Effects Of Proinflammatory Cytokines And Renin-Angiotensin System In Hypothalamic Paraventricular Nucleus In Rats With Heart Failure

Congestive heart failure (CHF) is the common outcome of a variety of cardiovascular diseases, including myocardial infarction. Increased sympathetic drive and activation of the renin-angiotensin system (RAS), with vasoconstriction and volume retention, are hallmarks of the CHF syndrome. The mechanism (s) of CHF are not completely understood. It has been considered that the overaction of sympathetic drive and neuroendocrine system are the important factors in the development and progression of CHF. It has been reported recently that the proinflammatory cytokines is overexpressed and the RAS is overaction in CHF. And there is interaction between the proinflammatory cytokines and the RAS in the periphery circulation and the heart tissues. Then the theory of neuroendocrine-cytokines in CHF has been proposed. But only a few studies have focused on the importance of central neural mechanisms to the heart failure syndrome. The hypothalamic paraventricular nucleus (PVN) is a principal central site mediating neurohumoral responses. PVN is also a principal critical brain site to regulate extracellular fluid volume and sympathetic drive. In rats with ischemia-induced congestive heart failure, metabolic activity within the PVN is increased. The rate of PVN neurons from rats with ischemia-induced HF was higher. Since these findings, it is considered that the cardiovascular centers,include PVN,has been implicated in the development and progression of CHF. RAS generally exists not only in circulation and the periphery tissues but also in brain sites. Proinflammatory cytokines increased in the corresponding brain sites during brain diseases or injuries. Then we supposed that whether there exist some changes in the expression of the proinflammatory cytokines and the RAS within the PVN, and whether there exists interaction between the proinflammatory cytokines and the RAS in the PVN, and then together promote the proceeding of the CHF.In addition, a number of excitatory and inhibitory neurotransmitters in the central nervous system play a key role in the regulation of cardiovascular activity, including glutamate, norepinephrine (NE), and gamma-aminobutyric acid (GABA). It has been reported that functional glutamate receptors expressed in the PVN are involved in the control of cardiovascular reflexes. It has also been shown that sympathetic hyperactivity in rats with HF is associated with increased extracellular NE in the PVN. GABA-mediated inhibitory mechanism within the PVN contributing to sympathoexcitation in HF rats. We hypothesized that increased proinflammatory cytokines in the brains of HF rats caused an imbalance in the excitatory and inhibitory neurotransmitters in the PVN, contributing to increased sympathoexcitation in HF.In the present study, we observed the expression changes of the proinflammatory cytokines and the RAS within the PVN in CHF rats, and explored the interaction between the proinflammatory cytokines and the RAS in the PVN by the application of cytokine synthesis blocker and the RAS blocking agents. We also observed the NF-κB expression in PVN in CHF rats to explored the mechanism of the interaction between the proinflammatory cytokines and the RAS in the PVN. We also observed the changes of excitatory and inhibitory neurotransmitters in the PVN and the effects on them after the application of the blockers of the proinflammatory cytokines, to explore the possible roles that the imbalance between them plays in the increased sympathoexcitation in heart failure, and the contributions that the proinflammatory cytokines modulate neurotransmitters in the PVN.Chapter I Effects of Proinflammatory Cytokines and Renin-Angiotensin System in the PVN of CHF Rats24 Sprague-Dawly rats underwent coronary ligation (CL) to induce HF or sham CL (SHAM). After 4 weeks, LV function was measured by hemodynamic measurements, the renal sympathetic nerve activity (RSNA) was recorded on a pair of silver recording electrodes. Wet lung weight and right ventricular weight, with respect to body weight, were measured as indices of pulmonary congestion and right ventricular remodeling, two indices of the severity of HF. The histopathology changes of heart tissues were observed. Immunohistochemical studies were performed to assess the PVN neuronal activation and the expressions of IL-1βand TNF-αin the PVN. Expression of Fra-like (Fra-LI) (fos family gene) activity was used as an indicator of chronic neuronal activation. The levels of IL-1βand AngII in plasma and the PVN and the plasma levels of NE were quanti?ed using ELISA kit. The Fra-like, TNF-α, COX-2 and AT1-R protein levels in the PVN were measured with Western blot technique. We found that the heart function of HF rats was lower than SHAM, and accompanied by myocardium hypermyotrophy and ventricular reconstruction. HF rats have higher LVEDP and HR and lower±dp/dt max than SHAM rats. The right ventricular weight/body weight and wet lung weight/body weight ratios were substantially higher in HF rats compared with SHAM rats. Myocardium hypermyotrophy can be observed in noninfarct-zone of the LV of HF rats. The RSNA and the plasma norepinephrine levels of HF rats were signi?cantly increased than those of SHAM rats. Fra-like immunostaining and protein expression in the PVN in HF rats were higher than SHAM, and confirmed that the PVN neurons has been significantly activised. Compared with SHAM rats, HF rats have higher plasma IL-1βlevels by ELISA analysis, higher IL-1βand TNF-αimmunostainings in the PVN,higher TNF-αand COX-2 protein expressions by Western blot analysis. These results suggest that the proinflammatory cytokines were clearly increased not only in circulation but also in the PVN in CHF. Compared with SHAM rats, HF rats have higher plasma and PVN AngII levels by ELISA, and higher AT1-R protein expression by western blot analysis. These results suggest that the RAS in periphery circulation and the PVN was significantly activiated in CHF. Then we concluded that the PVN neurons has been significantly activised, and the proinflammatory cytokines and the RAS within the PVN were both significantly increased in CHF. We considered that the PVN participated in the progression of CHF, and the proinflammatory cytokines and the RAS influence the development and progression of CHF not only on the periphery circulation level but also on the cardiovascular center level.Chapter II Interaction between the Proinflammatory Cytokines and the Renin-Angiotensin System in the PVN of CHF Rats120 Sprague-Dawly rats underwent coronary ligation (CL) to induce HF or sham CL (SHAM). After 24 hours, all rats underwent intracerebroventricular (ICV) cannula surgery, an Alzet osmotic mini-pump was implanted subcutaneously in the back of the rats neck and attached to the cerebral ventricle cannula for chronic ICV infusion of the angiotensin type 1 receptor (AT1-R) antagonist valsartan (VAL, 150μg/h), or enalapril (ENP, angiotensin converting enzyme inhibitor, 1.25μg/h), or spironolactone (SL, a mineralocorticoid receptor (MR) antagonist, 100ng/h), or pentoxifylline (PTX, the production inhibitor of cytokines, 40μg/h), or vehicle (Veh, 0.25μl/h). After 4 weeks, LV function was measured by hemodynamic measurements, the renal sympathetic nerve activity (RSNA) was recorded on a pair of silver recording electrodes. Wet lung weight and right ventricular weight, with respect to body weight, were measured as indices of pulmonary congestion and right ventricular remodeling, two indices of the severity of HF. Immunohistochemical studies were performed to assess PVN neuronal activation and the expression of IL-1βand TNF-αin the PVN. Expression of Fra-like (Fra-LI) (fos family gene) activity was used as an indicator of chronic neuronal activation. IL-1βand AngII levels in plasma and the PVN and the plasma NE levels were quanti?ed using ELISA kit. The Fra-like,TNF-α,COX-2, AT1-R and NF-κB protein levels in the PVN were measured with Western blot technique. We found that the heart function of HF rats was lower than SHAM, and accompanied by myocardium hypermyotrophy. HF rats have higher LVEDP and HR and lower±dp/dt max than SHAM rats. The right ventricular weight/body weight and wet lung weight/body weight ratios were substantially higher in HF rats compared with SHAM rats. HF rats treated with either VAL or ENP or SL or PTX had improved heart function indexes. The RSNA and the plasma norepinephrine levels of HF rats were signi?cantly increased than those of SHAM rats. In HF rats with chronic ICV infusion of either VAL or ENP or SL or PTX, the increased RSNA and the plasma norepinephrine levels were clearly down than HF+Veh rats. Fra-like immunostaining and protein expression in the PVN in HF rats were higher than SHAM, and confirmed that the PVN neurons has been significantly activised. In HF rats with either VAL or ENP or SL or PTX-treated, the Fra-like immunostaining and protein expression were clearly reduced than HF+Veh rats, but still higher than SHAM rats. Compared with SHAM rats, HF rats have higher plasma IL-1βlevels by ELISA analysis, higher IL-1βand TNF-αimmunostaining in the PVN,higher TNF-αand COX-2 protein expression by Western blot analysis. The HF rats with either VAL or ENP or SL or PTX-treated, had lower IL-1βplasma level and immunostaining in the PVN, and lower TNF-αimmunostaining and protein expression, and lower COX-2 protein expression in the PVN than HF+Veh rats in different degrees. These results suggest that the proinflammatory cytokines was clearly increased not only in circulation but also in the PVN in CHF. And the central blocked of RAS or cytokines can inhibit these effects. Compared with SHAM rats, HF rats have higher plasma and PVN AngII levels by ELISA, and higher AT1-R protein expression in the PVN by Western blot analysis. The HF rats with ICV infusion of either VAL or ENP or SL or PTX, these increases were significantly inhibited in different extents. These results suggest that the RAS in periphery circulation and the PVN was significantly activiated in CHF, and the central blocked of RAS or cytokines can inhibit these effects. HF rats have higher NF-κB protein expression in the PVN by Western blot analysis than the SHAM rats, and the HF rats treated with either VAL or ENP or SL or PTX have lower NF-κB protein expressions in the PVN than HF+Veh rats. Then we concluded that the proinflammatory cytokines and the RAS within the PVN were both significantly increased in CHF, the blocked of the RAS on central level could induced the decrease production of proinflammatory cytokines not only in circulation but also in the PVN in CHF, and the blocked of proinflammatory cytokines on central level also could result in a decreased activity of the RAS, and accompanied the attenuated RSNA and the improvement of the heart function and the ventricular reconstruction. In CHF, the proinflammatory cytokines and the RAS could be facilitated by each other in the PVN and mediated by the nuclear factorκB. The interaction between them contribute to the development and progression of CHF. Chapter III Brain Tumor Necrosis Factor-Alpha Modulates Neurotransmitters in Hypothalamic Paraventricular Nucleus in Heart FailureAims: Increased proinflammatory cytokines (PICs) after myocardial infarction augment the progression of heart failure (HF) and are of prognostic significance. Recently we demonstrated that increased PICs in the brains of HF rats increased paraventricular nucleus (PVN) superoxide and down-regulated neuronal nitric oxide synthase (nNOS) contributing to sympathoexcitation. In this study, we explored the possible roles of brain PICs and their effects on modulating PVN neurotransmitters in the exaggerated sympathetic activity in HF. Methods: Sprague-Dawley rats with HF or sham-operated control (SHAM) rats were treated for 4-weeks with a continuous intracerebroventricular (ICV) infusion of the cytokine blockers, pentoxifylline (PTX, 10μg/h and 40μg/h) or etanercept (ETN, 5μg/h and 10μg/h), or artificial cerebrospinal fluid (aCSF). Another set of HF and SHAM rats were treated with intraperitoneal (IP) infusion of a similar dose of PTX or ETN, or saline. Results: HF rats had increased neuronal excitation accompanied by higher levels of glutamate, norepinephrine (NE) and tyrosine hydroxylase (TH), and lower levels of gamma-aminobutyric acid (GABA), nNOS and 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN compared to SHAM rats. Plasma cytokines, NE, epinephrine and angiotensin II, and renal sympathetic nerve activity (RSNA) were also increased in HF rats. ICV treatment with low doses of PTX or ETN attenuated, and high doses prevented these changes in HF rats. IP treatment with similar doses of PTX or ETN did not affect glutamate, NE, TH, GABA, nNOS and GAD67 in the PVN and RSNA of HF rats. Conclusion: This study, for the first time, demonstrates that PICs modulate neurotransmitters in the PVN and contribute to sympathoexcitation in HF.