bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of weight‐reducing chronic vagal nerve stimulation on cardiovascular, autonomic, and respiratory functions (1126.12)
Authors: Stauss Harald , Morgan Donald , Rahmouni Kamal
Published: June 2021

We assessed potential cardiovascular, autonomic, and respiratory adverse effects of chronic vagal nerve stimulation (VNS). In normotensive rats on standard diet, cervical vagal nerve stimulators were turned on 24 h after surgical implantation or remained off (sham) for the remainder of the study (up to 29 days). After one week of VNS, body weight was reduced by 27% while sham‐treated rats had restored pre‐surgical weight. Chronic VNS caused bradycardia that turned into tachycardia when the stimulators were temporarily turned off. VNS also resulted in hypertension that persisted when the stimulators were turned off. Compared to sham rats, high frequency heart rate variability (parasympathetic cardiac modulation) was markedly increased and low frequency systolic blood pressure variability (sympathetic modulation of vascular tone) was reduced when the stimulators were on, but not when they were off. In rats with chronic VNS a gasping‐like breathing pattern was occasionally noted. Furthermore, respiratory effort assessed by high frequency (respiration‐linked) spectral power of systolic blood pressure, was enhanced in rats with chronic VNS when the stimulators were temporarily turned off. In conclusion, weight‐reducing chronic VNS potentially causes reversible bradycardia, sustained hypertension that cannot be explained by elevated sympathetic modulation of vascular tone, and respiratory distress.

Grant Funding Source: Supported by NIH (HL084207 KR), ADA (1‐11‐BS‐127, KR), UI Fraternal Order of Eagles Diabetes Center
Circadian variation in gene expression in the medulla of DOCA/salt hypertensive rats (1126.14)
Authors: Hernandez Morgan , Erdos Benedek , Hayward Linda
Published: June 2021

Salt intake has been linked to changes in HSD2 expression in the medulla. Hypocretin (HCRT), neuropeptide FF(NPFF), and brain‐derived neurotrophic factor (BDNF) have also been implicated in feeding behavior. Since DOCA/salt treated rats primarily exhibit elevated salt intake during the night, the present study aimed to evaluate circadian related changes in gene expression in the medulla associated with DOCA/salt treatment. Adult 12 wk old Sprague Dawley (SD) and SD DOCA/salt rats (n=3‐5/group) were sacrificed at 9pm (previously identified as peak active period) or 11am (peak inactive period), and the medulla was removed for RT‐PCR analysis of gene expression. In the SDs there was a strong trend (HSD2, NPFF) or a significant (HCRT‐receptor (R1), HCRT‐R2, BDNF) increase in gene expression when transitioning from inactive to active period (100% vs 150‐869%, respectively). Gene expression in the DOCA/salt animals was significantly elevated during the inactive period when compared to SD controls (194‐558%) but DOCA/salt treated rats showed no change in gene expression between the active and inactive period. As a result, gene expression in DOCA/salt rats did not differ significantly from SDs during the active period. These data suggest DOCA/salt treatment may be involved in the interruption of normal circadian rhythms of gene expression, especially with regard to ingestive behavior and salt appetite.

Grant Funding Source: Supported by UF‐CVM
Relief of endoplasmic reticulum stress in the brain subfornical organ rescues high fat diet‐induced non‐alcoholic fatty liver disease (1126.16)
Authors: Horwath Julie , Young Colin , Butler Scott , Mark Allyn , Davisson Robin
Published: June 2021

NAFLD is a hallmark of metabolic syndrome and endoplasmic reticulum (ER) stress has been suggested as an underlying mechanism. However, the role of the brain in mediating NAFLD remains unclear. Importantly, brain ER stress has emerged as the basis of a number of chronic diseases, and we have recently demonstrated robust ER stress in the subfornical organ (SFO) of high fat diet (HFD)‐fed mice. Therefore, we tested the hypothesis that ER stress in the SFO mediates HFD‐induced NAFLD. Male C57Bl/6 mice were fed HFD (60% fat, n=13) for 20 weeks. SFO‐targeted microinjections of an adenovirus encoding the ER chaperone GRP78 (AdGRP78) to reduce ER stress, or control vector (AdLacZ), were then performed. Selective reductions in SFO‐ER stress resulted in a significant decrease in HFD‐induced hepatomegaly (2.25±0.06 vs 1.71±0.12 g, HFD AdLacZ vs HFD AdGRP78, p<0.05), with no changes in body weight (49±1 vs 48±2 g, HFD AdLacZ vs HFD AdGRP78, p>0.05), food intake or adiposity. SFO‐targeted AdGRP78 also reduced HFD‐mediated lipid droplet accumulation in the liver (histological examination) and hepatic triglyceride accumulation (203±22 vs. 96±16 mmol; HFD AdLacZ vs. HFD AdGRP78; p<0.05). These findings demonstrate a novel role for brain SFO ER stress as a contributor to the NAFLD, independent of an influence on body weight, food intake and adiposity.

Grant Funding Source: Supported by HL63887, HL84207, AHA13POST14410020, K99HL166776
WFS1‐deficient mice have alterations in energy metabolism: role of TRPM8 receptors (1126.17)
Authors: Koks Sulev , Ivask Marilin , Ehrlich Marite
Published: June 2021

Wolfram syndrome, an autosomal recessive disorder characterized by juvenile‐onset diabetes mellitus and optic atrophy, is caused by mutations in the wolframin (Wfs1) gene. Wfs1 negatively regulates endoplasmic reticulum (ER) stress signalling and therefore have a role in the pathogenesis of diabetes and regulation of metabolism. One cardinal feature of Wfs1 mutant mice is their significantly reduced body weight. The real mechanism of weight loss is not known, but these mice develop normally until the age of 8 weeks. After this period weight loss starts. We recently found significant up‐regulation of TRPM8 gene in the hippocampus of Wfs1 mutant mice. TRPM8 is menthol receptor and is involved in the regulation of energy metabolism. In present study we analysed metabolic activity and sensitivity of Wfs1 mutant mice to the menthol stimulation. We found that Wfs1 mutant mice have elevated metabolic activity in metabolic cages. Moreover, menthol induced significant increase in basal metabolic activity in Wfs1 mutant mice. Over‐activation of TRPM8 receptor can explain increased metabolic rate in Wfs1 mutant mice and their reduced body weight.

Grant Funding Source: Supported by The European Regional Development Fund
Neuro‐glio‐vascular dysfunction and epigenetic remodeling in diabetic stroke (1126.19)
Authors: Kalani Anuradha , Kamat Pardip , Tyagi Suresh , Metreveli Naira , Tyagi Neetu
Published: June 2021

Clinical studies demonstrate that stroke in diabetes leads to exacerbated brain damage and functional decline but the nature and mechanisms are still undefined. Therefore, we hypothesize that stroke in diabetes severely disturbed neuro‐glio‐vascular unit and cognitive functions instigated by epigenetic remodeling. To test this hypothesis, we employed 8‐10 week‐old male C57BL/6J (WT) sham, WT+ ischemia reperfusion (I/R), Akita (Ins2+/‐, diabetic) sham, and Akita+I/R, mice groups. Ischemia was performed for 60 min and reperfusion for 24 h. Infarct area and edema were measured with 2,3,5‐triphenyltetrazolium chloride and Evans blue extravasation, respectively. We found that infarct area, edema, and brain functions (memory, hearing) are severely affected in Akita I/R as compared to I/R group. DNMTs and HDACs activity/expressions were highly altered in Akita I/R, as compared to I/R and sham operated groups, signifies epigenetic alterations. Molecular assessments exemplified Akita I/R severity, as compared to IR group, in terms of increase in white matter damage (amyloid beta), matrix metalloproteinase (MMP‐9), glutamatergic response (NMDA), and permeability of blood vessels. On contrary, Akita I/R exhibited decrease in neural differentiation (netrin‐1, nestin) and tight junctions (ZO‐1, claudin‐5) protein/transcript expressions as compared to I/R and sham operated groups. Immunohistochemistry studies determined deep alterations of vascular connections (Cx45, CD31, GFAP) and ion channels (Na+‐K+, Ca++) in Akita I/R as compared to I/R group. These results suggest stroke severity in diabetic brain due to intense damage in neuro‐glio‐vascular unit with epigenetic remodeling and stipulate treatments much needed to be explored.

Grant Funding Source: HL107640
Glucose responsive neurons in the nucleus of the solitary tract are responsive to nesfatin‐1 (1126.3)
Authors: Mimee Andrea , Ferguson Alastair
Published: June 2021

Nesfatin‐1 (nes‐1) exerts important effects on food intake and glucose handling and is expressed in the nucleus of the solitary tract (NTS), a critical medullary autonomic control center. Since hypoglycemia induces cFos expression in nes‐1 immunoreactive NTS neurons, here we examined effects of nes‐1 on the excitability of glucose responsive NTS cells using whole‐cell patch clamp recordings. A total of 44% of neurons tested (7/16) responded to a decrease from 3 to 0.2 mM glucose, 4 with a depolarization (glucose inhibited (GI), mean 6.1 ± 1.6 mV), and 3 with a hyperpolarization (glucose excited (GE), mean ‐5.7 ± 1.4 mV). Bath applied nes‐1 (10 nM) influenced the excitability of all but one of these glucose responsive neurons. All GI neurons responded to nes‐1, 3 with a depolarization (mean 7.6 ± 2.4 mV) and 1 with a hyperpolarization (‐8.5 mV). Moreover, 2/3 GE cells were depolarized by nes‐1 (mean 3.3 ± 1.0 mV) while 1 did not respond. NTS neurons which did not respond to changes in glucose (9/16) were also nes‐1 responsive, with 5 showing a depolarization (mean 3.5 ± 0.4 mV) and 4 a hyperpolarization (mean ‐5.8 ± 0.5 mV). While our findings reveal clear actions of nes‐1 on glucose sensing NTS neurons, they do not suggest functional differentiation between the effects of nes‐1 on GI and GE neurons. Our observations lend further support to the hypothesis that nes‐1 may act as a locally released modulator of intrinsic neuronal function.

Grant Funding Source: NSERC, FQRNT, HSFO
Neuropeptide Y1 receptor is a key element in stress response to food deprivation (1126.7)
Authors: Dimitrov Eugene , DeJoseph M. , Urban Janice
Published: June 2021

The hypothalamic‐pituitary adrenal (HPA) axis functions as a default pathway for the maintenance of energy homeostasis. Food restriction or deprivation leads to prolonged HPA axis activation but the exact mechanism of this continuous HPA activation is not yet well understood. Our experiments were designed to determine whether neuropeptide Y (NPY) Y1 receptors participate in the stress response to food deprivation since NPY expression is elevated during periods of fasting. Food deprivation of male rats for 24 hours caused weight loss, adrenal gland hypertrophy and increased plasma ACTH and corticosterone concentrations. Food deprivation increased NPY mRNA expression in arcuate and brainstem C2/A2 nuclei areas that are known to project to the hypothalamic paraventricular nucleus (PVN). While the total area of CRH mRNA positive cells in the PVN decreased during food deprivation, the CRH mRNA signal intensity increased in the remaining active cells. Food deprivation also increased pCREB‐ir expression in PVN, an indication for neuronal activation. A majority of pCREB‐positive cells were CRH/Y1‐ir neurons. Infusion into the PVN of 1 nmol BIBP3226, a specific Y1 receptor antagonist, eliminated the elevated corticosterone blood levels in food‐deprived animals. Our observations support the notion that hypothalamic NPY Y1 receptors expressed by CRH neurons are pivotal for triggering a strong stress response during periods of food deprivation.

Grant Funding Source: Supported by NIMH 62621
Evidence of decreased dopamine receptor‐mediated control of gap junction coupling in the inner nuclear layer of the diabetic Ins2Akita mouse retina (1129.1)
Authors: Fortepiani Lourdes , Pyarali Monika , Akimov Nikolay , Zhang Zhijing , Ribelayga Christophe , Rentería René
Published: June 2021

Dopamine (DA) is a light‐adaptive factor that plays an important role in daytime vision. DA controls the gain of retinal signal transmission partly by controlling gap junction coupling of retinal neurons. Because preliminary data suggests that DA levels are lower in the diabetic Ins2Akita mouse retina, we hypothesized that gap junction coupling would be altered in diabetic retina. We assayed coupling using a “cut‐loading” technique, in which whole‐mounted retinas are cut with a razor dipped in gap junction‐permeable tracer Neurobiotin (NB) and quantified NB‐labeled cells in the inner nuclear layer (INL) using fluorescence microscopy. Experiments were performed under dark‐adapted conditions. The extent of coupling in control (C57Bl6) retinas was tested in the presence of the D2‐like receptor (D2R) agonist quinpirole, the D2R antagonist sulpiride, or the gap junction antagonist beta‐glycyrrhizic acid (BGA). As expected, D2R activation and BGA reduced gap junction coupling of the NB‐labeled cells in the INL, while D2R antagonism increased tracer coupling. In the diabetic Ins2Akita mice, we found that neurons in the INL showed increased tracer coupling compared to non‐diabetic littermates. We propose that this increase in gap junction coupling between retinal neurons reflects a decrease in DA levels. Decreased DA levels and an altered coupling state of retinal neurons may be hallmarks of diabetic retinas

Grant Funding Source: Supported by American Heart Association (12BGIA12040362)
Expression of NaV1.9 channels by muscle afferent neurons (1129.10)
Authors: Marler Tyler , Elmslie Keith
Published: June 2021

Background: The exercise pressor reflex helps regulate cardiovascular responses to exercise. The voltage‐gated sodium channel (Nav) 1.9 has unique properties that could help to generate excessive neuronal activity. We have previously demonstrated the presence of Nav1.9 channels using antibody staining of identified neurons, but were not able to demonstrate functional activity (J Neurophysiol. 108: 2230, 2012). Methods: We voltage‐clamped identified muscle afferent neurons isolated from adult male Sprague Dawley rats and used a high fluoride internal solution to help separate Nav1.9 from Nav1.8. Results: We found a Nav1.9‐like current that activated at voltages 20 mV hyperpolarized to the main Nav1.8 current in 69% of muscle afferent neurons (n=13). This current exhibited significantly slower activation (8 ± 3 ms; n=9) compared with cells that showed only Nav1.8 (4 ± 1 ms; n=4). The Nav current in cells with Nav1.9‐like current inactivated with multiple components compared with a single component inactivation of neurons that showed only Nav1.8. Unexpectedly, the size of the Nav1.9 current declined (ran down) with time during whole‐cell recording, while the Nav1.8 current level appeared to be invariant with time. The current density of the Nav1.9‐like current declined 43% ± 35% (mean ± SD, n=5) over approximately 5 min. We are investigating the role of dialyzable molecules such as ATP in the maintenance of Nav1.9 current. Conclusion: Nav1.9 channels are functionally expressed in muscle afferent neurons. The kinetic properties of Nav1.9 (e.g. slow inactivation) could make this channel an important regulator of muscle afferent activity to exacerbate the exercise pressor reflex.

Grant Funding Source: NIH grant AR059397 (KSE) and ATSU BMS grant (TLM)
Calcium signaling and neuropeptide secretion in the expulsion step of the Caenorhabditis elegans defecation motor program (1129.6)
Authors: McCright Sam , Peters Maureen
Published: June 2021

A periodic posterior to anterior intestinal calcium wave initiates the defecation motor program of Caenorhabditis elegans (C. elegans). innexin‐16 (inx‐16) encodes an intestinal pannexin gap junction subunit. inx‐16 mutants have faulty calcium waves and mostly lack the expulsion step of the motor program. Our work investigates how these defective calcium waves result in loss of expulsion. The anterior‐ventral neuron L (AVL) and dorsal‐ventral neuron B (DVB) are responsible for expulsion through a two‐step signaling process. An intestinal neuropeptide‐like protein, NLP‐40, activates AVL and DVB which in turn release neurotransmitters to stimulate expulsion. NLP‐40 release likely depends on intestinal calcium flux, as vesicular localization of NLP‐40 requires synaptotagmin‐2.

We propose that the expulsion defects in the inx‐16 mutant are due to improper NLP‐40 release. To establish if inx‐16’s AVL and DVB neurons are functional, we used optogenetic methods. Activation of a light‐gated ion channel channelrhodopsin‐2 (ChR2) in AVL and DVB of inx‐16 animals resulted in normal expulsion 90% of the time. This demonstrates that an inx‐16 animal’s AVL and DVB neurons are able to signal expulsion and supports our hypothesis that defects in inx‐16’s intestinal NLP‐40 release inhibit expulsion. Current work addresses whether NLP‐40 is properly synthesized and released.
Effects of pregabalin on D‐serine content and NMDA receptor‐mediated synaptic transmission in the mouse spinal cord (1129.9)
Authors: Matsuzawa Rie , Takasusuki Toshifumi , Maekawa Masao , Yamaguchi Shigaki , Hori Yuuichi
Published: June 2021

The anticonvulsant pregabalin (PGB) is used to relieve neuropathic pain. It has been shown that PGB binds to presynaptic calcium channels and subsequently inhibits transmitter release. Additionally, a preponderance of the evidence raises the possibility of other mechanisms independent of binding to calcium channels. In the present experiments, we have studied the effects of PGB on D‐serine content and NMDA receptor‐mediated synaptic transmission in the spinal cord dorsal horn.

Experiments were performed on adult ICR mice. The sciatic nerve was partially ligated under halothane anesthesia. Mechanical allodynia was assessed by von Frey filaments. PGB (30‐50 mg/kg body weight) was intraperitonealy injected daily for 5‐7 days. The lumbar spinal cord was removed, homogenized, and ultrafiltrated. Supernatant samples were treated with Marfey's reagent and analyzed with liquid chromatography‐mass spectrometry for D‐serine concentration. Tight‐seal whole‐cell recordings were made from neurons in the superficial dorsal horn of spinal cord slices. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation.

D‐serine content was 9.9±1.0 nM/g of wet tissue in sciatic nerve‐ligated mice (n=6), which was significantly larger than that in sham‐operated mice (7.9±0.8 nM/g, p < 0.05). PGB considerably decreased D‐serine content to 6.0±0.9 nM/g (n=6, p < 0.05, vs. sciatic nerve‐ligated mice). PGB decreased the NMDA/non‐NMDA ratio of the EPSC amplitude, and shortened the decay phase of the NMDA component of the EPSCs. These observations suggest that PGB decreases D‐serine concentration and thereby affects synaptic plasticity.

Grant Funding Source: Supported by a grant from the Japan Society for the Promotion of Science (23500468 to Y.H.).
Renal denervation attenuates the enhanced activation of the paraventricular nucleus in heart failure: restoring nNOS! (1130.10)
Authors: Patel Kaushik , Liu Xuefei , Llewellyn Tamra , Zheng Hong
Published: June 2021

Previously we have shown that activated paraventricular nucleus (PVN) contributes to the sympatho‐excitation in chronic heart failure (HF). Afferent renal nerves project to neurons in several brain areas, including the PVN, that are involved in the regulation of the cardiovascular system. The purpose of the present study was to determine if the activation of the PVN was mediated by afferent renal nerves. Chronic HF was induced by left coronary ligation surgery for 3‐4 weeks and rats were surgically renal denervated (RDN) for 2 weeks. RDN resulted in reduced levels of urinary norepinephrine excretion in rats with HF compared with sham‐operated control rats. RDN resulted in reduced levels of FosB staining in the PVN in rats with HF. RDN also resulted in restoration of nNOS levels (NOS histochemistry and nNOS immunostaining) in the PVN of rats with HF. We observed that microinjections of L‐NMMA (NOS inhibitor) into the PVN induced a significant smaller increase in lumbar sympathetic nerve activity in anaesthetized rats with HF compared with sham rats. In contrast, RDN restored the L‐NMMA responses in rats with HF. These results suggest that intact renal nerves contribute to the reduction of nNOS within the PVN resulting in activation of the neurons in the PVN of rats with HF. RDN restores nNOS within the PVN and thus attenuates the enhanced neuronal activation as well as the sympatho‐excitation commonly observed in HF. Supported by NIH grant HL62222 & DK82956.

Grant Funding Source: NIH grant HL62222 & DK82956.
Neuronal plasticity within the medullary dorsal horn: the nasopharyngeal response returns 3 days after bilateral sectioning of the anterior ethmoidal nerve (1130.19)
Authors: McCulloch Paul , DiNovo Karyn
Published: June 2021

Stimulation of the nasal passages with ammonia vapors can reflexly produce bradycardia, apnea, and increased peripheral resistance. However, after acute bilateral sectioning of the anterior ethmoidal nerve (AEN), the nerve that innervates the nasal passages, this nasopharyngeal response consists of only a slight apnea and an increase in arterial pressure. In the present experiments, in urethane‐anesthetized Sprague‐Dawley rats, we recorded the cardiorespiratory responses to ammonia vapor stimulation of the nasal passages 3, 5, 7 and 9 days after bilateral sectioning of the AEN, and compared these responses to those from rats with sham and acute (0 day) AEN sectioning. After nasal stimulation, heart rate (HR) decreased 62% in Sham rats but only 12% in Acute rats. After waiting 3 days following AEN sectioning, HR decreased 26%, and when waiting 9 days by 32%. The re‐establishment of the cardiorespiratory responses to nasal stimulation after bilateral AEN sectioning suggests that other nerves that innervate the nasal passages are able to provide an afferent signal sufficient to initiate the nasopharyngeal reflex. Additionally, because the bradycardia is strongly attenuated after acute AEN sectioning, our research suggests that neuronal plasticity within the medullary dorsal horn, the brainstem termination site of the AEN, may be necessary before the re‐establishment of the nasopharyngeal response can occur.

Grant Funding Source: Supported by MWU Office of Research and Sponsored Programs.
Monoamine innervation of vagal motor neurons retrogradely labelled from the subdiaphragmatic oesophagus (1131.3)
Authors: Llewellyn‐Smith Ida , Travis Lee , Watson David , Dinning Phil , Spencer Nick , Fong Angelina , Menuet Clement , Allen Andrew
Published: June 2021

Neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus pars compacta (NAc) innervate the subdiaphragmatic oesophagus in rodents but little is known about the central circuitry that controls the activity of these vagal motor neurons. We assessed the monoamine innervation of these two groups of esophageal motor neurons with immunohistochemistry, conventional retrograde tracing and anterograde tracing using lentiviral vectors that drive GFP expression in either catecholamine or 5‐HT neurons. Close appositions from axons immunoreactive for 5‐HT or catecholamine synthetic enzymes (tyrosine hydroxylase or phenylethanolamine N‐methyltransferase) occurred on many esophageal motor neurons in DMV that had been retrogradely labelled with cholera toxin B subunit (CTB) from the oesophagus. In contrast, all three types of axons very rarely apposed CTB‐labelled motor neurons in NAc. In rats with lentiviral‐induced selective GFP expression in C1, C3 or midline 5‐HT neurons, GFP‐immunoreactive axons were found in close apposition to choline acetyltransferase (ChAT)‐positive DMV neurons, but not ChAT‐positive NAc neurons. This marked difference in innervation indicates that monoamine axons of brainstem origin are likely to be important for controlling the activity of esophageal motor neurons in DMV but not those in NAc. This conclusion is consistent with control of oesophageal smooth muscle by DMV neurons and of oesophageal striated muscle by NAc neurons.

Grant Funding Source: NHMRC of Australia
Sensory re‐innervation of the rat uterus postpartum (1131.4)
Authors: Llewellyn‐Smith Ida , Gnanamanickam Greta
Published: June 2021

Sympathetic, parasympathetic and sensory nerves almost completely disappear from the pregnant rat uterus at term but return of nerves post‐partum is not well characterized. Using immunoperoxidase staining for calcitonin gene related peptide (CGRP), we assessed post‐partum (P) sensory re‐innervation of rat uterus in whole mount preparations from days P1, P3, P5, P7, P10 and P14 (n=4/group). At P1, there were few CGRP‐immunoreactive axons, mostly at the mesometrium, where a few blood vessels were lightly innervated. CGRP‐positive axons increased at P3 and P5. There were many growth cones at both times. Muscle innervation was sparse. At P7, innervation was denser, with slightly more axons in the muscle and rare CGRP‐IR axons deep in the endometrium. There were CGRP‐immunoreactive axons in the linea uteri at the ovarian end in half the rats. At P10, the pattern of CGRP innervation was similar to P7. More CGRP‐immunoreactive axons occurred in all regions but re‐innervation of the linea uteri continued to lag behind. CGRP innervation was heaviest at P14, with blood vessels moderately to heavily innervated. Nevertheless, CGRP innervation density in all uterine layers was still much less at P14 than at oestrus. These results show that sensory innervation of the uterus has begun to return at P1. Uterine CGRP innervation increases to P14 but innervation density is still lower than in the non‐pregnant state.

Grant Funding Source: Supported by NHMRC Australia
Cystitis‐induced bladder overactivity is prevented by inhibition of N‐methyl‐d‐aspartate receptor‐mediated inflammatory responses in the urinary bladder (1131.5)
Authors: Liu Miao , Shen Shanwei , Grider John , Qiao Liya
Published: June 2021

We previously show that NMDAR‐mediated signaling pathways play a significant role in regulating spinal plasticity during cyclophosphamide (CYP) ‐induced cystitis. Here we show that administration of a specific NMDAR antagonist MK801 (3mg/kg body weight, i.v.) to cystitis rats reversed bladder overactivity and inflammation at 48 h post drug treatment. In metabolic tests, MK801‐treated cystitis group had an average of 3.3 ± 1.0 voiding per hour whereas the group treated with CYP alone showed an average of 15.1 ± 5.8 voiding per hour (n=5, p<0.05). Cystometry results show that MK801 treatment of cystitis animals had a significant longer intermicturition intervals (192.0 ± 15.9 seconds(s)) when compared to CYP treatment alone (48.8 ± 4.6s, n=3, p<0.05). MK801 treatment also decreased cystitis‐induced increases in the ratio of bladder weight to body weight (CYP: 0.73 ± 0.09 mg/g; CYP+MK801: 0.35 ± 0.02 mg/g; n=5, p<0.05), and restored the integrity of urothelium damaged by CYP treatment. The effects of MK801 on cystitis‐induced bladder inflammation was also examined by its ability in reversing the elevated expression levels of pro‐inflammatory factors interlukin (IL)‐1alpha, IL‐6 and TNFalpha in the urinary bladder during cystitis. Taken together, we show that blockade of NMDAR‐mediated pathways can be an effective regimen in treatment of cystitis‐associated bladder overactivity and inflammation.

Grant Funding Source: NIH DK077917
The number of chewing affects postprandial splanchnic blood flow (1131.8)
Authors: Hamada Yuka , Kashima Hideaki , Hayashi Naoyuki
Published: June 2021

The present study was to examine an effect of the number of chewing on postprandial splanchnic blood flow (BF). After baseline measurements, ten healthy normal‐weight subjects chewed 100‐kcal or 300‐kcal solid food as long and many times as they could before swallowing in S trial, while they swallowed the same but fractured food as fast as they could in R trial. Splanchnic BF values were calculated from the diameter and mean blood velocity in the celiac artery and superior mesenteric artery, which were recorded until 90 min after eating. The number of chewing was significantly greater in the S trials than the R trials (194±22 vs. 40±3 times in 100‐kcal trials; 702±108 vs. 137±15 times in 300‐kcal trials, respectively, P<0.05). Sums of postprandial both splanchnic BFs were significantly greater in S trials than in R trials (23±5 vs. ‐5±6 L / 90 min in 100‐kcal trials; 36±11 vs. 9±4 L / 90min in 300‐kcal trials, respectively, P<0.05). These findings suggest that the number of chewing affects the postprandial splanchnic blood flow.
Intermittent hypoxia induces neuroplasticity changes in the nucleus of the solitary tract (1132.1)
Authors: Ciriello John , Moreau Jason
Published: June 2021

Intermittent hypoxia (IH) is known to alter responses of neurons in NTS to cardiovascular afferent inputs. The mechanisms related for these IH effects in NTS may involve pre‐ or post‐synaptic neuroplasticity changes. To investigate this possibility, adult male Sprague‐Dawley rats were exposed to either 1‐, 7‐ or 95‐days of IH (8 h/day) or normoxia. Protein was extracted from NTS in each group of rats and analyzed by western blot for expression of brain‐derived neurotrophic factor (BDNF), tropomyosin kinase B (TrkB), synaptophysin and growth‐associated protein‐43 (GAP‐43). Arterial pressure (AP) and heart rate were not different between the IH and normoxic animals at 1‐ or 7‐days, but AP was elevated after 95‐days of chronic IH. After 1‐day IH, TrkB protein expression was higher and synaptophysin was lower than normoxic controls. After 7‐days of IH, BDNF and TrkB protein expression was higher in NTS. After 95‐days of chronic IH exposure, BDNF, synaptophysin, and GAP‐43 protein expression was lower in NTS. These results suggest that neuroplasticity changes occurring within NTS may be associated with the elevated AP induced by chronic IH activation of chemoreceptor afferents. Additionally, these data suggest that changes in neuroplasticity within NTS may contribute to the development of autonomic dysregulation often seen in patients with chronic obstructive sleep apnea. Supported by HSFO.

Grant Funding Source: Heart and Stroke Foundation of Ontario
Acute mild stress induces cardiac arrhythmias and sudden death in sarcoglycan delta deficient mice, a model of muscular dystrophy (1132.11)
Authors: Sabharwal Rasna , Chapleau Mark
Published: June 2021

Muscular dystrophy is a catastrophic neuromuscular disease characterized by muscle wasting and weakness and dilated cardiomyopathy (DCM). We recently demonstrated that sarcoglycan delta deficient (Sgcd‐/‐) mice, an established model of muscular dystrophy, exhibit sympathovagal imbalance at a young age prior to development of DCM and premature death [Clin Auton Res 20(5):300, 2010]. We hypothesized that the combination of autonomic dysregulation and DCM in middle‐aged Sgcd‐/‐ mice confers susceptibility to acute stress‐induced cardiac arrhythmias and sudden death. Blood pressure (BP), heart rate (HR), electrocardiogram and HR variability (HRV, SD of R‐R interval) were measured by telemetry in control C57BL/6 and Sgcd‐/‐ mice (50‐60 wks of age). Acute mild psychosocial stress was induced by two methods: brief (<1min) hand‐held restraint and ~15 minute exposure to an intruder mouse. In control mice, both types of stress transiently increased HR without affecting mean BP or HRV. In contrast, Sgcd‐/‐ mice immediately assumed a supine position after hand‐held restraint for several seconds accompanied with marked increase in HR (760±20 vs. 616±20 bpm at baseline, P<0.05) and arrhythmias. Exposure of Sgcd‐/‐ mice to an intruder mouse initially increased HR mice, decreased HRV, and triggered arrhythmias (see Table). These responses were not associated with a postural change and were followed by progressive bradyarrhythmias, hypotension, ventricular fibrillation and death in 40% of Sgcd‐/‐ mice. We conclude: 1) Acute mild stress induces cardiac arrhythmias and sudden death in Sgcd‐/‐ mice; and 2) Therapeutic targeting of both autonomic dysregulation and stress disorders may yield better clinical outcomes in muscular dystrophy.

*vs. Baseline± vs. Control
Control Mice (n=4)
Sgcd‐/‐ Mice (n=5)

Baseline
Intruder Stress
Baseline
Intruder Stress

BP (mmHg)
113±1
117±2
100±3±
94±2*±

HR (bpm)
572±21
628±19*
611±10±
743±15*±

HRV (ms)
9.4±1.3
8.8±1.6
6.2±1.6±
2.9±1.1*±
Transient acidosis induces a preconditioning chloride conductance that protects mouse nodose neurons from NMDA‐induced apoptosis (1132.13)
Authors: Lu Yongjun , Wang Runping , Cicha Michael , Chapleau Mark , Abboud Francois
Published: June 2021

Glutamate is a primary neurotransmitter expressed in nodose ganglia where the soma of baroreceptor and vagal afferent neurons are located. Excessive N‐methyl‐D‐aspartate (NMDA) receptor activation by glutamate initiates excitotoxicity and may cause nodose neuronal damage and apoptosis. In previous studies we showed that brief exposure to low pH induces activation of a novel prolonged Cl‐ conductance in nodose neurons. A similar Cl‐ conductance has been shown in cardiac myocytes to be protective from ischemic reperfusion damage (Diaz et al. 1999) . Here we tested the hypothesis that vagal sensory neurons may be protected from NMDA induced excitotoxicity and apoptosis by low pHo preconditioning.

Nodose neurons of 3 months old mice were cultured and exposed to 3 brief pulses of low pH (6.0) solution each lasting 15 seconds with intervals of 10 seconds. After 20 minutes, 50 μM NMDA plus 10 μM Glycine (NMDA receptor co‐activator) were added to the cultures. Apoptotic nodose neurons were fluorescently detected by using Alexa Fluor 488 annexin V. Without preconditioning acidosis, the increases in apoptotic neurons as percentages of viable neurons during exposures to NMDA for 20, 40 and 60 minutes were 16 ± 1 (n=2346), 33 ± 4 (n=2056) and 40 ± 8% (n=2093) (p<0.01). After preconditioning acidosis at pH6.0 the increases in NMDA‐induced neuronal apoptosis became less, with levels of 6 ± 2% (n=1764), 10 ± 3% (n=1438) and 16 ± 3% (n=1678) of the total viable neurons. This pHo preconditioning effect was restored partially by 10 μM Tamoxifen, a non‐specific Cl‐ channel blocker, resulting in apoptosis levels of 12 ± 3% (n=831), 22 ± 2% (n=858) and 27 ± 2% (n=804). Thus a transient preconditioning Cl‐ conductance activated by low pHo reduced significantly the NMDA‐induced neuronal damage ( ANOVA, p<0.01). This work provides a putative molecular ionic basis for a neuronal protection during ischemic damage.

Grant Funding Source: Supported by NIH with HL14388
Fear‐induced bradycardia is abnormal in rats with heart failure (1132.3)
Authors: Koba Satoshi , Hisatome Ichiro , Watanabe Tatsuo
Published: June 2021

We examined autonomic responses to fear in conscious rats with heart failure (HF) after myocardial infarction and central neural mechanisms for the responses. Rats exposed to 5‐min white noise sound (90 dB) displayed freezing behavior (an index of fear), renal sympathoexcitation, and bradycardia. The bradycardia was parasympathetically mediated since atropine injected intravenously prevented the response in normal rats (N5). In HF rats (N7, fractional shortening<35%), the fear‐induced bradycardia was significantly (P<0.05) larger than that in normal rats (N9). Of note, in HF rats, arrhythmia appeared in association with the reduction in heart rate seen during fear. Moreover, in normal rats (N7), microinjection into the lateral/ventrolateral periaqueductal gray (l/vlPAG) of muscimol, a GABAA receptor agonist, significantly suppressed the bradycardia response to fear. Lastly, in HF rats (N6), microinjection into the l/vlPAG of Tempol, a superoxide dismutase mimetic, significantly suppressed the bradycardia response to fear. Taken together, these results suggest that fear stimulates the l/vlPAG and parasympathetically causes bradycardia in rats, and that brain oxidative stress in HF leads to dysfunction of the l/vlPAG, thereby exaggerating parasympathetic effects on heart rate and elevating risks for cardiovascular events during fear. Supported by Shimane Pref Grant for regional medicine.
Heart failure alters skeletal muscle afferent acid‐sensing ion channel subunit composition (1132.4)
Authors: Gibbons David , Gautam Mamta , Benson Christopher
Published: June 2021

Acid‐sensing ion channels (ASICs) are thought to sense metabolic changes in skeletal muscle caused by ischemia and exercise. Heart failure (HF) causes alterations in muscle afferent function and exercise capacity. To identify whether ASICs are involved, we studied the biophysical characteristics of DiI‐labeled isolated afferent neurons innervating the gastrocnemius muscle in C57Bl/6 mice with and without HF (induced by myocardial infarction) using whole‐cell patch‐clamp. The amplitudes of acid‐evoked currents and the pH dose response curves in isolated skeletal muscle afferents did not differ between control and HF mice. On the other hand, HF significantly altered the kinetics of desensitization of these afferents. Currents evoked by pH 6.8 desensitized at a slower rate, currents evoked by pH 6 desensitized faster, and the rate of recovery from desensitization was faster in muscle afferents from HF mice. We also performed Q‐PCR reactions on RNA collected from lumbar DRG from control and HF mice and found a significant decrease in TRPV1 expression, as well as altered expression of specific ASIC subunits. These results indicate that the subunit composition of ASICs in skeletal muscle afferents is altered in mice with HF, which leads to functional changes in channel properties.

Grant Funding Source: Supported by Veterans Administration
Genetic regulation of glutathione concentrations and redox balance in kidney (1134.5)
Authors: Zhou Yang , Harrison David , Love‐Myers Kimberly , Chen Yi , Grider Arthur , Wickwire Kathie , Pazdro Robert
Published: June 2021

Glutathione is an essential component of the endogenous antioxidant defense system. A decline in glutathione redox balance ― the ratio of reduced (GSH) to oxidized (GSSG) glutathione ― suggests oxidative stress and is associated with kidney disease. Despite the importance of GSH/GSSG, the genetic regulation of this trait is not well defined. In this study, we measured kidney concentrations of GSH and GSSG in mice from 30 genetically‐diverse inbred mouse strains. Genetic background caused an approximately three‐fold difference in kidney GSH/GSSG among the most disparate strains. Haplotype association mapping revealed single nucleotide polymorphisms associated with glutathione concentrations and redox balance. We narrowed the number of significant loci by placing emphasis on those located within protein‐coding genes, and we compared these candidate genes to the results of our previous genetic analysis of hepatic GSH/GSSG. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue‐specific manner. To our knowledge, this is the first quantitative trait loci study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of renal redox biochemistry and possibly inform future therapies for kidney disease.

Grant Funding Source: Supported by the following NIH grants: GM101723 (R.P.) and AG032333 (D.E.H.).
Protective effects of docosahexaenoic acid metabolites against homocysteine‐induced podocyte injury by inhibition of NLRP3 inflammasomes (1134.8)
Authors: Abais Justine , Xia Min , Boini Krishna , Li Pin‐Lan
Published: June 2021

Our recent studies have provided substantial evidence that elevated levels of homocysteine (Hcys) induce podocyte injury through activation of NLRP3 inflammasomes, importantly contributing to the initiation of the inflammatory response and eventual progression to glomerular sclerosis. Ω‐3 fatty acid docosahexaenoic acid (DHA) is widely investigated for its anti‐inflammatory properties, and DHA metabolite resolvin D1 (RvD1) is reported to aid in the resolution of inflammation. The present study tested whether RvD1 and the intermediate metabolite 17‐hydroxy docosahexaenoic acid (17‐HDHA) could prevent Hcys‐induced podocyte injury via inhibition of the NLRP3 inflammasome. By confocal microscopy, Hcys treatment was found to induce inflammasome formation in cultured podocytes, shown by an increase in colocalization between the 3 major inflammasome proteins, namely, NLRP3, apoptosis‐associated speck‐like protein (ASC), and caspase‐1. However, pretreatment of podocytes with either 17‐HDHA (100 nM) or RvD1 (60 nM) was able to inhibit this colocalization, indicating prevention of Hcys‐induced inflammasome formation. Hcys also increased caspase‐1 activation by 1.7‐fold and IL‐1β secretion by 2.2‐fold, two hallmarks of NLRP3 inflammasome activation, which was also significantly attenuated by both 17‐HDHA and RvD1. In addition to suppression of Hcys‐induced NLRP3 inflammasome formation and activation, 17‐HDHA and RvD1 treatment protected podocytes from Hcys‐induced injury and dysfunction. This was demonstrated in the restoration of VEGF secretion as well as the expression of podocyte specific marker podocin, both of which are impaired in Hcys‐treated podocytes. These results conclude that 17‐HDHA and RvD1 block Hcys‐induced podocyte damage through inhibition of the NLRP3 inflammasome, demonstrating a potential new mechanism for the protective effects of DHA‐derived metabolites 17‐HDHA and RvD1.

Grant Funding Source: supported by NIH grants DK54927 and 1F31AG043289
Increase in renal proximal tubule GLUT5 and ketohexokinase in male mice, but not female mice, in response to high‐fructose feeding may contribute to sex differences in renal responses (1135.3)
Authors: Sharma Nikhil , Li Lijun , Ecelbarger Carolyn
Published: June 2021

High‐fructose corn syrup is a highly cost‐effective sweetener in which the intake of has been associated with increased incidence of renal disease, hypertension, and other pathologies. Most fructose is metabolized by the liver; however systemic levels of fructose can rise with increased consumption. We tested whether there were sex differences in the renal responses to a high fructose (Hfr) diet. Male and female mice (n = 6/sex/diet) were fed control (cornstarch, CS) or a Hfr (65%) diet for 3 months. No differences due to diet in weight gain were observed; however, female mice had increased urine volume, plasma K+, and decreased plasma Na+ with Hfr. This effect was not apparent or markedly attenuated in the male mice. Likewise female mice showed a 2‐3 fold reduction in the expression of the thick ascending limb Na+K+2Cl cotransporter, NKCC2, and the collecting duct, aquaporin‐2 with Hfr, relative to CS‐treated females; while males had no change. In contrast, male mice showed greater changes in proteins directly involved in fructose metabolism, e.g., cortical expression of GLUT5, the facilitative fructose transporter of the proximal tubule (PT) was increased by 54% relative to CS‐treated males. Females showed a significant decrease in this protein (63%, p = 0.02 as compared to CS‐treated females). Likewise, cortical ketohexokinase, the first enzyme in fructose metabolism was increased 198% in HFr males, p = 0.0005 versus CS males, but not changed significantly in females. Moreover, cortical protein levels of the insulin receptor (β‐subunit) was also increased by 77% (p = 0.0001) in males, and not changed in females by Hfr. Overall, our results show marked sex differences in the renal response to fructose feeding. Additional studies are warranted, but one interpretation is that females had a more maladaptive response, with diuresis, changes in distal transporters, and electrolyte disturbances, while males were better able to metabolize fructose in the PT, and avoided downstream effects.

Grant Funding Source: Supported by a Marriott Fellowship and NIH DK082507
Mutation of SH2B3 attenuates Dahl SS hypertension via inflammatory signaling (1136.15)
Authors: Rudemiller Nathan , Lund Hayley , Priestley Jessica , Geurts Aron , Mattson David
Published: June 2021

Previous studies implicate infiltrating immune cells in the kidney in the pathogenesis of Dahl salt sensitive hypertension. SH2B3, an adaptor protein shown to participate in vascular and inflammatory signaling, is associated with hypertension in human GWAS (Nature Gen 41:667), though the mechanisms underlying the association are unclear. To address this issue, zinc finger nuclease targeting of the SH2 domain of SH2B3 was performed in the Dahl SS rat (SS/JrHsdMcwi) and resulted in an in‐frame, 6 base pair deletion. Following salt stress, the SH2B3 mutants (SS‐Sh2b3em1Mcwi) had significantly lower blood pressure (135 ± 1 mmHg vs 168 ± 8 mmHg, n=4‐6/group) and attenuated kidney disease as indicated by albumin excretion rate (38 ± 7 mg/day vs 104 ± 9 mg/day) than the Dahl SS. Infiltration of leukocytes in the kidneys was significantly blunted in the SH2B3 mutant compared to the Dahl SS (5.4 ± 0.5 106/kidney vs 2.7 ± 0.3 106/kidney), and qPCR results show a significant reduction of IL‐6 (2 fold) and MCP‐1(1.8 fold) gene expression in the SH2B3 cortex. Vascular reactivity of mesenteric resistance arteries via wire myography in response serotonin or acetylcholine was not different between the SH2B3 mutant and Dahl SS. The mutation in SH2B3 appears to attenuate Dahl SS hypertension via inflammatory signaling and may reflect an underlying role for inflammation in human hypertension.

Grant Funding Source: Supported by HL116264 and DK96859
The glucagon‐like peptide‐1 receptor antagonist exendin‐9 elevates blood pressure and worsens renal function in SHR (1136.18)
Authors: Aragao Gabriela , Shimizu Maria , Davel Ana , Seguro Antonio , McDonough Alicia , Girardi Adriana
Published: June 2021

Pharmacological administration of glucagon‐like peptide‐1 receptor (GLP‐1R) agonists exert cardiorenal beneficial effects beyond blood glucose lowering in type II diabetes and non‐diabetic patients, including reduction of blood pressure. The present study aimed to test the hypothesis that baseline levels of GLP‐1R signaling play a role on blood pressure regulation in hypertensive rats. To this end, 5‐week‐old SHRs were treated with the GLP‐1R antagonist exendin‐9 (EX‐9; 10 µg/rat/day), the GLP‐1R agonist exendin‐4 (EX‐4; 2.5 µg/rat/day) or saline (control) via osmotic mini‐pumps during 4 weeks. Direct recording of BP at the end of the treatment demonstrated that mean blood pressure (MBP) was elevated by exendin‐9 treatment (182±4 mmHg) and lowered by exendin‐4 treatment (161±4 mmHg) when compared to controls (172±1 mmHg, all P<0.05). Exendin‐9 treated SHR also exhibited significantly elevated proteinuria, renal fibrosis, renal TGF‐beta and angiotensinogen expression and macrophage infiltration versus controls. Conversely, exendin‐4 administration exerted anti‐proteinuric, anti‐fibrotic and anti‐inflammatory effects. These findings support the hypothesis that endogenous GLP‐1 levels exert blood pressure lowering and renoprotective effects in hypertensive rats.

Grant Funding Source: Supported by FAPESP and USC.