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Comparative Study
. 2010 Mar 10:1319:21-32.
doi: 10.1016/j.brainres.2009.12.092. Epub 2010 Jan 11.

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

Chenbo Zeng  1 , Fenghui Pan, Lynne A Jones, Miranda M Lim, Elizabeth A Griffin, Yvette I Sheline, Mark A Mintun, David M Holtzman, Robert H Mach
Affiliations
Comparative Study

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

Chenbo Zeng et al. Brain Res. .

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.

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Figures

Fig. 1
Fig. 1
Chemistry of EdU detection. A: Chemical structures of BrdU and EdU. B: Click reaction between EdU and azide–modified dye. EdU contains an alkyne group which can be reacted with an azide–containing detection reagent to form a stable triazole ring.
Fig. 2
Fig. 2
EdU dose–response experiment. Five groups (n=6) of two month old female mice received single injection of EdU intraperitoneally at a dose of 10, 20, 50, 100 or 200 mg/kg body weight. Four hours after EdU injection, brains were processed for EdU staining. EdU positive cell numbers slightly increased in a dose–dependent manner both in control and running mice. The data were fitted by Eq. A (see methods) to obtain a solid line from which Nmax and D50 values were calculated. SE stands for standard error. Bars represent mean ± SEM. * p < 0.05 in comparison to the control mouse group is considered significant using a two–tailed Student’s t–test.
Fig. 3
Fig. 3
Comparison of EdU staining and BrdU staining. Two groups (n = 6) of mice were injected with EdU (200 mg/kg) or BrdU (243.5 mg/kg). Four hours after injection, brains were processed for EdU or BrdU staining. A: Representative images showing that running mice exhibited more EdU positive cells than control mice. EdU (red), Hoechst 33342 (blue). B: Representative images showing that running mice exhibited more BrdU positive cells than control mice. BrdU (green). C: Quantitative data showing that EdU and BrdU positive cell numbers are comparable. The bars represent mean ± SEM. * p < 0.05 in comparison to the control mouse group is considered significant using a two–way ANOVA. Scale bar = 100 μm.
Fig. 4
Fig. 4
EdU and BrdU co–localize within the same cells of the DG. A: Representative images showing that the anti–BrdU antibody from Sigma did not recognize EdU on the DG sections from the mouse injected with only EdU (200 mg/kg); in contrast, the anti–BrdU antibody from Accurate had cross–reactivity to EdU. EdU (red), BrdU (green). Scale bar = 50 μm. B: Representative images showing that “Click” reaction recognized EdU (red) but not BrdU. Scale bar = 50 μm. C: Representative images showing that EdU and BrdU co–localize within the same cells (arrows) of the DG in control and running mice, both of whom received a single injection of EdU (200 mg/kg) and BrdU (243.5 mg/kg). a and e are images in low magnification. Scale bar = 50 μm. b-d are high magnification images for the region delineated by the box in a. Scale bar = 20 μm. f-h are high magnification images for the region delineated by the box in e. Scale bar = 20 μm.
Fig. 5
Fig. 5
Voluntary exercise significantly increases the survival of the EdU positive cells and the EdU positive cells differentiated into neurons. Six control and running mice received a single injection of EdU (100 mg/kg) and continued in the respective conditions for 30 days. A: Quantitative data showing that voluntary exercise significantly increased the number of EdU positive cells. The bars represent mean ± SEM. p = 0.000029. * p < 0.05 in comparison to the control mouse group is considered significant using a two-tailed Student’s t–test. B: Representative confocal images showing the nucleus (arrows) triple–labeled with EdU (red), NeuN (green) and DAPI (blue). Scale bar = 10 μm.
Fig. 6
Fig. 6
Restraint stress significantly decreases EdU positive cells. Control and stressed mice were injected i.p. with 100 mg/kg of EdU four hours prior to sacrifice. A: Representative images showing that the mice under stress show fewer numbers of EdU positive cells than control mice. EdU (red), Hoechst 33342 (blue). B: Quantitative data showing that restraint stress significantly decreases the number of EdU–labeled cells in the dentate gyrus. The bars represent mean ± SEM. * p < 0.05 in comparison to the control mouse group is considered significant using a two–tailed Student’s t–test. Scale bar = 100 μm.
Fig. 7
Fig. 7
EdU staining is compatible with GFAP immunostaining. Representative images show that EdU (red) staining is compatible with GFAP (green) immunostaining. DAPI (blue) stains nuclei. Scale bar = 50 μm.
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