Manipulation of neuronal nitric oxide synthase within the paraventricular nucleus using adenovirus and antisense technology.
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.
Li
Yi-Fan
YF
Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, NE, USA.
Wang
Yu
Y
Channon
Keith M
KM
Schultz
Harold D
HD
Zucker
Irving H
IH
Patel
Kaushik P
KP
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001794:Blood Pressure / Q000187:drug effects; D015153:Blotting, Western; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018014:Gene Transfer Techniques; D006339:Heart Rate / Q000187:drug effects; D007150:Immunohistochemistry; D008297:Male; D009252:NADPH Dehydrogenase / Q000032:analysis / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000494:pharmacology; D010286:Paraventricular Hypothalamic Nucleus / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D013564:Sympathetic Nervous System; D013686:Telemetry; D001616:beta-Galactosidase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D009252:NADPH Dehydrogenase; D001616:beta-Galactosidase