title: A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.
abstract: A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania. Electronic address: dainius.pauza@lsmuni.lt.
authors.forename: Dainius H
authors.initials: DH
authors.lastname: Pauza
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Kristina
authors.initials: K
authors.lastname: Rysevaite-Kyguoliene
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Jurgita
authors.initials: J
authors.lastname: Vismantaite
authors.affiliation: Department of Cardiovascular Sciences, University of Leicester, UK; NIHR, Biomedical Research Unit, University of Leicester, UK.
authors.forename: Kieran E
authors.initials: KE
authors.lastname: Brack
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Hermanas
authors.initials: H
authors.lastname: Inokaitis
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Audrys G
authors.initials: AG
authors.lastname: Pauza
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Viktorija
authors.initials: V
authors.lastname: Rimasauskaite-Petraitienė
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Jaune I
authors.initials: JI
authors.lastname: Pauzaite
authors.affiliation: Institute of Anatomy, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Neringa
authors.initials: N
authors.lastname: Pauziene
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D000818:Animals; D017776:Autonomic Pathways / Q000737:chemistry* / Q000166:cytology*; D005260:Female; D006321:Heart / Q000294:innervation*; D008297:Male; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep; D013194:Staining and Labeling / Q000379:methods; D013552:Swine
chemical_list: D009419:Nerve Tissue Proteins; D000110:Acetylcholinesterase
keywords: AChE histochemistry; Cardiac ganglia; Heart; Immunofluorescent labeling; Intrinsic cardiac neural plexus; Parasympathetic nerves; Sympathetic nerves