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Brazilian Journal of Otorhinolaryngology 2022Inner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear....
INTRODUCTION
Inner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells.
OBJECTIVE
To investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation.
METHODS
Cochlear neural progenitor cells were cultured and induced in DMEM/F12+RA (10M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu.
RESULTS
The nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p<0.05). The ratios of Brdu-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased.
CONCLUSION
Retinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.
Topics: Humans; Tretinoin; Bromodeoxyuridine; Cells, Cultured; Neural Stem Cells; Cell Differentiation
PubMed: 33707121
DOI: 10.1016/j.bjorl.2021.01.005 -
PloS One 2015The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to...
The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.
Topics: Animals; Biomarkers; Bromodeoxyuridine; Cell Culture Techniques; Cell Line; Fluorescent Antibody Technique; Immunoblotting; Liver; Myofibroblasts; Portal System; Rats; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 25822334
DOI: 10.1371/journal.pone.0121161 -
Ecotoxicology and Environmental Safety Jun 2023Chronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive...
BACKGROUND
Chronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear.
METHODS
Mice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice.
RESULTS
Mice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL microglia and GSDMD microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1β) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU, BrdU-DCX, and BrdU-NeuN cells in the hippocampal dentate gyrus, as well as the percentages of BrdU cells that were NeuN in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX cells correlated negatively with the percentage of ameboid microglia, TUNEL microglia and GSDMD microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro.
CONCLUSION
Chronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.
Topics: Mice; Animals; Microglia; Aflatoxin B1; Depressive Disorder, Major; Pyroptosis; Bromodeoxyuridine; Hippocampus; Sucrose
PubMed: 37172405
DOI: 10.1016/j.ecoenv.2023.114991 -
Stem Cell Research & Therapy Jul 2022Impairment in neurogenesis correlates with memory and cognitive dysfunction in AD patients. In the recent decade, therapies with stem cell bases are growing and proved...
BACKGROUND
Impairment in neurogenesis correlates with memory and cognitive dysfunction in AD patients. In the recent decade, therapies with stem cell bases are growing and proved to be efficient. This study is a preliminary attempt to explore the impact of NTF-SCs on hippocampal neurogenesis mediated by the Wnt/β-catenin signaling cascade in AD-like mouse brain parenchyma.
METHODS
The BALB/c mice were divided into four groups: Control, AD +Vehicle, AD+ TF-SCs-CM and AD+NTF-SCs (n = 10). For AD induction, 100 µM Aβ was injected into lateral ventricles. The AD-like model was confirmed via passive avoidance test and Thioflavin-S staining 21 days following Aβ injection. Next, NTF-SCs were differentiated from ADMSCs, and both NTF-SCs and supernatant (NTF-SCs-CM) were injected into the hippocampus after AD confirmation. Endogenous neural stem cells (NSCs) proliferation capacity was assessed after 50 mg/kbW BrdU injection for 4 days using immunofluorescence (IF) staining. The percent of BrdU/Nestin and BrdU/NeuN positive NSCs were calculated. Real-time RT-PCR was used to detect genes related to the Wnt/β-catenin signaling cascade. The spatial learning and memory alternation was evaluated using the Morris water maze (MWM).
RESULTS
Data showed the reduction in escape latency over 5 days in the AD mice compared to the control group. The administration of NTF-SCs and NTF-SCs-CM increased this value compared to the AD-Vehicle group. Both NTF-SCs and NTF-SCs-CM were the potential to reduce the cumulative distance to the platform in AD mice compared to the AD-Vehicle group. The time spent in target quadrants was ameliorated following NTF-SCs and NTF-SCs-CM transplantation followed by an improved MWM performance. IF imaging revealed the increase in BrdU/Nestin and BrdU/NeuN in AD mice that received NTF-SCs and NTF-SCs-CM, indicating enhanced neurogenesis. Based on real-time PCR analysis, the expression of PI3K, Akt, MAPK, ERK, Wnt, and β-catenin was upregulated and coincided with the suppression of GSK-3β after injection of NTF-SCs-CM and NTF-SCs. In this study, NTF-SCs had superior effects in AD mice that received NTF-SCs compared to NTF-SCs-CM.
CONCLUSIONS
The activation of Wnt/β-catenin pathway via NTF-SCs can be touted as a possible therapeutic approach to restore neurogenesis in AD mice.
Topics: Alzheimer Disease; Animals; Bromodeoxyuridine; Glycogen Synthase Kinase 3 beta; Hippocampus; Mice; Nerve Growth Factors; Nestin; Neurogenesis; Transforming Growth Factor beta; Wnt Signaling Pathway; beta Catenin
PubMed: 35883119
DOI: 10.1186/s13287-022-03024-6 -
Journal of Neuroinflammation Oct 2023Some patients show persistent cognitive decline for weeks, months or even years after surgery, which seriously affects their long-term prognosis and quality of life....
Inhibition of adult hippocampal neurogenesis induced by postoperative CD8 + T-cell infiltration is associated with cognitive decline later following surgery in adult mice.
BACKGROUND
Some patients show persistent cognitive decline for weeks, months or even years after surgery, which seriously affects their long-term prognosis and quality of life. However, most previous basic studies have focused mainly on the mechanisms of early postoperative cognitive decline, whereas cognitive decline in the longer term after surgery is less well-understood. The subgranular zone of the dentate gyrus exhibits life-long neurogenesis, supporting hippocampus-dependent learning and memory.
MAIN TEXT
The aim of this study was to investigate whether adult hippocampal neurogenesis (AHN) involves in cognitive decline later following surgery and to further explore the roles of CD8 + T lymphocytes infiltrating the hippocampal parenchyma after surgery in this pathological process. Cognitive function was examined in adult mice that underwent laparotomy combined with partial hepatectomy, and the results showed that cognitive decline persisted in mice who underwent surgery during the first postoperative month, even though there was a trend toward continuous improvement over time. Significantly decreased numbers of DCX + cells, BrdU + cells, and BrdU + /DCX + cells were observed on day 8 after surgery, and a significantly decreased number of NeuN + /BrdU + cells was observed on day 28 after surgery, which indicated inhibition of AHN. After surgery, T lymphocytes, the majority of which were CD8 + T cells, infiltrated the hippocampus and secreted Interferon-γ (IFN-γ). Depletion of CD8 + T cells could inhibit the increase of IFN-γ synthesis, improve hippocampal neurogenesis, and improve postoperative cognitive function. Hippocampal microinjection of IFN-γ neutralizing antibody or adeno-associated virus to knock down IFN-γ receptor 1 (IFNGR1) could also partially attenuate the inhibition of AHN and improve postoperative cognitive function.
CONCLUSIONS
These results demonstrate that postoperative infiltration of CD8 + T cells into the hippocampus and subsequent secretion of IFN-γ contribute to the inhibition of AHN and cognitive decline later following surgery.
Topics: Mice; Humans; Animals; Adult; Bromodeoxyuridine; Quality of Life; Hippocampus; Neurogenesis; Cognitive Dysfunction; Interferon-gamma; CD8-Positive T-Lymphocytes
PubMed: 37798730
DOI: 10.1186/s12974-023-02910-x -
PloS One 2015Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an...
Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an increase in the proportion of cells in the S phase. The DNA content was reduced in the treated cells, and BrdU incorporation diminished. However, neither ammonia nor ammonia plus glutamine had any effect on DNA polymerase activity. iTRAQ analysis showed that exposure to ammonia induced a significant reduction in histone and heterochromatin protein 1 expression. A reduction in cell viability was also noted. The ammonia-induced reduction of proliferative activity in these cultured astroglial cells seems to be due to a delay in the completion of the S phase provoked by the inhibition of chromatin protein synthesis.
Topics: Ammonia; Animals; Astrocytes; Bromodeoxyuridine; Cell Count; Cell Proliferation; Cell Survival; Cells, Cultured; Chromatin; DNA-Directed DNA Polymerase; Gene Expression; Histones; Rats; S Phase Cell Cycle Checkpoints
PubMed: 26421615
DOI: 10.1371/journal.pone.0139619 -
Translational Vision Science &... Aug 2020The retina is a commonly used model for angiogenesis research due to its special characteristics. Oxygen-induced retinopathy (OIR) provides a useful model to study...
PURPOSE
The retina is a commonly used model for angiogenesis research due to its special characteristics. Oxygen-induced retinopathy (OIR) provides a useful model to study ischemia-induced neovascularization (NV) and to develop anti-angiogenic therapeutics. The purpose of this study was to develop a simple, accurate, and less-subjective quantification method for retinal NV in the OIR model.
METHODS
To address this challenge, we combined the conventional vascular staining and BrdU labeling of newly formed vascular cells to detect and analyze retinal NV. With daily injections of BrdU, which was incorporated into the DNA of newly formed retinal vessels under the OIR condition, ischemia-induced retinal neovasculature with BrdU labeling was distinguished from pre-existing vasculature and accurately quantified using the ImageJ program.
RESULTS
Compared with conventional quantification methods using isolectin B4 staining of the entire vascular network, BrdU labeling allowed us to distinguish newly formed vessels from the pre-existing vessels and to objectively quantify the newly formed vessels, which was verified in OIR mice with intravitreal injections of an antibody-neutralizing vascular endothelial growth factor.
CONCLUSIONS
BrdU labeling provides a useful and sensitive method for studying retinal NV and evaluating the therapeutic effects of medical interventions against pathological angiogenesis.
TRANSLATIONAL RELEVANCE
Quantitative, straightforward, and objective observation and evaluation of pathologic neovasculature are important to study the pathogenesis of NV and therapeutic effects using animal models.
Topics: Animals; Bromodeoxyuridine; Disease Models, Animal; Mice; Retinal Neovascularization; Retinal Vessels; Vascular Endothelial Growth Factor A
PubMed: 32879761
DOI: 10.1167/tvst.9.9.4 -
The Journal of Endocrinology Dec 2019Intrauterine growth-restricted (IUGR) fetuses are born with reduced skeletal muscle mass. We hypothesized that reduced rates of myogenesis would contribute to fewer and...
Intrauterine growth-restricted (IUGR) fetuses are born with reduced skeletal muscle mass. We hypothesized that reduced rates of myogenesis would contribute to fewer and smaller myofibers in IUGR fetal hindlimb muscle compared to the normally growing fetus. We tested this hypothesis in IUGR fetal sheep with progressive placental insufficiency produced by exposing pregnant ewes to elevated ambient temperatures from 38 to 116 days gestation (dGA; term = 147 dGA). Surgically catheterized control (CON, n = 8) and IUGR (n = 13) fetal sheep were injected with intravenous 5-bromo-2′-deoxyuridine (BrdU) prior to muscle collection (134 dGA). Rates of myogenesis, defined as the combined processes of myoblast proliferation, differentiation, and fusion into myofibers, were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. Total myofiber number was determined for the entire cross-section of the FDS muscle. In IUGR fetuses, the number of BrdU+ myonuclei per myofiber cross-section was lower in BF, TA, and FDS (P < 0.05), total myonuclear number per myofiber cross-section was lower in BF and FDS (P < 0.05), and total myofiber number was lower in FDS (P < 0.005) compared to CON. mRNA expression levels of cyclins, cyclin-dependent protein kinases, and myogenic regulatory factors were lower (P < 0.05), and inhibitors of the cell cycle were higher (P < 0.05) in IUGR BF compared to CON. Markers of apoptosis were not different in IUGR BF muscle. These results show that in IUGR fetuses, reduced rates of myogenesis produce fewer numbers of myonuclei, which may limit hypertrophic myofiber growth. Fewer myofibers of smaller size contribute to smaller muscle mass in the IUGR fetus.
Topics: Animals; Apoptosis; Bromodeoxyuridine; Female; Fetal Growth Retardation; Fetus; Insulin; Muscle Development; Muscle, Skeletal; Placental Insufficiency; Pregnancy; Pregnancy, Animal; Sheep; Temperature
PubMed: 31751294
DOI: 10.1530/JOE-19-0273 -
Frontiers in Immunology 2022TNBC, whose clinical prognosis is poorer than other subgroups of breast cancer, is a malignant tumor characterized by lack of estrogen receptors, progesterone hormone...
BACKGROUND
TNBC, whose clinical prognosis is poorer than other subgroups of breast cancer, is a malignant tumor characterized by lack of estrogen receptors, progesterone hormone receptors, and HER2 overexpression. Due to the lack of specific targeted drugs, it is crucial to identify critical factors involved in regulating the progression of TNBC.
METHODS
We analyzed the expression profiles of TNBC in TCGA and the prognoses values of GLDC. Correlations of GLDC and tumor immune infiltration were also identified. CCK8 and BrdU incorporation assays were utilized to determine cell proliferation. The mRNA and protein levels were examined by using Real-time PCR and Western blot analysis.
RESULTS
In the present study, we analyzed the mRNA expression profiles of TNBC in TCGA and found that GLDC, a key enzyme in glycine cleavage system, was significantly up-regulated in TNBC tissues and higher expression of GLDC was correlated with a worse prognosis in TNBC. Moreover, the expression of GLDC was negatively correlated with macrophage and monocyte and positively correlated with activated CD4 T cell and type 2 T helper cell in TNBC. Overexpression of GLDC facilitated the proliferation of TNBC cells, whereas GLDC knockdown had the opposite effects. Additionally, miR-30e acts as a functional upstream regulator of GLDC and the inhibitory effects of miR-30e on cell proliferation were mitigated by the reintroduction of GLDC.
CONCLUSIONS
These results imply that miR-30e-depressed GLDC acts as a tumor suppressive pathway in TNBC and provides potential targets for the treatment of TNBC.
Topics: Humans; Triple Negative Breast Neoplasms; Gene Expression Regulation, Neoplastic; MicroRNAs; Receptors, Progesterone; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; RNA, Messenger; Estrogens
PubMed: 36275705
DOI: 10.3389/fimmu.2022.1033367 -
Cells Aug 2019Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine...
Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.
Topics: Antimetabolites, Antineoplastic; Bromodeoxyuridine; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Drug Screening Assays, Antitumor; Humans; Neuroblastoma; Ribose-Phosphate Pyrophosphokinase; Structure-Activity Relationship; Tumor Cells, Cultured
PubMed: 31443513
DOI: 10.3390/cells8090955