Effects Of Corticotrophin Releasing Hormone-containing Neurons In Hypothalamic Paraventricular Nucleus On Sympathoexcitation In Congestive Heart Failure

Chronic congestive heart failure (CHF) is the end-stage manifestation of cardiac syndromes causing left ventricular dysfunction. The overreaction sympathetic drive is a cardinal manifestation of the heart failure syndrome, closely correlated with disease severity and adverse prognosis. It has been considered that augmented sympathetic drive is the important mechanism responsible for the development and progression of CHF. In addition, in many clinical trails, it has been demonstrated that the therapy for CHF targeting sympathetic nervous system was beneficial. However, the mechanisms of sympathoexcitation are still not fully understood. The hypothalamic paraventricular nucleus (PVN) is a principal central site mediating neurohumoral responses. It is also a chief critical brain site to regulate extracellular fluid volume and sympathetic drive. Recent studies of double-labeling for corticotropin release hormone (CRH) and Fra-like (Fra-LI) (fos family gene, as an indicator of chronic neuronal activation) activity demonstrated an increase of CRH expression in the PVN of rats with heart failure contributes to sympathoexcitation and heart deterioration. Furthermore, numerous active neurochemistry factors regulating cardiovascular activity convey in the PVN, indicating that CRH neuronal activation is involved in the pathogenesis of CHF. Most of the CRH neurons in the PVN projecting to median eminence play a neuroendocrine function, while others maybe modulate the sympathetic nervous system projecting directly to the rostral ventrolateral medulla (RVLM) and to the intermediolateral cell column (IML) of the spinal cord which are two important central nuclei containing presympathetic neurons. As a result, we suppose that the activation of CRH-containing neurons within the PVN maybe contribute to the exaggerated sympathetic activity and aggravate cardiac function in CHF.Recent literatures have implicated that the upregulated prostaglandin E2 (PGE2) and norepinephrine (NE) in the brains of heart failure maybe two important activators for CRH-containing PVN neurons. In addition, the CRH-containing neurons in the PVN receive direct innervations from several extrahypothalamic pathways. Some of these projections are largely GABAergic, some are glutamatergic or adrenergic, whereas others contain mixed populations of cells. The gamma-aminobutyric acid (GABA) is a dominant inhibitory neurotransmitter; glutamate and NE are two important excitatory neurotransmitters within the PVN. Therefore, we hypothesize that central elevated PGE2 and NE contribute to increased sympathoexcitation in CHF by activating CRH-containing PVN neurons directly or modulating the balance of neurotransmitters in the PVN.In the present study, we firstly observed the expression changes of CRH within the PVN and explored the relationship between the activated CRH-containing neurons and sympathetic hyperactivity in rats with HF. And then, we investigated whether the central elevated PGE2 and NE contribute to the activation of CRH-containing PVN neurons via chronic intracerebroventricular (ICV) infusion of the specific cyclooxygenase-2 inhibitor celecoxib (CEL) and anαl adrenergic receptor antagonists prazosin (PRA). Subsequently, we also observed the changes of GABA, glutamate and NE and their rate-limiting enzymes tyrosine hydroxylase (TH) and 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN and explored their possible roles in the process of activation of CRH-containing PVN neurons in CHF. As a gaseous neurotransmitter, nitric oxide (NO) is known to play a role in sympathetic regulation by interacting with some other neurotransmitters within the PVN, so we also observed the changes and roles of neuronal nitricoxide synthase (nNOS) in the PVN. The study, focusing on effects and mechanisms of CRH-containing PVN neurons in the development and progression of CHF, will be helpful for the therapy of CHF.Part One: Activation of Corticotrophin Releasing Hormone-containing Neurons in Hypothalamic Paraventricular Nucleus Contributes to Sympathoexcitation in Congestive Heart FailureAims: To observe the changes of CRH expression within the PVN and explore the relationship between the activated CRH-containing neurons and sympathetic hyperactivity in rats with HF. Methods: The experiment contains two parts. Firstly, Sprague-Dawly (SD) rats, subjected to coronary artery ligation to induce heart failure (HF), or sham surgery without ligating the vessel (SHAM), were treated with intracerebroventricular (ICV) cannula surgery, an Alzet osmotic mini-pump implanted subcutaneously in the back of the rats neck and attached to the cerebral ventricle cannula for chronic ICV infusion of CRH competitive inhibitorαh-CRH (15μg/h) or vehicle (VEH, artificial cerebrospinal fluid 0.25μl/h) for 4 weeks. Secondly, Lewis rats with impaired CRH production and Fischer (F344/N) rats with normal CRH systems to perform control study also underwent coronary ligation to induce HF or sham surgery. At the end of the experiment, at least 12 animals were ensured to survive in each group. After 4 weeks, their left ventricular function parameters, such as left ventricular end-diastolic pressure (LVEDP) and maximum change in pressure over time (±dp/dtmax) were determined by hemodynamic measurements. The right ventricular-to-body weight (RV/BW) and lung-to-body weight (lung/BW) ratios, indices of pulmonary congestion and right ventricular remodeling, were calculated to find the severity of HF. The renal sympathetic nerve activity (RSNA) was recorded and the plasma NE level was measured using an ELISA kit, revealing the severity of sympathetic nervous excitation. Immunohistochemical study and western blot technique determining the expressions of CRH were combined with the plasma adrenocorticotrophic hormone (ACTH) measurement using an ELISA kit to assess the CRH neuronal activation in the PVN. Results: (1) Compared with SHAM-SD rats, HF-SD rats had greater number of CRH positive neurons and higher level of CRH protein in the PVN (accordingly the plasma ACTH levels were increased) (p<0.05), accompanied by decreased±dp/dtmax (p<0.05) and increased RSNA, plasma NE, LVEDP, lung/BW and RV/BW (p<0.05). However, ICV treatment withαh-CRH attenuated these changes in HF-SD rats (p<0.05). (2) In comparison with SHAM-Fisher rats, HF-Fisher rats had higher level of CRH immunostaining and protein expression in the PVN (p<0.05), which confirmed that the CRH-containing PVN neurons have been significantly activated. In addition, they had significantly increased levels of the RSNA and the plasma NE (p<0.05), higher LVEDP, RV/BW and lung/BW (p<0.05), and lower±dp/dtmax (p<0.05). (3) Compared with SHAM-Lewis rats, HF-Lewis rats had no significantly change in CRH immunostaining and protein expression in the PVN, the plasma ACTH levels (p>0.05), the RSNA and the plasma NE levels (p>0.05), LVEDP and±dp/dtmax (p>0.05), RV/BW and lung/BW (p>0.05). Conclusion: This study demonstrates that in CHF, the CRH-containing PVN neurons are activated, which aggravates cardiac function by increasing sympathoexcitation.Part two: The Relationship between Central Prostaglandin E2 and the Activation of Corticotrophin Releasing Hormone-containing Neurons in Hypothalamic Paraventricular Nucleus in Congestive Heart FailureAims: To investigate the effect of central PGE2 on the activation of CRH-containing PVN neurons in CHF and explore its possible mechanism. Methods: SD rats underwent coronary ligation to induce HF or SHAM control. And then, all rats with HF or SHAM were treated for 4-weeks with a continuous ICV infusion of the specific cyclooxygenase-2 inhibitor celecoxib (CEL, 20μg/h), or vehicle. After 4 weeks, LVEDP and±dp/dtmax were measured by hemodynamic measurements. The RV/BW and lung/BW ratios were calculated. The RSNA was recorded. The levels of plasma NE and ACTH and PGE2 in cerebrospinal fluid (CSF) were tested using ELISA kit. Immunostaining and western blot were performed to determine the expressions of CRH, TH, GAD67 and nNOS in the PVN. The concentrations of NE, glutamate and GABA within the PVN were measured using HPLC with electrochemical detection. Results: (1) Compared with SHAM rats, HF rats had upregulated PGE2 in the CSF and increased CRH-containing PVN neuronal activation (CRH immunostaining and protein expression in the PVN and the plasma ACTH were elevated in HF rats) (p<0.05). In addition, they also had elevated RSNA and plasma NE, LVEDP, RV/BW and lung/BW, and decreased±dp/dtmax than SHAM rats (p<0.05). In contrast, ICV-infusion CEL for 4 weeks reduced the concentration of PGE2 in CSF, and decreased the activity of CRH-containing PVN neurons (accordingly the plasma ACTH levels) (p<0.05). Treatment with CEL also attenuated the sympathetic nervous excitation by reducing the RSNA and plasma NE (p<0.05). The HF rats with ICV-infusion CEL had a better cardiac function with reduced LVEDP and elevated±dp/dtmax than HF-VEH rats (p<0.05). The RV/BW and lung/BW were also decreased after ICV CEL (p<0.05). (2) Compared with SHAM rats, HF rats had higher levels of glutamate, NE and TH (p<0.05), and lower levels of GABA, nNOS and GAD67 in the PVN (p<0.05). Treatment with CEL lessened the levels of glutamate, NE and TH, and raised the levels of GABA, nNOS and GAD67 in the PVN (p<0.05). Conclusion: These findings indicate that in CHF, the increased central PGE2 can activate CRH-containing PVN neurons and contribute to the augmented sympathetic drive possibly by modulating the neurotransmitters within the PVN.Part three: The Relationship between Central Norepinephrine and the Activation of Corticotrophin Releasing Hormone-containing Neurons in Hypothalamic Paraventricular Nucleus in Congestive Heart FailureAims: To investigate the effect of central norepinephrine (NE) on the activation of CRH-containing PVN neurons in CHF and explore its possible mechanism.Methods: SD rats underwent coronary ligation to induce HF or SHAM control. And then, all rats with HF or SHAM were treated for 4-weeks with a continuous ICV infusion of anαl adrenergic receptor antagonists prazosin (PRA, 10nmol/h), or vehicle. After 4 weeks, LVEDP and±dp/dtmax were measured to ascertain the cardiac function. The RV/BW and lung/BW ratios were calculated. The RSNA was recorded and the plasma NE level was measured, determining the severity of sympathetic nervous excitation. In addition, the levels of plasma ACTH were tested using ELISA kit. Immunostaining and western blot were performed to determine the expressions of CRH, TH, GAD67 and nNOS in the PVN. The concentrations of NE, glutamate and GABA within the PVN were measured using HPLC with electrochemical detection. Results: (1) Compared with SHAM rats, HF rats had significantly increased concentrations of NE and its rate-limiting enzyme TH within the PVN (p<0.05). Besides, CRH immunostaining and protein expression in the PVN and the plasma ACTH were elevated in HF rats (p<0.05). The RSNA and the plasma NE levels of HF rats were also significantly upregulated than those of SHAM rats (p<0.05). In addition, HF rats had higher LVEDP, RV/BW and lung/BW, and lower±dp/dtmax than SHAM rats (p<0.05). However, ICV-infusion PRA for 4 weeks decreased the activity of CRH-containing PVN neurons (accordingly the plasma ACTH levels) (p<0.05) and attenuated the sympathetic nervous excitation by reducing the RSNA and plasma NE (p<0.05). Treatment with PRA also ameliorated cardiac function by reducing LVEDP and elevating±dp/dtmax compared with HF-VEH rats (p<0.05). The RV/BW and lung/BW were also decreased after ICV-PRA (p<0.05). (2) Compared with SHAM rats, the concentration of glutamate was increased (p<0.05), and the contents of GABA, nNOS and GAD67 were decreased in the PVN in HF rats (p<0.05). In contrast, ICV-infusion PRA for 4 weeks reduced the concentration of glutamate and elevated the contents of GABA, nNOS and GAD67 in the PVN (p<0.05).Conclusion: These results suggest that central increased NE, especially within the PVN, may also activate CRH-containing PVN neurons possibly by regulating the neurotransmitters within the PVN and contribute to the augmented sympathetic drive in CHF.