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• W2007060410 abstract "Adult neurogenesis research has made enormous strides in the last decade but has been complicated by several failures to replicate promising findings. Prevalent use of highly sensitive methods with inherent sources of error has led to extraordinary conclusions without adequate crossvalidation. Perhaps the biggest culprit is the reliance on molecules involved in DNA synthesis and genetic markers to indicate neuronal neogenesis. In this Protocol Review, we present an overview of common methodological issues in the field and suggest alternative approaches, including viral vectors, siRNA, and inducible transgenic/knockout mice. A multipronged approach will enhance the overall rigor of research on stem cell biology and related fields by allowing increased replication of findings between groups and across systems. Adult neurogenesis research has made enormous strides in the last decade but has been complicated by several failures to replicate promising findings. Prevalent use of highly sensitive methods with inherent sources of error has led to extraordinary conclusions without adequate crossvalidation. Perhaps the biggest culprit is the reliance on molecules involved in DNA synthesis and genetic markers to indicate neuronal neogenesis. In this Protocol Review, we present an overview of common methodological issues in the field and suggest alternative approaches, including viral vectors, siRNA, and inducible transgenic/knockout mice. A multipronged approach will enhance the overall rigor of research on stem cell biology and related fields by allowing increased replication of findings between groups and across systems. Stem cell biology is one of the fastest growing research areas in biomedicine and attracts considerable attention due to the potential for regenerative therapies of otherwise irreplaceable tissues. Embryonic stem cells have been isolated from many species, including humans. Also, resident adult precursor/stem cells have been identified in tissues such as the brain, bone marrow, intestine, and skin, and there is increasing evidence of their presence in other regions such as the muscle, kidney, and lung (Nystul and Spradling, 2006Nystul T.G. Spradling A.C. Breaking out of the mold: diversity within adult stem cells and their niches.Curr. Opin. Genet. Dev. 2006; 16: 463-468Crossref Scopus (35) Google Scholar). Particular interest has been devoted to neural precursor/stem cells and the regions displaying neurogenesis in adult mammals (Gage, 2000Gage F.H. Mammalian neural stem cells.Science. 2000; 287: 1433-1438Crossref PubMed Scopus (2654) Google Scholar, Sohur et al., 2006Sohur U.S. Emsley J.G. Mitchell B.D. Macklis J.D. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1477-1497Crossref Scopus (80) Google Scholar). This is partially due to the often poor clinical prognosis of neurodegenerative disease and neurotrauma as well as the difficulty in deriving and transplanting neural tissue. Multipotent neural precursor cells (NPCs) have been derived from many central nervous system (CNS) regions (Gage, 2000Gage F.H. Mammalian neural stem cells.Science. 2000; 287: 1433-1438Crossref PubMed Scopus (2654) Google Scholar). Furthermore, these findings that the brain adds new neurons to mature circuits in selected regions provides a model to assess how lost neurons might be replaced. As the identity of “true” CNS stem cells, defined as being capable of giving rise to all types of CNS neurons, oligodendroglia, and astroglia, is not yet established, we use the term “precursor cell” to encompass the entire lineage of neural stem and more restricted progenitor cells (Sohur et al., 2006Sohur U.S. Emsley J.G. Mitchell B.D. Macklis J.D. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006; 361: 1477-1497Crossref Scopus (80) Google Scholar). The recent explosion in the study of postnatal precursor cells has developed in parallel with increasingly complex techniques that allow for their precise labeling and manipulation. However, some of these reports have not been exempt from controversy, and the high scientific and social expectations for this field may have led to the acceptance of spectacular findings without sufficient supporting evidence (Aldhous, 2006Aldhous P. Stem cells: miracle postponed?.New Sci. 2006; 189: 38-41Google Scholar, Nowakowski and Hayes, 2000Nowakowski R.S. Hayes N.L. New neurons: extraordinary evidence or extraordinary conclusion?.Science. 2000; 288: 771Crossref PubMed Google Scholar, Rakic, 2002Rakic P. Neurogenesis in adult primate neocortex: an evaluation of the evidence.Nat. Rev. Neurosci. 2002; 3: 65-71Crossref PubMed Scopus (214) Google Scholar, Shaywitz, 2006Shaywitz, D. (2006). “Stem Cell Hype and Hope.” The Washington Post 12 Jan. 2006: A21.Google Scholar). Indeed, some findings have been difficult to reproduce—sometimes even by the same lab (Aldhous, 2006Aldhous P. Stem cells: miracle postponed?.New Sci. 2006; 189: 38-41Google Scholar)—and many claims of outstanding basic and clinical achievements have been refuted due to methodological issues (see Vogel, 2003Vogel G. Stem cell research. Same results, different interpretations.Science. 2003; 299: 324Crossref Scopus (5) Google Scholar and references therein) or bias in the selection of patients and experimental design (Aldhous, 2006Aldhous P. Stem cells: miracle postponed?.New Sci. 2006; 189: 38-41Google Scholar). Furthermore, data from the initial transplants of tumor-derived and immortalized progenitors or partially differentiated neural cells into clinical stroke patients do not show significant functional improvements, in stark contrast with the results from animal models on which they are based (see Bakay, 2005Bakay R.A. Neural transplantation.J. Neurosurg. 2005; 103: 6-8Crossref Scopus (6) Google Scholar for discussion and associated references; Kondziolka et al., 2005Kondziolka D. Steinberg G.K. Wechsler L. Meltzer C.C. Elder E. Gebel J. Decesare S. Jovin T. Zafonte R. Lebowitz J. et al.Neurotransplantation for patients with subcortical motor stroke: a phase 2 randomized trial.J. Neurosurg. 2005; 103: 38-45Crossref PubMed Google Scholar). The field of adult neurogenesis is particularly controversial. There have been reports of constitutive neurogenesis in the primate and rodent neocortex (Dayer et al., 2005Dayer A.G. Cleaver K.M. Abouantoun T. Cameron H.A. New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors.J. Cell Biol. 2005; 168: 415-427Crossref PubMed Scopus (196) Google Scholar, Gould et al., 1999Gould E. Reeves A.J. Graziano M.S. Gross C.G. Neurogenesis in the neocortex of adult primates.Science. 1999; 286: 548-552Crossref PubMed Scopus (783) Google Scholar, Gould et al., 2001Gould E. Vail N. Wagers M. Gross C.G. Adult-generated hippocampal and neocortical neurons in macaques have a transient existence.Proc. Natl. Acad. Sci. USA. 2001; 98: 10910-10917Crossref Scopus (242) Google Scholar, Kaplan, 1981Kaplan M.S. Neurogenesis in the 3-month-old rat visual cortex.J. Comp. Neurol. 1981; 195: 323-338Crossref PubMed Google Scholar), the amygdala (Bernier et al., 2002Bernier P.J. Bedard A. Vinet J. Levesque M. Parent A. Newly generated neurons in the amygdala and adjoining cortex of adult primates.Proc. Natl. Acad. Sci. USA. 2002; 99: 11464-11469Crossref Scopus (229) Google Scholar), area CA1 of the rodent (Rietze et al., 2000Rietze R. Poulin P. Weiss S. Mitotically active cells that generate neurons and astrocytes are present in multiple regions of the adult mouse hippocampus.J. Comp. Neurol. 2000; 424: 397-408Crossref Google Scholar), the dorsal vagal complex of the brainstem (Bauer et al., 2005Bauer S. Hay M. Amilhon B. Jean A. Moyse E. In vivo neurogenesis in the dorsal vagal complex of the adult rat brainstem.Neuroscience. 2005; 130: 75-90Crossref Scopus (55) Google Scholar), the spinal cord (Yamamoto et al., 2001Yamamoto S. Yamamoto N. Kitamura T. Nakamura K. Nakafuku M. Proliferation of parenchymal neural progenitors in response to injury in the adult rat spinal cord.Exp. Neurol. 2001; 172: 115-127Crossref PubMed Scopus (156) Google Scholar), and the substantia nigra (Zhao et al., 2003Zhao M. Momma S. Delfani K. Carlen M. Cassidy R.M. Johansson C.B. Brismar H. Shupliakov O. Frisen J. Janson A.M. Evidence for neurogenesis in the adult mammalian substantia nigra.Proc. Natl. Acad. Sci. USA. 2003; 100: 7925-7930Crossref Scopus (329) Google Scholar). To a large degree, these reports remain unconfirmed or have been directly challenged with negative findings, some identifying a possible cause of the original artifactual findings (Ackman et al., 2006Ackman J.B. Siddiqi F. Walikonis R.S. LoTurco J.J. Fusion of microglia with pyramidal neurons after retroviral infection.J. Neurosci. 2006; 26: 11413-11422Crossref PubMed Scopus (42) Google Scholar, Ekdahl et al., 2001Ekdahl C.T. Mohapel P. Elmer E. Lindvall O. Caspase inhibitors increase short-term survival of progenitor-cell progeny in the adult rat dentate gyrus following status epilepticus.Eur. J. Neurosci. 2001; 14: 937-945Crossref Google Scholar, Frielingsdorf et al., 2004Frielingsdorf H. Schwarz K. Brundin P. Mohapel P. No evidence for new dopaminergic neurons in the adult mammalian substantia nigra.Proc. Natl. Acad. Sci. USA. 2004; 101: 10177-10182Crossref Scopus (155) Google Scholar, Hoglinger et al., 2007Hoglinger G.U. Breunig J.J. Depboylu C. Rouaux C. Michel P.P. Alvarez-Fischer D. Boutillier A.-L. DeGregori J. Oertel W.H. Rakic P. et al.The pRb/E2F cell-cycle pathway mediates cell death in Parkinson's disease.Proc. Natl. Acad. Sci. USA. 2007; 104: 3585-3590Crossref Scopus (107) Google Scholar, Horner et al., 2000Horner P.J. Power A.E. Kempermann G. Kuhn H.G. Palmer T.D. Winkler J. Thal L.J. Gage F.H. Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord.J. Neurosci. 2000; 20: 2218-2228PubMed Google Scholar, Koketsu et al., 2003Koketsu D. Mikami A. Miyamoto Y. Hisatsune T. Nonrenewal of neurons in the cerebral neocortex of adult macaque monkeys.J. Neurosci. 2003; 23: 937-942PubMed Google Scholar, Kornack and Rakic, 2001Kornack D.R. Rakic P. Cell proliferation without neurogenesis in adult primate neocortex.Science. 2001; 294: 2127-2130Crossref PubMed Scopus (415) Google Scholar, Lie et al., 2002Lie D.C. Dziewczapolski G. Willhoite A.R. Kaspar B.K. Shults C.W. Gage F.H. The adult substantia nigra contains progenitor cells with neurogenic potential.J. Neurosci. 2002; 22: 6639-6649PubMed Google Scholar, Magavi et al., 2000Magavi S.S. Leavitt B.R. Macklis J.D. Induction of neurogenesis in the neocortex of adult mice.Nature. 2000; 405: 951-955Crossref PubMed Scopus (870) Google Scholar). In some cases, even positive findings of neurogenesis are not mutually crossvalidating, e.g., Gould and colleagues reported the genesis of projection neurons in the association neocortex, but not in the visual cortex (Gould et al., 1999Gould E. Reeves A.J. Graziano M.S. Gross C.G. Neurogenesis in the neocortex of adult primates.Science. 1999; 286: 548-552Crossref PubMed Scopus (783) Google Scholar), whereas Kaplan reported neurogenesis in the rat neocortex only in the visual cortex (Kaplan, 1981Kaplan M.S. Neurogenesis in the 3-month-old rat visual cortex.J. Comp. Neurol. 1981; 195: 323-338Crossref PubMed Google Scholar). In addition, Gould and colleagues indicated that new projection neurons could be generated in situ or potentially from SVZ cells, whereas Cameron and colleagues detected interneurons generated only in situ (Dayer et al., 2005Dayer A.G. Cleaver K.M. Abouantoun T. Cameron H.A. New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors.J. Cell Biol. 2005; 168: 415-427Crossref PubMed Scopus (196) Google Scholar). With regard to human neurogenesis, a recent report describing a rostral migratory stream in humans (Curtis et al., 2007Curtis M.A. Kam M. Nannmark U. Anderson M.F. Axell M.Z. Wikkelso C. Holtas S. van Roon-Mom W.M. Bjork-Eriksson T. Nordborg C. et al.Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension.Science. 2007; 315: 1243-1249Crossref PubMed Scopus (407) Google Scholar) is inconsistent with earlier studies by Alvarez-Buylla and coworkers that did not detect migrating neuron chains (Quinones-Hinojosa et al., 2006Quinones-Hinojosa A. Sanai N. Soriano-Navarro M. Gonzalez-Perez O. Mirzadeh Z. Gil-Perotin S. Romero-Rodriguez R. Berger M.S. Garcia-Verdugo J.M. Alvarez-Buylla A. Cellular composition and cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells.J. Comp. Neurol. 2006; 494: 415-434Crossref PubMed Scopus (244) Google Scholar, Sanai et al., 2004Sanai N. Tramontin A.D. Quinones-Hinojosa A. Barbaro N.M. Gupta N. Kunwar S. Lawton M.T. McDermott M.W. Parsa A.T. Manuel-Garcia Verdugo J. et al.Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration.Nature. 2004; 427: 740-744Crossref PubMed Scopus (672) Google Scholar). The conclusions of this new report by Curtis et al. have now been directly challenged (Sanai et al., 2007Sanai N. Berger M.S. Garcia-Verdugo J.M. Alvarez-Buylla A. Comment on “Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension”.Science. 2007; 318: 393Crossref Scopus (48) Google Scholar). Lesion-induced neurogenesis is similarly confused by unconfirmed or disputed findings (Frielingsdorf et al., 2004Frielingsdorf H. Schwarz K. Brundin P. Mohapel P. No evidence for new dopaminergic neurons in the adult mammalian substantia nigra.Proc. Natl. Acad. Sci. USA. 2004; 101: 10177-10182Crossref Scopus (155) Google Scholar, Hoglinger et al., 2007Hoglinger G.U. Breunig J.J. Depboylu C. Rouaux C. Michel P.P. Alvarez-Fischer D. Boutillier A.-L. DeGregori J. Oertel W.H. Rakic P. et al.The pRb/E2F cell-cycle pathway mediates cell death in Parkinson's disease.Proc. Natl. Acad. Sci. USA. 2007; 104: 3585-3590Crossref Scopus (107) Google Scholar, Lie et al., 2002Lie D.C. Dziewczapolski G. Willhoite A.R. Kaspar B.K. Shults C.W. Gage F.H. The adult substantia nigra contains progenitor cells with neurogenic potential.J. Neurosci. 2002; 22: 6639-6649PubMed Google Scholar, Zhao et al., 2003Zhao M. Momma S. Delfani K. Carlen M. Cassidy R.M. Johansson C.B. Brismar H. Shupliakov O. Frisen J. Janson A.M. Evidence for neurogenesis in the adult mammalian substantia nigra.Proc. Natl. Acad. Sci. USA. 2003; 100: 7925-7930Crossref Scopus (329) Google Scholar). Taken together, it appears that the individual techniques currently employed to study neural precursor cells have important caveats and limitations. Perhaps the most significant constraint is the observer effect: namely, the impact of the experimental manipulation itself on the processes being studied. It is essential that these caveats be adequately addressed by the experimental design and considered during the interpretation of results. A major contributor to the controversies noted above is the lack of a reliable, definitive method to label new neurons. In the present article, we will describe a range of available methods for the labeling and modification of neural precursor cells. Special emphasis is given to the limitations and confounding factors that may lead to ambiguous or inaccurate results and contribute to misinterpretation of the obtained data. It is hoped that the use of a combination of methods will circumvent the weaknesses of single methods and allow for crossvalidation. As the same techniques are used throughout related fields of stem cell biology, we hope to give insight as to how these methods might be applied rigorously to other systems. Replication of data is one of the foundations of scientific advancement, and we believe that by raising awareness of several key issues, the rigor and reliability of this maturing field can be optimized. The first evidence of the existence of postnatal precursor cells in the brain was obtained with tritiated thymidine (3H-dT). This nucleotide analog is incorporated during DNA synthesis and therefore labels all cells that pass through the S phase of the cell cycle during 3H-dT exposure. The resulting signal can be detected by autoradiography of tissue sections and is proportional to the amount of DNA synthesized, permitting the observation of the origin, migration, and fate of newly born cells. This method allowed for the initial observation of precursor cells in the subependymal zone of the lateral ventricles in the postnatal brain and in the dentate gyrus (DG) of the hippocampus in rodents and primates (Rakic, 2002Rakic P. Neurogenesis in adult primate neocortex: an evaluation of the evidence.Nat. Rev. Neurosci. 2002; 3: 65-71Crossref PubMed Scopus (214) Google Scholar). However, the radioactivity of this label, the time-consuming nature of autoradiography, and the inability to sample beyond the upper few microns of a tissue section were inherent limitations to this technique (Table 1). In addition, 3H-dT is toxic under certain circumstances, causing mutations, DNA strand breaks, chromosomal abnormalities, and cell death (Ehmann et al., 1975Ehmann U.K. Williams J.R. Nagle W.A. Brown J.A. Belli J.A. Lett J.T. Perturbations in cell cycle progression from radioactive DNA precursors.Nature. 1975; 258: 633-636Crossref Scopus (13) Google Scholar). Of note, these authors warned of several pitfalls in the use of thymidine analogs that often go unheeded, even some 30 years later, as discussed below.Table 1Summary of Methods: Advantages and Warnings for Thymidine Analogs and Viral VectorsMethodMeans of DeliveryCell Types TargetedAdvantagesCaveatsThymidine AnalogsTritiated thymidinePeripheral or local injectionCells synthesizing DNAWidespread labeling of proliferating cellsTaken up by cells undergoing abortive mitosis and by cells repairing DNAStoichiometric detection ratioCauses DNA strand breaksDilutes during every replication cycleBrdU, CldU, IdUPeripheral or local injectionCells synthesizing DNAWidespread labeling of proliferating cellsTaken up by cells undergoing abortive mitosis and by cells repairing DNADetection methods amplify signalCauses DNA strand breaks, DNA transcription errors, mutagenicAllows for phenotyping of labeled cells in thick tissue sectionsHighly toxicDilutes during every replication cycleAmplification in detection method obscures nature of DNA synthesisViral VectorsAdenovirusStereotaxic, focal injectionBroad rangeWidespread injection of most tissuesTransient transductionPantrophicSubcloning of transgenes typically requires shuttle∼8 kb insert (helper virus permits larger inserts)Not specific for newborn cellsFocal injection by its nature causes lesionLentivirusStereotaxic, focal injectionBroad rangePersistent genetic alteration of most tissuesGenomic integration disrupts host DNA at insertion sitePantrophicNot specific for newborn cells∼8 kb insertFocal injection by its nature causes lesionRetrovirusStereotaxic, focal injectionDividing cellsPersistent genetic alteration of dividing transduced cellsGenomic integration disrupts host DNA at insertion sitePantrophicInduced fusion reported∼7.5 kb insertFocal injection by its nature causes lesion Open table in a new tab To counter the limitations specific to 3H-dT, the analog bromodeoxyuridine (BrdU) was adapted for use in neural tissues (Miller and Nowakowski, 1988Miller M.W. Nowakowski R.S. Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system.Brain Res. 1988; 457: 44-52Crossref PubMed Google Scholar). Detection of BrdU via immunocytochemistry using a specific monoclonal antibody yields an amplifiable signal and expands the depth of tissue sections that can be imaged relative to 3H-dT autoradiography. Of course, amplification techniques can exaggerate the magnitude of DNA synthesis (Rakic, 2002Rakic P. Neurogenesis in adult primate neocortex: an evaluation of the evidence.Nat. Rev. Neurosci. 2002; 3: 65-71Crossref PubMed Scopus (214) Google Scholar) or lead to false-positive readings (Bak and Panos, 1997Bak P.M. Panos R.J. Protease antigen recovery decreases the specificity of bromodeoxyuridine detection in formalin-fixed tissue.J. Histochem. Cytochem. 1997; 45: 1165-1170Crossref Google Scholar), emphasizing the importance of appropriate controls during BrdU analysis (McGinley et al., 2000McGinley J.N. Knott K.K. Thompson H.J. Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas.J. Histochem. Cytochem. 2000; 48: 355-362Crossref PubMed Google Scholar) (Table 1). In addition to the improved sensitivity over 3H-dT, fluorescently labeled secondary antibodies used in BrdU detection can be combined with up to three additional primary antibodies for simultaneous cell characterization (Kornack and Rakic, 2001Kornack D.R. Rakic P. Cell proliferation without neurogenesis in adult primate neocortex.Science. 2001; 294: 2127-2130Crossref PubMed Scopus (415) Google Scholar, Magavi et al., 2000Magavi S.S. Leavitt B.R. Macklis J.D. Induction of neurogenesis in the neocortex of adult mice.Nature. 2000; 405: 951-955Crossref PubMed Scopus (870) Google Scholar). However, precise colocalization remains challenging in regions with a high density of cell bodies such as the granular layer of the DG. This is particularly difficult if the cell marker is not expressed in the nucleus, as is the case with the astrocytic marker glial fibrillary acidic protein (Gfap). BrdU labeling of DNA was pivotal in the report of neurogenesis in the human hippocampus and the lack of neurogenesis in the human neocortex (Bhardwaj et al., 2006Bhardwaj R.D. Curtis M.A. Spalding K.L. Buchholz B.A. Fink D. Bjork-Eriksson T. Nordborg C. Gage F.H. Druid H. Eriksson P.S. et al.Neocortical neurogenesis in humans is restricted to development.Proc. Natl. Acad. Sci. USA. 2006; 103: 12564-12568Crossref PubMed Scopus (156) Google Scholar, Eriksson et al., 1998Eriksson P.S. Perfilieva E. Bjork-Eriksson T. Alborn A.M. Nordborg C. Peterson D.A. Gage F.H. Neurogenesis in the adult human hippocampus.Nat. Med. 1998; 4: 1313-1317Crossref PubMed Scopus (2834) Google Scholar). Despite its advantages, the use of BrdU to track dividing cells can also introduce cellular changes due to the presence of the incorporated BrdU molecules. The molecular structure of BrdU is significantly different from the natural structure of thymidine (Stetson et al., 1988Stetson P.L. Maybaum J. Wagner J.G. Averill D.R. Wollner I.S. Knol J.A. Johnson N.J. Yang Z.M. Preiskorn D. Smith P. et al.Tissue-specific pharmacodynamics of 5-bromo-2′-deoxyuridine incorporation into DNA in VX2 tumor-bearing rabbits.Cancer Res. 1988; 48: 6900-6905Google Scholar) (Figure 1) and may cause steric hindrance when present in high quantities. The resulting impact of high doses of BrdU on the natural conformation of the DNA can alter transcription and translation and may lead to mutation and cell toxicity (Table 1), compromising the cellular function and even the overall health of the subject. Indeed, BrdU is well known for its ability to sensitize cancer cells to radiation (Djordjevic and Szybalski, 1960Djordjevic B. Szybalski W. Genetics of human cell lines. III. Incorporation of 5-bromo- and 5-iododeoxyuridine into the deoxyribonucleic acid of human cells and its effect on radiation sensitivity.J. Exp. Med. 1960; 112: 509-531Crossref PubMed Google Scholar). In vitro, BrdU can be selectively toxic to neurons when used at currently recommended concentrations (Caldwell et al., 2005Caldwell M.A. He X. Svendsen C.N. 5-Bromo-2′-deoxyuridine is selectively toxic to neuronal precursors in vitro.Eur. J. Neurosci. 2005; 22: 2965-2970Crossref Scopus (26) Google Scholar) and can also induce aberrant neuronal differentiation (Qu et al., 2004Qu T.Y. Dong X.J. Sugaya I. Vaghani A. Pulido J. Sugaya K. Bromodeoxyuridine increases multipotency of human bone marrow-derived stem cells.Restor. Neurol. Neurosci. 2004; 22: 459-468Google Scholar). In vivo, high doses of BrdU can induce abnormal proliferation (Goldsworthy et al., 1992Goldsworthy T. Dunn C. Popp J. Dose effects of bromodeoxyuridine (BRDU) on rodent hepatocyte proliferation measurements.Toxicologist. 1992; 12: 265Google Scholar) and act as a mutagen, teratogen, and carcinogen. The toxicity is expected to correlate with the number of cells that incorporate BrdU and with the percentage of thymidine nucleotides that are replaced (Bannigan and Langman, 1979Bannigan J. Langman J. The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.J. Embryol. Exp. Morphol. 1979; 50: 123-135Google Scholar, Bannigan et al., 1990Bannigan J.G. Cottell D.C. Morris A. Study of the mechanisms of BUdR-induced cleft palate in the mouse.Teratology. 1990; 42: 79-89Crossref Scopus (12) Google Scholar, Kolb et al., 1999Kolb B. Pedersen B. Ballermann M. Gibb R. Whishaw I.Q. Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.J. Neurosci. 1999; 19: 2337-2346PubMed Google Scholar, Kuwagata et al., 2004Kuwagata M. Muneoka K.T. Ogawa T. Takigawa M. Nagao T. Locomotor hyperactivity following prenatal exposure to 5-bromo-2′-deoxyuridine: neurochemical and behavioral evidence of dopaminergic and serotonergic alterations.Toxicol. Lett. 2004; 152: 63-71Crossref Scopus (13) Google Scholar, Nagao et al., 1998Nagao T. Kuwagata M. Saito Y. Effects of prenatal exposure to 5-bromo-2′-deoxyuridine on the developing brain and reproductive function in male mouse offspring.Reprod. Toxicol. 1998; 12: 477-487Crossref Scopus (16) Google Scholar). Therefore, increasing dosages and frequency of injection are thought to exacerbate the cellular toxicity and adverse side effects, likely causing toxicity in the very population that is under examination (Goldsworthy et al., 1992Goldsworthy T. Dunn C. Popp J. Dose effects of bromodeoxyuridine (BRDU) on rodent hepatocyte proliferation measurements.Toxicologist. 1992; 12: 265Google Scholar, Goldsworthy et al., 1993Goldsworthy T.L. Butterworth B.E. Maronpot R.R. Concepts, labeling procedures, and design of cell proliferation studies relating to carcinogenesis.Environ. Health Perspect. 1993; 101: 59-65Crossref PubMed Google Scholar). Although it has been recommended that doses of up to 300 mg/kg should be employed (Cameron and McKay, 2001Cameron H.A. McKay R.D. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus.J. Comp. Neurol. 2001; 435: 406-417Crossref PubMed Scopus (872) Google Scholar), lower 50–100 mg/kg doses may minimize toxicity and are adequate to reach near-saturation labeling (Burns and Kuan, 2005Burns K.A. Kuan C.Y. Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus.Eur. J. Neurosci. 2005; 21: 803-807Crossref Scopus (48) Google Scholar). It should also be noted that numerous, small doses that in total exceed 100–150 mg/kg per day can also lead to adverse effects. For example, in attempting to label cerebellar cell types, Mugnaini and colleagues noted that BrdU produced striking defects on the proliferation, migration, and localization of Purkinje neurons, along with defects in the patterning of foliation (Sekerkova et al., 2004Sekerkova G. Ilijic E. Mugnaini E. Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat.J. Comp. Neurol. 2004; 470: 221-239Crossref Scopus (34) Google Scholar). The distinct structure of the cerebellum, with a single layer of Purkinje cell bodies that exhibit a characteristic dendritic tree, made the defect evident. Postnatal neurogenic regions of the forebrain do not exhibit such precise cellular lamination and organization, opening the possibility that similar effects could be missed. Recently, several groups have reported that chlorodeoxyuridine and iododeoxyuridine (both thymidine analogs with a similar structure to BrdU [Figure 1]) can be detected individually by different monoclonal antibodies (Burns and Kuan, 2005Burns K.A. Kuan C.Y. Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus.Eur. J. Neurosci. 2005; 21: 803-807Crossref Scopus (48) Google Scholar). Using two temporally segregated injections of these two molecules, two cohorts of S phase cells may be tracked over the course of time. The recommended precautions for BrdU studies also apply to these related compounds. An underappreciated phenomenon is the abortive cell cycle that some neurons enter after insult (Burns et al., 2007Burns K.A. Ayoub A.E. Breunig J.J. Adhami F. Weng W.L. Colbert M.C. Rakic P. Kuan C.Y. Nestin-CreER mice reveal DNA synthesis by nonapoptotic neurons following cerebral ischemia-hypoxia.Cereb. Cortex. 2007; 17: 2585-2592Crossref PubMed Scopus (47) Google Scholar), during disease processes (Hoglinger et al., 2007Hoglinger G.U. Breunig J.J. Depboylu C. Rouaux C. Michel P.P. Alvarez-Fischer D. Boutillier A.-L. DeGregori J. Oertel W.H. Rakic P. et al.The pRb/E2F cell-cycle pathway mediates cell death in Parkinson's disease.Proc. Natl. Acad. Sci. USA. 2007; 104: 3585-3590Crossref Scopus (107) Google Scholar, Yang et al., 2001Yang Y. Geldmacher D.S. Herrup K. DNA replication precedes neuronal cell death in Alzheimer's disease.J. Neurosci. 2001; 21: 2661-2668Crossref PubMed Google Scholar), or prior to death (Kruman et al., 2004Kruman I.I. Wersto R.P. Cardozo-Pelaez F. Smilenov L. Chan S.L. Chrest F.J. Emokpae Jr., R. Gorospe M. Mattson M.P. Cell cycle activation linked to neuronal cell death initiated by DNA damage.Neuron. 2004; 41: 549-561Abstract Full Text Fu" @default.
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• W2007060410 date "2007-12-01" @default.
• W2007060410 modified "2023-10-16" @default.
• W2007060410 title "Everything that Glitters Isn't Gold: A Critical Review of Postnatal Neural Precursor Analyses" @default.
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