title: Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.
abstract: Recently, a novel method for detection of DNA synthesis has been developed based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analogue, into cellular DNA and the subsequent reaction of EdU with a fluorescent azide in a copper-catalyzed [3+2] cycloaddition ("Click" reaction). In the present study, we evaluated this method for studying cell proliferation in the adult central nervous system in comparison with the "gold standard" method of 5-bromo-2'-deoxyuridine (BrdU) staining using two behavioral paradigms, voluntary exercise and restraint stress. Our data demonstrate that the number of EdU-positive cells in the dentate gyrus of the hippocampus (DG) slightly increased in an EdU dose-dependent manner in both the control and voluntary exercise (running) mouse groups. The number of EdU-labeled cells was comparable to the number of BrdU-labeled cells in both the control and running mice. Furthermore, EdU and BrdU co-localized to the same cells within the DG. Voluntary exercise significantly increased the number of EdU- and BrdU-positive cells in the DG. In contrast, restraint stress significantly decreased the number of EdU-positive cells. The EdU-positive cells differentiated into mature neurons. EdU staining is compatible with immunohistochemical staining of other antigens. Moreover, our data demonstrated EdU staining can be combined with BrdU staining, providing a valuable tool of double labeling DNA synthesis, e.g., for tracking the two populations of neurons generated at different time points. In conclusion, our results suggest that EdU staining is a fast, sensitive and reproducible method to study cell proliferation in the central nervous system.
authors.affiliation: Department of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., St. Louis, MO 63110, USA.
authors.forename: Chenbo
authors.initials: C
authors.lastname: Zeng
authors.forename: Fenghui
authors.initials: F
authors.lastname: Pan
authors.forename: Lynne A
authors.initials: LA
authors.lastname: Jones
authors.forename: Miranda M
authors.initials: MM
authors.lastname: Lim
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Griffin
authors.forename: Yvette I
authors.initials: YI
authors.lastname: Sheline
authors.forename: Mark A
authors.initials: MA
authors.lastname: Mintun
authors.forename: David M
authors.initials: DM
authors.lastname: Holtzman
authors.forename: Robert H
authors.initials: RH
authors.lastname: Mach
mesh_terms: D000375:Aging; D000818:Animals; D001973:Bromodeoxyuridine; D002452:Cell Count; D049109:Cell Proliferation; D003584:Cytological Techniques / Q000379:methods*; D018891:Dentate Gyrus / Q000166:cytology* / Q000502:physiology; D003857:Deoxyuridine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D004305:Dose-Response Relationship, Drug; D005260:Female; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology / Q000502:physiology; D010805:Physical Conditioning, Animal / Q000502:physiology; D011897:Random Allocation; D012149:Restraint, Physical; D013194:Staining and Labeling; D013315:Stress, Psychological / Q000473:pathology / Q000503:physiopathology
chemical_list: D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine