Tritiated Thymidine Autoradiography Research Articles

Methods for Inferring Cell Cycle Parameters Using Thymidine Analogues.

Tritiated thymidine autoradiography, 5-bromo-2'-deoxyuridine (BrdU) 5-chloro-2'-deoxyuridine (CldU), 5-iodo-2'-deoxyuridine (IdU), and 5-ethynyl-2'-deoxyiridine (EdU) labeling have been used for identifying the fraction of cells undergoing the S-phase of the cell cycle and to follow the fate of these cells during the embryonic, perinatal, and adult life in several species of vertebrate. In this current review, I will discuss the dosage and times of exposition to the aforementioned thymidine analogues to label most of the cells undergoing the S-phase of the cell cycle. I will also show how to infer, in an asynchronous cell population, the duration of the G1, S, and G2 phases, as well as the growth fraction and the span of the whole cell cycle on the base of some labeling schemes involving a single administration, continuous nucleotide analogue delivery, and double labeling with two thymidine analogues. In this context, the choice of the optimal dose of BrdU, CldU, IdU, and EdU to label S-phase cells is a pivotal aspect to produce neither cytotoxic effects nor alter cell cycle progression. I hope that the information presented in this review can be of use as a reference for researchers involved in the genesis of tissues and organs.

Postnatal Neurogenesis Beyond Rodents: the Groundbreaking Research of Joseph Altman and Gopal Das.

An integral component of neural ontogeny and plasticity is the ongoing generation of new neurons from precursor cells throughout the lifespan in virtually all animals with a nervous system. In mammals, postnatal neurogenesis has been documented in the cerebellum, olfactory bulb, hippocampus, striatum, substantia nigra, hypothalamus, and amygdala. Germinal centers of new neuron production in the adult brain have been identified in the neuroepithelium of the subventricular zone and the dentate gyrus. One of the earliest lines of evidence gathered came from studies on the production of cerebellar microneurons in the external germinal layer of rodents and carnivores in the 1960s and 1970s. The undeniable pioneer of that research was the insightful developmental neurobiologist Joseph Altman (1925-2016). This Cerebellar Classic is devoted to the groundbreaking work of Altman and his graduate student and, subsequently, fellow faculty member, Gopal Das (1933-1991), on postnatal neurogenesis using tritiated thymidine autoradiography to tag newly formed neurons in the cerebellum of cats. Perseverant to their ideas and patiently working in West Lafayette (Indiana), they were the founders of two fields that brought about paradigm shifts and led to an explosive growth in brain research: adult neurogenesis and neural tissue transplantation.

Developmental timetables and gradients of neurogenesis in cerebellar Purkinje cells and deep glutamatergic neurons: A comparative study between the mouse and the rat.

The aim of this report is to determine whether the times of neuron origin and neurogenetic gradients of PCs and Deep cerebellar nucli (DCN) glutamatergic neurons are different between mice and rats. Purkinje cells (PCs) were analyzed in each compartment of the cerebellar cortex (vermis, paravermis, medial, and lateral hemispheres), and deep glutamatergic neurons at the level of the medialis, interpositus, and lateralis nuclei. Tritiated thymidine ([3 H]TdR) autoradiography was applied on sections. The experimental rodents were the offspring of pregnant dams injected with [3 H]TdR on embryonic days (E) 11-12, E12-13, E13-14, E14-15, E15-16, and E16-17. Our results indicate that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar PCs and deep glutamatergic neurons between mice and rats. In mice, PCs and deep glutamatergic neurons neurogenesis extend from E10 to E14, with a predominance of neurogenesis on E12 for PCs, and on E12, E11, and E10 for the medialis, interpositus, and lateralis neurons, respectively. When neurogenesis in rats was considered, the data reveal that PCs and deep glutamatergic neurons production extends from E12 to E16, with a peak of production on E14 for PCs, and on E14, E13, and E12 for the medialis, interpositus, and lateralis neurons, respectively. Current data also indicate that, both in mice and rats, both types of macroneurons are generated according to a lateral-to-medial gradient. Thus, the lateral hemisphere and the lateralis nucleus present more early-generated neurons than the vermis and the medialis nucleus, which in their turn have more late-produced neurons.

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

As exogenous markers of DNA synthesis, 5-bromo-2′-deoxyuridine (BrdU) and tritiated thymidine ([3H]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 [3H]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 [3H]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 [3H]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 [3H]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.

The heterochromatin of Rhoeo discolor

Q-banding and C-banding techniques as well as tritiated thymidine autoradiography were used to characterise the heterochromatin of Rhoeo discolor. The heterochromatin which is situated around the centromere in all the 12 chromosomes, is brightly fluorescent, strongly stainable with Giemsa, following denaturation and renaturation, as well as late replicating. Utilizing these techniques, the behaviour of the heterochromatic segments during male meiosis was studied. The role of heterochromatin in the pairing organization of multivalents are discussed.

Generation and survival of midbrain dopaminergic neurons in weaver mice

Generation and survival of midbrain dopaminergic (DA) neurons were investigated using tyrosine hydroxylase (TH) immunocytochemistry combined with tritiated thymidine autoradiography at appropriate anatomical levels throughout the anteroposterior (A/P) axes of the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). The wild-type (+/+) and homozygous weaver (wv/wv) mice used here were the offspring of pregnant dams injected with the radioactive precursor when the mesencephalic neurons were being produced (gestational days 11–15). Data reveal that, at postnatal day 90, depletion of TH-stained cells in the wv/wv presented an A/P pattern of increasing severity and, therefore, the DA cells located in posterior parts of the SNc or the VTA appear to be more vulnerable than the settled anterior neurons. When the time of neuron origin is inferred for each level of these cell groups, it is found that the neurogenesis span is similar for both experimental groups, although significant deficits in the frequency of wv/wv late-generated neurons were observed in any level considered. On the other hand, it has been found that TH-positive neurons were settled along the extent of the SNc and the VTA following precise and differential neurogenetic gradients. Thus, the acute rostrocaudal increase in the proportion of late-generated neurons detected in both +/+ DA-cell groups is disturbed in the weaver homozygotes due to the indicated A/P depletion.

Quantitative Analysis of In Vivo Cell Proliferation

Injection and immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU) has become the standard method for studying the birth and survival of neurons, glia, and other cell types in the nervous system. BrdU, a thymidine analog, becomes stably incorporated into DNA during the S-phase of mitosis. Because DNA containing BrdU can be specifically recognized by antibodies, this method allows dividing cells to be marked at any given time and then identified at time points from a few minutes to several years later. BrdU immunohistochemistry is suitable for cell counting to examine the regulation of cell proliferation and cell fate. It can be combined with labeling by other antibodies, allowing confocal analysis of cell phenotype or expression of other proteins. The potential for nonspecific labeling and toxicity are discussed. Although BrdU immunohistochemistry has almost completely replaced tritiated thymidine autoradiography for labeling dividing cells, this method and situations in which it is still useful are also described.

A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-

Early development of the hypothalamus of a wallaby (Macropus eugenii).

We have studied the development of the hypothalamus of an Australian marsupial, the tammar wallaby (Macropus eugenii), to provide an initial anatomic framework for future research on the developing hypothalamus of diprotodontid metatheria. Cytoarchitectural (hematoxylin and eosin), immunohistochemical (CD 15 and growth associated protein, GAP-43), tritiated thymidine autoradiography, and carbocyanine dye tracing techniques were applied. Until 12 days after birth (P12), the developing hypothalamus consisted of mainly a ventricular germinal zone with a thin marginal layer, but by P25, most hypothalamic nuclei were well differentiated, indicating that the bulk of hypothalamic cytoarchitectural development occurs between P12 and P25. Strong CD 15 immunoreactivity was found in radial glial fibers in the rostral hypothalamus during early developmental ages, separating individual hypothalamic compartments. Immunoreactivity for GAP-43 was used to reveal developing fiber bundles. The medial forebrain bundle was apparent by P0, and the fornix appeared at P12. Tritiated thymidine autoradiography revealed lateral-to-medial and dorsal-to-ventral neurogenetic gradients similar to those seen in rodents. Dye tracing showed that projections to the posterior pituitary arose from the supraoptic nucleus at P5 and from the paraventricular nucleus at P10. Projections to the medulla were first found from the lateral hypothalamic area at P0 and paraventricular nucleus at P10. In conclusion, the pattern of development of the wallaby hypothalamus is broadly similar to that found in eutheria, with comparable neurogenetic compartments to those identified in rodents. Because most hypothalamic maturation takes place after birth, wallabies provide a useful model for experimentally manipulating the developing mammalian hypothalamus.

Genetics of Childhood Disorders: XXXVI. Stem Cell Research, Part 1: New Neurons in the Adult Brain

Until recently, it was assumed that neurogenesis, or the production of new neurons, occurs only during development and never in the adult organism. The famous neuroanatomist Cajal stated that “Once development was ended, the fonts of growth and regeneration of the axons and dendrites dried up irrevocably. In adult centers, the nerve paths are something fixed and immutable: everything may die, nothing may be regenerated.” This statement holds true for most of the regions of the adult brain. However, there are two adult brain areas in which neurogenesis is observed: the subventricular zone of the anterior lateral ventricles gives rise to cells that become neurons in the olfactory bulb, and the subgranular zone in the dentate gyrus generates new granule cell neurons in the hippocampus, a brain region that is important for learning and memory. The initial studies that suggested that the adult brain could generate new neurons were largely ignored. In the 1960s Joseph Altman and coworkers published a series of papers reporting that some dividing cells in the adult brain survived and differentiated into cells with morphology similar to neurons. They used tritiated thymidine autoradiography to label the cells. Tritiated thymidine is incorporated into the DNA of dividing cells. They found that the highest density of labeling was in the subventricular zone and in the dentate gyrus of the hippocampus. It was known that the dentate gyrus of the hippocampus is essentially devoid of glia. Therefore, Altman attributed the labeling in this region to the uptake of thymidine by dentate granule cells. However, he could not prove that the adult-generated cells were neurons rather than glia, since no phenotypic markers were available that could be used in conjunction with thymidine autoradiography. The absence of specific markers for neurons and glia and continued skepticism surrounding the novel concept of adult neurogenesis limited further development of the research. In the mid 1970s and the early 1980s, Michael Kaplan and his colleagues reexamined the initial observations using the electron microscope and added substantial confidence that neurogenesis could occur in the adult brain. Combining electron microscopy and tritiated thymidine labeling, they showed that

Characterization of steroid hormone sensitivity in human breast cancers maintained ex vivo under organotypical culture conditions

The methodology we propose combines the immunohistochemical determination of the oestrogen and progesterone receptors (ER and PgR) with the characterization of the oestradiol- and progesterone-induced influence on cell proliferation in breast cancers in order to characterize their steroid hormone sensitivity at both the "static" and "dynamic" level. ER and PgR have been immunohistochemically quantified by means of computer-assisted microscopy. Cell proliferation has been determined by means of tritiated thymidine autoradiography in tumour samples maintained in vitro as organotypic cultures. A series of 14 patients was investigated. Of the 14 breast cancers under study, one with an unequivocally "very ER-rich"/"very PgR-rich" immunohistochemical phenotype totally failed to exhibit any modification in its cell proliferation level after both oestradiol and progesterone stimulation. Two cases definitively associated with an "ER-poor"/"PgR-poor" immunohistochemical phenotype nevertheless responded noticeably to the dynamic stimulation of their cell proliferation by oestradiol and progesterone. While our series of cases covers 14 patients only, it suffices to demonstrate the limits of ER and PgR determination in characterizing steroid hormone sensitivity in breast cancer. The present work therefore presents an in vitro approach to test growth regulation of human breast cancer by steroid hormones. The clinical value of the present approach should be further determined by showing that steroid hormone-induced modifications in cell proliferation level are actually associated with clinical response.

Ontogeny of the tectorotundal pathway in chicks (Gallus gallus): Birthdating and pathway tracing study

The avian tectorotundal system has been suggested as a homologue of the mammalian colliculopulvinar system. In the tectorotundal system, neurons of the stratum griseum centrale (SGC) of the optic tectum send their axons bilaterally to the nucleus rotundus (Rt). In transit to the Rt, the axons of the SGC neurons collateralize in the nuclei posteroventralis thalami (PV), subpretectalis (SP), and interstitiopretectosubpretectalis (IPS) of the tectothalamic tract (TT). The current study used birthdating and pathway-tracing methods to investigate the neurogenesis and time course of neuronal connections of the tectorotundal pathway in chicks during embryogenesis. By using tritiated thymidine autoradiography, we observed that the SGC neurons of the tectum were generated by embryonic days 3.0–5.5 (E3.0–E5.5), the Rt by E3.5–E5.0, and the nuclei of TT by E3.5–4.5. To trace the tectorotundal pathway, we injected cholera toxin B subunit (CTb) into the tectum, and the CTb-like immunoreactivity was examined. By E4.5–E5.5, some CTb-like immunoreactive (CTb-LI) axons terminated in the ipsilateral SP/IPS. By E6.0–E6.5, CTb-LI axon bundles were seen ipsilaterally in the TT. Increased numbers of labeled axons were seen terminating in the SP/IPS. By E7.0–E7.5, heavily labeled axons in the TT were observed with diffuse terminals in areas ventral to the presumptive Rt and PV. By E7.5–E8.0, the tectal axons innervated the ipsilateral Rt, in which some of the collaterals crossed the midline to the contralateral diencephalon. The crossed tectorotundal projection was seen first by E8.0–E8.5. Also, during this stage, a few CTb-LI collaterals terminated in the contralateral SP/IPS. Between E10 and E13, the pattern of bilateral tectorotundal projections became more regionalized, whereas labeling continued to increase in the SP/IPS. At E16, the labeling pattern of all tectorecipient structures resembled that of the hatchling. The current study revealed the temporal order of development of the tectorotundal pathway during embryogenesis. The SGC cells first innervate ipsilaterally the SP/IPS and then the Rt/PV. The schedule of the crossed tectorotundal connections coincides with the schedule of tectal projections onto the contralateral intrinsic nuclei of the TT. We conclude that E8.0 (±E0.5) is a critical stage for the development of the tectofugal pathway. Moreover, the current study provides important insights into the relative ontogeny of the mammalian tectofugal pathway. J. Comp. Neurol. 417:115–132, 2000. ©2000 Wiley-Liss, Inc.

Ontogeny of the tectorotundal pathway in chicks (Gallus gallus): Birthdating and pathway tracing study

The avian tectorotundal system has been suggested as a homologue of the mammalian colliculopulvinar system. In the tectorotundal system, neurons of the stratum griseum centrale (SGC) of the optic tectum send their axons bilaterally to the nucleus rotundus (Rt). In transit to the Rt, the axons of the SGC neurons collateralize in the nuclei posteroventralis thalami (PV), subpretectalis (SP), and interstitiopretectosubpretectalis (IPS) of the tectothalamic tract (TT). The current study used birthdating and pathway-tracing methods to investigate the neurogenesis and time course of neuronal connections of the tectorotundal pathway in chicks during embryogenesis. By using tritiated thymidine autoradiography, we observed that the SGC neurons of the tectum were generated by embryonic days 3.0-5.5 (E3.0-E5.5), the Rt by E3.5-E5.0, and the nuclei of TT by E3.5-4.5. To trace the tectorotundal pathway, we injected cholera toxin B subunit (CTb) into the tectum, and the CTb-like immunoreactivity was examined. By E4.5-E5.5, some CTb-like immunoreactive (CTb-LI) axons terminated in the ipsilateral SP/IPS. By E6.0-E6.5, CTb-LI axon bundles were seen ipsilaterally in the TT. Increased numbers of labeled axons were seen terminating in the SP/IPS. By E7.0-E7.5, heavily labeled axons in the TT were observed with diffuse terminals in areas ventral to the presumptive Rt and PV. By E7.5-E8.0, the tectal axons innervated the ipsilateral Rt, in which some of the collaterals crossed the midline to the contralateral diencephalon. The crossed tectorotundal projection was seen first by E8.0-E8.5. Also, during this stage, a few CTb-LI collaterals terminated in the contralateral SP/IPS. Between E10 and E13, the pattern of bilateral tectorotundal projections became more regionalized, whereas labeling continued to increase in the SP/IPS. At E16, the labeling pattern of all tectorecipient structures resembled that of the hatchling. The current study revealed the temporal order of development of the tectorotundal pathway during embryogenesis. The SGC cells first innervate ipsilaterally the SP/IPS and then the Rt/PV. The schedule of the crossed tectorotundal connections coincides with the schedule of tectal projections onto the contralateral intrinsic nuclei of the TT. We conclude that E8.0 (+/- E0.5) is a critical stage for the development of the tectofugal pathway. Moreover, the current study provides important insights into the relative ontogeny of the mammalian tectofugal pathway.

Neurogenesis and identification of developing layers in the visual cortex of the wallaby (Macropus eugenii)

Birthdates of the neurons that comprise the layers of the mature visual cortex in the wallaby (Macropus eugenii) have been determined with the aid of tritiated thymidine autoradiography. The laminar positions of cells, identified by their birthdates, have then been followed at early stages during development and compared with previously published data on the distribution of thalamocortical afferents and corticothalamic projecting cells (Sheng et al. [1991] J. Comp. Neurol. 307:17-38). Neurons are born in a deep to superficial sequence typical of other mammals. The loosely packed zone of cells, which develops at the base of the thin compact zone of cells at the superficial margin of the cortical plate early in development, was identified as being part of the cortical plate. Afferents did not wait below this zone but grew into the developing cortical layers immediately after the cells that form these layers began accumulating in the loosely packed zone, starting with layer 6 on postnatal day 22 (P22). The genesis of layer 4 did not begin until P32, and these cells reached the superficial cortical plate at P54 and entered the loosely packed zone by P65. Cells of layers 5 and 6 formed the initial projection to the thalamus. Despite the protracted development of the wallaby and the large discrepancy between the time of thalamic ingrowth and genesis of layer 4, there was no extended waiting period for afferents in the subplate.

The repopulation of murine Langerhans cells after depletion by mild heat injury.

We have developed a model of focal Langerhans cell depletion by mild heat injury and used it to investigate the mechanisms of Langerhans cell repopulation in the injured epidermis. The possibility whether repopulation occurred by recruitment of precursor cells from the circulation or dermis or, alternatively, by migration from the surrounding normal epidermis into the injured area was considered. Repopulation was studied by evaluating the pattern of Langerhans cell reappearance and calculating the rate of change in the density. Heat injury followed by whole-body irradiation with shielding of the injured skin was used to assess repopulation in the absence of bone marrow precursors. Using tritiated thymidine autoradiography, we also investigated whether the newly arrived Langerhans cells (be they from circulating precursors or surrounding normal epidermis) actually divide. The results showed that heat injury completely eliminated the Langerhans cells within the area delineated by the injury. Two hours after injury, the Langerhans cells were fragmented and 2 days later, they could not be detected. Regeneration of the epidermis occurred 2 days after injury and Langerhans cells reappeared scattered somewhat sparsely in the centre of the lesion on day 3. These cells were small and slender, bearing one or two short dendrites. As the dendrites increased in number and in length, the cells became similar morphologically and phenotypically to normal Langerhans cells. The rate of repopulation increased dramatically between days 5 and 7 and reached normal density on day 11. The pattern of Langerhans cell repopulation in the injured area and the lack of repopulation in the irradiated animals indicated that repopulation occurs by immigration of precursors from the circulation or dermis. There was no indication of migration of Langerhans cells from surrounding normal epidermis. Lastly, the newly arrived Langerhans cells failed to divide at the site of injury.

Immunocytochemical and Morphological Evidence for Intracellular Self-Repair as an Important Contributor to Mammalian Hair Cell Recovery

Although recent studies have provided evidence for hair cell regeneration in mammalian inner ears, the mechanism underlying this regenerative process is still under debate. Here we report immunocytochemical, histological, electron microscopic, and autoradiographic evidence that, in cultured postnatal rat utricles, a substantial number of hair cells can survive gentamicin insult even their stereocilia are lost. These partially damaged hair cells can survive for a prolonged time and regrow the stereocilia. Although the number of stereocilia-bearing hair cells increases over time after gentamicin insult, hair cell and supporting cell numbers remain essentially unchanged. Tritiated thymidine autoradiography and bromodeoxyuridine immunocytochemistry of the cultures demonstrate that cell proliferation in the sensory epithelium is very limited and is far below the number of recovered hair cells. Furthermore, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling analysis indicates that gentamicin-induced apoptosis in the sensory epithelium occurs mainly during a 2 d treatment period, and additional cell death is minimal 2-11 d after treatment. Considered together, intracellular repair of partially damaged hair cells can be an important contributor to spontaneous hair cell recovery in mammalian inner ears.

Radiation-induced apoptosis and gene expression in neonatal kidney and testis with and without protein synthesis inhibition

Purpose: To analyse the incidence of radiation-induced apoptosis, expression of two apoptosis-related genes,Bcl-2 and p53, and post-radiation levels of cell proliferation in the neonatal rat (4-5 days old) kidney and testis. Materials and methods : Apoptosis was quantified in control or treated kidney or testis at 2, 4, 6, 8 and 24h after 5Gy of whole body X-irradiation (n=4 per group). Morphology (light and electron microscopy) and DNA gel electrophoresis were used to assess apoptosis. Temporal and spatial expression of Bcl-2 or p53 were analysed using immunohistochemistry. Administration of cycloheximide (1.5mg/kg) was used to determine whether new protein synthesis had a role in induction of apoptosis. Tritiated thymidine uptake and autoradiography were used to indicate alterations in cell proliferation (radiolabel administered 1h prior to tissue collection) or S-phase cells undergoing radiation-induced apoptosis (radiolabel administered 1h prior to irradiation). Results: Apoptosis peaked at 4h in the testis and 6h in the kidney and was significantly higher in the renal nephrogenic zone than in the testis (p Alt; 0.05). Mitosis was almost completely negated after irradiation in both tissues. A higher proportion (almost fivefold) of the apoptotic cells died in S phase in the kidney than in the testis. Cycloheximide negated induction of apoptosis in the kidney, and markedly decreased apoptosis in the testis. Bcl-2 expression was highest in the differentiated zone of control kidneys and increased after irradiation in the nephrogenic zone, particularly near foci of apoptosis in developing nephrons. In the control testis, Sertoli cells had moderate expression of Bcl-2. After irradiation, there was complete absence of Bcl-2 expression in apoptotic Sertoli cells, with surviving cells increasing Bcl-2 expression. Irradiated kidney had more intense nuclear p53 expression compared with controls. In the testis, p53 that was present in controls continued to be expressed in surviving cells but not apoptotic cells in radiation-treated animals. Conclusions: Unique differences can be identified between the incidence and biomolecular control of radiation-induced apoptosis in the normal neonatal kidney and testis. These results may find application for minimizing damage to these normal neonatal tissues in the development of, for example, cancer treatment regimens.

Epiphyseal and physeal cartilage vascularization: a light microscopic and tritiated thymidine autoradiographic study of cartilage canals in newborn and young postnatal rabbit bone.

The vascular pattern of newborn and early postnatal epiphyseal and physeal cartilage is integral to long bone development and differs from later postnatal patterns. In the present study, we supplement light microscopic histology with tritiated thymidine autoradiography to help assess the position of cartilage canals and the dynamics of cartilage vascularity in relation to growth. Tritiated thymidine labeling studies to assess cell proliferation activity were done by using 2 microc/g body weight intraperitoneal injections into newborn and 3-, 4-, and 7-day-old New Zealand white rabbits that were killed 1 hr after the injection. Proximal humeral, distal femoral, and third metatarsal epiphyses were assessed by routine histology and serial section autoradiography. Cartilage canals were seen in each epiphysis. Transphyseal vessels were seen in each epiphysis continuous from the epiphysis to the metaphysis or were present within the physis traversing the proliferating and hypertrophic cell zones. Histologic sections showed vessels from the perichondrium continuous with those of the epiphyseal cartilage canals at proximal humeral, distal femoral, and metatarsal epiphyses. Serial sections showed vascular buds and connective tissue cells lying in indentations at the periphery of and present within the epiphyseal cartilage. Autoradiographic studies showed extensive labeling of vessel wall cells and surrounding connective tissue cells of the cartilage canals (a) within the epiphyseal cartilage, (b) traversing the physis, and (c) within the epiphyseal cartilage but continuous with the perichondrial vessels. The labeling was always far more extensive than in the surrounding chondrocytes and was always present throughout the entire extent of the canals. In conclusion, the cell labeling activity strongly supports an active dynamic phenomenon underlying the vascularization of epiphyseal and physeal cartilage.

Estrogen receptor-negative epithelial cells in mouse mammary gland development and growth

The mouse mammary gland undergoes rapid proliferation during puberty, then cyclical proliferation and involution during adulthood within a 5-day estrous cycle. Although proliferation of mammary epithelial cells is directed by elevated serum levels of estrogen acting via the estrogen receptor (ER), the ER status of the proliferating cells remains unknown. We examined the ER expression of proliferating epithelial cell types during pubertal development and normal adult growth using simultaneous immunohistochemistry for ER and tritiated thymidine ( 3H-Tdr) autoradiography. These studies demonstrate that during pubertal growth (4–6 weeks) ER-negative cells comprise more than 50% of the epithelial cell populations in the terminal end buds (TEBs) and ducts. Furthermore, the majority of proliferating cells in both TEBs and ducts are ER-negative. These findings indicate that proliferation of cells within both the TEBs and the mammary ducts contribute to pubertal growth of the mammary gland and that the greater proportion of dividing cells are ER-negative. Similar patterns of cell growth were observed in the normal estrous cycle when the majority of dividing cells were ER-negative during both pro-estrous and estrous. Intensive labelling of cells with 3H-Tdr was used to identify long-lived mammary epithelial cells which retained 3H-Tdr 2 weeks following labelling (i.e., following 3 estrous cycles). Of the small number of mammary epithelial cells retaining 3H-Tdr label, most were ER-positive luminal cells and only a few were ER-negative basal cells. This study indicates that pubertal growth of the mammary gland comprises division of ER-negative cap cells and of both ER-negative and ER-positive cells in the body of the TEBs and elongating mammary ducts. Similarly, estrogen-driven proliferation of ER-negative and ER-positive luminal cells and ER-negative basal cells maintains the differentiated mammary gland in the adult mouse.

The identification of myogenic cells in skeletal muscle, with emphasis on the use of tritiated thymidine autoradiography and desmin antibodies.

The identification of myogenic precursor cells (mpc) is a key factor in determining the early events in the myogenesis and regeneration of skeletal muscle. Although satellite cells have long been established as the providers of myoblastic cells, very little is really known (apart from their anatomical location in relation to muscle fibres and their ability to migrate) about the precise role of satellite cells in myogenesis. Numerous techniques for labelling mpc have been devised, but none of these has proven to be completely reliable in firmly establishing the origin of myogenic cells. The use of tritiated thymidine to label DNA in proliferating mpc (which are not specifically distinguishable at the time) and the subsequent location of their labelled progeny in myotube nuclei has revealed a great deal of data on the timing of myogenesis, but not about the nature of mpc themselves. DNA synthesis can also be detected by antibodies to the thymidine analogue, bromodeoxyuridine, and also by antibody staining for proliferating nuclear cell antigen. Like tritiated thymidine, these other markers are not specific for muscle but are general markers for DNA synthesis. In situ hybridisation of various muscle-specific genetic markers and their products has been informative, as has immunolabelling of myogenin, MyoD1 and desmin. Desmin labelling has been particularly instructive in identifying mpc because it is one of the first muscle-specific proteins to be produced in mpc. This review covers some of the techniques mentioned above and their usefulness in determining the early events in myogenesis.