bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS‐induced systemic inflammation impairs phrenic long‐term facilitation via okadaic acid‐sensitive protein phosphatase activity
Authors: Tadjalli Arash , Perim Raphael , Satriotomo Irawan , Santiago‐Moreno Juan , Seven Yasin , Mitchell Gordon S.
Published: November 2023

Moderate acute intermittent hypoxia (mAIH; 3, 5‐min hypoxic episodes, 5‐min normoxic intervals) elicits a form of respiratory motor plasticity known as phrenic long‐term facilitation (pLTF). Although mild systemic inflammation elicited by a low dose of lipopolysaccharide (LPS; 100 ug/kg ip) undermines the capacity for mAIH‐induced pLTF 24 hours post‐injection (Huxtable et al., 2013), cellular mechanisms of this impairment are unknown. We previously demonstrated that inflammation undermines pLTF by a p38 MAP kinase dependent mechanism. In our working model, p38 MAP kinase activates serine/threonine protein phosphatases that dephosphorylate and inactivate kinases critical for mAIH‐induced pLTF expression, particularly the ERK MAP kinases. Here, we tested the hypothesis that LPS impairs pLTF by a mechanism that requires spinal okadaic acid‐sensitive serine/threonine protein phosphatase activity (eg. PP1 and/or PP2A). In anesthetized, paralyzed and ventilated adult male Sprague Dawley rats, we confirmed that mAIH‐induced pLTF is abolished 24 hours post LPS (LPS: 1 ± 4% baseline at 90‐min post hypoxia; vs. vehicle: 49 ± 12%; p = 0.003). We extend prior reports with the observation that other forms of mAIH‐induced respiratory motor plasticity are also impaired; specifically, hypoglossal LTF was also abolished 24 hours post‐LPS (LPS: −12 ± 5% baseline 90‐min post‐AIH; vs. vehicle controls: 30 ± 11% baseline; p = 0.02). In support of our hypothesis, spinal intrathecal (C3) pre‐treatment with okadaic acid, a PP1/2A inhibitor (25 nM, 30‐min before mAIH) restored pLTF in LPS treated rats (33 ± 8% baseline; p = 0.01). Thus, okadaic acid‐sensitive protein phosphatases constrain pLTF expression with systemic inflammation. As expected, hypoglossal LTF was not restored since okadaic acid distribution was limited to the spinal cord (1 ± 5% baseline; p = 0.79). Preliminary data from separate rats demonstrate that: 1) LPS increased phosphorylated p38 MAP kinase expression within phrenic and hypoglossal motor neurons (immunofluorescence) consistent with a role in LPS impairment of phrenic and hypoglossal LTF; and 2) diminished ERK MAP kinase phosphorylation/activation following mAIH in phrenic motor neurons. Thus, a likely target for the relevant okadaic acid‐sensitive phosphatase is ERK, a key enzyme in the cellular mechanism of mAIH‐induced pLTF. Since moderate AIH has demonstrated therapeutic potential to restore lost respiratory and non‐respiratory motor function in neuromuscular disorders that compromise movement, and the high prevalence of systemic and neuroinflammation in those same disorders, greater understanding of mechanisms whereby inflammation undermines mAIH‐induced motor plasticity may guide combinatorial therapies to enhance the therapeutic potential of this promising new treatment.

Support or Funding Information

NIH HL111598 and the McKnight Brain Institute
The effect of urethane anesthesia on central coordination of cardiorespiratory coupling
Authors: Howard Courtney , Nall Andrea C , Zhu Vivian , Denson Heather B , Baekey David M
Published: November 2023

Rodent models are commonly used to study respiratory and cardiovascular control systems and this research is often confounded by the unknown effects of commonly used anesthetics. A primary example of this challenge is seen in in vivo urethane anesthetized rats which, when surgically anesthetized, demonstrate appropriate respiratory rate, blood pressure, and heart rate. Less apparent is the attenuated coordination of respiratory and cardiovascular rhythms expressed as respiratory modulation of heart rate (respiratory sinus arrhythmia ‐ RSA) and blood pressure (Traube‐Hering waves). In contrast, in situ decerebrate rodent preparations (unanesthetized) have well‐coordinated cardiorespiratory coupling with a robust RSA and respiratory modulation of perfusion pressure. This influence of the respiratory rhythm on cardiovascular parameters can be measured after both removal of the lungs and neuromuscular blockade indicating that sufficient coupling occurs at the neural level to express the coordination. Based on these observations, we hypothesized that urethane anesthesia interferes with brainstem circuitry linking respiratory and cardiovascular control networks.

To test our hypothesis, we performed experiments in decerebrate artificially perfused rodents (n=12) expressing baseline RSA and Traube‐Hering waves while monitoring phrenic nerve activity (PNA), thoracic sympathetic nerve activity (tSNA), ECG, and perfusion pressure. All preparations received increasing concentration of urethane anesthetic (1.5μg/ml: 0.1– 4.7ml/200ml perfusate) added to the perfusion bath and we subsequently characterized its influence on the monitored respiratory and cardiovascular signals. Atropine and hexamethonium were added to the perfusion bath in a subset of experiments (n=3) to confirm parasympathetic and sympathetic modulation was responsible for transmission of the respiratory pattern on the heart and vasculature. Additionally, in an overlapping subset (n=7), a multielectrode array was used to record ensembles of respiratory modulated neurons in the ventrolateral brainstem during application of the aforementioned drugs.

Urethane anesthesia attenuated cardiorespiratory coupling in a dose dependent manner which corresponded with a substantial reduction in the number of active respiratory modulated neurons in the ventrolateral brainstem. In concert with the uncoupling, the overall phrenic amplitude was reduced and showed a loss of its ramp patterned bursting. As respiratory rate remained relatively stable prior and subsequent to complete uncoupling, we conclude that urethane acts on a respiratory population downstream of the respiratory central pattern generator which relays the respiratory rhythm to the autonomic efferents responsible for RSA and Traube Hering waves.

Support or Funding Information

We thank the University of Florida College of Veterinary Medicine and the Veterinary Scholars Program for their generous support.
Distributed Networks of Medullary Tonic Expiratory Neurons Regulate Cough Motor Drive
Authors: Shen Tabitha Y. , Morris Kendall F. , Baekey David M. , Musselwhite M. Nicholas , Segers Lauren S. , Nuding Sarah , Rose Melanie J. , Lindsey Bruce G. , Bolser Donald C.
Published: November 2023

Recently, we showed that ventral medullary tonic E neurons participate in regulatory networks that control respiratory network excitability in response to alterations in chemical drive. The objective of this study was to determine the response patterns during cough of medullary neurons with continuous discharge during the breathing cycle that increased in the expiratory phase (tonic E). We hypothesized that dorsal and ventral medullary tonic E neurons would have activity patterns during coughing consistent with participation in the neurogenesis of this behavior. Further, we speculated that these neurons would participate in functionally‐identified neuronal circuits that regulate the excitability of cranial and spinal motor pathways to respiratory muscles. Using multiple electrode arrays, we recorded from many neurons simultaneously in the regions of the nucleus of the tractus solitarius (dorsal medullary), raphe nuclei, and ventral respiratory column (VRC) in 26 decerebrated, paralyzed, and artificially ventilated cats. A total of 106 tonic E neurons were recorded. During fictive cough, the discharge rates of 42 neurons increased, 21 decreased, and 43 did not change. Cross correlation analysis of simultaneously recorded dorsal and ventral medullary neurons revealed evidence for: a) circuits composed of tonic E neurons that excite one another and provide excitatory drive to expiratory laryngeal premotor and motoneurons, b) evidence for shaping of laryngeal motor discharge by suppression of tonic E neurons by phasic E decrementing neurons, and c) networks of cough‐responsive tonic E and non‐breathing modulated neurons that modulated the activities of recruited cough inspiratory neurons in the dorsal medulla and recruited cough expiratory neurons in the caudal VRC. The results support an important role of dorsal and ventral medullary tonic E neurons in controlling the excitability of cranial and spinal motoneurons that produce cough.

Support or Funding Information

NIH HL 103415 and 1OT20D001983
Sex Differences in Cough Responses of Cats
Authors: Rose Melanie J , Shen Tabitha Y , Musselwhite M. Nicholas , Pitts Teresa G , Bolser Donald C
Published: November 2023

In humans, women are more debilitated by chronic coughing, report to physicians for cough more frequently, and have a greater cough sensitivity than men. These sex differences between men and women begin to emerge at the end of puberty. We conducted a retrospective analysis in anesthetized cats to determine if sex differences could be identified in an animal model that is capable of intense repetitive coughing. Anesthetized cats (13 males, 10 females) were tracheotomized and allowed to breathe spontaneously. Electromyograms of parasternal, transversus abdominis/internal oblique, and laryngeal adductor muscles were recorded. Esophageal pressure (Pes) was recorded via small balloon placed in the midthoracic esophagus. Cough was induced by mechanical stimulation of the intrathoracic trachea with a small polyethylene tube via the trachea cannula. We evaluated the cough response before and after a protocol consisting of 20 consecutive mechanical stimulation trials separated by approximately one minute. This adaptation protocol typically results in a depression of coughing by approximately 50% with the subsequent cough response remaining stable for extended periods of time. There was no difference in the number of trials in the adaptation protocol between males and females (20±1 males, 20±1 females). Likewise, end tidal CO2, as an index of anesthetic level, did not differ between the groups (males 31±0.5, females 32±0.8). Cough frequency (CF‐number of coughs per second) declined significantly in both male and female cats during the adaptation protocol (males control CF 1.3±0.2, post adaptation CF‐0.8±0.1, P<0.001; females control CF‐1.0±0.06, post adaptation CF‐0.5±0.04, P<0.001). Control CF was not significantly different between males and females (P<0.2), but the post adaptation CF was higher in males (P<0.05). Pes declined significantly during the adaptation protocol in both males and females (males control 50±8 cm H2O, post adaptation 26±4 cm H2O, P<0.001; females control 22±3 cm H2O, post adaptation 14±3 cm H2O, P<0.04). Pes was higher in males both in the control period (P<0.05) and post adaptation (P<0.02). However, there was no significant difference between males and females when these changes were expressed as percentages or when normalized to body weight. The results indicate that sex differences in cough responses exist in this model of cough. Differences in cough intensity between sexes can be explained by body weight. CF is a metric of peripheral sensory and/or central excitability and females exhibit greater depression of coughing in a repetitive trial protocol than males. The mechanisms responsible for this sex difference are unknown.

Support or Funding Information

NIH HL 103415 and 1OT20D001983
IL‐1R Activation Undermines Respiratory Plasticity
Authors: Hocker Austin Dean , Huxtable Adrianne G
Published: November 2023

Despite a pervasive role for inflammation in ventilatory control disorders, we are only just beginning to understand the mechanisms by which inflammation undermines respiratory plasticity. Interleukin‐1 (IL‐1) signaling regulates plasticity in other central nervous system regions; promoting central pain in the spinal dorsal horn and inhibiting plasticity in the hippocampus. Thus, we tested the hypothesis that IL‐1 receptor (IL‐1R) activation during systemic inflammation undermines phrenic long‐term facilitation (pLTF), a model of respiratory plasticity induced by acute intermittent hypoxia. pLTF is reduced 24 hours after lipopolysaccharide (LPS; 100 mg/kg; i.p.) compared to control (12 ±8%, n=5; 57 ± 10%, n=6; respectively), similar to previously published studies. Importantly, pLTF is restored by peripheral IL‐1R antagonism (64 ± 6%; n=6; AF‐12198, i.p. 0.5 mg/kg, 24 hours) after LPS. Acute, spinal IL‐1R antagonism (1 mM AF‐12198 in aCSF; 15 mL; intrathecal) also restored pLTF (52 ± 16%; n=2) compared to LPS treated rats (9 ± 8%; n=3). These data suggest both peripheral and spinal IL‐1Rs play important roles in undermining pLTF after systemic inflammation. Understanding the inflammatory mechanisms inhibiting respiratory plasticity is crucial to developing treatment strategies to promote breathing during ventilatory control disorders.

Support or Funding Information

Supported by Francis Families Foundation (AGH) and University of Oregon.
Effects of 5‐HT2 and A2A Antagonists on pERK and pAKT Levels in the NTS and Spinal Cord of Rats Exposed to Chronic Sustained Hypoxia
Authors: Moya Esteban A , Powell Frank L
Published: November 2023

Different patterns of hypoxic exposure can result in long‐term changes of ventilation and the hypoxic ventilatory response. For example, acute intermittent hypoxia (AIH) causes long term facilitation (LTF) of ventilation and phrenic nerve activity, with an increased acute HVR and persistent increase in ventilation in normoxia after AIH. Others have shown that LTF involves plasticity in serotonin 5HT2 neurotransmission (Gq pathway) and A2A adenosine neurotransmission (Gs pathway) in the spinal cord. Chronic sustained hypoxia (CSH) also increases the acute HVR and ventilation remains greater than control when normoxia is restored, which is called ventilatory acclimatization to hypoxia (VAH). We previously showed VAH involves plasticity in the nucleus tractus solitarius (NTS). Here we tested the hypothesis that Gq and Gs signaling play similar roles in LTF and VAH. Systemic inhibition of the Gq pathway (5‐HT2 antagonist Ketanserin, 1 mg/Kg per day) and the Gs pathway (A2A receptor antagonist MSX‐3, 0.5 mg/Kg per day) administered with subcutaneous osmotic pumps during 7 days of CSH (inspired PO2 = 70 mm Hg) decreased but did not eliminate VAH in male adult rats. This is different than previous reports from other laboratories showing LTF is abolished by blocking the Gq pathway with ketanserin but increased by blocking the Gs pathway with MSX‐3 administered before exposure to moderate AIH. To understand the site of action of Gq and Gs effects on VAH, we collected tissue from (1) the NTS, and (2) the spinal cord (between C3 and C5). We used Western blots of pERK and pAKT to measure Gq and Gs activation, respectively, in 4 groups of rats (n≥6 each) administered drugs or vehicle (DMSO) for 7 days as previously described above for ventilation studies: CON = DMSO in normoxia, CSH = DMSO in hypoxia, KET = ketanserin in CSH, MSX = MSX‐3 in CSH. CSH decreased pERK and pAKT in the spinal cord but had no effect on them in the NTS, indicating Gq and Gs pathways respond differently to CSH in the NTS versus spinal cord. In the NTS, pERK decreased with KET but not MSX during CSH. In the spinal cord, MSX during CSH restored pERK and pAKT to normoxic CON levels. Effects of KET on pERK and pAKT in the spinal cord during CSH are not available yet. The data indicate the Gq and Gs signaling with CSH is different than previously reported for AIH in the spinal cord. Also, Gq signaling in the NTS may contribute to VAH. Further study is necessary to determine how such differences in Gq and Gs signaling in the NTS and spinal cord depend on specific neurons in these locations.

Support or Funding Information

NIH RO1 HL‐081823
Hypoxic ventilatory response of developing rats exposed to chronic hypoxia at various ages
Authors: Song Monata , Benevides Ethan S. , Bavis Ryan W.
Published: November 2023

The effects of chronic hypoxia on the hypoxic ventilatory response (HVR) reportedly differs in newborn versus adult rats: chronic hypoxia blunts the HVR in newborn rats but may enhance it in adult rats. To investigate the age at which this transition occurs, we exposed rats to 12% O2 for approximately 7 days beginning at birth (P0), P7, P14, or P21 and measured ventilation by head‐body plethysmography (P7 and P14) or whole‐body plethysmography (P21‐22 and P28‐29). Normoxic ventilation was greater after chronic hypoxia at all ages. Likewise, when expressed as a percentage of baseline ventilation, the HVR was significantly reduced by chronic hypoxia regardless of the age at which chronic hypoxia began. In the youngest groups (i.e., P7 and P14 when measured), we confirmed that the blunted HVR was at least partially explained by diminished carotid body responses to hypoxia (assessed from in vitro single‐unit chemoafferent activity). When initiated at birth, chronic hypoxia appears to delay the postnatal transition from a biphasic HVR to the sustained HVR characteristic of mature animals; in contrast, this effect was not observed when hypoxia began at P7 (i.e., after the HVR was nearly mature). We conclude that chronic hypoxia blunts the HVR across a wide age range in developing rats, but hypoxia may have age‐dependent effects on maturation of the HVR.

Support or Funding Information

Supported by NIH grant P20 GM‐103423 (Maine INBRE).
Does reduced carotid body BDNF contribute to developmental hyperoxia‐induced respiratory plasticity?
Authors: Muscato Gwen M. , Kim Maria S. , McDonough Amy B. , Bavis Ryan W.
Published: November 2023

Chronic exposure to moderate hyperoxia (60% O2) reduces carotid body expression of brain‐derived neurotrophic factor (BDNF) in neonatal rats. Reduced BDNF expression correlates with abnormal carotid body development and blunted hypoxic ventilatory responses, but a causal relationship has not been established. First, we used ELISA to confirm that both sustained and intermittent exposures to 60% O2 reduced carotid body BDNF levels; intermittent hypercapnic hypoxia had the opposite effect. Next, we attempted to block the effects of developmental hyperoxia by administering LM22A4 (50 mg kg−1, i.p., daily), an agonist for BDNF's TrkB receptor. After 7 days of treatment with 60% O2, carotid body volume and the hypoxic ventilatory response were significantly reduced in P7 rats that received either LM22A4 or vehicle (saline) from birth; the drug treatment did not attenuate this effect. Similar results were obtained using an alternate TrkB agonist, 7,8‐dihydroxyflavone (5 mg kg−1, i.p., daily). Although carotid body BDNF expression appears to be regulated by environmental O2, our data do not support the hypothesis that reduced BDNF is directly responsible for hyperoxia‐induced developmental plasticity in respiratory control.

Support or Funding Information

Supported by NIH grant R15 HL‐114001.
Prenatal Nicotinic Exposure (PNE) Upregulates Pulmonary 5‐HT and Prolongs Bronchopulmonary C‐fiber (PCF)‐mediated Apnea via Acting on PCF 5‐HT3 in Rat Pups
Authors: Zhao Lei , Zhuang Jianguo , Gao Xiuping , Xu Fadi
Published: November 2023

Our previous studies have shown a prolonged PCF‐mediated apnea by PNE in rat pups (J. Zhuang et al., Am J Physiol Lung Cell Mol Physiol. 2015), but the relevant mechanisms are not fully elucidated. It is already known that PCFs heavily express 5‐HT3 and its activation induces apneic response (B. J. Undem et al., J physiol. 2012). Thus, we asked if PNE would upregulate 5‐HT in BALF associated with 5‐HT3 upregulation in vagal pulmonary C‐neurons and if so, if these changes are involved in the PNE‐induced prolongation of the PCF‐mediated apnea. In P11– 14 rat pups, we compared: 1) 5‐HT content in BALF; 2) 5‐HT3 protein expression in the nodose ganglion and mRNA level in vagal pulmonary C‐neurons by using single cell RT‐PCR; and 3) the apneic response to right atrial bolus injection of phenylbiguanide (PBG, a selective 5‐HT3 agonist, 10 μg/kg) in Ctrl and PNE pups. Our results showed that PNE obviously increased 5‐HT in BALF and 5‐HT3 mRNA level in pulmonary C neurons (retrogradely labeled). Importantly, the apneic duration in response to PBG was significantly longer in PNE than Ctrl pups. We further tested the dependency of the 5‐HT3‐mediated apnea on PCFs and found that the apneic responses to PBG in both Ctrl and PNE pups were blocked by inactivating PCFs with perineural capsaicin treatment (500 μg/ml, 10 min). Our results suggest that PNE is capable of upregulating pulmonary 5‐HT that prolongs the PCF‐mediated apnea via acting on PCF 5‐HT3.

Support or Funding Information

Supported by HL‐107462 and HL‐119683
Unintended Consequences: Renal Denervation Attenuates Hypertension Development but Impairs Glucose Metabolism in Female Fawn‐Hooded Hypertensive Rats
Authors: Banek Christopher T , Gauthier Madeline , Van Helden Dusty A , Asirvatham‐Jeyaraj Ninitha , Osborn John W
Published: November 2023

For both men and women, hypertension (HTN) is the highest risk factor for cardiovascular disease and stroke – the two leading causes of morbidity and mortality throughout the world. Though HTN etiology remains debated, altered sympathetic nervous activity, particularly to the kidney (RSNA), is regarded as a primary contributor to the development and maintenance of HTN. Ablation of renal sympathetic nerves (RDNx) reportedly prevents and/or reverses several forms of experimental hypertension. Several off‐target improvements are also reported, such as glucose metabolism. However, an overwhelming majority of these experimental models and mechanisms focus only on responses in male subjects. Moreover, the sex differences in sympathetic control of blood pressure in hypertension remain poorly defined. In this study, we assessed the role of RDNx to prevent hypertension development and metabolic dysfunction in the Fawn‐Hooded Hypertensive (FHH) rat model. We hypothesized that RDNx would attenuate the development of HTN, and improve glucose handling in female and male rats. FHH rats (10 weeks old) underwent sham or bilateral RDNx surgeries (n=5/group). For the following six weeks, 24‐hour mean arterial pressure (MAP) was monitored by radiotelemetry. Glucose handling was assessed biweekly by glucose tolerance test (GTT). Cardiovascular data was averaged weekly, and analyzed by two‐way ANOVA with a Bonferroni post‐hoc test (α=.05). Data presented as mean ± SEM. Six weeks post‐treatment, RDNx attenuated the HTN in the females (p<.05) vs. sham controls (107±5 vs. 118±3). Interestingly, no difference in MAP between RDNx and sham males was observed (129±2 vs. 125±1mmHg). Regarding glucose metabolism, baseline fasting blood glucose (FBG) and glucose tolerance (GT) were similar between males and females prior to RDNx. After six weeks, FBG in female sham FHH (100.9±2.3mg/dL) was higher (p<.05) than male shams (89.2±2.7mg/dL). Likewise, GT was impaired in female rats compared to male shams. In contrast to our hypothesis, RDNx worsened the FBG (110±2.6mg/dL) and GT in female vs. female shams. No effect of time or RDNx on glucose handling was observed males. In summary, RDNx attenuated HTN development in female FHH rats; however, glucose metabolism was impaired by RDNx in females but not in males. These results suggest that sex may need to be considered in the use of RDNx to treat HTN. These findings are clinically important in that RDNx responses may be partially dependent on sex, and may result in negative outcomes such as impaired glucose metabolism in females. Further examination of sex differences in the responses to RDNx is underway.

Support or Funding Information

NIH 5R01HL116476; NIH 2T32HL7741
Is there a relationship between resting sympathetic nerve activity and leg vascular conductance in aerobically‐trained older adults?
Authors: O'Brien Myles W. , Ramsay Diane J. , Mekary Said , Dogra Shilpa , O'Neill Carley , Kimmerly Derek S.
Published: November 2023

Inactivity is associated with elevated resting muscle sympathetic nerve activity (MSNA) that directly relates to corresponding reductions in leg vascular conductance (LVC) as one ages. Aerobic training further increases resting MSNA but also augments nitric oxide (NO) bioavailability assessed via the flow‐mediated dilation (FMD) response. How these competing vascular mechanisms contribute to sympathetic‐transduction in older active adults is unclear. We tested the hypothesis that parallel increases in aerobic fitness, lower limb MSNA and FMD would negate the inverse relationship between MSNA and LVC in older active adults. We measured fibular MSNA (microneurography), popliteal artery blood flow (PBF) and FMD (Doppler ultrasound) in 16 (10♂, 25.8 ± 3.1 kg•m−2) older (64 ± 5 years), aerobically‐trained adults (152 ± 37% age‐predicted VO2peak). LVC was calculated as PBF/mean arterial pressure. Resting MSNA burst frequency and relative popliteal artery (PA)‐FMD were 31 ± 15 burst•min−1 and 9.2 ± 3.1%, respectively. Aerobic fitness correlated positively with both MSNA (r=0.56; p=0.03) and relative PA‐FMD (r=0.87; p=0.01). MSNA correlated with PA‐FMD (r=0.60; p=0.02) but not with resting LVC (r=0.02; p=0.94). Similarly, PA‐FMD did not relate to resting LVC (r=−0.01; p=0.96). The main findings include: 1) resting MSNA and PA‐FMD were significantly associated with aerobic fitness in active older adults, and 2) unlike their sedentary peers, LVC was not inversely related to resting MSNA in aerobically‐trained older adults. This latter finding may be due to reduced transduction of the sympathetic vasoconstrictor signal mediated, in part, by a corresponding increase in basal NO bioavailability

Support or Funding Information

Support provided by: Canadian Foundation for Innovation: Leaders Opportunity Fund, Faculty of Health Professions Research Development, and Nova Scotia Health Research Foundation Development/Innovation grants.
Effect of Sex on Muscle Sympathetic Nerve Responses to Static and Dynamic Exercise
Authors: Notay Karambir , Doherty Connor J. , Incognito Anthony V. , Burns Matthew J. , Nardone Massimo , Seed Jeremy D. , Millar Philip J.
Published: November 2023

Previous studies investigating sex differences in sympathetic outflow during exercise have produced conflicting results, demonstrating either similar or attenuated increases in muscle sympathetic nerve activity (MSNA) during static handgrip in women. Further, to our knowledge, no studies have investigated whether MSNA responses to low‐to‐moderate intensity dynamic leg exercise, a stimulus which decreases sympathetic outflow, differs between men and women. Therefore, the purpose of the present study was to examine the effect of sex on MSNA responses to static handgrip and dynamic one‐legged cycling. Heart rate (HR), mean arterial pressure (MAP), and MSNA (left fibular nerve; microneurography) were measured continuously in 9 men (22±3 yrs) and 9 women (21±3 yrs) at rest and during 2 minutes of static handgrip (30% maximal voluntary contraction), unloaded one‐legged cycling, and moderate intensity one‐legged cycling (30% of peak power output) in the recumbent position. All cycling was performed with the right leg using a modified arm ergometer. Static and dynamic exercise were separated by 10 minutes of rest. All variables were assessed as the change (Δ) from baseline to the second minute of exercise. Resting HR and MAP were similar between men and women (Both p>0.05) whereas MSNA burst frequency was higher in men (28±6 vs. 23±4; p=0.03). During static handgrip, the increases in HR (Δ11±8 vs. 9±7 bpm; p=0.55), MAP (Δ21±7 vs. 16±4 mmHg; p=0.10), MSNA burst frequency (Δ4±5 vs. 8±9 bursts/min, p=0.26), and total MSNA (Δ11±12 vs. 4±5 a.u./min; p=0.18) were similar between men and women. During one‐legged cycling, changes in HR (unloaded: Δ10±7 vs. 8±5 bpm, p=0.49; loaded: Δ23±6 vs. 23±5 bpm, p=0.94), MAP (unloaded: Δ8±4 vs. 6±5 mmHg, p=0.45; loaded: Δ12±6 vs. 11±4 mmHg, p=0.86), MSNA burst frequency (unloaded: Δ−5±4 vs. −3±4 bursts/min, p=0.39; loaded: Δ−3±8 vs. −1±6 bursts/min, p=0.70), and total MSNA (unloaded: Δ0±3 vs. −3±3 a.u./min, p=0.07; loaded: Δ1±4 vs. 0±4 a.u./min, p=0.36) were comparable between men and women. In summary, these results suggest that sex does not impact muscle sympathetic or blood pressure responses to moderate intensity static handgrip or dynamic one‐legged cycling.

Support or Funding Information

This research was supported by a Natural Science and Engineering Research Council (NSERC) of Canada Discovery Grant (P.J.M), the Canada Foundation for Innovation (P.J.M.), and a Canadian Institutes of Health Research Fredrick Banting and Charles Best Canada Graduate Scholarship (A.V.I.).
A loss of central serotonin in middle‐aged rats leads to increased blood pressure solely in males
Authors: Magnusson Jennifer , Li Aihua , Hodges Matthew , Cummings Kevin
Published: November 2023

Serotonin (5‐Hydroxytryptamine, 5‐HT) neurons within the midline raphe project widely to autonomic regions in the brainstem, but the specific role of 5‐HT in arterial blood pressure (BP) regulation is unresolved. In addition, while the role of sex steroids in BP regulation has been extensively characterized, the extent to which they interact with 5‐HT signaling has not been investigated. Furthermore, 5‐HT neurons have a relatively high firing rate during wakefulness (QW) and non‐rapid eye movement sleep (NREM), but are virtually quiescent during rapid eye movement sleep (REM). Thus, 5‐HT's role in BP regulation may also be sleep‐state dependent. Therefore, this study seeks to clarify the specific role of central 5‐HT on BP regulation during sleep in young (3–4 months) and middle‐aged (12–13 months), male and female rats across vigilance states. We compared BP in wild‐type (WT; young: n=9 males, 9 females; middle aged: n=8 males, 7 females) and rats deficient in tryptophan hydroxylase 2 (TPH2−/−; young: n=8 males, 6 females; middle aged: n=5 males, 4 females), that lack central 5‐HT. Based on data from female adult TPH2−/− mice, we hypothesized that TPH2−/− rats would have lower BP than WT, and that the effects of 5‐HT deficiency on BP regulation would be most obvious in wakefulness and NREM sleep, when these neurons retain activity. Electroencephalography and electromyography were used to assess sleep state. Femoral arterial catheters were used to measure BP and heart rate. We also performed power spectral analysis of systolic BP and calculated the ratio of low to high frequency components (LF/HF) as a measure of sympathovagal balance to the vasculature. A loss of central 5‐HT had no effect on BP in young rats. However, middle‐aged male TPH2−/− rats had a significantly higher BP (128, 126, and 132 mmHg in wakefulness, NREM and REM, respectively) than male WT littermates (116, 117 and 122 mmHg) (p=0.015). In contrast, the BP of middle‐aged female TPH2−/− rats (102, 100, 105 mmHg in wakefulness, NREM and REM, respectively) was the same as female WT littermates (104, 105, 107 mmHg). The higher BP of middle‐aged male TPH2−/− rats was due to an increased diastolic BP (p=0.003). In addition, middle‐aged male TPH2−/− rats had an increased LF/HF ratio compared to WT controls (p<0.05). Overall, our data suggests that a loss of central 5‐HT leads to increased BP in male rats, possibly due to increased sympathetic tone to the vasculature. These findings also suggest that female sex steroids serve to protect against the deleterious effects of central 5‐HT deficiency on BP regulation.

Support or Funding Information

Funding for this research was provided by an American Heart Association Scientist Development Grant (14SDG18560022; PI: KJC), and a University of Missouri, College of Veterinary Medicine Faculty Research Grant.
Acute Inhibition of HDACs after MI Leads to Low Inflammatory Profile: Implications for Post‐MI Angiogenesis
Authors: Veeranki Sudhakar , Tyagi Suresh C
Published: November 2023

Introduction

Although the histone deacetylase inhibitors (HDACi) confer protection after myocardial injury (MI), the changes in the profiles of the angiogenic regulators and neoangiogenesis are unknown.

Hypothesis

We hypothesized that pan‐HDACi treatment confers positive angiogenic responses after MI, thereby improving the myocardial repair process.

Methods

All the mice underwent permanent LAD ligation. The mice were treated with PXD101, a HDACi, for a period of 3 days at a dose of 100 mg/kg, I.P., daily after the creation of MI. Mice were sacrificed after 72 hrs. of MI and the ischemic and non‐ischemic portions of heart tissue were collected. Relative levels of 53 angiogenesis‐related proteins were quantified by using angiogenic arrays. Heart function was evaluated before and after 72 hrs. of MI injury. Markers of inflammatory cells (macrophages and neutrophils) and endothelial progenitor cells (EPCs) were assayed in the ischemic tissues.

Results

PXD101 treatment did not ameliorate heart function after the 72 hrs. of treatment. There were specific changes in the levels of inflammatory markers such as CXCL1 and MCP‐1 after the treatment. Further assaying of various markers revealed that less inflammatory cell presence with moderate changes in the markers of EPCs in the ischemic tissues after the treatment. Deacetylation of non‐histone proteins were implicated in suppression of the inflammatory cell chemoattractants.

Conclusions

Our study underscored the role of HDACi in regulation of myocardial angiogenic responses after MI injury. Acute inhibition of HDACs led to lower inflammatory profile. This study has pinpointed underlying epigenetic changes responsible for the neoangiogenesis and tissue regeneration after MI.

Support or Funding Information

NIH grants: HL108621, HL074185 and DK104653.
Heterogeneous Stock Rats: A Model To Study The Genetics Of Despair And Fluoxetine Resistance in Adolescence
Authors: Woods Leah C Solberg , Holl Katie , He Hong , Wedemeyer Michael , Clopton Larissa , Wert Stephanie , Meckes Jeanie , Kastner Abigail , Palmer Abraham , Redei Eva
Published: November 2023

Major depressive disorder is a complex illness caused by both genetics and the environment. Antidepressant resistance also has a genetic component. To date, very few genetic variations have been identified for major depression or antidepressant resistance. In the current study, we investigated whether outbred heterogeneous stock (HS) rats would be a suitable model to uncover the genetics of depression and its connection to antidepressant resistance. The WKY rat, one of the founders of the HS colony, is a known animal model of depression, in both adults and adolescents, and has been found to be resistant to fluoxetine anti‐depressant treatment. We therefore hypothesized that adolescent HS rats would exhibit variation in both despair behavior and response to fluoxetine treatment. We assessed despair behavior using a modified Forced Swim Test (FST) in four‐week old male and female HS rats. A separate group of rats were given sub‐acute treatment with either saline or fluoxetine prior to the FST and immobility, climbing and swimming behaviors were scored. Finally, we tested whether or not blood transcript levels previously identified to be differentially expressed in depressed human and rat models correlate with despair behavior in the HS rats. We demonstrate that 4‐week old HS rats have large variation in despair behavior in the FST and that most HS rats are resistant to treatment with fluoxetine. We also demonstrate that four genes previously identified as differentially expressed in the blood of depressed rats and humans are also negatively correlated with despair behavior in the HS rats. Taken together, these data demonstrate that the HS rat is a suitable model for investigating the underlying genetic basis of despair behavior as well as resistance to fluoxetine and that these studies will likely translate to depression in humans.

Support or Funding Information

P50 DA037844, Davee Foundation
Metabolomic Analysis of Microdialysis Samples from the Frontal Association Cortex (FrA) of C57BL/6J (B6) Mouse during Isoflurane Anesthesia
Authors: Baer Aaron , Bourdon Allen K , Spano Giovanna , Bellesi Michele , Bradley Abram , Tononi Giulio , Cirelli Chiara , Serra Pier Andrea , Baghdoyan Helen A. , Campagna Shawn R. , Lydic Ralph
Published: November 2023

Preliminary data have begun to characterize the metabolome in mouse FrA during sleep (FASEB J 30.1, 2016). Anesthesia and sleep are different states of consciousness that share a number of trait similarities. This ongoing study is quantifying the similarities and differences in the FrA metabolome during sleep and anesthesia. Microdialysis samples have been collected from the FrA of adult, male B6 mice (n=12) anesthetized with isoflurane. All experiments have adhered to FASEB policies on humane use of animals in research. The samples are being analyzed using ultra‐performance liquid chromatography coupled with high‐resolution mass spectrometry on an Exactive Plus Orbitrap instrument. An untargeted metabolomics method is being used to identify brain metabolites (Anal Chem 82:3212, 2010). To date we have analyzed 15 samples from 3 mice during isoflurane (1.1%) anesthesia. The results revealed approximately 90 known molecules in each 25 μL microdialysis sample. In FrA, 44 analytes were common to states of sleep and anesthesia, including myo‐inositol, tryptophan, homovanillic acid, uracil, and pyruvate. State‐specific differences in metabolomic measures included 37 metabolites present during anesthesia that were not observed during sleep, and absence of 6 metabolites during anesthesia that were found during sleep. Known metabolites common to both sleep and anesthesia were analyzed by partial least squares discriminant analysis (PLS‐DA). Variable importance projection scores calculated from PLS‐DA analyses highlighted 20 of the 44 metabolites present during sleep and anesthesia that contributed significantly to the observed separation in the PLS‐DA. Histological comparisons of FrA microdialysis sites during sleep and anesthesia are pending. These emerging results support the interpretation that metabolomic analyses provide a powerful approach for developing a neurochemical cartography of lower level phenotypes putatively regulating states of consciousness.

Support or Funding Information

NIH grants HL65252 & Kavli Foundation (RL and HAB); MH099231 & 1P01NS083514 (CC and GT)
Early Life Exposure to Intermittent Hypoxia Induces Long‐Lasting Changes in Microglial Inflammatory Responses
Authors: Kiernan Elizabeth A , Ewald Andrea , Watters Jyoti
Published: November 2023

Chronic intermittent hypoxia (IH), a hallmark of sleep‐disordered breathing, is prevalent in pediatric and pre‐term infant populations. IH increases reactive oxygen species formation, causes neuronal apoptosis, promotes peripheral and CNS inflammation, and results in long‐term neurocognitive deficits. However, little is known about cellular mechanisms underlying long‐term alterations in CNS function following early life IH exposure, nor if the activities of adult microglia, CNS resident innate immune cells, are also affected by early‐life IH. Previously, we observed that early life IH increased the microglial inflammatory response to a peripheral immune challenge the day following the last IH exposure, and that this occurred without changes in basal levels of microglial inflammation, suggesting that IH primes microglial responses acutely. It is unknown if these effects persist into adulthood, or if they are sex‐specific. To test the hypothesis that early life IH exposure will exert long‐lasting, sex‐specific effects on microglial inflammatory gene responses to an immune challenge in adulthood, we exposed male and female rat pups to 8 nights of IH (2 min cycles of 10.5% O2 for 8hrs/day) beginning on the first postnatal day (P1). We assessed microglial responses to a low‐dose peripheral lipopolysaccharide challenge (LPS; 0.1 mg/kg for 3hrs) either the day after the last IH exposure, or 6 weeks after. Using RNA sequencing (RNA‐Seq) and qRT‐PCR, we found in microglia from 9 day‐old neonates that IH alone downregulated multiple genes involved in metabolism, RNA processing and chromosome regulation, without changing basal expression of inflammatory genes such as IL‐1b and iNOS. Interestingly, microglial responses to acute LPS challenge in neonates and adults were different; microglial responses to an LPS challenge at 6 weeks of age were impaired in males. To begin to dissect potential chromatin changes (i.e. histone modifications) that may contribute to hypoxia‐induced alterations in microglial immune responses, we performed chromatin immunoprecipitation‐sequencing (ChIP‐Seq) in immortalized male microglia to evaluate genome‐wide histone 3 lysine 4 trimethylation (H3K4me3) changes, a mark of active gene transcription. We found increases in this mark at multiple genes involved in hypoxic and inflammatory responses, and decreases at genes downregulated by IH in vivo. Ongoing studies are aimed at resolving mechanisms whereby early life exposure to hypoxic challenges can exert epigenetic alterations that modulate the adult microglial inflammatory response. Understanding these mechanisms may enable us to identify and harness cellular mechanisms that can restore proper microglial immune responses in adult CNS disorders, many of which display sexual dimorphisms.

Support or Funding Information

This work was supported by NIH R01 NS085226
Altered DNA Methylation Patterns Associated With Clinically Relevant Increases in PTSD symptoms and PTSD Symptom Profiles in Military Personnel
Authors: Cho Young‐Eun , Martin Christiana , Kim Hyungsuk , Yun Sijung , Kanefsky Rebekah , Olivera Anlys , Mulukutla Shilpa , Mulukutla Vincent , Lee Young‐Ho , Cashion Ann , Gill Jessica
Published: November 2023

In military personnel, post‐traumatic stress disorder (PTSD) occurs and is associated with differential DNA methylation. But, the relationship between these DNA methylation patterns and clinically relevant increases in PTSD SYMPTOMATOLOGY are not understood. The purpose of this study was to identify differences in DNA methylation associated with PTSD symptoms, and IF DNA methylation changes ARE related to an increase in PTSD severity. Symptom measures were obtained and methylated DNA was measured from peripheral blood mononuclear cells (PBMC) at baseline and 3 months later. All measures were compared between subjects with PTSD (n=8) and combat matched controls without PTSD (n=6) at follow‐up. Additionally, changes in DNA methylation in military personnel with clinically relevant increases in PTSD symptoms were determined and compared to controls with no clinical changes in PTSD. Compared to controls at follow‐up, military personnel with PTSD had 10 genes that were significantly hypermethylated and 18 that were significantly hypomethylated, including USP17, SDHAP3, and HISTH4C. In military personnel with clinically elevated PTSD symptoms 3 months following baseline, 119 genes exhibited reduced methylation, and 8 genes exhibited increased methylation. Genes with reduced methylation in the PTSD onset group relate to the canonical pathways of netrin signaling, Wnt/Ca+ pathway, and axonal guidance signaling. These gene‐pathways relate to neurological disorders, and the current findings suggest that these epigenetic changes potentially relate to PTSD symptomology. This study provides some novel insights into the role of epigenetic changes in PTSD symptoms and the progression of PTSD symptoms in military personnel.
Characterization of Mago Homology B (Magohb) in Skeletal Muscle
Authors: Zubair Samira , Waddell David
Published: November 2023

Skeletal muscle atrophy is a physiological condition that occurs due to immobilization, denervation, spinal cord transection and aging. Muscle wasting causes significant loss of muscle size and measurable reduction is strength. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Mago Homology B (Magohb), an exon junction core complex component involved in splicing and mRNA surveillance, is expressed in skeletal muscle and is significantly induced in response to denervation. In order to characterize the transcriptional regulation of Magohb, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with MyoD1 or myogenin expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Magohb has revealed canonical E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. The discovery that Magohb is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of mRNA processing, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.

Support or Funding Information

This work was supported by University of North Florida Transformational Learning Opportunity grants to D.W.
Preference, dependence, and pharmacokinetic outcomes in an opioid two bottle choice paradigm
Authors: Grim Travis Wiliam , Schmid Cullen L. , LaPrairie Robert B. , Park Jin‐Hong , Lin Li , Cameron Michael D. , Bohn Laura M.
Published: November 2023

Non‐oral routes of administration are most common for illicit opioid abusers, while oral consumption remains the predominant method of intake for most prescription opioid abusers. Accordingly, preclinical oral self‐administration of opioids by rodents should be investigated to discern abuse liability in addition to the classical intravenous models. Two bottle choice (TBC) represents an assay which can quantify preference for one solution over another, and is one possible option to investigate oral abuse liability of opiates. Historically, C57BL6 and DBA strains have been compared against one another to assess the genetic determinants of oral self‐administration of morphine. Between strains, C57BL6J mice have been shown to prefer saccharin‐morphine solutions to tap water, and morphine solutions to quinine solutions, while DBA mice display the opposite phenotype. Additionally, C57BL6 mice can consume over 150 mg/kg/day of morphine versus quinine and naloxone (i.p.) following TBC can precipitate withdrawal. Here, we hypothesized that C57BL6 mice would prefer opioid‐saccharin to quinine‐saccharin solutions, and that naloxone would precipitate withdrawal after TBC. The potent opioid analgesic fentanyl was included to control for taste effects, and the non‐psychoactive, brain‐penetrant non‐steroidal anti‐inflammatory drug (NSAID) ketoprofen was included as a control analgesic which should lack reinforcing properties. To facilitate solubility of potential test compounds, a 1% DMSO and 0.5% Captisol solution was used in addition to other solutes. Using naloxone, withdrawal was precipitated, and plasma and brain levels of each test drug was quantified via mass spectrometry.

Under these conditions, and similar to previous results, C57BL6 mice preferred morphine to quinine in a saccharin vehicle, but developed no preference when the concentration of quinine was lowered or included in both bottles. Similarly, fentanyl in the absence of quinine did not induce preference, and ketoprofen was preferred at the low dose but not the high dose. Naloxone precipitated withdrawal in morphine and fentanyl groups, but not ketoprofen, and all drugs were detected in plasma and brain. Together, these data demonstrate a reliable method to induce dependence via the oral self‐administration, and indicate the novel vehicle is amenable to TBC procedures. The preference of C57BL6 mice for morphine is likely driven by the enhancing palatability via saccharin in the morphine solution or via quinine in the opposite solution. Further optimization could reveal conditions in which morphine and/or fentanyl is preferred without enhancing palatability.

Support or Funding Information

This research was supported by NIH grant R01‐DA038964.