Bromodeoxyuridine Labeling Research Articles

Deletion of the Igf1 Gene: Suppressive Effects on Adult Leydig Cell Development

Deletion of the insulin-like growth factor 1 (Igf1) gene was shown in previous studies to result in reduced numbers of Leydig cells in the testes of 35-day-old mice, and in reduced circulating testosterone levels. In the current study, we asked whether deletion of the Igf1 gene affects the number, proliferation, and/or steroidogenic function of some or all of the precursor cell types in the developmental sequence that leads to the establishment of adult Leydig cells (ALCs). Decreased numbers of cells in the Leydig cell lineage (ie, 3β-hydroxysteroid dehydrogenase-positive cells) were seen in testes of postnatal day (PND) 14-90 Igf1(-/-) mice compared with age-matched Igf1(+/+) controls. The development of ALCs proceeds from stem Leydig cells (SLCs) through progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). The bromodeoxyuridine labeling index of putative SLCs was similar in the Igf1(-/-) and Igf1(+/+) mice. In contrast, the labeling index of PLCs was reduced in the Igf1(-/-) mice on each day of PND 14 through PND 35, and that of more mature Leydig cells (referred to herein as LCs, a combination of ILCs plus ALCs) was reduced from PND 21 through PND 56. In Igf1(-/-) mice that received recombinant IGF-I, the labeling indices of PLCs and LCs were similar to those of age-matched Igf1(+/+) mice, indicating that the reductions in the labeling indices seen in the PLCs and LCs of the Igf1(-/-) mice were a consequence of reduced IGF-I. On each day of PND 21 through PND 90, testicular testosterone concentrations were significantly reduced in the Igf1(-/-) mice, as were the expressions of testis-specific mRNAs involved in steroidogenesis, including Star, Cyp11a1, and Cyp17a1. The increased expression of the gene for 5α-reductase (Srd5a1) in adult Igf1(-/-) testes suggests that the depletion of Igf1 might suppress or delay Leydig cell maturation. These observations, taken together, indicate that the reduced numbers of Leydig cells in the adult testes of Igf1(-/-) mice result at least in part from altered proliferation and differentiation of ALC precursor cells, but not of the stem cells that give rise to these cells.

Bigger brains cycle faster before neurogenesis begins: a comparison of brain development between chickens and bobwhite quail

The chicken brain is more than twice as big as the bobwhite quail brain in adulthood. To determine how this species difference in brain size emerges during development, we examined whether differences in neurogenesis timing or cell cycle rates account for the disparity in brain size between chickens and quail. Specifically, we examined the timing of neural events (e.g. neurogenesis onset) from Nissl-stained sections of chicken and quail embryos. We estimated brain cell cycle rates using cumulative bromodeoxyuridine labelling in chickens and quail at embryonic day (ED) 2 and at ED5. We report that the timing of neural events is highly conserved between chickens and quail, once time is expressed as a percentage of overall incubation period. In absolute time, neurogenesis begins earlier in chickens than in quail. Therefore, neural event timing cannot account for the expansion of the chicken brain relative to the quail brain. Cell cycle rates are also similar between the two species at ED5. However, at ED2, before neurogenesis onset, brain cells cycle faster in chickens than in quail. These data indicate that chickens have a larger brain than bobwhite quail mainly because of species differences in cell cycle rates during early stages of embryonic development.

Effects of Axial Stretch on Cell Proliferation and Intimal Thickness in Arteries in Organ Culture

Intimal hyperplasia (IH) remains the major cause of intermediate and long-term failure of vascular grafts and endovascular interventions. Arteries are subjected to a significant longitudinal stress in addition to the shear stress and tensile stress from the blood flow. The aim of this study was to determine the effect of axial stretch on cell proliferation and IH in arteries. Porcine carotid arteries, intact or endothelial cell (EC) denudated, were maintained ex vivo at different stretch ratios (1.3, 1.5, and 1.8) and flow rates (16 or 160 mL/min) while remaining at physiologic pressure for 7 days. The viability of the arteries was verified with norepinephrine, carbachol, and sodium nitroprusside stimulations, and the cell proliferation was detected using bromodeoxyuridine labeling and immunostaining. Our results showed that the axial stretch ratio did not significantly affect intimal thickness and cell proliferation in normal arteries. However, axial stretch increased the neointimal thickness in EC denudated arteries cultured under low flow conditions. The cell proliferation increased significantly in the intima and inner half of the media of the EC denudated arteries under normal or elevated axial stretch in comparison to intact arteries at the same stretch ratio. These results demonstrated that axial stretch with EC denudation and low flow increases neointimal formation and cell proliferation in the arteries.

Effect of Melatonin on Proliferative Activity and Apoptosis in Spermatogenic Cells in Mouse under Chemotherapy

Objective To investigate the possible protective role of melatonin on the proliferative activity, and apoptosis of germ cells in chemotherapy-induced spermiotoxicity. Methods Male adult NMRI mice were divided into four groups. Then each group was divided into two subgroups of a and b. Group A (control): mice received vehicle (1% ethanol) for 5 d. Group B (busulfan): mice received a single dose of 20 mg/kg busulfan. Group C (melatonin): mice received melatonin (10 mg/kg) for 5 d. Group D (busulfan+melatonin): mice received a 5-day course of melatonin (10 mg/kg) following an initial dose of busulfan (20 mg/kg). Evaluations were made using bromodeoxyuridine (BrdU) labeling and in situ TUNEL assay after 5 d (subgroup a) or 35 d (subgroup b). Results Busulfan-treated mice both in subgroups a and b, showed a significant increase in the number of apoptotic cells (P Conclusion Melatonin may have a protective effect against busulfan-induced testicular damage, partly by decreasing of apoptosis and alteration in proliferative activity of germ cells.

Enhancement of vinorelbine-induced cytotoxicity and apoptosis by clomipramine and lithium chloride in human neuroblastoma cancer cell line SH-SY5Y

The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.

Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling

Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis.A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation.These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth.

Reepithelialization of the Uterine Surface Arises from Endometrial Glands: Evidence from a Functional Mouse Model of Breakdown and Repair

The human endometrium is highly regenerative undergoing monthly cycles of growth and regression. Endometrial repair after menses is a critical component of the cycle; however, little is understood about the mechanisms behind this rapid process. Adult stem/progenitor cells identified in human and mouse endometrium may be responsible for its remarkable regenerative capacity; however, a functional role for stem/progenitor cells in menstruation is yet to be established. This study aimed to identify label retaining cells as candidate epithelial stem or progenitor cells involved in the rapid reepithelization of the uterine surface in our functional mouse model of endometrial breakdown and repair. Adult mice were pulse labeled with bromodeoxyuridine before endometrial breakdown and repair was induced. Throughout endometrial breakdown and repair, very rapid dilution of bromodeoxyuridine label was observed in the luminal epithelium, whereas label within the glandular epithelium remained constant. Importantly, glandular epithelial cells were shown to proliferate selectively in response to endometrial repair, and the majority strongly expressed estrogen receptor-alpha at this time. This is the first study to demonstrate a functionally diverse response during endometrial repair from the anatomically connected luminal and glandular epithelium and highlights the likelihood that the endometrial glands are the residence of epithelial progenitor cells contributing to reepithelialization of the uterine surface after menses.

Post-MPTP Treatment with Granulocyte Colony-Stimulating Factor Improves Nigrostriatal Function in the Mouse Model of Parkinson’s Disease

The neuroprotective effects of granulocyte colony-stimulating factor (G-CSF) were reported in several neurological disease models, including Parkinson's disease (PD). In the present study, we investigated the therapeutic effect of G-CSF after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD was established. G-CSF was subcutaneously administered into C57BL/6 mice that had undergone systemic MPTP injections. We found that G-CSF treatment markedly increased the number of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the G-CSF-treated group. Consistent with this finding, we found a significant increase in dopamine release under high K(+) stimulation in the striatum of the G-CSF-treated animals compared to the MPTP-exposed mice. Finally, we observed a persistent recovery of locomotor function in the G-CSF-treated animals. These results suggest the potential therapeutic value of G-CSF in treating PD. However, our bromodeoxyuridine labeling experiment failed to identify any newly generated dopaminergic neurons in SNpc. This might indicate an indirect effect of G-CSF on cell proliferation. The underlying mechanism of G-CSF is under further investigation.

Carvedilol in glioma treatment alone and with imatinib in vitro

The purpose of the study was to investigate whether carvedilol has an antiproliferative effect alone and whether carvedilol provides an additive, synergistic or antagonistic effect on imatinib mesylate-induced cytotoxicity in both C6 glioma monolayer and spheroid culture. The C6 rat glioma chemoresistant experimental brain tumour cell line, that is notoriously difficult to treat with combination chemotherapy, was used both in monolayer and spheroid cultures. We treated C6 glioma cells with carvedilol alone and a combination of carvedilol and imatinib mesylate at a concentration of 10 microM. Following treatment, we evaluated cell proliferation index, bromodeoxyuridine labelling index (BrDU-LI), cell cycle distributions, apoptotic cell percentages, cAMP levels and three dimensional cell morphology at monolayer cultures. In addition BrDU-LI, volume and morphology of spheroids were also assessed. Carvedilol and imatinib mesylate alone reduced cell number, BrDU-LI, cAMP levels and spheroid volume. Carvedilol and imatinib mesylate arrested cells at G0/G1 phase in a time-dependent manner and time-independent manner, respectively. Carvedilol increased apoptosis rate only at the 24th h, but imatinib mesylate did for all time intervals. Interestingly carvedilol, drug with well-known protective effect on mitochondria, induced severe mitochondria damage, and imatinib mesylate induced autophagy confirmed only by transmission electron microscopy. These results suggest that carvedilol showed antitumour activity against rat C6 glioma cells and a combination of carvedilol with imatinib mesylate resulted in enhanced in vitro antitumour activity.

Notch1 signaling related hippocampal neurogenesis in adult poststroke depression rats: a valid index for an efficient combined citalopram and WAY100635 pharmacotherapy

We investigated the hypothesis that hippocampal neurogenesis related to Notch1 signaling could be a valid index for a combined citalopram and WAY100635 pharmacotherapy for the treatment of depression arising after stroke. Adult rats were exposed to a chronic mild stress paradigm after ischemic surgery. Behavioral tests included the open-field test and a sucrose consumption test. Proliferating cells in the hippocampus ipsilateral to ischemia and their fate were monitored by bromodeoxyuridine labeling and confocal laser scanning microscopy for up to 28 days (day 28) after ischemia. Expression of the Notch1 signaling cascade, including its ligand and downstream target genes, was also examined. WAY100635 shortened the onset of citalopram action to less than the day 21 required with citalopram alone and also proved more effective. The activity of the Notch1 signaling pathway in the hippocampus fluctuated in its function in proliferation (day 21) and differentiation (day 28) of newly formed cells in animals receiving the combination treatment. This indicated that augmentation of citalopram by cotreatment with a selective 5-hydroxytryptamine 1A antagonist would be an efficacious strategy for poststroke depression. The observed effects are most likely because of enhanced poststroke neurogenesis mediated by the Notch1 signaling cascade.

Nogo-A Regulates Neural Precursor Migration in the Embryonic Mouse Cortex

Although Nogo-A has been intensively studied for its inhibitory effect on axonal regeneration in the adult central nervous system, little is known about its function during brain development. In the embryonic mouse cortex, Nogo-A is expressed by radial precursor/glial cells and by tangentially migrating as well as postmigratory neurons. We studied radially migrating neuroblasts in wild-type and Nogo-A knockout (KO) mouse embryos. In vitro analysis showed that Nogo-A and its receptor components NgR, Lingo-1, TROY, and p75 are expressed in cells emigrating from embryonic forebrain–derived neurospheres. Live imaging revealed an increased cell motility when Nogo-A was knocked out or blocked with antibodies. Antibodies blocking NgR or Lingo-1 showed the same motility-enhancing effect supporting a direct role of surface Nogo-A on migration. Bromodeoxyuridine (BrdU) labeling of embryonic day (E)15.5 embryos demonstrated that Nogo-A influences the radial migration of neuronal precursors. At E17.5, the normal transient accumulation of radially migrating precursors within the subventricular zone was not detectable in the Nogo-A KO mouse cortex. At E19, migration to the upper cortical layers was disturbed. These findings suggest that Nogo-A and its receptor complex play a role in the interplay of adhesive and repulsive cell interactions in radial migration during cortical development.

Involvement of serotonin neurotransmission in hippocampal neurogenesis and behavioral responses in a rat model of post-stroke depression

Ischemia-stimulated dentate gyrus (DG) neurogenesis is hypothesized to be an etiological factor of post-stroke depression (PSD) and a potential target of selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitors (SSRIs) in PSD. Clinical investigations have explored the strategy of augmenting SSRIs action by combination with a 5-HT1A receptor antagonist. We investigated the relative importance of the effects on ischemia-stimulated neurogenesis and depressive-like behavior of WAY-100635 versus citalopram at different dose levels in PSD animals. Adult rats were exposed to a chronic mild stress paradigm after ischemic surgery. Decreased sucrose consumption was indicative of the core depressive syndrome anhedonia. Proliferating cells and their fate were monitored by bromodeoxyuridine labeling protocols up to 28 days after ischemia. Expression of the 5-HT1A receptor in DG was also examined. The current findings confirmed the ability of WAY-100635 to augment SSRIs pharmacological efficacy and SSRIs-induced elevation of post-stroke DG neurogenesis. Specifically, WAY-100635 and citalopram in different dose combinations display their relative importance in ischemia-stimulated neurogenesis probably through reinforcing serotonergic neurotransmission and/or density of 5-HT1A receptor in DG. The present data extend our understanding that increase of ischemia-induced DG neurogenesis can be interpreted as a valid index, to an extent, or even a prerequisite for an efficient co-treatment strategy.

10.1007/s11174-008-2008-y

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15–20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.

Bromodeoxyuridine labeling of swine adipose-derived stem cellsin vitro

Bromodeoxyuridine labeling of swine adipose-derived stem cells<i>in vitro</i>

Orphan nuclear receptor TLX activates Wnt/β-catenin signalling to stimulate neural stem cell proliferation and self-renewal

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.

Effect of thrombopoietin on platelet counts and liver regeneration after partial hepatectomy in a rat model

We investigated the effects of thrombopoietin (TPO) on platelet counts and liver regeneration after partial hepatectomy in rats. We performed 70% partial hepatectomy on 60 rats given either TPO or a vehicle (controls). The rats were killed 6 h, 24 h, 72 h, or 168 h after the procedure, and we examined the platelet counts, weight of the regenerated liver, hepatocyte proliferation by bromodeoxyuridine (BrdU) labeling index, and expression of hepatocyte growth factor (HGF) mRNA by reverse transcription-polymerase chain reaction. In the controls, the platelet counts were significantly lower than the basal levels, by about 20%, at 6, 24, and 72 h; then recovered to the basal level at 168 h. Conversely, in the TPO-treated rats, the platelet counts increased significantly by 17%, 35%, and 60%, at 24, 72, and 168 h, respectively. The regenerated liver regained 80% of the pre-hepatectomy weight by 72 h in the controls, whereas reconstitution of the liver was accomplished by 72 h in the TPO-treated rats. Thrombopoietin significantly enhanced the BrdU labeling index of hepatocytes and expression of HGF mRNA 24 h post hepatectomy in TPO-treated rats versus controls. Thrombopoietin increased platelet counts; thereby accelerating liver regeneration after partial hepatectomy with enhanced induction of HGF.

Oral carcinogenesis induced by 4-nitroquinoline 1-oxide in lecithin:retinol acyltransferase gene knockout mice

Lecithin:retinol acyltransferase ( LRAT) regulates retinol (vitamin A) metabolism by esterifying retinol. LRAT expression is decreased in cultured human squamous cell carcinoma cells of the head and neck relative to normal epithelial cells. We investigated whether the carcinogen 4-nitroquinoline 1-oxide (4-NQO) induced a higher incidence of oral cancer in LRAT knockout ( LRAT −/− ) than in wild-type (Wt) mice. We also investigated retinol deprivation during 4-NQO treatment in LRAT −/− mice as a model for rapid retinol deficiency. We observed higher levels of secreted frizzled-related protein ( Sfrp) 2, an inhibitor of WNT signaling, in tongue tumors in LRAT −/− versus Wt. LRAT −/− embryonic stem cells also expressed higher Sfrp2 transcripts, indicating an interaction between retinol and WNT signaling. Cox-2, Cyclin D1, p21, Trop2 and RARβ 2 were not differentially expressed in Wt versus LRAT −/− tongue tumors. Wt and LRAT −/− mice fed a retinol-sufficient diet showed the same oral tumor incidence after 4-NQO treatment. In contrast, tongue tumors developed in 60% of Wt mice and in 100% of LRAT −/− mice fed a retinol-deficient diet during 4-NQO treatment ( P=.22); moreover, the bromodeoxyuridine labeling index was 21.0±2.4% in LRAT −/− normal tongue epithelium as compared to 9.9±0.8% in Wt normal tongue epithelium ( P<.001). Thus, partial retinol deficiency during carcinogen treatment (achieved in LRAT −/− ) resulted in more proliferating cells in tongue epithelia from LRAT −/− mice and, ultimately, a greater probability of carcinogenesis.

Potential effects of the herbicide Diuron on mammary and urinary bladder two-stage carcinogenesis in a female Swiss mouse model

The potential promoting effect of Diuron was investigated in a mouse model of mammary and urinary bladder carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Four-week old female Swiss mice were allocated to five groups: Groups G1-G3 received DMBA (5 x 1.5 mg/mouse) and BBN (8 x 7.5 mg/mouse) and G4 and G5 groups received only vehicles during the first 6 weeks. At week 7, G1 and G5 groups received basal diet and G2, G3 and G4 groups were fed a diet containing Diuron at 1,250, 2,500 and 2,500 ppm, respectively, during 13 weeks. At week 20, the animals were euthanized and the gross tumors were registered. Mammary glands and urinary bladder were processed for histopathological analysis. Samples from non-tumor areas were evaluated for cell proliferation by 5-bromodeoxyuridine labeling index (BrdU-LI%) and apoptosis. Dietary treatment with Diuron at 1,250 and 2,500 ppm significantly increased BrdU-LI% (P < 0.05) and the incidence of simple/nodular urothelial hyperplasia in the urinary bladder from DMBA/BBN-initiated groups (G2 and G3 vs. G1, P < 0.02) and in the non-initiated group (G4 vs. G5, P = 0.042). Two transitional cell carcinomas were observed in the group initiated and fed Diuron 2,500 ppm (G3). In contrast, in the mammary gland, Diuron feeding for 13 weeks did not significantly alter cell proliferation and apoptosis indexes or the incidence of hyperplastic lesions or neoplasms in the DMBA/BBN-initiated groups. These findings suggest that Diuron is a promoting agent to the urinary bladder but not to the mammary gland in female Swiss mice submitted to a medium-term two-stage carcinogenesis bioassay.

Effects of Pioglitazone, a Peroxisome Proliferator–Activated Receptor Gamma Agonist, on the Urine and Urothelium of the Rat

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which belong to the nuclear receptor superfamily. Some PPARgamma agonists, such as pioglitazone, and dual PPARgamma/PPARalpha agonists, such as muraglitazar, induced urothelial bladder tumors in rats but not in mice. In this study, we investigated the early effects in the urine and bladder of rats treated with pioglitazone to evaluate the possible relation between urinary solids formation and urothelial cytotoxicity and regenerative proliferation. In a 4-week experiment, treatment of rats with 16 mg/kg pioglitazone induced cytotoxicity and necrosis of the urothelial superficial layer, with increased cell proliferation measured by bromodeoxyuridine labeling index and hyperplasia by histology. It also produced alterations in urinary solid formation, especially calcium-containing crystals and calculi. PPARgamma agonists (pioglitazone and troglitazone) in vitro reduced rat urothelial cell proliferation and induced uroplakin synthesis, a specific differentiation marker in urothelial cells. Our data support the hypothesis that the bladder tumors produced in rats by pioglitazone are related to the formation of urinary solids. This strongly supports the previous conclusion in studies with muraglitazar that this is a rat-specific phenomenon and does not pose a urinary bladder cancer risk to humans treated with these agents.

Birthdays of retinal amacrine cell subtypes are systematically related to their molecular identity and soma position

The mammalian retina contains six major cell types, several of which are divided into multiple molecularly and morphologically distinct subtypes. To understand how subtype diversity arises during development, we focused on amacrine interneurons in the mouse retina; approximately 30 amacrine subtypes have been identified in mammals. We used antibody markers to identify the two main amacrine subsets--GABAergic and glycinergic--and further subdivided these groups into smaller subsets based on expression of neurotransmitter and transcription factor markers. We then used bromodeoxyuridine (BrdU) labeling to see whether amacrine subsets are born (become postmitotic) at different times, as is the case for lamina-specified subsets of cortical projection neurons. We found that GABAergic amacrines are generated on average 2-3 days before glycinergic amacrines. Moreover, subsets of GABAergic amacrines are born at distinct times. We also found a strong correlation between amacrine cell birthday and soma position in the mature retina, another point of similarity with cortical projection neurons. This relationship raised the possibility that amacrine subtype identity is determined by signals that uncommitted cells receive after they migrate to their destinations. However, cells labeled with BrdU in vivo, then dissociated and allowed to develop in vitro, acquired the amacrine subtype-specific markers appropriate for their birthdays, supporting the idea that they become specified near the time and place of their birth. Together, our results suggest that the birthdays of amacrine cells independently specify their destinations and subtype identities.