Acute Stimuli Research Articles

Abstract 051: Exploiting Angiotensin Sensitive Vagal Afferents To Understand And Alleviate Hypertension: A Closed Loop System For Blood Pressure Control

Cardiovascular homeostasis is maintained by neural signals arising from arterial baroreceptors (BRs) that apprise the brain of vascular stretch. Here, we test the overall hypothesis that angiotensin-II (AngII) type 1a receptor expressing neurons within the nodose ganglia (NDG AT1aR ) serve as BR that undergo plasticity during chronic elevations in BP. We found that NDG AT1aR receive input from the aortic arch, project to the caudal nucleus of the solitary tract (cNTS), and synthesize Piezo-1/-2. AT1aR-GCaMP6s mice were used to determine whether NDG AT1aR couple their firing to elevated BP. Two-photon intravital imaging revealed that phenylephrine (PE; i.v.) increased GCaMP6s fluorescence within NDG AT1aR (ΔF/F max = 0.65±0.05*, Time ΔF/Fmax = 92s) and this response was delayed in mice given DOCA-salt (Time ΔF/Fmax = 124s) or chronic AngII (Time ΔF/Fmax = 155s). To determine if optical perturbation of NDG AT1aR affects the baroreflex, expression of excitatory channelrhodpsin2 (ChR2) or inhibitory halorhodopsin (Halo) was directed to AT1aR cells. Optical excitation of NDG AT1aR at their axon terminals in the cNTS elicited frequency-dependent decreases in BP (ΔSBP @ 0.5, 1, 2, 3 Hz = -6.74±3.42 # , -9.75±2.33 # , -23.24±4.39 # , -25.27±4.38 # ) and HR (ΔHR @ 0.5, 1, 2, 3 Hz = -7.81±3.14 # , -14.86±3.22 # , -23.39±5.02 # , -23.66±4.13 # ). Conversely, optical inhibition of NDG AT1aR at axon terminals in the cNTS blunted pressor responses to PE (ΔHR PE(Min) = -36.15±5.31, ΔHR PE+Light(Min) = -23.11±3.33 + ). ChR2-expressing mice were then subjected to DOCA-salt. Optical excitation elicited hypotensive (ΔSBP Min = -21.11±4.05 + ) and bradycardic (ΔHR Min = -235.6±46.69 + ) responses; however, the duration of these effects was altered. We developed a closed loop system to target NDG AT1aR at axon terminals in the cNTS of ChR2-expressing mice to determine if these neurons can be exploited to alleviate hypertension. Mice were subjected to acute and chronic stimuli to elevate BP and stimulated by the system until BP returned below threshold. Collectively, these studies reveal the structure and function of NDG AT1aR and suggest that such neurons may be exploited to discern and relieve hypertension. ( p < 0.05, * time by treatment, # time by frequency, or + time by light interaction)

Independent Effects Of Hypoxia And Altitude On Human Physiology

PURPOSE: Decreased fraction of inspired oxygen (FiO2) is often used to simulate the atmospheric partial pressure of oxygen decreases experienced during high altitude sojourns. Therefore, we aimed to independently investigate hypoxia and altitude by isolating oxygen concentration and barometric pressure. METHODS: 18 subjects completed 3 trials (sea level, hypoxia, altitude). 90-minute duration and intensity matched hypoxic stimuli were induced via decreased FiO2 or 4,200 m ascent. Relative tissue oxygenation change and cardiovascular variables were measured during rest and a 3-minute step-test. RESULTS: Muscle oxygenated hemoglobin (O2Hb) and muscle deoxygenated hemoglobin (HHb) were not different across environments during rest or exercise (p > 0.339) with alterations noted during rest to exercise transitions (p < 0.001). Resting brain O2Hb at hypoxia and altitude were lower than sea level (p < 0.012), but not different than each other (p = 0.278). Exercising brain O2Hb was lower at altitude than sea level (p = 0.007) similarly trending compared to hypoxia (p = 0.066). Exercising brain O2Hb was not different between hypoxia and sea level (p = 0.158). Brain HHb at hypoxia and altitude were higher than sea level (p < 0.001), but not different than each other (p = 0.158). Exercise HR at hypoxia (141 ± 4 beats·min-1) and altitude (141 ± 3 beats·min-1) were lower than sea level (127 ± 44 beats·min-1, p < 0.002), but not different than each other (p > 0.208). Exercise stroke volume at altitude (109.6 ± 4.1 mL) was higher than hypoxia (97.8 ± 3.3 mL) and sea level (99.8 ± 3.9 mL, p < 0.010) with no differences between hypoxia and sea level (p = 0.481). Exercise cardiac output at hypoxia (13.8 ± 0.6 L) and altitude (15.5 ± 0.7 L) were greater than sea level (12.6 ± 0.5 L, p < 0.006) with altitude greater than hypoxia (p = 0.001). CONCLUSIONS: During acute hypoxic stimuli, skeletal muscle maintains oxygenation while the brain does not. Tissue oxygenation may be mediated by environmentally driven cardiovascular compensation. FiO2 decreases may not satisfactorily simulate all physiological outcomes experienced during altitude induced barometric pressure decreases. Funding: Department of Defense United States Army Medical Research and Materiel Command (DOD USAMRMC: W81XWH-15-2-0075).

Survey of extracellular communication of systemic and organ-specific inflammatory responses through cell free messenger RNA profiling in mice

SummaryBackgroundInflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signalling pathways of innate and adaptive immune systems. There is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses in circulation and in inaccessible solid organs.MethodsHere we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling and computational biology to temporally assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples.FindingsCf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. We further identified the dysregulation of liver-specific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this generally inaccessible biological compartment.InterpretationUsing a preclinical mouse model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses.FundingMolecular Stethoscope.

Time Course of Recovery Following CrossFit® Karen Benchmark Workout in Trained Men.

The establishment of fatigue following the acute exercise stimulus is a complex and multi-factorial process, that might arise due to a range of distinct physiological mechanisms. However, a practical method of assessing CrossFit® athletes’ recovery status has been neglected entirely in real-world sporting practice. The study describes the acute and delayed time course of recovery following the CrossFit® Benchmark Workout Karen. Eight trained men (28.4 ± 6.4 years; 1RM back squat 139.1 ± 26.0 kg) undertook the Karen protocol. The protocol consists of 150 Wall Balls (9 kg), aiming to hit a target 3 m high. Countermovement jump height (CMJ), creatine kinase (CK), and perceived recovery status scale (PRS) (general, lower and upper limbs) were assessed pre, post-0h, 24, 48 and 72 h after the session. The creatine kinase concentration 24 h after was higher than pre-exercise (338.4 U/L vs. 143.3 U/L; p = 0.040). At 48h and 72 h following exercise, CK concentration had returned to baseline levels (p > 0.05). The general, lower and upper limbs PRS scores were lower in the 24-h post-exercise compared to pre-exercise (general PRS: 4.7 ± 1.5 and 7.7 ± 1.7; p = 0.013; upper limbs PRS: 6.6 ± 1.3 and 7.5 ± 1.3; p = 0.037; lower limbs PRS: 3.9 ± 2.5 and 7.3 ± 0.1; p = 0.046). Our findings provide insights into the fatigue profile and recovery in acute CrossFit® and can be useful to coaches and practitioners when planning training programs. Moreover, recovery status can be useful to optimize training monitoring and to minimize the potential detrimental effects associated with the performance of repeated high-intensity sessions of CrossFit®.

Cellular and circuit diversity determines the impact of endogenous opioids in the descending pain modulatory pathway.

The descending pain modulatory pathway exerts important bidirectional control of nociceptive inputs to dampen and/or facilitate the perception of pain. The ventrolateral periaqueductal gray (vlPAG) integrates inputs from many regions associated with the processing of nociceptive, cognitive, and affective components of pain perception, and is a key brain area for opioid action. Opioid receptors are expressed on a subset of vlPAG neurons, as well as on both GABAergic and glutamatergic presynaptic terminals that impinge on vlPAG neurons. Microinjection of opioids into the vlPAG produces analgesia and microinjection of the opioid receptor antagonist naloxone blocks stimulation-mediated analgesia, highlighting the role of endogenous opioid release within this region in the modulation of nociception. Endogenous opioid effects within the vlPAG are complex and likely dependent on specific neuronal circuits activated by acute and chronic pain stimuli. This review is focused on the cellular heterogeneity within vlPAG circuits and highlights gaps in our understanding of endogenous opioid regulation of the descending pain modulatory circuits.

Involvement of neurosteroids in the control of prolactin secretion in sheep under basal, stressful and pregnancy conditions

Prolactin (PRL) secretion by the anterior pituitary (AP) is responsive to changes in physiological conditions and many external factors that also affect brain neurosteroid levels. This study tested the hypothesis that neurosteroids can affect PRL secretion in sheep under basal, stressful and advanced pregnancy conditions. In Experiment 1, luteal-phase sheep were subjected to a three-day series of intracerebroventricular (icv.) control (n = 12) or allopregnanolone (AL, 4 × 15 μg/60 μL/30 min at 30-min intervals, n = 12) infusions. Acute stressful stimuli, isolation and partial movement restriction were applied on the third day of infusion to half of the animals in each group. In Experiment 2, pregnant sheep were subjected to a three-day series of icv. control (n = 6) or finasteride (4 × 25 μg/60 μL/30 min at 30-min intervals, n = 6) infusions during the 16th week of pregnancy. As a result, the relative abundance of PRL transcript increased in the AP of luteal-phase sheep treated with stress, AL and AL in combination with stress (P < 0.05 - P < 0.01) compared to controls. The level of PRL mRNA in stressed-AL-treated sheep was higher (P < 0.01) than in sheep only subjected to stress. The PRL protein content in the AP decreased in stressed-only sheep compared to controls (P < 0.05) and increased in stressed-AL-treated sheep compared to controls and other groups (P < 0.05 - P < 0.01). Plasma PRL concentration increased (P < 0.05 - P < 0.01) in stressed-only sheep compared to controls; AL infusion counteracted the stress-induced increase in PRL levels (P < 0.05 - P < 0.01) and had no effect in non-stressed animals. Inhibition of neurosteroid synthesis in the brain of pregnant sheep by finasteride caused transient increases (P < 0.05 - P < 0.001) in plasma PRL concentration compared to controls. In conclusion, the presented results indicate a bimodal effect of AL on PRL secretion in sheep: first at the molecular level – stimulation of PRL mRNA expression; second – inhibition of hormone release from pituitary lactotrophs. Both AL activities may involve various mechanisms regulating PRL secretion. In general, cerebral neurosteroids can affect the supply of pituitary PRL in the body under certain conditions, such as stress and pregnancy.

Central Gαi2 Protein Mediated Neuro-Hormonal Control of Blood Pressure and Salt Sensitivity.

Hypertension, a major public health issue, is estimated to contribute to 10% of all deaths worldwide. Further, the salt sensitivity of blood pressure is a critical risk factor for the development of hypertension. The hypothalamic paraventricular nucleus (PVN) coordinates neuro-hormonal responses to alterations in plasma sodium and osmolality and multiple G Protein-Coupled Receptors (GPCRs) are involved in fluid and electrolyte homeostasis. In acute animal studies, our laboratory has shown that central Gαi/o subunit protein signal transduction mediates hypotensive and bradycardic responses and that Gz/q, proteins mediate the release of arginine vasopressin (AVP) and subsequent aquaretic responses to acute pharmacological stimuli. Extending these studies, our laboratory has shown that central Gαi2 proteins selectively mediate the hypotensive, sympathoinhibitory and natriuretic responses to acute pharmacological activation of GPCRs and in response to acute physiological challenges to fluid and electrolyte balance. In addition, following chronically elevated dietary sodium intake, salt resistant rats demonstrate site-specific and subunit-specific upregulation of Gαi2 proteins in the PVN, resulting in sympathoinhibition and normotension. In contrast, chronic dietary sodium intake in salt sensitive animals, which fail to upregulate PVN Gαi2 proteins, results in the absence of dietary sodium-evoked sympathoinhibition and salt sensitive hypertension. Using in situ hybridization, we observed that Gαi2 expressing neurons in parvocellular division of the PVN strongly (85%) colocalize with GABAergic neurons. Our data suggest that central Gαi2 protein-dependent responses to an acute isotonic volume expansion (VE) and elevated dietary sodium intake are mediated by the peripheral sensory afferent renal nerves and do not depend on the anteroventral third ventricle (AV3V) sodium sensitive region or the actions of central angiotensin II type 1 receptors. Our translational human genomic studies have identified three G protein subunit alpha I2 (GNAI2) single nucleotide polymorphisms (SNPs) as potential biomarkers in individuals with salt sensitivity and essential hypertension. Collectively, PVN Gαi2 proteins-gated pathways appear to be highly conserved in salt resistance to counter the effects of acute and chronic challenges to fluid and electrolyte homeostasis on blood pressure via a renal sympathetic nerve-dependent mechanism.

Regional hypothalamic, amygdala, and midbrain periaqueductal gray matter recruitment during acute pain in awake humans: A 7-Tesla functional magnetic resonance imaging study

Over the past two decades, magnetic resonance imaging (MRI) studies have explored brain activation patterns during acute noxious stimuli. Whilst these human investigations have detailed changes in primarily cortical regions, they have generally not explored discrete changes within small brain areas that are critical in driving behavioural, autonomic, and endocrine responses to pain, such as within subregions of the hypothalamus, amygdala, and midbrain periaqueductal gray matter (PAG). Ultra-high field (7-Tesla) MRI provides enough signal-to-noise at high spatial resolutions to investigate activation patterns within these small brain regions during acute noxious stimulation in awake humans. In this study we used 7T functional MRI to concentrate on hypothalamic, amygdala, and PAG signal changes during acute noxious orofacial stimuli. Noxious heat stimuli were applied in three separate fMRI scans to three adjacent sites on the face in 16 healthy control participants (7 females). Images were processed using SPM12 and custom software, and blood oxygen level dependent signal changes within the hypothalamus, amygdala, and PAG assessed. We identified altered activity within eight unique subregions of the hypothalamus, four unique subregions of the amygdala, and a single region in the lateral PAG. Specifically, within the hypothalamus and amygdala, signal intensity largely decreased during noxious stimulation, and increased in the lateral PAG. Furthermore, we found sex-related differences in discrete regions of the hypothalamus and amygdala. This study reveals that the activity of discrete nuclei during acute noxious thermal stimulation in awake humans.

Fucoxanthin decreases lipopolysaccharide-induced acute lung injury through the inhibition of RhoA activation and the NF-κB pathway.

Fucoxanthin is a natural pigment widely distributed in macroalgae and microalgae. An orange-colored xanthophyll, it has several bioactive effects, including anticancer, anti-obesity, oxidative stress reduction, and anti-inflammation. Acute lung injury (ALI) caused by acute infections or injurious stimuli to the lung tissues is a severe pulmonary inflammatory disease. To date, no evidence has shown ALI to be reduced by fucoxanthin through activation of Ras homolog family member A (RhoA) and the nuclear factor (NF)-κB pathway in lipopolysaccharide (LPS)-treated mice. Pretreatment with fucoxanthin inhibited histopathological changes in lung tissues and neutrophil infiltration into bronchoalveolar lavage fluid induced by LPS in ALI mice. Moreover, LPS-induced proinflammatory cytokine expression and neutrophil infiltration were inhibited by fucoxanthin in a concentration-dependent manner. Pretreatment of mice with fucoxanthin inhibited NF-κB phosphorylation and IκB degradation in the lungs of mice with LPS-induced ALI. We further found that phosphorylation of Akt and p38 mitogen-activated protein KINASE (MAPK) was inhibited by fucoxanthin. By contrast, the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase was not inhibited by fucoxanthin. Furthermore, we found that the activation of RhoA was inhibited by fucoxanthin in LPS-induced ALI. On the basis of these results, we propose that fucoxanthin disrupts the RhoA activation-mediated phosphorylation of Akt and p38 MAPK, leading to NF-κB activation in mice with LPS-induced ALI.

A non-invasive 13CO2 breath test detects differences in anabolic sensitivity with feeding and heavy resistance exercise in healthy young males: a randomized control trial.

There are limited tools to measure anabolic sensitivity non-invasively in response to acute physiological stimuli, which represents a challenge for research in free-living settings and vulnerable populations. We tested the ability of a stable isotope breath test to detect changes in leucine oxidation (OX) and leucine retention (intake-OX) across a range of anabolic sensitivities. Healthy males ingested a beverage containing 0.25 g·kg-1 protein and 0.75 g·kg-1 carbohydrate with the leucine content enriched to 5% with l-[1-13C]leucine at rest (FED) or after a bout of resistance exercise (EXFED), with a parallel group consuming only the tracer (FAST). Concurrent primed-constant infusions of l-[5,5,5-2H3]leucine revealed high peripheral bioavailability for FED (∼81%), EXFED (∼80%), and FAST (∼117%). After beverage ingestion, whole-body protein synthesis was greater in FED and EXFED than FAST. OX was greater in FED and EXFED than FAST, with OX lower in EXFED than FED. Leucine retention demonstrated expected physiological differences in anabolic sensitivity (EXFED>FED>FAST). We demonstrated that a non-invasive breath test based on an amino acid (leucine) that is preferentially metabolized in peripheral (muscle) tissues can detect differences in anabolic sensitivity. Future studies could examine this test within a variety of populations experiencing muscle growth or atrophy. This study was registered as a Clinical Trial at ClinicalTrials.gov (no. NCT04887727). Novelty: An oral l-[1-13C]leucine breath test can detect greater anabolic sensitivity after feeding and resistance exercise. This tool may be applied in growing (e.g., children) or wasting (e.g., aging) populations where invasive procedures are not possible.

The Disordered Amino Terminus of the Circadian Enzyme Nocturnin Modulates Its NADP(H) Phosphatase Activity by Changing Protein Dynamics.

Endogenous circadian clocks control the rhythmicity of a broad range of behavioral and physiological processes, and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes, and the NOCT protein exhibits phosphatase activity, catalyzing the removal of the 2'-phosphate from NADP(H). In addition to its daily nighttime peak of expression, it is also induced by acute stimuli. Loss of Nocturnin (Noct-/-) in mice results in resistance to high-fat diet-induced obesity, and loss of Noct in HEK293T cells confers a protective effect to oxidative stress. Modeling of the full-length Nocturnin protein reveals a partially structured amino terminus that is disparate from its CCR4 family members. The high sequence conservation of a leucine zipper-like (LZ-like) motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. In vitro biochemical and biophysical techniques demonstrate that the LZ-like domain within the flexible N-terminus is necessary for preserving the active site cleft in an optimal conformation to promote the efficient turnover of the substrate. This modulation occurs in cis and is pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity to provide fine-tuned control over cellular levels of NADPH.

E74-like factor 1 contributes to nerve trauma-induced nociceptive hypersensitivity through transcriptionally activating matrix metalloprotein-9 in dorsal root ganglion neurons.

Nerve trauma-induced alternations of gene expression in the neurons of dorsal root ganglion (DRG) participate in nerve trauma-caused nociceptive hypersensitivity. Transcription factors regulate gene expression. Whether the transcription factor E74-like factor 1 (ELF1) in the DRG contributes to neuropathic pain is unknown. We report here that peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve or unilateral fourth lumbar spinal nerve ligation led to the time-dependent increases in the levels of Elf1 mRNA and ELF1 protein in injured DRG, but not in the spinal cord. Preventing this increase through DRG microinjection of adeno-associated virus 5 expressing Elf1 shRNA attenuated the CCI-induced upregulation of matrix metallopeptidase 9 (MMP9) in injured DRG and induction and maintenance of nociceptive hypersensitivities, without changing locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking this increase through DRG microinjection of AAV5 expressing full-length Elf1 upregulated DRG MMP9 and produced enhanced responses to mechanical, heat, and cold stimuli in naive mice. Mechanistically, more ELF1 directly bond to and activated Mmp9 promoter in injured DRG neurons after CCI. Our data indicate that ELF1 participates in nerve trauma-caused nociceptive hypersensitivity likely through upregulating MMP9 in injured DRG. E74-like factor 1 may be a new target for management of neuropathic pain.

Age-Dependent FOSB/ΔFOSB Response to Acute and Chronic Stress in the Extended Amygdala, Hypothalamic Paraventricular, Habenular, Centrally-Projecting Edinger-Westphal, and Dorsal Raphe Nuclei in Male Rats.

FOS proteins are early-responding gene products that contribute to the formation of activator protein-1. Several acute and chronic stimuli lead to Fos gene expression, accompanied by an increase of nuclear FOS, which appears to decline with aging. FOSB is another marker to detect acute cellular response, while ΔFOSB mirrors long-lasting changes in neuronal activity upon chronic stress. The notion that the occurrence of stress-related mood disorders shows some age dependence suggests that the brain’s stress sensitivity is also a function of age. To study age-dependent stress vulnerability at the immediate-early gene level, we aimed to describe how the course of aging affects the neural responses of FOSB/ΔFOSB in the acute restraint stress (ARS), and chronic variable mild stress (CVMS) in male rats. Fourteen brain areas [central, medial, basolateral (BLA) amygdala; dorsolateral- (BNSTdl), oval- (BNSTov), dorsomedial-, ventral- (BNSTv), and fusiform- (BNSTfu) divisions of the bed nucleus of the stria terminalis; medial and lateral habenula, hypothalamic paraventricular nucleus (PVN), centrally-projecting Edinger-Westphal nucleus, dorsal raphe nucleus, barrel field of somatosensory cortex (S1)] were examined in the course of aging. Eight age groups [1-month-old (M), 1.5 M, 2 M, 3 M, 6 M, 12 M, 18 M, and 24 M] of rats were exposed to a single ARS vs. controls. In addition, rats in six age groups (2, 3, 6, 12, 18, and 24 M) were subjected to CVMS. The FOSB/ΔFOSB immunoreactivity (IR) was a function of age in both controls, ARS- and CVMS-exposed rats. ARS increased the FOSB/ΔFOSB in all nuclei (except in BLA), but only BNSTfu, BNSTv, and PVN reacted throughout the examined lifespan. The CVMS did not increase the FOSB/ΔFOSB in BLA, BNSTov, BNSTdl, and S1. PVN showed a constantly maintained FOSB/ΔFOSB IR during the examined life period. The maximum stress-evoked FOSB/ΔFOSB signal was detected at 2–3 M periods in the ARS- and at 6 M, 18 M in CVMS- model. Corresponding to our previous observations on FOS, the FOSB/ΔFOSB response to stress decreased with age in most of the examined nuclei. Only the PVN exerted a sustained age-independent FOSB/ΔFOSB, which may reflect the long-lasting adaptation response and plasticity of neurons that maintain the hypothalamus-pituitary-adrenal axis response throughout the lifespan.

Young Healthy Men and Women Have Similar Arterial Stiffness at Rest and Post‐Acute Inflammation

The cardioprotective effects of estrogen on arterial function and inflammation are well‐documented; however, it is unclear whether arterial stiffness and compliance vary between healthy premenopausal women and age‐matched men following an acute inflammation. The objective of the current study was to evaluate central and local arterial stiffness and compliance at rest and in response to an acute inflammatory stimulus, specifically, when estrogen levels are thought to be similar between young healthy women and men. It was hypothesized that sustained effects of estrogen throughout the lower‐hormone phase of a natural menstrual or oral contraceptive pill cycle would induce lower central and local stiffness and higher compliance in women as compared to men at rest and in response to an acute inflammatory stimulus. Concentrations of interleukin‐6 (IL‐6) and assessments of central and local arterial stiffness and compliance were obtained from 29 young healthy adults (21±3 y; female, n=15) at baseline (BL), and 24‐ (24H) and 48‐hours (48H) following administration of a seasonal influenza vaccine. To capture the lower‐hormone phases of female participants, women were assessed within the early follicular phase if naturally menstruating or the placebo pill phase if using oral contraceptives. Central stiffness was assessed via carotid‐to‐femoral pulse wave velocity (cfPWV) and local stiffness was measured using ultrasonography at the carotid artery to obtain β‐stiffness, elastic modulus (Ep) and arterial compliance (AC). IL‐6 concentrations were expressed as change‐values from BL and were compared between sexes and timepoints (24H‐BL vs. 48H‐BL). Indices of central and local stiffness and AC were compared between sexes and individual timepoints (BL vs. 24H vs. 48H). Men and women exhibited similar changes in concentrations of IL‐6 (effect of sex: P=0.60), with significant elevations at 24H‐BL as compared to 48H‐BL (effect of time: P=0.0002), indicating that the influenza vaccine successfully induced an acute inflammatory response in both groups. However, there were no effects of sex or time on cfPWV, β‐stiffness or Ep (P&gt;0.05 for all). While men displayed greater AC as compared to women at BL (1.09±0.25 vs. 0.88±0.17 mm2/kPa, P=0.01), there were no sex‐differences at 24H and 48H (P=0.10 and 0.70, respectfully), suggesting that the women were able to compensate for their lower resting AC values when exposed to an acute inflammatory stimulus. In conclusion, when estrogen levels are similar between young healthy women and men, there are no sex‐differences in central or local arterial stiffness at rest nor in response to an acute inflammatory stimulus.

A Potential Link Between Negative Affective Responsivity to Daily Stress and Blood Pressure Reactivity to Acute Stress in College‐Aged Adults

Daily stress, defined as the routine challenges of day‐to‐day living and the unexpected small hassles that disrupt everyday life, is predictive of future cardiovascular disease (CVD)‐related health outcomes; however, the underlying mechanisms remain unclear. Given that heightened blood pressure (BP) reactivity to stress is also purported to increase future CVD risk, we examined a potential link between daily stress and acute stress‐induced increases in BP. We hypothesized that increased frequency of daily stressor exposures and greater negative emotional responsiveness to daily stress (i.e., negative affective responsivity) would be positively related to increased BP reactivity to acute stressors in the laboratory setting. Thirty adults (23±5 yrs; 21 female) completed the Daily Inventory of Stressful Events (DISE) interview every day for 8 consecutive days to quantify objective (e.g., frequency) and subjective appraisal characteristics (e.g., emotions) of naturally occurring daily stressors (e.g., stressful event at home/work). On DISE Day 8, mean arterial pressure (MAP; finger photoplethysmography) was measured during two distinct laboratory‐based acute stress stimuli: the cold pressor test (CPT; generalized sympathoexcitatory stimulus) and the Stroop Color Word Test (SCWT; cognitive stress stimulus). Daily stress exposure was calculated as the total number of stressors across the 8‐day protocol, and negative affective responsivity was calculated as the magnitude of the increase in negative affect on stressor exposure days (at least 1 stressor reported) compared to negative affect on non‐stressor exposure days (no stressors reported). BP reactivity was quantified as the peak acute stress‐induced increase in MAP to the CPT and SCWT. Participants experienced a total of 3±3 stressors (range: 0‐9) over the 8‐day interview timeframe. Negative affect was greater on stressor exposure days (0.68±0.50 a.u.) compared to non‐exposure days (0.37±0.36 a.u.; p&lt;0.01). Both the CPT (∆21±7 mmHg) and the SCWT (∆9±6 mmHg) elicited increases in MAP (both p&lt;0.05). The total number of stressors was not related to the peak increase in MAP during either the CPT (slope=‐0.10; r=0.04; p=0.85) or the SCWT (slope=‐0.43; r=19; p=0.31). Negative affective responsivity was positively related to the peak increase in MAP during the CPT (slope=12.41; r=0.45; p=0.03) but not the SCWT (slope=3.00; r=0.14, p=0.50). These preliminary data demonstrate that daily stressor exposure by itself is not related to BP reactivity to laboratory stressors in college‐aged adults. However, greater negative affective responsivity is associated with greater acute stress‐induced increases in BP during the CPT, but not the SCWT, perhaps suggesting that the link between daily stress processes and BP reactivity may be dependent on the stress‐responsive neurocircuitry being activated.

Characterization of skeletal muscle wasting pathways in diaphragm and limb muscles of cystic fibrosis mice.

Patients with cystic fibrosis (CF) often suffer from skeletal muscle atrophy, most often attributed to physical inactivity and nutritional factors. CF is also characterized by abnormally elevated systemic inflammation. However, it is unknown whether the lack of a functional CF transmembrane conductance regulator (CFTR) gene predisposes to exaggerated inflammation-induced muscle proteolysis. CF mice (CFTR-/-) and their wild-type (WT = CFTR+/+) littermate controls were systemically injected with Pseudomonas-derived lipopolysaccharide (LPS). After 24 h, the diaphragm and limb muscles (fast-twitch tibialis anterior, and slow-twitch soleus) were assessed for induction of inflammatory cytokines (TNFα, IL1β, and IL6), oxidative stress, canonical muscle proteolysis pathways (Calpain, Ubiquitin-Proteasome, Autophagy), muscle fiber histology, and diaphragm contractile function. At baseline, CF and WT muscles did not differ with respect to indices of inflammation, proteolysis, or contractile function. After LPS exposure, there was significantly greater induction of all proteolysis pathways (calpain activity; ubiquitin-proteasome: MuRF1 and Atrogin1; autophagy: LC3B, Gabarapl-1, and BNIP3) in CF mice for the diaphragm and tibialis anterior, but not the soleus. Proteolysis pathway upregulation and correlations with inflammatory cytokine induction were most prominent in the tibialis anterior. Diaphragm force normalized to muscle cross-sectional area was reduced by LPS to an equivalent degree in CF and WT mice. CF skeletal muscles containing a high proportion of fast-twitch fibers (diaphragm, tibialis anterior) exhibit abnormally exaggerated upregulation of multiple muscle wasting pathways after exposure to an acute inflammatory stimulus, but not under basal conditions.

Specificity of California mouse pup vocalizations in response to olfactory stimuli.

To investigate flexibility in vocal signaling by rodent pups, we examined whether olfactory stimuli influence characteristics of pup calls and how these calls may be affected by sex and litter size in California mice (Peromyscus californicus). Pups were isolated and recorded during a 3-min baseline period followed by a 5-min exposure to bedding containing scent from their home cage, scent from the home cage of an unfamiliar family, coyote urine, or no scent (control). Latency to call, call rate, and call characteristics (duration, frequency, and amplitude) were compared between the baseline and scent-exposure periods and among olfactory conditions. Compared with the control condition, pups from two-pup litters called more quietly when exposed to odor from a predator, whereas pups from three-pup litters called more loudly. Additionally, pups showed nonsignificant tendencies to reduce call rates in response to odors from their home cage and to increase call rates when exposed to predator urine. Last, males produced higher-frequency calls and more ultrasonic vocalizations than females. These results indicate that pup calling behavior in this species can be influenced by acute olfactory stimuli as well as litter size and sex. The flexibility of pup calling in response to these three variables potentially increases the communication value of pup calls and helps shape the parents' responses.

Contextual control of conditioned pain tolerance and endogenous analgesic systems.

The mechanisms underlying the transition from acute to chronic pain are unclear but may involve the persistence or strengthening of pain memories acquired in part through associative learning. Contextual cues, which comprise the environment in which events occur, were recently described as a critical regulator of pain memory; both male rodents and humans exhibit increased pain sensitivity in environments recently associated with a single painful experience. It is unknown, however, how repeated exposure to an acute painful unconditioned stimulus in a distinct context modifies pain sensitivity or the expectation of pain in that environment. To answer this question, we conditioned mice to associate distinct contexts with either repeated administration of a mild visceral pain stimulus (intraperitoneal injection of acetic acid) or vehicle injection over the course of 3 days. On the final day of experiments, animals received either an acid injection or vehicle injection prior to being placed into both contexts. In this way, contextual control of pain sensitivity and pain expectation could be tested respectively. When re-exposed to the noxious stimulus in a familiar environment, both male and female mice exhibited context-dependent conditioned analgesia, a phenomenon mediated by endogenous opioid signaling. However, when expecting the presentation of a painful stimulus in a given context, males exhibited conditioned hypersensitivity whereas females exhibited endogenous opioid-mediated conditioned analgesia. These results are evidence that pain perception and engagement of endogenous opioid systems can be modified through their psychological association with environmental cues. Successful determination of the brain circuits involved in this sexually dimorphic anticipatory response may allow for the manipulation of pain memories, which may contribute to the development of chronic pain states.

Altered Pain Processing Associated with Administration of Dopamine Agonist and Antagonist in Healthy Volunteers.

Striatal dopamine dysfunction is associated with the altered top-down modulation of pain processing. The dopamine D2-like receptor family is a potential substrate for such effects due to its primary expression in the striatum, but evidence for this is currently lacking. Here, we investigated the effect of pharmacologically manipulating striatal dopamine D2 receptor activity on the anticipation and perception of acute pain stimuli in humans. Participants received visual cues that induced either certain or uncertain anticipation of two pain intensity levels delivered via a CO2 laser. Rating of the pain intensity and unpleasantness was recorded. Brain activity was recorded with EEG and analysed via source localisation to investigate neural activity during the anticipation and receipt of pain. Participants completed the experiment under three conditions, control (Sodium Chloride), D2 receptor agonist (Cabergoline), and D2 receptor antagonist (Amisulpride), in a repeated-measures, triple-crossover, double-blind study. The antagonist reduced an individuals’ ability to distinguish between low and high pain following uncertain anticipation. The EEG source localisation showed that the agonist and antagonist reduced neural activations in specific brain regions associated with the sensory integration of salient stimuli during the anticipation and receipt of pain. During anticipation, the agonist reduced activity in the right mid-temporal region and the right angular gyrus, whilst the antagonist reduced activity within the right postcentral, right mid-temporal, and right inferior parietal regions. In comparison to control, the antagonist reduced activity within the insula during the receipt of pain, a key structure involved in the integration of the sensory and affective aspects of pain. Pain sensitivity and unpleasantness were not changed by D2R modulation. Our results support the notion that D2 receptor neurotransmission has a role in the top-down modulation of pain.

Tracking the Temporal Footprint Effect of Thermonociception and Denervation on the Brain's Pain Matrix: fMRI and BOLD Study in Rats.

ObjectivePain constitutes an essential alarm for preserving the organism’s integrity. Damage to the nervous system produces a pathological condition known as neuropathic pain.PurposeBlood oxygenation level-dependent (BOLD) and functional magnetic resonance imaging (fMRI) have been widely used to map neuroanatomy and the active regions of interest (ROI) of nociceptive processing. Our study explored the brain’s BOLD response in rats after thermal noxious stimulation, immediately after sciatic nerve damage and during 75 minutes after surgical lesion of the sciatic nerve.MethodsNine male Wistar rats were tested; the experiments were performed on a 7-Tesla /21-cm Varian Agilent system. This approach allowed, for the first time, to measure in vivo the BOLD changes in brain regions involved with the pain process: cingulated (ACC), somatosensory (S1), and insular cortices (IC), as well as thalamus (Th) and ventral tegmental area (VTA) related with acute thermal pain and during the early stages of sciatic denervation that produce neuropathic pain.ResultsDuring thermonociception scan, all subjects showed BOLD activation in the ROIs determined as ACC, S1, Th, IC and VTA. After denervation, these regions continued to show activation with a slow decrement in intensity for the duration of the experiment. The results suggest that these brain structures are overactive during the genesis of neuropathic pain.ConclusionThe study shows for the first time continuous activation of the pain matrix following an acute thermal nociceptive stimulus followed by neuropathic damage. These results have given insight into the early stages of the development of neuropathic pain in vivo.