Tabela com principais espec.
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|A||FC, ICC, IHC||M||Purified||Monoclonal Antibody|
|Presentation||Liquid in 10 mM Phosphate buffer, pH 7.4 containing 150 mM NaCl and 0.1% sodium azide|
|Safety Information according to GHS|
|Material Size||50 µg|
Referências | 33 Disponível | Ver todas as referências
|Visão geral das referências||Aplicação||Pub Med ID|
|Hemodynamic forces regulate developmental patterning of atrial conduction. |
Bressan, MC; Louie, JD; Mikawa, T
PloS one 9 e115207 2014
Anomalous action potential conduction through the atrial chambers of the heart can lead to severe cardiac arrhythmia. To date, however, little is known regarding the mechanisms that pattern proper atrial conduction during development. Here we demonstrate that atrial muscle functionally diversifies into at least two heterogeneous subtypes, thin-walled myocardium and rapidly conducting muscle bundles, during a developmental window just following cardiac looping. During this process, atrial muscle bundles become enriched for the fast conduction markers Cx40 and Nav1.5, similar to the precursors of the fast conduction Purkinje fiber network located within the trabeculae of the ventricles. In contrast to the ventricular trabeculae, however, atrial muscle bundles display an increased proliferation rate when compared to the surrounding myocardium. Interestingly, mechanical loading of the embryonic atrial muscle resulted in an induction of Cx40, Nav1.5 and the cell cycle marker Cyclin D1, while decreasing atrial pressure via in vivo ligation of the vitelline blood vessels results in decreased atrial conduction velocity. Taken together, these data establish a novel model for atrial conduction patterning, whereby hemodynamic stretch coordinately induces proliferation and fast conduction marker expression, which in turn promotes the formation of large diameter muscle bundles to serve as preferential routes of conduction.
|Inducing and characterizing liver regeneration in mice: Reliable models, essential "readouts" and critical perspectives. |
Mastellos, DC; Deangelis, RA; Lambris, JD
Current protocols in mouse biology 3 141-170 2013
Elucidating the molecular circuitry that regulates regenerative responses in mammals has recently attracted considerable attention because of its emerging impact on modern bioengineering, tissue replacement technologies, and organ transplantation. The liver is one of the few organs of the adult body that exhibitsa prominent regenerative capacity in response to toxic injury, viral infection, or surgical resection. Over the years, mechanistic insights into the liver's regenerative potential have been provided by rodent models of chemical liver injury or surgical resection that faithfully recapitulate hallmarks of human pathophysiology and trigger robust hepatocyte proliferation leading to organ restoration. The advent of mouse transgenics has undeniably catalyzed the wider application of such models for researching liver pathobiology. This article provides a comprehensive overview of the most reliable and widely applied murine models of liver regeneration and also discusses helpful hints, considerations, and limitations related to the use of these models in liver regeneration studies.
|Periadolescent ethanol vapor exposure persistently reduces measures of hippocampal neurogenesis that are associated with behavioral outcomes in adulthood. |
Ehlers, CL; Liu, W; Wills, DN; Crews, FT
Neuroscience 244 1-15 2013
Excessive alcohol consumption is prevalent among adolescents and may result in lasting neurobehavioral consequences. The use of animal models to study adolescent alcohol exposure has the advantage of allowing for the control necessary in order to evaluate the effects of ethanol on the brain and separate such effects from genetic background and other environmental insults. In the present study the effects of moderate ethanol vapor exposure, during adolescence, on measures of neurogenesis and behavioral measures were evaluated at two different times following ethanol withdrawal, in adulthood. The two groups of Wistar rats were both exposed to intermittent ethanol vapor (14 h on/10h off/day) for 35-36 days from PD 23 to PD 58 (average blood ethanol concentration: 163 mg%). In the first group, after rats were withdrawn from vapor they were subsequently assessed for locomotor activity, conflict behavior in the open field, and behaviors in the forced swim test (FST) and then sacrificed at 72 days of age. The second group of rats were withdrawn from vapor and injected for 5 days with Bromo-deoxy-Uridine (BrdU). Over the next 8 weeks they were also assessed for locomotor activity, conflict behavior in the open field, and behaviors in the FST and then sacrificed at 113/114 days of age. All rats were perfused for histochemical analyses. Ethanol vapor-exposed rats displayed hypoactivity in tests of locomotion and less anxiety-like and/or more "disinhibitory" behavior in the open field conflict. Quantitative analyses of immunoreactivity revealed a significant reduction in measures of neurogenesis, progenitor proliferation, as indexed by doublecortin (DCX), Ki67, and increased markers of cell death as indexed by cleaved caspase-3, and Fluoro-Jade at 72 days, and decreases in DCX, and increases in cleaved caspase-3 at 114 days in the ethanol vapor-exposed rats. Progenitor survival, as assessed by BrdU+, was reduced in the vapor-exposed animals that were sacrificed at 114 days. The reduction seen in DCX labeled in cell counts was significantly correlated with hypoactivity at 24h after withdrawal as well as less anxiety-like and/or more "disinhibitory" behavior in the open field conflict test at 2 and 8 weeks following termination of vapor exposure. These studies demonstrate that behavioral measures of disinhibitory behavior correlated with decreases in neurogenesis are all significantly and persistently impacted by periadolescent ethanol exposure and withdrawal in Wistar rats.
|Delayed hyperbaric oxygen therapy induces cell proliferation through stabilization of cAMP responsive element binding protein in the rat model of MCAo-induced ischemic brain injury. |
Mu, J; Ostrowski, RP; Soejima, Y; Rolland, WB; Krafft, PR; Tang, J; Zhang, JH
Neurobiology of disease 51 133-43 2013
Treatments that could extend the therapeutic window of opportunity for stroke patients are urgently needed. Early administration of hyperbaric oxygen therapy (HBOT) has been proven neuroprotective in the middle cerebral artery occlusion (MCAo) in rodents. Our aim was to determine: 1) whether delayed HBOT after permanent MCAo (pMCAo) can still convey neuroprotection and restorative cell proliferation, and 2) whether these beneficial effects rely on HBO-induced activation of protein phosphatase-1γ (PP1-γ) leading to a decreased phosphorylation and ubiquitination of CREB and hence its stabilization. The experiments were performed in one hundred thirty-two male Sprague-Dawley rats with the body weight ranging from 240 to 270 g. Permanent MCAo was induced with the intraluminal filament occluding the right middle cerebral artery (MCA). In the first experiment, HBOT (2.5 ATA, 1h daily for 10 days) was started 48 h after pMCAo. Neurobehavioral deficits and infarct size as well as cyclic AMP response element-binding protein (CREB) expression and BrdU-DAB staining in the hippocampus and the peri-infarct region were evaluated on day 14 and day 28 post-MCAo. In the second experiment, HBOT (2.5 ATA, 1h) was started 3h after pMCAo. The effects of CREB siRNA or PP1-γ siRNA on HBO-induced infarct size alterations and target protein expression were studied. HBOT started with 48 h delay reduced infarct size, ameliorated neurobehavioral deficits and increased protein expression of CREB, resulting in increased cell proliferations in the hippocampus and peri-infarct region, on day 14 and day 28 post-MCAo. In the acute experiment pMCAo resulted in cerebral infarction and functional deterioration and reduced brain expression of PP1-γ, which led to increased phosphorylation and ubiquitination of CREB 24h after MCAo. However HBOT administered 3h after ischemia reversed these molecular events and resulted in CREB stabilization, infarct size reduction and neurobehavioral improvement. Gene silencing with CREB siRNA or PP1-γ siRNA reduced acute beneficial effects of HBO. In conclusion, delayed daily HBOT presented as potent neuroprotectant in pMCAo rats, increased CREB expression and signaling activity, and bolstered regenerative type cell proliferation in the injured brain. As shown in the acute experiment these effects of HBO were likely to be mediated by reducing ubiquitin-dependent CREB degradation owing to HBO-induced activation of PP1γ.
|Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells. |
Huh, YH; Cohen, J; Sherley, JL
Proceedings of the National Academy of Sciences of the United States of America 110 16862-7 2013
Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.
|Effect of rosiglitazone on capillary density and angiogenesis in adipose tissue of normoglycaemic humans in a randomised controlled trial. |
O Gealekman,N Guseva,K Gurav,A Gusev,C Hartigan,M Thompson,S Malkani,S Corvera
Diabetologia 55 2012
Recent reports of decreased capillary density in the adipose tissue of obese individuals suggest that an imbalance of angiogenesis and adipogenesis may, in part, underlie insulin resistance. This study aimed to determine whether the insulin-sensitising peroxisome proliferator-activated receptor γ (PPARγ) activator rosiglitazone affects adipose tissue vascularisation in normal humans.
|Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1. |
Li, J; Jiang, TX; Hughes, MW; Wu, P; Yu, J; Widelitz, RB; Fan, G; Chuong, CM
The Journal of investigative dermatology 132 2681-90 2012
To examine the roles of epigenetic modulation on hair follicle regeneration, we generated mice with a K14-Cre-mediated loss of DNA methyltransferase 1 (DNMT1). The mutant shows an uneven epidermal thickness and alterations in hair follicle size. When formed, hair follicle architecture and differentiation appear normal. Hair subtypes exist but hair fibers are shorter and thinner. Hair numbers appear normal at birth but gradually decrease to less than 50% of control in 1-year-old mice. Sections of old mutant skin show follicles in prolonged telogen with hyperplastic sebaceous glands. Anagen follicles in mutants exhibit decreased proliferation and increased apoptosis in matrix transient-amplifying cells. Although K15-positive stem cells in the mutant bulge are comparable in number to the control, their ability to proliferate and become activated to form a hair germ is reduced. As mice age, residual DNMT activity declines further, and the probability of successful anagen reentry decreases, leading to progressive alopecia. Paradoxically, there is increased proliferation in the epidermis, which also shows aberrant differentiation. These results highlight the importance of DNA methylation in maintaining stem cell homeostasis during the development and regeneration of ectodermal organs.
|Molecular mechanisms of basic fibroblast growth factor effect on healing of ulcerative colitis in rats. |
Paunovic, B; Deng, X; Khomenko, T; Ahluwalia, A; Tolstanova, G; Tarnawski, A; Szabo, S; Sandor, Z
The Journal of pharmacology and experimental therapeutics 339 430-7 2011
We demonstrated previously that basic fibroblast growth factor (bFGF) accelerated the healing of experimental duodenal ulcers, and we now hypothesize that bFGF might also accelerate the healing of experimental ulcerative colitis (UC). We also explored the potential molecular mechanisms involved in the accelerated healing of UC in rats treated with bFGF. The results demonstrated that colonic lesions were significantly reduced by bFGF treatment, whereas neutralization of bFGF aggravated iodoacetamide-induced UC. Protein expression of bFGF was increased during the healing stage of UC. Tumor necrosis factor-α levels and myeloperoxidase activity were significantly decreased in UC rats treated with bFGF, whereas they increased in rats treated with anti-bFGF antibody. Real-time polymerase chain reaction and immunohistochemistry showed decreased levels of p27 in the UC rats compared with the healthy controls, which was reversed by bFGF treatment in a dose-dependent manner. By immunohistochemistry and double labeling of Ki-67 and CD34, prominent positive staining of Ki-67 and CD34 was seen after bFGF treatment, indicating the enhanced proliferation of fibroblasts and epithelial and endothelial cells, i.e., angiogenesis. We conclude that bFGF plays a beneficial role in the healing of UC in rats. The molecular mechanisms of bFGF in UC healing not only involve the expected increased cell proliferation, especially angiogenesis, but also encompass the reduction of inflammatory cytokines and infiltration of inflammatory cells. Thus, bFGF enema may be a new therapeutic option for UC.
|Effects of MDMA (ecstasy) during adolescence on place conditioning and hippocampal neurogenesis. |
Briony J Catlow,Kimberly A Badanich,Ashley E Sponaugle,Amanda R Rowe,Shijie Song,Igor Rafalovich,Vasyl Sava,Cheryl L Kirstein,Juan Sanchez-Ramos
European journal of pharmacology 628 2010
The use of 3,4,methylenedioxymethamphetamine (MDMA), the active agent in ecstasy, during adolescence is widespread yet the effects on adolescent behavior and brain development are unknown. The aim of the present study was 1) to evaluate effects of MDMA in adolescent rats using the conditioned place preference (CPP) paradigm to measure MDMA-induced reward and 2) assess effects of MDMA administration on cellular proliferation, survival and neurogenesis in the dentate gyrus of the hippocampus. During the adolescent period, MDMA CPP was measured in adolescents [postnatal day (PND) 28-39] by training rats to associate 1.25, 2.5, 5.0mg/kg MDMA or saline administration with environmental cues. After CPP ended, bromodeoxyuridine (BrdU) was injected and rats were euthanized either 24h (to evaluate cell proliferation) or 2 weeks (to assess neurogenesis) after the last MDMA injection. Adolescents expressed a CPP for 2.5mg/kg MDMA. Repeated exposure to 5.0mg/kg MDMA during adolescence increased cell proliferation, yet diminished neurogenesis, an effect that was replicated using flow cytometry. These findings suggest differential dose effects of adolescent MDMA exposure on reward related behaviors and hippocampal neurogenesis.
|Long-term suppression of forebrain neurogenesis and loss of neuronal progenitor cells following prolonged alcohol dependence in rats. |
Hansson AC, Nixon K, Rimondini R, Damadzic R, Sommer WH, Eskay R, Crews FT, Heilig M
Int J Neuropsychopharmacol 2010
Alcohol dependence leads to persistent neuroadaptations, potentially related to structural plasticity. Previous work has shown that hippocampal neurogenesis is modulated by alcohol, but effects of chronic alcohol on neurogenesis in the forebrain subventricular zone (SVZ) have not been reported. Effects in this region may be relevant for the impairments in olfactory discrimination present in alcoholism. Here, we examined the effects of prolonged alcohol dependence on neurogenesis. Rats were sacrificed directly after 7 wk of intermittent alcohol vapour exposure, or 3, 7 or 21 d into abstinence. Proliferation was assessed using BrdU and Ki67 immunoreactivity, newly differentiated neurons (neurogenesis) as doublecortin-immunoreactivity (DCX-IR), and neural stem cells using the SOX2 marker. In the dentate gyrus, chronic dependence resulted in a pattern similar to that previously reported for acute alcohol exposure: proliferation and neurogenesis were suppressed by the end of exposure, rebounded on day 3 of abstinence, and returned to control levels by days 7 and 21. In the SVZ, proliferation was also suppressed at the end of alcohol exposure, followed by a proliferation burst 3 d into abstinence. However, in this area, there was a trend for reduced proliferation on days 7 and 21 of abstinence, and this was accompanied by significant suppression of DCX-IR, indicating a long-term suppression of forebrain neurogenesis. Finally, a decrease in the SOX2 stem cell marker was detected at days 7 and 21, suggesting long-term reduction of the SVZ stem cell pool. While suppression of hippocampal neurogenesis by alcohol dependence is transient, the suppression in the forebrain SVZ appears long-lasting.
|c-myc and N-myc promote active stem cell metabolism and cycling as architects of the developing brain. |
Alice Wey,Paul S Knoepfler
Oncotarget 1 2010
myc genes are associated with a wide variety of human cancers including most types of nervous system tumors. While the mechanisms by which myc overexpression causes tumorigenesis are multifaceted and have yet to be clearly elucidated, they are at least in part related to endogenous myc function in normal cells. Knockout (KO) of either c-myc or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre impairs mouse brain growth and mutation of N-myc also causes microcephaly in humans in Feingold Syndrome. To further define myc function in NSC and nervous system development, we created a double KO (DKO) for c- and N-myc using nestin-cre. The DKO mice display profoundly impaired overall brain growth associated with decreased cell cycling and migration of NSC, which are strikingly decreased in number. The DKO brain also exhibits specific changes in gene expression including downregulation of genes involved in protein and nucleotide metabolism, mitosis, and chromatin structure as well as upregulation of genes associated with differentiation. Together these data support a model of nervous system tumorigenesis in which excess myc aberrantly locks in a developmentally active chromatin state characterized by overactive cell cycling, and metabolism as well as blocked differentiation.
|c- and N-myc regulate neural precursor cell fate, cell cycle, and metabolism to direct cerebellar development. |
Wey, A; Martinez Cerdeno, V; Pleasure, D; Knoepfler, PS
Cerebellum (London, England) 9 537-47 2010
Separate murine knockout (KO) of either c- or N-myc genes in neural stem and precursor cells (NSC) driven by nestin-cre causes microcephaly. The cerebellum is particularly affected in the N-myc KO, leading to a strong reduction in cerebellar granule neural progenitors (CGNP) and mature granule neurons. In humans, mutation of N-myc also causes microcephaly in Feingold Syndrome. We created a double KO (DKO) of c- and N-myc using nestin-cre, which strongly impairs brain growth, particularly that of the cerebellum. Granule neurons were almost absent from the Myc DKO cerebellum, and other cell types were relatively overrepresented, including astroglia, oligodendrocytes, and Purkinje neurons. These findings are indicative of a profound disruption of cell fate of cerebellar stem and precursors. DKO Purkinje neurons were strikingly lacking in normal arborization. Inhibitory neurons were ectopic and exhibited very abnormal GAD67 staining patterns. Also consistent with altered cell fate, the adult DKO cerebellum still retained a residual external germinal layer (EGL). CGNP in the DKO EGL were almost uniformly NeuN and p27KIP1 positive as well as negative for Math1 and BrdU at the peak of normal cerebellar proliferation at P6. The presence of some mitotic CGNP in the absence of S phase cells suggests a possible arrest in M phase. CGNP and NSC metabolism also was affected by loss of Myc as DKO cells exhibited weak nucleolin staining. Together these findings indicate that c- and N-Myc direct cerebellar development by maintaining CGNP and NSC populations through inhibiting differentiation as well as directing rapid cell cycling and active cellular metabolism.
|Type 1 insulin-like growth factor receptor signaling is essential for the development of the hippocampal formation and dentate gyrus. |
Liu W, Ye P, O'Kusky JR, D'Ercole AJ
J Neurosci Res 87 2821-32. 2009
Type 1 insulin-like growth factor receptor (IGF1R) signaling in neuronal development was studied in mutant mice with blunted igf1r gene expression in nestin-expressing neuronal precursors. At birth [postnatal (P) day 0] brain weights were reduced to 37% and 56% of controls in mice homozygous (nes-igf1r(-/-)) and heterozygous (nes-igf1r(-/Wt)) for the null mutation, respectively, and this brain growth retardation persisted postnatally. Stereological analysis demonstrated that the volumes of the hippocampal formation, CA fields 1-3, dentate gyrus (DG), and DG granule cell layer (GCL) were decreased by 44-54% at P0 and further by 65-69% at P90 in nes-igf1r(-/Wt) mice. In nes-igf1r(-/-) mice, volumes were 29-31% of controls at P0 and, in the two mice that survived to P90, 6-19% of controls, although the hilus could not be identified. Neuron density did not differ among the mice at any age studied; therefore, decreased volumes were due to reduced cell number. In postnatal nes-igf1r(-/Wt) mice, the percentage of apoptotic cells, as judged by activated caspase-3 immunostaining, was increased by 3.5-5.3-fold. The total number of proliferating DG progenitors (labeled by BrdU incorporation and Ki67 staining) was reduced by approximately 50%, but the percentage of these cells was similar to the percentages in littermate controls. These findings suggest that 1) the postnatal reduction in DG size is due predominantly to cell death, pointing to the importance of the IGF1R in regulating postnatal apoptosis, 2) surviving DG progenitors remain capable of proliferation despite reduced IGF1R expression, and 3) IGF1R signaling is necessary for normal embryonic brain development.
|In vivo expansion of the mammary stem/ progenitor cell population by xanthosine infusion. |
Anthony V Capuco, Christina M Evock-Clover, Andrea Minuti, David L Wood, Anthony V Capuco, Christina M Evock-Clover, Andrea Minuti, David L Wood, Anthony V Capuco, Christina M Evock-Clover, Andrea Minuti, David L Wood
Experimental biology and medicine (Maywood, N.J.) 234 475-82 2009
Mammary stem cells provide for growth and maintenance of the mammary gland and are therefore of considerable interest as determinants of productivity and efficiency of dairy animals and as targets of carcinogenesis in humans. Xanthosine treatment was previously shown to promote expansion of hepatic stem cells in vitro. The objective of this study was to determine if in vivo treatment with xanthosine can increase the mammary stem cell population. Xanthosine was infused into the right mammary glands of four female Holstein calves for 5 consecutive days. Immediately after each xanthosine treatment, calves were injected intravenously with 5-bromo-2-deoxyuridine (BrdU). Forty days after the final treatment, calves were euthanized and mammary tissue harvested. BrdU-label retaining epithelial cells (LREC) were detected immunohistochemically and quantified. Retention of BrdU was used as a marker for putative bovine mammary stem cells. Infusion of xanthosine into the bovine mammary gland significantly increased the number of LREC in treated glands compared to contralateral control glands (P 0.05). LREC averaged 0.4% of epithelial cells in control glands and 0.8% in xanthosine-treated glands. The increase in LREC in xanthosine-treated glands was supported by a concomitant increase in telomerase activity (P 0.01) and a correlation between LREC and telomerase (P 0.05; r (2) = 0.7). Data indicate that in vivo treatment with xanthosine can be used to increase the number of mammary stem cells. This is the first demonstration of an in vivo treatment to increase the endogenous population of mammary stem cells, with utility for biomedical research and dairy management.
|Recruitment of podocytes from glomerular parietal epithelial cells. |
Appel, D; Kershaw, DB; Smeets, B; Yuan, G; Fuss, A; Frye, B; Elger, M; Kriz, W; Floege, J; Moeller, MJ
Journal of the American Society of Nephrology : JASN 20 333-43 2009
Loss of a critical number of podocytes from the glomerular tuft leads to glomerulosclerosis. Even in health, some podocytes are lost into the urine. Because podocytes themselves cannot regenerate, we postulated that glomerular parietal epithelial cells (PECs), which proliferate throughout life and adjoin podocytes, may migrate to the glomerular tuft and differentiate into podocytes. Here, we describe transitional cells at the glomerular vascular stalk that exhibit features of both PECs and podocytes. Metabolic labeling in juvenile rats suggested that PECs migrate to become podocytes. To prove this, we generated triple-transgenic mice that allowed specific and irreversible labeling of PECs upon administration of doxycycline. PECs were followed in juvenile mice beginning from either postnatal day 5 or after nephrogenesis had ceased at postnatal day 10. In both cases, the number of genetically labeled cells increased over time. All genetically labeled cells coexpressed podocyte marker proteins. In conclusion, we demonstrate for the first time recruitment of podocytes from PECs in juvenile mice. Unraveling the mechanisms of PEC recruitment onto the glomerular tuft may lead to novel therapeutic approaches to renal injury.Texto completo do artigo
|Abstinence from moderate alcohol self-administration alters progenitor cell proliferation and differentiation in multiple brain regions of male and female P rats. |
Jun He, David H Overstreet, Fulton T Crews
Alcoholism, clinical and experimental research 33 129-38 2008
BACKGROUND: Acute and chronic ethanol exposure has been found to decrease hippocampal neurogenesis, reduce dendritic differentiation of new neurons, and increase cell death. Interestingly, abstinence from such treatment increases hippocampal neurogenesis and microglial genesis across several brain regions. The goal of the current investigation was to study cellular alterations on neuro- and cell-genesis during abstinence following alcohol self-administration using alcohol-preferring rats (P rats). METHODS: Male and female P rats were given the choice of drinking 10% alcohol in water or pure water for 7 weeks. Social interaction behavioral assessments were conducted at 5 hours upon removal of alcohol, followed by bromo-deoxyuridine (BrdU, 150 mg/kg x 1/d x 14 d) injections to label proliferating cells. Animals were then killed 4 weeks later to conduct immunohistochemical and confocal analyses using antibodies against BrdU and other phenotypic markers (NeuN for mature neurons; Iba-1 for microglia; GFAP for astrocytes; and NG(2) for oligodendrocyte progenitors). RESULTS: Mild alcohol withdrawal anxiety was detected by reduction in social interactions. The number of hippocampal BrdU(+) cells was increased approximately 50% during alcohol abstinence (26 +/- 2.8 in controls vs. 39 +/- 4 in alcohol group). BrdU(+) cells were also increased in the substantia nigra (SN) approximately 65% in the alcohol abstinent group (12 +/- 1 in controls vs. 19 +/- 1.5 in alcohol group). No gender differences were found. Confocal analyses indicated that approximately 75% of co-localization of BrdU(+) cells with NeuN in the hippocampal dentate gyrus (DG) resulting a net increase in neurogenesis in the alcohol abstinent group compared to controls. In cingulum, greater proportion of BrdU(+) cells were co-localized with NG(2) in the alcohol abstinent group indicating increased differentiation toward oligodendrocyte progenitors in both genders. However, the phenotype of the BrdU(+) cells in SN and other brain regions were not identified by NeuN, Iba-1, GFAP, or NG(2) suggesting that these BrdU(+) cells probably remain in a nondifferentiated stage. CONCLUSIONS: These data indicate that abstinence from moderate alcohol drinking increases hippocampal neurogenesis, cingulate NG(2) differentiation, and SN undifferentiated cell proliferation in both males and females. Such cellular alteration during abstinence could contribute to the spontaneous partial restoration of cognitive deficits upon sobriety.
|Effects of dietary supplementation with flax during prepuberty on fatty acid profile, mammogenesis, and bone resorption in gilts. |
Farmer, C; Petit, HV; Weiler, H; Capuco, AV
Journal of animal science 85 1675-86 2007
The possible role of dietary flax on pre-pubertal development of mammary glands and bone resorption was investigated in gilts. Fifty-seven gilts were fed 1 of 4 diets from 88 d of age until slaughter (d 212 +/- 1). Diets were control without flax (n = 14); 10% flaxseed supplementation (n = 13); 6.5% flaxseed meal supplementation (n = 15); and 3.5% flaxseed oil supplementation (n = 15). All diets were isonitrogenous and isocaloric. Jugular blood samples were obtained on d 78 and 210 to establish the fatty acid profile and to determine the concentrations of prolactin, estradiol, and cross-linked N-telopeptides of type I collagen. At slaughter, the mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and the composition of the parenchymal tissue (protein, fat, DM, and DNA) was determined. Histochemical analyses of the mammary parenchyma were performed, and fatty acid profiles in the extraparenchymal tissue were evaluated. Dietary flax increased (P less than or = 0.001) the concentrations of PUFA and decreased those of SFA (P less than 0.01) and MUFA (P less than or = 0.001) in plasma and extraparenchymal tissues, which was largely due to the inclusion of 10% flaxseed or 3.5% flaxseed oil (P less than or = 0.01) but not 6.5% flaxseed meal. Circulating concentrations of prolactin and estradiol were unaltered by treatments (P greater than 0.1), but concentrations of cross-linked N-telopeptides of type I collagen tended to be greater (P less than 0.1) in flax-supplemented gilts. The DM content of parenchymal tissue was the only mammary compositional value affected, showing an increase with flax addition (P less than 0.05). No change (P greater than or = 0.1) in the bromodeoxyuridine labeling index or estrogen receptor localization was observed with treatments. Dietary supplementation with flax as seed, meal, or oil, therefore, brought about the expected changes in the fatty acid profile but had no beneficial effects on mammary development or bone resorption.
|Cell proliferation measurement in cecum and colon of rats using scanned images and fully automated image analysis: validation of method. |
E Persohn,W Seewald,J Bauer,J Schreiber
Experimental and toxicologic pathology : official journal of the Gesellschaft für Toxikologische Pathologie 58 2007
The purpose of this study was to establish and validate fully automatic measurement of cell proliferation on scanned images of rat cecum and colon. Tissue slides were taken from a 4-week mechanistic study and processed for BrdU immunohistochemistry. Four sections of the cecum and colon per slide were scanned with the Zeiss MIRAX SCAN and transferred to the Definiens eCognition Analyst LS5.0 system for evaluation. Two rule sets for automatic counting of BrdU-positive and negative nuclei from mucosal cells on the image tiles were created by Definiens, one for cecum, one for colon. For validation, manual counting of 16 randomly selected tiles from five different slides of colon and cecum was performed. Negative and positive cell nuclei were counted in each image tile by four different people. Comparison of results from manual counting with the automatic counting showed that the sum as well as single tile data and labeling index (LI) from automatic counting were within the range of manual counting results +/-10%. Automatic counting included only cell nuclei within the mucosa whereas muscularis and lymphoid tissue as well as wrinkles from tissue preparation were excluded. In addition, two data sets from automatic counting of the same image tile were compared: (1) data where image tiles with incorrect detection of mucosa were excluded from further calculation of LI and area, and (2) data where no visual check was performed and all measurements were included. Results were very similar for both data sets. The necessity of the manual correction may therefore be doubted.
|Identification of putative bovine mammary epithelial stem cells by their retention of labeled DNA strands. |
Anthony V Capuco
Experimental biology and medicine (Maywood, N.J.) 232 2007
Stem cells appear to retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. In this study, proliferating cells in the prepubertal bovine mammary gland were labeled using five daily injections of 5-bromo-2-deoxyuridine (BrdU). Five weeks later, BrdU-labeled mammary epithelial cells were still evident. The percentage of BrdU-labeled epithelial cells was greatest in the lower region of the mammary gland, near the gland cistern, and was decreased toward the periphery of the parenchymal region, where the ducts were invading the mammary fat pad. Increased numbers of BrdU-labeled epithelial cells in basal regions of the gland are likely a consequence of decreased proliferation rates and increased cell cycle arrest in this area. In peripheral regions of mammary parenchyma, the percentage of heavily labeled epithelial cells averaged 0.24%, a number that is consistent with estimates of the frequency of stem cells in the mouse mammary gland. Epithelial label-retaining cells seemingly represent a slowly proliferating population of cells, as 5.4% of heavily labeled cells were positive for the nuclear proliferation antigen Ki67. Because epithelial label-retaining cells contain estrogen receptor (ER)-negative and ER-positive cells, they apparently comprise a mixed population, which I suggest is composed of ER-negative stem cells and ER-positive progenitors. Continuing studies will address the usefulness of this technique to identify bovine mammary stem cells and to facilitate studies of stem cell biology.
|The extrachromosomal East protein of Drosophila can associate with polytene chromosomes and regulate gene expression. |
Wasser, M; Chia, W
PloS one 2 e412 2007
The EAST protein of Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations.
|Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion. |
Helge Rask-Andersen, Marja Boström, Bengt Gerdin, Anders Kinnefors, Gunnar Nyberg, Thomas Engstrand, Josef M Miller, Dan Lindholm
Hearing research 203 180-91 2005
Time lapse video recordings of cultured adult human and guinea pig spiral ganglion (hSG and gpSG) show that mitogen responsive progenitor/stem cells develop in the form of spheres that proliferate and differentiate into mature neurons and glia cells. Neurospheres, cultured with EGF and bFGF showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). Newly formed BrdU labelled cells were positive for beta-tubulin, and also for GFAP demonstrating that neuronal cells were derived from a dividing population of progenitor cells. Dissociated spheres cultured either with glia cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), induced differentiation of the progenitor cells. Video microscopy showed that neurons develop from subcultured spheres maintained for up to four weeks. Neurons showed fasciculation and migration with a speed of 10-30 microm/h, and some cells had up to 6 mm long neurites coexpressing TrkB and TrkC receptors. Precise dissection suggests that the neurons formed are cochlea-specific. The results suggest that the mammalian auditory nerve has the capability for self-renewal and replacement. Transplantation of progenitor cells together with established means to induce neural differentiation and fiber growth may facilitate strategies for better repair and treatment of auditory neuronal damage.
|Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex. |
Demyanenko, Galina P, et al.
Neuron, 44: 423-37 (2004) 2004
We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.
|Temporally specific burst in cell proliferation increases hippocampal neurogenesis in protracted abstinence from alcohol. |
Nixon, K; Crews, FT
The Journal of neuroscience : the official journal of the Society for Neuroscience 24 9714-22 2004
Adult neurogenesis is a newly considered form of plasticity that could contribute to brain dysfunction in psychiatric disease. Chronic alcoholism, a disease affecting over 8% of the adult population, produces cognitive impairments and decreased brain volumes, both of which are partially reversed during abstinence. Clinical data and animal models implicate the hippocampus, a region important in learning and memory. In a model of alcohol dependence (chronic binge exposure for 4 d), we show that adult neurogenesis is inhibited during dependence with a pronounced increase in new hippocampal neuron formation after weeks of abstinence. This increase is attributable to a temporally and regionally specific fourfold increase in cell proliferation at day 7 of abstinence, with a majority of those cells surviving and differentiating at percentages similar to controls, effects that doubled the formation of new neurons. Although increases in cell proliferation correlated with alcohol withdrawal severity, proliferation remained increased when diazepam (10 mg/kg) was used to reduce withdrawal severity. Indeed, those animals with little withdrawal activity still show a twofold burst in cell proliferation at day 7 of abstinence. Thus, alcohol dependence and recovery from dependence continues to alter hippocampal plasticity during abstinence. Because neurogenesis may contribute to hippocampal function and/or learning, memory, and mood, compensatory neurogenesis and the return of normal neurogenesis may also have an impact on hippocampal structure and function. For the first time, these data provide a neurobiological mechanism that may underlie the return of human cognitive function and brain volume associated with recovery from addiction.
|alpha6beta1 integrin directs migration of neuronal precursors in adult mouse forebrain. |
Emsley, J G and Hagg, T
Exp. Neurol., 183: 273-85 (2003) 2003
New neuroblasts are constantly generated in the adult mammalian subventricular zone (SVZ) and migrate via the very-restricted rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into functional neurons. Several facilitating and repulsive molecules for this migration have been identified, but little is known about chemoattractive molecules involved in the directed nature of this migration in vivo. Here, we investigated the role of the alpha6beta1 integrin, and its ligand, laminin, in controlling guidance of the migrating neuroblasts in adult mice. Immunostaining for the alpha6beta1 integrin was present in neuroblasts and their processes in the anterior/rostral SVZ and the RMS. Inhibition of the endogenous alpha6 or beta1 subunit with locally injected antibodies disrupted the cohesive nature of the RMS, but did not kill the neuroblasts. Infusion of a 15 a.a. peptide, representing the E8 domain of the laminin alpha chains that bind alpha6beta1 integrin, into the neostriatum redirected the neuroblasts away from the RMS towards the site of infusion. Injection of a narrow tract of intact laminin also drew the neuroblasts away from the RMS, but in a more restricted localization. These results suggest a critical role for integrins and laminins in adult SVZ-derived neuroblast migration. They also suggest that integrin-based strategies could be used to direct or restrict neuroblasts to CNS regions where they are needed for cell replacement therapies in the nervous system.
|Double and triple staining methods for studying the proliferative activity of human B and T lymphoid cells. |
Campana, D, et al.
J. Immunol. Methods, 107: 79-88 (1988) 1988
New methods for double and triple colour labelling using monoclonal antibodies to the proliferation-associated markers 5-bromo-2'-deoxyuridine (BrdU) and Ki67 are described. In order to make incorporated BrdU accessible to most anti-BrdU antibodies, mild denaturation of the DNA is needed, and this is usually obtained by exposing the cells to acid or base. This procedure destroys most cellular antigens, including nuclear TdT and Ki67. In this study we show that fixation in cold methanol, instead of 70% ethanol, for 30 min followed by immersion in 7 X 10(-3) N NaOH for 10-15 s allows BrdU staining with the simultaneous detection of nuclear, cytoplasmic and membrane antigens as well as preservation of morphological detail. This method is optimal for detection of nuclear Ki67 and TdT. These reagents together with antibodies to membrane antigens can be included in triple colour labelling using second layers conjugated to FITC, TRITC and colloidal gold. With these methods it is now possible to characterize the phenotype of dividing cell populations such as precursors in central lymphoid tissues and germinal centre blasts in peripheral lymphoid organs.
|Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue. |
Hayashi, Y, et al.
J. Histochem. Cytochem., 36: 511-4 (1988) 1988
We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.
|Effect of tissue fixation on anti-bromodeoxyuridine immunohistochemistry. |
Schutte, B, et al.
J. Histochem. Cytochem., 35: 1343-5 (1987) 1987
We investigated the influence of various fixatives on monoclonal anti-BrdUrd antibody binding of BrdUrd-substituted DNA in tissue sections of routinely processed mouse small intestine after in vivo administration of BrdUrd. For denaturing fixatives such as ethanol or Carnoy's fluid, a standard denaturation protocol showed specific crypt cell labeling. With cross-linking agents such as formalin and glutaraldehyde, a remarkable increase in staining intensity was obtained after tissue digestion with pepsin before acid denaturation. The optimal pepsin concentration was determined for maximal immunoreactivity combined with acceptable morphology.
|Improved high-affinity monoclonal antibody to iododeoxyuridine. |
Vanderlaan, M, et al.
Cytometry, 7: 499-507 (1986) 1986
Three monoclonal antibodies (Mabs), IU-1, IU-4, and B-44 were evaluated in enzyme immunoassays (ELISA) and by flow cytometry for their abilities to recognize bromodeoxyuridine (BrdUrd)- and iododeoxyuridine (IdUrd)-substituted DNA's, nucleotides, and nucleosides. IU-4 is a new Mab, derived from mice immunized with 5-iodo-2'-deoxyuridine-5'-monophosphate (IdUMP) conjugated (IdUMP) conjugated through the phosphate group to albumin. This immunogen was selected to resemble IdUMP in DNA. In competition ELISA assays, IU-4 prefers IdUrd to BrdUrd and prefers halogenated nucleotides over the corresponding nucleosides. In both ELISA and flow analysis, IU-4 recognizes IdUrd in DNA at substitution frequencies at least as low as one IdUrd one per 1,000 normal bases. The high affinity of IU-4 for IdUrd-DNA contrasts with IU-1 and B-44, which show a strong binding dependency on the frequency of base substitution and require DNA that is essentially fully substituted with BrdUrd for binding in both flow and ELISA assays. The high affinity of IU-4 for IdUrd in DNA and its independence of IdUrd residue spacing make it a superior reagent for the quantitative labeling of halogenated thymidine analogues in whole cells.
|S-phase fraction of human brain tumors in situ measured by uptake of bromodeoxyuridine. |
Hoshino, T, et al.
Int. J. Cancer, 38: 369-74 (1986) 1986
One hundred fifty-four patients with brain tumors of various types were given an intravenous infusion of the thymidine analogue bromodeoxyuridine (BUdR), 200 mg/m2, at the time of surgery but before biopsy of the tumor to label S-phase cells. Excised tumor specimens were fixed, sectioned, and stained by immunoperoxidase methods to detect BUdR. The labelling index (LI), or percentage of BUdR-labelled cells, was calculated for each tumor specimen. The LIs of glioblastomas, medulloblastomas, and most highly anaplastic astrocytomas were 5% to 20%. The majority of moderately anaplastic astrocytomas showed LIs of less than 1%, but 30% of them had LIs similar to those of highly malignant gliomas. Most pituitary adenomas and neurinomas showed LIs of less than 1%. Nonmalignant meningiomas had LIs of less than 1%, whereas malignant meningiomas had LIs higher than 2.7%. This is an important observation, because malignant meningiomas are not well-defined histopathologically and their growth rate and rate of recurrence cannot be predicted by current diagnostic procedures. By estimating the proliferative potential of individual tumors more precisely, the BUdR LI supplements histopathological diagnosis, allowing a more accurate estimate of prognosis and facilitating the design of treatment regimens for individual patients.
|The proliferative potential of human pituitary tumors in situ. |
Nagashima, T, et al.
J. Neurosurg., 64: 588-93 (1986) 1986
At the start of transsphenoidal microsurgery for removal of various types of pituitary adenomas, 21 patients received a 1-hour intravenous infusion of 5-bromodeoxyuridine (BUdR, 200 mg/sq m) to label tumor cells in the deoxyribonucleic acid (DNA) synthesis phase (S-phase). Excised tumor specimens were fixed in 70% ethanol and stained by the indirect peroxidase method using anti-BUdR monoclonal antibody as the first antibody. The percentage of BUdR-labeled cells, or S-phase fraction, was calculated for each specimen. The S-phase fraction was less than 0.1% in nine cases, 0.1% to 0.5% in seven, and greater than 0.5% in five. Except in two cases of Nelson's syndrome, in which it was greater than 1%, the S-phase fraction did not correlate with any other variable, including patient age, tumor size, or the duration of signs and symptoms. The small S-phase fraction of most of the pituitary adenomas correlates well with the clinical behavior of these tumors, which grow much more slowly than other kinds of brain tumors such as gliomas. However, the S-phase fractions varied by as much as one order of magnitude. The higher S-phase fractions may reflect aggressive and invasive growth. These results indicate that immunohistochemical studies of cell kinetics using BUdR and anti-BUdR monoclonal antibodies may provide information about the biological characteristics of pituitary adenomas which could lead to the design of appropriate treatment regimens (including surgery, radiation therapy, and chemotherapy) for individual patients.
|Immunocytochemical demonstration of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in human brain tumor tissues. |
Nagashima, T, et al.
Acta Neuropathol., 67: 155-9 (1985) 1985
Five patients with various brain tumors received bromodeoxyuridine (BrdU), 150-200 mg/m2 i.v., at the time of craniotomy. Biopsied materials were fixed in 70% ethanol, sectioned, denatured with hydrochloric acid, and reacted with monoclonal antibodies against BrdU. Immunofluorescence and immunocytochemical methods were used to visualize BrdU-labeled nuclei. Our results showed that both methods demonstrated BrdU-labeled nuclei satisfactorily in tissue sections. Thus, BrdU can be used to measure the proliferative potential of human tumors in situ.
|Immunofluorescent plasma cell labeling indices (LI) using a monoclonal antibody (BU-1). |
Greipp, P R, et al.
Am. J. Hematol., 20: 289-92 (1985) 1985
Tritiated thymidine labeling indices (LI), although useful in diagnosis and prognosis of multiple myeloma, have not found wide-spread application because autoradiographic analysis is difficult and time consuming. Using a monoclonal antibody (BU-1) reactive with 5-bromo-2-deoxyuridine (BrdUrd), we have developed an immunofluorescent procedure that allows DNA S-phase measurements to be determined in 4 hr. Plasma cells are easily identified by reactivity with a fluorescein isothiocyanate-conjugated antihuman immunoglobulin, and cells in DNA S phase are detected via BU-1 and a rhodamine-conjugated antimouse immunoglobulin. Results using this method on 12 patients with multiple myeloma compare favorably (correlation coefficient 0.84), with those obtained by tritiated thymidine. This immunofluorescent slide method will facilitate application of labeling indices as a clinical test to measure disease activity in patients with multiple myeloma and other hematologic neoplasms.
|Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody. |
Morstyn, G, et al.
J. Clin. Invest., 72: 1844-50 (1983) 1983
Using a monoclonal antibody to bromodeoxyuridine (BUdR) and immunohistochemistry, we measured the incorporation of this thymidine analogue into the DNA of human normal and malignant cells exposed in vivo. BUdR given as a constant intravenous infusion for 12 or 24 h daily for up to 13 d resulted in a steady-state plasma level of 10(-6) M during the infusion. We demonstrated extensive incorporation of BUdR into both normal skin, normal bone marrow, and malignant melanoma cells. In addition, this infusion of BUdR was adequate to identify sister chromatid exchanges from human marrow chromosomes exposed in vivo. Using this constant infusion, significant but reversible (acute) toxicity was observed with myelosuppression and skin photosensitivity. These techniques, which are considerably less cumbersome and time-consuming than the use of radioactive isotopes of thymidine, can be used for further human studies of cell kinetics and chromosomal replication in both normal and malignant cells.