bluuebunny/crossref_metadata_2025 huggingface dataset:
Vagal nerve stimulation suppressed the inflammatory response in chronic heart failure before improving hemodynamics
Authors: Nishizaki Akiko , Saku Keita , Ide Tomomi , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background

Although vagal nerve stimulation (VNS) significantly improves chronic heart failure (CHF) independent of heart rate (HR) reduction, the mechanism which benefits CHF remains unknown. It is also well known that VNS exerts the anti‐inflammatory response. In this study we investigated how VNS regulates the inflammatory response of CHF after myocardial infarction (MI) in rats.

Methods

In 8 weeks Sprague‐Dawley rats, we created MI. We implanted a device for VNS and started stimulation from 2 weeks after MI (CHF: n=15, VNS: n=15). We titrated VNS below its bradycardia threshold. To elucidate the pure impact of VNS on inflammatory response, we evaluated invasion of macrophages and inflammatory cytokines in 3 days after VNS. In some rats, we evaluated hemodynamics and infarct size in 4 weeks after VNS.

Results

In 3 days, VNS did not change LVEF (CHF: 33.4±5.6, VNS: 34.8±6.9%) or LVEDP (CHF: 15.8±7.5, VNS: 12.1±7.2 mmHg). In contrast, VNS significantly reduced the invasion of macrophages in the left ventricle (LV) (CHF: 295.1±142.1, VNS: 110.7±59.6 counts/mm2, p=0.001) and plasma interleukin‐1β(IL‐1β)(CHF: 75.7±27.6, VNS: 32.9±6.8 pg/mL, p=0.0002) as well as that of heart and spleen. In 4 weeks, VNS markedly decreased LVEDP (CHF: 24.2±5.0, VNS: 17.5±7.1 mmHg, p=0.002) and heart weight (CHF: 3.7±0.5, VNS: 3.2±0.5 g/kg, p=0.0005), whereas the infarct size remained unchanged.

Conclusion

VNS showed the anti‐inflammatory responses 3 days after the beginning of stimulation. The VNS induced anti‐inflammatory response is by far earlier than its impact on hemodynamics and LV systolic function. The VNS induced anti‐inflammatory response may play a major role in preventing cardiac remodeling and improving LV function in the long term.
Treatment with Losartan Improves Baroreflexes in Obese Zucker Rats Coincident with Enhanced Response of Nucleus Tractus Solitarius (NTS) to Increased Arterial Pressure
Authors: Chaudhary Parul , Dohi Akiko , Schreihofer Ann
Published: June 2021

Obese Zucker rats (OZR) have impaired baroreflex‐mediated responses to acute rises in mean arterial pressure (MAP) coincident with the development of hypertension. In addition, the NTS becomes less responsive, with reduced phenylephrine (PE)‐induced c‐Fos expression and smaller physiological responses to glutamate in the NTS. Here, we determined if treatment with the angiotensin‐ AT1 receptor antagonist, losartan (LOS) improves baroreflexes and the PE‐induced c‐Fos expression in NTS of OZR. Baroreflex‐mediated changes in HR to PE‐evoked rises in MAP (40 mmHg) were measured in conscious rats implanted with femoral catheters. Rises in MAP were sustained with PE, and then rats were perfused after 90 min to examine NTS c‐Fos expression. Treatment with LOS (for 5 wks) normalized MAP in OZR vs. LZR (untreated: 128±2 vs. 115±3 mmHg, with LOS: 111±3 vs. 112±2 mmHg; at 14.1 wks). LOS treatment enhanced PE‐induced reductions in HR in OZR but not LZR. (untreated: 43±10 vs. ‐101±9 ‐bpm; with LOS:. ‐75±12 vs. ‐112±11bpm). Although LOS improved PE‐induced reductions in HR in OZR, responses in LOS‐treated OZR were still smaller vs. LOS‐treated LZR, suggesting a partial restoration of baroreflexes. PE‐induced c‐Fos expression in NTS was less in untreated OZR vs. LZR (30±5 vs. 50±6 counts). Treatment with LOS enhanced PE‐induced c‐Fos expression in OZR (54±11 counts), but not LZR (58±11 counts), rendering comparable PE‐induced c‐Fos expression in LOS‐treated OZR and LZR. These data suggest that LOS improves hypertension, baroreflexes, and NTS function in OZR. Further study is needed to determine whether improved function by LOS is explained by normalization of MAP in OZR. Support: AHA GRNT18880005.
Sympathetic Tone Differentially Regulates MHC‐II Expression by Macrophages and Enteric Glia in the Gastrointestinal Tract
Authors: Vanderkamp Robert , Chow Aaron , Garver Hannah , Fink Gregory , Gulbransen Brian
Published: June 2021

The sympathetic nervous system modulates essential gut functions including motility, blood flow, and gut immunity. Yet how sympathetic innervation regulates the function of specific immune cell types in the gut is poorly understood. Norepinephrine regulates expression of major histocompatibility complex (MHC) class II molecules in astrocytes and microglia in the brain. We aimed to test the hypothesis that sympathetic innervation of their counterparts in the gut, enteric glia and macrophages, regulates their antigen‐presenting capacity by modulating MHC‐II expression.

We tested our hypothesis by assessing MHC‐II expression by enteric glia or macrophages before and after removal of sympathetic tone by surgery or treatment with 6‐hydroxydopamine. Expression of MHC‐II by enteric glia or macrophages was assessed by dual‐label immunohistochemistry and mean fluorescence compared with Student's t‐test.

Sympathectomy increased expression in macrophages by 260% (P<.005). In contrast, sympathectomy had no effect on MHC‐II expression by enteric glia. Elevated MHC‐II expression in macrophages was not due to increased macrophage numbers because macrophage densities were not altered by denervation. Our results suggest that sympathetic tone suppresses expression of MHC‐II in macrophages. Expression of MHC‐II by enteric glia may require other cues.

Funded by the Larry D. Fowler Undergraduate Research Stipend
Chronic Salt‐Loading Alters Pre‐Autonomic Neuropeptide Expression in the Paraventricular Nucleus
Authors: Smith Justin , Pati Dipanwita , Hiller Helmut , Wang Lei , Frazier Jason , de Kloet Annette , Krause Eric
Published: June 2021

A high‐salt diet and disregulated stress‐responsiveness are known risk‐factors for the development of hypertension and heart disease. Corticotrophin‐releasing factor (CRF) initiates the neuroendocrine response to stressful stimuli and is situated in close proximity to osmosensitive arginine‐vasopressin (AVP) and oxytocin producing neurons in the paraventricular nucleus of the hypothalamus (PVN). To investigate the effect salt‐loading has on CRF neurons, we utilized the Cre‐lox system to generate mice that express red fluorescent protein driven by CRF transcription. These CRF‐reporter mice had water replaced with 2% NaCl for 5 days ending in a 30 min restraint challenge. Compared with mice maintained on water, salt‐loaded mice drank up to 150% more fluid and had significantly higher plasma sodium, but food intakes and body weights were similar. In situ hybridization for CRF mRNA resulted in salt‐loaded mice exhibiting a decreased restraint‐induced CRF expression in the PVN specifically in neurosecretory regions, but an increase in pre‐autonomic areas. Analysis of cells labeled with retrograde tract tracer injected into the ventral‐lateral medulla confirmed an increase in the frequency of pre‐autonomic neurons expressing AVP and CRF in the PVN with salt‐loading. Whole‐cell patch clamp recording of CRF positive PVN cells found significantly fewer spontaneous excitatory postsynaptic currents; however, glutamatergic synapses onto these cells were upregulated. Collectively, these results show the plasticity of stress‐responsive neurons which may contribute to the anxiolytic and cardiovascular effects of high‐salt diets.

Support: HL‐096830 (EGK), HL116074 (ADK)
Selective inhibition of the adrenergic C1 neurons reduces the hypoxic ventilatory response in unanesthetized rats
Authors: Malheiros‐Lima Milene , Takakura Ana , Moreira Thiago
Published: June 2021

The catecholaminergic (TH) neurons that reside in the rostral half of the ventrolateral medulla (VLM), also called C1 neurons, regulate blood pressure, the CRF/ACTH/corticosterone cascade and autonomic glucoprivic responses. The stimuli that most commonly activates the C1 neurons (nociception, hypotension, and hypoxia) also increases breathing. Although pharmacological evidence suggests that catecholaminergic neurons regulate breathing, a specific contribution of the C1 neurons to respiratory control has not been demonstrated, particularly in vivo. Here, we evaluate the role of the C1 cells in the control of breathing under hypoxia (8% O2) or hypercapnia (7% CO2) condition in unanesthetized rats. Bilateral injection of the immunotoxin anti‐dopamine β‐hydroxylase‐saporin (anti‐DβH SAP) was done in adult unanesthetized male Wistar rats (270‐300g, N = 4‐7/group). Bilateral injections of anti‐DβH SAP (2.1 ng/100 nl) into VLM destroyed TH neurons. Selective inhibition of the C1 cells with bilateral injection of anti‐DβH‐SAP into the VLM reduced the increase in respiratory frequency (fR) (110 ± 5, vs. saline 136 ± 17 bpm), tidal volume (VT) (2.3 ± 0.2, vs. saline: 2.9 ± 0.3 ml/kg), and minute ventilation (MV) (244 ± 16, vs. saline: 402 ± 90 ml/kg/min) elicited by hypoxia (8% O2). Hypercapnia challenges (7% CO2) caused graded increases in fR, VT, and MV, however, the changes in breathing elicited by hypercapnia was not altered after selective destruction of C1 cells in the VLM. The present study indicates that C1 neurons could be involved in the regulation of breathing and provide evidence that they may contribute to the hypoxic ventilatory response.

Financial support: FAPESP, CNPq, CAPES/PROEX.
P2X3 receptor‐mediated chemoreceptor hypersensitivity in young spontaneous hypertensive rats
Authors: Moraes Davi , Machado Benedito , Ford Anthony , Paton Julian
Published: June 2021

Sympathetic overactivity has been related to the pathogenesis of hypertension. Enhanced peripheral chemoreceptor sensitivity has been suggested as a possible mechanism for sympathetic overactivity. We compared both baseline and chemoreflex evoked carotid sinus nerve (CSN) activity, and the electrophysiological properties of petrosal ganglion cells (PGc) innervating the carotid body in in situ preparations of young spontaneously hypertensive (SH) and Wistar rats. Since ATP plays a major role in chemosensory transduction in the carotid body, we also evaluated the contribution of ATP and P2X receptors in PGc and their involvement in sympathetic overactivity observed in SH rats. SH rats (n=22) presented: increased basal and chemoreflex evoked CSN discharge, and the electrical excitability of PGc was elevated relative to Wistar rats (n=25). A significant increase in the depolarizing effects of ATP and α,β‐methylene ATP in SH (n=18) versus Wistar (n=16) PGc was observed. This was consistent with raised single‐cell mRNA expression of ionotropic P2X3 channels (n=12) in SH rats. Moreover, both the increased baseline and chemoreflex evoked sympathetic nerve response in SH rats (n=12) was markedly reduced after perfusion of the PG with AF‐353, a selective antagonist of P2X3 receptors. Our results suggest that ATP and P2X3 receptors play an important role as a novel mechanism for increased carotid body tone and hyper‐chemosensitivy that contributes to the sympathetic overactivity in SH rats.

Support: British Heart Foundation and FAPESP
Sympathetic Axonal Recruitment During Severe Chemoreflex Stress in Humans
Authors: Badrov Mark , Usselman Charlotte , Shoemaker J Kevin
Published: June 2021

We tested the hypothesis that severe chemoreflex stress stimulates the recruitment of sub‐populations of larger, faster conducting sympathetic neurones that are otherwise silent at baseline. Muscle sympathetic nerve activity (MSNA; microneurography) was measured in 11 healthy individuals (6 females; 25 ± 3 yrs) at baseline and during a maximal end‐inspiratory apnea, which was preceded by a rebreathing period designed to maximize the chemoreflex stress during apnea (End‐apnea: PO2 = 58 ± 4 Torr; PCO2 = 55 ± 5 Torr). Compared to baseline, action potential (AP) frequency (174 ± 151 to 1083 ± 487 spikes/min; P < 0.001) and mean AP content per burst (11 ± 4 to 24 ± 12 spikes/burst; P < 0.01) were elevated during apnea. When detected APs were binned according to their peak‐to‐peak amplitude, the number of total ‘clusters' of APs detected (13 ± 4 to 20 ± 8 total clusters; P < 0.001), as well as the number of distinct ‘clusters' per burst (5 ± 2 to 8 ± 2 clusters/burst; P < 0.001), were increased during apnea. At baseline (R2 = 0.91; P < 0.001) and during apnea (R2 = 0.98; P < 0.001), a pattern emerged whereby AP cluster latency decreased as cluster amplitude increased. Furthermore, the cluster latency‐size profile was shifted downwards for every corresponding cluster during apnea versus baseline (range −14.9 to −99.4 ms; mean = −53.1 ms), such that APs with similar amplitudes were detected approximately 53.1 ms faster during apnea. In conclusion, there appears a unique ability of the sympathetic nervous system to recruit sub‐populations of previously dormant larger, faster conducting sympathetic neurones, as well as acutely modify reflex latency of all APs during severe chemoreflex stress. Supported by NSERC and CIHR.
Activation of Enteric Glia Contributes to Enteric Neuron Death in Inflammation
Authors: Brown Isola , McClain Jonathon , Patel Bhavik , Gulbransen Brian
Published: June 2021

Enteric glia (EG), a type of peripheral astroglia, surround enteric neurons and are critical for the maintenance of enteric circuits. Loss of glial maintenance is thought to lead to neuron death and gut dysfunction during inflammation (Aube et al 2006). However, this theory is at odds with data showing that astroglial activation drives neuron death during neuroinflammation (Mayo et al 2014) and our data showing that enteric neuron death is driven by activation of neuronal P2X7 purine receptors (P2X7Rs) (Gulbransen et al 2012). We hypothesized that EG activation promotes neuronal death via P2X7R pathways. We activated EG in situ with P2Y1 receptor (P2Y1R) agonists, measured glial ATP release with biosensors and neuronal survival with immunohistochemistry. Glial activity was monitored using Ca2+ imaging and glial ATP release pathways involving connexin‐43 (Cx43) hemichannels were modulated by altering nitric oxide (NO) levels with drugs or selectively ablated in EG using inducible Cx43 null mice. P2Y1R agonists primarily activated EG in the myenteric plexus. Glial activation drove ATP release through Cx43 channels and reduced neuron density by 23%. Inhibition or ablation of glial Cx43 prevented P2X7R‐driven neuron death in situ and in vivo (p < 0.01). Cx43‐dependent glial ATP release was potentiated by NO (p<0.05) and Cx43 inhibition protected against NO‐mediated neuron death (p<0.05). Our data demonstrate a novel pathogenic role of enteric glia in enteric neuron loss during colitis. Direct glial activation is sufficient to cause enteric neuron loss in a Cx43 and NO dependent manner. This research was supported by NIH/PHS K12 HD065879.
CB1 receptors into the Prelimbic Cortex modulate food intake in rats.
Authors: Scopinho América , Resstel Leonardo , Corrêa Fernando
Published: June 2021

The endocannabinoid system is involved in the central regulation of feeding behavior. Systemic administration of exogenous cannabinoids or endocannabinoids stimulates eating, and systemic administration of CB1 antagonist attenuates agonists' stimulatory effects on food intake, suggesting the involvement of these receptors in food intake modulation. Although CB1 receptors are present principally in the central nervous system (CNS), little is known about which areas are involved in this modulation and the role of endocannabinoids system. Therefore, the aim of the present study is to investigate the effects evoked by CB1 receptors antagonist (AM251) microinjected in the prelimbic cortex (PL) on the food intake regulation.

CB1 receptor antagonist AM251 (10, 50, 100pmol/100nl) or aCSF‐ artificial cerebrospinal fluid were microinjected into the PL of fed or fasted Wistar rats and 10 min later, the food intake test was performed during one hour for food intake determination.

The amount of food ingested by fasted animals was significantly higher than fed animals (F(1, 48) = 482,2; P<0.001). Moreover, significant effects of treatment with doses of AM251 (F(3, 48) = 74,41; P<0.001) and interaction between the two factors F(3, 48) = 78,26; P<0.001) for total food intake (n=6‐8 each group) were observed only in fasted rats.

The blockade of PL CB1 receptors inhibited food intake in fasted animals, suggesting that the PL endocannabinoid system modulated food intake behavior and satiation.

FINANCIAL SUPPORT: FAPESP
Hypothalamic AgRP‐mediated energy metabolism in skeletal muscle is a critical regulatory system in feeding rhythm‐induced insulin resistance
Authors: Shiuchi Tetsuya , Otsuka Airi , Chikahisa Sachiko , Shimizu Noriyuki , Sei Hiroyoshi
Published: June 2021

Hypothalamic control of peripheral energy metabolism is an important regulatory system in whole body energy homeostasis, although whether feeding rhythm affect the hypothalamic regulation system of energy metabolism has not elucidated. In this study C57BL/6J mice were separated for 3 feeding scheduled groups; given lab chows freely during dark phase (ZT12‐24; Control group), first 4‐hour in dark phase (ZT12‐16; Morning group), and last 4‐hour in dark phase (ZT20‐24; Evening group) for 8weeks. Mice in Evening group showed impaired whole body insulin sensitivity while mice in Morning group showed normal insulin sensitivity compared to Control group. We observed that higher lipid accumulation and impaired insulin signals in skeletal muscle in Evening group compared to other group. On the other hand, mRNA expression of agouti‐related protein (AgRP) was increased in hypothalamus in Evening group. We found that intracerebroventricular injection of AgRP increased gene expression of lipid accumulation in skeletal muscle of mice in Control group. Inhibition of central AgRP expression by antisense oligo improved insulin resistance in skeletal muscle of Evening group. These results indicate that usual energy ingestion mainly after the evening impairs insulin sensitivity in skeletal muscle mediated by hypothalamic AgRP.
Fatty Acid Preference is Differentially Mediated by Nutritional Status and Lingual CD36 in Obesity‐Prone and Resistant Rats
Authors: Primeaux Stefany
Published: June 2021

Fatty acid receptors on the tongue mediate oral sensing of fat/fatty acids and are proposed mechanisms contributing to the susceptibility to obesity. The first goal of the current experiments was to investigate the role of nutritional status on lingual CD36 expression in obesity‐prone Osborne‐Mendel (OM) and obesity‐resistant S5B/Pl (S5B) rats. The second goal was to assess the effects of nutritional status on linoleic acid (LA) preference in these rat models. In Exp. 1A, lingual CD36 protein expression was measured in OM and S5B rats fed a high fat diet (HFD). In Exp.1B, OM and S5B rats were fed a HFD and the preference threshold for increasing concentrations of LA was evaluated. In Exp. 2A, lingual CD36 mRNA expression was measured in OM and S5B rats fed a chow diet and fasted overnight. In Exp. 2B, the preference threshold for increasing concentrations of LA was determined following an overnight fast. In Exp. 3, the effect of lingual CD36 siRNA application on LA preference following an overnight fast in OM and S5B rats was assessed. Consumption of HFD decreased lingual CD36 protein levels and LA preference threshold in S5B rats, compared to OM rats. Following an overnight fast, lingual CD36 mRNA levels were elevated in S5B rats, compared to OM rats. LA preference threshold was not differentially altered in fasted OM and S5B rats. Application of CD36 siRNA decreased LA preference following an overnight fast in S5B rats, compared to OM rats. These data suggest that nutritional status leads to more pronounced alterations in lingual CD36 expression and LA preference in resistant S5B rats, compared to prone OM rats and supports a role for orosensory fat detection in the susceptibility to developing obesity. LSUHSC to SDP
Central QRFP‐26 in Female Rats: Effects on High Fat Intake, Meal Patterns and Hypothalamic Feeding Peptides
Authors: Primeaux Stefany , Lowe Mallory , Braymer H Douglas
Published: June 2021

Central administration of QRFP increases high fat diet (HFD), but not low fat diet (LFD) intake in male and cycling female rats. In males, QRFP's effects on HFD intake are regulated by neuropeptide Y (NPY) and the melanocortin system. The current experiments examined the effects of acute and sub‐chronic QRFP administration in female rats in the absence of estradiol. HFD intake, gene expression of hypothalamic NPY, agouti‐related peptide (AgRP) and pro‐opiomelanocortin (POMC) and meal patterns were assessed. In Experiment 1, HFD and LFD intake was measured following administration (icv) of QRFP in ovariectomized (OVX) rats. One week later, rats were administered QRFP and brains were harvested for the assessment of prepro‐NPY, AgRP and POMC mRNA in the mediobasal hypothalamus (MBH). In Experiment 2, following OVX, QRFP was administered centrally via an osmotic pump and food intake and meal patterns were continuously monitored using metabolic chambers for one week. At the end of one week, brains were harvested for the assessment of prepro‐NPY, AgRP and POMC mRNA in the MBH. Acute administration of QRFP increased HFD intake following OVX and increased the expression of AgRP mRNA in the MBH. Sub‐chronic administration of QRFP following OVX increased total HFD intake and HFD intake during the dark cycle and meal duration during the light cycle. An interaction between diet and QRFP was detected for prepro‐NPY expression in the MBH. These data support a role for QRFP as a mediator of HFD intake in the absence of estradiol and suggest that QRFP increases HFD intake by altering meal duration and hypothalamic expression of AgRP. LSUHSC to SDP; DK072476 PBRC
Neonatal Growth Restriction Heightens Leptin‐Evoked Arterial Blood Pressure and Renal Sympathetic Nerve Responses in Adult Mice
Authors: Peotta Veronica , Morgan Donald , Rahmouni Kamal , Movva Vani , Volk Kenneth , Roghair Robert
Published: June 2021

Neonatal growth restricted (GR) mice are leptin deficient and obesity resistant, but have adult hypertension, potentially due to enhanced central leptin sensitivity. We hypothesized that neonatal GR increases hypothalamic leptin receptor expression and leptin‐triggered sympathetic activation in adult mice. C57 male mice were fostered in litters of 12 (GR) or 6 (Ctrl), and GR was confirmed by weanling weights below the 10th percentile. At 12 wks, arterial pressure by radiotelemetry (BP) or renal sympathetic nerve activity (RSNA) were measured at baseline and following saline or leptin administration. Compared to normal weight controls, adult GR mice had significantly increased leptin receptor mRNA expression within the arcuate nucleus of the hypothalamus (ARC), decreased adult food intake (0.09±0.01 vs. 0.13±0.02g/g/d; p<0.05), decreased adult body weight, and exaggerated leptin‐triggered increases in both renal sympathetic nerve activity (29+/‐11 vs. ‐9+/‐6% change from baseline, p<0.01 for GR versus control) and systolic blood pressure (8+/‐3 vs. ‐1+/‐6% change from baseline, p<0.05 for icv leptin versus icv saline in GR mice). By confocal immunofluorescence, this GR‐induced leptin hyperresponsiveness was associated with enhanced leptin‐stimulated STAT3 activation within ARC POMC‐positive neurons. We conclude that neonatal GR‐associated leptin deficiency leads to enhanced ARC leptin sensitivity with subsequent hypertension and renal sympathetic hyperactivity.

R01 HL102659
Activation of Leptin's Signaling Cascade Results in the Endocytosis of Mu Opioid Receptors
Authors: Barnes Maria
Published: June 2021

Activation of mu opioid receptors (MOR) has been correlated with increased food intake. Obese animals show an increase in MOR receptor density in the hypothalamus. The possibility exist that changes in the expression and/or activity of MOR in obesity plays a role in the feeding behavior. Factors that contribute to the increased MOR are unknown. Thus, we sought to test the hypothesis that leptin can regulate the trafficking of MOR. We propose that under normal circumstances, leptin plays a role in the internalization of MOR which may be reflected in satiation. However, in the case of obesity, when leptin resistance develops, leptin's role in the internalization of MOR is decreased or lost such that more MOR remain at the cellular membrane and are available for activation. Human Embryonic Kidney (HEK 293) cells transfected with MOR and the leptin signaling cascade was used in these experiments. We observed that activation of leptin's signaling cascade resulted in the internalization of mu receptors. Internalization occurred after phosphorylation of serine 375. Taken together, these data demonstrate that leptin can regulate the trafficking of mu opioid receptors. Supported by DK R34‐32089
Evaluation of the Nrf2‐mediated anti‐oxidative and anti‐inflammatory responses in phrenic motoneurons following C2 hemisection in adult rat
Authors: Deramaudt T , Vinit S , Efthimiadi L , Keomani E , Petitjean M , Bonay M
Published: June 2021

The consequences of high cervical spinal cord injuries are permanent respiratory deficits and deleterious inflammatory and oxidative reactions, which may prevent any spontaneous functional recovery. Our laboratory has successfully mastered a preclinical model of respiratory insufficiency consisting in a C2 partial injury which leads to unilateral phrenic and diaphragm paralysis in the adult rat. This injury induces a gradual microglial activation and a macrophage infiltration around the deafferented phrenic motoneurons, identified by Cholera Toxin B‐fragment back‐labeling, hours to days post‐injury. iNOS and C/EBP δ protein levels, two transcriptional factors involved in pro‐inflammatory response, were increased overtime within the phrenic motoneurons. Interestingly, the injury induced a transient reduction in Nrf2 (Nuclear Factor E2‐Related Factor 2) protein, a key regulator in the anti‐inflammatory and anti‐oxidative responses, in the phrenic motoneurons, that was restored to control level at 7 days post‐injury. Our results show that the C2 injury negatively modulates the Nrf2 and the protective mechanisms within the phrenic motoneurons and their vicinity. This suggests a potential therapeutic role of Nrf2 in ameliorating the functional recovery of phrenic motoneurons by reducing the inflammatory and oxidative damages following spinal cord injury.
Differential Roles of Mammalian Target of Rapamycin (mTOR) in Phrenic Long‐Term Facilitation
Authors: Dougherty Brendan , Fields Daryl , Mitchell Gordon
Published: June 2021

Phrenic long‐term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). This form of spinal plasticity is expressed via distinct cell signaling cascades that depend on the severity of hypoxia during intermittent exposures. Each mechanism requires spinal protein synthesis, though the newly synthesized proteins differ (e.g. BDNF vs immature TrkB). Translational regulation of new protein synthesis is often regulated by mammalian Target of Rapamycin (mTOR)‐dependent translation. Here we tested the hypothesis that mTOR is necessary for severe AIH (sAIH; PaO2 25‐30mmHG), but not moderate AIH (mAIH; PaO2 35‐45mmHG) induced spinal protein synthesis. Phrenic nerve activity was recorded in anesthetized, paralyzed and ventilated male Sprague‐Dawley rats before, during and 60 min after mAIH or sAIH. Rats were pre‐treated with intrathecal injections of vehicle or Rapamycin (0.1mM, 12µl), a highly selective inhibitor of mTOR complex 1. Consistent with our hypothesis, Rapamycin blunted sAIH, but not mAIH induced pLTF. Thus, spinal activation of mTOR and/or its down‐stream targets are necessary for sAIH‐, but not mAIH‐induced pLTF. These results provide further evidence that mAIH and sAIH elicit pLTF via distinct cellular cascades despite differing only in the severity of hypoxemia within hypoxic episodes.
Pontine A7 and A5 noradrenergic neuronal groups mediate long‐term facilitation of hypoglossal motor activity induced by episodic airway obstruction
Authors: Song Gang , Poon Chi‐Sang
Published: June 2021

In obstructive sleep apnea (OSA), decreased noradrenergic activation of genioglossus muscle during REM sleep results in repetitive upper airway occlusion, a neurodegenerative process that aggravates with age. Episodic end‐expiratory airway occlusion simulating OSA in anesthetized rats has been shown to induce a vagally‐mediated and noradrenergic‐dependent long‐term facilitation (LTF) of genioglossus muscle activity (Tadjalli et al, J Neurosci, 2010). To identify the brainstem noradrenergic pathways mediating this effect, we applied episodic end‐expiratory tracheal occlusion (10 sec x 12 episodes) in 6 urethane‐anesthetized rats to induce genioglossus LTF and reveal corresponding cFos expressions in the affected brainstem pathways, in comparison to 3 control rats in which tracheal occlusion was not performed. We found that the percentage of cFos immunopositive neurons was significantly higher in A7 and A5 noradrenergic groups in experimental rats than that in control rats (32.9±5.3% vs. 0.7±0.7% for A7 and 34.8±7.2% vs. 5.4±5.4% for A5, P<0.01), while the expression of cFos was not significantly changed in A6 (locus coeruleus) and other medullary noradrenergic groups. In the second group of experiments, we examined the LTF induction after inhibition of A7 or A5 by microinjection of α2‐adrenoreceptor agonist clonidine. We found that after inhibition of either A7 (n=8) or A5 (n=4), the induction of genioglossus LTF was blocked. These studies demonstrated that the pontine noradrenergic neuronal groups A7 and A5 played a key role in the vagally‐mediated genioglossus LTF which, once fully developed, may help to defend against recurrent OSA. (Supported by HL093225)
Inflammation Enhances Spinal BDNF‐Induced Phrenic Motor Facilitation
Authors: Agosto Ibis , Mitchell Gordon
Published: June 2021

Long‐lasting phrenic motor facilitation (pMF) can be elicited by multiple, distinct cellular mechanisms. For example, acute intermittent hypoxia triggers a form of pMF known as phrenic long‐term facilitation (pLTF); pLTF requires new synthesis of brain derived neurotrophic factor (BDNF) and activation of its high affinity receptor (TrkB) in the cervical spinal cord (Baker‐Herman et al. 2004). Since systemic inflammation abolishes AIH‐induced pLTF (Huxtable et al. 2013), we tested the hypothesis that the functional impairment is upstream from BDNF/TrkB signaling by testing the impact of systemic inflammation on pMF elicited by cervical spinal injections of BDNF. We assessed phrenic nerve activity before and after intrathecal C4 BDNF (100ng, 12uL) injections in anesthetized, paralyzed and ventilated adult rats 24 hrs post‐LPS (100ug/kg, i.p.) or control (saline) injections. To our surprise, LPS pretreatment enhanced BDNF‐induced pMF (LPS: 105% baseline; versus control: 58% baseline; both n=4; p < 0.05). These results enable two

Conclusions

1) LPS induced inflammation most likely impairs AIH‐induced pLTF upstream from BDNF/TrkB signaling; and 2) unknown downstream mechanisms attempt to compensate, enhancing the BDNF/TrkB induced pMF. These conclusions advance our understanding concerning the impact of even mild inflammation on important forms of spinal respiratory motor plasticity.

[Supported by: NIH HL111598]
Sex Differences in Microglial Responses to Chronic Intermittent Hypoxia
Authors: Kimyon Rebecca , Smith Stephanie , Watters Jyoti
Published: June 2021

Chronic intermittent hypoxia is a hallmark of obstructive sleep apnea (OSA) that causes neuroinflammation and impairs one form of respiratory motor plasticity, phrenic nerve long‐term facilitation (pLTF). Interestingly, the prevalence of OSA is lower in women than in men, although this difference greatly decreases after menopause, suggesting that ovarian hormones may play a protective role. Although estrogen is well known to be anti‐inflammatory following ischemic/hypoxic injury, little is known about its ability to control neuroinflammation as it impacts respiratory neuroplasticity, or in response to inflammation induced by aspects of sleep disordered breathing. Our pilot data suggest that compared to males, microglia (CNS resident immune cells) from females display reduced inflammatory activities as evidenced by reduced cytokine expression (interleukins (IL)‐1b and ‐6), and decreased upregulation of surface CD14 levels. We evaluated differences in estrogen receptor alpha (ERa) expression in male and female microglia because ERa is well‐known to mediate anti‐inflammatory effects of estrogen in the CNS. Although we found no detectable differences in basal ERa expression, we did find that CIH reduced ERa in male microglia, but not in female. These preliminary observations suggest that males may not receive the same benefit of ERa‐mediated anti‐inflammatory activities that females do. Current studies are aimed at understanding the role of gonadal estrogen in creating these sexual dimorphisms in microglial neuroinflammatory responses to CIH. Results from this work will provide significant insight into mechanisms that can be exploited to minimize destabilization of respiratory plasticity in both sexes. (Supported by NIH R01 HL111598).
Preferential Neuropathology in Respiratory‐Related Motor Nuclei in Pompe Mice
Authors: Turner Sara , ElMallah Mai , Falk Darin , Byrne Barry , Fuller David
Published: June 2021

Pompe disease, caused by deficiency of acid alpha‐glucosidase (GAA), leads to glycogen accumulation, disruption of cellular architecture and neuromuscular impairments. Respiratory and lingual motor systems often decline first. We hypothesized neuropathology and neuroinflammation first develop in respiratory‐related motoneurons. Murine Pompe (Gaa‐/‐) and 129 SVE wildtype (WT) tissues were stained for periodic acid‐Schiff (PAS) and ionized calcium binding adaptor molecule 1 (IBA‐1) to assess glycogen accumulation and microglial morphology, respectively. Strikingly, in 6 week old Gaa‐/‐ tissues, medullary pathology was restricted to the hypoglossal motor nucleus, and spinal pathology was most extensive in phrenic and intercostal motor nuclei. Gaa‐/‐ tissues had greater co‐localization of microglia and neurons. With aging, PAS‐positive neurons became prevalent in sensory‐related medullary and spinal regions. By 12 months, most neurons showed neuropathology and overt glycogen accumulation. Furthermore, in adult Gaa‐/‐ tissues, TUNEL positive staining was identified in respiratory‐related motor nuclei, suggesting apoptosis contributes to neuromotor impairments. These data indicate respiratory neurons are particularly susceptible in Gaa‐/‐ mice, and show neuroinflammation and apoptosis in and around Gaa‐/‐ respiratory neurons for the first time. Funding: 2R01HD052682‐06A1
Differential Focal Adhesion Kinase (FAK) Expression Accounts for the Developmental Regulation of Pulmonary Artery Endothelial Cell(PAEC) Barrier Function
Authors: Ying Lihua , Barnes Elizabeth , Alvira Cristina , Cornfield David
Published: June 2021

Objective

Deferential FAK expression accounts for the developmental regulation of PAEC Barrier Function

Methods

Lung injury was induced in 6day old (neonatal) and 8week old (adult) mice by Intraperitoneal injection of lipopolysaccharide (LPS) administration. Evans Blue Dye (EBD) was injected in the retro‐orbital sinus vein 24h after LPS, and optical density (OD) of EBD measured in mouse lungs. Barrier function in vitro was assessed in PAEC from neonatal and adult mice using Electric Cell‐substrate Impedance Sensing (ECIS). Gene expression and cytokine profile were evaluated using microarray analysis. FAK expression and gap junction of VE‐Cadherin immuno‐staining were assessed in PAEC with/without FAKsiRNA.

Results

IP LPS increases lung permeability more in adult, than in neonates. With LPS, barrier function is more well preserved in neonatal, compared to adult PAEC. With LPS, FAK mRNA and protein expression increases more in neonatal, than in adult, PAEC. VE Cadherin membrane localization persists longer in neonatal, than in adult PAEC after LPS. VE cadherin membrane expression was decreased in the adult after 24h FAKsiRNA PAEC transfection. Pharmacologic FAK inhibition compromises barrier function more in adult than in neonatal PAEC. With LPS, adult PAEC FAK overexpression improves and depletion worsens barrier function.

Conclusions

In response to injury, PAEC barrier function is more well preserved in neonatal, compared to adult lungs. Deferential expression FAK may account for superior barrier function in neonatal PAEC and, perhaps, the survival advantage for children with ARDS. This observation may have important implications for novel cell‐based therapies in adults with ARDS.
HIF‐1α Expression is Decreased and Contractility is Enhanced in PASMC from PAH Patients
Authors: Barnes Elizabeth , Chen Chihhsin , Alvira Cristina , Cornfield David
Published: June 2021

OBJECTIVE

To determine if hypoxia inducible factor‐1α (HIF‐1α) expression is decreased and myosin light chain (MLC) phosphorylation and contractility are enhanced in pulmonary artery smooth muscle cells (PASMC) from patients with pulmonary arterial hypertension (PAH).

METHODS

PASMC were isolated from patients diagnosed with PAH and controls. PASMC were examined by Western, RT‐PCR, and immunofluorescence to assess HIF‐1α and phosphorylated MLC levels. Prolyl‐hydroxylase domain (PHD) activity was examined by oxygen, iron (II) and ascorbate cellular content. Metabolism was assessed via formazan and lactate production. The relative contractile response was determined by collagen gel assay. Rho kinase and myosin light chain kinase (MLCK) activities were quantified by ELISA. Ca2+ imaging was employed to assess intracellular Ca2+ levels in HIF‐1α‐/‐ PASMC.

RESULTS

In PAH SMC, HIF‐1α protein expression is decreased and MLC phosphorylation is increased. PHD inhibition increases HIF‐1α expression in PAH SMC. Furthermore, cellular oxygen and ascorbate levels are higher in PAH SMC. Formazan production is increased and lactate production is decreased in PAH SMC. The relative SMC contractile response is greater in PAH. MLCK activity is increased in PAH SMC. Loss of HIF‐1α in PASMC potentiates the hypoxia‐induced increase in cytosolic Ca2+.

CONCLUSIONS

In PASMC from patients with PAH, HIF‐1α protein expression is decreased and MLC phosphorylation and functional contraction are increased via MLCK activation. Overall, these results demonstrate that PASMC contractility is increased in PAH, perhaps as a direct result of decreased HIF‐1α expression. Altered HIF‐1α expression may play an etiologic role in the development of PAH.
Regulation of Formyl Peptide Receptor 2 During Chronic Kidney Disease
Authors: Labes Robert , Neymeyer Hanna , Saez Fara , Reverte Virginia , Salazar Javier , Bachmann Sebastian , Paliege Alexander
Published: June 2021

The G‐protein coupled receptor formyl peptide receptor 2 (FPR2) integrates signals of multiple anti‐inflammatory and anti‐fibrotic mediators including lipoxin A4, resolvin D1, and annexin A1. Previous studies have demonstrated protective effects of FPR2 in animal models of renal disease but the localization of FPR2 in the healthy kidney and its regulation in chronic kidney disease (CKD) have not been elucidated. Aim of this study was to determine the renal localization of FPR2 under control conditions and in a rat model of CKD. Newborn rats were treated with the AT1R antagonist Candesartan from postnatal d1‐14 to impair nephrogenesis and examined at 11 moth of age (CKD rats). Expression of FPR2 was studied using immunohistochemistry (IHC) and double labeling immunofluorescence (IF) for FPR2 and markers for fibroblasts, myofibroblasts, macrophages, and endothelial cells. CKD rats showed signs of renal inflammation and fibrosis with a focal accumulation of macrophages, myofibroblasts and extracellular matrix components. IHC revealed expression of FPR2 in the tubulointerstitium of controls and a strong accumulation of FPR2 expressing cells in fibrotic areas of CKD animals. IF identified fibroblasts and endothelial cells as the predominant FPR2 expressing cell types in both groups. Abundant signal was further detected in macrophages and myofibroblasts in the fibrotic areas of CKD animals. Our results show that FPR2 is abundantly expressed in the kidneys of control and CKD animals. It may therefore play an important role in the regulation of normal renal function and exert anti‐inflammatory and anti‐fibrotic effects during CKD. FPR2 may thus provide a promising target for pharmacological intervention in the treatment of CKD.
Elevated Aldosterone at Birth Predicts Renal Function Decline in Low Birth Weight Rats
Authors: Ojeda Norma , Alexander Barbara , Bhatt Abhay
Published: June 2021

Epidemiological and experimental studies reported elevated aldosterone levels in low birth weight individuals. However, the effect of this finding on renal function after ischemic events is unknown. The objective of this study was to investigate the effects of elevated aldosterone at birth on renal function after ischemia in later life. We used low birth weight rats exposed to bilateral mild renal ischemia (15 min) and treated with acute infusion of angiotensin converter enzyme inhibitor Enalaprilat. At 12 weeks of age male and female, control and low birth weight rats were exposed to renal ischemia or sham, and treated with Enalaprilat or vehicle. Systemic and renal hemodynamic were measured 2 hours after ischemia, followed by blood and tissue collection. GraphPad software was used for statistical analysis with significances set at P<0.05. Aldosterone levels measured at birth and 12 weeks of age were elevated (P<0.05) in male low birth weight rats compared to females counterparts and controls. Mean arterial pressure was elevated (P<0.05) in male low birth weight rats compared to female counterpart and controls. Mild renal ischemia exacerbated increases in Aldosterone and mean arterial pressure; and induced reductions in glomerular filtration rate and renal blood flow (P<0.05), and increases in renal vascular resistance (P<0.05) only in male low birth weight rats. Enalaprilat reverted changes observed in male low birth weight rats, while did not affect female counterpart and controls. This study suggest that elevated aldosterone at birth predicts renal function decline after ischemia event in low birth weight rats with sex dimorphism and involvement of the renin angiotensin system
Supervised and Unsupervised Learning Methods for the Automation of Glomerular Scoring
Authors: Bukowy John , Evans Louise , Broadway Elizabeth , Dayton Alex , Cowley Allen
Published: June 2021

A single rat kidney can contain in excess of 30,000 glomeruli whose function is to act as a filter at the initial interface between the bloodstream and tubular fluid. Sufficient damage to glomeruli and their surrounding Bowman's space leads to altered fluid handling within the kidney and disease states. Scoring of glomeruli from histological preparations with Gomori's One‐Step Trichrome staining can quantify glomerular damage but requires a tedious and time consuming process of manual scoring individually selected glomeruli.

In order to decrease inter/intra‐observer variability, analysis time, and resource dedication we propose a method of feature selection for the automation of glomerular scoring. The dataset was comprised of over 1,300 previously human‐scored bright‐field 40x histological images of rat glomeruli obtained under varying experimental conditions. The images consisted of glomeruli surrounded by tubular segments. Using an approach that considers the center of mass of salient features from the images, regions containing glomeruli were located and digitally “masked”, or isolated, within individual images. Unsupervised k‐means clustering was then implemented for color segmentation of the masked glomerular region to select features emphasized from trichrome staining. By training with the described feature set, the scores predicted by the classifier were found to be not significantly different than the human observer, thereby automating a subjective workflow while maintaining precision of scores. While the specific feature set construction described here may only be applicable to the scoring of glomerular damage assayed through trichrome staining, this approach may be applied to a variety of histological assays and stains.

(HL116264)
Development of a Sensitive and Specific Urinary Plasmin Activity Assay
Authors: Svenningsen Per , Østergaard Søberg Anders , Jensen Boye
Published: June 2021

Several human proteinuric conditions are characterized by excretion of plasmin in the urine, and urinary plasmin activates the epithelial sodium channel ENaC. Thus, urinary plasmin activity could potentially be used as a causal biomarker for proteolytic ENaC activity in patients with proteinuria. Current assays are immunologic (ELISA) that detects all moieties of plasmin (plasminogen, plasmin, anti‐plasmin‐plasmin complexes) or they are chromogenic activity assays with substrates and detection methods that are not very sensitive and also not specific. We therefore developed a luminiscense‐based assay. The assay is based on a circularly permuted firefly luciferase with a plasmin recognition site, which serves as a plasmin substrate. The uncleaved substrate is inactive, but cleavage at the plasmin site leads to reconstitution of luciferase activity. The substrate was synthesized through in vitro transcription and translation. After optimization of the assay conditions, the assay detected plasmin concentration as low as 1 ng/ml, and was not activated by urokinase‐type plasminogen activator and kallikrein. The substrate could be frozen and thawed without loss of activity, allowing the substrate to be produced in large batches. In conclusion, we have developed a sensitive and specific assay for plasmin activity allowing a rapid screening of plasmin activity in human urine samples.
Left Ventricle Cardiac Fibroblast Transcriptome Regulation by Aldosterone In Vivo
Authors: Ball Jana , Syed Maryam , Romero Damian
Published: June 2021

Excess aldosterone (ALDO) causes cardiac hypertrophy, inflammation and fibrosis that lead to cardiac dysfunction. We previously reported that ALDO/SALT time‐dependently modulates left ventricle (LV) transcriptome in vivo. However, the cardiac fibroblast (CF)‐specific transcriptome regulation by ALDO in vivo is unknown. Even the role of CFs in ALDO‐mediated cardiac injury has been questioned.

Uninephrectomized male Sprague Dawley rats were treated with ALDO (0.15 µg/h) and SALT (1.0% NaCl + 0.3% KCl) or vehicle for 2 weeks (N=6/group). LV CFs were isolated and CD90+ cells sorted by flow cytometry. Whole genome transcriptome was probed with Rat Gene 2.0‐ST Affymetrix arrays. Gene expression was analyzed with SAM software and DAVID and Ingenuity software used for bioinformatics analysis.

Isolated cells were positive for CF markers (Thy1, vimentin, Fsp1, Ddr2) while negative for cardiomyocyte (Tnnt2) and endothelial cell (CD31) markers. ALDO/SALT regulated (FC蠅1.5‐fold, FDR=10) 950 genes (740 UP and 210 DOWN). Upregulated genes were significantly enriched in the following gene ontology categories: immune response (68 genes), cell cycle (52), chemotaxis (22) and migration (32). Surprisingly, downregulated genes were significantly enriched in the gene ontology category “detection of chemical stimulus involved in sensory perception of smell” with 49 distinct GPCR olfactory receptors downregulated.

Our results show that the LV CFs are an ALDO target in vivo despite recent controversy in the field. Furthermore, we identified not only novel genes but completely new gene families that are targeted by ALDO in CFs in vivo, suggesting novel therapeutic approaches for patients with excess ALDO‐mediated cardiac injury.