Evaluation of 5-ethynyl-2′-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system

• Published in: Brain research Vol. 1319; pp. 21 - 32
• Main Authors: Zeng, Chenbo, Pan, Fenghui, Jones, Lynne A, Lim, Miranda M, Griffin, Elizabeth A, Sheline, Yvette I, Mintun, Mark A, Holtzman, David M, Mach, Robert H
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 10.03.2010; Elsevier
• Subjects: 5-bromo-2′-deoxyuridine; 5-ethynyl-2′-deoxyuridine; adult neurogenesis; Aging; Animals; Biological and medical sciences; BrdU; Bromodeoxyuridine; Cell Count; Cell cycle, cell proliferation; Cell physiology; Cell Proliferation; Cytological Techniques - methods; Dentate Gyrus - cytology; Dentate Gyrus - physiology; Deoxyuridine - administration & dosage; Deoxyuridine - analogs & derivatives; Dose-Response Relationship, Drug; Female; Fundamental and applied biological sciences. Psychology; hippocampus; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Molecular and cellular biology; Neurogenesis; Neurology; Neurons - cytology; Neurons - physiology; Physical Conditioning, Animal - physiology; Random Allocation; Restraint, Physical; Staining and Labeling; Stress, Psychological - pathology; Stress, Psychological - physiopathology; 5-ethynyl-2′-deoxyuridine; subventricular zone; adult neurogenesis; dentate gyrus; DG; hippocampus; SVZ; EdU; SGZ; subgranular zone; 5-bromo-2′-deoxyuridine; BrdU; Human; Cell proliferation; 5-bromo-2'-deoxyuridine; Evaluation; Rodentia; Central nervous system; Nervous system; 5-ethynyl-2'-deoxyuridine; Neurogenesis; Encephalon; Vertebrata; Mammalia; Mouse; Animal; Adult; Hippocampus
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/j.brainres.2009.12.092

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.