Conscious Wistar Rats Research Articles

Piezo1-pannexin-1-P2X3 axis in odontoblasts and neurons mediates sensory transduction in dentinal sensitivity.

According to the "hydrodynamic theory," dentinal pain or sensitivity is caused by dentinal fluid movement following the application of various stimuli to the dentin surface. Recent convergent evidence in Vitro has shown that plasma membrane deformation, mimicking dentinal fluid movement, activates mechanosensitive transient receptor potential (TRP)/Piezo channels in odontoblasts, with the Ca2+ signal eliciting the release of ATP from pannexin-1 (PANX-1). The released ATP activates the P2X3 receptor, which generates and propagates action potentials in the intradental Aδ afferent neurons. Thus, odontoblasts act as sensory receptor cells, and odontoblast-neuron signal communication established by the TRP/Piezo channel-PANX-1-P2X3 receptor complex may describe the mechanism of the sensory transduction sequence for dentinal sensitivity. To determine whether odontoblast-neuron communication and odontoblasts acting as sensory receptors are essential for generating dentinal pain, we evaluated nociceptive scores by analyzing behaviors evoked by dentinal sensitivity in conscious Wistar rats and Cre-mediated transgenic mouse models. In the dentin-exposed group, treatment with a bonding agent on the dentin surface, as well as systemic administration of A-317491 (P2X3 receptor antagonist), mefloquine and 10PANX (non-selective and selective PANX-1 antagonists), GsMTx-4 (selective Piezo1 channel antagonist), and HC-030031 (selective TRPA1 channel antagonist), but not HC-070 (selective TRPC5 channel antagonist), significantly reduced nociceptive scores following cold water (0.1ml) stimulation of the exposed dentin surface of the incisors compared to the scores of rats without local or systemic treatment. When we applied cold water stimulation to the exposed dentin surface of the lower first molar, nociceptive scores in the rats with systemic administration of A-317491, 10PANX, and GsMTx-4 were significantly reduced compared to those in the rats without systemic treatment. Dentin-exposed mice, with somatic odontoblast-specific depletion, also showed significant reduction in the nociceptive scores compared to those of Cre-mediated transgenic mice, which did not show any type of cell deletion, including odontoblasts. In the odontoblast-eliminated mice, P2X3 receptor-positive A-neurons were morphologically intact. These results indicate that neurotransmission between odontoblasts and neurons mediated by the Piezo1/TRPA1-pannexin-1-P2X3 receptor axis is necessary for the development of dentinal pain. In addition, odontoblasts are necessary for sensory transduction to generate dentinal sensitivity as mechanosensory receptor cells.

A method to evaluate dynamic cerebral pressure-flow relationships in the conscious rat.

The classic dogma of cerebral autoregulation is that cerebral blood flow is steadily maintained across a wide range of perfusion pressures. This has been challenged by recent studies suggesting little to no "autoregulatory plateau" in the relationship between cerebral blood flow and blood pressure (BP). Therefore, the mechanisms underlying the cerebral pressure-flow relationship still require further understanding. Here, we present a novel approach to examine dynamic cerebral autoregulation in conscious Wistar rats (n = 16) instrumented to measure BP and internal carotid blood flow (iCBF), as an indicator of cerebral blood flow. Transient reductions in BP were induced by occluding the vena cava via inflation of a chronically implanted intravascular silicone balloon. Falls in BP were paralleled by progressive decreases in iCBF, with no evidence of a steady-state plateau. No significant changes in internal carotid vascular resistance (iCVR) were observed. In contrast, intravenous infusions of the vasoactive drug sodium nitroprusside (SNP) produced a similar fall in BP but increases in iCBF and decreases in iCVR were observed. These data suggest a considerable confounding influence of vasodilatory drugs such as SNP on cerebrovascular tone in the rat, making them unsuitable to investigate cerebral autoregulation. We demonstrate that our technique of transient vena cava occlusion produced reliable and repeatable depressor responses, highlighting the potential for our approach to permit assessment of the dynamic cerebral pressure-flow relationship over time in conscious rats.NEW & NOTEWORTHY We present a novel technique to overcome the use of vasoactive agents when studying cerebrovascular dynamics in the conscious rat. Our method of vena cava occlusion to reduce BP was associated with decreased iCBF and no change in iCVR. In contrast, comparable BP falls with intravenous SNP increased iCBF and reduced iCVR. Thus, the dynamic cerebral pressure-flow relationship shows a narrower, less level autoregulatory plateau than conventionally thought. We confirm our method allows repeatable assessment of cerebrovascular dynamics in conscious rats.

Effects of intracerebroventricular leptin and orexin-A on the baroreflex control of renal sympathetic nerve activity in conscious rats fed a normal or high-fat diet.

This study examined the effect of leptin and orexin-A on autonomic baroreflex control in conscious Wistar rats exposed to high-fat (45% fat) or normal (3.4%) diet for 4weeks. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were monitored during the generation of baroreflex gain curves and acute volume expansion (VEP). Intracerebroventricular (ICV) leptin (1μg/min) increased RSNA in the normal diet group (0.31±0.04 vs 0.23±0.03mV/s) and MAP in the high-fat diet group (115±5 vs 105±5mm Hg, P<.05). Orexin-A (50ng/min) increased RSNA, HR and MAP in the high-fat diet group (0.26±0.03 vs 0.22±0.02mV/s, 454±8 vs 417±12beats/min, 117±1 vs 108±1mm Hg) and the normal diet group (0.18±0.05 vs 0.17±0.05mV/s, 465±10 vs 426±6beats/min, 116±2 vs 104±3 mmHg). Baroreflex sensitivity for RSNA was increased during ICV leptin by 50% in the normal diet group, compared to 14% in the high-fat diet group (P<.05). Similarly, orexin-A increased baroreflex sensitivity by 56% and 50% in the high-fat and normal diet groups, respectively (all P<.05). During ICV saline, VEP decreased RSNA by 31±5% (P<.05) after 10minutes and the magnitude of this response was blunted during ICV infusion of leptin (17±2%, P<.05) but not orexin-A in the normal diet group. RSNA response to VEP was not changed during ICV leptin or orexin-A in the high-fat diet group. These findings indicate possible central roles for leptin and orexin-A in modulating the baroreflexes under normal or increased fat intake in conscious rats and potential therapeutic approaches for obesity associated hypertension.

Neuroprotection by Insulin-like Growth Factor-1 in Rats with Ischemic Stroke is Associated with Microglial Changes and a Reduction in Neuroinflammation

We and others have shown that insulin-like growth factor-1 (IGF-1) is neuroprotective when administered systemically shortly following stroke. In the current study, we addressed the hypothesis that microglia mediate neuroprotection by IGF-1 following ischemic stroke. Furthermore, we investigated whether IGF-1 modulates pro- and anti-inflammatory mediators in ischemic brain with a special reference to microglia. Ischemic stroke was induced in normal conscious Wistar rats by infusing the vasoconstrictor, endothelin-1 (Et-1), next to middle cerebral artery (MCA). IGF-1 (300 μg) was injected subcutaneously (SC) at 30 and 120 min following stroke. Microglial inhibitor, minocycline, was injected intraperitoneally (IP) at 1 h before stroke (25 mg/kg) and 11 h after stroke (45 mg/kg). Post-stroke IGF-1 treatment reduced the infarct size and increased the sensorimotor function which coincided with an increase in the number of ameboid microglia in the ischemic cortex. Minocycline treatment abrogated the increase in ameboid microglia by IGF-1, while the effect of IGF-1 in the reduction of infarct size was only partially affected. IGF-1 suppressed mRNA expression of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β in the ischemic hemisphere, while in purified microglia, only iNOS expression levels were reduced. Our findings show that microglia are a target for IGF-1 and that neuroprotection by IGF-1 coincides with down-regulation of inflammatory mediators which could be instrumental to the beneficial effects.

Phase synchronization of baroreflex oscillations of blood pressure and pulse interval in rats: the effects of cardiac autonomic blockade and gradual blood loss

Objective: Phase synchronization of arterial pressure (AP) and pulse interval (PI) oscillations in the low-frequency band (around 0.4 Hz in rats) is governed by baroreflex activity. In long-term stationary data recordings, such synchronization can be estimated by the coherence. The phase synchronization index (PSI) can be used as well. The aim of this study was to correlate PSI and the coherence of AP and PI under stationary conditions and to estimate the informativity of PSI as a measure of baroreflex activity during transient processes. Approach: AP and PI were recorded in conscious Wistar rats using femoral artery catheters. To study the hemodynamics during hemorrhage, blood was gradually withdrawn (20 ml × kg−1 over 30 min) through a catheter in the carotid artery. Main results: PSI and coherence spectra calculated from 30-minute AP and PI recordings demonstrated distinct peaks at the frequency of 0.4 Hz; these indicators correlate well with each other (Pearson r = 0.920, p < 0.0001). Both PSI and coherence were markedly suppressed by vagal blockade (methylatropine) and tended to reduce after sympathetic blockade (atenolol). Importantly, PSI demonstrated dynamic alterations during gradual hemorrhage. During the initial approx. 10 min of hemorrhage, AP did not change but PI was noticeably shortened, and PSI increased, which indicates the activation of the baroreflex. With further blood loss, baroreflex influences were not enough to prevent blood pressure from falling, and under such conditions PSI decreased. Significance: PSI, like coherence, is an informative measure of baroreflex activity under stationary conditions. In addition, PSI permits us to follow the coupling between the baroreflex oscillations of AP and PI during transient processes, which strengthens its informative value.

Role of Vasopressin on Intrinsic Cardiac Activity in Conscious Wistar Rats.

BackgroundThe effects of vasopressin on cardiac chronotropism are controversial in different species.ObjectiveTo investigate if vasopressin can influence the cardiac intrinsic activity in conscious Wistar rats.MethodsAll the procedures of this study were approved by the Animal ethics committee (protocol #02/2016). Adult male Wistar rats underwent to a cannulation of the femoral artery and vein for mean arterial pressure (MAP) and heart rate (HR) recordings and drug infusions, respectively, in conscious rats, 24 h after the surgical procedures. After baseline recordings, i.v. atropine 2 mg/kg and i.v. metoprolol 1.7 mg/kg were injected with 5 min interval. After 10 min, i.v. vasopressin 0.0625 mg/mL/5 min (N=6) or saline (1 mL/5 min) (N=6) were infused and the variables were recorded for additional 10 min. Gene and protein expression of V1a, V1b and V2 subtype receptors for AVP were also carried in the right atrium by qPCR and Western Blotting, respectively. The AVP receptor subtypes were also labeled by immunofluorescence technique in the right atrium.ResultsVasopressin evoked bradycardic (−51±5 bpm) and pressor responses (39±6 mmHg) compared to baseline post‐autonomic blockade (130 ±8 mmHg and 406±16 bpm). Saline did not change HR (1±4 bpm) and MAP (−3±1 mmHg) compared to baseline after autonomic blockade (120±5 mmHg and 411±52 bpm). All subtypes of AVP receptors were expressed in the right atrium by qPCR, Western Blotting and immunofluorescence.ConclusionThe bradycardia elicited by vasopressin after cardiac autonomic blockade suggests that vasopressin can affect the cardiac intrinsic activity binding to the receptors in the right atrium.Support or Funding InformationFinancial support: FAPESP, CNPq and NEPAS‐FMABC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypothalamic paraventricular nucleus neuronal nitric oxide synthase activity is a major determinant of renal sympathetic discharge in conscious Wistar rats.

What is the central question of this study? Does chronic reduction of neuronally generated nitric oxide in the hypothalamic paraventricular nucleus affect the set-point regulation of blood pressure and sympathetic activity destined to the kidneys? What is the main finding and its importance? Within the hypothalamic paraventricular nucleus, nitric oxide generated by neuronal nitric oxide synthase plays a major constitutive role in suppressing long term the levels of both ongoing renal sympathetic activity and arterial pressure in conscious Wistar rats. This finding unequivocally demonstrates a mechanism by which the diencephalon exerts a tonic influence on sympathetic discharge to the kidney and may provide the basis for both blood volume and osmolality homeostasis. The paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in cardiovascular and neuroendocrine regulation. Application of nitric oxide donors to the PVN stimulates GABAergic transmission, and may suppress sympathetic nerve activity (SNA) to lower arterial pressure. However, the role of endogenous nitric oxide within the PVN in regulating renal SNA chronically remains to be established in conscious animals. To address this, we used our previously established lentiviral vectors to knock down neuronal nitric oxide synthase (nNOS) selectively in the PVN of conscious Wistar rats. Blood pressure and renal SNA were monitored simultaneously and continuously for 21days (n=14) using radio-telemetry. Renal SNA was normalized to maximal evoked discharge and expressed as a percentage change from baseline. The PVN was microinjected bilaterally with a neurone-specific tetracycline-controllable lentiviral vector, expressing a short hairpin miRNA30 interference system targeting nNOS (n=7) or expressing a mis-sense as control (n=7). Recordings continued for a further 18days. The vectors also expressed green fluorescent protein, and successful expression in the PVN and nNOS knockdown were confirmed histologically post hoc. Knockdown of nNOS expression in the PVN resulted in a sustained increase in blood pressure (from 95±2 to 104±3mmHg, P<0.05), with robust concurrent sustained activation of renal SNA (>70%, P<0.05). The study reveals a major role for nNOS-derived nitric oxide within the PVN in chronic set-point regulation of cardiovascular autonomic activity in the conscious, normotensive rat.

Comparison between spectral analysis and symbolic dynamics for heart rate variability analysis in the rat

Spectral analysis of heart rate (HR) has been widely used to assess the autonomic cardiovascular control. A nonlinear approach, known as symbolic analysis, has been reported to be very useful to assess the autonomic control of cardiovascular system in humans, but very few studies reported on the differences between these two approaches on experimental models. Two distinct approaches were used to elicit autonomic changes in conscious Wistar rats: (1) pharmacological blockade of cardiac autonomic receptors with atenolol (ATE, N = 9) or methylatropine (ATR, N = 9) and (2) mild changes in arterial pressure (AP) induced by phenylephrine (PHE, N = 9) or sodium nitroprusside (NPS, N = 9). Series of cardiac interval (CI) and systolic AP (SAP) were assessed using spectral analysis and symbolic dynamics. Results show that, for spectral analysis, the power in high frequency band of CI and the power in low frequency band of SAP are the most reliable indices of vagal and sympathetic modulation, respectively. For symbolic analysis, results point 0V% and 1V% to be related to sympathetic and 2UV% to vagal modulation. Interestingly, the incidence of 1V patterns, hitherto with unknown meaning, was revealed the best index of sympathetic modulation in the rat and should be accounted for in the future studies.

Physical Properties and Effect in a Battery of Safety Pharmacology Models for Three Structurally Distinct Enteric Polymers Employed as Spray-Dried Dispersion Carriers.

Establishing a wide therapeutic index (TI) for pre-clinical safety is important during lead optimization (LO) in research, prior to clinical development, although is often limited by a molecules physiochemical characteristics. Recent advances in the application of the innovative vibrating mesh spray-drying technology to prepare amorphous solid dispersions may offer an opportunity to achieve high plasma concentrations of poorly soluble NCEs to enable testing and establishment of a wide TI in safety pharmacology studies. While some of the amorphous solid dispersion carriers are generally recognized as safe for clinical use, whether they are sufficiently benign to enable in vivo pharmacology studies has not been sufficiently demonstrated. Thus, the physical properties, and effect in a battery of in vivo safety pharmacology models, were assessed in three classes of polymers employed as spray-dried dispersion carriers. The polymers (HPMC-AS, Eudragit, PVAP) displayed low affinity with acetone/methanol, suitable for solvent-based spray drying. The water sorption of the polymers was moderate, and the degree of hysteresis of HPMC-AS was smaller than Eudragit and PVAP indicating the intermolecular interaction of water-cellulose molecules is weaker than water-acrylate or water-polyvinyl molecules. The polymer particles were well-suspended without aggregation with a mean particle size less than 3 μm in an aqueous vehicle. When tested in conscious Wistar Han rats in safety pharmacology models (n = 6–8/dose/polymer) investigating effects on CNS, gastrointestinal, and cardiovascular function, no liabilities were identified at any dose tested (30–300 mg/kg PO, suspension). In brief, the polymers had no effect in a modified Irwin test that included observational and evoked endpoints related to stereotypies, excitation, sedation, pain/anesthesia, autonomic balance, reflexes, and others. No effect of the polymers on gastric emptying or intestinal transit was observed when measured using a barium sulfate tracer material. Finally, in telemetry-instrumented rats the polymers had no effect on acute or 24-h mean blood pressure and heart rate values at doses up to 300 mg/kg. Thus, the properties of the three enteric polymers are appropriate as spray-dried dispersion carriers and were benign in a battery of safety pharmacology studies, demonstrating their applicability to enable in vivo safety pharmacology profiling of poorly soluble molecules during LO.

High Salt Intake Increases Blood Pressure in Normal Rats: Putative Role of 20-HETE and No Evidence on Changes in Renal Vascular Reactivity

Background/Aims. High salt (HS) intake may elevate blood pressure (BP), also in animals without genetic salt sensitivity. The development of salt-dependent hypertension could be mediated by endogenous vasoactive agents; here we examined the role of vasodilator epoxyeicosatrienoic acids (EETs) and vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). Methods. In conscious Wistar rats on HS diet systolic BP (SBP) was examined after chronic elevation of EETs using 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB), a blocker of soluble epoxide hydrolase, or after inhibition of 20-HETE with 1-aminobenzotriazole (ABT). Thereafter, in acute experiments the responses of renal artery blood flow (Transonic probe) and renal regional perfusion (laser-Doppler) to intrarenal acetylcholine (ACh) or norepinephrine were determined. Results. HS diet increased urinary 20-HETE excretion. The SBP increase was not reduced by c-AUCB but prevented by ABT until day 5 of HS exposure. Renal vasomotor responses to ACh or norepinephrine were similar on standard and HS diet. ABT but not c-AUCB abolished the responses to ACh. Conclusions. 20-HETE seems to mediate the early-phase HS diet-induced BP increase while EETs are not engaged in the process. Since HS exposure did not alter renal vasodilator responses to Ach, endothelial dysfunction is not a critical factor in the mechanism of salt-induced blood pressure elevation.

Effect of pancreatic polypeptide on gastric accommodation and gastric emptying in conscious rats.

Pancreatic polypeptide (PP) is an anorexigenic hormone released from pancreatic F cells upon food intake. We aimed to determine the effect of PP on gastric accommodation and gastric emptying in conscious Wistar HAN rats to investigate whether effects on motor function could contribute to its anorexigenic effects. Intragastric pressure (IGP) was measured through a chronically implanted gastric fistula during the infusion of a nutrient meal (Nutridrink; 0.5 ml/min). Rats were treated with PP (0, 33 and 100 pmol·kg(-1)·min(-1)) in combination with N(G)-nitro-L-arginine methyl ester (L-NAME; 180 mg·kg(-1)·h(-1)), atropine (3 mg·kg(-1)·h(-1)), or vehicle. Furthermore, the effect of PP was tested after subdiaphragmal vagotomy of the stomach. Gastric emptying of a noncaloric and a caloric meal after treatment with 100 pmol·kg(-1)·min(-1) PP or vehicle was compared using X-rays. PP significantly increased IGP during nutrient infusion compared with vehicle (P < 0.01). L-NAME and atropine significantly increased IGP during nutrient infusion compared with vehicle treatment (P < 0.005 and 0.01, respectively). The effect of PP on IGP during nutrient infusion was abolished in the presence of L-NAME and in the presence of atropine. In vagotomized rats, PP increased IGP compared with intact controls (P < 0.05). PP significantly delayed gastric emptying of both a noncaloric (P < 0.05) and a caloric (P < 0.005) meal. PP inhibits gastric accommodation and delays gastric emptying, probably through inhibition of nitric oxide release. These results indicate that, besides the well-known centrally mediated effects, PP might decrease food intake through peripheral mechanisms.

Buccal absorption of propofol when dosed in 1-perfluorobutylpentane to anaesthetised and conscious Wistar rats and Göttingen mini-pigs

The purpose of this study was to evaluate whether propofol could be absorbed buccally when administered in semifluorinated alkanes (SFAs), here specifically perfluorobutylpentane (F4H5). This was evaluated in anesthetised and conscious rats and mini-pigs, to measure the relative bioavailability of propofol following buccal administration, but also partly to evaluate the animal models used for this investigation. The absolute bioavailability in the conscious animals was approximately 10% for both species and approximately 50% and 30% in the anesthetised rats and mini-pigs, respectively. This clearly demonstrates that propofol can be absorbed buccally, and F4H5 appears to be a relevant excipient for buccal administration of lipophilic drugs like propofol. The lower absorption in the conscious animals clearly indicates the need for an optimisation of the formulation.

Role of angiotensin II type 1 receptors in the subfornical organ in the pressor responses to central sodium in rats

Central infusion of Na+-rich artificial cerebro-spinal fluid (aCSF) activates the brain renin–angiotensin system and causes sympatho-excitatory and pressor responses. We evaluated the role of the subfornical organ (SFO) and angiotensin II type 1 (AT1) receptors in the SFO in mediating the central Na+-induced pressor response. In conscious Wistar rats, intra SFO infusions of Na+-rich aCSF containing 0.45 and 0.6M Na+ at 10nl/min or injection of angiotensin II (Ang II) at 80ng increased blood pressure (BP) by 15–22mmHg, whereas mannitol with the same osmolarity as the Na+-rich aCSF had no effects. Intra SFO infusion of the AT1 receptor blocker candesartan abolished the pressor response induced by intra SFO administration of Na+-rich aCSF or Ang II. Intra cerebro-ventricular (icv) infusion of Na+-rich aCSF (0.3M Na+) at 3.8μl/min for 10min increased BP by 15–20mmHg. Electrolytic lesion of the SFO attenuated these BP increases by 50–70%. Intra SFO infusion of candesartan also prevented 50% of these pressor responses. These data suggest that SFO neurons are indeed sensitive to Na+, the SFO is a major – but not only – site in the brain to sense an increase in CSF [Na+], and activation of AT1 receptors in the SFO mediates the SFO component of the Na+-induced pressor response.

Abstract 421: The Subfornical Organ (SFO) Mediates CSF Na+ -induced Pressor Response via Activation of AT1 Receptors

In salt-sensitive rats on high salt or rats with icv infusion of Na + , the increase in CSF [Na + ] leads to activation of the brain renin-angiotensin-aldosterone system and thereby to sympatho-excitation and hypertension. We tested whether the SFO and AT 1 receptors in the SFO play a crucial role in mediating the Na + -induced responses. In conscious Wistar rats, intra-SFO infusion of Na + -rich aCSF increased BP in a dose-related manner, whereas mannitol with the same osmolarity had no effects. Intra-SFO infusion of the AT 1 receptor blocker candesartan (cand.,10 μg) abolished pressor responses to intra-SFO infusion of Ang II (80 ng) or Na + -rich aCSF (0.45-0.6 M NaCl), and prevented 50% of the BP increase induced by icv infusion of Na + -rich aCSF (0.3 M NaCl, 4 μl/min for 6 min). In another set of Wistar rats, electrolytic lesion of the SFO prevented 50-65% of BP increases induced by icv infusion of Na + -rich aCSF or Ang II (5 ng/min). These data suggest that the SFO neurons are Na + -sensitive and via AT 1 receptors mediate a major part of the pressor response to CSF Na + . Data=means±SE (n=5-7). *p&lt;.05 vs vehicle or sham lesion.

Abstract 420: Role of Ang II and Glutamate in the PVN of Rats with Ang II-induced Hypertension

A chronic increase in circulating Ang II increases neuronal activation in hypothalamic nuclei such as the paraventricular nucleus (PVN) and causes progressive hypertension. These effects can be largely prevented by central infusion of blockers of an aldosterone-ouabain neuromodulatory pathway that enhances the activity of angiotensinergic sympathoexcitatory pathways. A number of models of chronic sympathetic hyperactivity are associated with an increase in glutamate receptor and AT 1 -receptor activation in the PVN. The present study evaluated whether these mechanisms in the PVN also contribute to the elevated BP from a chronic increase in plasma Ang II by chronic subcutaneous (sc) infusion of Ang II for 2 weeks. One week following bilateral stereotaxic cannulation of the PVN, bilateral infusion (300 nL/min/side) of AT 1 and glutamate receptor blockers, candesartan and kynurenate were performed, and BP and HR were recorded in conscious Wistar rats. Data are mean±SE; (n=4-5/group). *p&lt;0.05 vs. baseline Kynurenate or candesartan in the PVN both fully reversed the increase in BP from sc infusion of Ang II for 2 weeks. Infusion of candesartan after kynurenate or kynurenate after candesartan did not further lower BP. These findings suggest that enhanced AT 1 -receptor and glutamate receptor activation in the PVN both contribute to the elevated BP in Wistar rats with chronically elevated plasma Ang II. The effects of increased AT 1 -receptor activation in the PVN appear to be mediated by local glutamate release.

Correlation of renal solute-free water reabsorption and energy of interaction of vasotocin analogs with V2 receptor

The nonapeptide-receptor interaction energy calculated from docking algorithms was compared with the effect of the same peptides upon solute-free water reabsorption in rat kidney in vivo. Vasotocin and some of its analogs were injected intramuscularly into conscious female Wistar rats in doses from 0.1 pmol to 0.5 nmol/kg of body weight after oral water load (50 ml/kg of body weight). A significant correlation was found between the calculated energy of peptide interaction with V2 receptors and an increase in the renal solute-free water reabsorption stimulated by injection of nonapeptides. The results evidence that physiological changes in rat kidney in vivo induced by vasotocin analogs can be accurately simulated by virtual modeling of their interactions with V2 receptors.

Regulation of hypothalamic renin-angiotensin system and oxidative stress by aldosterone

In rats with salt-induced hypertension or postmyocardial infarction, angiotensin II type 1 receptor (AT(1)R) densities and oxidative stress increase and neuronal NO synthase (nNOS) levels decrease in the paraventricular nucleus (PVN). The present study was designed to determine whether these changes may depend on activation of the aldosterone -'ouabain' neuromodulatory pathway. After intracerebroventricular (i.c.v.) infusion of aldosterone (20 ng h(-1)) for 14 days, blood pressure (BP) and heart rate (HR) were recorded in conscious Wistar rats, and mRNA and protein for nNOS, endothelial NO synthase (eNOS), AT(1)R and NADPH oxidase subunits were assessed in brain tissue. Blood pressure and HR were significantly increased by aldosterone. Aldosterone significantly increased mRNA and protein of AT(1)R, P22phox, P47phox, P67phox and Nox2, and decreased nNOS but not eNOS mRNA and protein in the PVN, as well as increased the angiotensin-converting enzyme and AT(1)R binding densities in the PVN and supraoptic nucleus. The increases in BP and HR, as well as the changes in mRNA, proteins and angiotensin-converting enzyme and AT(1)R binding densities were all largely prevented by concomitant i.c.v. infusion of Digibind (to bind 'ouabain') or benzamil (to block presumed epithelial sodium channels). These data indicate that aldosterone, via 'ouabain', increases in the PVN angiotensin-converting enzyme, AT(1)R and oxidative stress, but decreases nNOS, and suggest that endogenous aldosterone may cause the similar pattern of changes observed in salt-sensitive hypertension and heart failure postmyocardial infarction.

Chronic knockdown of nNOS in the paraventricular nucleus (PVN) produces persistent increases in arterial pressure and renal sympathetic nerve activity (RSNA) in the rat

The PVN plays a crucial role in cardiovascular and neuroendocrine regulation. Nitric oxide (NO) stimulates GABAergic transmission in the PVN, and may tonically suppress sympathetic outflow. However its chronic role in regulating RSNA remains to be established. To address this, we used a lentiviral vector system to knock down nitric oxide synthase (nNOS) in the PVN of conscious Wistar rats. Blood pressure (BP) and RSNA were monitored continuously via telemetry. RSNA was normalised to the maximal response elicited by activation of the nasopharyngeal reflex (100 normalised units, nu) and background noise level (0 nu). Following recovery and baseline recording, the PVN was microinjected bilaterally with a neurone-specific tetracycline-controllable lentiviral vector, expressing a microRNA 30 based short hairpin (shRNA) interference system targeting endogenous nNOS (n=5) or a negative control sequence (n=5). Recordings continued for 21 days. The construct was GFP-tagged, and successful placement in the PVN confirmed post-hoc. Knock-down of the nNOS gene resulted in a modest but sustained increase in BP (96±3 to 105±3 mmHg, Days 14–21; p<0.05), with a concurrent increase in RSNA (9±2 to 15±2 nu; p<0.05). Control rats showed no change. These results suggest that NO activity in the PVN is a critical determinant of the chronic level of BP and RSNA in the conscious rat. Royal Society, BHF and NIH funded research.

Baicalein, an active component of Scutellaria baicalensis Georgi, improves cardiac contractile function in endotoxaemic rats via induction of heme oxygenase-1 and suppression of inflammatory responses

To evaluate the protective effect of baicalein on myocardial dysfunction caused by endotoxaemia in rats and to explore the possible mechanisms. Baicalein (10mg/kg, intravenous) was administered to conscious Wistar rats 30min after lipopolysaccharide (LPS; 10mg/kg, intravenous) challenge. Six hours after LPS administration, the contractile function of the isolated heart was examined using the Langendorff technique. Cardiac protein expression related to inflammatory responses, superoxide anion production and caspase-3 activity were measured. Post-treatment with baicalein significantly attenuated the LPS-induced hypotension with accompanying tachycardia. The contractile function of isolated heart was significantly preserved 6h after LPS administration, following treatment with baicalein. Furthermore, baicalein induced the expression of heme oxygenase-1 protein and reduced superoxide anion formation in the myocardium of LPS-treated rats. Cardiac levels of inducible nitric oxide synthase, monocyte chemoattractant protein-1, phospho-IκBα and phospho-p65 protein and caspase-3 activity significantly increased 6h after LPS challenge but baicalein significantly attenuated these LPS-induced changes. Baicalein improves myocardial contractility in LPS-induced sepsis, which may be related to reductions in oxidative stress, myocardial inflammatory responses and apoptosis.

CATALASE INHIBITION INTO THE FOURTH CEREBRAL VENTRICLE AFFECTS BRADYCARDIC PARASYMPATHETIC RESPONSE TO INCREASE IN ARTERIAL PRESSURE WITHOUT CHANGING THE BAROREFLEX

Exogenous catalase influences neural control of cardiovascular system; however, we do not know yet if its inhibition into the fourth cerebral ventricle (4(th) V) influences baroreflex regulation. We evaluated the effects of central catalase inhibition on baroreflex in conscious Wistar rats. We used males Wistar rats (320-370 g), which were implanted with a stainless steel guide cannula into 4(th) V. The femoral artery and vein were cannulated for mean arterial pressure (MAP) and heart rate (HR) measurement and drug infusion, respectively. After basal MAP and HR recordings, the baroreflex was tested with a pressor dose of phenylephrine (PHE, 8 μg/kg, bolus) and a depressor dose of sodium nitroprusside (SNP, 50 μg/kg, bolus). Baroreflex was evaluated before 5, 15, 30 and 60 minutes after 3-amino-1, 2, 4-triazole (ATZ, 0.001 g/100 μL) injection into the 4(th) V. Vehicle treatment did not change baroreflex responses. ATZ attenuated bradycardic peak and reduced HR range at 30 minutes. ATZ into the 4(th) V reduced bradycardic and tachycardic reflex responses to increase and decrease MAP, respectively (p<0.05) 30 minutes after its microinjection without significantly changing the basal MAP and HR. In conclusion, central catalase inhibition influenced the highest parasympathetic response to MAP increase in conscious Wistar rats without change baroreflex gain.