bluuebunny/crossref_metadata_2025 huggingface dataset:
Repetitive Transcranial Magnetic Stimulation (rTMS) Elicits Long Lasting Phrenic Motoneuron Excitability Increase in the Rat: Possible Treatment for Respiratory Insufficiency?
Authors: Vinit Stéphane , Michel‐Flutot Pauline , Zholudeva Lyandysha V , Randelman Margo L , Mansart Arnaud , Deramaudt Thérèse B , Lane Michael A , Petitjean Michel , Bonay Marcel
Published: June 2021

Repetitive transcranial magnetic stimulation (rTMS) is a cortical neuromodulatory technique shown to be effective in a clinical setting. However, the underlying mechanisms are poorly understood, thus emphasizing the need to reverse translate the technique to prelinical animal models. We have recently demonstrated the feasibility of using TMS to evaluate diaphragmatic corticomotor pathways by recording specific motor evoked potentials from the diaphragm (MEPdia) in naïve rats as well as in a preclinical rat model of neurological respiratory deficit (C2 hemisection, Vinit et al., PLoS One 2014, 2016). Based on these encouraging preliminary results, we evaluated the evolution of the phrenic motoneuron excitability (by assessing MEPdia) following acute high frequency rTMS protocol applied to the cortical and subcortical areas in a respiratory neurobiology animal model. In an anesthetized adult rat preparation, we applied a repetitive high frequency rTMS protocol (9 trains of 100 biphasic pulses at 10Hz, with 30s intervals, under the motor threshold stimulation of the animal) and evaluated the phrenic motoneuron excitability by recording MEPdia before and up to 60 min post‐rTMS session. This acute high frequency rTMS protocol led to a robust and long lasting increase in phrenic motoneuron excitability (89.9 ± 21 % increase from baseline compared to time control animals 12.6 ± 14.6 %; p<0.002) at 60 min post rTMS protocol. Interestingly, pretreatment with Clonazepam (GABAA agonist) or Baclofen (GABAB agonist) before rTMS blocked this increase in respiratory excitability (9.1 ± 9.2 % and −2.3 ± 10.6 % respectively; p<0.001). Trans‐synaptic retrograde labeling with pseudorabies virus from the diaphragm revealed pre‐phrenic interneurons with GABAA and GABAB receptors, supporting the notion that GABAergic modulation of respiratory excitability could be induced by rTMS. To conclude, one single rTMS protocol induces a long‐lasting increase in respiratory excitability in naïve animals, by disinhibiting GABAergic receptors. Ongoing experiments investigating whether chronic daily rTMS might extend this acute effect. These data serve as foundations for future applications of rTMS to further enhance functional recovery of respiration after high cervical injury.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distribution of brain derived neurotrophic factor (BDNF) immunostaining along the C2–C5 spinal cord in adult rats
Authors: Benevides Ethan S , Sunshine Michael D , Wollman Lila B , Jorgensen Marda L , Fuller David D
Published: June 2021

BDNF is a powerful modulator of neuroplasticity and can be found across the rostral‐caudal extent of the spinal neuroaxis in neurons, astrocytes and microglia. BDNF is necessary and sufficient to drive plasticity in the phrenic motor system, likely through either pre‐ and/or post‐synaptic actions on phrenic motoneurons. To our knowledge, the relative distribution of BDNF expression across this region of the mid‐cervical spinal cord has not been systematically “mapped”. Accordingly, in ongoing experiments, we are quantitatively evaluating the distribution of BDNF positive neurons in the C2–C5 spinal cord in adult Sprague‐Dawley rats. The guiding hypothesis is that BDNF will be most prevalent in the C3‐5 ventral horn in the region of the phrenic motor nucleus. Tissues were harvested from adult male rats and processed with standard immunochemistry methods using a BDNF antibody (Alomone, 1:500 dilution). Control experiments showed an absence of spinal cord staining when tissues were incubated without the primary antibody and while keeping all other aspects constant. A custom MATLAB code was used to quantify the number of BDNF positive cells throughout C2–C5. Specifically, BDNF expressing soma were identified as areas with fluorescent intensity above a common threshold. To separate putative neurons, identified regions were sorted by size and only regions with an area of 250–1500 μm2 were included for further analysis. We observed BDNF expression within neurons in laminae I, II, VII, VIII, and IX across the mid‐cervical spinal cord. Further, the number of BDNF positive neurons increased in a rostral to caudal manner from C2 to C5 (P<0.05). There were more BDNF expressing neurons located in the ventral horn when compared to the dorsal horn across C2–C5 (p< 0.01). Finally, smaller BDNF positive cells were evident throughout the gray and white matter which is consistent with microglial distribution in the spinal cord, however, this staining has not been verified at this time. These initial results suggest that naturally occurring levels of BDNF vary across the cervical spinal cord, and are greatest in the region of the phrenic motor nucleus.

Support or Funding Information

1R01HL139708‐01A1 (DDF), T32‐HD043730 (MDS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Impact of Disrupting BDNF/TrkB Signaling on Diaphragm Muscle Force Generation across Motor Behaviors
Authors: Pareja‐Cajiao Miguel , Cole Naomi A. , Gransee Heather M. , Sieck Gary C. , Mantilla Carlos B.
Published: June 2021

Brain‐derived neurotrophic factor (BDNF) binds with high affinity to tropomyosin‐related kinase receptor subtype B (TrkB), and plays an important role in neuromuscular junction (NMJ) function (regulating neuromuscular transmission and muscle innervation). We previously showed impaired neuromuscular transmission following acute inhibition of TrkB kinase activity in TrkBF616A mice harboring a point mutation that renders the kinase domain susceptible to inhibition by 1NMPP1 treatment. Neuromuscular transmission failure is expected to predominantly affect the ability of more fatigable motor units to generate force and thus primarily impair higher force motor behaviors. Accordingly, we hypothesized that acute administration of 1NMPP1 will impair the ability of the diaphragm muscle to accomplish higher force behaviors in adult, 6‐month old TrkBF616A mice. Transdiaphragmatic pressure (Pdi) measurements were conducted across a range of motor behaviors in lightly anesthetized mice during eupnea, exposure to hypoxia (10% O2)/hypercapnia (5% CO2), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Adult male and female TrkBF616A mice at 6 months were injected with 1NMPP1 or vehicle (DMSO) one hour prior to Pdi measurements. The Pdi generated during eupnea and hypoxia/hypercapnia was ~8 cm H20 compared to ~70 cm H20 during maximal stimulation. There was a significant reduction in Pdi during maximal force with bilateral phrenic nerve stimulation in the 1NMPP1 group compared to the vehicle control (p=0.01). There was no effect of 1NMPP1 on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. The reduction in Pdi is generally similar to the impairment in neuromuscular transmission consistent with a predominant effect on motor units innervating fast‐twitch fatigable muscle fibers in the diaphragm muscle. Thus, impaired trophic BDNF/TrkB signaling can be expected to limit the range of motor behaviors accomplished by the diaphragm muscle and may be an important contributor to neuromuscular dysfunction.

Support or Funding Information

This study was supported by NIH grant R01 AG057052.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adenosine 2A Receptor Inhibition Promotes Neuroprotection Following Toxic Insult to Phrenic Motor Neurons
Authors: Sajjadi Elaheh , Seven Yasin B , Simon Alec K , Zwick Amanda , Satriotomo Irawan , Mitchell Gordon S
Published: June 2021

Motor neuron death is the hallmark of certain diseases, such as amyotrophic lateral sclerosis (ALS). Neuroprotective strategies promoting respiratory motor neuron survival are critical to avoid respiratory insufficiency and improper airway clearance. Adenosine 2A (A2A) receptors contribute to excitotoxic motor neuron death in vitro. Since different motor neuronal populations have differential susceptibilities in neurodegenerative disorders, we explored the role of A2A receptors in phrenic motor neuron cell death in vivo. We used an experimental model of phrenic motor neuron death developed in our laboratory (CtB conjugated Saporin; CtB‐Sap) injected into the intrapleural space. We tested the hypothesis that A2A receptor expression is upregulated in phrenic motor neurons under toxic insult. We also tested the hypothesis that A2A receptor inhibition promotes phrenic motor neuron survival and preserves diaphragm activity. Rats were randomly assigned to control, CtB‐Sap + Vehicle and CtB‐Sap + Istradefylline (A2A receptor antagonist) groups. CtB and CtB‐Sap were injected intrapleuraly bilaterally. Following transcardial perfusion, spinal cords were fixed, cryoprotected, and sectioned. Immunohistochemistry was performed to count CtB‐labeled phrenic motor neurons and assess A2A receptor expression within these motor neurons. Diaphragm EMG activity was recorded via intramuscular electrodes during normoxia, hypoxia (10.5%) – hypercapnia (7%), and spontaneous sighs during hypoxia‐hypercapnia. A2A receptors were upregulated in the spared phrenic motor neurons of CTB‐Sap treated rats (p<0.05). CtB‐Sap selectively killed nearly all phrenic motor neurons within a week and caused diaphragm paralysis (p<0.01). With A2A receptor inhibition, phrenic motor neuron loss was reduced (p<0.05) and diaphragm EMG activity partially preserved (p<0.05). A2A receptors, which contribute to motor neuron death during toxic insults, are upregulated in spared phrenic motor neurons of CTB‐Sap treated rats. This is an important finding since A2A receptor upregulation may accelerate motor neuron death in neurodegenerative diseases like ALS.

Support or Funding Information

NIH HL69064 and the McKnight Brain Institute

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Respiratory Deficiency in the Optn−/−Mouse, A Novel Model of Amyotrophic Lateral Sclerosis
Authors: McCall Angela L , Strickland Laura M , Pucci Logan , Dhindsa Justin , Tseng Henry , ElMallah Mai K
Published: June 2021

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease with no current cure. Patients with ALS die 3–5 years after diagnosis when they ultimately succumb to inadequate ventilation, hypoxia, and respiratory failure. Several genes associated with ALS have recently been discovered. One of these genes encodes optineurin (OPTN) which is associated with neurodegeneration in both ALS and glaucoma. Optineurin has multiple roles in various biochemical pathways such as regulation of inflammation and autophagy. Through these functions, OPTN appears to be neuroprotective but the exact mechanism by which loss of OPTN results in progressive neural degeneration and respiratory failure in ALS is still unclear.

Since respiratory pathology is a significant cause of morbidity and mortality in ALS, this study sought to characterize the respiratory pathology in a novel ALS mouse model – the Optn−/− mouse. The hypothesis driving this work is that the Optn−/− mouse has respiratory insufficiency due to degeneration of the respiratory motor unit – motor neuron, nerve, neuromuscular junction and muscle. Whole body plethysmography (WBP) was used to assess breathing at baseline and during a respiratory challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). During the respiratory challenge, compared to the WT mice, Optn−/− mice had significantly lower peak inspiratory flow and peak expiratory flow. Peak inspiratory flow and peak expiratory flow are indirect measures of inspiratory and expiratory muscle strength. Furthermore, throughout the challenge period Optn−/− mice spent a greater amount of time in apnea indicating pathology in the respiratory control centers. The weakness and pathology in control of breathing during the respiratory challenge progresses as the mice age. Post mortem immunohistochemical studies of the neuromuscular junctions within the diaphragm showed that Optn−/− mice had fragmented synaptotagmin staining indicating presynaptic degeneration. Finally, the hypoglossal nerves of Optn−/− mice had significantly reduced g‐ratio which indicates pathology and decompaction of myelin sheaths. In conclusion, the Optn−/− ALS mouse model displays respiratory insufficiency, aberrant control of breathing, and increased nerve demyelination.

Support or Funding Information

R21 NS098131‐02 (MKE) and K08HD077040‐07 (MKE)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PHD2 Deficiency Induces Nitrative Stress via Suppression of Caveolin‐1 in Pulmonary Arterial Hypertension
Authors: Dai Zhiyu , Zhao Youyang
Published: June 2021

Nitrative stress is evident in human pulmonary arterial hypertension (PAH) patients. However, the role of nitrative stress in the pathogenesis of obliterative vascular remolding and severe PAH remains unclear. Our recent studies identified a novel mouse model (Egln1Tie2Cre) with obliterative vascular remodeling and right heart failure, which provides us an excellent model to study nitrative stress in PAH. We found that nitrative stress was elevated whereas endothelial Caveolin‐1 (Cav1) was suppressed in the lung of Egln1Tie2Cre mice in HIF‐2 a dependent manner. Genetically overexpression of endothelial Cav1 restored nitrative stress and inhibited right ventricular (RV) systolic pressure and RV hypertrophy, as well as improved right heart function Egln1Tie2Cre mice. ROS scavenger MnTmPyp treatment also suppressed severe PAH in Egln1Tie2Cre mice. These data suggest that suppression of endothelial Cav1 expression depended on PHD2 deficiency and HIF‐2 a activation mediated upregulation of nitrative stress, which leading to elevation of pulmonary pressure and severe vascular remodeling in PAH. Our studies provide evidences that restoration of Cav1 and ROS scavenger might be therapeutic potentials for the inhibition of obliterative vascular remodeling and severe PAH.

Support or Funding Information

This work was supported in part by NIH grants R01HL123957, R01HL125350, R01HL133951, and P01HL077806 (Project 3) to Y.Y.Z, and K99HL138278 to Z.D.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DDAH1 siRNA Knockdown in a Human Pulmonary Vascular Co‐Culture Cell Model
Authors: Milton Avante D , Almazroue Hanadi , Nelin Leif D , Trittmann Jennifer K
Published: June 2021

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of premature infants. Pulmonary hypertension (PH) develops in 25–40% of BPD patients, contributing to morbidity and mortality. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made in the pulmonary endothelium by nitric oxide synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous competitive NOS inhibitor. Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA and DDAH1 is the most important for ADMA metabolism in vivo. Translational studies from our group have identified DDAH1 single nucleotide polymorphisms that are differentially expressed in BPD‐PH patients compared to BPD patients. We hypothesized that inhibiting DDAH1 in human pulmonary microvascular endothelial cells (hPMVECs) will lead to decreased apoptosis and increased proliferation of human pulmonary artery smooth muscle cells (hPASMCs) via effects in hPMVECs. We utilized a co‐culture model where hPMVECs were transfected with DDAH1 siRNA or scramble for 24h and then incubated in co‐culture with hPASMCs for 24h. Analysis included 1) trypan blue exclusion for viable cell numbers and 2) western blot analysis for cleaved caspase‐3 and 8, total caspase‐3 and 8, PCNA, and β‐actin. After DDAH1 siRNA transfection of hPMVECs and co‐culture with hPASMCs, we found greater hPASMC viable cell numbers compared to scramble hPASMC controls (N=3, p<0.05). After DDAH1 siRNA transfection of hPMVECs and co‐culture with hPASMCs, we found lower hPASMC levels of cleaved caspase‐3 protein (N=3, p<0.05) and cleaved caspase‐8 (N=3, p<0.05), with greater hPASMC protein levels of PCNA (N=3, p<0.05) compared to scramble. DDAH1 siRNA knockdown in hPMVEC results in greater hPASMC proliferation and less cleaved caspase‐3 mediated apoptosis. We speculate that DDAH1 is a potential therapeutic target for BPD‐PH. We speculate that these findings support the concept that DDAH1 regulates vascular remodeling by minimizing ADMA‐inhibition of eNOS, resulting in maintained NO production which would support angiogenesis and prevent/attenuate vascular remodeling. Future studies would include in vivo experiments in transgenic neonatal mice exposed to a BPD‐like lung injury.

Support or Funding Information

This work was funded by NIH‐K08 HL129080

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of various solvents on the cytotoxic activity and stability of Bleomycin for in vitro cell culture based assays
Authors: De La Paz Rosa , Gupta Dipankar , Shenoy Anitha , Kolli ChandraSekhar , Shenoy Vinayak
Published: June 2021

Background

Bleomycin sulfate is a metal‐complexing glycopeptide that is commonly used as an experimental model for pulmonary fibrosis. Pulmonary fibrosis induced by Bleomycin sulfate is mediated via DNA damage and cytotoxic actions on the alveolar epithelial cells. Among various factors, the in vitro actions of Bleomycin sulfate depend considerably on its chemical stability and the solvent used to dissolve the drug. In this study, we evaluated the effects of various solvents [water, dimethyl sulfoxide (DMSO), saline and Dulbecco's Modified Eagle Medium (DMEM)] on the in vitro cytotoxic activity and stability of Bleomycin.

Methods

The cytotoxic effect of Bleomycin sulfate was evaluated on alveolar epithelial cells (A549) using various concentration (10, 15 and 20 mcg/mL) of the drug dissolved in different solvents. A549 cells were seeded at a concentration of 5 × 103 cells per well in a 96 well‐plate and incubated for 24 hours. These cells were then exposed to various concentrations of Bleomycin sulfate in the different solvents for the next 24 hours. The cell viability was determined using the MTT (3‐(4, 5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide) reduction assay.

Results

Significant cytotoxic response was observed with all the doses of Bleomycin sulfate (10, 15 and 20 mcg/mL) when it was dissolved in either water or DMSO. However, this was not the case with DMEM or saline since lower concentration of Bleomycin sulfate (10 mcg/mL) failed to induce cell death. We noticed that treatment of A549 cells with any of the solvents by themselves (vehicle control) did not induce cell death. With regards to DMSO, we used a final concentration of 0.025% v/v in the cell culture media.

Conclusion

We conclude that water or DMSO (at a final concentration of 0.025% v/v) may serve as suitable solvents to dissolve Bleomycin sulfate for carrying out in vitro cell culture assays. Also, it appears that higher concentrations of Bleomycin sulfate are required to show cytotoxic activity when DMEM or saline is used as a solvent. Further studies are warranted using high performance liquid chromatography (HPLC) to explore drug stability in the various solvents used.

Support or Funding Information

Seed Funding from California Health Sciences University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deconstructing pulmonary fibrosis at single‐cell resolution
Authors: Kropski Jonathan A , Calvi Carla L , Habermann A. Chris , Guitierrez Austin , Winters Nichelle , Rodenberry John C , McDonnell Wyatt , Shaver Ciara M , Ware Lorraine B , Mallal Simon , Blackwell Timothy S , Banovich Nicholas E
Published: June 2021

Rationale

Despite years of research, the fundamental mechanisms driving the pathogenesis of pulmonary fibrosis remain unclear. Numerous histopathologic patterns of pulmonary fibrosis have been identified in association with different patterns of risk factors, but to date it remains unclear as to what mechanisms are shared across different forms of pulmonary fibrosis and which drive distinct pathologies and outcomes. We hypothesize that using single‐cell transcriptomic approaches, we can decipher both the conserved and distinct mechanisms driving pulmonary fibrosis phenotypes.

Methods

At the time of lung transplantation, single‐cell suspensions were generated from the lung parenchyma of pulmonary fibrosis patients and from declined donor lungs (controls). Unsorted single‐cell suspensions and CD45 depleted fractions were used for scRNA‐seq. Single‐cell RNA‐sequencing library preparation was performed using the 10X Genomics 3′ or 5′ kit, and sequencing was performed on an Illumina hiSeq4000 or Novaseq. Following alignment, demultiplexing was performed using Cell Ranger. Graph‐based clustering and scRNA‐seq analysis was performed using the Seurat package in R. Developmental lineage reconstruction was performing using Monocle and p‐Creode. Diagnoses were assigned based on clinical interpretation of explant pathology.

Results

Joint graph‐based clustering and canonical correlation analysis of scRNA‐seq profiles from >30,000 cells from control (n=7), IPF (n=7), chronic hypersensitivity pneumonitis (cHP, n=4), and nonspecific interstitial pneumonia (NSIP, n=3) identified 21 distinct clusters, representing the major known subtypes in the lung, as well as numerous intermediate/transitional cell types and/or states. Strikingly, across pulmonary fibrosis phenotypes, collagen and ECM gene expression was highly enriched in ACTA2low, PDGFRa+ fibroblasts, while collagen and ECM gene expression were lower in ACTA2hi myofibroblasts; few collagen‐expressing inflammatory or epithelial cells were identified. A series of cell types co‐expressing AT1 and AT2 markers were identified in both fibrotic and control lungs. Compared to NSIP and cHP, IPF epithelial cells demonstrated increased senescence markers.

Conclusions

Across different histopathologic forms of pulmonary fibrosis, both shared and divergent pathologic gene expression programs can be identified. Further study is needed to more comprehensively define the common gene expression programs that drive progressive pulmonary fibrosis.

Support or Funding Information

This work was supported by NIH/NHLBI, the Doris Duke Charitable Foundation, and Boehringer Ingleheim.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delta‐like 1 Homolog (DLK1) Identifies a Specific Subset of Orexin Neurons and Contributes to Anxiety Behavior
Authors: Harris Tatiyana , Bugescu Raluca , Kelly Jaylyn R. , Leinninger Gina
Published: June 2021

Lateral hypothalamic area (LHA) neurons expressing the neuropeptide orexin (OX) are implicated in diverse physiology including energy homeostasis, arousal, reward processing and anxiety. It has long been speculated that there may be separate populations of OX neurons to control distinct functions. Indeed, slice electrophysiology studies identified two functionally distinct subtypes of OX neurons, but there has been no molecular “marker” to differentiate them as needed to parse their respective functions. We examined whether the transmembrane protein Delta‐like 1 homolog (DLK1) distinguishes subsets of OX neurons, since it is expressed in the hypothalamus and has been implicated in at least two physiologic processes regulated by OX neurons: energy balance and anxio‐depression. Using immunofluorescence, we found that DLK1 is selectively expressed in ~50% of OX neuronsin mice: we refer to these to as OXDLK1 neurons to differentiate them from the OX neurons that lack DLK1 (OXOnly neurons). We also observed OXDLK1 and OXOnly subsets in human hypothalami, indicating that both populations are relevant to understanding human physiology. Intriguingly, DLK1 protein is present in the cleaved biologically active form in the adult mouse LHA. Moreover, DLK1 expression in the LHA is regulated by circadian timing, but not by alterations in energy status (e.g. fasting, leptin), suggesting that DLK1 protein might contribute to specific physiologic processes. While DLK1 is vital for normal development, its role in the established nervous system, and in OX neurons, is unclear. We therefore used an “adult‐onset deletion” method to explore the contribution of DLK1 to behavior. Briefly, adult DLK1flox/flox mice were injected in the LHA with either AAV‐GFP (Control mice) or AAV‐Cre‐GFP to induce Cre‐mediated deletion of DLK1 selectively from the OX‐DLK1 neurons (DLK1‐Null mice). Intriguingly, the DLK1‐Null mice exhibited increased locomotor activity, but decreased anxiety compared to Control mice via the forced swim and elevated plus maze tests. Collectively, our data identify DLK1 as a protein marker that can be used to distinguish OX subpopulations and suggest a role for DLK1 protein in regulating anxio‐depressive behavior. Going forward, understanding the function of DLK1, and the OXDLK1 neurons may provide insight into how to modulate specific aspects of OX neuron‐regulated physiology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiorespiratory and Cerebrovascular Responses to Leg Cycling Exercise during Muscle Metaboreflex Activation with Blood Flow Restriction in Moderate Normobaric Hypoxia
Authors: Alsalahi Sultan , Huynh Richard , Oldham Cleo , Pitman Ben , Thomas Callum , Lucas Rebekah , Fisher James P
Published: June 2021

Skeletal muscle blood flow restriction and normobaric hypoxia are strategies adopted during exercise training because the associated reductions in local or systemic oxygenation may enhance athletic performance. Both manoeuvres evoke cardiorespiratory and cerebrovascular responses but have not been studied in combination. Blood flow restriction during exercise activates metabolically sensitive group III and IV skeletal muscle afferents (i.e., muscle metaboreflex activation), while systemic hypoxia activates the peripheral chemoreflex. We sought to determine whether the cardiorespiratory and cerebrovascular response to muscle metaboreflex activation with blood flow restriction during leg cycling exercise is augmented in moderate normobaric hypoxia.

Nine healthy men (age 23±2 years; mean±SE) participated in two experimental trials performed under normoxic (fraction of inspired O2 [FiO2] = 21 %) and hypoxic (FiO2 = 13.5 %; equivalent of altitude of 3500 m) conditions whilst seated in a semi‐recumbent position in an environmental chamber. Trials consisted of a resting baseline period (3 min), followed by low intensity leg cycling exercise (64±7 Watts) under free‐flow conditions (~3½ min), and then bilateral thigh cuff inflation to 100 mmHg to induce blood flow restriction and muscle metaboreflex activation (~3 ½ min). Peripheral capillary oxygen saturation (SpO2; finger pulse oximetry), mean arterial pressure (MAP; Finometer and SunTech), heart rate (HR; electrocardiogram), the partial pressure of end‐tidal carbon dioxide (PETCO2; capnography), minute ventilation (VE; pneumotachometer) and middle cerebral artery mean blood flow velocity (MCAV; transcranial Doppler) were recorded.

At baseline, SpO2 was lower during hypoxia (87±1 %) than normoxia (97±1 %; P<0.05), but MAP, HR, PETCO2, VE and MCAv were not different between trials (P>0.05). Leg cycling under free‐flow conditions significantly increased MAP, HR, PETCO2, VE and MCAv (P<0.05 vs. baseline), with no differences observed between hypoxic and normoxic trials (P>0.05). MAP (Δ14±2 vs. Δ16±2 mmHg; P=0.215), HR (Δ10±2 vs. Δ8±3 b·min−1; P=0.251), PETCO2 (Δ−1.1±0.3 vs. Δ−3.4±1.8 mmHg; P=0.596), VE (Δ4±1 vs. Δ4±1 L·min−1; P=0.446) and MCAv (Δ4±2 vs. Δ4±1 cm·s−1; P=0.408) responses to blood flow restriction during leg cycling (i.e., muscle metaboreflex activation) were not different in the hypoxic and normoxic trials.

These preliminary findings suggest that moderate normobaric hypoxia (FiO2 = 13.5 %) does not augment the cardiorespiratory and cerebrovascular responses to muscle metaboreflex activation with blood flow restriction during low intensity leg cycling.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrical stimulation of the cervical Sympathetic Chains decreases upper airway resistance in conscious Zucker‐Fat Rats
Authors: Sun Melissa H , Zenebe Woineshet J , Lewis Stephen J , Hsieh Yee‐Hsee
Published: June 2021

BACKGROUND

The cervical sympathetic chains (CSC) provide sympathetic drive to carotid sinus baroreceptor afferents and chemoreceptive glomus cells within the carotid bodies. Zucker‐fat rats are a model of sleep apnea as they present with both obstructive and central apneas. We have previously demonstrated that CSC stimulation improves airway patency in anesthetized Zucker‐fat rats.

AIM

We sought to investigate (1) whether electrical stimulation of the cervical sympathetic chain in conscious Zucker‐fat rats will lead to decrease in specific airway resistance, and (2) whether intermittent or constant stimulation was more effective.

METHODS

In Zuckerfat rats (n=6), both CSC were implanted with cuff electrodes, which were trocared to a headcap cemented on the skull. Two weeks after recovery, the rats were placed in double chambered plethysmography (DCP) chamber where specific airway resistance was measured. Two protocols (intermittent (30s on/30s off for 15 min) or constant (3 min every 10 min) were performed using the stimulation parameters of 0.8 mA, 7.5 Hz, and 0.2 ms to assess efficacy on airway resistance.

RESULTS

We present two novel findings. First, bilateral CSC stimulation reduced upper airway resistance by 12 ± 5%. Second, intermittent stimulation progressively decreased upper airway resistance by 30 ± 8% whereas sustained stimulation progressively increased upper airway resistance by 34 ± 11%.

CONCLUSION

For the first time, we have demonstrated that CSC stimulation reduces upper airway resistance in conscious Zucker‐fat rats and the pattern of stimulation is critical to its efficacy.

Support or Funding Information

Supported by NIH1OT2OD023860‐01, Galvani Bioelectronics

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Symptom Severity Impacts Inflammation and Sympathetic Over‐activity in Post‐Traumatic Stress Disorder (PTSD)
Authors: Fonkoue Ida T , Jones Toure N , DaCosta Dana , Kankam Melanie , Park Jeanie
Published: June 2021

Patients with post‐traumatic stress disorder (PTSD) are at significantly higher risk of developing hypertension and cardiovascular disease. Low‐grade inflammation and overactive sympathetic nervous system (SNS) activity are suggested as contributing mechanisms. Our prior studies showed that while SNS reactivity during stress is heightened in PTSD, resting MSNA and inflammation was not elevated in young combat Veterans with PTSD as a group; however, prior results did not take into account the potential influence of PTSD symptom severity on neural cardiovascular function. The aim of the current study was to determine if PTSD patients with greater PTSD symptoms measured via the gold‐standard clinician administered PTSD scale (CAPS) have higher SNS activity and inflammation compared to PTSD patients with less severe symptoms. To answer this question, we recruited 21 veterans clinically diagnosed with PTSD (mean CAPS score, 68±4). We measured muscle sympathetic nerve activity (MSNA) via microneurography and blood pressure using standard technique. We collected blood samples to measure serum levels of two inflammatory biomarkers: highly sensitive C‐reactive protein (hsCRP) and interleukin‐6 (IL‐6). For data analysis, the participants were grouped based on the median CAPS score of 70, with the low CAPS group (L‐CAPS) having a score ≤ 70 and the high CAPS group (H‐CAPS) having a score > 70. The two groups (L‐CAPS, n=11; H‐CAPS, n=10) were matched for age, body mass index (BMI) and selective serotonin reuptake inhibitor (SSRI) prescription. Resting blood pressure and heart rate were similar between the two groups. However, the H‐CAPS group had a significantly greater MSNA at baseline when quantified as both burst frequency (26±5 vs 13±2 bursts/min, p= 0.011) and burst incidence (35±6 vs 21±3 bursts/100hb, p= 0.022) compared to the L‐CAPS group. Similarly, hsCRP (2.02±0.71 vs 0.74±0.18, p= 0.036) and IL‐6 (5.44±1.86 vs 2.35±0.73, p= 0.055) levels were higher in the H‐CAPS group compared to the L‐CAPS group. Furthermore, the levels of hsCRP (r= 0.766, p< 0.001) and IL‐6 (r= 0.594, p=0.007) were positively correlated with MSNA. When the groups were analyzed separately, the correlation between MSNA and hsCRP was stronger in the H‐CAPS group (r=0.822, p=0.012) than the L‐CAPS group (r=0.604, p=0.065). These results suggest that the severity of PTSD is associated with chronically increased MSNA and inflammation, and SNS overactivity may be mechanistically linked to inflammation in severe PTSD.

Support or Funding Information

Supported by NIH HL‐098744, NIH DK‐00756, VA Merit I01CX001065, AHA 15CSA24340001

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Bacterial Endotoxin (LPS) on Cardiac and Neural Function in Various Animal Models
Authors: Cooper Robin Lewis , McNabb Micaiah , Saelinger Christa , Greenhalgh Abigail , Istas Oscar , Stanback Maddie , Stanback Stanback , Anyagaligbo Ogechi , Bernard Jate , Danley Melody L , Bierbower Sonya M , Ghoweri Adam , Thibault Olivier
Published: June 2021

The endotoxic effect from gram negative bacteria is primarily due to the lipopolysaccharides (LPS). LPS activates the innate immune response through a Toll‐like receptor 4 (TLR4) known as the CD14/TLR4/MD2 receptor complex in mammals. The Toll receptors are conserved from primates to insects. However, in insects the peptidoglycan recognition proteins (PGRPs) are the receptors which respond to LPS from gram negative bacteria. These PGRPs activate the Immune Deficient (IMD) signaling cascade. There is a family of these receptors known in Drosophila melanogaster but their expression profiles in different tissues has yet to be fully elucidated. We examined a variety of model preparations to better understand the acute actions of exposure to LPS of Serratia marcescens to better understand the varied mechanisms of action. There is a differential, dose dependent effect of LPS in increasing and decreasing HR heart rate (HR) in the larval medicinal blow fly (Phaenicia sericata) and a fruit fly (Drosophila melanogaster). LPS depressed evoked and miniature (quantal) EJPs while hyperpolarizing the skeletal muscle in larval Drosophila, but increased EJPs with no effect on muscle membrane potential at the crayfish NMJ. Both NMJs are glutamatergic. LPS had no effect on sensory transduction of proprioceptive sensory neurons in crayfish and blue crab. LPS at the cholinergic frog NMJ depressed synaptic transmission with no effect on muscle membrane potential. LPS depressed sensory‐CNS‐motor nerve circuits in both crayfish and larval Drosophila. Synaptically‐evoked population spikes in field CA1 of the mouse hippocampus were also significantly reduced by acute LPS applications. The varied effects of LPS in different model systems paves the way to examining differential cellular mechanisms induced by acute exposure to LPS.

Support or Funding Information

Dept of Biology, Univ. of KY laboratory funds and personal funds (RLC)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Central Command Increases Muscle Sympathetic Nerve Activity More to Contracting than Non‐Contracting Muscle during Unloaded Rhythmic Exercise in Humans
Authors: Taylor Chloe E , Boulton Daniel , Howden Erin , Siebenmann Christoph , Macefield Vaughan G
Published: June 2021

We have previously shown that the increase in muscle sympathetic nerve activity (MSNA) to contracting muscle during sustained isometric exercise is due primarily to central command, and that the contracting muscle does not exhibit a metaboreflex. Here, we tested the hypothesis that MSNA increases to the contracting muscle also during rhythmic isotonic exercise, in which muscle metabolites will not accumulate because the contraction is performed without external load. MSNA was recorded from the common peroneal nerve in 10 participants and negative‐going sympathetic spikes extracted during 50 cycles of sinusoidal (0.15 Hz) isotonic dorsiflexions of the ipsilateral or contralateral ankle. Electromyographic activity (EMG) was recorded from the tibialis anterior muscle on both sides. Cross‐correlation analysis between the MSNA and EMG revealed a marked cyclic modulation of MSNA to the contracting (ipsilateral) muscle. This modulation, in which MSNA increased during the contraction phase, was three times greater than that to the non‐contracting muscle (modulation index = 27.4 ± 3.2 vs 9.2 ± 1.5 %; P<0.001). There were no differences in either the intensity or magnitude of modulation of EMG during ipsilateral and contralateral contractions. We conclude that, in addition to static isometric exercise, central command increases MSNA to the contracting muscle during rhythmic isotonic exercise.

Support or Funding Information

This work was supported by the National Health & Medical Research Council (Grants 1029782 and 1100040).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tandem Requirement for Renal D1R and D5R Functionality
Authors: Rozyyev Selim , Crusan Annabelle P , Tiu Andrew C , Jurgens Julie A , Quion Justin Michael B , Asico Laureano D , Felder Robin A , Jose Pedro A , Villar Van Anthony M
Published: June 2021

The peripheral dopaminergic system assists in maintaining blood pressure homeostasis by engendering natriuresis mainly through the D1R and D5R receptors. These receptors share structural features and pharmacological profiles; there is no specific agonist that can discriminate between these two receptors. Genetic ablation of one receptor in the kidney results in the loss of activity of the other. Therefore, we evaluated the mechanisms for the cooperative and co‐dependent interaction between these two receptors. Both receptors share the same anatomical distribution, specifically at the brush border and luminal membranes of renal proximal tubules and collecting ducts, respectively. These receptors dimerized (~110 kDa) and coimmunoprecipitated each other, basally and after fenoldopam (D1R/D5R agonist, 1 μM/15 min) treatment. Bimolecular fluorescence complementation showed that heterologously expressed D1R and D5R tagged with the C and N termini of the fluorescent protein EYFP, respectively, heterodimerized basally in HEK‐293 cells. This was corroborated by confocal microscopy of HEK‐293 cells that were transiently transfected with D1R::RFP and D5R::GFP, which showed colocalization basally at the plasma membrane, most abundantly at the apical membrane in polarized cells. The D1R and D5R partitioned to lipid and non‐lipid rafts; agonist treatment enhanced the lipid raft segregation of D1R and D5R. Both receptors were abundantly expressed in renal proximal tubules of human and C57Bl/6 mouse kidneys, where they colocalized basally that was increased by fenoldopam (1 μM/15 min). D1R/D5R stimulation is known to increase cAMP production and inhibit renal sodium transport. Receptor gene silencing via antisense technology in human renal proximal tubule cells (hRPTCs) decreased the cAMP response to fenoldopam (−71±2% for D1R, vs. −38±7% for D5R, vs. −90±2% for both, *P<0.05). In hRPTCs grown in Transwells, fenoldopam or ouabain (500 nM/30 min) given at the basolateral compartment inhibited Na+/K+‐ATPase (NKA) activity and increased intracellular Na+. siRNA‐mediated gene silencing of DRD1/DRD5 prevented the inhibition of NKA activity by fenoldopam (*P<0.05). Our observations demonstrate the requirement for D1R/D5R to form heterodimers to achieve their full functionality.

Support or Funding Information

R01DK039308, R01HL092196, and P01HL074940

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diverse nephron cell type‐specific adaptation to furosemide by morphometry and single cell RNA sequencing
Authors: Schaffert Steven , Krauson Aram , Walczak Elisabeth , Nizar Jonathan , Holdgate Gwendolyn , Iyer Sonali , Elsayed Ragwa , Qiao Yanli , Khatri Purvesh , Bhalla Vivek
Published: June 2021

Furosemide, a commonly prescribed diuretic to treat hypertension and edema‐forming states, inhibits sodium reabsorption in the thick ascending limb (TAL) of the loop of Henle. Tubular adaptation to diuretics has been documented but not fully explored. Using morphometry and single cell RNA sequencing in furosemide vs. vehicle‐treated mice, we found that furosemide induces renal tubular epithelial hyperplasia selectively in distal, but not proximal, nephron segments after 1 week that subsides by 3 weeks. We measured an increase in kidney size in furosemide‐treated mice, but in contrast to hyperplasia, we measured cellular hypertrophy in both proximal and distal nephron segments, after 1 week treatment that progresses at 3 weeks. We detected an increase in receptor tyrosine kinase pathways and IGF‐1 protein in whole kidney with furosemide, and inducible deletion of renal tubular Igf1r, diminished overall kidney growth and proximal but not distal tubular hypertrophy. In TAL, furosemide enhanced TUNEL‐positive nuclei with selective depletion of Cldn10(+) / Cldn16(−) cells that were enriched for pro‐apoptotic genes Hist1h1c and Fxyd2. Each of the 15 identified nephron segments / cell‐types had diverse changes in relative gene expression with a small cluster of precursor Wnt4(+) cells demonstrating the largest reactivity and a transition toward a sodium reabsorptive phenotype. We demonstrate that furosemide‐treated mice undergo cell type‐specific forms of tubular remodeling including selective changes in hyperplasia, distinct IGF‐1 receptor‐dependent and independent hypertrophy, Cldn10(+) cell death, and significant perturbations of the transcriptional programme of Wnt4(+) cells. These findings highlight mechanisms of diuretic resistance and greatly expand the implications of long‐term diuretic use for human disease.

Support or Funding Information

NIH / NIDDK

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polycystin 1 is an atypical adhesion GPCR that responds to non‐canonical WNT signals and inhibits GSK3β
Authors: Gresko Nikolay , Merrick David M , Mistry Kavita M , Caplan Michael J
Published: June 2021

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, which encode PC1 and PC2, respectively. PC1 is a 460kD multi‐spanning membrane protein. PC1 undergoes multiple proteolytic cleavages, one of which is an autocatalytic cleavage that produces a large N terminal fragment (NTF) that remains non‐covalently attached to the membrane‐embedded C terminal fragment (CTF). A recent study demonstrates that PC1 serves as a receptor for Wnt ligands through the direct binding of these ligands to the PC1 NTF. We find that PC1 responds to Wnt9b by shedding its 350kD NTF, which bears a striking resemblance to the activation mechanism of an adhesion GPCR. For aGPCRs, NTF removal liberates a short peptide sequence near the GPS cleavage site called stachel, which then interacts with an internal binding site and engages a G protein signalling cascade. We find that Wnt9b induces a PC1‐dependent phosphorylation and inhibition of the GSK3β kinase through the RhoA GTPase‐ROCK kinase pathway. Expression of a “constitutively active” aGPCR form of PC1 that lacks its NTF leads to profound suppression of GSK3β activity that is dependent upon RhoA and ROCK. Interestingly, GSK3β is an important negative regulator of the HIPPO/non‐canonical Wnt signaling target TAZ(WWTR1), lack of which leads to the development severe renal cysts. Furthermore, recent data show that pharmacological inhibition of GSK3β is beneficial in mouse models of ADPKD. We find that exogenous expression of an active form of TAZ in PC1 null cells or in PKD1a/b morphant zebrafish suppresses the development of relevant phenotypes. We also demonstrate that pharmacological inhibition of GSK3β leads to accumulation of the TAZ protein and to reduced cystogenesis in a 3D matrigel assay employing PC1 null cells. TAZ abundance and activity have been shown to be upregulated through the non‐canonical Wnt signalling pathway. We find that HEK293 cells that express PC1 and PC2 respond to Wnt9b treatment by significantly increasing TAZ abundance as compared to the wild type HEK293 cells treated in the same manner. Taken together, our data suggest that PC1 is an aGPCR‐like receptor for non‐canonical Wnt ligands that participates in a novel signalling pathway by linking non‐canonical Wnt ligands to the GSK3β‐dependent regulation of TAZ, a multifaceted signalling molecule whose absence is sufficient to induce renal cystic disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.