bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐Acetyl cysteine improves dystrophic (mdx) mouse diaphragm muscle quality and strength
Authors: Burns David P , Drummond Sarah E , Bolger Dearbhla , Murphy Kevin H , Coiscaud Amélie P , Edge Deirdre , O'Halloran Ken D
Published: June 2021

Respiratory muscle weakness occurs as a consequence of dystrophin deficiency in Duchenne muscular dystrophy (DMD), with implications for breathing and airway protective behaviours. The mdx mouse model of DMD shows evidence of impaired respiratory muscle performance with attendant inflammation and oxidative stress. N‐Acetyl cysteine (NAC) is a dietary antioxidant commonly used in the treatment of respiratory disease. We sought to examine the effects of NAC intervention on respiratory system performance in the mdx mouse.

Young adult male wild‐type and mdx mice were studied; a subset of mdx received 1% NAC in the drinking water for 14 days. Ventilation was assessed in conscious mice using whole‐body plethysmography. Inspiratory pressure and respiratory muscle (diaphragm and external intercostal) EMG activity were assessed in anaesthetised mice during ventilatory and non‐ventilatory behaviours. Diaphragm ex vivo force‐generating capacity, muscle structure, cytokine concentrations, glutathione status and mRNA expression were determined. Data were statistically compared by unpaired Student's t test.

Diaphragm function was severely impaired in mdx, evidenced by reductions in force‐generating capacity (40–160Hz range), total muscle shortening and maximal shortening velocity compared to wild‐type. Extensive diaphragm myofibre remodelling and damage was observed for mdx, characterised by increased muscle fibrosis, immune cell filtration, muscle fibre central nucleation and embryonic myosin expression. The pro‐inflammatory cytokines IL‐1β and IL‐6 were elevated in mdx diaphragm and plasma samples. mRNA studies indicate an increase in gene expression related to muscle atrophy, regeneration and inflammation in mdx diaphragm, and a reduction in anti‐oxidant related gene expression. NAC supplementation increased mdx diaphragm force (60–160Hz), maximal shortening and shortening velocity. Collagen deposition and the infiltration of putative inflammatory cells in mdx diaphragm was decreased following NAC. Mdx diaphragm and plasma IL‐1β and IL‐6 concentrations were decreased following NAC. Glutathione reductase and peroxidase activities were increased in mdx diaphragm; both were unaffected by NAC. mRNA expression of Nrf2 was increased in mdx diaphragm and increased further with NAC supplementation. No adverse effects of NAC intervention were observed on ventilation, inspiratory pressure and EMG, and growth measures.

We reveal that NAC treatment improved mdx diaphragm force‐generating capacity by way of beneficial anti‐inflammatory and anti‐fibrotic effects. These data support the potential use of NAC as an adjunctive therapy in the dystrophinopathies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparing the Efficacy of Adeno‐associated Virus Serotype 9 (AAV9) Mediated Retrograde Transgene Delivery to Hypoglossal (XII) Motor Neurons (MNs) in Mouse Versus Rat Models
Authors: Doyle Brendan M , Jorgensen Marda , Falk Darin J , Mandel Ron J , Fuller David D
Published: June 2021

Previous work by our group shows that AAV9 can robustly transduce XII MNs following direct intralingual injection and subsequent retrograde transport along the XII nerve. The current experiments compared the effectiveness of this approach in commonly studied strains of mouse and rat. In the adult C57BL/6 mouse, delivery of AAV9‐CBA‐GFP (1.20e10vg/μL, 8.33μL) via a unilateral or bilateral injection to the base of the tongue both produced a robust transgene expression in XII MNs as determined by immunohistochemistry (tissues harvested 6 weeks post‐injection). The extent of XII MN transduction was similar with uni‐ or bilateral tongue injection, presumably owing to the small volume of the mouse tongue. In contrast to the murine data, following intralingual delivery of AAV9‐CMV‐mCherry in adult Sprague‐Dawley (SD) rats (1.42e10vg/μL, 1.5 μL or 7.0μL), transgene expression in XII MNs was not detected using either fluorescence microscopy or antibodies against mCherry. Intralingual delivery was repeated in 3 separate cohorts (N=28 total) and MN transduction was never detected. We next tried a direct unilateral injection of AAV9‐mCherry into the main trunk of the XII nerve of adult SD rats (1.42e10vg/μL, 1.5μL). The nerve was injected in the neck, proximal to the point where it bifurcates into medial and lateral branches. At 6 wks post‐injection, we observed clear expression of mCherry in XII MNs detected using immunochemistry. Unexpectedly, MN transgene expression was seen in both XII motor nuclei, despite the unilateral nature of the AAV delivery. We are unaware of any neuroanatomical evidence to suggest that retrograde transport along a single XII nerve would result in bilateral targeting of XII motoneurons, and ongoing experiments are aimed at determining the mechanisms underlying this result. Collectively, these results indicate that retrograde transport of AAV9 following intramuscular delivery may show species variability. Problems with intralingual delivery in the rat were circumvented by direct delivery of AAV9 to the XII nerve.

Support or Funding Information

Funding: 1R01HL139708‐01A1 (DDF)

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 Antagonism in Acute Cervical Contusion/Compression Injury Preserves Serotonin‐Dependent Phrenic Motor Plasticity
Authors: Seven Yasin B. , Allen Latoya L. , Tadjalli Arash , Zwick Amanda , El‐Chami Mohamad , Perim Raphael R. , Gonzalez‐Rothi Elisa J. , Mitchell Gordon S.
Published: June 2021

Cervical spinal cord injury (cSCI) disrupts descending respiratory neural pathways, causing respiratory muscle paralysis and decreased breathing capacity. Immediately following acute trauma, a suite of pathogenic mechanisms contributes to secondary degeneration and respiratory failure including spinal ischemia, excitotoxicity and inflammation. These events are accompanied by increased extracellular adenosine that activates adenosine receptor signaling cascades. Adenosine 2A receptor (A2AR) antagonism is shown to preserve neurons in neurodegenerative diseases and ischemic injury, likely due to reduced excitotoxicity and inflammation. However, little is known concerning how A2AR activation immediately following cSCI impacts the respiratory control system and its neuromodulatory inputs. Thus, the fundamental hypothesis guiding this study is that A2ARs exert pathogenic effects on respiratory function and plasticity immediately following cSCI. Serotonergic plasticity plays a significant role in the respiratory control network. For example, the phrenic motor system exhibits phrenic long‐term facilitation (pLTF) following moderate acute intermittent hypoxia (AIH), a form of phrenic motor plasticity that requires raphe serotonergic neuron activation. Spinal trauma induces neurodegeneration in descending raphe serotonergic axons, undermining the capacity for pLTF following cSCI. Here, we tested the hypothesis that A2AR antagonism in acute cSCI preserves serotonin‐dependent, AIH‐induced pLTF in adult male Sprague‐Dawley rats with mid‐cervical spinal contusion with compression (cSCC). Addition of a brief compression with dwell time exacerbated the impact of C4/5 contusion as demonstrated by: 1) high death rates (~50% vs <10% without compression); 2) severe paralysis in all limbs for up to 5 days; 3) delays in regaining of the righting reflex; 4) ventilatory deficits when challenged with 7% CO2 + 10.5% O2; and 5) profound neurodegeneration extending rostrally to the brainstem. One week post‐cSCC, pLTF is abolished, consistent with degeneration in raphe serotonergic neurons. However, even a single administration of istradefylline 1‐hour post‐cSCC preserved and even augmented pLTF 1‐week post‐cSCC. Respiratory and locomotor functional studies are in progress. These exciting preliminary data suggest that plasticity outcomes are improved by A2AR antagonism in acute cSCC.

Support or Funding Information

NIH OT2OD023854 (SPARC), HL69064, and K12 HD055929 (EGR), 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.
The Role of TrkB Kinase Activity in Stabilization of Presynaptic Terminals Wanes in Old Age
Authors: Gransee Heather M. , Zhan Wen‐Zhi , Sieck Gary C. , Mantilla Carlos B.
Published: June 2021

Brain derived neurotrophic factor (BDNF) acting through the high affinity tropomyosin‐related kinase receptor B (TrkB) plays an important role in synaptic plasticity and function at the neuromuscular junction (NMJ) as well as maintenance of neuromuscular transmission. Our previous studies showed that susceptibility to age‐related changes in diaphragm muscle (DIAm) NMJ morphology and denervation depends on trophic influences exerted by BDNF/TrkB signaling. Indeed, TrkB kinase activity is necessary to stabilize DIAm NMJ pre‐synaptic terminals in 18 month old mice (representing early old age; ~90% survival). However, by 24 months of age (~75% survival), BDNF/TrkB signaling has no effect on DIAm neuromuscular transmission, suggesting reduced TrkB kinase activity in the older age groups. We hypothesized that the NMJs that are resilient to old age do not depend on TrkB kinase activity for stabilization of pre‐synaptic terminals. Male and female 24 month old TrkBF616A mice were used to examine the structural properties of NMJs across various muscles with varying susceptibility to age‐related effects (DIAm, EDLm, and SOLm). Confocal microscopy was used to compare pre‐ and post‐synaptic morphology and denervation following a 7 day treatment with the phosphoprotein phosphatase‐1 derivative 1NMPP1, which inhibits TrkB kinase activity in TrkBF616A mice vs. vehicle treatment. Inhibition of TrkB kinase activity had no effect on pre‐synaptic or post‐synaptic terminal volume in DIAm, EDLm, and SOLm. Similarly, the overlap of pre‐ and post‐synaptic structures and the proportion of denervated NMJs did not change with 1NMPP1 treatment in all muscle types. The average percent of denervated NMJs was 8, 22, and 7% in DIAm, EDLm, and SOLm, respectively. Collectively these results suggest that the requirement of TrkB kinase activity for stabilization of NMJs in DIAm, EDLm and SOLm wanes in old age.

Support or Funding Information

Supported by 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.
Distribution of Ipsilateral and Contralateral Glutamatergic Synaptic Inputs to Phrenic Motor Neurons
Authors: Rana Sabhya , Zhan Wen‐Zhi , Sieck Gary C. , Mantilla Carlos B.
Published: June 2021

Excitatory descending glutamatergic (Glu) synaptic input to phrenic motor neurons (PhMNs) emanates from the ventrolateral medulla and is responsible for inspiratory drive and a range of higher force behaviors. Disruption of this excitatory input following unilateral C2 hemisection (C2SH) results in cessation of inspiratory‐related diaphragm muscle (DIAm) activity ipsilateral to injury, but DIAm activity during higher force behaviors persists, reflecting the contribution of spared contralateral inputs. In a model of PhMN (DIAm motor unit) recruitment, we proposed that smaller PhMNs innervating more fatigue resistant DIAm motor units are recruited to accomplish ventilatory behaviors, whereas larger PhMNs innervating more fatigable motor units are recruited to accomplish more forceful expulsive and straining behaviors. We hypothesized that differences in the distribution of ipsilateral and contralateral Glu synaptic inputs to PhMNs contribute to the effects of C2SH on the range of motor behaviors accomplished by the DIAm. Accordingly, we hypothesized that C2SH primarily disrupts ipsilateral Glu synaptic inputs to smaller PhMNs, whereas Glu synaptic inputs to larger PhMNs are minimally impacted after C2SH. To address this hypothesis, we examined changes in Glu presynaptic input onto retrogradely labeled PhMNs using immunohistochemistry for VGLUT1 and VGLUT2. We found that following C2SH (by 7 days), there was a ~45% reduction in ipsilateral Glu synaptic input to PhMNs compared to a ~20% reduction contralaterally. In addition, ipsilateral to C2SH, Glu synaptic input to smaller PhMNs was reduced to a greater extent compared to that to larger PhMNs (55 vs. 35% reduction, respectively). Contralateral to C2SH, inputs to PhMNs were similarly reduced independent of motor neuron size. These results are consistent with more uniformly distributed contralateral inputs to all PhMNs and predominant input to smaller PhMNs ipsilaterally. These results also indicate that the source of Glu synaptic input to PhMNs varies depending on motor neuron size and likely reflects different motor control, including possibly distinct central pattern generator and premotor circuits.

Support or Funding Information

Grant Support: NIH Grant R01 HL96750 and Mayo Clinic Graduate School of Biomedical Sciences

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracellular Vesicle Sphingolipids from Stored Platelets Mediate Transfusion Related Acute Lung Injury
Authors: McVey Mark John , Maishan Mazharul , Spring Chris , Semple John W , Kuebler Wolfgang M
Published: June 2021

BACKGROUND

Transfusion‐related acute lung injury (TRALI) leads to chest infiltrates and hypoxia after 0.02–1% of transfusions with an associated mortality up to 15%. Sphingolipid ceramide weakens endothelial barriers while sphingosine‐1‐phosphate (S1P) is barrier protective. Aged stored platelets elicit TRALI by acid sphingomyelinase (ASM) dependent ceramide formation, which causes lung injury (in vivo) and impairs pulmonary endothelial cell (EC) barrier integrity (in vitro). Concomitantly, aged platelets form extracellular vesicles (EVs) which can shuttle hydrophobic lipids such as sphingolipids to target cells. Combining these concepts, we hypothesized that 5‐day but not 1‐day stored platelets generate EVs which mediate TRALI in a sphingolipid‐dependent manner.

METHODS

EVs from male C57BL/6 wild type (WT) or Smpd1−/− (ASM knockout) platelets stored for 1‐ or 5‐days duration were isolated by differential centrifugation and filtration prior to enumeration (flow cytometry) and sphingolipid analysis (mass spectroscopy). For TRALI induction, BALB/c male mice were primed with 2 mg/kg intraperitoneal lipopolysaccharide (LPS) two hours prior to intravenous infusion of 10 ml/kg EVs (≈ 5×106) or saline (sham) and were assessed (6 hours later) by measuring lung tissue wet‐to‐dry (W/D) weight ratios, bronchoalveolar lavage fluid protein (BAL‐p) and lung tissue myeloperoxidase activity (MPO). Transendothelial electrical resistance (TEER) was measured in cultured human microvascular pulmonary ECs treated with PBS (sham) or 5×105 EVs (from 1‐ vs 5‐day stored platelets) for 4 hours.

RESULTS

Compared with 1‐day EVs, 5‐day EVs were more numerous, ceramide enriched and S1P depleted. 5‐day EVs from ASM KO platelets were less numerous than 5‐day WT EVs, but had similar sphingolipid profiles. LPS primed mice receiving 5‐day EVs had increased W/D (p < 0.05), MPO and BAL‐p (p<0.05) relative to mice receiving LPS priming in addition to saline (sham), 1‐day EVs or ASM KO 5‐day EVs. 5‐day EVs but not 1‐day EVs led to reductions in TEER (< 2 hours) in vitro in keeping with EC loss of barrier integrity which persisted for > 4 hours. 5‐day EVs could be made to resemble 1‐day EVs (no injury) with addition of exogenous S1P at levels equivalent to those found in 1‐day EVs.

CONCLUSIONS

Ceramide formation in aged platelets contributes to EV formation and impairs human EC barrier integrity resulting in TRALI. Removal or modification of aged EVs from stored platelets may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.

Support or Funding Information

This work was jointly funded by Canadian Blood Services and the Canadian Institute of Health Research (CIHR). MJM is supported by a CIHR Vanier Scholarship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DIY Video Laryngoscopy in Rats–Safe, Fast and Reliable
Authors: Balzer Claudius , Salzman Michele M. , Cleveland William J. , Riess Matthias L.
Published: June 2021

Introduction

The airway management of rats in the field of basic research is of great importance. Intubation is needed for general anesthesia, ventilation, and the use of neuromuscular blocking agents. For survival studies, especially, a less invasive intubation technique is preferred over tracheostomy. In the past, several different methods with high success rates have been described, but most need a high level of technical effort, training, and financial expenditure. In humans, video laryngoscopy for intubation is the most successful technique; it may also be feasible in rats with simple and cost‐effective devices.

Hypothesis

By using a commercial flexible video endoscope (VE), with small modifications for direct visualization, and the Seldinger‐Technique, intubation in rats can be quick, easy and reliable.

Methods

A cost‐effective and waterproof VE (GiraffeCam, 5.5 mm diameter) with USB‐connection and an integrated light source was purchased. Due to its softness, and for fixation on the board, a 20 cm wire attached with surgical tape was added to the VE for more rigidity. After anesthesia was induced with an intraperitoneal injection of pentobarbital (45mg/kg), each rat was placed on its back on the board and fixed in position with surgical tape. The board was then secured in place on the bench at a 45° angle head up. The VE was hooked up to the board and connected to a computer. The tip of the VE was gently introduced into the mouth, on one side of the upper incisors, until the vocal cords could be identified on the video screen. A 14‐gauge catheter, used as endotracheal tube (ET), was prepared with a stylet (15 cm PE50 tubing) that was inserted and advanced 1 cm past the catheter tip. The ET was inserted into the trachea, and once past the vocal cords, the stylet was pulled back. One hundred (100) rats (30 Zucker Diabetic Fatty, 70 Sprague Dawley; weight between 350–450g) were intubated by this technique and used for experiments with cardiac arrest and resuscitation.

Results

All intubation attempts were successful on the first attempt, with a proper placement of the ET. No complications, such as bleeding occurred, and all rats were ventilated without problems. Furthermore, this technique can be performed with little previous experience and in less than one minute.

Conclusion

With estimated costs of approximately $30, this VL intubation procedure is a safe, fast and reliable technique for rat intubation.

Support or Funding Information

Dr. Balzer is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number 397561247. Additional support provided by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veteran Affairs Biomedical Laboratory R&D Service awarded to Dr. Riess.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Both Dietary Salt Restriction and Excess Dietary Salt Intake Exaggerate Cardiovascular Reflex Responses to Activation of the Sciatic Afferents, Aortic Depressor Nerve, and Vagal Afferents
Authors: Katayama Pedro Lourenco , Wenner Megan M , Farquhar William B , Browning Kirsteen N , Stocker Sean D
Published: June 2021

Excess dietary salt intake exaggerates sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses to several cardiovascular reflexes in salt‐resistant animals. Recently, observational cohort studies suggest that salt restriction increases cardiovascular morbidity/mortality in several cardiovascular diseases, and the relationship between cardiovascular morbidity and dietary salt intake resembles a J‐shaped curve. However, little evidence exists to document the impact of salt restriction on cardiovascular reflex responses and whether this relationship resembles a J‐shaped curve. Thus, the aim of the present study was to determine whether salt restriction affects SNA and ABP responses to activation of sciatic afferents, the aortic depressor nerve, and cervical vagal afferents. To address this hypothesis, male Sprague‐Dawley rats were fed low (0.01% NaCl, D17010, Research diets), normal (0.1% NaCl, D17020, Research Diets), and high (4.0% NaCl, D17013, Research Diets) salt diet for 14–21 days. Then, reflex responses were measured in Inactin‐anesthetized animals. Baseline mean ABP was not different across groups (low: 112±4, normal: 101±5, high: 109±3mmHg; P>0.1 overall ANOVA). Electrical stimulation of sciatic afferents (1ms pulse, 500uA, 5s duration, 2–20Hz) produced frequency‐dependent increases in ABP. Interestingly, the pressor response to sciatic nerve stimulation was significantly greater in rats fed low and high versus normal NaCl (5Hz; low: 22±4, normal: 12±2, high: 27±2 mmHg, n=5–10, P<0.05). Activation of the aortic depressor nerve (2ms pulse, 500uA, 15s duration, 2–20Hz) produced frequency‐dependent decreases in ABP. The depressor response to aortic nerve stimulation was significantly greater in rats fed low and high versus normal NaCl (5Hz; low: −29±6, normal: −11±3, high: −29±4 mmHg; n=3–7, P<0.05). Finally, electrical stimulation of the cervical vagal afferents (1ms pulse, 500uA, 5s duration, 2–20Hz) produced frequency‐dependent decreases in ABP. Again, the depressor response to vagal nerve stimulation was significantly greater in rats fed low and high versus normal NaCl (5Hz; low: −25±3, normal: −15±2, high: −29±2 mmHg; n=5–10, P<0.05). Simultaneous recordings of lumbar, renal, and splanchnic SNA followed the changes in ABP across low, normal, and high NaCl diets. The present findings showed that both dietary salt restriction and excess dietary salt intake exaggerates sympathetic and cardiovascular responses. These cardiovascular responses to activation of sciatic afferents, the aortic depressor nerve, and vagal afferents as a function of dietary salt resemble a J‐shaped curve.

Support or Funding Information

NIH R01 HL128388 (WBF, SDS)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microglia regulate RVLM‐projecting PVN neuronal activity
Authors: Shi Peng , Bi Qianqian , Wang Chao , Wei Bo , Shen Xiao Z , Liu Xiaoli , Gu Yan
Published: June 2021

Microglia, the primary resident immune cells in the CNS, communicate with neurons very dynamically. Our previous studies show that activated microglia are involved in neurogenic hypertension. In the present study, we are investigating the mechanism by which microglia regulate RVLM‐projecting PVN neuronal activity in resting state. Adult male transgenic CD11b‐DTR mice were randomly assigned into two groups: sham control and diphtheria toxin (DT) groups, which received ICV infusion of saline or DT (depletion of microglia) respectively. Sympathetic neurons in the hypothalamic PVN are retrograde labeled by fluorescent microsphere from the brain stem RVLM. The electronic profile of RVLM‐PVN neurons are recorded in voltage clamp mode. Our results indicate that depletion of naïve microglia did not alter resting membrane potential nor membrane resistance. However, loss of microglia significantly inhibited outward potassium currents in RVLM‐PVN neurons. The frequency of mEPSP were also decreased in the PVN neurons in the absence of microglia. By confocal microscopy analysis, we found that there was a significantly decrease of spine density in the PVN neurons without microglia compared to the ones from the sham controls. Our results suggest that microglia directly regulate both intrinsic and extrinsic activities of the PVN sympathetic neurons.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamics of Ventilatory Pattern Variability (VPV), Cardio‐Respiratory Coupling (CRC) and Cytokines in an E. coli Sepsis Model
Authors: Tackett Shiloh H , Solanki Neha , Nethery David E , Horton Kofi‐Kermit A , Bonfield Tracey L , Jacono Frank J , Dick Thomas E
Published: June 2021

Cardio‐respiratory control is disrupted in sepsis. We hypothesized that increases in inflammatory cytokines that are expressed in the brainstem in a rat model of Escherichia coli (E. coli) peritonitis would correlate with decreases in VPV and the uncoupling of CRC. We implanted fibrin clots containing either 25×106 or 0 E. coli in the peritoneal cavity of chronically instrumented, adult Sprague‐Dawley Rats (n=19). We recorded cardiorespiratory patterns of awake, freely moving rats at 3, 6, 12, 24, and 48 hours after implantation. At each time point, blood, bronchoalveolar lavage fluid (BALF), peripheral and central nervous system (CNS) tissues were collected to measure cytokine concentrations. We quantified VPV and CRC using a set of complementary measures sensitive to both linear and nonlinear dynamics. We measured interleukin 1β (IL‐1b), interleukin 6 (IL‐6) and tumor necrosis factor α (TNF‐a) concentrations in serum, BALF and tissue supernatants by Luminex assay. Compared to the sham group, infected rats developed a systemic inflammatory response by 3h [increased serum IL‐6 (p<0.05), IL‐1b (p=0.05)], which peaked at 6h and then decreased progressively. Also, brainstem inflammation was evident at 3h post inoculation, with increases in IL‐1β in the medulla (p<0.01) and pons (p<0.05). Brainstem inflammation was associated with increased predictability in VPV, indicated by increases in mutual information (p<0.05) and nonlinear complexity index (NLCI). In our initial analysis of CVC (n=8 rats at 24h), the X2 values of the distribution of R‐wave timings relative to the onset of inspiration were highly significant but were not different between sham and septic groups. We conclude that decreases in VPV (i.e., increased pattern predictability) were present early in the course of systemic inflammation following E. coli infection in rats and these changes were associated with elevated serum and brainstem cytokines. In contrast, the persistence of CVC in the inoculated rats was unexpected; however, at 24h the NLCI was decreasing indicating that these rats may be recovering from the peritonitis.

Support or Funding Information

VA Research Service (I01BX004197); NIH (U01 EB021960)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of mRNA Expression in Peripheral Tissue in a Rodent E. coli Sepsis Model
Authors: Horton Kofi‐Kermit A , Clifford Caitlyn , Tackett Shiloh H , Nethery David E , Dick Thomas E , Jacono Frank J , Strohl Kingman P
Published: June 2021

The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates pathophysiology in many pro‐inflammatory conditions, such as irritable bowel diseases, atherosclerosis and acute lung injury. Sepsis, a highly morbid condition, results from a dysregulated host response to infection. Despite extensive study the transcriptional regulation of cytokines and the ECS during sepsis remains to be established. We hypothesized that cannabinoid receptor (Cnr1 & 2) mRNA expression increases with systemic infection and these increases coincide with increases in pro‐inflammatory cytokines. We implanted a fibrin clot that contained either 25×106 or 0 E. coli in the peritoneal cavity of adult, male rats (n=8). Rats were euthanized at 3, 6, 12 or 24 h following the implant; lung, liver and serum were harvested. Total RNA was extracted using Trizol reagent, and cDNA was synthesized using reverse transcription PCR. Quantitative real‐time PCR was performed in duplicate for each sample using TaqMan Gene® Expression Assays specific for 18S ribosomal RNA (18S), Cnr1 & 2, interleukin (IL) IL‐4 & IL‐6 and tumor necrosis factor – alpha (TNF‐α). Each target gene was normalized to 18 S content, and relative quantitation of gene expression levels were performed using the comparative CT method (2−ΔΔCT method) with the 0 E. coli fibrin clot at each time point as the comparative control. The time course of cytokine expression depended on the cytokine and the tissue compartment. In liver, IL‐6 peaked at 3h (1086 ± 59% vs 0.36 ± 0.1%, p<0.01). In serum, IL‐4 peaked at 6h (168 ± 34% vs 86 ± 5%). In lung, both IL‐6 and TNF‐α expression levels peaked at 3h (IL‐6: 755 ± 33% vs 8 ± 0.5%, p<0.01; TNF‐a: 496 ± 230% vs 8 ± 0.2%); whereas, IL‐4 levels peaked at 24h (291 ± 31% vs 3 ± 2%, p<0.05). Interestingly, at 24h Cnr2 mRNA increased concomitantly with IL‐4 in the lung (360.0 ± 10% vs 30 ± 5%, p<0.01), whereas the expression levels of Cnr1 mRNA did not increase above sham surgical levels during the time course of this study. These preliminary data and analyses support our hypothesis that induction of ECS, as characterized by the increased transcription of Cnr2 mRNA, is associated with upregulation of mRNA of pro‐inflammatory cytokines.

Support or Funding Information

NIH 5T32HL007913‐18, NIH U01 EB021960, VA Research Service I01BX004197

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acid‐Sensing Ion Channels Blockade Attenuates Pressor and Sympathetic Responses to Skeletal Muscle Metaboreflex Activation in Humans
Authors: Fernandes Igor Alexandre , Campos Monique O , Mansur Daniel E , Mattos João , Paiva Adrielle , Videira Rogerio , Macefield Vaughan , Nóbrega Antonio CL
Published: June 2021

In animals, the blockade of acid‐sensing ion channels (ASICS), cation pore‐forming membrane proteins located in the free nerve endings of group IV afferent fibers that are activated by acidic pH transients, attenuates reflex increases in sympathetic nerve activity (MSNA) and blood pressure (BP) during muscle contraction. These findings indicate that ASICS have an important role in mediating the metabolic component (skeletal muscle metaboreflex) of the exercise pressor reflex in animal models. Nonetheless, it is still unclear whether these findings can be translated to humans. We then tested the hypothesis that ASICS play a role in evoking the skeletal muscle metaboreflex in humans, quantifying beat‐by‐beat mean BP (MBP, Finometer), MSNA (peroneal nerve, microneurography) and heart rate (HR, ECG) in 11 healthy young men (N = 11) during rest, static handgrip exercise (SHE, 2 min at 35% of the MVC) and post‐exercise muscle ischemia (PEMI, 2 min) before (BFA) and after local infusion of amiloride (AFA −0.75 mg/mL), an antagonist of ASICs, in saline (2.5% of total forearm volume) via modified Bier block (regional intravenous anesthesia). In an additional protocol, participants (N = 7) underwent the experimental procedures before (BFS) and after (AFS) venous infusion of a similar amount of saline via modified Bier block. MBP responses were slightly but significantly attenuated during SHE after amiloride infusion (BFA +29.6 ± 6.1 vs. AFA +24.3 ± 7.0 mmHg, p = 0.001). A smaller increase in MSNA (N = 9) was also observed as a consequence of ASICs blockade (BFA +14.5 ± 9.6 vs. AFA +10.7 ± 6.5 bursts/min, p = 0.004). Equivalent SHE‐induced increases in HR were observed before and after ASICs blockade (p > 0.05). Amiloride infusion attenuated the PEMI‐induced increases in both MBP (BFA +25.8 ± 6.9 vs. AFA +16.3 ± 6.4 mmHg, p = 0.0001) and MSNA (BFA +16.2 ± 9.2 vs. AFA +8.4 ± 8.0 bursts/min, p = 0.0001). In the additional protocol, SHE‐evoked increases in MBP (BFS +28.7 ± 12.2 vs. AFS +27.0 ± 9.8 mmHg, p = 0.477), MSNA (N = 5, BFS +10.0 ± 8.6 vs. AFS +13.0 ± 9.4 burst/min, p = 0.208) and HR (BFS +32.0 ± 12.4 vs. AFS +37.2 ± 22.5 bpm/min, p = 0.265) did not differ before and after saline infusion. Similar observations were obtained for MBP (BFS +24.2 ± 14.2 vs. AFS +23.7 ± 10.4 mmHg, p = 0.830) and MSNA (BFS +10.0 ± 8.6 vs. AFS +13.0 ± 9.4 burst/min, p = 0.208) during PEMI. Therefore, these findings indicate that ASICs play a role in evoking pressor and sympathetic responses to both SHE and isolated activation of the skeletal muscle metaboreflex in humans.

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, but not the Muscle Metaboreflex, is Responsible for Intensity‐Dependent Increases in Muscle Sympathetic Nerve Activity to Contracting Muscle during High‐Intensity Static Isometric Exercise in Humans
Authors: Boulton Daniel , Taylor Chloe , Green Simon , Macefield Vaughan
Published: June 2021

Post‐exercise ischaemia has been used to demonstrate muscle metaboreflex‐mediated increases in muscle sympathetic nerve activity (MSNA) to an inactive limb during static isometric exercise. In contrast, we have previously shown a rapid centrally‐mediated increase in MSNA to a contracting leg during sustained isometric exercise, which promptly returned to resting levels at the cessation of exercise despite the presence of post‐exercise muscle ischaemia of the ipsilateral limb. However, these observations have been made during relatively low‐intensity exercise. The present study tested the hypothesis that central command, but not the muscle metaboreflex, increases MSNA to the contracting muscle during sustained isometric contractions even at higher intensities. MSNA was recorded continuously (microneurography) from the right common peroneal nerve. Twelve subjects (seven males, five females) performed two‐minute ankle dorsiflexions at 10, 20, 30, 40 and 50% of maximum voluntary contraction (MVC) ipsilateral to the MSNA recording, which was immediately followed by six minutes of post‐exercise ischaemia. There was at least six minutes rest between contractions. MSNA was analysed by measuring negative‐going cardiac‐locked sympathetic spikes from the raw neurogram. Compared with MSNA at rest (82 ± 5 spikes/min), MSNA to contracting muscle increased in an intensity‐dependent manner within one‐minute of contraction at 10 %MVC (145 ± 27 spikes/min, P=0.04), 20 %MVC (219 ± 28 spikes/min, P=0.002), 30 %MVC (480 ± 92 spikes/min, P=0.002), 40 %MVC (716 ± 185 spikes/min, P=0.01) and 50 %MVC (934 ± 275 spikes/min, P=0.02). Blood pressure increased in an intensity‐dependent manner during contractions up to 50 %MVC (76 ± 5 vs 93 ± 7 mmHg, P=0.02). Within one‐minute after each contraction MSNA returned close to resting levels even in the presence of post‐exercise ischaemia. These findings indicate that central command, and not the metaboreflex, is responsible for the increase in MSNA to contracting muscle during sustained isometric contractions, even at intensities up to 50% of MVC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle Sympathetic Nerve Activity and Vascular Conductance Responses at the Onset of Arm and Leg Exercise in the Inactive Limb
Authors: King Trevor J , Doherty Connor J , Millar Philip J
Published: June 2021

Local mechanical and metabolic factors are known to regulate the blood flow response at the onset of exercise. However, little is known regarding the contribution of muscle sympathetic nerve activity (MSNA) to this onset blood flow response. The aim of the study was to examine the MSNA and vascular conductance response to exercise anticipation and onset in the inactive leg. Two identical visits were performed on 16 participants (n = 6 female). During visit 1, hemodynamic (Finometer) and MSNA (fibular nerve microneurography) measurements were acquired during rest (1‐minute), exercise anticipation (verbal preparation, 30‐seconds), and 1 minute of rhythmic handgrip (RHG, 1:1 duty cycle, 40% MVC) and one‐legged cycling (17 ± 3% peak power output). During visit 2, hemodynamic and superficial femoral artery (SFA) blood flow (Doppler ultrasound) measurements were acquired during the same exercise modes. Leg vascular conductance was calculated as SFA mean blood flow/mean arterial pressure. Total MSNA (burst frequency x burst area) was analyzed in 15‐second moving windows (5‐second progressions), and SFA blood flow was analyzed as 5‐second time bins aligned with the last 5 seconds of each MSNA moving window; both were expressed as relative change from baseline (%). Burst frequency, burst incidence, and total MSNA (height) followed the same pattern as total MSNA (area), and thus only total MSNA (area) is presented. Significance was set as p < 0.05, and data are mean ± SE. When participants were prompted to prepare for upcoming exercise (i.e. anticipation), total MSNA decreased from baseline prior to RHG (−40 ± 6%, p < 0.001) and cycling (−20 ± 6%, p = 0.03). During anticipation of RHG, this decrease in vasoconstrictor drive was followed 5 seconds later by a trend for increased SFA vascular conductance (+24 ± 12%, p = 0.07). At the onset of exercise, total MSNA decreased during RHG (−28 ± 6%, p = 0.002) and cycling (−39 ± 7%, p = 0.009). This was followed by an increase in SFA vascular conductance during both RHG (+34 ± 12%, p = 0.02) and cycling (+34 ± 20%, p = 0.005) conditions. In the absence of mechanical or metabolic factors in the inactive limb, these data suggest that a reduction in total MSNA can contribute to the exercise hyperemia that is observed at the onset of exercise. Furthermore, the anticipatory response of MSNA suggests a partial contribution of feedforward central command in this response.

Support or Funding Information

Natural Science and Engineering Research Council of Canada

Ontario Ministry of Research, Innovation, and Science

Canadian Foundation for Innovation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Sodium Excretion Consequent to Pharmacogenetic Activation of Gq‐DREADD in Principal Cells
Authors: Mironova Elena , Stockand James
Published: June 2021

Stimulation of Gq‐coupled metabotropic P2Y2 receptors decrease the activity of ENaC in renal principal cells of the distal nephron. The physiological consequences of disrupting P2Y2 receptor signaling in the P2Y2 receptor knockout mouse are decreased sodium excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and the distal nephron verses that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure are obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient alone to drive renal sodium excretion. Here we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase renal sodium excretion using a pharmacogenetic approach and selective agonism of the P2Y2 receptor. Selective stimulation of the P2Y2 receptor with the ligand, MRS2768, decreased ENaC activity in freshly isolated tubules as assessed with patch clamp electrophysiology; and rapidly increased urinary sodium excretion as assessed in metabolic cages. Similarly, selective agonism with clozapine N‐oxide (CNO) of hM3Dq‐DREADD restrictively expressed in principal cells of the distal nephron decreased ENaC activity with consequent increases in sodium excretion. CNO when applied to control littermates failed to impact ENaC and renal sodium excretion. These studies represent the first use of the pharmacogenetics DREADD technology in the kidney; and demonstrate that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.

Support or Funding Information

This research was supported by NIH/NIDDK grants R01DK98460, R01DK103758 and American Heart Association grant 17GRNT32920002 to JDS

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uncoupling of Sodium and Potassium Excretion in Kidney‐Specific BMAL1 Knockout Mice following Potassium Depletion
Authors: Crislip Gene Ryan , Douma Lauren G , Masten Sarah H , Cheng Kit‐Yan , Lynch I. Jeanette , Barral Dominique , Glasford Krystal , Esser Karyn A , Wingo Charles S , Gumz Michelle L
Published: June 2021

Blood pressure regulation is influenced by circadian clock factors. BMAL1 is a core mammalian circadian transcription factor responsible for the tissue‐specific regulation of thousands of genes. We have generated a kidney‐specific knockout of BMAL1 that exhibits lower blood pressure compared to control mice (CNTL). Additionally, qPCR data demonstrated basal differences in expression of potassium handling genes. The goal of this study was to determine if solute excretion in response to a potassium depleted diet was different in kidney‐specific BMAL1 knockout mice (KO) compared to CNTL. Our mouse model was generated using floxed exon 8 BMAL1 mice crossed with kidney‐specific cadherin Cre+ mice to generate KO. Floxed Cre− littermates were used as CNTL. Metabolic cages were used for urine collection. Urinalysis was performed via flame photometry. Basal urinary sodium and potassium excretion was similar between CNTL and KO (Na: 0.29±0.02 vs. 0.29±0.01 mmol; P=0.6; K: 0.65±0.05 vs. 0.64±0.02 mmol; P=0.2; N=5–6). After baseline measurements, mice were administered a potassium depleted diet for 5 days. There was a transient decrease in food intake in both genotypes (P<0.0001), however, body weight remained unchanged throughout treatment (P=1). Both body weight and food intake were comparable between CNTL and KO. Potassium excretion decreased within 24 hours of the start of treatment in both genotypes and remained low for all 5 days (CNTL: 0.012±0.002; KO: 0.015±0.004 mmol; PINTERACTION=0.3). Initially, sodium excretion decreased following potassium depletion in both groups which was matched with transient sodium retention. Interestingly, sodium excretion remained lower than baseline throughout treatment in CNTL whereas KO increased back to baseline levels by day 5 of treatment (0.19±0.01 vs. 0.26±0.01 mmol; PINTERACTION=0.0001). As expected, plasma aldosterone decreased following potassium depletion, however, levels were comparable between the groups (P=0.5). BMAL1 in the distal nephron and collecting duct play a role in regulating sodium handling in male mice in response to dietary potassium depletion. KO are able to uncouple sodium and potassium excretion during a potassium depleted diet. The mechanism responsible for this may shed light on the lower blood pressure phenotype that KO exhibit.

Support or Funding Information

5T32HL083810‐10 NIH/NIDDK 1R01DK109570 The Gatorade Trust through the UF Department of Medicine

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acidic Environment Facilitates Mitochondrial Fragmentation Induced by APOL1 Renal‐Risk‐Variants in Kidney Cells
Authors: Li DengFeng , Snipes James A. , Divers Jasmin , Freedman Barry I. , Ma Lijun , Petrovic Snezana
Published: June 2021

African Americans frequently carry G1 and G2 renal‐risk variants of the apolipoprotein L1 (APOL1) gene. Two copies of G1 and/or G2 confer 5–29 fold higher risk of chronic kidney disease (CKD); however, only 20% of those carrying risk‐genotype develop CKD. This indicates that an additional stressor may facilitate development of CKD in those carrying G1/G2 risk variants. Considering that a) G1/G2 variants elicit mitochondrial dysfunction in renal cells, b) APOL1 function is pH sensitive and c) renal interstitial pH is <7, we hypothesized that acidic interstitial environment in the kidney may amplify mitochondrial dysfunction induced by G1/G2 variants. To test the hypothesis, we incubated human embryonic kidney (HEK293) cells expressing APOL1 in pH=6.8 or 7.4; we estimated early mitochondrial dysfunction by measuring mitochondrial network morphology with confocal microscopy (Fig.1). HEK293 cells stably expressing APOL1 G0, G1, G2 or empty vector (EV) under doxycycline (Dox) control were exposed to Dox for 4, 6 or 8hr, while cultured in media pH=6.8 or pH=7.4. Confocal images were acquired at 1000 magnification in live cells kept in an environmentally‐controlled chamber at each time point; FIJI software was used to quantify relative mitochondrial length (ratio of “rods” vs. “network‐branches”). We also ascertained that a) APOL1 expression was comparable regardless of pH or APOL1 genotype for up to 8hr post Dox‐induction using real‐time PCR and b) cell viability was comparable under all conditions using lactate‐dehydrogenase‐based cytotoxicity assay. Without Dox, relative mitochondrial length was similar regardless of APOL1 genotype or pH. However, after 6hr of Dox‐induction in media pH=6.8, G2‐expressing cells had shorter mitochondrial length (6.54±0.40) vs. EV (7.65±0.72, p=0.02) or G0 cells (7.46±0.31, p=0.003), indicating early mitochondrial fragmentation. After 8hr of doxycycline‐induction in media pH=6.8, mitochondrial length in G1 (6.21±0.26) and G2 cells (6.46±0.34) was lower than EV (7.13±0.32, p=0.002 and p=0.008, respectively) and G0 cells (7.22±0.45, p=0.003 and p=0.01, respectively; Fig. 2). At media pH 7.4, mitochondrial length was comparable up to 8hr of Dox‐induction, regardless of APOL1 genotype. We concluded that acidic pH may facilitate early mitochondrial dysfunction induced by G1/G2 APOL1 risk‐variants in renal cells. We propose that an acidic interstitial pH may facilitate adverse effects of APOL1 renal‐risk variants in the kidney, but not other organs expressing APOL1.

Support or Funding Information

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