In the bullfrog (Rana catesbeiana), the process of metamorphosis culminates in the appearance of new visual and visuomotor behaviors reflective of the emergence of binocular vision and visually-guided prey capture behaviors as the animal transitions to life on land. Using several different neuroanatomical tracers, we examined the substrates that may underlie these behavioral changes by tracing the afferent and efferent connectivity of the midbrain optic tectum across metamorphic development. Intratectal, tectotoral, tectotegmental, tectobulbar, and tecto-thalamic tracts exhibit similar trajectories of neurobiotin fiber label across the developmental span from early larval tadpoles to adults. Developmental variability was apparent primarily in intensity and distribution of cell and puncta label in target nuclei. Combined injections of cholera toxin subunit β and Phaseolus vulgaris leucoagglutinin consistently label cell bodies, puncta, or fiber segments bilaterally in midbrain targets including the pretectal gray, laminar nucleus of the torus semicircularis, and the nucleus of the medial longitudinal fasciculus. Developmentally stable label was observed bilaterally in medullary targets including the medial vestibular nucleus, lateral vestibular nucleus, and reticular gray, and in forebrain targets including the posterior and ventromedial nuclei of the thalamus. The nucleus isthmi, cerebellum, lateral line nuclei, medial septum, ventral striatum, and medial pallium show more developmentally variable patterns of connectivity. Our results suggest that even during larval development, the optic tectum contains substrates for integration of visual with auditory, vestibular, and somatosensory cues, as well as for guidance of motivated behaviors.
Medial septum; Metamorphosis; Midbrain; Multisensory convergence; Nucleus isthmi; Optic tectum; Tadpole; Thalamus
On the basis of patterns of anterograde, retrograde, and bi-directional transport of tracers from both the superior olivary nucleus (SON) and the torus semicircularis (TS), we report anatomical changes in brainstem connectivity across metamorphic development in the bullfrog, Rana catesbeiana. In early and late stages of larval development (Gosner stages 25–37), anterograde or bi-directional tracers injected into the SON produce terminal/fiber label in the contralateral SON and in the ipsilateral TS. Between stages 38–41 (deaf period), only sparse or no terminal/fiber label is visible in these target nuclei. During metamorphic climax (stages 42–46), terminal/fiber label reappears in both the contralateral SON and in the ipsilateral TS, and now also in the contralateral TS. Injections of retrograde tracers into the SON fail to label cell bodies in the ipsilateral TS in deaf period animals, mirroring the previously-reported failure of retrograde transport from the TS to the ipsilateral SON during this developmental time. Bilateral cell body label emerges in the dorsal medullary nucleus and the lateral vestibular nucleus bilaterally as a result of SON transport during the late larval period, while cell body label in the contralateral TS emerges during climax. At all larval stages, injections into the SON produce anterograde and retrograde label in the medial vestibular nucleus bilaterally. These data show anatomical stability in some pathways and plasticity in others during larval development, with the most dramatic changes occurring during the deaf period and metamorphic climax. Animals in metamorphic climax show patterns of connectivity similar to that of froglets and adults, indicating the maturation during climax of central anatomical substrates for hearing in air.
Tadpoles; Anurans; Vestibular nucleus complex; Dorsal medullary nucleus; Superior olivary complex; Torus semicircularis; Lipophilic dyes; PHA-L; Cholera toxin; Metamorphosis
We examined the distribution of adult cell proliferation throughout the brain of an anuran amphibian using 5-bromo-2′-deoxyuridine (BrdU). BrdU, a thymidine analog, is a commonly used cellular marker that is incorporated into actively dividing progenitor cells. Adult green treefrogs, Hyla cinerea, received injections of BrdU and were sacrificed 2 hours, 2 days, 2 weeks, or 30 days later. Immunohistochemistry revealed BrdU-immunopositive (BrdU+) cells to be distributed in ventricular zones throughout the brain. The heaviest concentrations of cells were located in the telencephalon, primarily in the ventrolateral region of the lateral ventricles, and the ventricles of olfactory bulbs. Numerous BrdU+ cells were located around the preoptic and hypothalamic recesses and few around the third ventricle in the diencephalon. Proceeding caudally towards the midbrain, there was a marked decrease in BrdU-labeling and few BrdU+ cells were found in the hindbrain. Consistent with previous studies in ectothermic vertebrates, BrdU+ cells were found predominantly in the ventricular zone (VZ) and immediately adjacent to the VZ; at later time points (i.e., 30 days), the cells appeared to have migrated into parenchymal regions. The extent of cellular proliferation in anurans is similar to that of fishes and reptiles and thus is more widespread compared to mammals.
amphibian; BrdU; cell proliferation; cell migration; ventricular zone
Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of 3H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti–androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.
androgen receptor; amphibian; brain; PG-21
Computation of rate in auditory signals is essential to call recognition in anurans. This task is ascribed to a group of central nervous sytem nuclei in the dorsal midbrain or torus semicircularis, homologous to the inferior colliculus of mammals. We have mapped the connections of the subnuclei of the torus semicircularis in Xenopus laevis to determine which receive auditory and which receive lateral line information. Relative to terrestrial anurans, the torus of X. laevis is hypertrophied and occupies the entire caudal, dorsal midbrain. Auditory input to the torus, that arising directly from the dorsal medullary nucleus, is present only in the laminar nucleus. The principal and magnocellular nuclei receive their input from the lateral line nucleus of the medulla. All three nuclei of the torus also have reciprocal connections with the superior olive and the nucleus of the lateral lemniscus. Ascending efferents from all three nuclei of the torus innervate central and lateral thalamic nuclei, and all have a weak reciprocal connection with the posterior thalamus. The laminar and magnocellular nuclei have reciprocal connections with the ventral thalamus, and all three nuclei of the torus receive descending input from the anterior entopeduncular nucleus. The laminar and magnocellular nuclei also receive descending input from the preoptic area. Based on our identification of toral nuclei and these results we assign a major function for the detection of water-borne sounds to the laminar nucleus and a major function for the detection of near field disturbances in water pressure to the principal and magnocellular nuclei.
torus semicircularis; temporal processing; vocalizations; inferior colliculus
FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques.
The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2’-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half day post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 week of chase period, a substantial number of lacrimal gland cells were BrdU+ (11.98 ± 1.84 and 7.95 ± 1.83 BrdU+ cells/mm2, respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU+ cells (0.38 ± 0.06 BrdU+ cells/mm2), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU+ cells/mm2, injured: 2.91 ± 0.62 BrdU+ cells/mm2). Furthermore, during repair, among BrdU+ cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells, and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells.
Progenitor cells; BrdU-label retaining cells; Tissue repair; Lacrimal gland
Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody.
Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental - what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells? - to the detailed - what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25-500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 minutes, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the G2/M phase protein pHisH3 maximally colocalized 8 hr after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 hr. In addition, triple labeling with BrdU and PCNA and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.
BrdU; PCNA; pHisH3; Ki-67; mitosis; neurogenesis
We previously reported that neuronal numbers within adult nodose ganglia (NG) were restored to normal levels 60 days following the capsaicin-induced destruction of nearly half of the neuronal population. However, the nature of this neuronal replacement is not known. Therefore, we aimed to characterize neural proliferation, neurochemical phenotypes, and functional recovery within adult rat NG neurons following capsaicin-induced damage. Sprague-Dawley rats received intraperitoneal injections of capsaicin or vehicle solution, followed by 5-bromo-2-deoxyuridine (BrdU) injections to reveal cellular proliferation. NG were collected at multiple times post-treatment (up to 300 days) and processed for immunofluorescence, RT-PCR, and dispersed cell cultures. Capsaicin-induced cellular proliferation, indicated by BrdU/Ki-67-labeled cells, suggests that lost neurons were replaced through cell division. NG cells expressed the stem cell marker, nestin, indicating that these ganglia have the capacity to generate new neurons. BrdU-incorporation within β-III tubulin-positive neuronal profiles following capsaicin suggests that proliferating cells matured to become neurons. NG neurons displayed decreased NMDAR expression up to 180-days post-capsaicin. However, both NMDAR expression within the NG and synaptophysin expression within the central target of NG neurons, the NTS, were restored to pre-injury levels by 300 days. NG cultures from capsaicin-treated rats contained bipolar neurons, normally found only during development. To test the functional recovery of NG neurons, we injected the satiety molecule, CCK. The effect of CCK on food intake was restored by 300-days post-capsaicin. This restoration may be due to the regeneration of damaged NG neurons or generation of functional neurons that replaced lost connections.
neurogenesis; injury; BrdU; sensory; TRPV1; vagus
Measurements of cell proliferation can be used as biomarkers of preneoplastic change. In this study, two immunocytochemical methods that measure different components of the cell cycle were compared to assess cell proliferation on biopsy samples from human colonic mucosa. These methods are based on a monoclonal antibody against 5-bromo-2-deoxyuridine (BrdU), which is confined to S phase cells, and a more broad assessment of proliferation based on an antibody against proliferating cell nuclear antigen (PCNA, clone 19A2). In the PCNA assay, only strongly immunostained nuclei were included. The proliferation index was assessed in colonic mucosa from patients with no colonic disorders. Correlation between individual total proliferation indices determined by either method was significant with rs = 0.6 (p < 0.05). The mean proliferation index in the study group was 7.79% using BrdU and 7.64% using PCNA immunocytochemistry. Distribution of labelled cells within crypts was similar with respect to the two methods with a peak at the 18th and the 24th percentile in the case of BrdU and at the 23rd percentile for PCNA. Variance component analysis showed that at least two biopsy specimens should be evaluated per subject to allow a precise individual characterisation. It is concluded that PCNA (19A2) immunocytochemistry may be used as an operational marker of cell proliferation in normal colonic mucosa. A significant correlation and an agreement in the mean proliferation index between PCNA (19A2) and BrdU can only be achieved by a strictly standardised enumeration of labelled cells limited to strongly stained nuclei in the PCNA evaluation.
Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellular nuclei was compared in recut tissue sections from animals given BrdU by a single IP injection 2 hr before killing the animals. Ten-week-old male B6C3F1 mice and F344 rats were exposed to known mitogenic hepatocarcinogens, Wy-14,643 (WY) in the diet at 0.1% for 2 days or 1,4-dichlorobenzene (DCB) in corn oil by gavage for 2 days (600 mg/kg/day in mice; 300 mg/kg/day in rats). In mice, PCNA and BrdU hepatocyte LI were similar in control, WY-treated, and DCB-treated animals. In rats, PCNA and BrdU gave similar LI in controls and Wy-treated animals. Although PCNA LI was statistically lower than BrdU LI in DCB-treated rats, both PCNA and BrdU LI for DCB-treated rats was increased over LI in control rats. Different patterns of PCNA immunohistochemical staining, interpreted to represent different subpopulations of cells at various phases of the cell cycle, were quantitated using PCNA immunohistochemistry. The proliferating index (PI), defined as the percentage of cells in the cell cycle (G1 + S + G2 + M), was more sensitive than the LI (S phase only) in detecting a chemically induced cell proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)
Voluntary wheel running activates dentate gyrus granule neurons and increases adult hippocampal neurogenesis. Average daily running distance typically increases over a period of 3 weeks in rodents. Whether neurogenesis and cell activation are greater at the peak of running as compared to the initial escalation period is not known. Therefore, adult C57BL/6J male mice received 5 days of BrdU injections, at the same age, to label dividing cells during the onset of wheel access or after 21 days during peak levels of running or in sedentary conditions. Mice were sampled either 24 hours or 25 days after the last BrdU injection to measure cell proliferation and survival, respectively. Immunohistochemistry was performed on brain sections to identify the numbers of proliferating BrdU labeled cells, and new neurons (BrdU/NeuN co-labeled) in the dentate gyrus. Ki67 was used as an additional mitotic marker. The induction of c-Fos was used to identify neurons activated from running. Mice ran approximately half as far during the first 5 days as compared to after 21 days. Running increased Ki67 cells at the onset but after 21 days levels were similar to sedentary. Numbers of BrdU cells were similar in all groups 24 hours after the final injection. However, after 25 days, running approximately doubled the survival of new neurons born either at the onset or peak of running. These changes co-varied with c-Fos expression. We conclude that sustained running maintains a stable rate of neurogenesis above sedentary via activity-dependent increases in differentiation and survival, not proliferation, of progenitor cells in the C57BL/6J model.
exercise; wheel running; c-Fos; adult hippocampal neurogenesis; granule cell activation; C57BL/6J
The present study was designed to investigate the phenotypic transformation and migration of adventitial fibroblasts using 5-bromo-2′-deoxyuridine (BrdU) labeling following angioplasty and to explore the correlation between adventitial cells and post-angioplasty restenosis. A vascular restenosis model was established in 23 rats by injuring the common carotid artery with a wire. BrDU was used to label the fibroblasts followed by immunohistochemistry for α-actin. Blood vessels were observed under light microscopy and scanning electron microscopy followed by image analysis. The number of BrDU-positive fibroblasts in the intima, media and adventitia of the blood vessels was determined 3, 7, 41 and 28 days after injury. The results demonstrated that at different time points, the number of BrDU-positive cells was significantly different in the intima, media and adventia (P<0.05). Electron microscopy indicated that the fibroblasts were full of cytoplasm. In addition, many secretory granules were noted on the rough endoplasmic reticulum and a large amount of microfilament bundles were noted after angioplasty. The fibroblasts transformed into myofibroblasts. Seven and 14 days after injury, the myofibroblasts formed wide pseudopods stretching to the fenestrae of the external and internal elastic lamina, and cells had a tendency to migrate into the lumen. The fibroblasts in the adventitia underwent transformation after percutaneous transluminal angioplasty and secreted α-actin. In conclusion, the fibroblasts in the adventitia transformed into myofibroblasts, migrated into and proliferated in the intima and became a component of the newly generated intima. Adventitial cells are thus related to vascular restenosis.
angioplasty; vascular intima; myofibroblast; phenotype; migration
Cellular turnover rates in the immune system can be determined by labelling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) or deuterated glucose ((2)H-glucose). To estimate the turnover rate from such measurements one has to fit a particular mathematical model to the data. The biological assumptions underlying various models developed for this purpose are controversial. Here, we fit a series of different models to BrdU data on CD4(+) T cells from SIV(-) and SIV(+) rhesus macaques. We first show that the parameter estimates obtained using these models depend strongly on the details of the model. To resolve this lack of generality we introduce a new parameter for each model, the 'average turnover rate', defined as the cellular death rate averaged over all subpopulations in the model. We show that very different models yield similar estimates of the average turnover rate, i.e. ca. 1% day(-1) in uninfected monkeys and ca. 2% day(-1) in SIV-infected monkeys. Thus, we show that one can use BrdU data from a possibly heterogeneous population of cells to estimate the average turnover rate of that population in a robust manner.
To determine whether or not local, injury-induced aromatization and/orestrogen provision can affect cyto-or neuro-genesis following mechanical brain damage, two groups of adult male zebra finches sustained bilateral penetrating brain injuries. The first received contralateral injections of vehicle or the aromatase inhibitor fadrozole. The second group received contalateral injections of fadrozole, or fadrozole with 17β-estradiol. Subsequent to injury, birds were injected with the thymidine analog 5-Bromo-2′-deoxyuridine (BrdU). Two weeks following injury, the birds were perfused, and coronal sections were labeled using antibodies against BrdU and the neuronal proteins HuC/HuD. In a double blind fashion, BrdU positive cells and BrdU/Hu double-labeled cells in the subventricular zone (SVZ) and at the injury site (INJ) were imaged and sampled. The average numbers of cells per image were compared across brain regions and treatments using repeated measures ANOVAs and, where applicable, post-hoc, pairwise comparisons. Fadrozole administration had no detectable effect on cytogenesis or neurogenesis, however, fadrozole coupled with estradiol significantly increased both measures. The dorsal SVZ had the greatest proportion of new cells that differentiated into neurons, though the highest numbers of BrdU labeled and BrdU, Hu double-labeled cells were detected at the injury site. In the adult zebra finch brain, local estradiol provision can increase cytogenesis and neurogenesis, however, whether or not endogenous glial aromatization is sufficient to similarly affect these processes remains to be seen.
neurogenesis; estrogens; neurosteroids; neuroplasticity; songbirds
The adult hippocampal dentate gyrus (DG) exhibits cell proliferation and neurogenesis throughout life. We examined the effects of daily administration of eszopiclone (Esz), a commonly used hypnotic drug and GABA agonist, compared to vehicle, on DG cell proliferation and neurogenesis, and on sleep-wake patterns. Esz was administered during the usual sleep period of rats, to mimic typical use in humans. Esz treatment for 7 days did not affect the rate of cell proliferation, as measured by 5-bromo-2’-deoxyuridine (BrdU) immunostaining. However, twice daily Esz administration for two weeks increased survival of newborn cells, by 46%. Most surviving cells exhibited a neuronal phenotype, identified BrdU-NeuN double-labeling. NeuN (Neuronal nuclei) is a marker of neurons. NREM sleep was increased on day one, but not on days 7 or 14 of Esz administration. Delta EEG activity was increased on days 1 and 7 of treatment, but not on day 14.
There is evidence that enhancement of DG neurogenesis is a critical component of the effects of antidepressant treatments of major depressive disorder (MDD). Adult born DG cells are responsive to GABAergic stimulation which promotes cell maturation. The present study suggests that Esz, presumably acting as a GABA agonist, has pro-neurogenic effects in the adult DG. This result is consistent with evidence that Esz enhances antidepressant treatment response of MDD patients with insomnia.
sleep; hippocampus; adult neurogenesis; GABA; hypnotic; MDD
AIMS--To examine the correlation between bromodeoxyuridine (BrdU) labelling indices (LI) and tubular damage in renal biopsy specimens; to evaluate the diagnostic and prognostic potential of measuring cell proliferation in a variety of renal lesions. METHODS--In vitro BrdU labelling of renal biopsy specimens was undertaken and labelled cells were detected in routinely fixed, paraffin wax embedded sections by immunohistochemistry. The BrdU LI were calculated as percentages for the three types of tubular cells--proximal and distal convoluted tubules and medulla (LI/PCT, LI/DCT, LI/Med)--and a total tubular BrdU LI (LI/Tub) was also calculated for each biopsy specimen. Histological features indicative of tubular damage were also scored and a total tubular damage score obtained for each biopsy specimen. RESULTS--The one hour labelling process did not affect tissue morphology or impede subsequent diagnosis. Four biopsy specimens were obtained from three renal transplant recipients. Diagnosis of 19 non-transplant biopsy specimens revealed a variety of renal lesions. Total tubular damage scores ranged from 0 to 25 and the LI/Tub ranged from 0 to 3.68% in all 23 biopsy specimens. Analyses of variance showed highly significant correlations between the total tubular damage score and both LI/Tub (p = 0.004) and LI/PCT (p = 0.004); a weaker correlation was found between the total tubular damage score and LI/DCT (p = 0.013). CONCLUSIONS--A correlation was found between tubular damage and BrdU LI. This was most clearly seen in the proximal tubules. However, as the study was limited to a few examples of specific forms of glomerular or interstitial disease, firm conclusions about the value of BrdU labelling in routine diagnosis and prognosis could not be drawn.
Extensive efforts have been made to determine the status on neural progenitor cell proliferation in specific pathological conditions and to evaluate the therapeutic efficacy of drugs for preventing neurogenic deficits in neurodegenerative diseases. However, the most commonly used stereological analysis using 5-bromo-2′-deoxyuridine (BrdU) immuno-positive sections is a time consuming and labor intensive process and is often a bottle neck in neurogenic drug development, particularly when large sample sizes are needed. In addition, BrdU is toxic to new born neurons and also labels DNA damage in old cells. In this study, we established a method that quantitatively measures the number of Ki-67, an endogenous cell proliferation marker, positive cells by flow cytometry which analyzes extracted cell nuclei from rodent hippocampi in suspension. Our results demonstrate that this approach can be applied to a large number of rodent samples, can be accomplished in a short period of time (1-3 days), and can be completed in a more accurately objective manner than by using 3-D cell counting with immunohistochemically processed sections.
Neurogenesis; high throughput screen; hippocampus; flow cytometry; Ki-67
This work explores the distribution of various markers expressed by interstitial cells in rat kidneys after ischemic injury (35 minutes) during regeneration of S3 tubules of outer stripe of outer medulla (OSOM). Groups of experimental animals (n = 4) were sacrificed every two hours during the first 24 hours post-ischemia as well as 2, 3, 7, 14 days post-ischemia. The occurrence of lineage markers was analyzed on kidney sections by immunohistochemistry and morphometry during the process of tubular regeneration. In postischemic kidneys, interstitial cell proliferation, assessed by 5-bromo-2′-deoxyuridine (BrdU) and Proliferating Cell Nuclear Antigen (PCNA) labeling, was prominent in outer medulla and reach a maximum between 24 and 72 hours after reperfusion. This population was characterized by the coexpression of vimentin and nestin. The density of -Neural Cell Adhesion Molecule (NCAM) positive interstitial cells increased transiently (18–72 hours) in the vicinity of altered tubules. We have also localized a small population of α-Smooth Muscle Actin (SMA)-positive cells confined to chronically altered areas and characterized by a small proliferative index. In conclusion, we observed in the postischemic kidney a marked proliferation of interstitial cells that underwent transient phenotypical modifications. These interstitial cells could be implicated in processes leading to renal fibrosis.
Retinal Müller glia in higher vertebrates have been reported to possess progenitor cell properties and the ability to generate new neurons after injury. This study was conducted to determine the signals that can activate this dormant capacity of Müller glia in adult mice, by studying their behavior during glutamate stimulation.
Various concentrations of glutamate and its analogue α-aminoadipate, which specifically binds Müller glia, were injected subretinally in adult mice. Proliferating retinal cells were labeled by subretinal injection of 5′-bromo-2′-deoxyuridine (BrdU) followed by immunohistochemistry. Müller cell fates were analyzed in retinal sections by using double immunolabeling with primary antibodies against Müller and other retinaspecific cell markers. The effects of glutamate and α-aminoadipate were also determined in purified Müller cell cultures.
Although high levels of glutamate induce retinal damage, subtoxic levels of glutamate directly stimulate Müller glia to re-enter the cell cycle and induce neurogenesis in vivo and in purified Müller cell cultures. α-Aminoadipate, which selectively target glial cells, also induced expression of progenitor cell markers by Müller cells in vitro or stimulated Müller cell migration to the outer nuclear layer (ONL) and to differentiate into photoreceptors in vivo.
Mature Müller glia in adult mice can be induced to dedifferentiate, migrate, and generate new retinal neurons and photoreceptor cells by α-aminoadipate or glutamate signaling. The results of this study suggest a novel potential strategy for treating retinal neurodegeneration, including retinitis pigmentosa and age-related macular degeneration, without transplanting exogenous cells.
The purpose of this study is to investigate the mechanism of alternative responses to low dose irradiation for neuronal cell proliferation in the dentate gyrus of rats. To determine the effect of a single exposure to radiation, rats were irradiated with a single dose of 0.1, 1, 10 or 20 Gy. To determine the effect of the cumulative dose, the animals were irradiated daily with 0.01 Gy or 0.1 Gy from 1 to 4 days. The neuronal cell proliferation was evaluated using immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU), Ki-67 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. Four consecutive daily irradiations with a 0.01 Gy/fraction increased the number of BrdU-positive and Ki-67-positive cells in a dose dependent manner, but this did not affect the number of TUNEL-positive cells. However, there was not a dose dependent relationship for the 0.1 Gy/fraction irradiation with the number of BrdU, Ki-67 and TUNEL positive cells. Our data support the explanation that the adaptive response, induced by low-dose radiation, in the hippocampus of rats is more likely a reflection of the perturbations of cell cycle progression.
Adaptive Response; Radiation Effects; Hippocampus Neurons
Astrocytes and their precursors respond to spinal cord injury (SCI) by proliferating, migrating, and altering phenotype. This contributes to glial scar formation at the lesion border and gliosis in spared gray and white matter. The present study was undertaken to evaluate astrocyte changes over time and determine when and where interventions might be targeted to alter the astrocyte response. Bromodeoxyuridine (BrdU) was administered to mice three days after SCI, and cells expressing BrdU and the astrocyte marker, glial fibrillary acidic protein (GFAP), were counted at 3, 7, and 49 days post injury (DPI). BrdU-labeled cells accumulated at the lesion border by 7 DPI and approximately half of these expressed GFAP. In spared white matter, the total number of BrdU+ cells decreased, while the percentage of BrdU+ cells expressing GFAP increased at 49 DPI. Phenotypic changes were examined using the progenitor marker, nestin, the radial glial marker, brain lipid binding protein (BLBP), and GFAP. Nestin was upregulated by 3 DPI and declined between 7 and 49 DPI in all regions, and GFAP increased and remained above naïve levels at all time points. BLBP increased early and remained high along the lesion border and spared white matter, but was expressed transiently by cells lining the central canal and in a unique population of small cells found within the lesion and in gray matter rostral and caudal to the border. The results demonstrate that the astrocyte response to SCI is regionally heterogeneous, and suggests astrocyte populations which could be targeted by interventions.
progenitor cell; central canal; nestin; brain lipid binding protein
AIM: To examine the influence of ghrelin on the regenerative potential of gastrointestinal (GI) epithelium.
METHODS: Damage to GI epithelium was induced in mice by two intravenous injections of doxorubicin (10 and 6 mg/kg). Some of the doxorubicin-treated mice received a continuous subcutaneous infusion of ghrelin (1.25 μg/h) for 10 d via implanted mini-osmotic pumps. To label dividing stem cells in the S-phase of the cell cycle, all mice received a single intraperitoneal injection of 5’-bromo-2’-deoxyuridine (BrdU) one hour before sacrifice. The stomach along with the duodenum were then removed and processed for histological examination and immunohistochemistry using anti-BrdU antibody.
RESULTS: The results showed dramatic damage to the GI epithelium 3 d after administration of chemotherapy which began to recover by day 10. In ghrelin-treated mice, attenuation of GI mucosal damage was evident in the tissues examined post-chemotherapy. Immunohistochemical analysis showed an increase in the number of BrdU-labeled cells and an alteration in their distribution along the epithelial lining in response to damage by doxorubicin. In mice treated with both doxorubicin and ghrelin, the number of BrdU-labeled cells was reduced when compared with mice treated with doxorubicin alone.
CONCLUSION: The present study suggests that ghrelin enhances the regenerative potential of the GI epithelium in doxorubicin-treated mice, at least in part, by modulating cell proliferation.
Gastrointestinal cell proliferation; Gastro-intestinal mucosal damage; Ghrelin
The period of complex signals is encoded in the bullfrog’s eighth nerve by a synchrony code based on phase-locked responding. We examined how these arrays of phase-locked activity are represented in different subnuclei of the auditory midbrain, the torus semicircularis (TS). Recording sites in different areas of the TS differ in their ability to synchronize to the envelope of complex stimuli, and these differences in synchronous activity are related to response latency. Cells in the caudal principal nucleus (cell sparse zone) have longer latencies, and show little or no phase-locked activity, even in response to low modulation rates, while some cells in lateral areas of the TS (magnocellular nucleus, lateral part of principal nucleus) synchronize to rates as high as 90–100 Hz. At midlevels of the TS, there is a lateral-to-medial gradient of synchronization ability: cells located more laterally show better phase-locking than those located more medially. Pooled all-order interval histograms from short latency cells located in the lateral TS represent the waveform periodicity of a biologically relevant complex harmonic signal at different stimulus levels, and in a manner consistent with behavioral data from vocalizing male frogs. Long latency cells in the caudal parts of the TS (cell sparse zone, caudal magnocellular nucleus) code stimulus period by changes in spike rate, rather than by changes in synchronized activity. These data suggest that neural codes based on rate processing and time domain processing are represented in anatomically different areas of the TS. They further show that a population-based analysis can increase the precision with which temporal features are represented in the central auditory system.
auditory; midbrain; temporal coding; periodicity coding; synchrony; complex sounds