Adult Male Wistar-Kyoto Research Articles

Cholesterol lowering acitivity and vasorelaxant effect of ethanol extract Piper aduncum leaves in hypercholesterolemic wistar kyoto rats

Spike betel (Piper aduncum L.) is one of the plant widely used as traditional medicine. There are still many unknown recorded pharmacological activities of Piper aduncum.This study aimed to observe the cholesterol lowering and vasorelaxant effect of P. aduncum ethanol extract (PAEE) in hypercholesterolemic model. Total cholesterol, triglyceride, LDL, HDL, NO level, Atherogenic Plasma Index (API), Systolic and Diastolyc Blood Pressure of hypercholesterolemic Wistar Kyoto (WKY) rats were used as parameters. Thirty adult male WKY rats were divided into six groups; each group contained five animals. Five groups were given high-fat diet foods, and PTU induced the animals to obtain hypercholesterolemia with a frequency of 1 time a day orally for 14 days. At the same time, the normal group was given destiled water and standard feed. On the 15th day the normal group was given destiled water; the high-fat diet group was given high-fat feed, the atorvastatin group was given an atorvastatin dose of 0.26 mg/kg BW, and the treatment group was given PAEE dose of 25, 50 and 100 mg/kg body weight for 14 days orally. Ethanol extract of P. aduncum significantly reduce total cholesterol levels and LDL, increase HDL levels and increase NO levels significantly (p<0.05). Doses of 50 and 100 mg/kg BW had the best effect on lowering total cholesterol, LDL level and provides a vasorelaxant effect, confimed by systolic and diastolic blood pressure examination.

Systemic and local administration of vasopressin differently affect hemodynamic and respiratory parameters in normotensive and hypertensive rats

IntroductionUpregulation of vasopressinergic system and increased chemoreflex activity contribute to essential hypertension. Vasopressin (AVP) V1a receptors (V1aRs) were found in the brain stem structures involved in the control of breathing and in the carotid bodies (CBs). It has also been shown that in normotensive rats intravenous (i.v.) and local (intra‐carotid, i.a.) AVP administration results in different hemodynamic and respiratory response.PurposeWe aimed at finding out how systemic (i.v.) and local (i.a.) administration of AVP affects cardiorespiratory system via V1aRs in normo‐ and hypertensive rats and if V1aRs are expressed in CBs in spontaneously hypertensive (SHR) and normotensive Wistar‐Kyoto (WKY) rats.MethodsThe study was performed on urethane‐anesthetized adult male WKY (n=7) and SHR rats (n=7). Animals were implanted with vascular catheters into femoral vein (i.v. infusions), femoral artery (hemodynamic parameters) and external carotid artery (local infusions), as well as the tracheal tube (respiratory parameters) and subcutaneous ECG electrodes. After obtaining baseline parameters, the arterial chemoreflex was triggered with i.v. administration of potassium cyanide (KCN) (30 μg/100 μL). Bolus of AVP was given i.v. (10 ng/100 μL) and i.a. (5 ng/50 μL). After stabilization of hemodynamic and respiratory parameters, V1a receptor was blocked by administration of V1aR antagonist (d(CH2)51,Tyr(Me)2,Arg8)‐Vasopressin; 5 μg/100 μL) and AVP and KCN infusions were repeated. After measurements, carotid bodies (CBs) were collected and immunostained for V1aRs and visualized with confocal microscopy.ResultsResting mean arterial blood pressure (MABP), heart rate (HR) and minute ventilation (MV) were significantly higher in SHR rats than in WKY control. In both groups hemodynamic and respiratory response to pharmacological trigger of the reflex occurred, however increase in MABP and MV were significantly greater in SHR than in WKY rats. Intravenous AVP administration caused an increase in MABP, but ventilatory inhibition only in SHR rats. Intra‐arterial AVP infusion caused a modest increase in MABP in both groups, but inhibition of ventilation only in SHR rats. Blockade of V1aR abolished the effects of both systemic and local AVP administration. Furthermore, V1aR antagonist decreased the pressor component of the chemoreflex, however it did not influence ventilatory response. Immunostaining revealed V1aRs in CBs in both groups.ConclusionsOur findings show that enhanced cardiorespiratory responses to activation of arterial chemoreflex and augmented pressor and ventilatory effects of systemic and local administrations of AVP are present in hypertensive rats and depend on V1aRs. Presence of V1aRs in CBs suggests that AVP may control cardiorespiratory system via arterial chemoreceptors.

Diabetes associated with hypertension exacerbated oxidative stress-mediated inflammation, apoptosis and autophagy leading to erectile dysfunction in rats.

Diabetes or hypertension contributes to erectile dysfunction (ED). We hypothesized that excess reactive oxygen species (ROS) production evoked by diabetes combined with hypertension may further suppress endothelial nitric oxide (NO) expression/activity and promote oxidative stress in the ED penis. Twenty-four adult male Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were divided into four groups: normal WKY, diabetic WKY, normal SHR and diabetic SHR. Intraperitoneal streptozotocin (65 mg/kg) was applied to induce type I diabetes. After 4-week diabetes and/or hypertension induction, we determined the intra-cavernous pressure (ICP) using electrical stimulation of cavernous nerves, intra-cavernosum NO amount using an electrochemical NO probe, and blood ROS using an ultrasensitive chemiluminescence-amplified analyzer. Western blot analysis and immunohistochemistry were used to explore the pathophysiologic mechanisms of inflammation, apoptosis and autophagy in the penis. A novel NO donor, CysaCysd Lu-5 (CCL5, (RCH2CH2S)(R'R"CHCH2S)Fe(NO)2, 1-4 µg), was intravenously administered to these ED rats for evaluating their ICP responses. In the baseline status, the lucigenin- and luminol-amplified blood ROS were significantly enhanced in the diabetic SHR rats vs normal WKY rats. Significantly decreased ICP, eNOS expression and NO amount were found in the normal SHR, diabetic WKY, and diabetic SHR vs normal WKY rats. Intravenous NO donor L-Arginine markedly increased ICP and NO amount, whereas eNOS inhibitor, Nω-Nitro-L-Arginine methyl ester hydrochloride depressed ICP in all four groups. Diabetes and/or hypertension alone increased fibrosis, proinflammatory NF-kB/ICAM-1 expression, mast cell numbers, CD68 expression and infiltration, Caspase 3-mediated apoptosis, Beclin-1/LC3-II-mediated autophagy and mild Nrf-2/HO-1 expression and depressed eNOS expression in the ED penis. The novel NO donor, CCL5, was more efficient than L-arginine to improve diabetes and/or hypertension-induced ED by the significant increase of ICP. Diabetes combined with hypertension synergistically exacerbated ED through enhanced oxidative stress, inflammation, apoptosis and autophagy and depressed eNOS activity and NO production.

Abstract MP39: Metabolic And Gene Expression Profiling Reveal Disparities In Absorption And Metabolism Of Butyrate And Lysine In The Colon Of Spontaneously Hypertensive Rodents

Introduction: Metabolic dysregulation and gut dysbiosis are linked to hypertension. The proximal colon is the main site of metabolism and absorption of short chain fatty acid butyrate. Reduced circulating butyrate concurrently with elevated fecal levels have been reported in human and rodent hypertension. We tested the hypothesis that both the transport and metabolism of butyrate as well as the overall metabolic profile are dysregulated in the colon of the Spontaneously Hypertensive rats (SHR) compared to the Wistar Kyoto (WKY) rats. Methods: Proximal colons from adult male WKY and SHR were placed in oxygenated warmed Ussing chamber buffer containing dextrose, and physiologically relevant concentration of butyrate (40mM) was applied to the luminal side of the colon. Following 1hr incubation, butyrate levels from the luminal and basolateral sides of the Ussing chamber were measured with high performance liquid chromatography to establish butyrate colonic transport and metabolism. Global metabolomics was performed in Ussing chamber media and in the plasma of WKY and SHR to determine the overall metabolic profile. RNA-seq data in the gut epithelium of WKY and SHR was used to elucidate putative signaling pathways altered in rodent hypertension. Results: Both the colonic transport and metabolism of butyrate were significantly impaired in the SHR. In addition, global metabolomics identified reduced levels of essential amino acid L-lysine, among other metabolites, on the basolateral side of the Ussing chamber and in the plasma of the SHR compared to WKY. Enriched metabolite pathways disrupted in the SHR included lysine degradation. RNA-seq revealed differential expression of several fatty acid and amino acid transporters in the colonic epithelium of the SHR. Conclusion: Colonic transport and metabolism of butyrate are reduced in the SHR, accompanied by dysregulation in the metabolism of the essential amino acid lysine in the gut. This, coupled with reduced expression levels of several fatty acid and amino acid transporters in the gut, suggest aberrant host-microbiota communication and colonic metabolic dysfunction in the context of hypertension.

A Novel Assessment of Baroreflex Activity Through the Similarity of Ternary Codes of Oscillations Between Arterial Blood Pressure and R–R Intervals

Arterial baroreflex plays a crucial role in the homeostasis of blood pressure. Elevated blood pressure stimulates baroreceptors and slows the heart rate consequently. Baroreflex activity has been quantified as the relationship between systolic blood pressure (SBP) and ECG R–R intervals (RRI), which is called baroreflex sensitivity (BRS). The purpose of this study is to define a new parameter of BRS and verify the reproducibility and validity of this indicator as compared with a popular BRS estimated by sequence method and a traditional BRS evaluated by pharmacological intervention. At first, the spontaneous oscillations of SBP and RRI were defined by ternary arithmetic codes: decrease as 0, unchanged as 1 and increase as 2. The new BRS was quantified as the similarity between these two ternary coded series after decimalization with different digits from 3 to 5. The validity and reproducibility of this method were verified in vivo in 10 adult male Wistar Kyoto rats. This new parameter correlated well with the BRS estimated by the sequence method and pharmacological intervention. The reproducibility of this novel method was good according to the Bland–Altman plot. This study offered a simple and reliable algorithm to estimate BRS, which can be embed in current instruments for clinical research.

Abstract P171: Gut Microbiota-Derived Hydrogen Sulfide is Reduced in Spontaneously Hypertensive Rats

Introduction: Gut bacteria play a significant role in host homeostasis, and gut dysbiosis has been associated with many conditions including hypertension (HTN). Endogenous hydrogen sulfide (H 2 S) is an important freely-diffusing molecule that can modulate neural, cardiovascular and immune system activity, and circulating levels of H 2 S are reduced in animals and humans with HTN. While most research on the role of H 2 S in HTN has focused on H 2 S produced by host tissues, H 2 S produced by gut bacteria may also enter the host circulation. We investigated whether the spontaneously hypertensive rat (SHR), an established model of HTN, has a disruption in gut bacteria-derived H 2 S production. Methods: Bacterial DNA from fecal samples of adult male normotensive Wistar-Kyoto (WKY) and SHR was isolated for 16s bacterial genomic sequencing. WKY and SHR fecal and plasma samples were analyzed for H 2 S levels using modified methylene blue assay. Blood pressures were determined in all rats at several time points using a tail cuff. Data are expressed as mean±SEM, with P&lt;0.05 considered statistically significant. Results: We found a significant reduction in the abundance of two H 2 S-producing gut bacteria in the SHR compared to WKY: Enterobacteriaceae (1.423e-005±1.056e-005 vs. 0.0002329±7.853e-005, n=6, P&lt;0.05), and Clostridiaceae (0.01134±0.00431 vs. 0.06408±0.01416, n=6, P&lt;0.01). This decrease in the H 2 S-producing gut bacteria was also associated with a significant reduction in fecal H 2 S levels (SHR: 0±0.01703 AU vs. WKY: 0.094±0.03385 AU, n=5; P&lt;0.05) and lower plasma H 2 S levels (SHR: 0.3265±0.07817 AU vs. WKY: 0.6850±0.1790 AU, n=2) in the SHR compared to the WKY. Conclusion: These results suggest that diminished gut bacterial production of H 2 S may contribute to the reduced H 2 S observed in host circulation in established HTN.

Abstract 084: Gut Dysbiosis Impairs Serotonergic Gut-Brain Axis and Increases Blood Pressure

Introduction: Fecal microbiota transfer (FMT) from hypertensive to normotensive rats elevates blood pressure (BP). As gut microbiota can affect synthesis of serotonin (5-HT), a major neurotransmitter predominantly synthesized in the gut, our objective was to investigate the effect of gut dysbiosis on gut-derived 5-HT in control of BP. Methods: Gut microbiota was depleted in normotensive adult male Wistar Kyoto (WKY) rats (N=24) using a cocktail of Vancomycin/Meropenem/Omeprazole at 50mg/kg/day for 5 days, followed by FMT from either adult male spontaneously hypertensive rats (SHR) (SHR-WKY, N=12) or age-, sex-matched WKY (WKY-WKY, N=12). At 8 weeks post-FMT, the following parameters were measured: BP by tailcuff and radiotelemetry; autonomic variables by spectral analysis of waveform BP; activity of cardioregulatory brain regions by manganese-enhanced MRI; fecal and proximal colonic 5-HT levels by HPLC; and levels of 5-HT 3 receptors in the proximal colon, nodose ganglia and the nucleus of the solitary tract (NTS) by qPCR. Lastly, BP and heart rate (HR) responses to intra-mesenteric i.v. injection of 5-HT 3 receptor agonist 1-phenylbigunide (35μg) were determined in anesthetized rats. Results: Compared with WKY-WKY, SHR-WKY showed a significant increase in systolic BP (Δ=34mmHg, P&lt;0.05). This was associated with increased low frequency (by 13%, P&lt;0.05) and decreased high frequency of BP (by 14%, P&lt;0.05), indicative of sympathetic and parasympathetic variables respectively. A suppression of neural activity in the NTS (by 2 fold, P=0.06), and a reduction in 5HT 3 receptors in the NTS (by 1.5 fold, P&lt;0.01), nodose ganglia (by 2 fold, P&lt;0.05) and proximal colon (by 1.5 fold, P=0.09) in SHR-WKY resembled those in the SHR. Lower level of 5-HT in the proximal colon (by 4 fold, P&lt;0.01), but not in the feces of SHR-WKY were observed. Similar to SHR, WKY-SHR showed significantly blunted drop in BP (by 5 fold, P&lt;0.05) and HR (by 2 fold, P&lt;0.05) in response to intra-mesenteric i.v. injection of 1-phenylbigunide, compared with WKY-WKY. Conclusions: FMT from SHR to WKY is associated with imbalanced autonomic variables and perturbations in serotonergic gut-brain axis that may contribute to hypertension partly via a dampened afferent vagal feedback to the NTS.

Activation of the kynurenine pathway and mitochondrial respiration to face allostatic load in a double-hit model of stress

Allostasis is the process by which the body’s physiological systems adapt to environmental changes. Chronic stress increases the allostatic load to the body, producing wear and tear that could, over time, become pathological. In this study, young adult male Wistar Kyoto rats were exposed to an unpredictable chronic mild stress (uCMS) protocol to increase allostatic load. First, physiological systems which may be affected by extended uCMS exposure were assessed. Secondly, 5 weeks of uCMS were used to investigate early adaptations in the previously selected systems. Adverse experiences during developmentally sensitive periods like adolescence are known to severely alter the individual stress vulnerability with long-lasting effects. To elucidate how early life adversity impacts stress reactivity in adulthood, an additional group with juvenile single-housing (JSH) prior to uCMS was included in the second cohort. The aim of this work was to assess the impact of chronic stress with or without adversity during adolescence on two domains known to be impacted in numerous stress-related disorders: mitochondrial energy metabolism and the immune system. Both, uCMS and adolescence stress increased kynurenine and kynurenic acid in plasma, suggesting a protective, anti-oxidant response from the kynurenine pathway. Furthermore, uCMS resulted in a down-regulation of immediate early gene expression in the prefrontal cortex and hippocampus, while only rats with the double-hit of adolescent stress and uCMS demonstrated increased mitochondrial activity in the hippocampus. These results suggest that early life adversity may impact on allostatic load by increasing energetic requirements in the brain.

Piperazine ferulate exerts antihypertensive effect and improves endothelial function in vitro and in vivo via the activation of endothelial nitric oxide synthase

To investigate the effect of piperazine ferulate (PF) on hypertension and endothelial function, and to assess the possible underlying mechanism. Human umbilical vein endothelial cells (HUVEC), adult male Wistar Kyoto (WKY) rats aged 12 to 14 weeks, and spontaneously hypertensive (SH) and Sprague Dawley (SD) rats were used for this study. Cell viability, activities of angiotensin-converting enzyme (ACE) and heme oxygenase-1 (HO-1), in vivo NO synthesis, arterial systolic blood pressure, vascular function, expressions of endothelial NO synthase (eNOS) and phosphorylated-eNOS (p-eNOS) were determined or assessed as appropriate. The results of MTT assay showed the number of viable cells were significantly increased with increase in PF concentration (p &lt; 0.05). The level of expression of ACE was significantly reduced with increase in PF concentration (p &lt; 0.05), while the level of HO-1 expression significantly increased (p &lt; 0.05). Results of DAF-FM fluorescent staining showed that the amounts of NO synthesized in vivo was significantly higher in aortic rings of SH and SD rats treated with PF than in the corresponding control groups (p &lt; 0.05). Treatment with PF in vivo significantly improved impaired acetylcholine-induced aortic relaxation in SH rats. Total eNOS expression was significantly increased after treatment with PF (p &lt; 0.05). The expressions of total eNOS and p-eNOS in both groups were not affected by PF when compared to the control group. These results indicate that PF exerts antihypertensive effect and improves endothelial function in vitro and in vivo via the activation of eNOS.

In vivo assessment of endothelial function in small animals using an infrared pulse detector.

Objective:Endothelial dysfunction is the earliest change in atherosclerosis. Flow-mediated dilatation (FMD) is used to assess endothelial function in humans. However, this assessment is not easy in small animals. This study demonstrated the reliability and reproducibility of a proposed instrument for in vivo assessment of FMD in a rodent model using infrared pulse sensors.Materials and Methods:We used 24 adult male Wistar Kyoto rats randomly divided into three groups. FMD was measured under continuous infusion of normal saline followed by intra-arterial infusion of acetylcholine (Ach; n = 8), sodium nitroprusside (SNP; n = 8), or Nω-nitro-L-arginine methyl ester (L-NAME; n = 8).Results:The dilatation indices (DIs) of all three groups were similar before application of the vasoactive agents (1.82 ± 0.46, 1.81 ± 0.44, and 1.93 ± 0.40, P = 0.877, by one-way analysis of variance). The DI was significantly increased during infusion of Ach (2.97 ± 1.03 vs. 1.82 ± 0.46, P = 0.015), unchanged during infusion of SNP (1.81 ± 0.44 vs. 1.98 ± 0.40, P = 0.574), and attenuated during infusion of L-NAME (1.91 ± 0.40 vs. 1.42 ± 0.35; P = 0.028).Conclusion:The results of this study correlated well with those of human studies, suggesting that this method can be used for in vivo evaluation of endothelial function in small animals.

24-Hour Profile of Blood Pressure, Heart Rate, Excretion of Electrolytes, and Locomotor Activity in Wistar-Kyoto and SHR Rats Under Conditions of Free-Run Rhythm.

We presented the results of our study of chronostructure of BP, HR, electrolyte excretion, and locomotor activity under conditions of "free-run rhythm" (light deprivation). In adult male Wistar-Kyoto (normotensive) and SHR (spontaneously hypertensive) rats, BP, biopotentials of the heart (ECG), and locomotor activity were recorded over 24 h by telemetric monitoring and the rate of excretion of electrolytes (Na+, K+, Ca2+, and Mg2+) during the nighttime and daytime hours was measured. It was found that under free-run rhythm, 24-h profiles of BP, HR, excretory function of the kidneys, and locomotor activity underwent more considerable changes in normotensive Wistar-Kyoto rats in comparison with hypertensive SHR rats. However, hypertensive rats demonstrated pronounced changes in rhythmic characteristics of HR, which can restrict adaptation reserves of the cardiovascular system.

Uric Acid Treatment After Stroke Prevents Long-Term Middle Cerebral Artery Remodelling and Attenuates Brain Damage in Spontaneously Hypertensive Rats.

Hypertension is the most important modifiable risk factor for stroke and is associated with poorer post-stroke outcomes. The antioxidant uric acid is protective in experimental normotensive ischaemic stroke. However, it is unknown whether this treatment exerts long-term protection in hypertension. We aimed to evaluate the impact of transient intraluminal middle cerebral artery (MCA) occlusion (90min)/reperfusion (1-15days) on brain and vascular damage progression in adult male Wistar-Kyoto (WKY; n= 36) and spontaneously hypertensive (SHR; n= 37) rats treated (i.v./120min post-occlusion) with uric acid (16mgkg-1) or vehicle (Locke's buffer). Ischaemic brain damage was assessed longitudinally with magnetic resonance imaging and properties of MCA from both hemispheres were studied 15days after stroke. Brain lesions in WKY rats were associated with a transitory increase in circulating IL-18 and cerebrovascular oxidative stress that did not culminate in long-term MCA alterations. In SHR rats, more severe brain damage and poorer neurofunctional outcomes were coupled to higher cortical cerebral blood flow at the onset of reperfusion, a transient increase in oxidative stress and long-lasting stroke-induced MCA hypertrophic remodelling. Thus, stroke promotes larger brain and vascular damage in hypertensive rats that persists for long-time. Uric acid administered during early reperfusion attenuated short- and long-term brain injuries in both normotensive and hypertensive rats, an effect that was associated with abolishment of the acute oxidative stress response and prevention of stroke-induced long-lasting MCA remodelling in hypertension. These results suggest that uric acid might be an effective strategy to improve stroke outcomes in hypertensive subjects.

Ultrafine Particulate Matter Combined With Ozone Exacerbates Lung Injury in Mature Adult Rats With Cardiovascular Disease.

Particulate matter (PM) and ozone (O3) are dominant air pollutants that contribute to development and exacerbation of multiple cardiopulmonary diseases. Mature adults with cardiovascular disease (CVD) are particularly susceptible to air pollution-related cardiopulmonary morbidities and mortalities. The aim was to investigate the biologic potency of ultrafine particulate matter (UFPM) combined with O3 in the lungs of mature adult normotensive and spontaneously hypertensive (SH) Wistar-Kyoto rats. Conscious, mature adult male normal Wistar-Kyoto (NW) and SH rats were exposed to one of the following atmospheres: filtered air (FA); UFPM (∼ 250 μg/m3); O3 (1.0 ppm); or UFPM + O3 (∼ 250 μg/m3 + 1.0 ppm) combined for 6 h, followed by an 8 h FA recovery period. Lung sections were evaluated for lesions in the large airways, terminal bronchiolar/alveolar duct regions, alveolar parenchyma, and vasculature. NW and SH rats were similarly affected by the combined-pollutant exposure, displaying severe injury in both large and small airways. SH rats were particularly susceptible to O3 exposure, exhibiting increased injury scores in terminal bronchioles and epithelial degeneration in large airways. UFPM-exposure groups had minimal histologic changes. The chemical composition of UFPM was altered by the addition of O3, indicating that ozonolysis promoted compound degradation. O3 increased the biologic potency of UFPM, resulting in greater lung injury following exposure. Pathologic manifestations of CVD may confer susceptibility to air pollution by impairing normal lung defenses and responses to exposure.

Increased Oxidative Parameters and Decreased Cytokine Levels in an Animal Model of Attention-Deficit/Hyperactivity Disorder.

Attention-deficit/hyperactivity disorder (ADHD) is a highly heterogeneous disorder characterized by impairing levels of hyperactivity, impulsivity and inattention. Oxidative and inflammatory parameters have been recognized among its multiple predisposing pathways, and clinical studies indicate that ADHD patients have increased oxidative stress. In this study, we aimed to evaluate oxidative (DCFH oxidation, glutathione levels, glutathione peroxidase, catalase and superoxide dismutase activities) and inflammatory (TNF-α, IL-1β and IL-10) parameters in the most widely accepted animal model of ADHD, the spontaneously hypertensive rats (SHR). Prefrontal cortex, cortex (remaining regions), striatum and hippocampus of adult male SHR and Wistar Kyoto rats were studied. SHR presented increased reactive oxygen species (ROS) production in the cortex, striatum and hippocampus. In SHR, glutathione peroxidase activity was decreased in the prefrontal cortex and hippocampus. TNF-α levels were reduced in the prefrontal cortex, cortex (remaining regions), hippocampus and striatum of SHR. Besides, IL-1β and IL-10 levels were decreased in the cortex of the ADHD model. Results indicate that SHR presented an oxidative profile that is characterized by an increase in ROS production without an effective antioxidant counterbalance. In addition, this strain showed a decrease in cytokine levels, mainly TNF-α, indicating a basal deficit. These results may present a new approach to the cognitive disturbances seen in the SHR.

Exposure to morphine-associated cues increases mu opioid receptor mRNA expression in the nucleus accumbens of Wistar Kyoto rats

The Wistar-Kyoto (WKY) rat has been proposed as a model of anxiety vulnerability as it exhibits pronounced behavioral inhibition, passive avoidance, exaggerated startle response, enhanced HPA-axis activation, and active avoidance that is resistant to extinction. Accumulating evidence suggests that WKY rats respond differently to rewarding stimuli when compared to outbred strains of rat. Conditioned responding to drug-associated cues is linked with alterations in the activation of mu opioid receptors (MOR) and kappa opioid receptors (KOR) in the nucleus accumbens (NAc). Furthermore, alterations in KOR expression/activation in the NAc of WKY rats are implicated in the regulation of some of the components that make up the unique behavioral phenotype of this strain. The purpose of this study was to extend upon previous work from our laboratory by investigating conditioned morphine reward in adult male WKY and SD rats, and to examine levels of KOR mRNA and MOR mRNA in the NAc at baseline and after acquisition of morphine CPP. Our results demonstrate that SD rats displayed morphine-induced CPP to each of the six doses of morphine tested (0.5, 1.25, 2.5, 5, 7.5, or 10mg/kg). Interestingly, WKY rats demonstrated CPP for only the 1.25, 2.5, and 5mg/kg doses, yet no preference at the lowest (0.5mg/kg) or highest (7.5 and 10mg/kg) doses. qPCR analysis of MOR and KOR in the NAc revealed no strain differences in basal levels of MOR, but higher levels of KOR in WKY rats compared to those of SD rats. Interestingly, after completion of the CPP task, WKY rats had overall higher levels of NAc MOR mRNA compared to SD rats; the initial basal differences in NAc KOR levels persisted without change due to CPP in either strain. These results demonstrate that the WKY rat exhibits a unique pattern of behavioral responding to morphine and implicates differences in NAc KOR signaling as a potential source of aversion to higher doses of morphine. Additionally, the CPP-induced upregulation of NAc MOR mRNA in WKY rats warrants further investigation in terms of its potential role as a factor constituting a unique vulnerability to subsequent drug exposure.

Abstract WP269: New Insights Into Worsened Cerebral Ischemia in Hypertensive Rats: Roles of WNK-SPAK/OSR1-NKCC1 Signaling

Background and Purpose: WNK kinases [with-no-lysine (K)], Ste20-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) are a family of evolutionarily conserved regulators of ion transporter proteins. The kinases alter the phosphorylation state of ion transporter proteins and so play an important role in renal salt handling and in the pathogenesis of hypertension. We previously reported that pharmacological inhibition or genetic knockout of Na+-K+-2Cl- cotransporter 1 (NKCC1) is neuroprotective against ischemic injury. Our current study investigated whether WNK-SPAK/OSR1-NKCC1 signaling is augmented in cerebral ischemia in hypertension. Methods: Cerebral ischemia was induced in adult male Wistar Kyoto (WKY) or spontaneously hypertensive rats (SHR) by 2-hour occlusion of the left middle cerebral artery (MCAO) followed by reperfusion. Brain tissues were harvested for immunoblot assessment of expression levels of NKCC1, SPAK/OSR1 or WNK1-4. Another two cohorts of WKY and SHR were treated with saline or NKCC1 inhibitor bumetanide (BMT, 10 mg/kg/day, i.p.) after MCAO. In these animals, ischemic infarct volume was determined by TTC staining and behavioral deficits were analyzed by neurological scoring and turning-alley test. Results: Basal levels of total NKCC1 protein were significantly higher in the contralateral (CL) cortical tissues in SHR brains than WKY brains. Ischemia was associated with increased phosphorylation of NKCC1 in the ipsilateral (IL) cortex of SHR, but not WKY at 6 hours post-ischemia. WNK1 increased 6.1±1.5 fold and WNK4 increased 8.7±1.7 fold in IL cortex of SHR, but not in that of WKY, and phosphorylated SPAK/OSR1 was unchanged. Lastly, sensitivity to pharmacological NKCC1 inhibition was higher in SHR than WKY rats. BMT blunted the MCAO-induced infarction and improved neurobehavioral score and sensory motor function in SHR rats. Conclusion: These findings suggest that augmented WNK1/4-NKCC1 signaling is responsible, at least in part, for the increased susceptibility to ischemic brain damage associated with hypertension. They highlight the potential for inhibition of WNK1/4-NKCC1 signaling as part of anti-ischemic stroke therapy. Supported by NIH Grant R01 NS38118 (D Sun).

WNK-Cab39-NKCC1 signaling increases the susceptibility to ischemic brain damage in hypertensive rats

With-no-lysine kinase (WNK) and Na+-K+-2Cl− cotransporter 1 (NKCC1) are involved in the pathogenesis of hypertension. In this study, we investigated the WNK-NKCC1 signaling pathway in spontaneously hypertensive rats (SHR) and their associated susceptibility to stroke injury. Basal NKCC1 protein levels were higher in SHR than in normotensive Wistar Kyoto (WKY) rat brains. After inducing ischemic stroke, adult male WKY and SHR received either saline or NKCC1 inhibitor bumetanide (10 mg/kg/day, i.p.) starting at 3-h post-reperfusion. NKCC1 inhibition blunted the extent of ischemic infarction in SHR and improved their neurobehavioral functions. Interestingly, ischemia led to increased NKCC1 phosphorylation in SHR but not in WKY rats. Pronounced elevation of WNK1, WNK2 and WNK4 protein and downregulation of WNK3 were detected in ischemic SHR, but not in ischemic WKY rats. Upregulation of WNK-NKCC1 complex in ischemic SHR brain was associated with increased Ca2+-binding protein 39 (Cab39), without increases in Ste20-related proline alanine-rich kinase or oxidative stress-responsive kinase-1. Moreover, subacute middle cerebral artery stroke human brain autopsy exhibited increased expression of NKCC1 protein. We conclude that augmented WNK-Cab39-NKCC1 signaling in SHR is associated with an increased susceptibility to ischemic brain damage and may serve as a novel target for anti-hypertensive and anti-ischemic stroke therapy.

Transcranial direct current stimulation improves short-term memory in an animal model of attention-deficit/hyperactivity disorder

Attention deficit hyperactivity disorder (ADHD) is characterized by impairing levels of hyperactivity, impulsivity and inattention. However, different meta-analyses have reported disruptions in short and long-term memory in ADHD patients. Previous studies indicate that mnemonic dysfunctions might be the result of deficits in attentional circuits, probably due to ineffective dopaminergic modulation of hippocampal synaptic plasticity. In this study we aimed to evaluate the potential therapeutic effects of a neuromodulatory technique, transcranial direct current stimulation (tDCS), in short-term memory (STM) deficits presented by the spontaneous hypertensive rats (SHR), the most widely used animal model of ADHD. Adult male SHR and Wistar Kyoto rats (WKY) were subjected to a constant electrical current of 0.5mA intensity applied on the frontal cortex for 20min/day during 8 days. STM was evaluated with an object recognition test conducted in an open field. Exploration time and locomotion were recorded, and brain regions were dissected to determine dopamine and BDNF levels. SHR spent less time exploring the new object when compared to WKY, and tDCS improved object recognition deficits in SHR without affecting WKY performance. Locomotor activity was higher in SHR and it was not affected by tDCS. After stimulation, dopamine levels were increased in the hippocampus and striatum of both strains, while BDNF levels were increased only in the striatum of WKY. These findings suggest that tDCS on the frontal cortex might be able to improve STM deficits present in SHR, which is potentially related to dopaminergic neurotransmission in the hippocampus and striatum of those animals.

Tissue distribution of CysAP activity and its relationship to blood pressure and water balance

To better understand the functional role of soluble (Sol) and membrane-bound (MB) cystinyl-aminopeptidase (CysAP) activities, we studied differentially their organ distribution in adult male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)with or without treatment with captopril.We searched for a possible tissue-specific association of CysAP with water balance and blood pressure. We used twenty WKY rats distributed in ten controls and ten captopril-treated, and sixteen SHR divided in eight controls and eight captopril-treated. Captopril (100 mg/kg/day) was administered in drinking water for 4 weeks. Systolic blood pressure, water intake and diuresis were measured individually. CysAP was assayed fluorometrically using L-cystine-di-β-naphthylamide as substrate. Sol or MB activities were generally higher in SHR compared to WKY notably in hypothalamus and kidney than in the other tissues. Captopril mainly decreased CysAP in SHR whereas it increased in WKY. The distribution of Sol CysAP was more homogeneous among tissues ofWKY than SHR. In contrast, the distribution of MB CysAP was more heterogeneous than Sol CysAP in both WKY and SHR. This suggests that MB CysAP activity acts in a more tissue-specific manner than Sol CysAP. The majority of the significant correlations between tissue activities and the measured physiological parameters were observed mostly in renal medulla and hypothalamus. Sol and MB CysAP activities, acting separately or in concert and mainly in renal medulla, regulate the function of their susceptible endogenous substrates, and may participate meaningfully in the control of blood pressure and fluid balance.

Coconut oil supplementation and physical exercise improves baroreflex sensitivity and oxidative stress in hypertensive rats.

The hypothesis that oral supplementation with virgin coconut oil (Cocos nucifera L.) and exercise training would improve impaired baroreflex sensitivity (BRS) and reduce oxidative stress in spontaneously hypertensive rats (SHR) was tested. Adult male SHR and Wistar Kyoto rats (WKY) were divided into 5 groups: WKY + saline (n = 8); SHR + saline (n = 8); SHR + coconut oil (2 mL·day(-1), n = 8); SHR + trained (n = 8); and SHR + trained + coconut oil (n = 8). Mean arterial pressure (MAP) was recorded and BRS was tested using phenylephrine (8 μg/kg, intravenous) and sodium nitroprusside (25 μg·kg(-1), intravenous). Oxidative stress was measured using dihydroethidium in heart and aorta. SHR + saline, SHR + coconut oil, and SHR + trained group showed higher MAP compared with WKY + saline (175 ± 6, 148 ± 6, 147 ± 7 vs. 113 ± 2 mm Hg; p < 0.05). SHR + coconut oil, SHR + trained group, and SHR + trained + coconut oil groups presented lower MAP compared with SHR + saline group (148 ± 6, 147 ± 7, 134 ± 8 vs. 175 ± 6 mm Hg; p < 0.05). Coconut oil combined with exercise training improved BRS in SHR compared with SHR + saline group (-2.47 ± 0.3 vs. -1.39 ± 0.09 beats·min(-1)·mm Hg(-1); p < 0.05). SHR + saline group showed higher superoxide levels when compared with WKY + saline (774 ± 31 vs. 634 ± 19 arbitrary units (AU), respectively; p < 0.05). SHR + trained + coconut oil group presented reduced oxidative stress compared with SHR + saline in heart (622 ± 16 vs. 774 ± 31 AU, p < 0.05). In aorta, coconut oil reduced oxidative stress in SHR compared with SHR + saline group (454 ± 33 vs. 689 ± 29 AU, p < 0.05). Oral supplementation with coconut oil combined with exercise training improved impaired BRS and reduced oxidative stress in SHR.