da Silva Melina P. , Machado Benedito H. , Paton Julian F.R.
Published: November 2023

The cardiovascular and respiratory reflex responses to activation of the arterial chemoreceptors or baroreceptors are dependent on the phase of respiratory cycle. To determine if the respiratory modulation of the chemoreflex and baroreflex occurs within the nucleus tractus solitarii (NTS), whole cell patch clamp recordings of NTS neurons were obtained in in situ preparations from juvenile rats. Thirty non‐respiratory NTS neurons that were depolarized following activation of the carotid body chemoreceptors (by intra‐arterial injection of sodium cyanide) also exhibited excitatory post‐synaptic currents (EPSCs) in response to electrical stimulation of the ipsilateral carotid sinus nerve (CSN; standard deviation of latency: 0.51 ± 0.02). These cells were also tested for the presence of convergent inputs from both the ipsilateral carotid sinus and aortic baroreceptors. Convergent post‐synaptic inhibition from both carotid sinus and aortic baroreceptors was observed in all chemoreceptive NTS neurons tested. CSN evoked EPSCs were averaged separately during different phases of respiratory cycle. Amplitude of carotid sinus‐evoked EPSCs were significant enhanced during expiration relative to inspiration (p<0.05). Additionally, fifty non‐respiratory NTS neurons were depolarized following activation of either ipsilateral carotid (carotid sinus pressure) or aortic baroreceptors (aortic depressor nerve stimulation; standard deviation of latency: 0.2 ± 0.01) and were found to be more sensitive to aortic than carotid sinus activation (p<0.05). Twenty one neurons depolarized only by activation of aortic baroreceptors, while eleven neurons depolarized only by activation of carotid sinus baroreceptors. Convergent post‐synaptic inhibition from arterial chemoreceptors was observed in all tested NTS baroreceptive neurons. Similar to chemoreceptive NTS neurons, amplitude of aortic baroreceptors‐evoked EPSCs were enhanced during expiration relative to inspiration (p<0.05). The results of the present study suggest that: i) there is respiratory modulation of arterial chemoreceptor and baroreceptors inputs by central respiratory drive at this early stage of the reflex arc and; ii) both baroreceptor modulation of arterial chemoreflex, and chemoreceptor modulation of arterial baroreflexes occurs at the level of the NTS. These data reveal a potential mechanism for the baroreflex enhancement observed when peripheral chemoreceptor afferents are denervated.

Support or Funding Information

FAPESP: SÃO PAULO RESEARCH FOUNDATION

BHF: BRITISH HEART FOUNDATION
Simulating Selected Human Hormonal Pathways During a 24‐Hour Period and Its Implications for Aging Research
Authors: Garan Steven A. , Efe Gizem , Cheung Kevin , Khurramb Maaz A. , Brooks George A.
Published: November 2023

A subset of compounds that regulate functions that are affected by aging and in turn affect the aging process in humans have been simulated in a quantitative temporal model using the Center for Research and Education in Aging Language (CREAL). CREAL is a language that can be used to describe biological systems designed by integrating data yielded from different research studies, and events that take place in a simulated 24‐hour period. It has been used to develop a simulation of glucose intake, absorption and and subsequent modulation of insulin and glucagon levels in humans. Example of the compounds that are simulated are hormones such as orexin, glucocorticoids and melatonin. The release of orexin, which modulates the human hunger response is simulated with respect to insulin, leptin, and ghrelin. This is achieved by modeling the concentrations of gut hormones based on food intake within 24 hours. The release of glucocorticoids, which is the primary modulator of the stress response is simulated with respect to actions that take place in the hypothalamic–pituitary–adrenal axis (HPA axis). Furthermore, the release of melatonin, which modulates the sleep cycle is simulated with respect to events that take place in the suprachiasmatic nucleus (SCN). The model also shows changes in many other compounds that are involved in regulating daily processes. Ultimately, such a program can be used to simulate whole organism homeostatic processes.

Support or Funding Information

Center for Research and Education in Aging, University of California at Berkeley, LBNL, CA, USA
GeneLab: Multi‐Omics Investigation of Rodent Research‐1 Biobanked Tissues
Authors: Chakravarty Kaushik , Lai San‐Huei , Boyko Valery , Berrios Daniel , Fogle Homer , Dueck Sandra , Timucin Linda , Galazka Jonathan , Reinsch Sigrid , Stotzky Olga , Skidmore Mike
Published: November 2023

NASA's Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS), it is imperative to use crew time efficiently while maximizing high‐quality science return. NASA's GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi‐omics, systems biology‐based approach, and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA, DNA, and protein extracted from archived RR‐1 tissue samples for epigenomic, transcriptomic, and proteomic assays. During the first RR spaceflight experiment, a variety of tissue types were harvested from subjects, snap‐frozen or RNAlater‐preserved, and then stored at least a year at −80°C after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio‐specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands, quadriceps, gastrocnemius, tibialis anterior, extensor digitorum longus, soleus, eye, and kidney. We report here protocols for and results of these tissue extractions, and thus, the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues, our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities.

Support or Funding Information

Support from the NASA Space Life and Physical Sciences Division and the International Space Station Program is gratefully acknowledged
Comprehensive Coverage of Rat Models for Human Diseases at the Rat Genome Database
Authors: Wang Shur‐Jen , Laulederkind Stanely JF , Smith Jennifer R , Hayman George Thomas , Petri Victoria , Dwinell Melinda , Shimoyama Mary
Published: November 2023

Rat Genome Database (RGD) is a model organism database where organized functional annotations for rat, mouse and human are integrated and displayed in a portal style. These annotations, either imported from other sources or manually curated at RGD are associated with genes, strains or QTL. Recently, curation efforts have been focused on diseases and 11 area‐specific disease portals have been created. In this poster RGD is presenting its curation effort on rat disease models. To facilitate searching and browsing registered rat strains are organized in a nomenclature structure (Rat Strain Ontology), where relationships with related strains are the basis of the structure. The rat strain‐disease association is made based on disease markers, genomic or genetic association curated from published literature or user submission. These curated disease annotations are organized by RGD Disease Ontology/ vocabulary to facilitate query in either very granular disease terms or a higher level broad disease branch. Three rat disease models are presented in this poster as examples to show how rats are used in human disease study and how these data are organized and displayed at RGD. We first discuss the naturally occurring rat model of Hirschsprung disease, the spotting lethal (Sl) rat. The deletion mutation of Ednrb causes aganglionic megacolon and pigmentary disorder in rats. Both the disease and coat color phenotypes were curated at RGD. Another example is the ENU‐induced malignant peripheral nerve sheath tumor (PMST) model between inbred BDIX and BDIV strains. From these two strains which exhibit different responses to ENU, several disease QTL and congenic rat strains were developed. The last example is the collection of genetically modified rats created by the “Gene Editing Rat Resource Center” by researchers at the Medical College of Wisconsin. The disease and phenotypes association of these genetic models, captured by qualitative disease and phenotype terms and the quantitative measurements of associated traits, are available at RGD. In conclusion RGD is a comprehensive data resource for the laboratory rat used in disease study. Disease and phenotype data from various sources have been integrated and organized by controlled vocabularies for easy querying and browsing by biomedical researchers.

Support or Funding Information

RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.
Selection and Characterization of Novel of RNA Aptamer Recognizing Homocysteine which Ameliorates Oxidative Stress in Mouse Brain Endothelial (bEND3) Cells
Authors: Bala Jyoti , Behera Jyotirmaya , Kelly Kimberly , Tyagi Neetu
Published: November 2023

Neurodegenerative diseases are characterized by a progressive deterioration of brain function, with a significant consequent decrease in the quality of patient's lives. Recently pathological mechanisms operating in cerebrovascular diseases mediated via homocysteine (Hcy) have gained increased attention. It has also been known that Hcy causes endothelial dysfunction by inducing oxidative stress in most cerebrovascular disorders. This dysfunction is highly correlated with reactive oxygen generation (ROS). Therefore, it is crucial to develop potential novel measures to slow down or prevent the onset of these diseases mediated by increased level of Hcy. Therefore, we proposed to select and characterize novel RNA aptamer recognizing Hcy to inhibit and modulates the Hcy activities involved in cerebrovascular disease. RNA Aptamers are specific bio‐molecules selected by the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamer folds into 3‐dimensional shapes which are capable of binding non‐covalently with high affinity and specificity to their target molecule. We have selected and characterized Hcy RNA aptamers using SELEX technology. The structural characterization was done by bioinformatics tools such as Clustal W, Mfold, IFold and HADDOCK. Binding characterization were done by CD and RNA EMSA studies. The best Hcy RNA aptamers were further investigated for their biological and functional characterization. Our results demonstrate that the Hcy RNA aptamer undergoes a conformation change upon ligand binding. These aptamers exhibit high specificity to ligand over nonspecific competitor. In our studies, bEND3 cells were exposed to Hcy treatment in the presence or absence of Hcy RNA aptamers for 24 hrs. We also demonstrated that these aptamers are novel class of RNA aptamer which ameliorates the effects of ROS thereby reducing the oxidative stress in bEND cell lines. Apoptotic and autophagic cell death were measured with 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐ Diphenyltetrazolium Bromide (MTT) and monodansylcadaverine (MDC) staining assay. ROS production was determined using fluorescent probes 2, 7‐dichlorofluorescein acetate (DCFH‐DA. Autophagy flux was assessed using a conventional GFP‐microtubule‐associated protein 1 light chain 3 (LC3) dot assay. Mitochondrial fusion and fission was evaluated using Mfn‐2 and Drp‐1 RT‐PCR. Treatment of bEnd3 with Hcy RNA aptamer attenuated the production of free radicals and protected the cells from oxidative damage. Altogether, our studies strongly suggest that Hcy‐RNA aptamer has beneficial effects on cerebrovascular diseases and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy).

Support or Funding Information

Financial support from National Institute of health grant AR‐067667 and HL‐107640 are greatly acknowledged.
Dynamic Bayesian Graphical Modeling to Predict Regulatory Networks in Hypertensive Rats
Authors: Dayton Alex , Ahn Kwang Woo , Liu Pengyuan , Laud Purushottam , Stingo Francesco , Vannucci Marina , Bukowy John D , Liang Mingyu , Cowley Allen W
Published: November 2023

We have previously used Bayesian graphical methods to detect networks of interacting genes relevant to the development of salt sensitive hypertension. However, these previous approaches are “static” or time‐insensitive methods that cannot infer causality to gene interactions. Here, we present a dynamic Bayesian graphical modeling approach which is used to connect potential target genes to other genes and networks.

Dynamic Bayesian graphical networking approaches require large amounts of time‐series data, in addition to being computationally complex. Data was generated by isolating medullary thick ascending limb (mTAL) cells from 3 rat strains: the Dahl Salt‐sensitive rat (SS), the Fawn Hooded Hypertensive rat (FHH) and the Spontaneously Hypertensive Rat (SHR). After isolation, the freshly isolated mTAL cells were exposed to one of two stimuli: H2O2 (500 nM) or TNF‐α (50 ng/mL). RNA was then collected at 30 minute intervals for 8 hours, resulting in a 16‐point time‐series for each strain and stimulus. Each time‐series was duplicated, and duplicates were pooled to reduce overall measurement noise. As there are 6 combinations of strains (3) and stimuli (2) this procedure resulted in 96 individual time‐points. RNA was then isolated and sequenced on an Illumina Hi‐Seq 2500.

The Dynamic Bayesian graphical model attempts to find connections between genes by looking at the sequence of activation/inactivation of genes across the entire 8 hour time‐series. For example, in a time‐series generated by treatment of SS mTAL cells with 500 nM H2O2, we find that the gene Dusp5 undergoes a 4‐fold increase in RNA expression one hour before a similar 4‐fold increase in the gene Rasgrp3, a GWAS gene for hypertension that has no known function in the mTAL. The Bayesian graphical modeling algorithm looks at such changes across each time‐series for the different rat strains and stimuli, and detects the most probable connections between the analyzed genes.

This analysis represents the first attempt to create a dynamic Bayesian graph in non‐cancer mammalian cells. The use of such a graphical modeling approach in mTAL, a cell type of particular relevance to the pathophysiology of hypertension, will give us insight into the regulation of critical genes in hypertension and allow us to better understand, and potentially treat, this important disease.

Support or Funding Information

A. Dayton supported by NIH Fellowship: 1F30HL127979‐01A1.Work funded by NIH grant: 4P01HL116264.
Modeling of calcium‐induced swelling in spatially divergent populations of cardiac mitochondria
Authors: Chapa Xavier R , Makarov Vladimir , Javadov Sabzali
Published: November 2023

In addition to ATP synthesis, mitochondria are involved in high cytosolic calcium (Ca2+) waves necessary for the regulation of the cardiac excitation‐contraction coupling, a focal point in the determination of cardio‐pathophysiology. Depending on mitochondrial location within subcellular compartments, mitochondrial functions expands from the buffering of cytosolic Ca2+ necessary for cellular signaling to the initiation of cell death via necrosis or apoptosis depending on ATP availability. The exposure of mitochondria to Ca2+ plays a dual role in determining metabolic activity; the sequestering of low Ca2+ concentration ([Ca2+]) accompanied by a minor matrix swelling enhances ATP production through the upregulation in activity of TCA cycle enzymes, ETC complexes and oxidative phosphorylation. However, excessive matrix swelling at high [Ca2+] can induce the opening of non‐selective permeability transition pores (PTP), sustained (irreversible) opening of which allow for the release Ca2+ to propagate from one mitochondria to the other leading to the wide spread of mitochondrial swelling, thus initiating cell death. Studies have suggested that different mitochondrial sub‐population differ in functional properties such as membrane potential, calcium retention capacity (CRC), and ROS production. In this study, we designed a biophysical model that can explain the kinetics of the transition from a reversible to an irreversible Ca2+‐induced swelling state in heterogenic mitochondrial population including subsarcolemmal (SSM) and intermyofibrillar mitochondria (IFM) of cardiac cells.

Model development

In this project, a heterogenic population of mitochondria were isolated and purified via differential centrifugation. CRC was determined by the application of numerous Ca2+ pulses until Ca2+ release via PTP opening measured through spectrophotometry. Kinetics model was developed utilizing the 4th order Runge‐Kutta approximation method and fitted to experimental mitochondrial swelling data using the least square method. Reversible and irreversible mitochondrial swelling induced by low and high [Ca2+] in isolated cardiac mitochondria have been measured. Reversibility of mitochondrial swelling was determined in the presence of EGTA, a Ca2+‐chelating agent. Based on experimental data, the development of a kinetic model that simulates the swelling dynamics at low and high [Ca2+] in cardiac mitochondria has been created with a correlation coefficient of r >0.95.

Conclusion

Our model describes transition from reversible to irreversible swelling in different subcellular populations of mitochondria that is important for understanding the role of mitochondrial heterogeneity in PTP‐induced cell death.

Support or Funding Information

Supported by NHLBI NIH (SC1HL118669)
Brain inflammation and oxidative stress in the developmental programming of metabolic syndrome in adult offspring to maternal nutrition insult
Authors: Chan Julie YH , Chao Yung‐Mei
Published: November 2023

Early life exposure to adverse environments can lead to a variety of adult diseases by a process referred to as developmental origin of adult health and disease (DOHaD) or developmental programming. Maternal high fructose diet (HFD) promotes programmed hypertension in adult offspring, although the underlying mechanism is not fully understood. Brain inflammation and oxidative stress have been reported to play pivotal roles in neural mechanism of hypertension. We tested in this study the hypothesis that brain inflammation and oxidative stress contribute to the programmed hypertension in adult offspring to maternal HFD. In normotensive Sprague‐Dawley rats, maternal HFD during gestation and lactation led to insulin resistance, hypertriglyceride, increased sympathetic activity and hypertension in the 3‐month‐old young offspring. The increase in blood pressure was associated with the augmented neurogenic sympathetic vasomotor tone, increase in plasma norepinephrine level, production of proinflammatory cytokines and high tissue levels of reactive oxygen species (ROS) at the brain stem. Maternal melatonin treatment or minocycline treatment beginning at the age of two months significantly ameliorated neuroinflammation and oxidative stress in the brain stem; alongside the delay in the programming of hypertension in adult offspring. At the same time, intracisternal infusion with a superoxide scavenger, tempol, abrogated sympathetic overexcitation and prevented the development of programmed hypertension in the 3‐month‐old offspring to maternal HFD. Together, these results suggest that brain inflammation and stem oxidative stress may contribute to sympathoexcitation and the programmed hypertension in offspring to maternal HFD.

Support or Funding Information

This study was supported by grant (CMRPG8C0051‐053) from Kaohsiung Chang Gung Memorial Hospital.
Effects of Culture Time, Fetal Bovine Serum, Progesterone, Testosterone and Estradiol on Alkaline Phosphatase Activity, Extracellular Matrix Mineralization and Osteoblast Viability
Authors: Lemos José E.S. , Brancaglião Lucas F.C. , Bonfá Ana L.O. , Rocha Nícolas F. , Gonçalves Vítor M. , Montrezor Luís H.
Published: November 2023

Understanding the interactions between ovarian and plasma changes induced by polycystic ovary syndrome (PCOS) and bone metabolism is essential for the comprehension of the pathophysiological mechanisms involving these organs. Ovarian steroids are involved in activities related to bone synthesis and reabsorption. Knowledge of the actions of the main ovarian steroids on the viability of osteoblasts in culture, which mimic the conditions of PCOS, is relevant to a better understanding of the functions of the cells in this endocrine disease that affects more than 10% of women of reproductive age. The goal of this study was to develop a cell culture model mimicking polycystic ovary syndrome that can be used for studies on culture conditions and the effects of steroids progesterone (P4), testosterone (T) and estradiol (E2) on cell viability, alkaline phosphatase activity and extracellular matrix mineralization of osteoblasts. We plated 5 × 103 cells in each well of 96‐well culture plates with 100 μL of modified Eagle's medium supplemented or not with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were maintained at 37° C under a humid atmosphere of 5% CO2 and 95% air for 72 h. P4, T and E2 were used in different concentrations: control (without hormones), 10−8 M and 10−7 M mimicking the normal plasma concentrations in adult female rats and 10−6 M and 10−5 M, mimicking plasma concentrations in adult female rats with induced PCOS. To this end, each of the steroids were added to the wells of culture both separately and together (all in the same well). Our results show the cell viability of undifferentiated and differentiated osteoblasts in the osteogenic medium, the alkaline phosphatase activity and mineralization of the extracellular matrix in cells cultured for different periods of time and challenged with different concentrations of P4, T, E2, and the combination of all three added to the culture medium. Furthermore, FBS influences the culture conditions. From these results, we propose that the culture system described can be used to study the viability of osteoblasts, the activity of alkaline phosphatase and mineralization of the extracellular matrix in conditions similar to PCOS and, thus, it can be suggested that ovarian steroids, in isolation and in combination, modulate bone formation in vitro.

Support or Funding Information

CNPq and FUNADESP
Effect of Perinatal Blockade of Androgen Receptors on Estrogen Receptor‐Levels in Specific Brain Regions and Spatial Working Memory in Male Rats
Authors: Hershkowitz Richie , Shah Binita , Nehrt Paige A , Sanchez Alejandra , Secor Tatum , Starr Blake , Toennies Shelby , Taylor George , Gopalan Chaya
Published: November 2023

Sex differentiation of the male rat brain occurs during the perinatal period (four days prior to and four days after birth) in the presence of testosterone (TS) which serves as a prehormone and gets metabolized to either 17β‐estradiol (E2) or dihydrotestosterone (DHT). E2 exerts its effect by binding to estrogen receptor (ER‐ and ER‐β) and DHT to androgen receptor (AR). Both ER and AR are shown to be important in the sexual differentiation of the brain. ER‐ is expressed in the hypothalamus, amygdala, and hippocampus in greater amounts and is associated with memory and sexual behavior. Whether blockade of AR during the perinatal period modulates the concentrations of ER‐ in the adult brain and, in turn, affects spatial working memory (SWM) was tested in this study. Two timed‐pregnant Long Evans rats were administered Flutamide (20mg/kg; SC) or vehicle during the last four days of pregnancy. Ten mg/kg (SC) of flutamide or vehicle was given to the newborn male pups for an additional four days. We tested SWM in these male rats when they reached postnatal day 65 and found that flutamide treatment improved SWM compared to control animals. At the end of the behavioral study, these animals were sacrificed and trunk blood was collected to measure serum E2 and TS levels. We found that the behavioral changes were associated with elevated serum E2 and reduced TS levels. The hypothalamus, amygdala, and hippocampus were isolated from the frozen brain tissue and ER‐ protein levels were measured using chemiluminescent western blots. Flutamide‐treated group showed an increase in ER‐ levels in the hypothalamus and the amygdala whereas concentrations in the hippocampus was decreased. Our results suggest that the increased ER‐ levels in the hypothalamus and amygdala along with increased serum E2 levels appear to improve SWM.
Six Weeks Inspiratory Muscle Strength Training Lowers Blood Pressure but Does Not Affect Baroreflex Sensitivity or Cardiac Output in Active Young Adults
Authors: DeLucia Claire M , De Asis Roxanne M , Bailey E. Fiona
Published: November 2023

We showed previously that inspiratory muscle strength training (IMST) lowers resting blood pressure in healthy, college‐aged adults (Vranish and Bailey 2015) and in adults with obstructive sleep apnea (Vranish and Bailey 2016). However, the mechanism underlying IMST‐related declines in blood pressure is unclear. Here, we explore the potential for IMST to modulate baroreflex sensitivity (BRS), cardiac output and /or heart rate in healthy young (19.9±0.3 years) adults (13 men, 12 women). Subjects were randomly assigned to IMST (n=12; 75% maximal inspiratory pressure) or sham training (n=13; 15% maximal inspiratory pressure) groups and underwent 6 weeks of training (30 breaths/day, 5 days/week). Pre‐ and post‐training measures included blood pressure, cardiac output, heart rate, BRS, and maximal inspiratory pressure. Additionally, all subjects completed the Cohen‐Hoberman inventory of physical symptoms survey and the Epworth Sleepiness Scale. Subjects in the IMST group exhibited significant declines in systolic (112.51±2.99 mmHg vs 108.25±3.72 mmHg, p=0.022) and diastolic blood pressures (67.73±2.45 mmHg vs 63.83±2.83 mmHg, p=0.018) whereas resting blood pressures in sham trained subjects were unchanged. IMST trained subjects reported significantly fewer physical symptoms pre vs post training (p=0.025) relative to sham trained individuals (p=0.135). There was no effect on cardiac output (p=0.722), heart rate (p=0.795), or BRS (p=0.776) pre vs post training for either group however, muscle sympathetic nerve activity (MSNA) recorded in four subjects highlights reduced sympathetic nerve traffic in IMST (25.0±6.0 vs 18.5±2.5 bursts/min) relative to sham‐trained (23.6±10.5 vs 23.0±10.0 bursts/min) subjects. Changes in central and /or other reflex modulation of MSNA may underpin IMST related reductions in mean arterial pressure in active young adults.
Effects of Perinatal Nicotine Exposure on Cholinergic Signaling in the Airway Epithelium
Authors: Borrero Nathan , Fregosi Ralph , Boitano Scott
Published: November 2023

Up to 20% of pregnant women in the USA smoke or use nicotine delivery devices (e‐cigarettes, water pipes, nicotine patches or gum). While addictive, many believe nicotine to be otherwise safe, causing the use of nicotine delivery devices to double between 2008–2012. However, there is growing evidence that nicotine can alter development of the lungs and airways, as well as pulmonary immune function and brainstem neurons that control breathing and heart rate. In this study, we propose to test the hypothesis that in utero exposure to nicotine leads to alterations in cholinergic signaling in the airway that are evident at birth and persist into adulthood. Importantly, dysfunction of acetylcholine (ACh) signaling in the airway epithelium can result in compromised airway surface liquid and subsequent loss of innate immune function in the airways. This signaling is mediated primarily by muscarinic acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs), both of which may be affected by developmental nicotine exposure. We have used rat neonate tracheal mounts and primary cultured rodent airway epithelium in Ussing Chamber studies to evaluate airway epithelial cholinergic signaling in animals with and without developmental nicotine exposure. Initial results show that transtracheal resistance (TTR), transepithelial resistances (TER), and short circuit current (Isc) responses to apical application of ACh are not altered by nicotine exposure. However, airway epithelial signaling is dominated by mAChRs‐dependent signaling. When preparations are exposed to nicotine, unexposed rat trachea and primary mouse cultures demonstrate limited, but clear responses. These responses appear to be altered in animals exposed to nicotine in utero. Our data suggest that perinatal nicotine exposure adversely affects the development of cholinergic signaling in the airway epithelium.

Support or Funding Information

1R25ES025494

NIEHS/SBRPP ES04940
Sex Differences and Role of Sex Hormones in the Expression of Inflammatory Mediators and Lung Function in Response to Ozone
Authors: Fuentes Nathalie , Cabello Noe , Nicoleau Marvin , Silveyra Patricia
Published: November 2023

Background

Breathing ground‐level ozone, a reactive oxidant gas, results in a number of negative health effects that affect men and women disproportionally. Previous studies have shown that ozone induces a differential inflammatory response in males vs. females, but the mechanisms associated with these differences remain unclear. Here, we hypothesized that circulating sex hormone levels can modulate lung function and the inflammatory response to air pollutants such as ozone.

Methods

To test this, we performed gonadectomy and hormone replacement (estradiol, 2 weeks) in adult male and female mice. We exposed these mice to 1 ppm of ozone or filtered air (FA) for 3 hours, and we compared lung function 24 h after exposure with the FlexiVent system. We also measured the levels of lipocalin, albumin, and cell differential counts in bronchoalveolar lavage fluid (BALF); as well as the expression of the pro‐inflammatory cytokine interleukin‐6 (IL‐6) in lung tissue by Real‐Time PCR.

Results

Our preliminary results indicate that males showed higher lung compliance and elasticity than females, and treatment with estradiol for two weeks in gonadectomized mice increased lung expansion in both sexes. Furthermore, gonadectomy decreases IL‐6 expression in the female lung, but not in the male lung. Estradiol replacement in females restored IL‐6 levels, but decreased it in males. Exposure to ozone resulted in higher neutrophil counts in BALF, with females higher than males. Gonadectomy and estradiol treatment had opposite effects in BALF cell counts in males vs females.

Conclusions

Together, our preliminary results suggest that both lung function and the inflammatory response to ozone can be influenced by circulating sex hormone levels (estradiol). Future studies will investigate the specific role of other sex hormones (testosterone, progesterone) in these responses.

Support or Funding Information

The authors thank the Pennsylvania State University College of Medicine Genome Sciences Core Facility for Bioanalyzer analysis and real‐time PCR equipment. The authors also thank Todd Umstead and Susan Di Angelo for assistance with training and experiments, and Dr. Joanna Floros for the ozone equipment. NIH Grant K01HL133520.
TNFα Induces Endoplasmic Reticulum Stress in Human Airway Smooth Muscle Cells
Authors: Yap John Qian , Delmotte Philippe , Sieck Gary C
Published: November 2023

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL‐13) enhance cytosolic Ca2+ concentration ([Ca2+]cyto) triggering airway smooth muscle (ASM) hyperreactivity (enhanced contractile state). Airway inflammation also induces ASM proliferation and remodeling (enhanced ASM synthetic state). In various diseases inflammation induces sarcoplasmic/endoplasmic reticulum (SR/ER) stress, which decreases SR/ER‐mitochondria interactions, with downstream effects on Ca2+ regulation. In vascular smooth muscle cells, SR/ER stress promotes smooth muscle contraction and proliferation by disrupting SR/ER‐mitochondrial coupling. We hypothesized that inflammation induces SR/ER stress in human ASM, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca2+ regulation and contractility as well as proliferation.

Human ASM cells were isolated from lung specimen incidental to patient surgery and exposed to TNFα or IL‐13 for 24h. Expression of SR/ER stress markers including PERK (protein kinase‐like endoplasmic reticulum kinase), Bip (Binding immunoglobulin protein also known as 78 kDa glucose‐regulated protein), PDI (protein disulfide isomerase), and mitochondrial fission/fusion proteins Drp1 and Mfn2 were evaluated by Western blot. The expression of the spliced isoform of the transcription factor XBP1 was investigated by quantitative real‐time PCR (RT‐PCR). For mitochondrial morphology (fission/fusion), ASM cells were loaded with Mitotracker Green and images analyzed using ImageJ and Matlab to quantify mitochondrial Form Factor and Aspect Ratio. The effect of inflammation induced SR/ER stress on human ASM proliferation was evaluated using CyQUANT assay.

We found that after 24 h exposure to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, BiP and XBP1) increased; 2) Drp1 expression increased and Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca2+]cyt and [Ca2+]mito responses to ACh stimulation were uncoupled; 5) net [Ca2+]cyt increased; 6) contractility increased; and 7) ASM cell proliferation increased.

Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca2+ regulation resulting in ASM hyperreactivity and remodeling.

Support or Funding Information

NIH grant HL126451 (GCS).
Reflex bronchoconstriction evoked by inhaled nicotine aerosol in guinea pigs: role of the nicotinic acetylcholine receptor
Authors: Lin Ruei‐Lung , Khosravi Mehdi , Xu Fadi , Lee Lu‐Yuan
Published: November 2023

We have previously reported that stimulation of vagal bronchopulmonary C‐fibers by nicotine via its action on neuronal nicotinic acetylcholine receptor (NnAChR) is primarily responsible for the acute bronchoconstriction evoked by inhaled cigarette smoke. Recent studies have reported that nicotine at high concentrations can also activate the transient receptor potential ankyrin 1 receptor (TRPA1) expressed in these sensory nerves. This study was carried out to investigate the mechanisms involved in the bronchoconstrictive effect of inhaled nicotine, and the relative roles of NnAChR and TRPA1 in this effect. Guinea pigs were anesthetized and mechanically ventilated; nicotine aerosol (2% solution; 1 × VT) was diluted by air to 2 × VT and delivered directly into the lung via trachea tube. Our results showed: 1) Delivery of one breath of nicotine aerosol immediately triggered a pronounced airway constriction; the increase in airway resistance (Raw) reached a peak of 588 ± 205 % in 10–40 sec, which gradually returned to baseline after 1–5 min. The response was reproducible in the same animal tested 20 min later. 2) Challenge with aerosolized alkaline solution of the same pH as that in the nicotine solution (10.5) did not cause any change in Raw. 3) Pretreatment with atropine (0.1 mg/kg; i.v.) and mecamylamine (Mec; an antagonist of NnAChR) (2%; 4 breaths of aerosol) almost completely abolished the nicotine‐induced bronchoconstriction: ΔRaw was reduced by 83.1 ± 8.2 % and 92.5 ± 1.9 %, respectively. The Mec pretreatment did not block the bronchoconstriction and bradycardia generated by electrical stimulation of the distal end of one sectioned vagus nerve, indicating its minimal systemic effects. 4) Pretreatment with HC‐030031 (0.5 mg/kg; i.v.), a selective TRPA1 antagonist, abolished the bronchoconstriction induced by allyl isothiocyanate (1.7 mg/kg, i.v.), a TRPA1 agonist, but did not render any attenuation of the nicotine‐evoked bronchoconstriction. In conclusion, inhalation of a single puff of nicotine aerosol evoked acute bronchoconstriction mediated through the cholinergic reflex pathway, which was triggered by activation of NnAChR located in airway sensory nerves. In contrast, TRPA1 receptor did not play a significant role in this response.

Support or Funding Information

Supported by NIH grants HL96914, HL107462 and UL1TR001998
Abnormalities in Excitability of Nucleus Tractus Solitarius (nTS) Neurons Following Neonatal Hypoxia Exposure: Implications for Sudden Infant Death Syndrome
Authors: Mayer Catherine , MacFarlane Peter
Published: November 2023

We showed previously that the respiratory neural control system exhibits a heightened vulnerability to sustained hypoxia (SH) exposure during a critical period of postnatal development, specifically between postnatal (P) day 11–15. The vulnerability was associated with a loss of the acute hypoxic ventilatory response (HVR) and ~85% unexpected and unexplained mortality that occurred not during SH per se, but ~3 days later (P18). These effects of SH, combined with increased microglia and decreased serotonin (5‐HT) expression found uniquely within the nucleus tractus solitarius (nTS), are key characteristics of the brainstem abnormalities associated with sudden infant death syndrome (SIDS). In the current study, therefore, we investigated whether these effects of following SH exposure are associated with altered excitability of neurons in a site of the nTS associated with hypoxic chemoafferent integration. Rats were exposed to SH (10% O2, 24hrs/day) between P11–15. Control rats received normoxia (NX, 21% O2). We used whole‐cell patch clamp recordings to examine the excitatory properties of nTS neurons monosynaptically connected to the solitary tract in P18 rats i.e. 3 days post‐SH and at a time leading up to the anticipated day of mortality. SH exposure decreased the frequency (3.0 ± 0.6 Hz) of spontaneous EPSCs in nTS neurons compared to NX (6.9 ± 1.4 Hz) rats; rise time, decay time, and area of the spontaneous EPSC's did not differ between groups. The amplitude of evoked (0.5 and 10 Hz stimulus train) EPSCs in monosynaptically connected nTS neurons was increased in the SH group compared to NX after being corrected for cells size, which was also significantly decreased in the SH group. We speculate that the increased evoked responses of the nTS neurons following SH could be a form of homeostatic plasticity, representing an attempt to compensate for disturbances in chemoafferent inputs. Further, the decreased spontaneous activity of the nTS neurons could imply a significant disturbance in spontaneous neurotransmitter release, which appears to worsen leading up to the day that prior SH exposure manifests as neonatal mortality. These data reveal the changes that occur in brainstem neural control regions following neonatal SH exposure and could provide important insight into the (cardio)‐respiratory abnormalities of SIDS.
Sex‐specific Effects of Nuclear and Membrane Progesterone Receptors on Breathing Stability in Newborn Rats
Authors: Boukari Ryma , Bairam Aida , Joseph Vincent
Published: November 2023

We tested the hypothesis that the nuclear (nPR) and membrane progesterone receptors α (mPRα) and β (mPRβ) have sex‐specific effects on respiratory control and breathing stability in newborn rats. The expression of nPR, mPRα or mPRβ in the brainstem region was attenuated by intracisternal injection of specific small interfering RNA (siRNA) to 10 days‐old male and female rats. An artificial cerebrospinal fluid was injected to the control animals. Twenty‐four hours after the injection, ventilation was recorded using whole body plethysmography during normoxia (21% O2, 50 min), hypoxia (12% O2, 5 min) and hypercapnia (5% CO2, 5 mim). The frequency of sighs and apneas was determined in normoxia and reported as events/hour. Treatments with siRNA against nPR, mPRα, or mPRβ had no effect on ventilation in normoxia, hypoxia, and hypercapnia. In male rats, the treatment with the siRNA against nPR had no effect on the frequency of sighs and apneas, while the treatment with the siRNA against mPRα increased the frequency of sighs (from 24±2 to 44±8, p=0.003) and the treatment with the siRNA against mPRβ increased the frequency of apneas (51±10 to 83±16, p=0.04). In female rats, the treatment with the siRNA against nPR increased the frequency of apneas (from 47±5 to 101±15, p=0.0003), but the treatment with the siRNAs against mPRα or mPRβ had no effect. We conclude that in newborn male rats mPRα and mPRβ, while in newborn female rats nPR contributes to breathing stability. These data could be useful to develop innovative approaches for pharmacological treatments of apnea of prematurity.

Support or Funding Information

Funded by CIHR (operating grants ‐ VJ, AB) and FRQ‐S (PhD fellowship ‐ RB).
Arousal defects and apnea in rat pups deficient in central 5‐HT: interactions with sleep state
Authors: Young Jacob O , Hodges Matthew , Cummings Kevin
Published: November 2023

Sudden Infant Death Syndrome (SIDS) occurs during sleep periods and is preceded by apnea and abnormal arousal from sleep. Histological abnormalities within serotonin (5‐HT) neurons, including reduced 5‐HT, are consistently identified in SIDS cases, but we have an incomplete understanding of how these abnormalities increase the risk of sudden death during sleep. Given that the activity of 5‐HT neurons varies with state of vigilance, so too may their influence on respiratory function and arousal. We previously showed that rat pups deficient in tryptophan hydroxylase 2 (TPH2−/−) had delayed arousal responses to increasing CO2 mainly in quiet sleep (QS). We hypothesized that the apnea displayed by TPH2−/− pups would also be most obvious in QS, and that the delayed arousal was not a generalized defect but rather was specific for increasing CO2. To address these hypotheses we used TPH2−/− and wild‐type (WT) rat pups between postnatal days (P)14–16. Two groups of pups were used: Group 1 (normoxia): 10 WT, 8 TPH2−/−; Group 2 (hypoxia): n= 8 WT, 8 TPH2−/−. Group 1 pups were allowed to sleep normally in room air over two hours. Group 2 pups were exposed to episodes mild hypoxia (~17% O2, balance N2) at the start of AS and QS until arousal. For group 1 pups we measured the number and duration of apneas occurring in each state, as well as the duration of AS and QS episodes. For Group 2 pups we measured the latencies to arousal in response to hypoxia. TPH2−/− pups displayed significantly more apneas than WT in both sleep states, but especially in AS: TPH2−/− pups experienced ~13 more apneas/hr than WT in QS, and this rose to ~75 more apneas/hr in AS (effect of genotype within AS: p<0.001). The overall % time spent in each sleep state was not influenced by central 5‐HT deficiency. However, TPH2−/− pups exhibited longer periods of QS and AS, with AS being particularly prolonged (genotype × state: p=0.003). In contrast to CO2, the arousal latencies of TPH2−/− pups in response to hypoxia were not significantly different than WT. These findings suggest that 5‐HT‐deficient infants are especially vulnerable during AS because they experience profound apnea and a severely compromised ability to arouse.

Support or Funding Information

Funding for this research was provided by the American Physiological Society Undergraduate Research Excellence Fellowship (to J.Y.) and by an American Heart Association Scientist Development Grant (14SDG18560022; PI: KJC).