Systematic differences in time of cerebellar‐neuron origin derived from bromodeoxyuridine immunoperoxidase staining protocols and tritiated thymidine autoradiography: A comparative study

Abstract

As exogenous markers of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU) and tritiated thymidine ([(3)H]TdR) have revolutionized our ability to identify proliferating neuroblasts and follow their fate during the development of the central nervous system. The effect of the incorporation of these molecules into DNA on cell proliferation, migration and differentiation is frequently neglected (Duque and Rakic, 2011. J. Neurosci. 31, 15205-15217). By a progressively delayed cumulative labeling method, the current paper analyzes the development of the cerebellum in mice exposed to either BrdU or [(3)H]TdR as embryos and collected at postnatal day 90. We observed that, in comparison to the saline group, several parameters of the cerebellum such as length of the cerebellar cortex, the area of the molecular layer, Purkinje cell (PCs) number, the areas of the cerebellar nuclei, and the number of the deep cerebellar nuclei (DCN) neurons were lower in the BrdU injected group. No consequence of [(3)H]TdR administration was observed. On the other hand, we also studied whether immunohistochemical methods, including BrdU antibodies from different vendors (Sigma and Dako), partial DNA denaturation procedures and trypsin pretreatments, alter the neurogenetic timetables of PC and DCN neurons that resulted from analysis of these tissue specimens. Our analysis revealed that the generative programs of these macroneurons were unrelated to differences in the sensibility of BrdU antibodies but were dependent on the partial denaturation of DNA and trypsin digestion protocols. Finally, we also compare the generation and spatial distribution of PC and DCN neurons in mice exposed to either BrdU or [(3)H]TdR to assess whether the results obtained by these two markers are quantitatively similar. The data presented here show that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar neurons between mice injected with BrdU or [(3)H]TdR. These findings have implications for the interpretation of results obtained by both exogenous makers as an index of the production, migration and settling of neurons in the developing central nervous system.

An Animal Model to Assess the Effects of Hydroxyurea Exposure Suggests That the Administration of This Agent to Pregnant Women and Young Infants May Not Be as Safe as We Thought<strong> </strong>

“…After this treatment, they were injected several times with 6 mg of 5bromo-2´-deoxyuridine (BrdU) in accordance with the procedure of Sekerkova et al [61]. This marker was delivered following a progressively delayed labeling comprehensive procedure [37,57] Our results revealed no signs of toxicity in the pregnant dams after HU treatment.…”

An Animal Model for Assessing the Effects of Hydroxyurea Exposure Suggests That the Administration of This Agent to Pregnant Women and Young Infants May Not Be as Safe as We Thought

“…These observations indicate that the degree of damage induced by HU may influence the level of abnormalities in the cerebellum. As PCs and DCN neurons are the first cells that arise from the neuroepithelium [36,41,42] and the time windows of HU exposure (from E13 to E15) correspond with the developmental timetables of these neurons [36,56], we propose that several PCs and DCN neurons may have never been produced.…”

“…After this treatment, they were injected several times with 6 mg of 5-bromo-2’-deoxyuridine (BrdU) in accordance with the procedure of Sekerkova et al [60]. This marker was delivered following a progressively delayed labeling comprehensive procedure [36,56] that consisted of injecting pregnant dams in an overlapping series, in accordance with the following time-windows: E13–14, E14–15, E15–16 … E19–20. The offspring were sacrificed at postnatal day 90 (P90) and several cerebellar features were quantified, per section, in each cerebellar cortex compartment (vermis, paravermis, and medial and lateral hemisphere), or alternatively in each deep nucleus (fastigial, interposed and dentate): The area of the cerebellum, length of the cerebellar cortex, ML area, PC number, GC number, area of the IGL, area of the white matter, the areas of the cerebellar nuclei, and number of DCN neurons.…”

Glioblastoma Multiforme and Adult Neurogenesis in the Ventricular‐Subventricular Zone: A Review

“…One of the most regular features of the CNS development is that each neuronal population is generated during a specific temporal window, the neurogenetic timetable, (Bayer and Altman, ; Martí et al, , ). It has now been demonstrated that neurogenesis does not stop abruptly at birth, but it continues in specific brain regions throughout life in many species, including human (Gage, ).…”