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. 2022 Jun 10;11(12):1888.
doi: 10.3390/cells11121888.

Synthetic Thymidine Analog Labeling without Misconceptions

Anna Ivanova  1   2 , Olesya Gruzova  1 , Elizaveta Ermolaeva  1 , Olga Astakhova  1   2 , Sheed Itaman  3   4 , Grigori Enikolopov  3 , Alexander Lazutkin  1   2   3
Affiliations

Synthetic Thymidine Analog Labeling without Misconceptions

Anna Ivanova et al. Cells. .

Abstract

Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach.

Keywords: BrdU; DNA labeling; EdU; cell division; neural stem cell; neurogenesis; proliferation; thymidine analog.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Different doses of thymidine analogs result in equivalent numbers of labeled cells in the SGZ of the DG. (a) Scheme of injections; EdU or BrdU at different doses (EdU: 20, 40, 80, or 123 mg/kg; BrdU: 25, 50, 100, or 150 mg/kg) were injected 2 h before euthanasia. (b) The number of EdU+ cells in the SGZ. (c) Number of BrdU+ cells in the SGZ. One-way ANOVA did not reveal a significant difference between groups (not marked). (d) Representative images of EdU- and BrdU-labeled cells in the sections of the DG; doses are indicated; bar is 100 µm.
Figure 2
Figure 2
Single injection of EdU does not affect the survival and subsequent division of hippocampal neural precursor cells. (a) Scheme of injections. Either saline or EdU at different doses (40 or 123 mg/kg) was injected. BrdU at a dose of 50 mg/kg was injected 46 h later, as well as 2 h before euthanasia. (b) Representative images of EdU- and BrdU-labeled cells. Filled arrowheads indicate EdU + BrdU+ cells, and empty arrowheads indicate EdU + BrdU- cells; the scale bar is 20 µm. (c) Number of EdU+ cells in the DG. (d) Number of BrdU+ cells in the DG. (e) Number of EdU + BrdU+ double-labeled cells, as a fraction of BrdU+ cells. (c,e) Mann–Whitney U-test, (d) one-way ANOVA; no significant difference between groups was found.
Figure 3
Figure 3
EdU and BrdU have different long-term effects on hippocampal neurogenesis. (a) Scheme of injections; mice were euthanized 6 weeks after a single EdU or BrdU injection. (b) Number of EdU+ (40 and 123 mg/kg) and BrdU+ (50 and 150 mg/kg) cells in the DG. (c) Total number of DCX+ cells in DG. (d) The proportion of young neurons (N) and neuroblasts (NB) among DCX+ cells. (e) Different subtypes of DCX+ cells; empty arrowheads indicate neuroblasts (NB) and filled arrowheads indicate young neurons (N), the scale bar is 20 µm. (f,g) Representative images of EdU+, BrdU+, and DCX+ cells, bar is 100 µm. (b–d) *** p < 0.0005, factor “Analog”, two-way ANOVA.
Figure 4
Figure 4
Effects of a single injection of EdU on somatic development and neurogenesis of newborn pups. (a) Scheme of injections. EdU at different doses (40, 60, or 123 mg/kg) or saline (SAL) was administered on PND1. BrdU (50 mg/kg) was injected on PND16, and pups were euthanized on PND17. (b) Weight change dynamics in control and EdU-injected pups. Two-way ANOVA, Tukey’s posthoc test, * p < 0.05. Differences between the SAL and EdU groups are shown. Blue, green, and red stars indicate p-values for the 40, 60, and 123 mg/kg groups, respectively. (c) Somatic development of control and EdU-injected pups. (d) Brain weight in PND17 pups. (e) Brain weight to whole bodyweight ratio in PND17 pups. (f) Number of EdU+ cells in the DG in PND17 pups (16 days after single injection). (g) Number of BrdU+ cells in the DG in PND17 pups (24 h after single injection). (d–g) One-way ANOVA, Sidak’s posthoc test, * p < 0.05, and ** p < 0.01. (h) Representative images of EdU- and BrdU-labeled cells; the scale bar is 100 µm.
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