bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Age on the Cerebrovascular Responses to the Initiation of Dynamic Exercise
Authors: Fisher James P , Ahmed Amar , Ainslie Phil N , Bain Anthony R , Wildfong Kevin W , Morris Laura , Flück Daniela
Published: November 2023

The rapid hemodynamic changes induced by the initiation of dynamic exercise robustly challenge the regulatory processes which ensure that cerebral perfusion is appropriately maintained. We sought to determine whether age alters the cerebrovascular responses to the initiation of dynamic exercise.

Ten young (mean±SD, 23±2 years, 2 women) and eight older (65±3 years, 1 women) individuals performed a 5‐minute bout of semi‐recumbent leg cycling exercise at 75W. Blood pressure, cardiac output, total peripheral resistance (Finometer) and middle cerebral artery blood flow velocity (MCAv: transcranial Doppler ultrasound) were monitored. Gosling pulsatility index (PI) was calculated as; (systolic MCAv–diastolic MCAv)/mean MCAv. Beat‐to‐beat data during exercise were linearly interpolated at 1 Hz.

At baseline, MAP (mean±SEM, young, 87±3; older, 93±4 mmHg), MCAv (young, 57±2; older, 56±3 cm/s) and PI (young, 0.88±0.4; older, 0.92±0.4 cm/s) were not different in young and older groups (P>0.05), while TPR tended to be lower (young, 16±1; older, 21±2 mmHg/L·min−1, p=0.08) and CO tended to be higher (young, 5.6±0.4; older, 4.6±4 L·min−1; p=0.13) in the young. The initiation of exercise evoked a rapid and sustained increase in cardiac output and fall in total peripheral resistance, that was blunted in the older group. Blood pressure fell transiently at exercise onset, reaching a similar nadir (young, −9.9±2.8; older, −10.0±2.9 mmHg; p>0.05) after a similar duration (15±2; older, 13±2 s; p>0.05) in both groups. MCAv also fell transiently at the initiation of exercise (young, −5.3±1.5; older, −5.1±1.9 cm/s, p>0.05), but the nadir was reached later in the older group (young, 20±2; older, 29±2 s, p<0.05). PI increased rapidly at the onset of exercise in young individuals, but was elevated more sluggishly in older individuals.

Collectively, these findings suggest that older individuals exhibit delayed cerebrovascular adjustments at the onset of exercise.

Support or Funding Information

This research was supported by a National Sciences and Engineering Research Council Discovery Grant and Canadian Research Chair in Cerebrovascular Physiology (PNA), the Swiss National Science Foundation (DF) and the British Heart Foundation (JPF).
The detection of the proteasomal proteolysis efficiency in C2C12 myoblast
Authors: Osana Shion , Suzuki Naoki , Aoki Masashi , Nagatomi Ryoichi
Published: November 2023

Background

The loss of skeletal muscle mass in older adults, which is called sarcopenia, is a rapidly growing global health issue. The balance of muscle protein synthesis and degradation is the key point of the regulation of skeletal muscle mass. Previous reports showed that muscle‐specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in sever skeletal muscle atrophy (Kitajima et al., 2014). Bortezomib and MG132, which are proteasome inhibitor induced a decrease in cellular amino acid levels (Suraweera et al., 2012, Zhang et al., 2014). We assume that proteasomal proteolysis play an important role in maintaining myocellular amino acid level. However, the effective detection of the proteasomal proteolysis method is not well established. One possibility of amino acids detection is click chemistry technology by using Azidohomoalanine (AHA). We therefore investigate whether click chemistry was able to detect the proteasomal proteolysis effectively. To examine this hypothesis, we used the AHA, which is methionine‐like chemical.

Methods

Mouse C2C12 myoblasts were cultured under proliferate conditions (37 °C under a humidified atmosphere containing 5% CO2) in high glucose DMEM supplemented with 10% fetal bovine serum and 100mg/ml penicillin streptomycin solution. C2C12 myoblasts stained using click chemistry technology. The medium was replaced with DMEM containing AHA without methionine for 3 hours. Next, We exposed proteasome inhibitor which is MG132 to C2C12 for 3h, 6h, 9h, 18h, 24h. Following fixation and permeabilization, cells were stained with green fluorescent Alexa Fluor 488 alkyne to detected the intracellular AHA intensity (arbitrary units).

Results

C2C12 myoblasts could be stained implied that click chemistry technology was available for C2C12 myoblasts. The intracellular AHA of normal C2C12 myoblasts decreased sequentially; on the other hand, the intracellular AHA level of proteasome inhibited C2C12 myoblasts did not change. Additionally the inhibition of proteasome induced cell death in C2C12 myoblasts. In summary, these data suggested that the AHA might be able to detect the proteasomal proteolysis effectively. This procedure may contribute to clarify more detail of the role of proteasomal proteolysis.

Conclusions

We concluded that the click chemistry technology is visualized sequential proteasomal proteolysis on C2C12 myoblasts.
MECHANISMS OF H2S ACTIVATION BY INTERMITTENT HYPOXIA
Authors: Yuan Guoxiang , Peng Ying‐Jie , Khan Shakil A. , Nanduri Jayasri , Kumar Ganesh K. , Prabhakar Nanduri R.
Published: November 2023

H2S generated by the enzyme cystathionine‐ϒ‐lyase (CSE) is important for the carotid body activation by hypoxia. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. An enhanced carotid body chemoreflex has been implicated in causing sympathetic activation and hypertension in sleep apnea patients and in CIH treated rodents. Rodents exposed to CIH exhibit markedly increased carotid body response to hypoxia, which was attributed to increased generation of reactive oxygen species (ROS). But, how ROS mediates the carotid body activation by CIH is not known. We tested the hypothesis that CIH‐induced ROS activates CSE‐derived H2S production in the carotid body. Experiments were performed on adult rats, wild‐type and CSE null mice exposed to CIH for 10 days. H2S levels were markedly elevated in CBs of CIH treated rats and wild‐type mice. This effect was absent in rats treated with L‐propargyl glycine, an inhibitor of CSE and in mice deficient in CSE. These findings suggested that CIH increases H2S production by activating CSE. MnTMPyP, a ROS scavenger also blocked CIH‐induced H2S generation, suggesting that ROS activates CSE. However, studies on human embryonic kidney cells (HEK)‐293 cells heterologously expressing CSE showed that CSE is not a direct target of ROS. Instead, IH‐induced ROS inactivates heme oxygenase‐2, an enzyme that generates carbon monoxide (CO) by interfering with Cys265 (C265A) residue in the heme regulatory motif of the enzyme. As a consequence, maximal CO synthesis (Vmax) is reduced leading to decreased CO generation. The reduced CO generation in turn activates CSE by inhibiting the serine phosphorylation and thus increases H2S production. (Supported by NIH‐PO1‐HL90554).

Support or Funding Information

NIH‐PO1‐HL90554
Bilateral Renal Cryodenervation Decreases Arterial Pressure and Plasma Renin Activity in Fructose‐Fed Sprague Dawley Rats
Authors: Soncrant Tyler , Beierwaltes William H. , Chen Haiping , Wu Min , Rossi Noreen F.
Published: November 2023

High consumption of fructose containing foods is increasingly prevalent. Fructose intake is associated with metabolic syndrome including hypertension. Fructose upregulates renal sodium and chloride reabsorption. When combined with high salt diet, fructose‐fed rats display increased arterial pressure and renal sympathetic nerve activity (RSNA). We tested the hypothesis that bilateral renal denervation using cryo‐techniques will decrease arterial pressure and plasma renin activity (PRA) in fructose‐fed rats on high salt diet. Male Sprague Dawley rats were instrumented with radiotransmitters for heart rate and arterial pressure. They were placed on 20% fructose (FF) in their drinking water and 0.4% NaCl diet. After 1 week they were maintained on 0.4 % (LS) or switched to 4% NaCl (HS) diet. One week later, rats were subjected either to bilateral denervation by freeze (−150°C)‐thaw × 3 (cryoDNX) or sham denervation (shamDNX). After 7–10 days, aortic blood was taken for PRA and renal cortex tested for norepinephrine content to verify denervation. Mean arterial pressure (MAP) increased by 9.6 ± 1.0 Hg in FF‐HS vs 0.8 ± 0.7 in FF‐LS rats. MAP decreased from 114.8 ± 2.9 to 108.5 ± 2.0 mmHg in FF‐HS‐cryo but did not change in FF‐HS‐sham rats, 115.6 ± 2.6 mmHg. PRA was higher in FF‐LS‐shamDNX 35.1 ± 13.2 vs FF‐HS‐shamDNX 10.9 ± 5.5 ng AngI/hr/ml and decreased significantly after cryodenervation in both groups. These findings support the concept that high fructose associated with high salt diet leads to renin dependent hypertension which is mediated, at least in part, by increased renal sympathetic nerve activity.

Support or Funding Information

VA Merit Award to NFR
The Effect Of Dietary Amino Acids On Chronic Kidney Disease Progression in Rats
Authors: Pillai Samyuktha Muralidharan , Verrey François
Published: November 2023

Chronic kidney disease (CKD) affects over 10% of the population (Troidle, 2014) and is on the rise. An increase in the protein intake, one of the characteristics of a western‐type diet, is thought to have a detrimental effect by increasing the Renal Plasma Flow (RPF), inducing hyperfiltration and increasing acid load. In addition to these, the source of the protein i.e. animal or vegetal (Goraya and Wesson, 2016) and consequently its amino acid content, might also play a role in the progression of the disease. The actual mechanisms by which these proteins / amino acids lead to increased Glomerular Filtration Rate (GFR) and to kidney function deterioration is not well understood.

In our experiments, we address the question of which amino acids, or groups of amino acids, might affect the progression of renal disease.

To address this question, we utilized the well‐established 5/6th Nephrectomy (5/6th Nx) model. 5/6th Nx Wistar Han rats were randomly divided into groups, receiving either the control diet (18% protein), or one of different diets, in each case containing 8% protein supplemented with 10% amino acid (aa) mix (Essential aas, Non‐essential aas, Branched chain aas or all aas in the same proportion as in the protein mix). Both GFR and RPF were measured in free moving animals, GFR transcutaneously using FITC‐sinistrin, and RPF by using radiolabelled para‐aminohippurate (PAH). None of the modified diets caused any decrease in body weight, food or water consumption in the different groups. However, the pace of RPF and GFR alteration was diet‐dependent, with the slowest change in animals receiving the EAA diet, and the most dramatic one in the animals on BCAAs. The kidney of these BCAA receiving rats also showed the strongest increase in smooth muscle actin and collagen mRNA expression in their kidney, as expected for a higher level of inflammation and fibrosis. The SMA levels were increased 2‐fold and the collagen levels were increased more than 3‐fold compared to animals on the control diet. Taken together, these results suggest that high levels of BCAAs contained in the diet have a deleterious effect on the progression of CKD.

Support or Funding Information

This research/project was supported by the NCCR Kidney. CH funded by the Swiss National Science Foundation
Novel CRISPR/Cas9 mouse knock‐in model containing an alpha‐actinin 4 mutation associated with focal segmental glomerulosclerosis recapitulated human kidney injury and provides insight into how aberrations in cytoskeleton lead to podocyte dysfunction
Authors: Feng Di , Krishnan Ramaswamy , Benjamin Ava , Pollak Martin R.
Published: November 2023

Alpha‐actinin 4 (ACTN4) is a cytoskeleton protein that crosslinks actin filaments, helps anchor the actin cytoskeleton to focal adhesions, and provides structural support for cells. Mutations in ACTN4 cause a highly penetrant form of focal segmental glomerulosclerosis (FSGS) in humans, a type of kidney injury characterized by proteinuria and podocyte dysfunction. We previously found that disease‐causing mutant K255E ACTN4 increases podocyte contractility in response to injurious circulating stimuli and increased extracellular matrix stiffness. We hypothesize that this increased contractility is pathologic, rendering mutant podocytes unable to adapt to the dynamic mechanical stresses that they experience in the glomerulus while maintaining the filtration barrier. Here we test this hypothesis by subjecting primary podocytes derived from a novel mutant mouse model to repetitive stretch and measuring their response. Using CRISPR/Cas9 technology, we first developed a new mouse model Actn4 K256E (a mutation analogous to the FSGS‐causing K255E mutation in humans) in a pure FVB background. Compared with WT littermates, homozygous mutant mice developed significant proteinuria consistent with the human phenotype. Traction force microscopy was then used to study the contractile forces exerted by mutant and WT podocytes on their underlying substrates in response to stretch. Each podocyte was subjected to five intermittent 4s stretches, with a 3 min recovery period between stretches during which contraction force was continuously measured. Whereas WT podocytes were able to consistently recover their initial contraction forces after each stretch, mutant podocytes on average demonstrated higher initial contraction forces that drastically deteriorated without recovery over time. Additionally, mutant podocytes developed obvious cracks in their cytoskeletons as visualized by immunofluorescence staining – a finding largely absent in WT podocytes. These results represent the net effect of aberrant cytoskeleton remodeling contributed by mutant ACTN4, leading to abnormalities of podocyte structure and function that may underlie the development of FSGS.

Support or Funding Information

R37DK059588 and T32DK007199
Chronic alterations in blood pH affect fasting‐state amino acid oxidation and myofibrillar and albumin protein synthesis in healthy young men
Authors: Moore Daniel R. , Lysecki Paul , Breen Leigh , Burd Nicholas A. , Smith Kenneth , Atherton Philip J. , Phillips Stuart M.
Published: November 2023

Blood pH is typically held within a narrow range in healthy individuals. However, pathological conditions (e.g. chronic kidney disease) or net acid loads associated with Western diets and/or aging can induce shifts in acid‐base homeostasis that may alter whole body and muscle protein kinetics that, over time, could lead to changes in lean body mass. Therefore, we aimed to determine the effect of chronic alterations in blood pH over physiological and pathophysiological ranges on muscle and whole body protein metabolism in vivo in humans. After 7‐d supplementation in a randomized, crossover fashion with 0.15g/kg/d of NH4Cl (acid, A), KHCO3 (base, B), or KCl (control, C), 8 healthy males (22±1y; BMI: 27.8±1.9) received a primed constant [13C]leucine infusion to measure whole body leucine metabolism and myofibrillar (MYO) and albumin (ALB) protein synthesis in the fasted state. Supplementation predictably altered venous blood pH (A<C0.05) on whole body leucine turnover, leucine rate of appearance, or non‐oxidative leucine disposal (estimate of protein synthesis). Although there were no differences (P>0.05) between conditions, leucine oxidation was negatively (r = −0.57; P<0.01) whereas leucine net balance was positively (r = 0.54; P<0.01) correlated with blood pH. There was a trend (P=0.07) towards differences in MYO between conditions. MYO was positively correlated with blood pH (r = 0.47; P<0.05). There was no effect of blood pH on mTORSer2448, 4E‐BP1Thr37/46, or eEF2Thr52 phosphorylation (P>0.05). S6K1Thr389 phosporylation was lower in A compared to B and C and its phosphorylated‐state was correlated with blood pH (r = 0.52; P=0.02) and tended to relate to MYO (r = 0.44; P=0.07). Given this, metabolic acidosis may suppress MYO in part via an impairment in mRNA translation initiation. There was no difference (P>0.05) between conditions in candidate markers of muscle protein breakdown (i.e. total ubiquitin‐conjugated proteins and 55‐kDa actin fragment). ALB was lower in A compared to B (P<0.05) and, similar to MYO, was correlated with blood pH (r = 0.51; P=0.01). Our data demonstrate that blood pH has a modifiable effect on whole body, muscle, and albumin protein kinetics. It is possible that normalization of acid‐base balance towards an alkolytic state will help mitigate the characteristic loss of lean body mass in populations at risk of clinical and subclinical metabolic acidosis through the attenuation of oxidative amino acid losses and an enhancement of myofibrillar protein synthetic rates.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council of Canada
CENTRAL TRPV4 AND WARMTH‐DEFENCE RESPONSE IN WISTAR RATS
Authors: Bícego Kenia Cardoso , Scarpellini Carolina Silveira , Almeida Maria Camila , Gargaglioni Luciane Helena
Published: November 2023

Peripheral TRPV4 is activated and triggers warmth‐defense responses at ambient temperatures (Tas) of 26–30°C in non‐genetically modified rats. TRPV4 also can be found in medial preoptic area (MPA), region known by being involved with activation of heat loss mechanisms. Then, the aim of this study was to verify if central TRPV4 in the MPA plays a role in the control of body temperature (Tb) affecting autonomic and behavioral thermoeffectors. To this end, under two different Tas (21 e 28°C), the body temperature (Tb) of rats was measured after TRPV4 in MPA were antagonized by HC‐067047. Metabolic rate, heat loss index (HLI) and preferred temperature were also recorded after central blockage of TRPV4. The antagonism of central TRPV4 caused a further increase in the Tb of rats (p< 0.001) exposed to 28°C, but not to those ones exposed to 21°C, compared to vehicle‐treated animals. The rise on Tb under 28°C was accompanied by an augment in oxygen consumption (p= 0.007) and by reduction of HLI (p=0.041). Lastly, the cold seeking behaviour observed in control animals after exposure to warm Tas for 2 hours (28–30°C) was inhibited by the microinjection of the antagonist of TRPV4 into MPA (p= 0.01). Thus, our results indicate that central TRPV4 plays a role in thermoregulation in warm condition, affecting both autonomic and behavioral heat loss responses.

Support or Funding Information

FAPESP (15/04849‐6)
Medullary reticular GABAergic neurons that mediate hunger responses induced by hypothalamic neuropeptide Y
Authors: Nakamura Yoshiko , Yanagawa Yuchio , Morrison Shaun F , Nakamura Kazuhiro
Published: November 2023

Hunger stimulates release of neuropeptide Y (NPY) in the paraventricular hypothalamus (PVH) from nerves derived from the arcuate nucleus. The NPY action in the PVH reduces energy expenditure through inhibition of brown adipose tissue (BAT) thermogenesis and stimulates food intake—hunger responses to survive starvation. However, the central circuit mechanism by which the NPY‐mediated hunger signaling from the hypothalamus coordinates the autonomic and somatic motor systems to reduce energy expenditure and to drive the consummatory responses is unknown. In this study, using rats and mice, we found that hypothalamic action of NPY activates GABAegic neurons in the intermediate and parvicellular reticular nuclei (IRt/PCRt) of the medulla oblongata that project to the rostral medullary raphe, which contains sympathetic premotor neurons controlling BAT thermogenesis. Selective stimulation of GABAergic neurons in the IRt/PCRt with a chemogenetic technique inhibited cooling‐induced BAT thermogenesis. Inactivation of IRt/PCRt neurons eliminated the inhibitory effect of NPY action in the PVH on cooling‐induced BAT thermogenesis. Intriguingly, the GABAergic IRt/PCRt neurons innervating the BAT sympathetic premotor region also innervated the masticatory motor region, and stimulation of the IRt/PCRt elicited mastication and increased feeding as well as inhibition of BAT thermogenesis. These results demonstrate a hunger response central circuit in which NPY‐triggered signaling from the hypothalamus activates GABAergic IRt/PCRt neurons, which then inhibit the thermogenic sympathetic premotor drive to BAT to reduce energy expenditure and stimulate a masticatory motor drive for feeding.

Support or Funding Information

Grants‐in‐Aid for Scientific Research from MEXT, Japan (16H05128, 15H05932 & 15K21744 to KN, 26290002 & 15H01415 to YY, 26860159 to YN); NIH grant (R01NS40987 to SFM); Takeda Science Foundation (KN & YY); Uehara Memorial Foundation (KN).
Influence of gender on blood brain barrier permeability and adenosine receptor signaling
Authors: Torres Luisa , Bynoe Margaret S
Published: November 2023

The blood brain barrier (BBB) is a highly selective barrier that separates the circulating blood from the brain extracellular fluid, acting as a protective wall that allows for the passage of nutrients into the brain while blocking the entry of toxic factors. In addition, transporters expressed on brain endothelial cells, such as P‐glycoprotein (Pg‐p), selectively regulate the influx of key molecules necessary for proper brain function, thus imposing additional restrictions on permeability. Although protective, the BBB is a major hurdle for drug delivery to the central nervous system (CNS). We have shown that adenosine, a molecule produced by the enzyme CD73 in response to CNS injury, increases BBB permeability and allows the entry of macromolecules into the brain. Activation of adenosine receptor A2A with an FDA‐approved adenosine receptor agonist (Lexiscan) potently induced BBB permeability in mice and in primary human brain endothelial cell barriers. However, previous studies have shown that the kinetics of BBB breakdown may be different between males and females. Here, we investigate whether adenosine receptor‐mediated permeabilization of the BBB is deferentially regulated in male and female mice. We showed that endothelial cells from the healthy female mouse brain have significantly higher protein levels of Pg‐p compared to endothelial cells from healthy male mice. In vitro treatment of brain endothelial cells with Lexiscan induces greater intracellular accumulation of the P‐gp substrate Rhodamine 123 in brain endothelial cells from male mice compared to those from female mice. We also observed faster and longer‐lasting accumulation of Epirubicin, another Pg‐p substrate, in the brains of healthy male mice following I.V treatment with both Epirubicin and Lexiscan. Our studies could have an impact on how we look at treatment for many disorders of the CNS that affect the BBB and may reveal whether we need to revisit our approach regarding proper drug dosing regimens for the treatment of neurological diseases in males and females.
Early Life Adversity in Rodent and Porcine Models Induces Enteric Cholinergic System Upregulation and Mast Cell Hyperplasia
Authors: Pohl Calvin Seneca , Fried David , Gulbransen Brian , Moeser Adam
Published: November 2023

Early life adversity (ELA) is a major risk factor for development of functional gastrointestinal (GI) disorders (FGID's) later in life. Disturbances in enteric nervous system function play a central role in stress‐related GI disease associated with ELA, but the alterations in ENS function and phenotype are poorly understood. We addressed this issue by characterizing the changes in intestinal secretomotor neuron function and motility induced by ELA in the murine neonatal maternal separation (NMS) model and the porcine early weaning stress (EWS) model. Compared with normal‐handed (NH) control mice, adult, C57BL/6 mice previously exposed to NMS exhibited alterations in colonic motility characterize by increased strength (130%), duration (130%) and frequency (200%) of migrating motor complexes, (p<0.001). Using a combination of Ussing Chambers and electrical field stimulation, we found that NMS mice exhibited heightened ileal and colonic secretomotor neuron activity measured as delta short circuit current (Isc) (2 fold increase relative to NH controls, p<0.05). Quantitative PCR and Western blotting showed an upregulated (by 1.3 fold) expression of choline acetyltransferase (CHAT) and vesicular choline acetyltransferase (VACHT), both involved in acetylcholine synthesis and transport (p<0.05). Choline transporter 1 (CHT1) and Acetylcholinesterase (ACHE) were unchanged in NMS mice. Mast cell tryptase was upregulated (by 1.6 fold) in colonic tissue of NMS mice compared to NH controls (p<0.05). Similarly, juvenile and adult pigs that were previously subjected to EWS, exhibited 2–3 fold heightened ileal cholinergic‐mediated Isc responses (p<0.05) that was more pronounced in female pigs compared with males. Coinciding with elevated cholinergic activity in EWS pigs were increased numbers of intestinal mast cells which were found in close‐proximity to submucosal and myenteric ganglia. Further, EWS pigs exhibited elevated mast cell mediator release compared to non‐stressed controls. Together, these studies demonstrate that ELA induces a persistent upregulation of proteins involved in acetylcholine synthesis and trafficking, which coincided with heightened motility, secretomotor function, and mast cell hyperplasia. The combination of heightened cholinergic signaling and mast cell hyperplasia and hyperactivity may contribute to the mechanism which links ELA to adult FGIDs. Future studies will investigate possible cholinergic nerve, mast cell nerve circuits that may develop and induce intestinal dysfunction and increased GI disease risk following ELA.

Support or Funding Information

Michigan State University Matilda Wilson Endowment; NICHD R01 HD072968 AJM; NIDDK R03 DK097462 AJM; NIDDK K08 DK084313 AJM
Genetic deletion of mu opioid receptors from Kölliker‐Fuse neurons reduces morphine‐induced respiratory depression
Authors: Levitt Erica S , Bissonnette John M , Kieffer Brigitte
Published: November 2023

Activation of mu opioid receptors in the pontine Kölliker‐Fuse (KF) causes a reduction in respiratory rate in anesthetized rats. We have previously shown that mu opioid agonists hyperpolarize a population of KF neurons (Levitt et al., J Physiol, 2015). The goal of these experiments was to determine the contribution of the opioid‐sensitive KF neurons to the reduction in respiratory rate caused by systemic administration of the opioid agonist morphine. Targeted deletion of mu opioid receptors was accomplished by viral expression of Cre recombinase in the KF of mice with floxed mu opioid receptors. Experiments were performed 4– 6 weeks later. In whole‐cell recordings from brain slices, infected KF neurons were no longer hyperpolarized by opioid agonist, confirming deletion of mu opioid receptors. Head‐out plethysmography was used to measure respiratory rate. Morphine (10 mg/kg, ip) caused a significantly smaller reduction in respiratory rate in mice with bilateral deletion of mu opioid receptors from KF, compared to uninjected or GFP injected controls. The time‐course for the effect of morphine was also delayed in mice lacking mu receptors in KF neurons. Thus, opioid‐sensitive KF neurons contribute to, but are not entirely responsible for, systemic morphine‐induced respiratory depression.

Support or Funding Information

NIH K99/R00 DA038069
Effects of Two‐Hit Lipopolysaccharide (LPS) Administration on Anoxic Gasping in Spontaneously Breathing Adult Male C57BL/6 Mice
Authors: Ahmad Mohammed S , Wadolowski Robert M , Wadolowski Steven B , Solomon Irene C
Published: November 2023

Ongoing studies designed to induce peripheral inflammation, and its accompanying neuroinflammation, have begun to examine various aspects of neural control of breathing. These studies typically administer the bacterial endotoxin lipopolysaccharide (LPS) systemically or in an airway/lung targeted manner in order to produce models of sepsis and acute lung injury (ALI). Recently, a ‘two‐hit’ LPS administration protocol in which systemic LPS (ip, 3 mg/kg) is administered ~24 hr prior to an airway/lung targeted (intratracheal (IT); 0.5 mg/kg) LPS injection has been used to better reflect chronic (lung/airway) disease states. Here we evaluate and characterize the impact of this two‐hit (ip‐IT) administration approach on the anoxic gasping response (recorded as EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles) in urethane‐anesthetized spontaneously breathing adult male C57BL/6 mice at 3–6 hr post LPS administration. A two‐hit protocol using ip‐IT saline injections served as a control. We found that following two‐hit LPS administration, the temporal characteristics of the gasping response were modified in LPS‐treated mice (compared to saline‐treated control mice), such that (1) the apneic pause that precedes the onset of gasping was eliminated, (2) the onset latency to gasping occurred earlier (expressed relative to anoxic duration; 39.2±2.8% vs 59.4±3.3%; P<0.001), (3) the duration of gasping was longer (109±8 s vs 51±7 s; P<0.001), (4) the total number of primary gasps was greater (23±2 vs 12±1; P<0.001), and (5) the number of terminal gasps increased (5.7±0.2 vs 4.8±0.3; P=0.021). In addition, changes in gasp pattern and duration were observed in LPS‐treated mice (compared to saline‐treated control mice), with the peak amplitude shifting to a slightly earlier time point in the inspiratory burst (P<0.001) albeit the decrease in gasp burst duration was attenuated (~10% vs ~40% decrease; P=0.029) and more highly variable. While our prior work found that ‘two‐hit’ LPS administration markedly alters basal ventilatory behaviors, the current work extends these observations to indicate that central neural mechanisms controlling gasping activities are also modified. Moreover, our data support using a ‘two‐hit’ administration approach as viable option for investigating the impact of LPS‐induced on ventilatory control.
Effects of the 5‐HT1A Agonist 8‐OH DPAT on Basal Inspiratory Motor Output and the Acute Hypercapnic Ventilatory Response in Urethane‐anesthetized Spontaneously Breathing Adult Male C57BL/6 Mice
Authors: Srichawla Bahadar , Solomon Irene C
Published: November 2023

Numerous studies have shown that activation of serotonin (5‐HT) receptors can exert marked effects on basal inspiratory motor activities as well as chemical control of breathing. While many of these studies have focused on the role of 5‐HT2A receptors in neural control of breathing, the roles of other 5‐HT receptors are only beginning to be appreciated. Here, we studied the effects of systemic application of the 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐npropylamino) tetralin (8‐OH DPAT) on basal inspiratory motor output and the acute hypercapnic ventilatory response (HCVR) in spontaneously breathing urethane‐anesthetized adult male C57BL/6 mice. For these experiments, EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles were recorded under baseline conditions (40% O2 in a balance of N2), during the acute HCVR (7% CO2/40% O2/balance of N2 for 5 min), and during recovery both before and 20–30 min after subcutaneous administration of 8‐OH DPAT (0.3 mg/kg). A similar recording protocol was used in control experiments in which mice received saline injections. We found that before administration of 8‐OH DPAT or saline, the HCVR was characterized by an ~10% increase in breathing frequency with variable effects on amplitude. Administration of 8‐OH DPAT produced an initial robust increase in inspiratory burst frequency (by 60.1±32.2%; P=0.035), which declined to a steady‐state level that was still elevated over the initial CO2 recovery level (by 31.1±7.4%; P=0.007); no persistent changes in breathing frequency were observed following saline administration. Following administration of 8‐OH DPAT, the HCVR frequency increase was attenuated or abolished. In contrast, saline‐treated mice exhibited a similar or enhanced HCVR frequency response. These findings indicate that 8‐OH DPAT‐induced activation of 5‐HT1A receptors not only enhances basal inspiratory burst frequency but also significantly alters the acute HCVR.

Support or Funding Information

Thomas Hartman Center for Parkinson's Disease Research at Stony Brook University
Cholinergic modulation of pedunculopontine tegmental nucleus in respiratory control
Authors: Lima Janayna D , Sobrinho Cleyton R , Santos Leonardo K , Takakura Ana C , Mulkey Daniel K , Moreira Thiago S
Published: November 2023

The pedunculopontine tegmental nucleus (PPTn) has been shown to have important functions relevant to the regulation of behavioral states and various motor control systems, including breathing control. The PPTn is considered an important nucleus in the mesopontine region with considerably cholinergic input to the ventral respiratory column. In addition, recent studies indicate that cholinergic innervation of the ventral respiratory column may play an important role in modulation of breathing. Here, we investigated a cholinergic projection from the PPTn to the retrotrapezoid nucleus (RTN) and if the stimulation of cholinergic neurons in the PPTn elicit changes in the respiratory pattern in conscious rats. Male Wistar rats (280–350g, N = 3–7/group) with unilateral stainless steel cannula implanted into the PPTn were used. Respiratory parameters (tidal volume (VT), respiratory frequency (fR) and ventilation (VE)) were analyzed by whole body pletismography. Retrograde tracer FluorGold (FG‐2%) injected into the RTN region showed a light direct cholinergic projection from PPTn to RTN. In unrestrained awake rats, unilateral injection of the cholinergic muscarinic agonist carbachol (10 mM − 100 nL) in the PPTn decreased fR (60 ± 8, vs. saline: 102 ± 8 breaths/min) and VE (315 ± 105, vs. saline: 604 ± 114 ml/kg/min), without changing VT. We conclude that cholinergic stimulation of PPTn has a significant impact on the breathing activity. Our data suggest that PPTn may influence breathing through a direct pathway from the mesopontine region to the RTN.

Support or Funding Information

FAPESP, CNPq, CAPES‐PROEX
REDUCTION OF PERIAQUEDUCTAL GRAY PROJECTING NEURONS TO THE RETROTRAPEZOID NUCLEUS IN A 6‐OHDA MODEL OF PARKINSON'S DISEASE
Authors: Lima Juliana C , Moreira Thiago C , Takakura Ana C
Published: November 2023

Parkinson's disease (PD) is characterized by a reduction in the number of dopaminergic neurons of the substantia nigra (SN) accompanied by motor and non‐motor deficiencies such as respiratory failure. Among the non‐motor symptoms, the disease has respiratory complications, and the one that is the leading cause of death in PD is obstructive sleep apnea. Our laboratory suggested an impairment in brain regions crucial for breathing automaticity such as the retrotrapezoid nucleus (RTN) and a decrease in respiratory rate in a model of PD. In addition, we also showed no direct projections from SN to RTN, but we identified a connection that bypass the periaqueductal gray (PAG), a region with important modulatory role in respiratory activity. Therefore, our aim was to investigate whether there is a reduction in the number of neurons in PAG and whether the neurons in the PAG that project to RTN are reduced in the animal's model of PD. male Wistar rats (250 – 300 g; N = 3–5/group, CEUA:99/22/03) with vehicle or 6‐hydroxydopamine (6‐OHDA) injection in the Caudate and Putamen (CPu) region were used. After 30 days, one group of animals received the injection of the retrograde tracer Fluorogold (2%) in the RTN. Fourty days after 6‐OHDA or vehicle injection, all animals were submitted to hypercapnia for 3 hours and perfused for immunohistochemistry to evaluate the neuronal marker (NeuN‐ir), and the presence of fos in PAG neurons that project to RTN. In 6‐OHDA‐lesioned rats, there is a reduction in the density of NeuN‐ir in the dorsomedial (10 ± 3 vs. vehicle: 23 ± 1%), dorsolateral (15 ± 6 vs. vehicle: 38 ± 2%) and the ventrolateral (14 ± 5, vs. vehicle: 30 ± 1%) regions of the PAG and a reduction in the number of TH‐ir varicosities in the PAG (778 ± 135 vs. vehicle: 1338 ± 133). Moreover, the number of neurons in PAG activated by hypercapnia that project to RTN is reduced in the dorsolateral (3 ± 1 vs. vehicle: 21 ± 12) and ventrolateral (4 ± 1, vs. vehicle: 116 ± 8) regions in 6‐OHDA‐lesioned rats. We conclude that there is a commitment of the pathway between PAG and RTN in the animal's model of PD.

Support or Funding Information

FAPESP, CNPQ, CAPES
Repetitive Acute Intermittent Hypoxia Affects Lesion Volume After Cervical Spinal Injury
Authors: Santiago‐Moreno Juan Gabriel , Satriotomo Irawan , Dougherty Brendan J , Springborn Sarah , Kopp Elizabeth , Sullivan Lydia , Mitchell Gordon S
Published: November 2023

After spinal cord injury (SCI), lesion volume increases progressively as axons withdraw from the site of injury. Strategies to promote growth through the injured spinal cord will require treatments to minimize or reverse such spinal degeneration. Repetitive exposure to acute intermittent hypoxia (rAIH) has emerged as a promising new treatment to restore motor function after chronic SCI. rAIH elicits spinalplasticity by increasing growth/trophic factor expression, including BDNF and VEGF; both trophic factors also promote neuroprotection and neural progenitor cell proliferation, differentiation and survival. To explore the potential for rAIH to preserve/restore spinal integrity after injury, double‐blinded studies were initiated on rats with cervical spinal hemisections (a single slice injury); two months post‐injury, the rats began six months exposures to rAIH (10, 5 min episodes of 10.5% O2 per day; 5 min normoxic intervals; 3 × per week) or sham normoxia (designated as groups A & C). Rats were sacrificed two months after rAIH had ended. Since our analyses are still ongoing, the blind has not been broken. Preliminary analyses demonstrate significant differences in lesion size and scar tissue volume between groups (A: 31±3% versus C: 49±6%; p = 0.02, n = 5). Astroglia penetrate the lesioned area and more proliferating neuronal somata surround injured area in group A versus C. We await analysis of additional animals and breaking of the blind to determine if rAIH exerts beneficial or deleterious effects on injury volume. Regardless of rAIH effects, our results will be of major interest as we attempt to develop this promising new treatment for chronic spinal injuries, an injury for which there is currently no effective therapy.

Support or Funding Information

Supported by NIH HL69064, DoD, Craig H. Neilsen Foundation, and McKnight Brain Institute.
Image‐Based Screening for Epigenetic Modifiers of Myofibroblast Activation
Authors: Haak Andrew J. , Ligresti Giovanni , Abbey Rachelle , Romanov Sergei , Chung Thomas D.Y. , Tschumperlin Daniel J.
Published: November 2023

Tissue fibrosis represents an increasing cause of morbidity and mortality worldwide, with fibroblast‐derived myofibroblasts playing an essential pathogenic role. Myofibroblasts are characterized by their increased expression of smooth muscle actin (α‐SMA), and contribute to tissue fibrosis through their increased abundance, matrix deposition and tissue contraction. Unlike in physiological wound repair, where fibroblast activation is spontaneously reversible, the fibroblast activation associated with pathological fibrosis is perpetuated, and fibroblasts maintain their activated phenotype even after the initial injury has been resolved. Previous studies have shown that aberrant epigenetic signaling may drive sustained fibroblast activation during fibrosis and several small‐molecule epigenetic modifiers have been shown to be effective in protecting against fibrosis in various animal models, however no systematic effort has been reported to identify epigenetic small molecule regulators of myofibroblast activation. Here we screened a library of 98 well‐annotated epigenetic modifying compounds from Mayo Clinic's collaboration with Nanosyn (Santa Clara, CA) for their ability to inhibit fibroblast to myofibroblast activation. To analyze compound effects, we designed a high‐content image‐based screening method to quantify α‐SMA expression, organization, and area coverage. For each metric we calculated an independent Z‐score, and to identify which compounds most effectively blocked myofibroblast activation we created a robust, multiparametric “Deep Z Analysis” which stacked together individual Z‐scores. In the initial screen each compound was run in triplicate using three concentrations (0.5, 2.5, and 10 μM). Screen hits were defined by any compound which displayed an estimated IC50 < 2.5μM, resulting in a hit rate of 12.2%. These 12 hits were then validated for their efficacy in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis. In support of our overall screening approach, among the primary screen hits were compounds previously shown to be antifibrotic, including: bromodomain inhibitors JQ1 and CPI‐0610, DNA methyl transferase inhibitor azacitidine, and Poly (ADP‐ribose) Polymerase (PARP) inhibitor ABT‐888. While several epigenetic therapies are already approved by the FDA for cancer treatment, no epigenetic drugs for the treatment of fibrotic diseases have reached the clinic. Our approach will help identify epigenetic modifiers attractive for further detailed research, with a long term goal of defining new targets to move forward towards eventual therapies for lung fibrosis.
Sex differences in hepatic MCT1 and CD147 expression
Authors: Cao Jieyun , Ng Michael , Felmlee Melanie A
Published: November 2023

Purpose

Monocarboxylate transporter 1 (MCT) belongs to the solute carrier super family, and transports monocarboxylates such as ketone bodies, lactate and pyruvate, as well as drugs such as gamma‐hydroxybutyric acid. CD147 interacts with MCT1 as an ancillary protein for trafficking to the plasma membrane. Previously, it has been shown that MCT expression changes under different sex hormone conditions in skeletal muscle and Sertoli cells. However, it is unknown if MCT1 or CD147 demonstrates sex differences in the liver where MCT1 play an important role in drug disposition. The purpose of this study is to evaluate hepatic MCT1 and CD147 mRNA and protein expression in males, over the estrus cycle in female rats and in the absence of female sex hormones.

Method

Liver samples were harvested from 6 groups of rats (N = 3 – 5 per group): females at the four stages of the estrus cycle (proestrus, estrus, metestrus, diestrus), ovariectomized (OVX) females and male Sprague‐Dawley rats. Estrus cycle stages of females were classified by vaginal lavage smear. Hepatic mRNA expression of MCT1 and CD147 were analyzed by qPCR. Whole cell and membrane protein expression of MCT1 and CD147 were evaluated by western blot.

Results

MCT1 mRNA expression was significantly lower (0.4 and 0.3 fold respectively) in proestrus and metestrus compared to control (male rats). OVX and male rats showed similar MCT1 mRNA and whole cell protein expression. Membrane MCT1 expression in proestrus, metestrus, and diestrus groups was significantly lower (21% – 42%) compared to OVX rats. MCT1 total and membrane protein expression were positively correlated (r = 0.69). Hepatic membrane bound ancillary protein CD147 in males was significantly higher (2.3 – 3 times) than other groups whereas mRNA and whole protein expression did not show any difference.

Conclusion

MCT1 mRNA expression is lower in the presence of female sex hormones indicating they play a role in reducing MCT1 mRNA expression in liver. Ovariectomy increased membrane MCT1 expression compared to female rats indicating female hormones negatively regulate MCT1 membrane expression. Significant sex differences were observed in membrane CD147 expression, whereas there was no difference in whole cell protein and mRNA levels demonstrating that androgens are involved in regulating CD147 membrane localization. This study reveals that hepatic MCT1 and CD147 expression and localization were regulated by sex hormones. Sex differences in hepatic MCT1 expression may lead to altered drug disposition of its substrates, so it is critical to understand the role of sex hormones and MCT1 regulation.
Dissecting the role of PLD2 signaling pathways in endothelial barrier recovery.
Authors: Shaaya Mark , Fu Panfeng , Vogel Stephen , Karginov Andrei , Natarajan Viswanathan
Published: November 2023

Rationale

Acute lung injury (ALI) is a life‐threatening form of hypoxemic respiratory failure, characterized by inflammation, damage to the alveolar‐capillary barrier, and accumulation of fluid in the lung resulting in pulmonary edema. In order for the pulmonary barrier to recover, endothelial cells at the injury site must migrate to reseal the inter‐endothelial gaps formed in the injury phase. Phospholipase D (PLD)‐mediated generation of phosphatidic acid (PA) plays an important role in cell migration; therefore, we hypothesize that PLD/PA signaling contributes to the pulmonary endothelial barrier recovery process in ALI resolution.

Methods

Human Lung Microvascular Endothelial Cells (HLMVECs) permeability to thrombin or edemagenic agents was determined by Electric Cell‐substrate Impedance Sensing system, and trans‐well permeability assay. Western blot was performed to assess changes in FAK, c‐Src and Paxillin phosphorylation in response to Thrombin. Epifluorescence imaging was performed and time‐lapse movies were taken for 3 h after thrombin stimulation at 2‐min time intervals in the absence or presence of PLD1 or PLD2 inhibitors. Metamorph and MATLAB softwares were used to quantify cell spreading, protrusive activity, and focal adhesions (FA) count. To analyze FAs spatiotemporal dynamics, we used total internal reflection fluorescence (TIRF) was used. The ex‐vivo role of PLD2 on PAR‐1 peptide‐mediated lung inflammation and injury was evaluated in wild type (WT) and PLD2 knockout (KO) mice. Lungs were isolated from WT or PLD2 KO mice under isoflurane anesthesia, ventilated (PIP 12 cm H2O), and perfused (2 ml/min at 37°C) for determinations of lung liquid permeability (microvessel filtration coefficient, Kfc).

Results

Inhibition of PLD2 catalytic activity in HLMVECs significantly increased thrombin‐induced endothelial permeability. Inhibiting PLD2 activity had no effect on thrombin‐induced protrusive activity; however, cells failed to spread and recover after 3‐h of thrombin treatment. This suggests that PLD2 is not essential for cells to send out protrusions, but is required for their adhesion to the matrix. PLD1 inhibition did not alter cell‐spreading dynamics. Further, inhibition of PLD2 activity destabilized focal adhesions resulting in less cell spreading. Genetic deletion of PLD2 in mice increased pulmonary leak in response to PAR‐1 agonist compared to WT mice. Also, PLD2 knockout mice had more lung injury in an intratracheal LPS‐induced ALI model.

Conclusions

PLD2/PA signaling plays an important role in pulmonary endothelial recovery from thrombin insult, and contributes to the resealing of the endothelium by stabilizing the protrusions of contiguous endothelial cells to reach out and form new adherent junctions.

Support or Funding Information

This work was supported by NIH grant HL P01 060678 Project 4 to VN.