bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of induced pluripotent stem cells from Epstein‐Barr Virus immortalized lymphoblastoid cell lines using Episomal vectors
Authors: Saito Kazuyoshi , Mayhew Christopher , Stanton Lori , Towbin Jeffrey Allen , Purevjav Enkhsaikhan
Published: November 2023

Introduction

Generating functional cardiomyocytes from human induced pluripotent stem cells (iPSCs) derived from fresh peripheral blood mononuclear cells (PBMCs) or skin fibroblasts has been widely implemented for drug discovery, cardiotoxicity screening, and modeling cardiac diseases and development in vitro. However, generating patient‐specific iPSCs using standard approaches has not been possible when patients are deceased or fresh blood or fibroblasts are unavailable. In these cases iPSC generation from alternative cell sources, such as Epstein‐Barr virus (EBV) immortalized lymphoblastoid B cell lines (LCLs), as unlimited sources, is desirable. Though several papers have been published that outline iPSCs generation from EBV‐LCLs, the efficiency remains low. The aim of this study was to establish a robust technique for iPSC generation from EBV‐LCLs.

Methods

Four EBV‐LCLs from control individuals and patients were used. Multiple assessments of different conditions such as 1) cell number, 2) plasmid combination, 3) transfection methods (chemical or electroporation using 4D‐Nucleofector™ X unit System (LONZA) machine) and design, 4) media and cell culture conditions, including small molecule combinations, with or without feeder‐free, media changing methods, and 5) expansion techniques were performed.

Results

We established the optimal conditions for generation of iPSCs from EBV‐LCLs included: a cell number of 1×10^5/100ul solution (P3 primary cell 4D‐Nucleofection Kit (Lonza)), the EO‐100 program on LONZA ZAP electroporation, and Thomson's plasmids combination (l‐Myc plasmid included) of total 10ug. We developed a specific media change approach using media containing small molecules. The harvesting time was around 20 days.

Conclusions

We improved and adjusted the conditions for establishing a robust system of generating iPSCs from EBV‐LCLs. Our approach resulted in 100% colony generation. We are in the process of improving the cell culture conditions in order to increase efficacy.

Support or Funding Information

Johnson & Johnson K.K. Codman Neurovascular Fellowship for Pediatric Interventional Cardiology in Japan (2012)
Examining the Functional Role of Fragile X Related Protein 1(FXR1P) in Adult Neurogenesis
Authors: Patzlaff Natalie , Nemec Kelsey , Zhao Xinyu
Published: November 2023

FXR1P is a member of the fragile X family of RNA binding proteins, which includes FMRP and FXR2P. Both FMRP and FXR2P regulate adult neural stem cells (aNSCs) and neurogenesis, a process affected in a number of neurological and neuropsychiatric disorders, including Fragile X syndrome. Although FXR1P has been implicated in diverse developmental processes and neuropsychiatric diseases, its role in neurodevelopment is not well understood. The goal of this study is to elucidate the function of FXR1P in adult neurogenesis. We used an inducible triple transgenic mouse model (Fxr1‐cKO; Nestin‐CreERT2; Ai14::tdTomato) that allows us to investigate how FXR1P deficiency in aNSCs affects proliferation and fate specification. Deletion of FXR1 in aNSCs resulted in fewer adult‐born cells and reduced populations of radial glia‐like cells, neuroblasts, immature neurons, neurons, and astrocytes. We hypothesize that this reduction in new cell numbers resulted from impaired proliferation, which we confirmed in vitro using primary aNSCs. In addition, we found that mRNAs related to cell cycle progression were downregulated in vitro. Ongoing work includes identification of mRNAs regulated by FXR1P in aNSCs. These results indicate that FXR1P plays an important role in regulating aNSC self‐renewal and maintenance in the adult brain.

Support or Funding Information

NIH (MH080434, MH07897) to X.Z.; Waisman Center core grant NIH(P30HD03352); Molecular and Cellular Pharmacology Training Grant NIH(T32 GM008688) to N.E.P.; a NIMH‐NIH NSRA (F31MH103945) to N.E.P.; the Wayne and Jean Roper Memorial Wisconsin Distinguished Graduate Student Fellowship to N.E.P and the Hilldale Undergraduate Fellowship to KN.
Sexually Dimorphic Expression of RNA Processing Genes in the Developing Mouse Cortex and Hippocampus
Authors: Franklin Michael Killen , Armoskus Chris , Donovan Courtney , Tsai Houng‐Wei
Published: November 2023

The cerebral cortex and hippocampus are important for the control of cognitive functions and social behaviors. Cortical and hippocampal dysfunction has been observed in a variety of neurological diseases and psychiatric disorders, many of which display gender bias in incidence and symptomatology, including autism and Alzheimer's disease. Some of these sex‐biased diseases have been linked to defects in pre‐mRNA splicing, suggesting that RNA processing factors may play a vital role in brain sexual differentiation at the posttranscriptional level. Therefore, we hypothesize that the expression of the genes associated with RNA splicing and processing might be sexually dimorphic in the mouse cortex and hippocampus during early development, which creates different mRNA variants between the sexes and then leads to differences in structure and function of these brain regions. To test our hypothesis, we first reanalyzed our gene expression microarray data in neonatal mouse cortex/hippocampus with Gene Ontology enrichment analysis and found that the genes with the annotation of “RNA binding” were over‐represented (13 out 89 candidate genes). With addition of 4 other genes with the same annotation displaying >1.2‐fold difference in expression between the sexes, we identified 17 candidate genes. We next used reverse transcriptase with quantitative PCR (RT‐qPCR) to measure and determine relative mRNA levels of the 14 candidate genes residing autosomal chromosomes in the cortex/hippocampus of male and female mice collected on the day of birth (PN0), and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. A two‐way ANOVA revealed a significant main effect of sex on mRNA levels of Khdrbs2, Nanos2, Rbm48, and Tdrd3. Among these genes, females expressed more Rbm48 and Tdrd3 mRNA on PN0 and PN7 while female‐biased Khdrbs2 expression was observed in neonates. In contrast, Nanos2 displayed an overall female‐biased expression among all ages, but there was no significant effect of sex on mRNA levels at a particular age. These results suggest that sex and age‐dependent change in expression of RNA processing factors might be the molecular mechanism underlying the development of sexual dimorphism in cortical and hippocampal structure and function as well as gender bias in risk and resilience to neurological diseases and psychiatric disorders associated with these brain regions.
Exposure to a Diet High in Saturated‐fat during Pre‐natal and Post‐weaning Brain Development Alters Hippocampal Gene Expression and Behavioral Indices for Anxiety and Depression
Authors: Page Kathleen C , Winkelmann Maegan E , Anday Endla K
Published: November 2023

Background

We have shown previously that high‐fat diets during critical periods of development perturb both hypothalamic and hippocampal function. More specifically, adult male rats in our studies that were exposed to dietary saturated fat during gestation and after weaning had perturbations in key hormones regulating metabolism, impaired spatial learning and memory, and alterations in both hypothalamic and hippocampal gene expression.

Objective

Our current hypothesis is that maternal and postweaning exposure to excess saturated fat promotes an increased emotionality which is associated with altered expression of genes mediating hippocampal synaptic function.

Design/Methods

Dams were fed control or high fat diet (45% kcal saturated fat) prior to mating, pregnancy, and lactation. Male offspring from control and high fat‐fed dams were weaned to control (CC or HFC) or to high‐fat diet (CHF or HFF). At 120 d the forced swim test was used to measure differences in time spent immobile which is an index for depressive‐like behavior and at 150 d they were evaluated using the elevated plus maze which is an index for anxiety‐like behavior. Animals were sacrificed 2 weeks after completion of behavioral testing. Serum leptin, insulin and corticosterone were measured by radioimmunoassay; RT‐PCR and ELISA methods were used to analyze hippocampal gene expression.

Results

Hippocampal synaptophysin mRNA level was significantly reduced in HFF offspring (maternal diet effect, P=0.044) and was susceptible to the modulating effects of the post‐weaning diet (post‐weaning diet effect, P=0.002). Reductions in the mRNA levels of ionotropic glutamate receptor subunit, AMPAR1 (P=0.003), glial‐derived neurotrophic factor, GDNF (P=0.034), microtubule‐associated protein 2, MAP2 (P=0.025) and synaptosomal‐associated protein, SNAP25 (P=0.011) were detected in offspring exposed to excess fat in the postweaning diet. Expression of the glutamate receptor subunit, AMPAR2, was not significantly different among the four groups. Protein expression for GDNF and SNAP‐25 was reduced in response to excess fat in both the maternal and postweaning diet, and an interaction between the maternal and the postweaning high fat diet was detected for MAP2 protein expression. Changes in hippocampal gene expression were associated with anxiety‐like and depressive‐like behavior using the elevated plus maze or forced swim test, respectively.

Conclusions

These data support the hypothesis that prenatal and postweaning exposure to dietary saturated fat during critical periods of brain development has a complex and detrimental effect on hippocampal synaptic plasticity that may lead to affective disorders in adult life.

Support or Funding Information

Supported by Bucknell University Biology Department
Optogenetic stimulation of distinct populations of anterior preoptic neurons reveals a locus for the reciprocal control of thirst
Authors: Abbott Stephen B , Geerling Joel C , Saper Clifford B
Published: November 2023

The median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT) regulates hydro‐mineral homeostasis in mammals. The neurochemical composition of this region is complex and the possibility of divergent roles for MnPO/OVLT neurons in thirst regulation has never been addressed. To this end, we injected a Cre‐dependent Channelrhodopsin2‐mCherry viral vector into the MnPO/OVLT of VGluT2‐ires‐Cre and VGAT‐ires‐Cre mice to selectively stimulate excitatory and inhibitory neurons respectively. Stimulation of excitatory MnPO/OVLT neurons caused water consumption in satiated mice (N=8). Water and physiological saline consumption during stimulation declined with successive trials over a 15 minute period, and fully recovered after 1 hour of rest, suggesting a pre‐systemic feedback gating of stimulus‐induced ”thirst”. Photostimulation of inhibitory MnPO/OVLT neurons reduced water consumption by 30% during 30 minutes of water access in dehydrated mice (N=8), but had no effect on food consumption following food deprivation. Axonal tracing of MnPO/OVLT excitatory and inhibitory neurons revealed overlapping innervation of areas important in hydro‐mineral homeostasis, including the paraventricular nucleus of the hypothalamus, sub‐fornical organ and supra‐optic nucleus. Together, these result indicate that the MnPO/OVLT region exerts a bi‐directional influence on thirst.

Support or Funding Information

National Health and Medical Research Council of Australia Early Career Fellowship (GNT1052674) (to S.B.A.)NIH‐NINDS R25 #NS070682 (to J.C.G.) NIH NS072337 (to C.B.S.)
Increases in Cerebrospinal Fluid NaCl Concentration Excites Neurons of the Organum Vasculosum of the Lamina Terminalis to Elevate Sympathetic Outflow and Blood Pressure
Authors: Simmonds Sarah S , Stocker Sean D
Published: November 2023

Accumulating evidence suggests salt‐sensitive hypertension is mediated partly by an increase in cerebrospinal fluid (CSF) NaCl concentration and elevation in sympathetic nerve activity (SNA). Increased NaCl concentration or osmolality is sensed by specialized neurons in the organum vasculosum of the lamina terminalis (OVLT). The present study investigated the contribution of these neurons to the SNA and ABP responses during acute increases in CSF NaCl concentrations. Male Sprague‐Dawley rats were anesthetized with Inactin (120mg/kg, IV) and prepared for SNA and ABP recordings. Lateral ventricle infusion of 1M NaCl (5uL over 10 min) increased CSF [Na+] by 5±1 mM and elevated mean ABP (9±1 mmHg), lumbar SNA (125±3%), adrenal SNA (121±5%) but decreased renal SNA (−9±1%, n=8) and did not alter splanchnic SNA (102±3%). Inhibition of the OVLT with injection of the GABAA agonist muscimol (2.5mM per 20nL, n=5) significantly attenuated the increase in ABP (2±1 mmHg), lumbar SNA (102±1%), adrenal SNA (103±2%) and decrease in renal SNA (−3±1%). Furthermore, direct injection of NaCl (100nL over 20s, n=3) into the OVLT produced dose‐dependent increases in mean ABP (0.15M: 0±1mmHg; 0.5M: 2±1mmHg; 1.0M: 4±1mmHg; 2.0M: 7±1mmHg) and lumbar SNA (0.15M: 101±2%; 0.5M: 107±1%; 1.0M: 112±3%; 2.0M: 118±4%). In vivo single‐unit recordings demonstrate that lateral ventricular infusion of 1M NaCl (5uL per 10 min) significantly increased the firing rate in 75% (3/4) of OVLT neurons from 1.2±0.4Hz to 5.1±1.2 Hz (P<0.05). Altogether, these findings suggest that increases in CSF NaCl concentrations excite OVLT neurons to elevate lumbar and adrenal SNA and ABP.
Vasodilation and Hyperemia during Passive Limb Movement: Impact of Acute Sympathetic Activation
Authors: Trinity Joel Douglas , Lee Joshua F , Garten Ryan S , O'Keefe Zachary Barrett , Layec Gwenael , Wray David Walter , Richardson Russell S
Published: November 2023

Heightened muscle sympathetic nerve activity (MSNA) is associated with impaired vasodilatory capacity and vascular dysfunction linked to aging and cardiovascular disease. The contribution of elevated MSNA to the vasodilatory response during continuous passive limb movement (PLM), a novel method to assess vascular function, has not been determined. This study sought to test the hypothesis that elevated MSNA will negatively impact the vasodilatory response to PLM in healthy young men (n=11, 25±2 yr). Post exercise circulatory occlusion (PECO) following 2 min of isometric handgrip (HG) exercise performed at 25% (PECO‐25%) and 40% (PECO‐40%) of maximum voluntary contraction (MVC) was utilized to incrementally engage the metaboreceptors and augment MSNA. Control (CON‐25% and CON‐40%) trials, without PECO, were performed to account for changes due to HG exercise. PLM was performed 2 min after the cessation of exercise and central (photoplethysmography) and peripheral hemodynamics (Doppler ultrasound) were assessed. MSNA was directly recorded by microneurography (n=8). MSNA was incrementally augmented during PECO‐25% (+ 8±3 burst/min) and PECO‐40% (+ 15±4 burst/min) and returned to pre‐HG levels during CON trials. Vasodilation, assessed by the overall change in leg vascular conductance (LVC) during PLM, was reduced following HG exercise in all conditions (Δ in peak LVC range; −15 to −41%). The peak change in LVC was not different between CON‐25% (7.2±1 ml/min/mmHg) and PECO‐25% (8.1±1.4 ml/min/mmHg, p <0.05) while the peak change in LVC during PECO‐40% (5.4±1.1 ml/min/mmHg) was significantly attenuated compared to PECO‐25% (p < 0.05). These findings indicate that alterations in MSNA can modify the vasodilatory response to PLM and that the magnitude of reduction in vasodilation during PLM appears to be graded in relation to the elevation in MSNA. In conclusion, elevations in MSNA that are typically exhibited with aging a cardiovascular disease, impact the vasodilatory response to PLM. Thus, basal sympathetic tone is important to consider when comparing vasodilatory capacity across heterogeneous groups.

Support or Funding Information

AHA SDG (1850039), VA RR&D CDA2 (1215)
Functional Changes of Baroreceptor Afferent, Central and Vagal Efferent Components in the Baroreflex Arc: Effects of SOD1 Overexpression in Mice
Authors: Chen Jin , Hatcher Jeff , Gu He , Cheng Zixi Jack
Published: November 2023

Overproduction of reactive oxygen species (ROS), such as the superoxide radical (O2‐.), is associated with diseases which compromise cardiac autonomic function. Overexpression of SOD1 may offer protection against ROS damage to the cardiac autonomic system, but reductions of O2‐. may interfere with normal cellular functions. Therefore, we need to consider whether or not SOD1 overexpression in animals can affect the neural components of the baroreflex loop in normal animals. We have selected the C57B6SJL‐Tg (SOD1)2 Gur/J mouse as a model to determine whether SOD1 overexpression alters baroreflex sensitivity (BRS), aortic depressor nerve (ADN) function, and heart rate (HR) responses to ADN and vagal nerve stimulation. Under isoflurane anesthesia, left femoral artery and vein of mice were catheterized and MAP and HR were measured. Baroreflex control of HR (baroreflex sensitivity: BRS) and aortic depressor nerve functions were assessed in response to hypotension and hypertension which were induced by sequential injections of sodium nitroprusside and phenylephrine. In addition, HR responses to electrical stimulation of aortic depressor nerves and vagal efferent nerves were measured. Our data indicated that SOD1 overexpression: 1) increased aortic depressor nerve function, 2) decreased HR responses to vagal efferent nerve stimulations, 3) did not alter HR responses to aortic baroreceptor afferent nerve stimulation, 4) did not change baroreflex control of HR. Collectively, our findings suggest that SOD1 overexpression preserved the normal BRS, but may alter the function of baroreceptor afferent, central, and vagal efferent components differently. While SOD1 overexpression enhanced aortic depressor nerve function and central component function, a decreased vagal efferent control over HR may explain an unchanged BRS in C57B6SJL‐Tg (SOD1)2 Gur/J mice.

Support or Funding Information

NIH RO‐1 HL‐58727 to Zixi (Jack) Cheng
The Effect of Oral Hygiene on Cardiac Health as Determined by Heart Rate Variability
Authors: Bujnoski Emily Elizabeth
Published: November 2023

Cardiac disease is the number one cause of death throughout the world (“The 10 Leading Causes of Death in the World, 2000 and 2012”, 2014). An indicator of physiological cardiac health is heart rate variability (HRV). HRV, determined using an electrocardiogram, measures the variation of time between R‐intervals of consecutive heartbeats due to autonomic influence. Higher variability between these R‐intervals indicates better cardiac health. HRV can be divided into low frequency and high frequency oscillations, whereas more low frequency indicates increased sympathetic activity and more high frequency indicate parasympathetic activity. This relationship is made possible via a Fast Fourier Transformation. The aim of this study is to relate these autonomic implications of the heart to oral hygiene, because little research in this field exists today. Over the course of five months, a random voluntary sample of college‐aged individuals completed oral hygiene surveys including information on brushing, flossing, and numbers of dental caries (cavities). HRVs were then taken from the sample group. Initial analysis shows significant positive correlation between low frequency HRV and number of cavities (>2) (n=55, r=0.71, p=0.014). The findings indicate a potential relationship between poor oral health and specific autonomic cardiac outcomes.

Support or Funding Information

Funding for this project provided by the Elon University Undergraduate Research Program, the Elon College Fellows Program, and Elon University's Summer Undergraduate Research Program.
Electrical Stimulation of the Carotid Sinus Nerve Improves Hemodynamics and Heart Rate Variability in L‐NAME Hypertensive Rats
Authors: Souza Gean Domingos , Silva Luiz Eduardo Virgilio , Dias Daniel Penteado Martins , Santos‐Almeida Fernanda Machado , Castania Jaci Airton , Salgado Helio Cesar , Fazan Rubens
Published: November 2023

Baroreflex activation by electrical stimulation of the carotid sinus (CS) has been recently used to treat resistant hypertensive patients. In the present study, we evaluated the effects of long‐term electrical stimulation (ES) of CS in L‐NAME hypertensive rats.

Hypertension was induced in male Wistar rats by administration of L‐NAME (70 mg/kg, per os) for 10 days. 7 days after initiating L‐NAME administration the animals were anesthetized and implanted with a catheter into the femoral artery and received, subcutaneously, a miniaturized battery‐operated electrical stimulator connected to electrodes placed around the left CS. On the following day, basal AP was directly recorded for 60 min and the stimulator was turned on. Electrical pulses (1 ms, 3 V, 30 Hz) were delivered to the CS, intermittently (20/20s ‐ on/off), for the next 48 h. Basal mean arterial pressure (AP) from L‐NAME hypertensive rats was 176±3 mmHg but showed a marked fall (D= −51±5 mmHg) at the onset of ES of the CS. At the end of the stimulation period only a mild rise in AP was seen; however, the AP recorded during the first 60 min after ES of the CS was lower (159±3 mmHg) than before the stimulation period. Spectral analysis showed that ES of the CS reduced the LF power of AP spectra (10.0±1.9 to 3.7±0.4 mmHg2, p=0.014) and also increased the power of PI spectra at HF range (1.8±0.4 to 3.4±0.8 ms2, p=0.028). Moreover, LF/HF ratio of PI spectra was also markedly reduced after the stimulation period (0.42±0.09 to 0.14±0.02, p<0.01). In conclusion, ES of the CS for 48 h was effective to reduce the AP lasting 1 h after the stimulation period. In addition, the AP and heart rate variability strongly suggests a lower sympathetic modulation upon the cardiovascular system after ES of the CS in L‐NAME hypertensive rats.

Support or Funding Information

FAPESP, CAPES, CNPq, FAEPA
Enhanced Carotid Body‐mediated Chemoreflex Drive in Heart Failure: a Matter of Flow
Authors: Andrade David C , Schultz Harold D , Marcus Noah J , Toledo Camilo , Lucero Claudia , Del Rio Rodrigo
Published: November 2023

Enhanced carotid body (CB) chemoreflex function in heart failure (HF) is strongly related to cardiorespiratory disorders and the progression of the disease. Importantly, HF patients with an increased CB chemoreflex drive exhibit a poor prognosis. The mechanisms underlying CB sensitization during HF are not fully understood. We hypothesized that blood flow restrictions to the CB region during the progression of HF severely impact CB chemoreflex function. We sought to determine whether blood flow reductions to the CB in the setting of HF induced chemoreflex potentiation. Myocardial infarcted Sprague‐Dawley rats were used as a low output HF model (LowHF) and aortocaval fistulated rats were used as a high output HF model (HighHF). All experiments were performed 8 weeks after HF induction. Blood flow to the CB region was estimated by carotid artery Doppler imaging. Autonomic function was measured by heart rate variability (HRV) and atropine‐propranolol test. The ventilatory CB‐mediated chemoreflex drive was measured by plethysmography allowing the rats to breathe hypoxic gas (FiO2 10%). Resting breathing recordings were visually inspected to scored apneas and hypopneas (AHI). Expression of the flow sensitive transcription factor, Krupper‐like factor‐2 (KLF2) was assessed by immunofluorescence and immunoblot. LowHF rats and HighHF rats displayed similar degrees of autonomic imbalance (P>.05, HRV and HR response to atropine/propranolol). LowHF rats exhibited a significantly enhanced CB‐mediated hypoxic ventilatory response compared to sham rats (222±20 vs. 112±6 ml/min/100g, LowHF vs. sham, respectively; P<.05) and compared to HighHF rats (222±20 vs. 55±7 ml/min/100g, LowHF vs. HighHF, respectively; P<.05). However, AHI was increased (P<.05) in both LowHF and HighHF rats compared to sham rats (9±1 vs. 16±2 vs. 20±2 events/h, sham vs. LowHF vs. HighHF, respectively). Carotid artery blood flow was decreased in LowHF (P<.05) compared to sham rats and HighHF rats (17±1 vs. 28±4 vs. 35±2 ml/min LowHF vs. sham vs. HighHF, respectively). KLF2 expression in the CB was markedly reduced in LowHF compared to sham rats while no changes in KLF2 expression was found in HighHF. Our results showed that carotid blood flow reductions during the progression of low output HF reduced CB KLF2 expression and enhanced the CB chemoreflex drive. CB chemoreflex sensitivity was not altered in high output HF in which carotid blood flow and KLF2 remained normal.

Support or Funding Information

Supported by FONDECYT 1140275 and NIH PO1‐HL62222.
Effect of Six Months of Cardiac Rehabilitation on Autonomic Function in Coronary Artery Disease Patients
Authors: Badrov Mark B , Norton Katelyn N , Lalande Sophie , Suskin Neville , Shoemaker J Kevin
Published: November 2023

We investigated the effects of a 6‐month exercise training‐based cardiac rehabilitation (CR) program on autonomic function in patients with coronary artery disease (CAD). Twenty‐two CAD patients (4 women; 62 ± 8 yrs, 172 ± 9 cm, 86 ± 13 kg) were studied prior‐to and following six months of aerobic and resistance training‐based CR. Seventeen healthy aged‐matched controls were also studied (6 women; 60 ± 9 yrs, 169 ± 8 cm, 73 ± 14 kg). We evaluated resting blood pressure (BP; manual sphygmomanometry), muscle sympathetic nerve activity (MSNA; microneurography; CAD, n = 14), heart rate variability (HRV; time‐ and frequency‐domain), and cardiovagal baroreflex sensitivity (BRS; sequence method). At baseline, prior to CR, CAD patients had greater burst frequency (48 ± 8 vs. 36 ± 8 bursts/min; P < 0.01) and burst incidence (81 ± 7 vs. 62 ± 10 bursts/100 heart beats; P < 0.01) in comparison to healthy controls. No differences at baseline between CAD patients and healthy controls were found for resting BP, any index of HRV, or cardiovagal BRS (all P > 0.05). Six months of exercise‐based CR lowered resting mean arterial BP (86 ± 10 to 83 ± 7 mmHg; P < 0.05) and systolic BP (122 ± 16 to 116 ± 11 mmHg; P < 0.05) in CAD patients. In CAD patients, CR reduced burst frequency (48 ± 8 to 39 ± 11 bursts/min; P < 0.01; d = 0.95) and burst incidence (81 ± 7 to 66 ± 17 bursts/100 heart beats; P < 0.01; d = 1.32). Also, following CR, and in contrast to pre‐CR, levels of MSNA were not significantly different between CAD and healthy controls for burst frequency (P = 0.43) and burst incidence (P = 0.39). No index of HRV (all P > 0.05) or cardiovagal BRS (P = 0.78) was changed in CAD patients following CR. In conclusion, six months of exercise training‐based CR reduced baseline BP and sympathetic outflow in CAD patients to levels observed in healthy controls.

Support or Funding Information

Canadian Institutes for Health Research (CIHR) Team Grant “Physical Activity, Mobility, and Neural Health” (Grant #217532).
Acute alcohol consumption blunts the muscle sympathetic nerve activity response to mental stress in humans
Authors: Fonkoue Ida T , Gervais Brett , Chen Qinghui , Carter Jason R
Published: November 2023

The sympathoexcitatory response of acute alcohol consumption on muscle sympathetic nerve activity (MSNA) at rest is well documented. Mental stress is a common laboratory stressor that elicits robust increases in blood pressure (BP) and heart rate (HR), and our laboratory has recently established that BP, HR, and MSNA reactivity to mental stress are significantly reproducible within an individual. Acute alcohol consumption has been reported to blunt BP and HR response to mental stress, often referred to as the stress dampening response (SDR), but this SDR response is not universal and mechanisms remain unclear. We hypothesized that acute alcohol consumption would significantly blunt MSNA reactivity and/or augment the forearm vasodilatory response to mental stress. Twenty two young, healthy subjects (23±1 years of age, BMI 26±1 kg/m2) volunteered to be part of this study. BP, HR, MSNA, and forearm blood flow (FBF) were recorded during 5 min of supine rest and five min of mental arithmetic before and 45 min after consumption of 2.5ml of vodka per kg body mass (i.e., a 4–5 drink equivalent). Successful nerve recordings were obtained in 19 of the 22 subjects, and maintained throughout both trials of mental stress (i.e., pre‐ and post‐alcohol) in 16 individuals. At baseline, acute alcohol consumption significantly increased HR (62±3 to 68±3 beats/min; p<0.001) and MSNA (12±2 to 19±2 bursts/min; P<0.001), but did not alter MAP (83±2 to 83±3 mmHg; p=0.848), FBF (2.1±0.1 to 2.3±0.2 ml/100 ml of tissue/min; p=0.223), or forearm vascular conductance (FVC; 0.026±0.002 to 0.028±0.002 ml/100 ml of tissue/min/mmHg; p=0.282). During mental stress, alcohol significantly reduced FBF (Δ1.7±0.3 to Δ1.0±0.3 ml/100 min of tissue/min; p=0.014) and FVC (Δ63±10% to Δ39±10%; p=0.016) reactivity. Acute alcohol consumption did not alter MAP (Δ9±2 to Δ8±2mmHg; p=0.195) and HR (Δ21±3 to Δ17±2 beats/min; p=0.123) reactivity to mental stress, but significantly blunted MSNA reactivity to mental stress when quantified as either changes in MSNA burst frequency (Δ0.1±2 to Δ−2.2±2 bursts/min; p=0.022) or total MSNA (Δ112±10% to Δ21±10%; p=0.005). In conclusion, our findings demonstrate a blunted MSNA reactivity to mental stress that was coupled with a paradoxical blunting of the classic and consistent forearm vasodilation associated with mental stress, suggesting that factors other than MSNA are influencing the alcohol‐mediated attenuation of forearm vascular reactivity to mental stress in humans.
Hypertension Impairs Peripheral and Cerebrovascular Reactivity to Isocapnic Hypoxia in Humans
Authors: Fernandes Igor Alexandre , Campos Monique Opuszcka , Mansur Daniel Elias , Mattos João Dario , Rocha Marcos Paulo , Nóbrega Antonio Claudio Lucas
Published: November 2023

Hypertensive animal models indicate impairment in both peripheral and cerebrovascular reactivity to isocapnic hypoxia. However, it is still uncertain whether these findings could be translated to a setting of human hypertension. Therefore, the main purpose of this investigation was to determine the impact of hypertension on peripheral and cerebrovascular reactivity to isocapnic hypoxia in humans. Six normotensive (NT, 44 ± 11yrs, mean arterial pressure (MAP) 87 ± 6 mmHg) and 5 hypertensive men (HT, 46 ± 11yrs, MAP 110 ± 9 mmHg) were exposed to 5‐min bouts of: (1) Normoxia (21% O2) and (2) isocapnic‐Hypoxia (10%O2). Oxygen saturation (pulse oxymetry), beat‐to‐beat MAP (photoplethysmography), muscle sympathetic nerve activity (MSNA [microneugraphy]) and partial pressure of end‐tidal carbon dioxide (PETCO2) were monitored throughout the study. Clamping of PETCO2 was also performed through a rebreathing system and blood flow in the femoral (FA), internal carotid (ICA) and vertebral arteries (VA) was simultaneously quantified via Doppler ultrasound at the last 30s of each condition. Both experimental groups showed similar reductions in oxygen saturation as a result of isocapnic‐hypoxia exposure (NT −25.7 ± 3.3 vs. HT −21.2 ± 4.0%, p > 0.05). No substantial changes on MAP and PETCO2 were observed during isocapnic hypoxia. While comparable increases in ICA perfusion were observed (NT +204.4 ± 32.7 vs. HT +134.8 ± 65.5 ml.min−1, p > 0.05), only NT exhibited a significant increment in VA blood flow (+78.1 ± 24.5 vs. HT +8.3 ± 7.9 ml.min−1, p < 0.05). At the peripheral circulation, HT presented a drastic drop in FA blood flow (−110.2±43.0 ml.min−1) while NT showed a significant increase in the FA perfusion (+73.9 ± 37.4 ml.min−1, p < 0.05). MSNA recordings revealed greater increases in sympathetic activation of hypertensive individuals (HT +12.7 ± 6.7 vs. NT +3.6 ± 1.6 bursts/min, p < 0.05). In conclusion, hypertension impairs both peripheral and cerebrovascular reactivity to isocapnic hypoxia and MSNA recordings may suggest a contribution of a sympathetic overdrive to this response.

Support or Funding Information

Grant Funding Source: CNPq, FAPERJ, CAPES e FINEP
Effects of Acute Hypoxia on Human Adipose Tissue Lipoprotein Lipase Activity and Lipolysis
Authors: Mahat Bimit , Chassé Étienne , Mauger Jean‐François , Imbeault Pascal
Published: November 2023

Context

Adipose tissue regulates postprandial lipid metabolism by storing dietary fat through lipoprotein lipase (LPL)‐mediated hydrolysis of dietary triglycerides, and by inhibiting non‐esterified fatty acid (NEFA) delivery to nonadipose tissues. Studies conducted in animal showed that acute intermittent hypoxia, a model of obstructive sleep apnea, reduce adipose tissue LPL activity and increase NEFA release, adversely affecting postprandial lipemia. These observations remain to be tested in humans.

Objective

To investigate the effects of acute hypoxia on human adipose tissue LPL activity and lipolysis.

Methods

Differentiated human preadipocytes were exposed to hypoxia (3% O2) for 24h and adipose tissue biopsies were obtained from 10 healthy men exposed for 6 hours to either normoxia or intermittent hypoxia following an isocaloric high‐fat meal.

Results

In differentiated human preadipocytes, acute hypoxia induced a 6‐fold reduction in LPL activity (p<0.001). In humans, the rise in postprandial triglyceride levels did not differ between normoxia and intermittent hypoxia. NEFA levels were significantly higher during intermittent hypoxia session (condition effect, p<0.05). Intermittent hypoxia did not affect subcutaneous abdominal adipose tissue LPL activity. No difference in lipolytic‐induced responses of isolated subcutaneous abdominal adipocytes were observed between normoxia and intermittent hypoxia sessions.

Conclusions

Acute hypoxia strongly inhibits LPL activity in differentiated subcutaneous abdominal preadipocytes. Acute intermittent hypoxia increases circulating plasma NEFA in young healthy men, but does not seem to affect postprandial triglyceride levels, nor subcutaneous abdominal adipose tissue LPL activity and adipocyte lipolysis.

Support or Funding Information

The study is dedicated to the memory of our colleague Michaël Babinsky. We are grateful to Mrs. Ann Beninato and Sabrina Ait‐Ouali for their technical assistance. Bimit Mahat is a recipient of a Ontario Graduate scholarship. Étienne Chassé is a recipient of a Institut de recherche de l'Hôpital Montfort scholarship. This study was supported by the Natural Sciences and Engineering Research Council of Canada as well as the University of Ottawa.
Beneficial Effects of Physical Training on the Vascular Dysfunction Induced by Intermittent Hypoxia during Apnea
Authors: ESTEVE Eric Jean‐Claude , El Dirani Zeinab , Lemarie Emeline , Daher Ahmad , Joumaa Wissam , Faury Gilles
Published: November 2023

Obstructive sleep apnea syndrome (OSA) is characterized by multiple breathing interruptions during sleep due to transient closing of the superior airways. This induces chronic intermittent hypoxia (IH), which enhances cardiovascular dysfunction and remodeling, potentially leading to hypertension and cardiac infarct. Since physical training has previously been described as limiting the extent and occurrence of several cardiovascular diseases, the aim of this study was to evaluate the beneficial effect of intensive training on the reactivity of arteries and calcium signaling in vascular smooth muscle cells (SMC) of rats submitted to IH.

Four groups of Wistar rats were randomly assigned: N (normoxic sedentary rats), NIE (normoxic intensively exercised rats), IH (intermittent hypoxic sedentary rats) and IHIE (intermittent hypoxic intensively exercised rats). IH consisted in alternating normoxia (21%O2) and hypoxia (5%O2) every 30 sec in the cages for 8 h/day during 3 weeks. Exercise sessions were conducted 5 times/week in NIE and IHIE rats as a double fast 24 min walk with a speed progressively rising from 16 to 30 m/min. After 21 days of normoxia or IH (with/without training), the aortic ring tone in response to increasing doses of vasoactive agonists has been investigated by tension arteriography. Also, calcium imaging of cultured aortic SMC and protein expression of calcium channels were performed to examine the effects of lack of oxygen and/or training at the cellular and molecular levels respectively.

21 days of exposure to IH had no impact on the aortic response to tested vasoconstrictors (10−11 to 10−4 M endothelin‐1 and 10−12 to 10−5 M phenylephrine). IH also reduced by up to 20% the vasorelaxant effect of acetylcholine (at 10−6 M), which is in accordance with the previously shown endothelial dysfunction generated by IH. On the contrary, intensive training increased by ~20% the vasodilatory effect of acetylcholine (10−10 to 10−3 M), compared to aortic responses of sedentary animals, while it decreased by ~50% the aortic response to the vasoconstrictors endothelin‐1 and phenylephrine in both normoxic and IH groups. Intensive training also increased the caffeine‐induced elevation of cytoplasmic calcium level in aortic SMC. At the molecular level, the protein expressions of major calcium channels like RyR or TRP were altered during hypoxia and/or exercise modulating SMC calcium signaling. Our results suggest that intensive exercise training could decrease the adverse effects of IH on the cardiovascular physiology trough calcium homeostasis. This could lead to improvements in the treatment of OSA patients.

Support or Funding Information

UGA, INSERM, Agir@Dom, AUF (PCSI)
Effect of Electrical Stimulation on Grasshopper Jumping Muscle
Authors: Yazdani Ariella A. , Broadwell Randi C. , Kirkton Scott D.
Published: November 2023

Skeletal muscles can undergo biochemical and physiological changes due to exercise training. For example, aerobic exercise training may lead to an increased mitochondria volume, a higher proportion of slow twitch myosin ATPase, and a reduced rate of lactic acid production. Similar changes in mammalian muscle have also been demonstrated using electrical stimulation. However, it is unclear if invertebrate muscle also responds to training or electrical stimulation. We examine whether Schistocerca americana grasshopper jumping muscles are affected by electrical stimulation. Grasshopper jumping muscle is one of the few invertebrate muscles to produce lactic acid during locomotion. Electrical probe leads are placed near the coxa on the ventral side of the animal. The muscle was stimulated with a single pulse width of 2 ms at 0.075 V and repeated at a frequency of 100 Hz. We electrically stimulate the entire jumping muscle, including both the flexor and the extensor, to elicit continuous muscle contractions in a single metathoracic leg. To invoke a training response, a grasshopper is stimulated for 5 hours daily for 5 consecutive days. The lactate production rate of trained and untrained grasshopper muscle will then be fluorometrically measured after 5 min, 30 min, 60 min, 180 min, or 300 min of electrical stimulation. We predict that repeated stimulation would alter the rate of lactic acid production. Since grasshopper jumping muscle is similar to human muscle, this research may provide insight into fundamental questions about muscle plasticity.

Support or Funding Information

Union College Undergraduate Research Committee
Age‐Related Decline of Anoxia Tolerance in Adult Drosophila melanogaster
Authors: Sargent James C. , Campbell Jacob B. , Harrison Jon F.
Published: November 2023

Cell death occurring from anoxia is considered to be the cause of many human diseases such as heart attack and stroke. Though much is known about the cause of anoxia in such conditions, we still have a poor understanding of the mechanisms causing cell death and the genetic and physiological processes responsible for variation in survival of anoxia. Drosophila melanogaster are particularly interesting models for studying responses to anoxia as they can survive many hours of anoxia and most of their metabolic pathways are similar to those of humans. We hypothesized, based on studies with humans, that younger adults would have a higher anoxia tolerance than older adults. We exposed adult Drosophila, ages 1, 3, 5, 7, 9, and 12 days old, to six hours of anoxia. Survival was assessed 24 hours post‐treatment; surviving individuals exhibited movement if stimulated. Survival declined with age in a linear fashion for females and in a more exponential fashion for males. Seventy nine percent of adults one day past eclosion survived six hours of anoxia; while only 10% of twelve‐day‐old adults survived. Additionally, we measured ATP in 1 and 12 day old Drosophila indifferent durations of anoxia. In anoxia, ATP levels declined rapidly to near‐zero levels in both 1 and 12 day old adults; thus the better capacities of young adults to survive anoxia are not due to a better capacity to keep ATPelevated. This age‐related decline in anoxia tolerance may be due to loss of capacities to prevent or repair anoxia‐related damage. Overall, these data show that patterns of gender‐ and age‐associated variation in tolerance to anoxiaare similar in Drosophila and mammals, suggesting that Drosophila may be underutilized models for studies of the genetic and biochemical mechanisms of pathology of stroke and heart disease.

Support or Funding Information

This research was partially supported by NSF IOS 1256745 and the School of Life Sciences Undergraduate Research (SOLUR) Program at Arizona State University, Tempe Campus.
Using confocal imaging to understand the effect of atmospheric oxygen on insect respiratory systems
Authors: Gstrein Gregory , Arthur Kathryn , Parker Greg , Friedman Jessica , Brooks John Vanden
Published: November 2023

Recent geochemical models suggest that over geologic time atmospheric oxygen has varied from as low as 12kPa and as high as 31kPa. These changes in atmospheric oxygen would have had strong effects on the physiology and evolution of organisms living at those times including insects and other arthropods. Insects transport oxygen completely in the gas phase through hollow tubes known as tracheae. We have hypothesized that this unique respiratory system of insects made them more susceptible to changes in atmospheric oxygen. Previously, it has been shown that as insects increase in body size, their tracheal system increases at a faster rate and may eventually limit the maximum body size of insect species. As a corollary, we have shown that rearing oxygen is inversely correlated with tracheal system density in some insects; with hyperoxia decreasing tracheal investment and hypoxia increasing tracheal investment. Therefore, a hyperoxic rearing environment may remove the spatial constraint of the tracheal system and allow insects to achieve larger body sizes. However, a limit to these studies has been a focus on the large conducting tracheae which drive bulk flow of gases through the system. Previous research has largely ignored the final oxygen delivery component of the tracheal systems.

Here we look at the effect of rearing oxygen on the smallest components of the tracheal system – the blind ended tracheoles across which the actual oxygen delivery to the tissues occurs. To this end, we have developed a new confocal imaging technique to image and analyze insect tracheoles in 3D, which allows for increased resolution and spatial imaging capability. The density of tracheoles, tracheolar diameters, and network properties of the tracheolar branching all affect oxygen delivery and may be a major driver in the control of insect body size. To understand these effects, we reared Drosophila melanogaster under three different oxygen partial pressures (12kPa ‐ hypoxia, 21kPa ‐ normoxia, and 31kPa hyperoxia) that match the range of variation seen over geologic time. We then carried out confocal imaging of the tracheolar network in the flight muscle dissected out of the thorax of these D. melanogaster. The tracheolar network auto‐fluoresces under the confocal microscope excitation allowing us to obtain high resolution 3D reconstructions of the respiratory system. By using ImageJ and AVIZO, we were able to measure tracheal and tracheolar diameters and characterize the network branching patterns of these D. melanogaster reared under the varying oxygen levels. By measuring these two components of the insect respiratory system, we have shown that oxygen does effect tracheolar diameters and has a strong effect on branching pattern and tracheolar investment in the flight muscle of D. melanogaster.

Support or Funding Information

Midwestern University College of Veterinary Medicine Fellowship Program
Metabolic and Genetic Implications of Survival in Anoxic Drosophila
Authors: Campbell Jacob B , Harrison Jon F
Published: November 2023

Anoxia or severe hypoxia is a fundamental component of pathologies associated with heart disease, stroke and many other human pathologies; yet we still lack a fundamental understanding of how anoxia/severe hypoxia kills and the mechanisms responsible for the wide variation in anoxia tolerance across animals. Drosophila larvae feed on the yeasts of rotting fruits in a semi liquid environment, and as a consequence, the larvae regularly experience episodes of hypoxia/anoxia whereas adults live in air and are likely to experience hypoxia on rare occasions. Perhaps surprisingly, larvae are much less tolerant of anoxia than adults; nearly 100% of adults survive up to 4 hours of anoxia while less than 50% of third in star larvae survive anoxic bouts greater than 90 minutes. We used biochemical and physiological methods to assess stage‐related variation in the ability to match ATP supply and demand, and in addition, we conducted a genome‐wide association study to find and then verify candidate genes responsible for variation in anoxia survival within populations of larvae and adults. Anoxia induced strong decreases in glycogen levels in both larvae and adults; however, both stages also showed strong increases in free glucose indicating that carbohydrate depletion is likely not a cause of death. Both larvae and adults accumulated lactate, but larvae did so at a higher rate during the first hour of anoxic exposure which suggests that high locomotion of larvae during initial exposure to hypoxia requires high rates of anaerobic metabolism that may be functionally linked to mortality. Preliminary measures of ATP show that ATP declines to near zero at the first measured time points—2 hours for adults and 30 minutes for larvae. Survival of a 1 hour anoxia exposure was highly repeatable but varied greatly across 178 lines from the Drosophila Genetics Reference Panel ranging from 25–95%. Thirty‐two SNPs and 23 genes were associated with the variation in survival across lines, all with P values less than 0.00001. Gene ontology enrichment analysis implicates differential genes in classes involved in vertebrate angiogenesis, cell proliferation, membrane/cytoskeletal remodeling, and fatty acid metabolism associated with differential survival across lines. Several of the identified genes linked to survival across the DGRP lines have human orthologs that have been linked to oxygen‐mediated cancer pathways and Alzheimer's progression in humans.

Support or Funding Information

This research was supported by NSF IOS 1256745.