Involvement Of Enhanced Cardiac Sympathetic Afferent Reflex In Sympathetic Activation In Early Stage Of Diabetes

BackgroundDiabetes mellitus, an endocrine disease, severely affects the quality of human lifearound the world. Autonomic neuropathy is one of the most common complicationsof diabetes and is characterized by sympathetic dominance and parasympatheticwithdrawal. The symptoms and signs of diabetic autonomic neuropathy carry a poorprognosis in diabetic patients. The cardiac sympathetic afferent reflex (CSAR), animportant cardiovascular reflex, can be induced with epicardial application ofbradykinin, capsaicin, adenosine or hydrogen peroxide, resulting in increasedsympathetic outflow and arterial pressure. Paraventricular nucleus (PVN) is a seniorcenter regulating sympathetic nerve activity, in which angiotensin II (Ang II) and AT1receptor (AT1R) play an important role.The sympathetic activity in diabetes is enhanced, so we hypothesized that theCSAR may be enhanced in diabetes and the enhanced CSAR may be important in thesympathetic over-excitation. The diabetes animal model induced by streptozotocin(STZ) was used to explore the change of CSAR and related mechanism in presentstudy, as to provide theoretic basis for rvealing smpathetic activation and andpathogenesis of diabetes.Objective1. To determine whether the CSAR is enhanced in STZ-induced diabetic rats aswell as the CSAR changing pattern in the pathogenesis of diabetes.2. To determine whether the enhanced CSAR and sympathetic overdrive can be attenuated by peripherally blocking the cardiac sympathetic afferent activities.3. To determine whether the enhanced CSAR and sympathetic overdride can beattenuated by inhibiting the activity of PVN neurons.4. To determine whether the Ang II and AT1R in PVN mediate the enhancedCSAR in diabetic rats.MethodsThe male Sprague-Dawley rats weighing between280and300g were used andrandomly divided into diabeic rats (Diabeic) and control rats (Control). STZ wasdissolved in0.1M of sodium citrate buffer. The diabeic and control rats weresubjected to a single injection of (60mg/kg) or vehicle via the tail vein separately.The cute experiments were carried out three weeks after the model was successful.The plasma norepinephrine levels measured by high pressure liquid chromatography(HPLC) and the maximum change of mean arterial pressure (MAP) after intravenousinjection of hexamethonium were used to evaluate the basic sympathetic level. Therenal sympathetic nerve activity (RSNA) and MAP were recorded by powerlabbiological signal acquisition system, and the changes of RSNA and MAP afterepicardial application of different doses of capsaicin and bradykinin were used toevaluate the CSAR. The bilateral PVN microinjections were carried out withstereotaxic apparatus and microinjector. The enzyme-linked immunosorbent assaywas used to meaure the plasma Ang II and left ventricular wall TRPV1level. TheWestern blot was used to measure AT1R protein level of PVN.Protocol1: For determination of the basic sympathetic activity, ganglionicblockade hexamethonium hydrochloridewas intravenously administered in Control(n=6) and Diabetic rats (n=6) to induce depressor effect. The maximal decrease in theMAP was considered as an index of sympathetic activity.Protoscol2: Blood samples were collected for measuring plasma norepinephrine and Ang II. The brain and heart were removed for determining the AT1R expression inthe PVN and the TRPV1expression in the left ventricle wall in control and diabeticrats, respectively (n=6for each group).Protocol3: To determine the CSAR change during the development of diabetes,the CSAR induced by epicardial application of capsaicin (1.0nmol) was respectivelydetermined in rats without STZ treatment and in rats at the3rd week, the6th weekand the9th week after STZ treatment (n=6for each group).Protocol4: The CSAR was evaluated by the RSNA and MAP responses torandomly epicardial applications of saline, capsaicin (0.1or1.0nmol) or bradykinin(0.03or0.3nmol) in control and diabetic rats (n=8for each group). The intervalbetween epicardial applications was at least30min.Protocol5: Resiniferatoxin (RTX) was used to determine the effects of cardiacafferent blocking on the RSNA and MAP. Either control or diabetic rats wererandomly divided into two groups, which were respectively subjected to injection ofRTX (60pmol) or saline into the pericardial cavity (n=6for each group). At the140thminute after administration of RTX or saline, the CSAR induced by capsaicin (1.0nmol) was determined to confirm the effectiveness of RTX on blocking the cardiacafferents.Protocol6: To determine the role of the PVN in the enhanced CSAR andsympathetic activation, the PVN microinjection of saline, lidocaine (8.5nmol) andsaline were in turn carried out in control and diabetic rats (n=6for each group). Theinterval between injections was at least30minutes for complete recovery. The CSARinduced by capsaicin (1.0nmol) was determined at the5th minute after PVNmicroinjection.Protocol7: To determine the influence of Ang II in PVN on baseline RSNA andMAP, the bilateral PVN microinjection of Ang II (0.03,0.3and3.0nmol) were randomly carried out in control and diabetic rats (n=6for each group). At least30minwere provided to ensure the total restoration between each microinjection.Protocol8: To determine the roles of Ang II and AT1receptors in the PVN, eithercontrol or diabetic rats were randomly divided into four subgroups, which weresubjected to the PVN microinjection of saline, Ang II (0.3nmol), losatran (50nmol)and same dose of Ang II pretreated with losartan (n=6for each group). The CSARinduced by capsaicin (1.0nmol) was determined at the5th minute after PVNmicroinjection.Results1. The plasma norepinephrine and Ang II levels were higher in diabetic rats thanthat in control rats. There was no significant difference in the baseline MAP betweenthe diabetic rats and control rats, but the maximal depressor response tohexamethonium hydrochloride was greater in diabetic rats than that in control rats.2. Compared with the rats without STZ treatment, the CSAR was significantlyenhanced in the rats at the3rd week and attenuated in the rats at the9th week afterSTZ treatment. Therefore, the present study was designed to explore whether theenchanced CASR in early stage of diabetes contributes to sympathetic activation.3. The CSAR induced by capsaicin was significantly enhanced in diabetic ratsthan that in control rats. High dose of capsaicin caused greater RSNA and MAPresponses than low dose of capsaicin. Bradykinin-induced CSAR was similar tocapsaicin-induced CSAR in both control and diabetic rats.4. Intrapericardial administration of RTX caused immediate and short-termincreases in both RSNA and MAP peaking at10th min in both control and diabeticrats. The RTX-caused RSNA and MAP changes in diabetic rats were greater than thatin control rats. Subsequent decreases in RSNA and MAP were only found in diabeticrats but not in control rats. The RTX treatment abolished the CSAR evoked by capsaicin in both control and diabetic rats. Intrapericardial administration of salinehad no significant effects on RSNA, MAP and CSAR. There was no significantdifference of TRPV1in the left ventricle wall between diabetic rats and control rats.5. The PVN microinjection of lidocaine decreased the baseline RSNA and MAP indiabetic rats but not in control rats. However, lidocaine in the PVN abolished theCSAR in both diabetic and control rats.6. PVN microinjection of Ang II can cause inrceases in RSNA and MAP in adose-dependent manner. The Ang II-caused RSNA and MAP changes in diabetic ratswere greater than that in control rats under the same dose.7. Microinjections of Ang II into the PVN caused more increases in the baselineRSNA and MAP and greater enhancement in the CSAR in diabetic rats than that incontrol rats, which was abolished by pretreatment with losartan in the PVN.Microinjections of losartan into the PVN decreased the RSNA and MAP andnormalized the enhanced CSAR in diabetic rats, but had no significant effects incontrol rats.8. The AT1receptor expression in the PVN was increased in diabetic ratscompared with control rats.Conclusions1. The CSAR is enhanced in the diabetic rats.2. Pericardial injection of resiniferatoxin to desensitize cardiac afferents or PVNmicroinjection of lidocaine attenuated the CSAR, and decreased the RSNA and MAPin diabetic rats, indicating that the enhanced CSAR in diabetic rats contributes to thesympathetic over-excitation.3. Ang II and AT1receptor of PVN play an important role in the enhancedcentral gain of the CSAR in diabetic rats.