Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population

SummaryA cumulative labelling protocol using 5-bromo-2′-deoxyuridine (BUdR) was followed to determine: (1) the growth fraction (i.e., the proportion of cells that comprise the proliferating population), (2) the length of the cell cycle, and (3) the length of the DNA-synthetic phase (S-phase) for proliferative cells in the dentate gyrus of the mouse. On postnatal day 20 (P20), C57BL/6J mice were injected with BUdR at two hour intervals for a total period of 12 hours. Animals were sacrificed at selected intervals, and the brains were processed for immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BUdR. The numbers of BUdR-labelled and unlabelled cells in sections through the hilus of the dentate gyrus were counted. The number of BUdR-labelled cells increased linearly from an initial value of about 12% of the total number of cells to a maximum value of just over 24% of the total. These findings indicate that, at P20, a maximum of 24.2 ± 1.2% of the cells in the dentate hilus are part of the proliferating population. The calculated length of the cell cycle of the cells comprising the intrahilar proliferative zone was estimated to be 16.1 ± 0.8h. The length of the S-phase was estimated at 8.0 ± 0.4 h. In addition, mathematical analysis, using one and two population models, indicates that over 90% of the proliferating cells in the dentate hilus at this age comprise a single population at least in terms of the lengths of the cell cycle and the S-phase. This protocol provides a convenient method for thein situ analysis of the cell cycle for anatomically defined proliferative populations.

[1]  M. Miller,et al.  Effect of prenatal exposure to ethanol on the development of cerebral cortex: I. Neuronal generation. , 1988, Alcoholism, clinical and experimental research.

[2]  R. Williams,et al.  The control of neuron number. , 1988, Annual review of neuroscience.

[3]  J W Gray,et al.  Cell cycle analysis using flow cytometry. , 1986, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[4]  S. Shackney,et al.  Percent Labeled Mitosis Curve Analysis , 1987 .

[5]  W. Cowan,et al.  The development of the hippocampus and dentate gyrus in normal and reeler mice , 1979, The Journal of comparative neurology.

[6]  H. Gratzner,et al.  Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication. , 1982, Science.

[7]  V. Mareš,et al.  The cellular kinetics of the developing mouse cerebellum. II. The function of the external granular layer in the process of gyrification. , 1970, Brain research.

[8]  Michael W. Miller,et al.  Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system , 1988, Brain Research.

[9]  R. Skalko,et al.  Incorportaiton of thymidine and its analogue, bromodeoxyuridine, into embryos and maternal tissues of the mouse. , 1973, Differentiation; research in biological diversity.

[10]  M. Shimada,et al.  Effect of fluorodeoxyuridine, colcemid, and bromodeoxyuridine on developing neocortex of the mouse. , 1973, The American journal of anatomy.

[11]  M. Nüsse,et al.  Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine. , 1985, Cytometry.

[12]  V. Bond,et al.  THE CELL GENERATION CYCLE OF THE ELEVEN-DAY MOUSE EMBRYO , 1965, The Journal of cell biology.

[13]  P. Rakić,et al.  The site of origin and route and rate of migration of neurons to the hippocampal region of the rhesus monkey , 1981, The Journal of comparative neurology.

[14]  H. Preisler,et al.  Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia. , 1985, Cytometry.

[15]  R. Sidman.  Autoradiographic Methods and Principles for Study of the Nervous System with Thymidine-H3 , 1970 .

[16]  Angevine Jb.  Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse. , 1965 .

[17]  P. Rakić,et al.  Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[18]  P. Rakić,et al.  The time of origin of neurons in the hippocampal region of the rhesus monkey , 1981, The Journal of comparative neurology.

[19]  M. Miller,et al.  Effects of alcohol on the generation and migration of cerebral cortical neurons. , 1986, Science.

[20]  A. Privat,et al.  Subgranular zone of the dentate gyrus of young rabbits as a secondary matrix. A high-resolution autoradiographic study. , 1982, Developmental neuroscience.

[21]  J. Bannigan.  Autoradiographic analysis of effects of 5-bromodeoxyuridine on neurogenesis in the chick embryo spinal cord. , 1987, Brain research.

[22]  Michael W. Miller,et al.  Transient expression of Alz-50 immunoreactivity in developing rat neocortex: a marker for naturally occuring neuronal death? , 1989, Brain Research.

[23]  R. Wimer,et al.  On the development of strain and sex differences in granule cell number in the area dentata of house mice. , 1988, Brain research.

[24]  S. Bayer,et al.  Development of the hippocampal region in the rat I. Neurogenesis examined with 3H‐thymidine autoradiography , 1980, The Journal of comparative neurology.

[25]  J. Langman,et al.  The cellular effect of 5-bromodeoxyuridine on the mammalian embryo. , 1979, Journal of embryology and experimental morphology.

[26]  S. Fujita.  Applications of Light and Electron Microscopic Autoradiography to the Study of Cytogenesis of the Forebrain , 1966 .

[27]  J. Trent,et al.  Cell cycle analysis using bromodeoxyuridine: comparison of methods for analysis of total cell transit time. , 1986, Cancer genetics and cytogenetics.

[28]  R. von Waechter,et al.  Generation times of the matrix cells during embryonic brain development: an autoradiographic study in rats. , 1972, Brain research.