title: 3-D imaging and illustration of mouse intestinal neurovascular complex.
abstract: Because of the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and three-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with micrometer-level resolution. Four examples are given: 1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intraganglionic at myenteric plexus; 2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface; 3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts; and 4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.
authors.affiliation: Connectomics Research Center, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Ya-Yuan
authors.initials: YY
authors.lastname: Fu
authors.forename: Shih-Jung
authors.initials: SJ
authors.lastname: Peng
authors.forename: Hsin-Yao
authors.initials: HY
authors.lastname: Lin
authors.forename: Pankaj J
authors.initials: PJ
authors.lastname: Pasricha
authors.forename: Shiue-Cheng
authors.initials: SC
authors.lastname: Tang
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000033:anatomy & histology / Q000294:innervation; D002196:Capillaries / Q000033:anatomy & histology; D017615:Enteric Nervous System / Q000033:anatomy & histology*; D005260:Female; D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D007422:Intestines / Q000098:blood supply* / Q000294:innervation*; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D009197:Myenteric Plexus / Q000033:anatomy & histology; D009412:Nerve Fibers / Q000502:physiology; D009414:Nerve Growth Factors / Q000378:metabolism; D009415:Nerve Net / Q000033:anatomy & histology; D009457:Neuroglia / Q000502:physiology; D012039:Regional Blood Flow / Q000502:physiology; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000378:metabolism; D012701:Serotonin / Q000502:physiology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579159:S100b protein, mouse; D012701:Serotonin; D014446:Tyrosine 3-Monooxygenase; D043222:Ubiquitin Thiolesterase; C496976:Uchl1 protein, mouse