Brain Inflammatory Cytokines And Angiotension-(1-7)Modulate Sympathetic Activity And Cardiac Sympathetic Afferent Reflex

Part1Inflammatory Cytokines in Paraventricular Nucleus Modulate Sympathetic Activity and Cardiac Sympathetic Afferent Reflex in RatsBackgroundThe excessive sympathetic activation plays a vital role in the pathogenesis and progression of many cardiovascular diseases, such as hypertension and chronic heart failure (CHF). Cardiac sympathetic afferent reflex (CSAR) is a sympatho-excitatory reflex with a feature of positive feedback, and can be induced by the stimulation of the cardiac sympathetic afferent endings with bradykinin, adenosine, and hydrogen peroxide released from the myocardium in myocardial ischemia. It has been found that the CSAR is enhanced in experimental hypertension and CHF, and the enhanced CSAR partially contributes to the increased sympathetic outflow in these diseases. Paraventricular nucleus (PVN) is an important integrative site of the sympathetic activity and a pivotal component of the central neurocircuitry of the CSAR. The PVN is involved in the enhanced CSAR and sympathetic activation in the animal models of hypertension and CHF. It has been reported that the pro-inflammatory cytokines (PIC) accumulates in the PVN in rats with acute myocardial infarction and heart failure. The increased PIC in the brain in those pathologic states contributes to the enhanced sympathetic nerve activity. Inhibition of the brain PIC synthesis attenuates sympatho-excitation in heart failure. However, the role of inflammatory cytokines in the PVN in regulating the CSAR is unknown. On the other hand, our previous studies have shown that angiotensin Ⅱ (Ang Ⅱ) and AT1receptors in the PVN regulate CSAR and are involved in the enhanced CSAR and sympathetic hyperactivity in heart failure. The interaction between inflammatory cytokines and the Ang Ⅱ has been found in peripheral tissue and brain. However, the interation of inflammatory cytokines with Ang Ⅱ-AT1R in the PVN in regulating the CSAR is still unknown.Objective1. To determine whether PIC, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (TL-1β) and anti-inflammatory cytokines (AIC), such as IL-4and IL-13in the PVN modulate sympathetic activity, blood pressure and the CSAR.2. To determine the interaction of TNF-α, IL-1β, IL-4and IL-13with the Ang II-ATIR in the PVN in modulating sympathetic outflow, blood pressure and the CSAR.3. To determine whether successive stimulation of cardiac sympathetic afferents increased the levels of TNF-α, IL-1β and IL-4in the PVN.MethodsIn anesthetized rats with sinoaortic denervation and cervical vagotomy, renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded in vivo on a PowerLab data acquisition system. The coordinates for PVN were determined in a stereotaxic instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into PVN. The CSAR was evaluated by the RSNA response to epicardial application of bradykinin (BK). The inflammatory cytokines were measured with ELISA. Specific fluorogenic probe dihydroethidium (DHE) was used to detect in situ superoxide anions in the RVLM. 1. To determine separately the effects of different doses of TNF-α, IL-1β, IL-4or IL-13(0,0.1,1and10ng for each cytokine) in the PVN on the CSAR and the baseline RSNA and MAP.2. To determine the possible synergetic effects of Ang Ⅱ and TNF-α or IL-1β in the PVN, the effects of Ang Ⅱ (0.3nmol or3nmol), TNF-α(1ng or10ng), IL-1β (1ng or10ng), TNF-α+Ang Ⅱ and IL-1β+Ang Ⅱ were respectively investigated. Furthermore, the synergetic effects were investigated in sub-response doses of Ang Ⅱ (0.0003nmol) and four inflammatory cytokines (0.1ng for each cytokine). Ang Ⅱ was immediately administered following TNF-α, IL-1β, IL-4or IL-13.3. To determine the effects of the PVN pretreatment with losartan. Ten minutes after saline or losartan (50nmol) pretreatment in the PVN, the Ang Ⅱ (3nmol), TNF-α, IL-1β, IL-4or IL-13(10ng for each cytokine) was administered into the PVN.4. To determine the effects of cardiac afferent stimulation on the levels of TNF-α, IL-1β and IL-4. The rats were separately treated with successive epicardial application of saline or BK three times lasting15minutes for each application. Then the PVN, cerebral cortex and blood samples from individual rats were quickly collected for the measurement of TNF-α, IL-1β or IL-4using ELIS A method.5. To determine superoxide anions level in PVN. Brain were removed after PVN microinjection of saline, TNF-α or IL-1β and cryostat sectioned (30μm, coronal). Then, the sections were incubated in the dark with DHE. DHE fluorescence was visualized under a fluorescence microscope.Results1. Microinjection of TNF-α or EL-1β into the PVN induced dose-related increases in the CSAR, baseline RSNA and MAP, while IL-4or IL-13had no influences on baseline RSNA and the CSAR although they caused significant increases in baseline MAP. There was a linear correlation between the doses of TNF-α or IL-1β and the responses.2. The PVN microinjection of TNF-α or IL-1β combined with Ang Ⅱ at low dose but not at high dose caused much greater responses in the CSAR, baseline RSNA and MAP than the same dose of TNF-α, IL-1β or Ang Ⅱ alone. The very low dose of Ang Ⅱ (0.0003nmol) had no significant effect on the RSNA, MAP and CSAR. Pretreated with very low dose of TNF-α or IL-1β but not with saline, IL-4or IL-13, the sub-response dose of Ang II caused significant increases in the MAP, RSNA and CSAR.3. Losartan pretreatment in the PVN attenuated the effects of Ang Ⅱ, TNF-a and IL-1β on the MAP, RSNA and CSAR but did not affect the MAP responses to IL-4and IL-13.4. Compared with epicardial application of saline, epicardial application of BK significantly increased the TNF-α and IL-1β levels but not IL-4level in the PVN. However, epicardial application of BK had no significant effect on the TNF-a and IL-1β levels in the cerebral cortex and plasma.5. Compared with saline, PVN administration of TNF-α or IL-1β enhanced the intensity of the red fluorescent signals which means superoxide anions were increased.ConclusionsThe PIC, TNF-α or IL-1β, in the PVN increases blood pressure and sympathetic outflow and enhances the CSAR, which is partially dependent on the AT1receptors, while IL-4or IL-13in the PVN increases blood pressure but not enhances the (?) sympathetic activity and CSAR. There is a synergetic effect of Ang Ⅱ with TNF-α or IL-1β on blood pressure, sympathetic activity and CSAR. Part2Angiotensin-(1-7) in the Rostral Ventrolateral Medulla Modulate the Cardiac Sympathetic Afferent Reflex and Sympathetic Activity in RatsBackgroundThe cardiac sympathetic afferent reflex (CSAR) is a sympathoexcitatory cardiovascular reflex and contributes to the enhanced sympathetic outflow in chronic heart failure and hypertension. The enhancement of the CSAR in these diseases is caused not only by the increased stimulation of cardiac sympathetic afferents by factors such as adenosine, bradykinin, and hydrogen peroxide released from the myocardium in myocardial ischemia, but also by the increased central gain of the CSAR. It is known that the presympathetic bulbospinal neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the control of sympathetic outflow and blood pressure. Projections from the paraventricular nucleus (PVN) to the RVLM and the spinal cord have been identified. Our recent studies have shown that the PVN is an important nucleus in control of the CSAR and reactive oxygen species (ROS) in the RVLM are necessary for the enhanced CSAR response caused by angiotensin (Ang) Ⅱ in the PVN suggesting an important role of the RVLM in control of the CSAR. Abundant Ang Ⅱ type1(AT1) receptors are found in the RVLM. Microinjection of Ang Ⅱ into the RVLM induces a pressor response, which is mediated by AT1receptors. However, it is unknown whether Ang Ⅱ in the RVLM is involved in regulating the CSAR. It is possible that Ang Ⅱ in the RVLM may play an important role in regulating the CSAR. The first goal of this study was to determine the effects of Ang Ⅱ on the CSAR. Ang-(1-7) is a biologically active peptide of the renin-angiotensin system (RAS) family. It is mainly indirectly formed from Ang Ⅰ or directly from Ang Ⅱ through the newly described enzyme, angiotensin-converting enzyme2. Most effects of Ang-(1-7) are mediated by Mas receptors, which are selective G protein-coupled receptors different from the classical AT1or AT2receptor subtypes. Mas receptors are selectively blocked by its selective antagonist D-Alanine-Ang-(1-7)(A-779). Although opposite effects of Ang Ⅱ and Ang-(1-7) on impulse propagation, excitability and cardiac arrhythmias have been found, microinjection of Ang-(1-7) into the RVLM elicits a similar pressor response to Ang Ⅱ. It is therefore interesting to further study whether Ang-(1-7) in the RVLM has a similar or opposite effect to Ang Ⅱ on the CSAR. The second goal of the study is to determine the effect of the Ang-(1-7) in the RVLM on the CSAR.Objective1. To determine the effect of microinjection of Ang Ⅱ into the RVLM on the CSAR.2. To determine the effects of microinjection of Ang-(1-7) into the RVLM on the baseline RSNA, MAP and CSAR and to investigate whether Ang-(1-7) in the RVLM has a similar or opposite effect to Ang Ⅱ.3. To determine the role of Mas receptor and AT1receptor in the RVLM in the tonic control of baseline RSNA, MAP and CSAR and in mediatng the effects of Ang Ⅱ and Ang-(1-7).MethodsIn anesthetized rats with sinoaortic denervation and cervical vagotomy, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in vivo on a PowerLab data acquisition system. The coordinates for RVLM were determined in a stereotaxic instrument according to the Paxinos and Watson rat atlas. All the drugs are bilaterally microinjected into RVLM. The CSAR was evaluated by the RSNA response to epicardial application of capsaicin.1. Effects of different doses of Ang Ⅱ and Ang-(1-7) on the baseline RSNA, MAP and CSAR in the RVLM. The rats were randomly divided into two groups (n=6for each) randomly subjected to the RVLM microinjection of Ang Ⅱ (0,0.03,0.3, and3nmol) and RVLM microinjection of Ang-(1-7)(0,0.03,0.3, and3nmol).0dose means saline.2. Effects of pretreatment with AT1receptor antagonist losartan or Mas receptor antagonist D-alanine-Ang-(1-7)(A-779). The rats were first randomly divided into nine groups (n=6for each group) to RVLM microinjection of saline, Ang Ⅱ, losartan, losartan+Ang Ⅱ, A-779+Ang Ⅱ, Ang-(1-7), A-779, losartan+Ang-(1-7) or A-779+Ang-(1-7) into the RVLM to determine the their effects on the baseline RSNA, MAP and CSAR.Results1. Microinjection of either Ang Ⅱ or Ang-(1-7) into the RVLM significantly enhanced the CSAR, and increased baseline RSNA and MAP in a dose-dependent manner. Furthermore, there was no significant difference between Ang Ⅱ-induced and Ang-(1-7)-induced responses at the same dose.2. Microinjection of the losartan into the RVLM had no significant effect on the CSAR, baseline RSNA, and MAP. Microinjection of Ang Ⅱ into the RVLM enhanced the CSAR and increased the baseline RSNA and MAP. This effect was abolished by pretreatment of the AT1receptor antagonist losartan but not by pretreatment with the Mas receptor antagonist A-779in the RVLM.3. Microinjection of A-779into the RVLM attenuated the CSAR and decreased baseline RSNA and MAP. Microinjection of Ang-(1-7) into the RVLM enhanced the CSAR and increased baseline RSNA and MAP. This effect was abolished by A-779, but not by losartan in the RVLM.ConclusionsAng-(1-7) is as effective as Ang Ⅱ in sensitizing the CSAR and increasing sympathetic outflow. In contrast to Ang Ⅱ, the effects of Ang-(1-7) are not mediated by AT1receptors but by Mas receptors.Mas receptors but not the AT1receptors in the RVLM are involved in the tonic control of the CSAR. Part3Role of Paraventricular Nucleus in Modulating White Adipose Tissue Afferent Reflex and Sympathetic ActivityBackgroundExcess weight gain may contribute to the risk for hypertension. Activation of the sympathetic nervous system (SNS) have been suggested to contribute to hypertension in obesity. There is sensory innervation of white adipose tissue (WAT). However, possible function of the WAT sensory innervation is not clear. Injection of leptin into WAT induced the activation of RSNA. The implied role of WAT afferent reflex may play an important role in the activation of the SNS and contribute to the development of hypertension in obesity. An anterograde transneuronal viral tract tracing found substantial overlap of the CNS sites with the central origins of the sympathetic outflow to WAT, including paraventricular nucleus (PVN), rostral ventrolateral medulla (RVLM), intermediolateral column of spinal cord (IML). The PVN is an important integrative region in the control of sympathetic outflow and arterial pressure through projections to the intermediolateral column of the spinal cord and the rostrol ventrolateral medulla. This study also examined whether PVN play a key role in the WAT afferent reflex.Objective1. To determine whether stimulation of WAT enhances sympathetic outflow and increases blood pressure.2. To determine whether stimulation of WAT increases afferent nerves activity that signaled to the PVN and whether chemical lesion of the PVN abolishes WAT afferent reflex.MethodsIn anesthetized rats, extracellular single-unit recording, adipose nerve afferent activity (ANAA), renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in vivo on a PowerLab data acquisition system. The coordinates for PVN were determined in a stereotaxic instrument according. to the Paxinos and Watson rat atlas and the glass micropipettes were advanced using a hydraulic microdrive into the PVN. In addition to the rats used for the PVN c-Fos-immunoreactivity detection by immunohistochemistry method were intraperitoneally anesthetized with pentobarbital sodium, the other rats were intraperitoneally anesthetized with urethane and a-chloralose. After stimulating the inguinal and/or retroperitoneal WAT using the capsaicin, c-Fos-immunoreactivity in the PVN, the activity of the PVN neurons, ANAA, RSNA and MAP were investigated separately. The rats were randomly divided into two groups to determine the effects of the PVN pretreatment with saline and Kainic acid (KA) on the RSNA and MAP responses induced by WAT application of capsaicin.Results1. Injection of capsaicin into the WAT increased RSNA and MAP.2. Injection of capsaicin into the WAT increased ANAA.3. Injection of capsaicin into the WAT not only increased the PVN neuronal activity but also induced c-Fos expression in the PVN.4. Pretreatment with microinjection of KA into the PVN abolished increased RSNA and MAP responses to the injection of capsaicin into the WAT.ConclusionsWAT activation could send afferent signals to the PVN via sensory afferent nerves to trigger increases in the SNS outflow.