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Congestive heart failure (CHF) is characterized by impaired cardiovascular reflexes and increased neurohumoral drive. The long-term sympatho-excitation increases the progression and risk of mortality during CHF. The paraventricular nucleus (PVN) of the hypothalamus is a very important central site for integration of sympathetic outflow and cardiovascular function. Within the PVN, nitric oxide (NO), mainly generated by neuronal nitric oxide synthase (nNOS), functions in inhibitory regulation of sympathetic outflow. Our previous study has indicated that in rats with experimental heart failure, the NO mechanism within the PVN is attenuated. We hypothesize that this alteration may contribute to the sympatho-excitation commonly observed in CHF. To investigate the role of NO within the PVN in sympathetic dysfunction in CHF, we have manipulated nNOS expression using adenoviral gene transfer of nNOS or nNOS antisense. These techniques have allowed us to observe the effects of alterations in nNOS on sympathetic outflow and cardiovascular function. In this chapter, we describe the methods for delivering nNOS adenoviral vector or nNOS antisense into the PVN using microinjection, as well as the protocols for detecting nNOS expression after these manipulations, using Western blot, NADPH-diaphorase staining, and immunofluorescent staining.

Type

Journal article

Journal

Methods Mol Med

Publication Date

2005

Volume

112

Pages

59 - 79

Keywords

Adenoviridae, Animals, Antibodies, Monoclonal, Blood Pressure, Blotting, Western, Fluorescent Antibody Technique, Indirect, Gene Expression Regulation, Enzymologic, Gene Transfer Techniques, Heart Rate, Immunohistochemistry, Male, NADPH Dehydrogenase, Nerve Tissue Proteins, Nitric Oxide Synthase, Nitric Oxide Synthase Type I, Oligonucleotides, Antisense, Paraventricular Hypothalamic Nucleus, Rats, Rats, Wistar, Sympathetic Nervous System, Telemetry, beta-Galactosidase