The halopyrimidine 5-bromo-2′-deoxyuridine(BrdU)is an exogenous marker of DNA synthesis.Since the introduction of monoclonal antibodies against BrdU,an increasing number of methodologies have been used for the immuno...The halopyrimidine 5-bromo-2′-deoxyuridine(BrdU)is an exogenous marker of DNA synthesis.Since the introduction of monoclonal antibodies against BrdU,an increasing number of methodologies have been used for the immunodetection of this synthesized bromine-tagged base analogue into replicating DNA.BrdU labeling is widely used for identifying neuron precursors and following their fate during the embryonic,perinatal,and adult neurogenesis in a variety of vertebrate species including birds,reptiles,and mammals.Due to BrdU toxicity,its incorporation into replicating DNA presents adverse consequences on the generation,survival,and settled patterns of cells.This may lead to false results and misinterpretation in the identification of proliferative neuroblasts.In this review,I will indicate the detrimental effects of this nucleoside during the development of the central nervous system,as well as the reliability of BrdU labeling to detect proliferating neuroblasts.Moreover,it will show factors influencing BrdU immunodetection and the contribution of this nucleoside to the study of prenatal,perinatal,and adult neurogenesis.Human adult neurogenesis will also be discussed.It is my hope that this review serves as a reference for those researchers who focused on detecting cells that are in the synthetic phase of the cell cycle.展开更多
This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats. The behavior of rats with experimental chronic neuropathic pai...This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats. The behavior of rats with experimental chronic neuropathic pain was observed, and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected. Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sham-operated group, sham-rTMS group, 1 Hz group and 20 Hz group (8 rats in each group). Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group. Then we applied different frequencies of rTMS to the primary motor cortex (M1) contralateral to the pain side once daily for 10 consecutive days. Pain behavior scores were observed before and after treatment. Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs. Double immunofluorescent labeling for glial fibrillary acidic protein (GFAP) and 5-bromo-2- deoxyuridine (BrdU) was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn. After rTMS treatment, the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group (P〈0.05). Moreover, the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group (P〈0.05), suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated. Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P〈0.01) after rTMS treatment. Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain. After rTMS treatment, the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P〈0.05). In addition, the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain (P〈0.05). It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.展开更多
Schwann cell proliferation,migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury.Lithium promotes remyelination by Schwann cells and improves peripheral ne...Schwann cell proliferation,migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury.Lithium promotes remyelination by Schwann cells and improves peripheral nerve regeneration.However,whether lithium modulates other phenotypes of Schwann cells,especially their proliferation and migration remains elusive.In the current study,primary Schwann cells from rat sciatic nerve stumps were cultured and exposed to 0,5,10,15,or 30 mM lithium chloride(LiCl)for 24 hours.The effects of LiCl on Schwann cell proliferation and migration were examined using the Cell Counting Kit-8,5-ethynyl-2′-deoxyuridine,Transwell and wound healing assays.Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays showed that 5,10,15,and 30 mM LiCl significantly increased the viability and proliferation rate of Schwann cells.Transwell-based migration assays and wound healing assays showed that 10,15,and 30 mM LiCl suppressed the migratory ability of Schwann cells.Furthermore,the effects of LiCl on the proliferation and migration phenotypes of Schwann cells were mostly dose-dependent.These data indicate that lithium treatment significantly promotes the proliferation and inhibits the migratory ability of Schwann cells.This conclusion will inform strategies to promote the repair and regeneration of peripheral nerves.All of the animal experiments in this study were ethically approved by the Administration Committee of Experimental Animal Center of Nantong University,China(approval No.20170320-017)on March 2,2017.展开更多
The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characte...The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. There was significant difference between the experimental group and the control group (P〈0.001). Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.展开更多
Although alpha-synuclein is generally thought to have a pathological role in Parkinson's disease, accumulative evidence exists that alpha-synuclein has a neuroprotective effect. The aim of this study was to evaluate ...Although alpha-synuclein is generally thought to have a pathological role in Parkinson's disease, accumulative evidence exists that alpha-synuclein has a neuroprotective effect. The aim of this study was to evaluate the effect of extracellular alpha-synuclein on dopaminergic cell survival. We assessed cell viability using the 3-(4,5-dimethyt-thiazol-2-yt)-2,5-diphenyltertazolium bromide (MTT) assay both in undifferentiated SH-SY5Y (SHSY) cells and neuronally-differentiated SH-SY5Y (ndSHSY) cells after 24 hour treatment with monomeric alpha-synuclein at various concentrations (0 [control], 50, 100 nmol/L, 1 IJmol/L). To determine whether cell viability assessed by MTT assay was affected by cell proliferation, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay was per- formed. Level of both Akt and phosphorylated Akt was measured using western blot method in ndSHSY cells with or without 24 hour alpha-synuclein treatment. Cell viability was increased in ndSHSY cells at the nanomolar concentration of alpha-synuclein, but not in SHSY cells. Proportion of BrdU-positive ndSHSY cells was decreased in alpha-synuclein-treated group compared with control group. Level of phosphorylated Akt in alpha-synuclein-treated group was higher compared with the control group. Our study shows that extracellular alpha-synuclein at nanomolar concentra- tion benefits dopaminergic cell survival via Akt pathway.展开更多
5-Bromo-2′-deoxyuridine(BrdU)is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle.BrdU is widely used in fate-mapping studies of embryonic and adult neu...5-Bromo-2′-deoxyuridine(BrdU)is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle.BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons,however side effects on neural stem cells and their progeny have been reported.In vivo astrocyte-to-neuron(AtN)conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons.The BrdU-labeling strategy has been used to trace astrocyte-converted neurons,but whether BrdU has any effect on the AtN conversion is unknown.Here,while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury,we accidentally discovered that BrdU inhibited AtN conversion.We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex.Although most NeuroD1-infected astrocytes were converted into neurons,the number of BrdU-labeled neurons was surprisingly low.To exclude the possibility that this BrdU inhibition was caused by the ischemic injury,we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU.Surprisingly,we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group.These results revealed an unexpected inhibitory effect of BrdU on AtN conversion,suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.展开更多
This article reported the distribution of DNase-1 sensitive regions in genomes of three Burkitt's lymphoma cell lines, P3HR-1, Raji and Ramos cell lines using a new method of in situ nick translation of chromosome...This article reported the distribution of DNase-1 sensitive regions in genomes of three Burkitt's lymphoma cell lines, P3HR-1, Raji and Ramos cell lines using a new method of in situ nick translation of chromosomes substituted completely by BrdU. The results showed that the Blym locus on chromosomes In three cell lines and the c-myc locus on chromosomes in P3HR-1 were the DNase-1 sensitive regions and found that the rearrangemental sites of chromosomes present in three Burkltt' s lymphoma cell lines were sensitive to DNase-1 digestion, Indicating that c-myc, bcl-1 genes located at the rearrangemental sites and the Blym gene in Burkltt' s lymphoma are the active genes having the capability of expression.展开更多
Background The neurogenesis in retina of adult mammals is generally abolished, and this renders the retina lack of regenerative capacity. Despite this, there is a small population of nestin-positive cells in the cilia...Background The neurogenesis in retina of adult mammals is generally abolished, and this renders the retina lack of regenerative capacity. Despite this, there is a small population of nestin-positive cells in the ciliary epithelium which retains neurogenic potential. The present study aimed at investigating the effect of two drugs, corticosterone and paroxetine, on the cell proliferation of the ciliary body. Methods Adult Sprague-Dawley rats were given vehicle, corticosterone, paroxetine, or both corticosterone and paroxetine treatment for 14 days. Cell proliferation in the ciliary body was quantified using 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. Co-labelling of BrdU and stem cell marker was used to phenotype the BrdU immunoreactive cells. Results Corticosterone treatment suppressed while paroxetine treatment increased the cell proliferation of the ciliary body. Co-labelling with cell markers revealed that the BrdU positive cells also showed nestin expression but not glial fJbrillary acidic protein (GFAP). Conclusions The results illustrate that proliferation of retinal progenitor cells situated in ciliary body are subjected to regulation by selective serotonin reuptake Jnhibitors (SSRI) and corticosteroJd, which is similar to our previous findings in neurogenic regions in central nervous system (CNS). Paroxetine treatment could reverse the suppressive effect of corticosterone on ciliary body cell proliferation. This provides information for future investigation of retinal stem cell biology and potential treatment of retinal degenerative diseases.展开更多
文摘The halopyrimidine 5-bromo-2′-deoxyuridine(BrdU)is an exogenous marker of DNA synthesis.Since the introduction of monoclonal antibodies against BrdU,an increasing number of methodologies have been used for the immunodetection of this synthesized bromine-tagged base analogue into replicating DNA.BrdU labeling is widely used for identifying neuron precursors and following their fate during the embryonic,perinatal,and adult neurogenesis in a variety of vertebrate species including birds,reptiles,and mammals.Due to BrdU toxicity,its incorporation into replicating DNA presents adverse consequences on the generation,survival,and settled patterns of cells.This may lead to false results and misinterpretation in the identification of proliferative neuroblasts.In this review,I will indicate the detrimental effects of this nucleoside during the development of the central nervous system,as well as the reliability of BrdU labeling to detect proliferating neuroblasts.Moreover,it will show factors influencing BrdU immunodetection and the contribution of this nucleoside to the study of prenatal,perinatal,and adult neurogenesis.Human adult neurogenesis will also be discussed.It is my hope that this review serves as a reference for those researchers who focused on detecting cells that are in the synthetic phase of the cell cycle.
文摘This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats. The behavior of rats with experimental chronic neuropathic pain was observed, and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected. Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sham-operated group, sham-rTMS group, 1 Hz group and 20 Hz group (8 rats in each group). Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group. Then we applied different frequencies of rTMS to the primary motor cortex (M1) contralateral to the pain side once daily for 10 consecutive days. Pain behavior scores were observed before and after treatment. Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs. Double immunofluorescent labeling for glial fibrillary acidic protein (GFAP) and 5-bromo-2- deoxyuridine (BrdU) was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn. After rTMS treatment, the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group (P〈0.05). Moreover, the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group (P〈0.05), suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated. Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P〈0.01) after rTMS treatment. Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain. After rTMS treatment, the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P〈0.05). In addition, the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain (P〈0.05). It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.
基金supported by the National Natural Science Foundation of China,No.81970820(to HX)
文摘Schwann cell proliferation,migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury.Lithium promotes remyelination by Schwann cells and improves peripheral nerve regeneration.However,whether lithium modulates other phenotypes of Schwann cells,especially their proliferation and migration remains elusive.In the current study,primary Schwann cells from rat sciatic nerve stumps were cultured and exposed to 0,5,10,15,or 30 mM lithium chloride(LiCl)for 24 hours.The effects of LiCl on Schwann cell proliferation and migration were examined using the Cell Counting Kit-8,5-ethynyl-2′-deoxyuridine,Transwell and wound healing assays.Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays showed that 5,10,15,and 30 mM LiCl significantly increased the viability and proliferation rate of Schwann cells.Transwell-based migration assays and wound healing assays showed that 10,15,and 30 mM LiCl suppressed the migratory ability of Schwann cells.Furthermore,the effects of LiCl on the proliferation and migration phenotypes of Schwann cells were mostly dose-dependent.These data indicate that lithium treatment significantly promotes the proliferation and inhibits the migratory ability of Schwann cells.This conclusion will inform strategies to promote the repair and regeneration of peripheral nerves.All of the animal experiments in this study were ethically approved by the Administration Committee of Experimental Animal Center of Nantong University,China(approval No.20170320-017)on March 2,2017.
基金This project was supported by a grant from National Natural Sciences Founcation of China (No. 30170911).
文摘The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. There was significant difference between the experimental group and the control group (P〈0.001). Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.
基金supported by the Seoul National University Hospital(SNUH)Research Fund,No.03-2010-0240
文摘Although alpha-synuclein is generally thought to have a pathological role in Parkinson's disease, accumulative evidence exists that alpha-synuclein has a neuroprotective effect. The aim of this study was to evaluate the effect of extracellular alpha-synuclein on dopaminergic cell survival. We assessed cell viability using the 3-(4,5-dimethyt-thiazol-2-yt)-2,5-diphenyltertazolium bromide (MTT) assay both in undifferentiated SH-SY5Y (SHSY) cells and neuronally-differentiated SH-SY5Y (ndSHSY) cells after 24 hour treatment with monomeric alpha-synuclein at various concentrations (0 [control], 50, 100 nmol/L, 1 IJmol/L). To determine whether cell viability assessed by MTT assay was affected by cell proliferation, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay was per- formed. Level of both Akt and phosphorylated Akt was measured using western blot method in ndSHSY cells with or without 24 hour alpha-synuclein treatment. Cell viability was increased in ndSHSY cells at the nanomolar concentration of alpha-synuclein, but not in SHSY cells. Proportion of BrdU-positive ndSHSY cells was decreased in alpha-synuclein-treated group compared with control group. Level of phosphorylated Akt in alpha-synuclein-treated group was higher compared with the control group. Our study shows that extracellular alpha-synuclein at nanomolar concentra- tion benefits dopaminergic cell survival via Akt pathway.
基金supported by the Natural Science Foundation of Guangdong Province of China,Nos.2021A1515011237(to WL),2020A1515010854(to QSW)the National Natural Science Foundation of China,Nos.U1801681(to GC),31701291(to WL)the Guangdong Province Science and Technology Planning Project of China,No.2018B030332001(to GC)。
文摘5-Bromo-2′-deoxyuridine(BrdU)is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle.BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons,however side effects on neural stem cells and their progeny have been reported.In vivo astrocyte-to-neuron(AtN)conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons.The BrdU-labeling strategy has been used to trace astrocyte-converted neurons,but whether BrdU has any effect on the AtN conversion is unknown.Here,while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury,we accidentally discovered that BrdU inhibited AtN conversion.We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex.Although most NeuroD1-infected astrocytes were converted into neurons,the number of BrdU-labeled neurons was surprisingly low.To exclude the possibility that this BrdU inhibition was caused by the ischemic injury,we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU.Surprisingly,we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group.These results revealed an unexpected inhibitory effect of BrdU on AtN conversion,suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.
文摘This article reported the distribution of DNase-1 sensitive regions in genomes of three Burkitt's lymphoma cell lines, P3HR-1, Raji and Ramos cell lines using a new method of in situ nick translation of chromosomes substituted completely by BrdU. The results showed that the Blym locus on chromosomes In three cell lines and the c-myc locus on chromosomes in P3HR-1 were the DNase-1 sensitive regions and found that the rearrangemental sites of chromosomes present in three Burkltt' s lymphoma cell lines were sensitive to DNase-1 digestion, Indicating that c-myc, bcl-1 genes located at the rearrangemental sites and the Blym gene in Burkltt' s lymphoma are the active genes having the capability of expression.
文摘Background The neurogenesis in retina of adult mammals is generally abolished, and this renders the retina lack of regenerative capacity. Despite this, there is a small population of nestin-positive cells in the ciliary epithelium which retains neurogenic potential. The present study aimed at investigating the effect of two drugs, corticosterone and paroxetine, on the cell proliferation of the ciliary body. Methods Adult Sprague-Dawley rats were given vehicle, corticosterone, paroxetine, or both corticosterone and paroxetine treatment for 14 days. Cell proliferation in the ciliary body was quantified using 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. Co-labelling of BrdU and stem cell marker was used to phenotype the BrdU immunoreactive cells. Results Corticosterone treatment suppressed while paroxetine treatment increased the cell proliferation of the ciliary body. Co-labelling with cell markers revealed that the BrdU positive cells also showed nestin expression but not glial fJbrillary acidic protein (GFAP). Conclusions The results illustrate that proliferation of retinal progenitor cells situated in ciliary body are subjected to regulation by selective serotonin reuptake Jnhibitors (SSRI) and corticosteroJd, which is similar to our previous findings in neurogenic regions in central nervous system (CNS). Paroxetine treatment could reverse the suppressive effect of corticosterone on ciliary body cell proliferation. This provides information for future investigation of retinal stem cell biology and potential treatment of retinal degenerative diseases.