bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the Central Renin‐Angiotensin System (RAS) Causes Selective Cerebrovascular Dysfunction
Authors: De Silva T Michael , Lynch Cynthia , Grobe Justin , Faraci Frank
Published: June 2021

Although the central RAS is known to play important roles in several homeostatic mechanisms, its impact on the cerebral vasculature is unknown. We tested the hypothesis that activation of the central RAS with deoxycorticosterone (DOCA)‐salt (which simultaneously suppresses the peripheral RAS) alters cerebrovascular function. We treated male C57Bl/6 mice with DOCA and gave them drinking water both with and without 0.9% salt for 3 weeks and measured reactivity of arteries and arterioles in vitro in a pressure myograph and small pial arterioles in vivo using a cranial window. Blood pressure was elevated modestly after DOCA‐salt treatment (79±2 vs 95±3 mmHg; P<0.01). Endothelium‐dependent dilation of middle cerebral arteries was markedly impaired in DOCA‐salt treated mice compared to shams whereas dilation in similarly sized mesenteric arteries was normal. These striking cerebrovascular effects extended to brain parenchymal arterioles and to pial arterioles in vivo. Endothelium‐independent vasodilation to nitroprusside, nifedipine, and papaverine was similar between groups. In pial arterioles in vivo, local inhibition of angiotensin type 1 (AT1R), mineralocorticoid receptors (MR) or Rho kinase reversed endothelial dysfunction in DOCA‐salt treated mice. These findings suggest that activation of the central RAS with DOCA‐salt profoundly, but selectively, impairs endothelial function throughout the brain. Our findings identify AT1R, MR and Rho kinase as key contributors to DOCA‐salt‐induced endothelial dysfunction in brain. Studies were supported by the NIH, Department of Veterans Affairs, Fondation Leducq, and NHMRC.
Optimizing the Microfil Dye Perfusion Technique will Allow for Rat Cerebral Vascular Network Analysis through uCT Image Quantification
Authors: McCarter Monika , Balaez Alexander , Yarrow Joshua , Zubcevic Jasenka , Raizada Mohan
Published: June 2021

Introduction

Vascular pathophysiology is a hallmark of hypertension (HTN)‐linked cardiovascular disease, as increased peripheral resistance leads to vascular remodeling. Consistent with this is the evidence of links between impaired cerebral blood flow and hypoperfusion of the brainstem in HTN (1‐2). Thus, establishment of a technique for the cast dye perfusion would allow for 3D quantification of the microvasculature in order to investigate cerebral vascular changes associated with HTN and stroke.

Methods

Male SHR and Wistar Kyoto (WKY) rats (3 and 12 week) were intracardially perfused using varying procedures. Focusing on limiting vessel damage and bubbling, Protocol 1 was as follows: 20 ml Microfil dye (47% dye, 51% diluent, 1% curing agent), 0.1 ml/min. Protocols 2,3,4 and 5 followed an identical procedure, however, perfusion speeds varied from 0.1 to 35 ml/min. A PBS/Nitroprusside/Heparin mixture was added to Protocol 6 to induce greater vessel dilation, prior to perfusion with a 1:4 dye to diluent ratio, with 0.2 ml of curing agent and 0.5 ml heparin at 50 ml/min. All procedures allowed for curing overnight at 4C.

Results

uCT imaging revealed that Protocol 6 provided the best perfusion with minimum damage to microvessels.

Conclusion

Thus, our study has established a protocol to generate 3D images of the brain vasculature and puts us in a unique position to compare the impact of HTN on microvessels in the autonomic brain regions.

Acknowledgements

This research was made possible through funding from the APS UGSRF program. Special thanks to Mohan Raizada and lab for all of their support and guidance.
Clinical Salt‐Sensitive Hypertension Induced by Chronic Aortic Denervation in Rat is Dependent on the Activation of Intrarenal RAS
Authors: Rodriguez‐Martínez Manuel , López‐Rodríguez Juan , Calvo‐Turrubiartes Miriam , Casillas‐Santana Miguel , Martínez‐Mendoza Itzel
Published: June 2021

We have reported in rats (BJMBR 46:868‐880:2013) that the interaction of chronic aortic denervation (AD) and high sodium diet (HNa, 8%) increases the likelihood of developing clinical salt‐sensitive hypertension (cSSHT). To assess whether intrarenal RAS activation is involved in cSSHT, mean arterial pressure (MAP, mmHg) and plasma total nitrogen oxides ([NOx]p, µM) were measured in two occasions in each rat: after 7 days on low sodium diet (LNa = 0.04%) and again after 21 days on high sodium diet (HNa, 8%) in three groups of conscious male Wistar rats: (SHM: n = 9) sham bilaterally chronic aortic denervated , (AD: n = 9) real bilaterally chronic aortic denervated and (AD + LOS: n = 9) AD plus Losartan (10 mg/kg/day) only during the 21 days on HNa. At the end, the renal medullary superoxide anion production (RMSAP, cpm/mg) and the urinary excretion of angiotensinogen (Uagt/Ucreat, µg/g) were measured too. SSi = Salt Sensitivity index = HNaMAP ‐ LNaMAP, mmHg; Δ[NOx]s = LNa[NOx]s ‐ HNa[NOx]s, µM. Values are mean ± SEM; ° p< 0.005, *p<0.0001 vs. LNaMAP; ♦p=0.03,# p=0.01 vs. SHM.

LNa
HNa
SSi
Δ[NOx]s
RMSAP

MAP
MAP

LNa‐HNa
HNa

SHM
116
122
6
16
1.8 x 106

n = 9
±2
±2
±1
±3.0
±0.2 x 106

AD
126#
140*#
14♦
14
4.8 x 106♦

n = 9
±2
±2
±2
±2.6
±0.6 x 106

AD+LOS
126#
132°#
6
17
4.5 x 106♦

n = 9
±2
±2
±1
±3.5
±0.5 x 106

Uagt/Ucreat samples are being processed and analyzed

Conclusions

The clinical Salt‐Sensitive Hypertension (cSSHT = SSi 蠅 10 and MAP 蠅 130, mmHg) induced by chronic aortic denervation in rat is dependent on the activation of intrarenal RAS. Supported by C13‐FAI‐03‐36.36
Intracardiac Ganglion Neuron Dysfunction in Type 2 Diabetes
Authors: Li Yulong , Liu Jinxu , Zhang Dongze , Tu Huiyin , Muelleman Robert
Published: June 2021

Reduced cardiac parasympathetic activity is involved in sudden cardiac death and is responsible for high mortality in patients with type 2 diabetes mellitus (T2DM). Nicotinic acetylcholine receptor (nAChR) mediates synaptic transmission in intracardiac ganglion (ICG) and regulates the excitability of postganglionic neurons. We hypothesize that changes in the responsiveness of ICG neuronal excitability and postsynaptic nAChR to nicotine (a nAChR agonist) might be involved in attenuated cardiac vagal tone in type 2 diabetic rats. Type 2 diabetic rats were induced by a combination of both high‐fat diet and low‐dose streptozotocin (30 mg/kg i.p.) injection. As an index of cardiac vagal tone, changes of heart rate in response to vagal efferent nerve stimulation were blunted in T2DM rats, compared to sham rats. In Langendorff‐perfused hearts, there was no significant different on sensitivity of the heart to acetylcholine in sham and T2DM rats. Whole‐cell patch‐clamp data showed that nicotine increases action potential frequency and Ca2+ currents in ICG neurons from sham and T2DM rats, which was completely abolished by hexamethonium (a nAChR antagonist). T2DM reduced the responsiveness of ICG neruonal excitability and Ca2+ channel to nicotine. Additionally, T2DM decreased nAChR currents and sensitivity of the nAChR channel to nicotine in ICG neurons. Data from immunoﬂuorescence staining studies showed that T2DM did not alter the protein expression of nAChR subunits in ICG neurons. These results suggest that the low sensitivity of ICG neurons to nicotine might contribute to impairment of the cardiac vagal activity in T2DM.
Sympathetic Overactivity to the Liver: A Possible Contributor to Hepatosteatosis in Diet‐Induced Obesity?
Authors: Young Colin , Morgan Donald , Rahmouni Kamal , Mark Allyn , Davisson Robin
Published: June 2021

Obesity is associated with hepatic pathologies, such as steatosis and fibrosis, but the involvement of the autonomic nervous system in these processes remains unclear. Given that elevations in sympathetic outflow play a role in other obesity‐induced diseases (e.g., hypertension), we tested the hypothesis that obesity is associated with hepatic sympathetic overactivity. Male C57Bl/6 mice were fed a high fat diet (HFD; 60% fat; n=5) or normal chow (5% fat, n=6) for 20 weeks and were then instrumented for direct anesthetized recordings of multifiber sympathetic nerve activity (SNA) to the liver. Hepatic SNA recordings revealed a near doubling of basal sympathetic outflow to the liver in HFD mice and this was evident when evaluated as a frequency (33±2 vs 63±5 spikes/s, normal chow vs HFD, p<0.05) or integrated voltage (0.28±0.02 vs 0.46±0.03 volts/s/min, normal chow vs HFD, p<0.05). Removal of afferent input, via cutting of the hepatic nerve distal to the recording electrode, indicated that elevated efferent sympathetic outflow was a large contributor to the increased basal liver SNA (efferent SNA: 0.23±0.02 vs 0.36±0.03 volts/s/min, normal chow vs HFD, p<0.05). These findings provide the first direct evidence of hepatic sympathetic overactivity in obesity and suggest that elevated liver SNA may play a role in obesity‐mediated hepatic disorders, including hepatosteatosis.

HL63887, HL84207, K99HL166776
Neuron‐Specific (Pro)renin Receptor Deletion Regulates Renin‐Angiotensin Components and Contributes to the Amelioration of DOCA‐salt Hypertension
Authors: Ali Asghar , Li Wencheng , Sullivan Michelle , Feng Yumei
Published: June 2021

We reported that neuronal deletion of the (pro)renin receptor (PRR) attenuates DOCA‐salt hypertension, but the underlying mechanisms are unclear. To test our hypothesis that changes in renin‐angiotensin system (RAS) components contribute to beneficial effects of PRR deletion during DOCA‐salt hypertension. Wildtype (WT) and neuron‐specific PRR knockout (PRRKO) mice underwent SHAM or DOCA (50 mg) pellet implantation and were given 0.9% NaCl water. After 3 weeks, real time PCR was performed on the paraventricular nucleus of the hypothalamus to compare mRNA levels of angiotensin II type 1 or type 2 receptors (AT1R, AT2R), Mas receptors (MasR), and angiotensin‐converting enzyme 2 (ACE2). In SHAM mice, PRR deletion increased AT2R (1.46 ± 0.03 vs. 1.0 ± 0.04), MasR (2.14 ± 0.07 vs. 1.0 ± 0.05), and ACE2 (1.25 ± 0.01 vs. 1.0 ± 0.05) mRNA levels compared with WT mice, but no difference in AT1R expression was observed (1.26 ± 0.24 vs. 1.0 ± 0.07). DOCA‐salt treatment increased AT1R (2.33 ± 0.19) but lowered ACE2 mRNA levels (0.69 ± 0.03) compared with SHAM treated WT mice. There was a slight, but statistically insignificant, decrease in AT2R (0.82 ± 0.12) and MasR (0.83 ± 0.06) mRNA following DOCA‐salt treatment compared to SHAM in WT mice. Importantly, neuronal PRR deletion attenuated increases in AT1R (1.59 ± 0.20) and normalized the reduction of ACE2 mRNA levels (0.90 ± 0.04) compared with WT following DOCA‐salt treatment. However, AT2R and MasR mRNA levels remained lower in DOCA‐salt treated PRRKO relative to WT mice. In summary, neuron‐specific PRR deletion improves the balance of vasoconstrictory and vasodilatory arms of the RAS that may contribute to the attenuation of DOCA‐salt hypertension.
PVN Neuropeptide Y Contributes to the Sympathoexcitatory Effect of Leptin
Authors: Shi Zhigang , Li Baoxin , Brooks Virginia
Published: June 2021

Intracerebroventricular (ICV) leptin administration increases sympathetic nerve activity (SNA) via a neuronal pathway that includes the paraventricular nucleus of the hypothalamus (PVN). PVN Neuropeptide Y (NPY) tonically inhibits SNA via type 1 receptors (Y1R). Leptin inhibits NPY neurons; however, whether leptin increases SNA in part by decreasing tonic PVN NPY inhibition is unknown. To test this hypothesis, we determined if blockade of PVN Y1R (Y1Rx) elicits smaller increases in lumbar SNA (LSNA) after ICV leptin (3 μg) in α‐chloralose anesthetized male SD rats. LSNA was increased 90 min after ICV leptin (to 158±8% of control, n=5; P<0.05), but not after ICV artificial cerebrospinal fluid (aCSF; to 112±2%, n=5). In rats given ICV aCSF, subsequent PVN Y1Rx (bilateral BIBO3304, 60 pmol/60 nL) increased LSNA to 146±5 % of control (P<0.05). However, after ICV leptin, neither PVN Y1Rx (to 171±10 %) nor PVN aCSF (160±18 to 177±19%, n=5) significantly increased LSNA. These data support our hypothesis that leptin reduces tonic PVN NPY inhibition of LSNA. Leptin increases SNA in part via activation of PVN melanocortin 3/4 receptors (MC3/4R), and PVN presympathetic neurons express both Y1R and MC4R. Therefore, to test if leptin‐induced decreases in PVN NPY inhibition unmask MC3/4R excitation, we next determined if the MC3/4R agonist MTII increases LSNA more after Y1Rx. Bilateral PVN MTII (60 pmol/60 nL) did not alter LSNA (110±5% to 112±10 %, n=3); however, after Y1Rx, MTII increased LSNA from 124±3% to 164±6% (P<0.05, n=4). In conclusion, leptin decreases tonic PVN NPY inhibition of SNA, which may contribute to leptin‐induced increases in SNA by exposing enhanced MC3/4R excitatory drive of PVN presympathetic neurons. Supported by NIH HL088552 & AHA.
PSD95 Coupling of NMDA Receptor Activation and Nitric Oxide Activity in the Hypothalamic PVN
Authors: Bardgett Megan , Andrade Mary Ann , Calderon Alfredo , Toney Glenn
Published: June 2021

PVN regulation of sympathetic nerve activity (SNA) and mean arterial pressure (MAP) strongly depends on the balance of glutamatergic excitation by NMDAR and GABAergic inhibition facilitated by nitric oxide (NO). The adaptor protein PSD95 regulates NO production by tethering nNOS to Ca2+‐permeable GluN2B‐containing NMDA receptors. To determine the importance of PSD95 in NMDA‐NO auto‐inhibitory feedback in PVN, lentivirus encoding PSD95 shRNA or scrambled shRNA (control) was injected into PVN (400 nL/side) of male rats. Rats were anesthetized 3‐4 weeks later and NMDA was injected into PVN. Consistent with diminished NMDA‐mediated stimulation of NO in LV‐PSD95‐shRNA rats, PVN NMDA evoked greater increases of renal SNA (PSD95 shRNA: 80±15% vs control: 28±5%) and MAP (PSD95 shRNA: 13±3 mmHg vs control: 10±1 mmHg) compared to controls. Whereas the non‐selective NOS inhibitor L‐NAME in PVN had no effect on baseline renal SNA or MAP, it reduced PVN NO as measured in controls by amperometry. During L‐NAME suppression of NO in controls, PVN NMDA caused greater increases of renal SNA (NMDA: 28±5% vs NMDA+L‐NAME: 70±12%) and MAP (NMDA: 10±1 mmHg vs NMDA+L‐NAME: 21±1 mmHg). Consistent with reduced capacity for NMDA stimulation of NO in LV‐PSD95‐shRNA rats, PVN L‐NAME did not enhance renal SNA (NMDA: 62±16% vs NMDA+L‐NAME: 59±13%) or MAP (NMDA: 13±1 mmHg vs NMDA+L‐NAME: 13±1 mmHg) responses to NMDA. Results indicate that PVN PSD95 couples NMDAR activation to NO production, which restrains NMDAR driven responses. In the context of neurogenic cardiovascular diseases, decreased PVN PSD95 expression/function could contribute to loss of NO inhibition and to exaggerated glutamatergic/NMDAR driven SNA. HL 088052 & 102310 (GMT), T32 07446 (MEB)
ARTERIAL CHEMOREFLEX FUNCTION IN RENOVASCULAR HYPERTENSIVE RATS
Authors: Melo Mariana , Lemes Eduardo , Gasparini Silvia , Lauar Mariana , Speretta Guilherme , Menani Jose , Colombari Debora , Zoccal Daniel , Colombari Eduardo
Published: June 2021

Baroreflex gain decreases and chemoreflex function increases in spontaneously hypertensive rats (McBryde et al., 2013). The two kidneys, one clip (2K1C) renovascular hypertension also produces an impairment of baroreflex, but peripheral chemoreflex sensitivity in 2K1C animals is unknown. Herein, we evaluated the cardiorespiratory responses elicited by hypoxia in 2K1C rats. Male Holtzman rats (150‐180 g) received a silver clip around the left renal artery to induce 2K1C hypertension (n = 16). Rats with sham surgery were used as normotensive control (NT; n = 12). Six weeks after surgery, the tidal volume (VT), respiratory frequency (fR) and minute ventilation (VE) were measured by whole‐body plethysmography together with mean arterial pressure (MAP) and heart rate (HR) during normoxia or hypoxia (7% O2, 2 min). MAP was 206 ± 6 mmHg in 2K1C hypertensive rats and 110 ± 4 mmHg in NT (p<0.05). Baseline VE was increased in 2K1C hypertensive rats (969 ± 49 vs. NT: 776 ± 38 ml/kg/min, p<0.05). In 2K1C hypertensive rats, hypoxia induced a larger increase of VT (14 ± 1 vs. NT: 12 ± 1 ml/kg, p<0.05) and VE (1578 ± 99 vs. NT: 1305 ± 70 ml/kg/min, p<0.05). The increase in fR was similar in both groups (2K1C: 115.2 ± 4.2 vs.NT: 112.1 ± 3.3 resp.min‐1). Hypoxia produced similar changes in MAP and HR in both groups. (Δ MAP: ‐6.5 ± 5 vs. NT: ‐0.9 ± 2.0 mmHg; Δ HR: ‐1.8 ± 10.8 vs. NT: ‐7.8 ± 21.8 bpm). These results show that 2K1C hypertensive rats present baseline hyperventilation and increased ventilatory response to hypoxia, which suggests peripheral chemoreflex overactivity similar to SHR. The mechanisms underlying these changes remain to be studied. Supported by: CAPES, CNPq, FAPESP, UNESP‐PROPE.
Pressor Component of the Chemoreflex Response Is Not Mediated by P2 Purinergic Receptors in the A5 Area
Authors: Haibara Andrea , Zebral Silvia
Published: June 2021

Previous studies have shown that the noradrenergic neurons of the A5 area have an important role in the regulation of the pressor component of the chemoreflex. The aim of the present study was to evaluate the role of ATP and P2 purinergic receptors in the A5 area in the cardiovascular regulation as well as in the pressor component of the chemoreflex response in conscious rats. Unilateral microinjection of α,β‐methylene ATP (1.25 nmol/100 nl, n = 7), a selective P2 purinergic agonist, into the A5 area produced pressor (+33 ± 7 mmHg) and tachycardic responses (+66 ± 10 bpm). Ipsilateral microinjection of PPADS (0.5 nmol/100 nl), a P2 receptors antagonist, reduced the cardiovascular responses produced by α,β‐methylene ATP (+14 ± 4 mmHg and +29 ± 12 bpm) microinjected into the A5 area. Bilateral microinjection of PPADS (0.5 nmol/100 nl, n = 5) into the A5 area produced no changes in the pressor (+38 ± 6 vs +39 ± 5 mmHg) and bradycardic responses (‐253 ± 8 vs ‐237 ± 25 bpm, n = 5) induced by chemoreflex stimulation (KCN, 40 µg, i.v.). Baseline mean arterial pressure and heart rate (111 ± 5 vs 116 ± 4 mmHg and 347 ± 16 vs 335 ± 22 bpm) were also not changed by bilateral microinjection of PPADS into the A5 area. These results suggest that the P2 purinergic receptors in the A5 area participate in the modulation of the cardiovascular control, but are not involved in the integration of cardiovascular responses of the chemoreflex. Supported by Capes, INCT/CNPq, Fapemig.
Bardet‐Biedl Syndrome (BBS) Proteins in POMC Neurons are Required for Energy Homeostasis
Authors: Guo Deng , Rahmouni Kamal
Published: June 2021

BBS is a pleiotropic autosomal recessive human disorder associated with several clinical features including obesity. We previously demonstrated that Bbs1 gene deletion from the leptin receptor expressing cells caused obesity in mice highlighting the importance of neuronal BBS proteins for energy homeostasis. Here, we investigated the role of Bbs1 gene in the anorexigenic pro‐opiomelanocortin (POMC) neurons. We crossed Bbs1fl/fl mice with POMCCre mice and confirmed Cre recombinase activity in POMC cells of POMCCre/td‐Tomato reporter mice. Interestingly, POMCCre/Bbs1fl/fl mice developed obesity as indicated by the increased body weight (28%) relative to control animals. Both increased energy intake and decreased energy expenditure (as indicated by the decreased O2 consumption and heat production) contributed to the development of obesity. The emerging importance of BBS proteins for the trafficking of receptors, particularly G protein‐coupled receptors (GPCR), led us to investigate the contribution of GPCR in POMC neurons to the obesity phenotype. Using immunohistochemistry, we noticed that in hypothalamic neurons of wild type mice neuropeptide Y2 receptor (NPY2R) localize exclusively to cilia. Strikingly, deletion of the Bbs1 gene from POMC neurons abolished the ciliary localization of NPY2R in these neurons, but not in other neuronal populations. Adenyl cyclase III was not affected by Bbs1 deletion suggesting that ciliary mistrafficking of NPY2R after Bbs1 ablation is specific. In HEK 293 cells, silencing the Bbs1 gene decreased NPY2R trafficking to the plasma membrane. These findings demonstrate that BBS1 in POMC cells is involved in the regulation of energy homeostasis by controlling the trafficking of receptors such as NPY2R.
Leptin in the Hindbrain Facilitates Phosphorylation of STAT3 in the Hypothalamus
Authors: Desai Bhavna , Harris Ruth
Published: June 2021

Leptin receptors (ObRb) in the forebrain and hindbrain are each independently recognized as important mediators of leptin‐induced changes in energy balance. We recently used sub‐threshold doses of leptin to show that chronic activation of both hypothalamic and brainstem ObRb is required to reduce body fat. The objective of this study was to identify selectively activated brain nuclei in rats receiving chronic infusions of sub‐threshold leptin in both forebrain and hindbrain. Male Sprague Dawley rats were fitted with 3rd and 4th ventricle cannulas. Alzet pumps were used to infuse either saline (S) or leptin (L) (0.1 µg/24h in 3rd, 0.6 µg/24h in 4th) for 6 days, resulting in four treatment groups SS, SL, LS, LL (3th‐4th). There was no effect of leptin in single ventricle infused groups (LS, SL) compared to controls (SS). Double ventricle infused rats (LL) showed a 50% inhibition of food intake, 30% loss in body fat, and an increase in ObRb activation marker phosphorylated STAT3 (pSTAT3‐ir) in the arcuate nucleus (Arc), ventromedial (VMH), dorsomedial (DMH) and posterior hypothalamus compared to other groups. No differences in pSTAT3‐ir were observed in midbrain or brainstem nuclei despite a 6 fold higher infusion of leptin into the 4th ventricle than the 3rd. Delta Fos B‐ir, a chronic neuronal activation marker, showed that multiple brain nuclei were chronically activated due to the process of infusion, but only the Arc, VMH, DMH and ventral tuberomammillary nucleus showed a significant increase in LL rats compared to other groups. These data imply that the simultaneous activation of both forebrain and hindbrain ObRb is required to reduce body fat and that leptin in the hindbrain facilitates phosphorylation of STAT3 in hypothalamic areas involved in the control of energy balance.
Apelin Icv Infusion Blunts Brain ACE2 Overexpression‐mediated Metabolic Abnormalities in Mice
Authors: Xia Huijing
Published: June 2021

We previously reported that transgenic mice overexpressing ACE2 in the brain (SA) exhibit age‐dependent impaired metabolic function. Apelin is involved in the central regulation of energy and glucose homeostasis. ACE2 cleaves Apelin‐13 and 36 in vitro, however, the effect in vivo is unknown. We hypothesized that ACE2 overexpression alters apelin signaling in the brain, leading to metabolic dysfunction. To test this, hypothalamus Apelin levels, daily food intake, body weight, fasting blood glucose (16 h fasting) and glucose tolerance (measure blood glucose at 0, 15, 30, 60, 120 min after IP injection of glucose 2 g/kg) were determined in SA and wildtype mice (24wks old, n=5). In another set of experiments, SA mice were infused with Apelin‐13 (icv, 40 ng/d, osmotic pump) for 4 wks, metabolic parameters were measured before and after infusion. Interestingly, a significant reduction in hypothalamus Apelin level was observed in SA mice compared to wildtype (1.2±0.16 vs. 2.0±0.14 fmol/mg). This was concomitant with increased food intake (0.12±0.01 vs.0.09±0.01 g/g), higher body weight (39±1.5 vs. 31±0.8 g), elevated fasting blood glucose (147.8±13 vs.106.5±7 mg/dl) and impaired glucose tolerance (AUC: 51.6±2 vs. 41.5±1.7) in these animals (P<0.05). Following Apelin‐13 infusion, although food intake in SA mice was not affected (P>0.05), body weight was significantly reduced compared to pre‐infusion (38±1.1 vs. 44±1.1 g, P<0.05). In addition, Apelin‐13 infusion significantly decreased fasting blood glucose level (113±7 vs. 168±22 mg/dl) and improved glucose tolerance (AUC: 43±8 vs. 72±4) in SA mice (P<0.05 vs. Pre‐infusion). These data suggest that central ACE2‐overexpression decreases Apelin levels in the brain, thus contributing to metabolic abnormalities. (NIH P20 GM103514)
Stimulation of 5‐HT1A receptors of the PVN with inhibition of HPA axis on feeding
Authors: Rito Domingo Melissa , López Alonso Veronica Elsa , Reyes Santos Karla Araceli , Ambrocio Segundo Gabriela , Cruz Garcia Karina , Mancilla Díaz Juan Manuel
Published: June 2021

5‐HT1A Receptor is expressed in the paraventricular nucleus of the hypothalamus (PVN), where corticotropin release hormone is located release and synthesis, stimulation of 5‐HT1A receptor of the PVN was described to produce an increase of CRH in rats due probably stimulation of HPA. The aim of the present study was characterize the stimulation of 5‐HT1A receptor of the PVN with inhibition of HPA axis on feeding. Male Wistar rats, with 250–300 g were accustomed to a diet of separate sources of protein, carbohydrate and fat, and then were implanted a cannula on right PVN, a second surgery to identified (sham) or remove the adrenal glands bilaterally (ADX) were done. After post‐surgery recovery (5 days), rats were assigned to a group and a treatment: sham (saline solution), sham+agonist 8‐OH‐DPAT, sham+WAY 100635, sham+WAY100635+8‐OH‐DPAT, ADX (saline solution), ADX+8‐OH‐DPAT, ADX+WAY 100635, ADX+WAY 100635+8‐OH‐DPAT. Continuous record of 60 min was made for analyse the behavioral satiety sequence (BSS). The results showed that ADX decrease food intake. Administration of 8‐OH‐DPAT decrease carbohydrates and fat intake in sham groups. Pretreatment with WAY 100635 prevent hypophagic effect. BSS reveal that 8‐OH‐DPAT delay the natural development pattern of satiety. Administration 8‐OH‐DPAT increases glucose serum concentration. Agonist 8‐OH‐DPAT decrease food intake of carbohydrates in ADX groups. ADX rats interrupt the natural pattern of BSS. Finally is possible to suggest that HPA axis must be intact for the integration of signals between serotoninergic and CRH to control food intake and BSS.
Reduced Regional Cerebral White Matter Perfusion in Middle‐Aged Hispanic Adults
Authors: Pasha Evan , Tarumi Takashi , Gonzales Mitzi , Pearman Miriam , Haley Andreana , Tanaka Hirofumi
Published: June 2021

Background

Hispanics are at least 1.5 times more likely to have mild cognitive impairment and exhibit dementia symptoms 7 years earlier than non‐Hispanic Caucasians. Regional cerebral hypoperfusion has been implicated as an initial event in the pathogenesis of dementia.

Aim

To compare cerebral perfusion in brain regions susceptible to vascular related hypoperfusion in Hispanic and non‐Hispanic Caucasian adults.

Methods

Apparently healthy middle‐aged Hispanic (n=16) and non‐Hispanic Caucasian (n=16) subjects were matched across age, gender, years of education, and cognitive status. Arterial spin labeled perfusion magnetic resonance imaging estimated cerebral blood flow in a priori brain regions of interest.

Results

Mean cerebral perfusion was significantly lower in the Hispanic cohort than matched non‐Hispanic Caucasian peers in the right (38±32 vs. 76±22 ml/100 g/min, p=0.005) and left posterior white matter (39±32 vs. 72±31 ml/100 g/min, p=0.047). These differences remained significant even after controlling for blood pressure (p<0.05).

Conclusions

Middle‐aged Hispanic adults demonstrated significantly lower cerebral perfusion to the right and left posterior white matter compared with matched non‐Hispanic Caucasian peers. Hypoperfusion in these regions may contribute to cerebrovascular changes that could lead to early cognitive declines in the Hispanic population.

Supported in part by grants from the American Heart Association (09BGIA2060722, APH), the National Institute of Neurological Disorders and Stroke (R01NS75565, APH), the National Institute on Aging (F31 AG040890, MMG), and American Federation for Aging Research (8A0024, APH).
AVP release induced by increased intrarenal NaCl through renal afferent nerves in conscious rats
Authors: Kitagata Yuta , Nagai Yuko , Yamaguchi Aoi , Obata Koji , Abe Chikara , Morita Hironobu
Published: June 2021

Previous study from our laboratory demonstrated that the renal sensory afferent fibers responded to NaCl concentration in the renal artery, i.e., the renal afferent nerve activity was increased by an intrarenal arterial infusion of hypertonic NaCl solution in a dose dependent manner. However, a physiological significance of the increased renal afferent nerve activity was still unclear. We hypothesized that the renal afferent nerves may contribute to body fluid homeostasis through controlling arginine vasopressin (AVP) release. To examine this, plasma AVP was measured, while intrarenal NaCl receptors were selectively stimulated by a small dose infusion of hypertonic NaCl in conscious rats. Under a gas anesthesia, a PE‐10 catheter for NaCl infusion was inserted into the right renal artery via the suprarenal artery and a PE‐50 catheter for blood sampling was inserted into the abdominal aorta via the left femoral artery; these catheters were exteriorized from the back of the neck and connected to a swivel. One day was elapsed to recover from anesthesia and the surgery. Hyperosmotic NaCl solution (616 mmol/L) was infused into the renal artery at a rate of 50 μL/min for 10 min. Plasma Na+ concentration was not affected by this infusion (from 142.8 ± 0.6 to 143.6 ± 0.4 mmol/L), while plasma K+ concentration was significantly decreased (4.3 ± 0.1 to 4.1 ± 0.1 mmol/L). The hypertonic NaCl infusion significantly increased plasma AVP concentration from 2.8 ± 0.2 to 3.4 ± 0.4 pg/mL. This response was completely abolished by renal denervation. These data indicate that the renal sensory afferent fibers might sense changes in the intrarenal arterial NaCl concentration and alter AVP release.
Estradiol Replacement Attenuates Central Body Fluid Regulatory Responses to Hyperosmolality and Central Angiotensin II in Ovariectomized Rats
Authors: Omura Natsumi , Nishimura Yuri , Igarashi Ayako , Takamata Akira
Published: June 2021

Estrogen replacement reportedly attenuates the drinking response to systemic hypertonic saline infusion and i.c.v. angiotensin II (ANGII) administration in ovariectomized rats. In order to elucidate the site of the action of estradiol, we examined the effect of estradiol replacement on c‐Fos expression in the circumventricular organs (CVOs), and vesopressinergic neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) in response to systemic hypertonic saline and i.c.v. ANGII administrations in ovariectomized rats. We also examined ANGII type I (AT1) receptor expression in the hypothalamus and kidney by use of Western blotting in estradiol replaced and not replaced ovariectomized rats. Estradiol replacement attenuated c‐Fos expression at the organum vasclosam laminar terminalis (OVLT), supraoptic nucleus (SON) and paraventricular nucleus (PVN) in response to systemic hypertonic saline infusion, and also attenuated c‐Fos expression at the OVLT, subfornical organ, median preoptic nucleus, SON and PVN after i.c.v. ANGII administration. We also found that the AT1 receptor expression in the hypothalamus and kidney was less in estradiol‐replaced rats than estrogen deficit rats. Our data suggest that estradiol possibly attenuates AT1 receptor expression in the hypothalamus, and the attenuated AT1 receptor expression may be involved in the attenuated body fluid regulatory responses to systemic hyperosmolality and central ANGII.
Influence of estrogen and 5‐HT1A receptors in electrolyte balance and endocrine in ovariectomized rats
Authors: Fonseca Fabricia , Mecawi Andre , Araujo Iracema , Pereira Gislaine , Antunes José , Reis Luís
Published: June 2021

Thus, we examined in the present study the endocrine response in ovariectomized (OVX) rats submitted to oil or estrogen treatment (OVX‐E2 group estradiol cypionate, 100microg/kg, SC, during 14 days) and to treatment with the 8‐OH‐DPAT(8‐hydroxy‐di‐propilamino tetraline, 5‐HT1A receptor agonist, 250microg/kg, IP for 7 days) or vehicle (0.9% NaCl) during 7 days). We also investigated a possible correlation between serotonergic functional status and E2 replacement on the plasma levels of angiotensin I and II (ANG I, II), oxytocin (OT), natriuretic peptide (ANP) and corticosterone (CORT) in basal condition and after sodium depletion. The ANG I and ANG II release and respective mechanisms seem to be depending of the condition of the animal or 8‐OH‐DPAT treatment. Chronic 8‐OH‐DPAT in basal condition increased the ANG I plasma concentration while the E2 seems to attenuate this response following sodium depletion. The OT and CORT plasma levels at basal condition were enhanced by association of the two treatments. E2 replacement, both at baseline and depletion conditions, increased OT and CORT plasma levels. Additionally we did not observe estrogen influence on plasma CORT concentrations following sodium depletion; on the other hand, the 8‐OH‐DPAT seems to be critical to enhance the ANP secretion following sodium depletion. The serotonergic system seems to recruit different endocrine mechanisms regarding the homeostatic condition of the animal.
Sympathetic and respiratory activities during increases in osmolarity in an in situ rat preparation.
Authors: Silva Elaine , Bassi Mirian , Menani Jose , Zoccal Daniel , Pedrino Gustavo , Colombari Debora , Colombari Eduardo
Published: June 2021

In the present study, we investigated the changes in the thoracic sympathetic (tSNA) and phrenic nerve activity (PNA) induced by hypertonic saline (HS) infusion in different concentrations in decorticated arterially‐perfused in situ rat preparations. Juvenile male Holtzman rats (P 28‐38, 60‐100 g, n = 6/group) were used. In one group of rats, intra‐arterial infusions of HS (0.175; 0.3; 0.7; 1.5 e 2 M NaCl; 200 µL each in 15 seconds) were made in ascending order and in another group only a single infusion of 2 M NaCl was done. Infusion of 2 M NaCl increased tSNA when combined with previous HS infusions in ascending concentrations (35.1 ± 5.6% vs. Ringer solution: ‐1.1 ± 2.1%, p<0.05), whereas single infusion of 2 M NaCl did not change tSNA (‐0.1 ± 2.1% vs. Ringer solution: 1.4 ± 1.9%). Infusion of 2 M NaCl did not induce significant change in the PNA either alone (3.0 ± 0.5 bpm, Ringer solution: ‐2.5 ± 1.5 bpm) or combined with previous HS infusions in ascending concentrations (4.9 ± 1.7 bpm vs. Ringer solution: 1.5 ± 0.4 bpm). The osmolality of the perfusate increased after HS infusion in ascending concentrations (306.5 ± 1.8 vs. pre infusion: 298.3 ± 1.6 mOsm/kg; p<0.05). These data show that acute 2 M NaCl infusion increases tSNA only when preceded by the administration of ascending concentrations of HS in the in situ rat preparations.

Supported by Capes; CNPq; FAPEG and FAPESP
Combined Effects of Intermittent Hyperoxia and Intermittent Hypercapnic Hypoxia on Respiratory Control
Authors: Millstrom Alexandra , Kim Song , Asklof Kendra , McDonough Amy , Bavis Ryan
Published: June 2021

We previously observed that chronic intermittent hyperoxia attenuates the hypoxic ventilatory response (HVR) in neonatal rats. In clinical situations, however, intermittent hyperoxia alternates with bouts of hypercapnic hypoxia. In the present study, we exposed rats from birth through 14 days of age to one of three treatments: control (21% O2/0% CO2), intermittent hypercapnic hypoxia (10% O2/6% CO2, 5 episodes h‐1; I21/10), or intermittent hyperoxia + hypercapnic hypoxia (30% O2/0% CO2 to 10% O2/6% CO2, 5 episodes h‐1; I30/10). I30/10 rats exhibited slightly greater baseline ventilation than controls (measured by head‐body plethysmography) at P14, but this corresponded to a greater resting metabolic rate (in both I30/10 and I21/10 vs. controls). Although I21/10 and I30/10 treatments reduced single‐unit carotid chemoafferent responses to hypoxia (measured in vitro), the HVR was not different from that of control rats in either of these groups. Similarly, I21/10 and I30/10 treatments did not alter the hypercapnic ventilatory response. I21/10 and I30/10 rats had larger mass‐specific dry lung weight compared to controls, while continuous hyperoxia (60% O2) decreased lung size. Our results suggest that intermittent hypercapnic hypoxia counteracts the effects of intermittent hyperoxia, either by presenting an opposing stimulus (e.g., carotid body activation vs. inhibition) or by eliciting a more prominent plastic response.
A Novel Method to Quantify Diaphragm Muscle Fiber Type Clustering in the Context of Sarcopenia
Authors: Vasdev Amrit , Greising Sarah , Medina Juan , Sieck Gary , Mantilla Carlos
Published: June 2021

Sarcopenia is the age‐related decline of skeletal muscle function. Sarcopenia involves loss of motor neurons and the subsequent denervation of muscle fibers. Denervated muscle fibers can be re‐innervated through terminal axonal sprouting of neighboring axons. Given the predominant role of motor neurons in establishing muscle fiber type, we hypothesized that sarcopenia (and the associated partial denervatin effects) result in clustering of muscle fibers of the same type (classified by myosin heavy chain isoform composition). Fiber type specific immunofluorescence histology was used to quantify fiber clustering in murine diaphragm muscle (n=15) at 6 and 24 months of age. Clustering was analyzed using the distance between fibers of the same fiber type as well as quantification of the number of type‐identified fibers in a cluster. There was a 14% decrease to the closest fiber for type I and a 24% increase for type IIx and/or IIb fibers (P<0.001) in old vs. young mice. Additionally, a 34% increase to the 3 closest type IIx and/or IIb fibers was found (P<0.001). Changes in the interfiber distances for type‐identified fibers reveals important effects that consider changes in fiber size and proportion across disease conditions. This novel method of analyzing fiber type clustering may be useful in examining pathophysiological conditions of motor unit loss in neuromuscular disorders, myopathies, dystrophies, injuries, or amyotrophic lateral sclerosis.

Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.
miRNA‐29b Directly Downregulates K+ Channel Expression and Function in IPAH‐PASMC
Authors: Ayon Ramon , Pohl Nicole , Yamamura Aya , Yamamura Hisao , Makino Ayako , Yuan Jason
Published: June 2021

Downregulation of K+ channel expression and function in pulmonary artery smooth muscle cells (PASMC) is linked to the development of pulmonary vascular remodeling and sustained vasoconstriction in patients with idiopathic pulmonary arterial hypertension (IPAH). However, the underlying mechanism involved in attenuating K+ current (IK) in IPAH‐PASMC remains unclear. MicroRNAs (miRNA), which are small non‐coding RNA that post transcriptionally regulate gene expression, have been implicated in the development and progression of IPAH. In this study, we sought to determine whether miR‐29b, which is upregulated in IPAH, attenuates K+ channel expression and function. In silico analysis revealed complimentary binding sites for miR‐29b on KCNA5 and KCNMA1. Western blot analysis and luciferase assays further showed that miR‐29b directly targets and regulates K+ channel expression in IPAH‐PASMC. Overexpression of miR‐29b in normal PASMC significantly decreased Kv1.5 expression and whole‐cell IK(V) while inhibition of miR‐29b in IPAH‐PASMC rescued Kv1.5 expression and IK(V). Additionally, overexpression of miR‐29b in normal PASMC was shown to decrease whole‐cell currents through Ca2+‐activated K+ channels (BKCa channels), while inhibition of miR‐29b in IPAH‐PASMC rescued IBK(Ca). These data suggest that upregulated miR‐29b is directly involved in downregulating K+ channel expression and attenuating IK in IPAH‐PASMC. NIH Grant: HL115014, HL066012 and HL098053.
Role for Bromodomain‐Containing Protein 4 in Pulmonary Arterial Hypertension
Authors: Vaillancourt Mylène , Meloche Jolyane , Ruffenach Grégoire , Provencher Steeve , Bonnet Sébastien
Published: June 2021

Background

Pulmonary arterial hypertension (PAH) is associated with sustained DNA damage accounting for a PARP‐1‐dependent downregulation of miR‐204, promoting the expression of oncogenes (Survivin, Pim‐1). Once activated through the transcription factor NFAT, they contribute to the pro‐proliferative and anti‐apoptotic phenotype of PAH‐pulmonary arterial smooth muscle cells (PAH‐PASMC). In cancer, the epigenetic reader Bromodomain‐containing protein 4 (BRD4) sustains cell proliferation. Interestingly, BRD4 is upregulated by DNA damage and is a predicted target of miR‐204. We hypothesized that the PARP‐1/miR‐204‐dependent upregulation of BRD4 triggers the pro‐proliferative and anti‐apoptotic phenotype seen in PAH‐PASMC.

Method/Results

BRD4 is upregulated (immunoblot; p<0.01) in distal PAs and PASMC of PAH patients (n=9) compared to control (n=5). This increase is miR‐204 dependent, as miR‐204 upregulation using mimic in PAH‐PASMC decreases BRD4 (p<0.05). BRD4 activation in PAH‐PASMC induces Survivin and Pim‐1 expression (immunoblot; p<0.05), leading to mitochondrial hyperpolarization (TMRM; p<0.05), decreasing mitochondrial respiration (Seahorse XFe 24) and increasing NFAT activation (p<0.05), all of which were reversed upon molecular (siRNA) and pharmacological ((+)JQ1) inhibition of BRD4. BRD4 inhibition reverses PAH‐PASMC proliferation (Ki67; p<0.05) and resistance to apoptosis (TUNEL; p<0.05). In vivo, BRD4 inhibition reverses sugen/hypoxia induced PAH in rats.

Conclusion

Through a miR‐204‐dependent mechanism, BRD4 upregulation triggers the expression of oncogenes contributing to the proliferative and anti‐apoptotic phenotype seen in PAH‐PASMC, whereas BRD4 inhibition improves PAH.
Reno‐protective Effects of Plerixafor (AMD3100) in an Angiotensin II‐Induced Chronic Kidney Injury Model
Authors: Nair Anand , Ebenezer Philip , Francis Joseph
Published: June 2021

Angiotensin II (AngII) is a well‐known mediator of kidney injury. Previous studies suggest that AngII induces inflammation via the production of proinflammatory cytokines and reactive oxygen species. In addition to the inflammatory response, AngII also facilitates the infiltration of leukocytes into the renal tissue. CXCR4 is a chemokine receptor for stromal‐derived factor 1 (SDF‐1), a molecule known to modulate the chemotactic effects of leukocytes. Plerixafor, a CXCR4 antagonist is known to attenuate the production of proinflammatory cytokines. We examined whether treatment with plerixafor ameliorates the renal function decline and protects against AngII‐induced chronic kidney injury. AngII was administered to male Sprague‐Dawley rats for 28 days. The rats were administered either AMD3100 (Plerixafor) or saline intraperitoneally. Compared to the AngII administered rats, the animals treated with AMD3100 exhibited improved glomerular filtration rate, increased renal blood flow and a reduced renal vascular resistance. A reduction in the rate of production of free radicals was observed in the AMD3100 treated animals. Gene and protein expressions for CXCR4, SDF‐1 and proinflammatory molecules were also attenuated in animals that were treated with AMD3100, as measured by real‐time PCR and western blotting respectively. AMD3100 treated rats had decreased renal pathology and leukocyte infiltration compared to the AngII‐administered counterparts. These results provide evidence that AngII‐induced renal injury are mediated, at least in part, by CXCR4 and that CXCR4 antagonism attenuates AngII‐induced inflammatory response, possibly via attenuation of proinflammatory cytokines and reactive oxygen species.
Podocyte Specific Deletion of Acid Ceramidase Predisposes Mice to Obesity‐Induced Glomerular Injury
Authors: Boini Krishna , Xia Min , Conley Sabena , Gehr Todd , Li Pin‐Lan
Published: June 2021

Acid ceramidase (AC) as a key regulatory enzyme involved in ceramide metabolism plays a critical role in the intracellular lipid metabolism and molecular trafficking. Given recent reports that obesity enhances ceramide production, leading to the activation of NADPH oxidase and consequent development of glomerular sclerosis, the present study was designed to test whether AC contributes to the development of glomerular injury associated with obesity. First, we generated and characterized the podocyte‐specific AC knockout mice by cross breeding loxP‐floxed AC‐/‐ and nephrin cre promoter mice. The podocyte specific AC knockout (Ac‐/‐/Nephcre) and wild type (AC+/+) mice were fed a high fat diet (HFD) or normal chow (ND) for 10 weeks to produce obesity. Immunohistochemical analysis demonstrated that AC expression was reduced in glomeruli of Ac‐/‐/Nephcre mice compared to AC+/+ mice. In contrast, the ceramide level, and desmin expression were higher in glomeruli of Ac‐/‐/Nephcre than AC+/+ mice. Furthermore, Western blot, real time RT‐PCR and Immunohistochemical analyses showed that HFD significantly decreased the AC expression in Ac‐/‐/Nephcre mice, but not in AC+/+ mice. Correspondingly, the urinary protein excretion was significantly higher in HFD fed Ac‐/‐/Nephcre than Ac+/+ mice. In in vitro studies of podocytes, AC inhibitor, D‐NMAPPD decreased the podocin expression, VEGF level and increased the desmin expression compared to control cells. In conclusion, our observations reveal that normal expression of AC contributes to the function of podocytes and the defect of this gene expression is a critical mechanism triggering podocyte injury and ultimately resulting in obesity‐associated end‐stage renal disease.
RNA Sequencing to Determine Gene Expression in Rat Nephron Segments
Authors: Yang Chun , Liu Pengyuan , Zheleznova Nadya , Dayton Alexander , Cowley Allen
Published: June 2021

Whole genome RNA sequencing (RNAseq) was carried out to characterize gene expression of defined nephron segments in rats. Four defined nephron segments were manually microdissected from Dahl salt‐sensitive rats (SS) after collagenase digestion: glomerulus (Glo), proximal tubule (PXT), medullary thick ascending limb (mTAL), and outer medullary collecting duct (OMCD), about 200 tubules (0.5‐1mm) for each segment. Marker genes traditionally used to validate the purity of tubular isolation were quantified by RT‐PCR: Nphs2, Aqp1, Slc12a1 and Aqp2. RNA was extracted from the four segments from three rats yielding 100 ng for library preparation. Genome wide RNA expression levels were determined by multiplexing six library samples per lane on a 300 Gb flow cell and read paired‐end with an Illumina HiSeq 2000. The reads were aligned to the Rn5 rat genome and gene‐level or isoform expression was presented as fragments per kilobase per million reads (FPKM) enabling direct comparison of each gene between nephron segments. Each library yielded 6‐7 Gb of data with ~ 70 million reads and a mean quality score of 35 with an average mapping rate of 90%. Data analysis found a highly uniform pattern of gene expression unique to each of the tubular segments. In addition to the traditional marker genes mentioned above, other unique sets of genes were found specifically expressed in each of the segments. Examples of unique genes for each segment were: Sema3g andMgp in Glo; Spink1andPck1 in PXT;Glycam1 andPpp1r1a in mTAL; Guca2a and Defb1 in OMCD. Since these may represent more than simply novel marker genes, their unique functional roles in the segments need to be explored. (HL082798)