Conscious Rats Research Articles

Divergent roles of angiotensin II upon the immediate and sustained increases of renal blood flow following unilateral nephrectomy.

Although the molecular and functional responses related to renal compensatory hypertrophy after unilateral nephrectomy (UNX) has been well described, many aspects of these events remain unclear. One question is how the remaining kidney senses the absence of the contralateral organ, and another is what the role of the renin-angiotensin system is in these responses. Both acute anesthetized and chronic unanesthetized experiments were performed using the angiotensin II type 1 receptor blocker losartan and the renin inhibitor aliskiren to determine the contribution of the renin-angiotensin system to immediate changes and losartan for chronic changes of renal blood flow (RBF) and the associated hypertrophic events in male Sprague-Dawley rats. Chronic experiments used implanted RBF probes and arterial catheters for continuous data collection, and the glomerular filtration rate was determined by noninvasive transcutaneous FITC-sinistrin measurements. The results of the acute experiments found that RBF increased nearly 25% (4.6 ± 0.5 to 5.6 ± 0.6 mL/min/g kidney wt) during the first 15 min following UNX and that this response was abolished by losartan (6.7 ± 0.7 to 7.0 ± 0.7 mL/min/g kidney wt) or aliskiren (5.8 ± 0.4 to 6.0 ± 0.4 mL/min/g kidney wt) treatment. Thereafter, RBF increased progressively over 7 days, and kidney weight increased by 19% of pre-UNX values. When normalized to kidney weight determined at day 7 after UNX, RBF was not significantly different from pre-UNX levels. Semiquantification of CD31-positive capillaries revealed increases of the glomeruli and peritubular capillaries that paralleled the kidney hypertrophy. None of these chronic changes was inhibited by losartan treatment, indicating that neither the compensatory structural nor the RBF changes were angiotensin II type 1 receptor dependent.NEW & NOTEWORTHY This study found that the immediate increases of renal blood flow (RBF) following unilateral nephrectomy (UNX) are a consequence of reduced angiotensin II type 1 (AT1) receptor stimulation. The continuous monitoring of RBF and intermittent measurement of glomerular filtration rate (GFR) in conscious rats during the 1-wk period of rapid hypertrophy following UNX provided unique insights into the regulation of RBF and GFR when faced with increased metabolic loads. It was found that neither kidney hypertrophy nor the associated increase of capillaries was an AT1-dependent phenomenon.

Heart Rate Fragmentation in Rats with Streptozotocin‐induced Diabetes

Heart rate fragmentation (HRF) is a recently proposed approach to evaluate sino‐atrial instability characterized by the presence of numerous inflections points in a series of successive values of RR intervals (RRi). HRF has been shown to be increased in aging and coronary diseases and seems to be linked to the risk of life‐threatening cardiovascular events and death. We recently demonstrated that cardiac autonomic modulation in involved in HRF in rats. Moreover, HRF is increased in a rat model of heart failure. Diabetes mellitus (DM) markedly alters HRV indices and might affect HRF as well. Therefore, we hypothesize that HRF is altered in streptozotocin‐ (STZ) induced diabetic rats. Male Sprague‐Dawley rats were injected with STZ (50 mg/kg), or vehicle (citrate buffer) into penile vein. Development of diabetes was confirmed 72h after STZ by the presence of hyperglycemia (> 350 mg/dL). Electrocardiographic (ECG) recordings were performed in conscious rats, 7 days (acute) or 4 weeks (chronic) after STZ or vehicle administration. Series of RR intervals were generated and processed by the customized software PyBios as follows: RRi values were symbolized as “‐1”, “0,” or “1” when the differences between successive RRi were negative, null, or positive, respectively. Transitions between symbols “‐1” and “1” were labeled as “hard” (H) inflection points, while those between “‐1” (or “1”) and “0” were labeled as “soft” (S) inflection points. The percentage of inflection points (PIP) was quantified, as well as sequences of 4 consecutive symbols, so‐called “words”, computing the occurrence of words with zero (W0), one (W1), two (W2), or three (W3) inflections points. The occurrence of words with only hard (WH) or only soft transitions (WS) were also quantified. Both groups of diabetic rats were bradycardic as compared to their control counterparts. Data of HRF are shown in the table below. Occurrences (%, mean ± SEM) of PIP, words with 0 to 3 inflection points, and words with only soft or hard transitions of symbols. As hypothesized, diabetes affects HRF, increasing fragmented indices (PIP, W3 and WH) and decreasing fluent ones (W1 and WS). Moreover, our results showed that changes in HRF due to diabetes in this model is time‐dependent. In addition to traditional methods of analyzing heart rate variability, HRF is a new method that can contribute to the diagnosis and prognosis of cardiovascular diseases in diabetic patients. Further studies are necessary to understand better the mechanisms involved in our findings.

Hypertensive Dahl Salt‐Sensitive Rats Demonstrate a Reduction in Central Blood Pressure Following the Short‐Term Treatment with Ivabradine and Spironolactone

IntroductionA proper management of central blood pressure (cBP) has been recently recognized as a novel therapeutic target to reduce cardiovascular risks among hypertensive (HT) patients. Considering that the hypotensive action of most antihypertensive drugs does not persist after treatment interruption, the search for compounds which would permanently reduce the level of cBP remains vital. It has been previously reported that a mineralocorticoid receptor antagonist spironolactone (SL) as well as a “pure” heart rate lowering drug ivabradine (IVA) can both affect the level of cBP and cardiac structure in hypertension.HypothesisIt was hypothesized that combined treatment with SL and IVA could permanently reduce cBP in chronically HT animals via alterations in myocardial properties.MethodsSelf‐sustaining hypertension was induced in 20 male (seven‐week‐old) Dahl salt‐sensitive rats by feeding them a high‐salt (HS) diet (8% NaCl) for 7 weeks. Then all HT rats were switched back to a normal‐salt (NS) diet (0.3% NaCl) and randomized in two experimental groups to receive ether a combined treatment (HT‐T) with IVA (10 mg/kg/day) and SL (20 mg/kg/day) or the vehicle alone (HT‐V) for 8 weeks via intraperitoneal ALZET osmotic pumps. The ten age‐matched male rats fed a NS diet only were used as normotensive controls (NT‐C). Heart rate and peripheral blood pressure were evaluated every week in conscious rats using a CODA tail‐cuff plethysmography system. One week following treatment termination, the central (aortic) and cardiac hemodynamic parameters were assessed in anesthetized rats using an intravascular/intracardiac Millar micro‐tip pressure catheter and a PowerLab data acquisition system. Subsequently, rats were euthanized, and their hearts were excised, weighed and processed to paraffin for histology and quantitative morphology.ResultsAfter 7 weeks on a HS diet, all experimental rats had markedly elevated mean arterial pressure (MAP) and heart rate. Subsequent replacement of a HS diet with a NS diet did reduce the level of MAP by ~12%, but in both HT groups it remained significantly higher than in NT‐C rats throughout the rest of the study on average by ~27% (P≤0.001). On the other hand, during eight weeks of treatment, heart rate was persistently reduced only in HT‐T rats on average by ~38% and ~27% (P≤0.001) compared to HT‐V and NT‐C groups, respectively. At the end of the study, the invasive hemodynamic measurements showed that although cBP remained markedly elevated in all HT rats compared to a NT‐C group, the level of cBP in HT‐T rats was significantly lower than that in HT‐V group (149.4±4.3 vs. 167.5±3.3 mmHg in MAP, P≤0.01). Moreover, a reduction in cBP among HT‐T rats was associated with decreased left ventricular (LV) contractility and delayed active relaxation as compared to HT‐V rats. At the same time, histological examination of the hearts has confirmed that sustained hypertension caused significant LV hypertrophy (P≤0.001) and myocardial fibrosis (P≤0.01) in both HT groups. Most important, compared to a HT‐V group, the HT‐T rats had thicker LV free wall (3.20±0.06 vs. 2.94±0.11 mm, P≤0.05) and a markedly higher myocardial density of monocytes/macrophages (228.8±12.5 vs 62.4±7.1 CD68‐positive cells/mm2, P≤0.0001).ConclusionOur findings demonstrated that concurrent treatment of chronically HT rats with IVA and SL led to a sustained reduction in cBP and that such hypotensive effect was associated with ongoing myocardial remodeling in treated animals.

Overproduction of reactive oxygen species in cardiac vagal postganglionic neurons contributes to myocardial infarction‐induced lethal ventricular arrhythmias in type 2 diabetes mellitus

Myocardial infarction (MI)‐evoked ventricular arrhythmia is the major cause of mortality in type 2 diabetes mellitus (T2DM) patients. Although intensive glycemic control over time have been noted in T2DM patients, it fails to reduce MI‐related mortality in T2DM patients. Withdrawal of cardiac vagal (parasympathetic) activity is a common complication and is associated with arrhythmia‐related sudden cardiac death in T2DM patients. Our recent study found that T2DM‐reduced cell excitability of CVP neurons contributes to the withdrawal of cardiac vagal activity and further exacerbates acute MI‐evoked fatal ventricular arrhythmias and high mortality rate in T2DM. However, the mechanisms responsible for T2DM‐reduced cell excitability of CVP neurons are unclear. Since reactive oxygen species (ROS) has been reported to modulate calcium (Ca2+) channel activity in peripheral postganglionic neurons, here we tested if and how hydrogen peroxide (H2O2, a type of ROS) inactivates CVP neurons and subsequently contributes to MI‐evoked fatal ventricular arrhythmias in T2DM rats. Rat T2DM was induced by a high‐fat diet plus streptozotocin injection. Adenoviral catalase gene (Ad.CAT) or its vector control (Ad.Empty, 2 μl, 1x1010 pfu/ml) was microinjected into CVP neurons. Local in vivo transfection of Ad.CAT successfully induced overexpression of catalase and attenuated H2O2 overproduction in CVP neurons in T2DM rats. Additionally, transfection of Ad.CAT not only restored protein expression and ion currents of N‐type Ca2+ channels, but also increased cell excitability of CVP neurons in T2DM rats. Using Fluo3/AM with a confocal microscope, we also demonstrated that Ad.CAT gene transfection significantly restored intracellular Ca2+ levels in isolated CVP neurons from T2DM rats. Moreover, data from 24‐hour continuous ECG recording in conscious rats demonstrated that transfection of Ad.CAT into CVP neurons alleviated the heterogeneity of ventricular electrical activity (a critical factor of ventricular arrhythmogenesis). More importantly, Ad.CAT transfection into CVP neurons significantly reduced the cumulative duration of VT/VF induced by MI in conscious T2DM rats (28.3 ± 6.9 sec/hour in the T2DM+AD.CAT group vs. 71.7 ± 7.4 sec/hour in the T2DM group, P<0.05). Ad.Empty gene transfection into CVP neurons had no effects on intracellular H2O2 and Ca2+ levels, protein expression and ion currents of N‐type Ca2+ channels, cell excitability of CVP neurons, and duration of VT/VF induced by MI in T2DM rats. Based on these data, we concluded that overproduction of H2O2 decreased protein expression and activation of N‐type Ca2+ channels and reduced cell excitability of CVP neurons, which further contributed to ventricular arrhythmogenesis in T2DM. Our current study suggests that targeting H2O2‐N‐type Ca2+ channel signaling pathway in CVP neurons is an effective intervention against MI‐evoked fatal ventricular arrhythmias in the T2DM state.

Difelikefalin, a peripherally restricted kappa opioid receptor agonist, evokes diuresis through a central kappa opioid receptor pathway

Kappa opioid receptor (KOR) agonists produce a variety of beneficial effects such as nonaddictive analgesia and diuresis, but their translation into the clinic has been hindered by central adverse effects (dysphoria). In clinical studies, difelikefalin, a KOR agonist recently FDA approved for treatment of pruritus in hemodialysis patients, was shown to be devoid of producing adverse CNS effects suggesting that this compound is peripherally restricted. This is of interest from a renal excretory perspective, since it would be predicted that difelikefalin would not produce diuresis, which is a classical centrally mediated response produced by KOR agonists. However, in preliminary studies in rats we have shown that IV administration of difelikefalin markedly increased urine output and decreased urinary sodium and potassium excretion similar to that produced by other KOR agonists. Therefore, we hypothesized that difelikefalin activates a central KOR pathway to cause diuresis. To test this hypothesis, Sprague‐Dawley rats were implanted with bladder, femoral artery, and femoral vein catheters and administered an isotonic saline infusion. To delineate the central and/or peripheral site of action of difelikefalin, renal excretory function was measured in rats pretreated centrally or peripherally with norBNI (KOR antagonist) or vehicle via an intracerebroventricular (ICV) cannula or IV catheter. Following stabilization, conscious rats were administered an IV bolus of difelikefalin (10 µg/kg) and urine output, mean arterial pressure (MAP), and heart rate (HR) were recorded for 90 minutes. The results demonstrated that ICV norBNI pretreatment significantly decreased the diuretic response to IV difelikefalin but had no effect on urinary sodium or potassium concentration (Table 1). In contrast, IV norBNI pretreatment prevented the diuresis to IV difelikefalin and increased urinary sodium/potassium concentration (Table 1). Notably, ICV and IV norBNI pretreatment abolished the difelikefalin‐induced decrease in MAP that was also seen in the vehicle pretreated groups. Together, these findings suggest that difelikefalin produces its diuretic and blood pressure effects through a central KOR pathway, while the sodium/potassium retaining effects of difelikefalin are mediated through peripheral KORs.

Central Adenosine A1 Receptors Arbitrate the Nicotine Counteraction of Cardiovascular and Autonomic Dysfunctions in Septic Rats

The cholinergic anti‐inflammatory pathway plays a crucial role in improving end‐organ damage provoked by inflammatory disorders. Given the interplay between cholinergic and adenosinergic neuronal circuits in the central nervous system, we tested the hypothesis that central adenosine A1 receptors (A1Rs) modulate the offsetting action of nicotine on cardiovascular and inflammatory insults induced by sepsis. Pharmacologic agonistic and antagonistic studies were conducted in conscious rats, pre‐instrumented with femoral vessel and intracisternal (i.c.) catheters. Sepsis was induced by cecal ligation and puncture (CLP) 24 hr before cardiovascular measurements. Nicotine (25 or 100 µg/kg i.v.) dose‐dependently reversed septic manifestations of hypotension and concomitant reductions in time and spectral measures of heart rate variability (HRV) and cardiac sympathovagal balance (low‐frequency/high‐frequency ratio). Like nicotine, the i.c. administration of N(6)‐cyclopentyladenosine (CPA, AR1 agonist) to CLP rats caused significant increases in indices of HRV and sympathovagal balance and greater surges in these parameters were noted upon simultaneous administration of CPA plus nicotine. We also show that the favorable influences of nicotine on blood pressure and HRV in septic rats were significantly inhibited after central blockade of A1Rs by i.c. 8‐Cyclopentyl‐1,3‐dipropylxanthine (DPCPX). Immunohistochemically, the alleviation by nicotine of the upregulated myocardial expression of NFκB in septic rats disappeared upon prior exposure to DPCPX. The data suggest that central adenosine A1Rs act tonically to facilitate the ameliorative effect of nicotine against the inflammatory response of sepsis and interrelated cardiomyopathic and neuropathic hitches.

Does Dietary Methionine Alter Perivascular Adipose Tissue Function?

Perivascular adipose tissue (PVAT) is an adipose depot surrounding blood vessels and is a paracrine regulator of vascular tone. Obesity is associated with the loss of PVAT‐mediated anti‐contractile properties, thus contributing to obesity‐associated hypertension. Hydrogen sulfide (H2S) is a gaseous signaling molecule that is produced in the vasculature and is important for regulating vascular tone. In PVAT, cystathionine gamma lyase (Cse) is the primary source of H2S which acts as an important adipose‐derived relaxing factor. Dietary methionine restriction recapitulates many of the metabolic effects of caloric restriction and leads to preferential expression of activating transcription factor 4 (ATF4) which facilitates Cse transcription. We hypothesized that dietary methionine restriction will prevent high fat diet‐induced loss of PVAT anti‐contractile properties, through activation of the integrated stress response (ISR) and increased ATF4‐mediated transcription of Cse. Male Wistar rats were fed either a control (C, 10% calories from fat) or a high fat diet (HFD) (H, 60% calories from fat) that was either methionine replete (R, 0.86% methionine) or methionine depleted (D, 0.12% methionine) for 12 weeks such that there were four groups: CR, CD, HR, and HD. Dietary methionine restriction prevented body weight gain, while rats fed a methionine replete diet gained weight over the 12 week feeding period. There was no difference in weight gain between control or HFD fed rats. Arterial blood pressure, measured in conscious rats using tail cuff plethysmography, was not different between the four groups over the 12 week feeding period. After 12 weeks on diets, arterial contractility was assessed using wire myography in 2nd order mesenteric arteries (MAs) with and without attached PVAT. In our hands, the presence of PVAT was associated with reduced contraction to norepinephrine (NE, 10 µM) and greater relaxation to acetylcholine (ACh,10 µM) in MAs from all groups, and HFD was not associated with a loss of PVAT anti‐contractile properties. In the presence of PVAT, dietary methionine restriction increased relaxation to ACh in NE‐precontracted MAs, while HFD had no effect on ACh‐induced relaxation. In conclusion, in male Wistar rats, while HFD had no effect on weight gain, blood pressure, or PVAT modulation of MA contractility, dietary methionine restriction for 12 weeks had profound effects on body weight gain, no effect on arterial blood pressure, and a small effect on MA ACh‐induced relaxation in the presence of PVAT. Future studies include examining gene and protein expression of Cse and other ATF4 responsive genes using real time qPCR, Western blot analysis, and immunohistochemical staining of arterial cross‐sections. These studies will provide further insights into dietary interventions to minimize the cardiovascular co‐morbidities associated with obesity.

M ethod for Measuring Cervical Vagus Nerve Activity in Conscious and Freely Moving Rats

The vagus nerve innervates most of the organs in the thoracic and abdominal cavity and plays a central role in inter‐organ networks through afferent and efferent vagal nerve activity. However, few attempts have been made to measure vagal nerve activity in the conscious state; thus, the nature of vagal nerve activity remains unclear. In this study, we attempted to develop a method to measure cervical vagal nerve activity in conscious, freely moving rats. Using male Wistar rats, the right cervical vagus nerve was dissected approximately 1 mm under anesthesia, and a bipolar electrode made of twisted stainless‐steel wire was hooked onto the vagus nerve and fixed with silicone gel. A microcatheter for administering a long‐acting local anesthetic (levobupivacaine hydrochloride 0.75%) was placed at least 5 mm peripherally from the electrode. Measurements were recorded under conscious and free‐living conditions. The right cervical vagal nerve activity (rcVNA) showed continuous activity of approximately 20 μV at peak‐to‐peak potential. The rcVNA was reduced by approximately 80% after administering 30 μL of levobupivacaine through the microcatheter. This allowed us to block the afferent VNA and selectively measure the efferent VNA. Using this method, we observed that efferent rcVNA decreased during REM sleep compared with NREM sleep. Moreover, we succeeded in measuring rcVNA and renal SNA simultaneously and continuously in conscious rats.

Contribution of Adenosine A2A Receptors in the Rostral Ventrolateral Medulla to Acupuncture Modulation of Hypertension

Acupuncture is used to manage high blood pressure (BP) in clinical practice. However, the mechanisms underlying its hypotensive effects remain unclear. Our previous studies have shown that electroacupuncture (EA) at the ST36‐37 acupoints, overlying the deep peroneal nerve, attenuates pressor responses through adenosine A2A receptors (A2AR) in the rostral ventrolateral medulla (rVLM). A2AR activation in the rVLM is associated with the upregulation of nitric oxide synthase (NOS) that contributes to sympathoinhibition and BP reduction. However, it is uncertain whether rVLM A2AR contributes to EA’s action in lowering high BP in sustained hypertension (HTN) in the rVLM. We hypothesized that EA lowers BP in HTN through adenosine A2AR mechanisms associated with the upregulation of neuronal NOS (nNOS) in the rVLM. The nNOS derived NO has been shown to exert central effects on BP. To mimic essential HTN in the clinic, we performed EA in conscious Dahl salt‐sensitive hypertensive rat (DSHR). EA (0.1‐0.4 mA, 2 Hz) was applied at ST36‐37 for 30 min twice weekly for four weeks while sham‐EA was conducted with the same procedures as EA except for no electrical stimulation. Elevated BP was reduced by EA (n=10) but not sham‐EA (n=8) and untreated ones (n=6). The A2AR level in the rVLM after EA treatment was increased (n=5; P < 0.05) compared to the sham‐EA (n=5). EA’s inhibitory effect on elevated BP was reversed transiently by unilateral microinjection of SCH 58261 (1 mM in 50 nl; an A2AR antagonist; n=7; P < 0.05) but not the vehicle (n=5) into the rVLM in EA‐treated DSHRs under anesthesia. Activation of rVLM A2AR in DSHRs treated with sham‐EA using an A2AR agonist, CGS‐21680 (0.4 mM in 50 nl; n=6), decreased BP. Furthermore, we found a co‐localization of A2AR with nNOS in the rVLM of DSHRs. The expression of rVLM nNOS was enhanced in DSHRs treated with EA compared to those treated with sham‐EA (n=5 in each group; P < 0.05). These data suggest that adenosine‐ A2AR in the rVLM contributes to EA’s inhibitory effect on elevated BP in sustained HTN, which is likely relevant to the upregulation of nNOS.

A High Salt Diet Does Not Alter the Kinetics or the Magnitude of Renal Blood Flow Autoregulation in Conscious Salt‐Resistant and Salt‐Sensitive Rats

Blood pressure (BP) salt‐sensitivity is a major public health problem that augments the risk of kidney disease. Both BP dependent and independent mechanisms are thought to contribute to the increased risk of kidney injury in salt‐sensitive (SS) populations. Previous studies have reported that high dietary salt intake impairs renal blood flow (RBF) autoregulation, which increases the susceptibility to hypertensive renal injury. However, in these studies, RBF autoregulation was assessed in anesthetized rats or in isolated renal resistance vessels, which do not fully characterize the dynamic nature of RBF autoregulation in the conscious state. Our group has recently developed novel analytical methods to characterize both the magnitude and kinetics of the RBF autoregulatory response to spontaneous BP fluctuations in conscious, chronically instrumented rats. The goal of this study was to assess the effects of a high salt diet on the dynamics of RBF autoregulation in conscious, salt‐resistant and SS rat strains. At 8‐13 weeks of age, male Sprague‐Dawley (SD), Dahl SS, Consomic SS.BN1, Spontaneously Hypertensive (SHR), and Stroke‐prone SHR (SHRsp) rats were instrumented with a BP radiotelemeter and an ultrasonic RBF probe was positioned on the left renal artery. Following 1 week of recovery, BP and RBF were assessed for 2‐3 hours/day over 2‐3 consecutive days while rats were maintained on normal salt diet (0.4‐1% NaCl). All rats were then administered a high salt diet (4‐8% NaCl) and 2‐3 days later, BP and RBF were assessed for 2‐3 hours/day over 3‐5 consecutive days. All recordings were divided into segments of varying length (0.5‐20 seconds), and adjacent segments which exhibited a BP difference of 5 mmHg or greater were used to calculate RBF autoregulatory indices (i.e., % change in RBF / % change in BP). Different segment lengths were used to infer the time required for the preglomerular vasculature to restore RBF to levels observed prior to the change in BP. As shown in Figure 1, BP was significantly different among strains during normal salt diet with SD < SS.BN1 < SS = SHR < SHRsp. A high salt diet significantly increased BP in SS and SHR rats. No significant differences in RBF were found among all strains during normal or high salt intake, and a high salt diet did not significantly alter the RBF autoregulatory response in any strain (Figure 2). As also shown in Figure 2, all strains exhibited > 60% RBF autoregulatory compensation by 2.5 seconds following a change in BP, and the response was significantly faster in SHRsp vs. SD, SS, and SS.BN1 strains. All strains except SS and SS.BN1 rats exhibited complete RBF compensation by 10‐20 seconds following a change in BP. We conclude that a high salt diet does not significantly alter RBF autoregulatory responses in the conscious state in the salt‐resistant or salt‐sensitive strains examined in this study. There are strain differences in the initial magnitude of the RBF autoregulatory response with those strains exhibiting higher ambient BP’s having a stronger RBF autoregulatory response. The implications of the weaker RBF autoregulatory responses at later time points (i.e., 10‐20 seconds) following a change in BP in SS and SS.BN1 rats requires further investigation.

Effects of sinoaortic denervation on hemodynamic perturbations of prolonged paradoxical sleep deprivation and rapid cold stress in rats.

Sleep disturbances and aversive cold stress (CS) are cardiovascular risk factors. This study investigates how homeostatic control autonomic baroreflex influences the hemodynamic perturbations evoked by paradoxical sleep deprivation (PSD) and CS. Conscious adult male rats were randomly divided into four groups (Sham/CON [control], Sham/PSD, sinoaortic denervation [SAD]/CON, and SAD/PSD). Spectral analysis and SAD were employed to evaluate the effects of a 72-hr PSD with 10-min CS on blood pressure variability and heart rate variability (BPV and HRV) at total power (TP) and three frequency power densities, very-low-frequency (VLF), low frequency (LF), and high frequency (HF). Key findings showed: (I) Compared with the control sham surgery (Sham/CON), in the natural baseline (PreCS) trial, SAD surgery (SAD/CON) causes high systolic blood pressure (SBP), heart rate (HR), increases LFBPV (low-frequency power of BPV), LF/HFHRV (the ratio LF/HF of HRV), and TPBPV (the total power of BPV), but decreases HFHRV (high-frequency power of HRV) and VLFHRV (very-low-frequency power of HRV) than the Sham/CON does. In the CS trial, SAD/CON increases the CS-induced pressor, increases the CS-elicited spectral density, LF/HFHRV, but decreases HFBPV than the Sham/CON does. (II) Compared with SAD/CON and Sham/PSD (PSD under sham surgery), in both PreCS and CS trials, SAD/PSD (PSD under SAD) causes high SBP and HR than both SAD/CON and Sham/PSD their SBP and HR. In PreCS, SAD-PSD also changes the spectral density, including increasing Sham-PSD's LFBPV, LF/HFHRV, VLFBPV, and TPBPV but decreasing Sham-PSD's VLFHRV and TPHRV. However, in CS, SAD-PSD changes the CS-elicited spectral density, including increasing Sham-PSD's VLFBPV, LF/HFHRV, and TPHRV but decreasing Sham-PSD's HFBPV and LFBPV. The results suggest baroreflex combined with other reflex pathways, such as inhibitory renorenal reflex, modulates the vascular and cardiorespiratory responses to PSD under PreCS and subsequent CS trials.

Regionally selective cardiovascular responses to adenosine A2A and A2B receptor activation.

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein‐coupled receptors (A1, A2A, A2B, A3). Here, we have investigated the effect of A2A and A2B‐selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague‐Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A2A‐selective agonist CGS 21680 (0.1, 0.3, 1 µg kg−1 min−1) or the A2B‐selective agonist BAY 60‐6583 (4,13.3, 40 µg kg−1 min−1) following predosing with the A2A‐selective antagonist SCH 58261 (0.1 or 1 mg kg−1 min−1), the A2B/A2A antagonist PSB 1115 (10 mg kg−1 min−1) or vehicle. The A2A‐selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B‐selective agonist BAY 60‐6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury.

Dopaminergic, cholinergic and nitrinergic pathways implicated in blood pressure lowering effects of Saccharum officinarum L. (Sugarcane) on rats.

BackgroundSaccharum officinarum L. (sugarcane), a grass belonging to the Poacea family, is widely cultivated in Brazil mainly to produce sugar and ethanol. According to ethnopharmacological data, the sugarcane is used in folk medicine for the treatment of cardiovascular diseases. Besides this, a previous study showed that there is a relationship between the pharmacological actions of sugarcane and the activation of dopaminergic and cholinergic pathways. Physiologically, it is well established that the blood pressure control could involve different neurotransmission systems, such as dopaminergic, cholinergic and nitrinergic. AimTo validate the cardiovascular actions of sugarcane preconized by traditional medicine and evaluate the possible participation of the dopaminergic, cholinergic and nitrinergic systems in its pharmacological mechanism of action. MethodsDried leaves were extracted with distilled water, concentrated, and lyophilized (AE). In vivo assays were performed in conscious rats to record systolic blood pressure (SBP) by the tail method after acute and subacute oral (gavage) administration of AE (10–500 mg/kg). The involvement of the cholinergic and dopaminergic pathways in AE responses was evaluated with administration (30 min) of atropine (5 mg/kg, i.p.) or haloperidol (0.5 mg/kg, i.p.), respectively. The participation of nitric oxide in the cardiovascular mechanism of action was evaluated in animals submitted to treatment with l-Name (60 mg/kg, p.o.). After installation of hypertension, animals were treated with AE. ResultsAcute and subacute treatment with AE reduced SBP in a dose-dependent manner, with greater pharmacological efficacy after administration of lower doses. Atropine and haloperidol inhibited the hypotensive effects of AE, and it was shown that in hypertensive animals, AE was ineffective in reducing SBP. ConclusionThis study revealed the blood pressure lowering effect of AE in a dose-dependent manner and demonstrated that the pharmacological cardiovascular effects of sugarcane involve dopaminergic, cholinergic and nitrinergic pathways.

Peptide fragments of bradykinin show unexpected biological activity not mediated by B1 or B2 receptors.

Bradykinin (BK-(1-9)) is an endogenous nonapeptide involved in multiple physiological and pathological processes. Peptide fragments of bradykinin are believed to be biologically inactive. We have now tested the two major peptide fragments of bradykinin in human and animals. BK peptides were quantified by MS in male rats. NO release was quantified from human, mouse and rat cells loaded with DAF-FM. Rat aortic rings were used to measure vascular reactivity. Changes in BP and HR were measured in conscious male rats. To evaluate pro-inflammatory effects both vascular permeability and nociception were measured in adult mice. BK-(1-7) and BK-(1-5) are produced in vivo from BK-(1-9). Both peptides induced NO production in all cell types tested. However, unlike BK-(1-9), NO production elicited by BK-(1-7) or BK-(1-5) was not inhibited by B1 or B2 receptor antagonists. BK-(1-7) and BK-(1-5) induced concentration-dependent vasorelaxation of aortic rings, without involvement of B1 or B2 receptors. Intravenous or intra-arterial administration of BK-(1-7) or BK-(1-5) induced similar hypotensive response in vivo. Nociceptive responses of BK-(1-7) and BK-(1-5) were reduced compared to BK-(1-9), and no increase in vascular permeability was observed for BK-(1-9) fragments. BK-(1-7) and BK-(1-5) are endogenous peptides present in plasma. BK-related peptide fragments show biological activity, not mediated by B1 or B2 receptors. These BK fragments could constitute new, active components of the kallikrein-kinin system.

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats.

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The Effect of Unilateral Nephrectomy on Arterial Blood Pressure Variability in Spontaneously Hypertensive Rats


 
 
 The aim of the current study was to investigate the effects of unilateral nephrectomy (ULN) on fast oscillation of arterial blood pressure in spontaneously hypertensive rats (SHR). Experiments were carried out on conscious normotensive Wistar rats and SHR divided in groups with intact kidneys: W, n = 10; SHR, n = 9 and with unilateral nephrectomy: WN, n = 10; SHRN, n = 10. ULN was performed under general anaesthesia (Nembutal 35 mg/kg b.w. i.p.), eight days before the experiments. Blood pressure variability was investigated by means of spectral analysis of systolic (SAP) diastolic (DAP) and mean (MAP) arterial blood pressure, estimated in directly registered arterial blood pressure wave. In SAP, DAP and MAP spectrograms, derived by Lab View software equipment, the spectral power (P) in low (LF, 20–195 mHz), mid (MF, 195–605 mHz) and high frequency (HF, 605—3000 mHz) bands was calculated. PLF in SAP spectrograms increased after ULN both in Wistar rats and SHR. ULN led to decrease of PMF in SAP, DAP and MAP spectrograms only in SHR. The ULN provokes changes in blood pressure variability in different manner in the normotensive and the spontaneously hypertensive rats. In addition to humorally-mediated low-frequency oscillations, ULN in SHR also af- fected the spectral characteristics of the arterial pressure in which sympathetic nervous activity operates.
 
 

The sympathetic nervous system exacerbates carotid body sensitivity in hypertension.

The carotid bodies (CBs) of spontaneously hypertensive (SH) rats exhibit hypertonicity and hyperreflexia contributing to heightened peripheral sympathetic outflow. We hypothesized that CB hyperexcitability is driven by its own sympathetic innervation. To test this, the chemoreflex was activated (NaCN 50-100 µL, 0.4 µg/µL) in SH and Wistar rats in situ before and after: (i) electrical stimulation (ES; 30 Hz, 2 ms, 10 V) of the superior cervical ganglion (SCG), which innervates the CB; (ii) unilateral resection of the SCG (SCGx); (iii) CB injections of an α1-adrenergic receptor agonist (phenylephrine, 50 µL, 1 mmol/L), and (iv) α1-adrenergic receptor antagonist prazosin (40 µL, 1 mmol/L) or tamsulosin (50 µL, 1 mmol/L). ES of the SCG enhanced CB-evoked sympathoexcitation by 40-50% (P < 0.05) with no difference between rat strains. Unilateral SCGx attenuated the CB-evoked sympathoexcitation in SH (62%; P < 0.01) but was without effect in Wistar rats; it also abolished the ongoing firing of chemoreceptive petrosal neurones of SH rats, which became hyperpolarized. In Wistar rats, CB injections of phenylephrine enhanced CB-evoked sympathoexcitation (33%; P < 0.05), which was prevented by prazosin (26%; P < 0.05) in SH rats. Tamsulosin alone reproduced the effects of prazosin in SH rats and prevented the sensitizing effect of the SCG following ES. Within the CB, α1A- and α1B-adrenoreceptors were co-localized on both glomus cells and blood vessels. In conscious SH rats instrumented for recording blood pressure (BP), the CB-evoked pressor response was attenuated after SCGx, and systolic BP fell by 16 ± 4.85 mmHg. The sympathetic innervation of the CB is tonically activated and sensitizes the CB of SH but not Wistar rats. Furthermore, sensitization of CB-evoked reflex sympathoexcitation appears to be mediated by α1-adrenoceptors located either on the vasculature and/or glomus cells. The SCG is novel target for controlling CB pathophysiology in hypertension.