TNFα Triggers an Augmented Inflammatory Response in Brain Neurons from Dahl Salt-Sensitive Rats Compared with Normal Sprague Dawley Rats.

Tumor Necrosis Factor (TNF)-α is a proinflammatory cytokine (PIC) and has been implicated in a variety of illness including cardiovascular disease. The current study investigated the inflammatory response trigged by TNFα in both cultured brain neurons and the hypothalamic paraventricular nucleus (PVN), a key cardiovascular relevant brain area, of the Sprague Dawley (SD) rats. Our results demonstrated that TNFα treatment induces a dose- and time-dependent increase in mRNA expression of PICs including Interleukin (IL)-1ß and Interleukin-6 (IL6); chemokines including C-C Motif Chemokine Ligand 5 (CCL5) and C-C Motif Chemokine Ligand 12 (CCL12), inducible nitric oxide synthase (iNOS), as well as transcription factor NF-kB in cultured brain neurons from neonatal SD rats. Consistent with this finding, immunostaining shows that TNFα treatment increases immunoreactivity of IL1ß, CCL5, iNOS and stimulates activation or expression of NF-kB, in both cultured brain neurons and the PVN of adult SD rats. We further compared mRNA expression of the aforementioned genes in basal level as well as in response to TNFα challenge between SD rats and Dahl Salt-sensitive (Dahl-S) rats, an animal model of salt-sensitive hypertension. Dahl-S brain neurons presented higher baseline levels as well as greater response to TNFα challenge in mRNA expression of CCL5, iNOS and IL1ß. Furthermore, central administration of TNFα caused significant higher response in CCL12 in the PVN of Dahl-S rats. The increased inflammatory response to TNFα in Dahl-S rats may be indicative of an underlying mechanism for enhanced pressor reactivity to salt intake in the Dahl-S rat model.

The Orexin System and Hypertension.

In this review, we focus on the role of orexin signaling in blood pressure control and its potential link to hypertension by summarizing evidence from several experimental animal models of hypertension. Studies using the spontaneously hypertensive rat (SHR) animal model of human essential hypertension show that pharmacological blockade of orexin receptors reduces blood pressure in SHRs but not in Wistar-Kyoto rats. In addition, increased activity of the orexin system contributes to elevated blood pressure and sympathetic nerve activity (SNA) in dark-active period Schlager hypertensive (BPH/2J) mice, another genetic model of neurogenic hypertension. Similar to these two models, Sprague-Dawley rats with stress-induced hypertension display an overactive central orexin system. Furthermore, upregulation of the orexin receptor 1 increases firing of hypothalamic paraventricular nucleus neurons, augments SNA, and contributes to hypertension in the obese Zucker rat, an animal model of obesity-related hypertension. Finally, we propose a hypothesis for the implication of the orexin system in salt-sensitive hypertension. All of this evidence, coupled with the important role of elevated SNA in increasing blood pressure, strongly suggests that hyperactivity of the orexin system contributes to hypertension.

Activation of CaMKIIδA promotes Ca2+ leak from the sarcoplasmic reticulum in cardiomyocytes of chronic heart failure rats.

Activation of the Ca2+/calmodulin-dependent protein kinase II isoform δA (CaMKIIδA) disturbs intracellular Ca2+ homeostasis in cardiomyocytes during chronic heart failure (CHF). We hypothesized that upregulation of CaMKIIδA in cardiomyocytes might enhance Ca2+ leak from the sarcoplasmic reticulum (SR) via activation of phosphorylated ryanodine receptor type 2 (P-RyR2) and decrease Ca2+ uptake by inhibition of SR calcium ATPase 2a (SERCA2a). In this study, CHF was induced in rats by ligation of the left anterior descending coronary artery. We found that CHF caused an increase in the expression of CaMKIIδA and P-RyR2 in the left ventricle (LV). The role of CaMKIIδA in regulation of P-RyR2 was elucidated in cardiomyocytes isolated from neonatal rats in vitro. Hypoxia induced upregulation of CaMKIIδA and activation of P-RyR2 in the cardiomyocytes, which both were attenuated by knockdown of CaMKIIδA. Furthermore, we showed that knockdown of CaMKIIδA significantly decreased the Ca2+ leak from the SR elicited by hypoxia in the cardiomyocytes. In addition, CHF also induced a downregulation of SERCA2a in the LV of CHF rats. Knockdown of CaMKIIδA normalized hypoxia-induced downregulation of SERCA2a in cardiomyocytes in vitro. The results demonstrate that the inhibition of CaMKIIδA may improve cardiac function by preventing SR Ca2+ leak through downregulation of P-RyR2 and upregulation of SERCA2a expression in cardiomyocytes in CHF.

Increased activity of the orexin system in the paraventricular nucleus contributes to salt-sensitive hypertension.

The orexin system is involved in arginine vasopressin (AVP) regulation, and its overactivation has been implicated in hypertension. However, its role in salt-sensitive hypertension (SSHTN) is unknown. Here, we tested the hypothesis that hyperactivity of the orexin system in the paraventricular nucleus (PVN) contributes to SSHTN via enhancing AVP signaling. Eight-week-old male Dahl salt-sensitive (Dahl S) and age- and sex-matched Sprague-Dawley (SD) rats were placed on a high-salt (HS; 8% NaCl) or normal-salt (NS; 0.4% NaCl) diet for 4 wk. HS intake did not alter mean arterial pressure (MAP), PVN mRNA levels of orexin receptor 1 (OX1R), or OX2R but slightly increased PVN AVP mRNA expression in SD rats. HS diet induced significant increases in MAP and PVN mRNA levels of OX1R, OX2R, and AVP in Dahl S rats. Intracerebroventricular infusion of orexin A (0.2 nmol) dramatically increased AVP mRNA levels and immunoreactivity in the PVN of SD rats. Incubation of cultured hypothalamus neurons from newborn SD rats with orexin A increased AVP mRNA expression, which was attenuated by OX1R blockade. In addition, increased cerebrospinal fluid Na+ concentration through intracerebroventricular infusion of NaCl solution (4 µmol) increased PVN OX1R and AVP mRNA levels and immunoreactivity in SD rats. Furthermore, bilateral PVN microinjection of the OX1R antagonist SB-408124 resulted in a greater reduction in MAP in HS intake (-16 ± 5 mmHg) compared with NS-fed (-4 ± 4 mmHg) anesthetized Dahl S rats. These results suggest that elevated PVN OX1R activation may contribute to SSHTN by enhancing AVP signaling.NEW & NOTEWORTHY To our best knowledge, this study is the first to investigate the involvement of the orexin system in salt-sensitive hypertension. Our results suggest that the orexin system may contribute to the Dahl model of salt-sensitive hypertension by enhancing vasopressin signaling in the hypothalamic paraventricular nucleus.

Long-Term High Salt Intake Involves Reduced SK Currents and Increased Excitability of PVN Neurons with Projections to the Rostral Ventrolateral Medulla in Rats.

Evidence indicates that high salt (HS) intake activates presympathetic paraventricular nucleus (PVN) neurons, which contributes to sympathoexcitation of salt-sensitive hypertension. The present study determined whether 5 weeks of HS (2% NaCl) intake alters the small conductance Ca2+-activated potassium channel (SK) current in presympathetic PVN neurons and whether this change affects the neuronal excitability. In whole-cell voltage-clamp recordings, HS-treated rats had significantly decreased SK currents compared to rats with normal salt (NS, 0.4% NaCl) intake in PVN neurons. The sensitivity of PVN neuronal excitability in response to current injections was greater in HS group compared to NS controls. The SK channel blocker apamin augmented the neuronal excitability in both groups but had less effect on the sensitivity of the neuronal excitability in HS group compared to NS controls. In the HS group, the interspike interval (ISI) was significantly shorter than that in NS controls. Apamin significantly shortened the ISI in NS controls but had less effect in the HS group. This data suggests that HS intake reduces SK currents, which contributes to increased PVN neuronal excitability at least in part through a decrease in spike frequency adaptation and may be a precursor to the development of salt-sensitive hypertension.

Determination of Cadmium in Shrimp and Shell Fish Samples by Coprecipitation Enrichment with Mn(â ¡)-5-Br-PADAP Flame Atomic Absorption Spectrometry.

A separation/preconcentration procedure with coprecipitation has been proposed for the flame atomic absorption spectrometric (FAAS) determination of cadmium at trace level in food and environmental samples. Manganese(Ⅱ) was used as a carrier which chelated with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol to detect the content of trace cadmium in shrimp and shell fish samples with flame atomic absorption spectrometry for the first time. The precipitate could be easily dissolved with concentrated nitric acid. The optimum coprecipitation of this new method including the amount of reagent, amount of manganese(Ⅱ), the pH, and the standing time of the precipitate had been confirmed for the quantitative recoveries of the analytes. The effect of matrix ions and the interference of co-existing ions were also evaluated. Under the experimental conditions established by the optimization step, the system of Mn(Ⅱ)-5-Br-PADAP was able to overcome the matrix interference which showed the effect of separation and enrichment well. The linear range of cadmium content was determined to be 0.1～1.0 mg·L-1. The sensitivity and the relative standard deviation(RSD) were found 0.147(mg·L-1)-1, 0.73%, respectively. The optimum procedure allows the determination of cadmium with limit of detection of 4.27 µg·L-1. The complexity of preprocessing was determined by the complexity of food samples. So the differences of cadmium content in the samples between the direct determination with atomic absorption spectrometry and the measurement after coprecipitation were examined, which providedevidences for the superiority of the system again. Cadmium in shell fish and shrimp samples were 1.85 mg·kg-1 and 1.74 mg·kg-1, which in line with international standards of the Codex Alimentarius Commission(CAC). The credibility of the method was evaluated by standard additional method and recovery experiments. The standard addition recoveries of sample and RSDs of the method were in the range of 99.9%～100.3% and 0.15%～0.83%. The results of recovery experiment showed that the presented coprecipitation procedure had good repetition, high accuracy. In addition, with the method, we could draw conclusions that the experiments were simple and rapid. The developed method described in the literature was successfully applied for the determination of trace cadmium in shrimp and shell fish samples with satisfactory results.

Activation of the (pro)renin receptor in the paraventricular nucleus increases sympathetic outflow in anesthetized rats.

Previous studies have indicated that hyperactivity of brain prorenin receptors (PRR) is implicated in neurogenic hypertension. However, the role of brain PRR in regulating arterial blood pressure (ABP) is not well understood. Here, we test the hypothesis that PRR activation in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA). In anaesthetized adult Sprague-Dawley (SD) rats, bilateral PVN microinjection of human prorenin (2 pmol/side) significantly increased splanchnic SNA (SSNA; 71 ± 15%, n = 7). Preinjection of either prorenin handle region peptide, the PRR binding blocker (PRRB), or tiron (2 nmol/side), the scavenger of reactive oxygen species (ROS), significantly attenuated the increase in SSNA (PRRB: 32 ± 5% vs. control, n = 6; tiron: 8 ± 10% vs. control, n = 5; P < 0.05) evoked by prorenin injection. We further investigated the effects of PRR activation on ROS production as well as downstream gene expression using cultured hypothalamus neurons from newborn SD rats. Incubation of brain neurons with human prorenin (100 nM) dramatically enhanced ROS production and induced a time-dependent increase in mRNA levels of inducible nitric oxide synthase (iNOS), NAPDH oxidase 2 subunit cybb, and FOS-like antigen 1 (fosl1), a marker for neuronal activation and a component of transcription factor activator protein-1 (AP-1). The maximum mRNA increase in these genes occurred 6 h following incubation (iNOS: 201-fold; cybb: 2 -fold; Ffosl1: 11-fold). The increases in iNOS and cybb mRNA were not attenuated by the AT1 receptor antagonist losartan but abolished by the AP-1 blocker curcumin. Our results suggest that PVN PRR activation induces sympathoexcitation possibly through stimulation of an ANG II-independent, ROS-AP-1-iNOS signaling pathway.

Sympathoexcitation in ANG II-salt hypertension involves reduced SK channel function in the hypothalamic paraventricular nucleus.

Hypertension (HTN) resulting from subcutaneous infusion of ANG II and dietary high salt (HS) intake involves sympathoexcitation. Recently, we reported reduced small-conductance Ca(2+)-activated K(+) (SK) current and increased excitability of presympathetic neurons in the paraventricular nucleus (PVN) in ANG II-salt HTN. Here, we hypothesized that ANG II-salt HTN would be accompanied by altered PVN SK channel activity, which may contribute to sympathoexcitation in vivo. In anesthetized rats with normal salt (NS) intake, bilateral PVN microinjection of apamin (12.5 pmol/50 nl each), the SK channel blocker, remarkably elevated splanchnic sympathetic nerve activity (SSNA), renal sympathetic nerve activity (RSNA), and mean arterial pressure (MAP). In contrast, rats with ANG II-salt HTN demonstrated significantly attenuated SSNA, RSNA, and MAP (P < 0.05) responses to PVN-injected apamin compared with NS control rats. Next, we sought to examine the individual contributions of HS and subcutaneous infusion of ANG II on PVN SK channel function. SSNA, RSNA, and MAP responses to PVN-injected apamin in rats with HS alone were significantly attenuated compared with NS-fed rats. In contrast, sympathetic nerve activity responses to PVN-injected apamin in ANG II-treated rats were slightly attenuated with SSNA, demonstrating no statistical difference compared with NS-fed rats, whereas MAP responses to PVN-injected apamin were similar to NS-fed rats. Finally, Western blot analysis showed no statistical difference in SK1-SK3 expression in the PVN between NS and ANG II-salt HTN. We conclude that reduced SK channel function in the PVN is involved in the sympathoexcitation associated with ANG II-salt HTN. Dietary HS may play a dominant role in reducing SK channel function, thus contributing to sympathoexcitation in ANG II-salt HTN.

Sympathoexcitation and pressor responses induced by ethanol in the central nucleus of amygdala involves activation of NMDA receptors in rats.

The central nervous system plays an important role in regulating sympathetic outflow and arterial pressure in response to ethanol exposure. However, the underlying neural mechanisms have not been fully understood. In the present study, we tested the hypothesis that injection of ethanol in the central nucleus of the amygdala (CeA) increases sympathetic outflow, which may require the activation of local ionotropic excitatory amino acid receptors. In anesthetized rats, CeA injection of ethanol (0, 0.17, and 1.7 µmol) increased splanchnic sympathetic nerve activity (SSNA), lumbar sympathetic nerve activity (LSNA), and mean arterial pressure (MAP) in a dose-dependent manner. A cocktail containing ethanol (1.7 µmol) and kynurenate (KYN), an ionotropic excitatory amino acid receptor blocker, showed significantly blunted sympathoexcitatory and pressor responses compared with those elicited by CeA-injected ethanol alone (P < 0.01). A cocktail containing ethanol and d-2-amino-5-phosphonovalerate, an N-methyl-d-aspartate (NMDA) receptor antagonist, elicited attenuated sympathoexcitatory and pressor responses that were significantly less than ethanol alone (P < 0.01). In addition, CeA injection of acetate (0.20 µmol, n = 7), an ethanol metabolite, consistently elicited sympathoexcitatory and pressor responses, which were effectively blocked by d-2-amino-5-phosphonovalerate (n = 9, P < 0.05). Inhibition of neuronal activity of the rostral ventrolateral medulla (RVLM) with KYN significantly (P < 0.01) attenuated sympathoexcitatory responses elicited by CeA-injected ethanol. Double labeling of immune fluorescence showed NMDA NR1 receptor expression in CeA neurons projecting to the RVLM. We conclude that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in CeA neurons projecting to the RVLM.

Coping with dehydration: sympathetic activation and regulation of glutamatergic transmission in the hypothalamic PVN.

Autonomic and endocrine profiles of chronic hypertension and heart failure resemble those of acute dehydration. Importantly, all of these conditions are associated with exaggerated sympathetic nerve activity (SNA) driven by glutamatergic activation of the hypothalamic paraventricular nucleus (PVN). Here, studies sought to gain insight into mechanisms of disease by determining the role of PVN ionotropic glutamate receptors in supporting SNA and mean arterial pressure (MAP) during dehydration and by elucidating mechanisms regulating receptor activity. Blockade of PVN N-methyl-D-aspartate (NMDA) receptors reduced (P < 0.01) renal SNA and MAP in urethane-chloralose-anesthetized dehydrated (DH) (48 h water deprivation) rats, but had no effect in euhydrated (EH) controls. Blockade of PVN α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors had no effect in either group. NMDA in PVN caused dose-dependent increases of renal SNA and MAP in both groups, but the maximum agonist evoked response (Emax) of the renal SNA response was greater (P < 0.05) in DH rats. The latter was not explained by increased PVN expression of NMDA receptor NR1 subunit protein, increased PVN neuronal excitability, or decreased brain water content. Interestingly, PVN injection of the pan-specific excitatory amino acid transporter (EAAT) inhibitor DL-threo-ß-benzyloxyaspartic acid produced smaller sympathoexcitatory and pressor responses in DH rats, which was associated with reduced glial expression of EAAT2 in PVN. Like chronic hypertension and heart failure, dehydration increases excitatory NMDA receptor tone in PVN. Reduced glial-mediated glutamate uptake was identified as a key contributing factor. Defective glutamate uptake in PVN could therefore be an important, but as yet unexplored, mechanism driving sympathetic hyperactivity in chronic cardiovascular diseases.

Acetic Acid: An Underestimated Metabolite in Ethanol-Induced Changes in Regulating Cardiovascular Function.

Acetic acid is a bioactive short-chain fatty acid produced in large quantities from ethanol metabolism. In this review, we describe how acetic acid/acetate generates oxidative stress, alters the function of pre-sympathetic neurons, and can potentially influence cardiovascular function in both humans and rodents after ethanol consumption. Our recent findings from in vivo and in vitro studies support the notion that administration of acetic acid/acetate generates oxidative stress and increases sympathetic outflow, leading to alterations in arterial blood pressure. Real-time investigation of how ethanol and acetic acid/acetate modulate neural control of cardiovascular function can be conducted by microinjecting compounds into autonomic control centers of the brain and measuring changes in peripheral sympathetic nerve activity and blood pressure in response to these compounds.

Induction of Neuroinflammation and Brain Oxidative Stress by Brain-Derived Extracellular Vesicles from Hypertensive Rats.

Neuroinflammation and brain oxidative stress are recognized as significant contributors to hypertension including salt sensitive hypertension. Extracellular vesicles (EVs) play an essential role in intercellular communication in various situations, including physiological and pathological ones. Based on this evidence, we hypothesized that EVs derived from the brains of hypertensive rats with salt sensitivity could trigger neuroinflammation and oxidative stress during hypertension development. To test this hypothesis, we compared the impact of EVs isolated from the brains of hypertensive Dahl Salt-Sensitive rats (DSS) and normotensive Sprague Dawley (SD) rats on inflammatory factors and mitochondrial reactive oxygen species (mtROS) production in primary neuronal cultures and brain cardiovascular relevant regions, including the hypothalamic paraventricular nucleus (PVN) and lamina terminalis (LT). We found that brain-derived DSS-EVs significantly increased the mRNA levels of proinflammatory cytokines (PICs) and chemokines, including TNFα, IL1ß, CCL2, CCL5, and CCL12, as well as the transcriptional factor NF-κB in neuronal cultures. DSS-EVs also induced oxidative stress in neuronal cultures, as evidenced by elevated NADPH oxidase subunit CYBA coding gene mRNA levels and persistent mtROS elevation. When DSS-EVs were injected into the brains of normal SD rats, the mRNA levels of PICs, chemokines, and the chronic neuronal activity marker FOSL1 were significantly increased in the PVN and LT. Furthermore, DSS-EVs caused mtROS elevation in brain PVN and LT, particularly in neurons. Our study reveals a novel role for brain-derived EVs from hypertensive rats in triggering neuroinflammation, upregulating chemokine expression, and inducing excessive ROS production. These findings provide insight into the complex interactions between EVs and hypertension-associated processes, offering potential therapeutic targets for hypertension-linked neurological complications.

Ventricular tachyarrhythmias in rats with acute myocardial infarction involves activation of small-conductance Ca2+-activated K+ channels.

In vitro experiments have shown that the upregulation of small-conductance Ca(2+)-activated K(+) (SK) channels in ventricular epicardial myocytes is responsible for spontaneous ventricular fibrillation (VF) in failing ventricles. However, the role of SK channels in regulating VF has not yet been described in in vivo acute myocardial infarction (AMI) animals. The present study determined the role of SK channels in regulating spontaneous sustained ventricular tachycardia (SVT) and VF, the inducibility of ventricular tachyarrhythmias, and the effect of inhibition of SK channels on spontaneous SVT/VF and electrical ventricular instability in AMI rats. AMI was induced by ligation of the left anterior descending coronary artery in anesthetized rats. Spontaneous SVT/VF was analyzed, and programmed electrical stimulation was performed to evaluate the inducibility of ventricular tachyarrhythmias, ventricular effective refractory period (VERP), and VF threshold (VFT). In AMI, the duration and episodes of spontaneous SVT/VF were increased, and the inducibility of ventricular tachyarrhythmias was elevated. Pretreatment in the AMI group with the SK channel blocker apamin or UCL-1684 significantly reduced SVT/VF and inducibility of ventricular tachyarrhythmias (P < 0.05). Various doses of apamin (7.5, 22.5, 37.5, and 75.0 µg/kg iv) inhibited SVT/VF and the inducibility of ventricular tachyarrhythmias in a dose-dependent manner. Notably, no effects were observed in sham-operated controls. Additionally, VERP was shortened in AMI animals. Pretreatment in AMI animals with the SK channel blocker significantly prolonged VERP (P < 0.05). No effects were observed in sham-operated controls. Furthermore, VFT was reduced in AMI animals, and block of SK channels increased VFT in AMI animals, but, again, this was without effect in sham-operated controls. Finally, the monophasic action potential duration at 90% repolarization (MAPD(90)) was examined in the myocardial infarcted (MI) and nonmyocardial infarcted areas (NMI) of the left ventricular epicardium. Electrophysiology recordings showed that MAPD(90) in the MI area was shortened in AMI animals, and pretreatment with SK channel blocker apamin or UCL-1684 significantly prolonged MAPD(90) (P < 0.05) in the MI area but was without effect in the NMI area or in sham-operated controls. We conclude that the activation of SK channels may underlie the mechanisms of spontaneous SVT/VF and susceptibility to ventricular tachyarrhythmias in AMI. Inhibition of SK channels normalized the shortening of MAPD(90) in the MI area, which may contribute to the inhibitory effect on spontaneous SVT/VF and inducibility of ventricular tachyarrhythmias in AMI.

Ethanol Metabolite, Acetate, Increases Excitability of the Central Nucleus of Amygdala Neurons through Activation of NMDA Receptors.

The central nucleus of the amygdala (CeA) is a key brain region involved in emotional and stressor responses due to its many projections to autonomic regulatory centers. It is also a primary site of action from ethanol consumption. However, the influence of active metabolites of ethanol such as acetate on the CeA neural circuitry has yet to be elucidated. Here, we investigated the effect of acetate on CeA neurons with the axon projecting to the rostral ventrolateral medulla (CeA-RVLM), as well as quantified cytosolic calcium responses in primary neuronal cultures. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. N-Methyl-d-aspartate receptor (NMDAR) antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability and memantine suppressed the direct activation of NMDAR-dependent inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. Computational modeling of acetic acid at NMDAR/NR1 glutamatergic and glycinergic sites suggests potential active site interactions. These findings suggest that within the CeA, acetate is excitatory at least partially through activation of NMDAR, which may underlie the impact of ethanol consumption on autonomic circuitry.

Role of small conductance calcium-activated potassium channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats.

Small conductance Ca(2+)-activated K(+) (SK) channels regulate membrane properties of rostral ventrolateral medulla (RVLM) projecting hypothalamic paraventricular nucleus (PVN) neurons and inhibition of SK channels increases in vitro excitability. Here, we determined in vivo the role of PVN SK channels in regulating sympathetic nerve activity (SNA) and mean arterial pressure (MAP). In anesthetized rats, bilateral PVN microinjection of SK channel blocker with peptide apamin (0, 0.125, 1.25, 3.75, 12.5, and 25 pmol) increased splanchnic SNA (SSNA), renal SNA (RSNA), MAP, and heart rate (HR) in a dose-dependent manner. Maximum increases in SSNA, RSNA, MAP, and HR elicited by apamin (12.5 pmol, n = 7) were 330 ± 40% (P < 0.01), 271 ± 40% (P < 0.01), 29 ± 4 mmHg (P < 0.01), and 34 ± 9 beats/min (P < 0.01), respectively. PVN injection of the nonpeptide SK channel blocker UCL1684 (250 pmol, n = 7) significantly increased SSNA (P < 0.05), RSNA (P < 0.05), MAP (P < 0.05), and HR (P < 0.05). Neither apamin injected outside the PVN (12.5 pmol, n = 6) nor peripheral administration of the same dose of apamin (12.5 pmol, n = 5) evoked any significant changes in the recorded variables. PVN-injected SK channel enhancer 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO, 5 nmol, n = 4) or N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidin]amine (CyPPA, 5 nmol, n = 6) did not significantly alter the SSNA, RSNA, MAP, and HR. Western blot and RT-PCR analysis of punched PVN tissue showed abundant expression of SK1-3 channels. We conclude that SK channels expressed in the PVN play an important role in the regulation of sympathetic outflow and cardiovascular function.

Response of different benthic biotic indices to eutrophication and sediment heavy metal pollution, in fujian coastal water, East China sea.

The types and intensity of anthropogenic pressure in the same sea area may differ spatially and may change as time passes, but response of benthic biotic indices to different pressure is different, which makes it unreasonable to use the same benthic biotic indices in a large sea area. We provided a new way of thinking as to selecting benthic biotic indices according to pressure type. The study took six bays under eutrophication and sediment heavy metal pollution to different levels in Fujian coastal water, East China sea, as examples, analysed the response of five benthic biotic indices, namely AZTI marine biotic index (AMBI), multivariate AMBI (M-AMBI), Shannon-Wiener diversity index (H'), benthic opportunistic polychaetes amphipods (BOPA) and benthic polychaetes amphipods (BPA), to eutrophication factors and sediment heavy metal pollution factors firstly. The result indicated that AMBI well responded to dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorus (DIP); M-AMBI responded soundly in the range of DIN >0.131 mg L-1 and DIP >0.022 mg L-1 and responded universally to heavy metals; H' responded to only Hg and Cd; BOPA has response to eutrophication condition of DIN >0.242 mg L-1; BPA had response to DIN, Cu and As. Then, suitable indices were selected based on the four pressure scenarios in the study area. AMBI was selected in no pressure scenario; M-AMBI was chosen under only eutrophication pressure and under dual pressure; H' was preferred in only heavy metal pressure scenario (mainly Hg pollution). At last, the density plot of the distribution of the selected indices in the evaluation grades under different pressure scenarios proved the proposal of selecting benthic biotic indices according to pressure types feasible. This study can offer some new insights into rapidly choosing indices to evaluate the coastal benthic ecological quality status.

Clinical characteristics of 68 children with atypical hand, foot, and mouth disease caused by coxsackievirus A6: a single-center retrospective analysis.

Background: Hand, foot, and mouth disease (HFMD) caused by coxsackievirus A6 (CV-A6) has become prevalent in many parts of the world. It is commonly referred to as atypical HFMD which more likely to present as bullous lesions. Compared with traditional HFMD, its misdiagnosis rate is relatively high, which brings difficulties to clinical diagnosis. We retrospectively analyze the clinical characteristics of children with HFMD with bullous lesions caused by CV-A6. Methods: The study included 68 children with atypical HFMD caused by CV-A6 who were hospitalized from 2018 to 2020. Data of the children including age, sex, month of HFMD onset, the morphologies and distribution of rashes, the details of fever, the presence or absence of onychomadesis, and laboratory test results were analyzed and compared between an infant group (<1 year), a toddler group (1-<3 years), and a preschool group (3-<6 years). Results: Of the 68 children, 67 were younger than 5 years old, with a male to female ratio of 1.62:1. The disease peaked in the period from June to September. With 75.0% of the infant group had more than three kinds of rashes; 95.0% of the preschool group had rashes in more than five locations. These differences were statistically significant (P<0.05). All children had fever. The peak fever in the toddler group was lower (P=0.033). No critical cases were observed in any of the groups. Of the 61 children who were successfully followed up, 68.9% developed onychomadesis within 2-3 weeks. The proportion of cases with abnormal liver function was 83.3%, 41.7%, and 10.0% in the infant, toddler, and preschool groups (P<0.001). The proportion of cases with increased serum creatine kinase MB isoenzyme (CK-MB) were significantly higher in the toddler group (P<0.05). Conclusions: Atypical HFMD caused by CV-A6 infection usually occurred in children under 5 years old. The morphologies of the rashes in the infant group changed more, while the rashes in the preschool group was more widely distributed. The incidence of critical cases was low. More than half of the cases can develop onychomadesis in the recovery period. Organ damage was relatively mild in the preschool group.

Maternal diabetes increases large conductance Ca2+-activated K+ outward currents that alter action potential properties but do not contribute to attenuated excitability of parasympathetic cardiac motoneurons in the nucleus ambiguus of neonatal mice.

Previously, we demonstrated that maternal diabetes reduced the excitability and increased small-conductance Ca(2+)-activated K(+) (SK) currents of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA). In addition, blockade of SK channels with apamin completely abolished this reduction. In the present study, we examined whether maternal diabetes affects large-conductance Ca(2+)-activated K(+) (BK) channels and whether BK channels contribute to the attenuation of PCMN excitability observed in neonates of diabetic mothers. Neonatal mice from OVE26 diabetic mothers (NMDM) and normal FVB mothers (control) were used. The pericardial sac of neonatal mice at postnatal days 7-9 was injected with the tracer X-rhodamine-5 (and 6)-isothiocyanate 2 days prior to the experiment to retrogradely label PCMNs in the NA. Whole cell current- and voltage-clamps were used to measure spike frequency, action potential (AP) repolarization (half-width), afterhyperpolarization potential (AHP), transient outward currents, and afterhyperpolarization currents (I(AHP)). In whole cell voltage clamp mode, we confirmed that maternal diabetes increased transient outward currents and I(AHP) compared with normal cells. Using BK channel blockers charybdotoxin (CTx) and paxilline, we found that maternal diabetes increased CTx- and paxilline-sensitive transient outward currents but did not change CTx- and paxilline-sensitive I(AHP). In whole cell current-clamp mode, we confirmed that maternal diabetes increased AP half-width and AHP, and reduced excitability of PCMNs. Furthermore, we found that after blockade of BK channels with CTx or paxilline, maternal diabetes induced a greater increase of AP half-width but similarly decreased fast AHP without affecting medium AHP. Finally, blockade of BK channels decreased spike frequency in response to current injection in both control and NMDM without reducing the difference of spike frequency between the two groups. Therefore, we conclude that although BK transient outward currents, which may alter AP repolarization, are increased in NMDM, BK channels do not directly contribute to maternal diabetes-induced attenuation of PCMN excitability. In contrast, based on evidence from our previous and present studies, reduction of PCMN excitability in neonates of diabetic mothers is largely dependent on altered SK current associated with maternal diabetes.

Activation of Orexin 1 Receptors in the Paraventricular Nucleus Contributes to the Development of Deoxycorticosterone Acetate-Salt Hypertension Through Regulation of Vasopressin.

Salt-sensitivity is a major factor in the development of hypertension. The brain orexin system has been observed to play a role in numerous hypertensive animal models. However, orexin's role in the pathology of salt-sensitive hypertension (SSH) remains to be adequately explored. We assessed the impact of orexin hyperactivity in the pathogenesis of the deoxycorticosterone acetate (DOCA) - salt rat model, specifically through modulation of Arginine Vasopressin (AVP). Adult male rats were separated into three groups: vehicle control, DOCA-salt, and DOCA-salt+OX1R-shRNA. DOCA-salt rats received subcutaneous implantation of a 21-day release, 75 mg DOCA pellet in addition to saline drinking water (1% NaCl and 0.2% KCl). DOCA-salt+OX1R-shRNA rats received bilateral microinjection of AAV2-OX1R-shRNA into the paraventricular nucleus (PVN) to knockdown function of the Orexin 1-Receptor (OX1R) within that area. Following 2-week to allow full transgene expression, a DOCA pellet was administered in addition to saline drinking solution. Vehicle controls received sham DOCA implantation but were given normal water. During the 3-week DOCA-salt or sham treatment period, mean arterial pressure (MAP) and heart rate (HR) were monitored utilizing tail-cuff plethysmography. Following the 3-week period, rat brains were collected for either PCR mRNA analysis, as well as immunostaining. Plasma samples were collected and subjected to ELISA analysis. In line with our hypothesis, OX1R expression was elevated in the PVN of DOCA-salt treated rats when compared to controls. Furthermore, following chronic knockdown of OX1R, the hypertension development normally induced by DOCA-salt treatment was significantly diminished in the DOCA-salt+OX1R-shRNA group. A concurrent reduction in PVN OX1R and AVP mRNA was observed in concert with the reduced blood pressure following AAV2-OX1R-shRNA treatment. Similarly, plasma AVP concentrations appeared to be reduced in the DOCA-salt+OX1R-shRNA group when compared to DOCA-salt rats. These results indicate that orexin signaling, specifically through the OX1R in the PVN are critical for the onset and maintenance of hypertension in the DOCA-salt model. This relationship is mediated, at least in part, through orexin activation of AVP producing neurons, and the subsequent release of AVP into the periphery. Our results outline a promising mechanism underlying the development of SSH through interactions with the brain orexin system.