Ginkgolide B promotes the proliferation and differentiation of neural stem cells following cerebral ischemia/reperfusion injury,both in vivo and in vitro

Neural stem cells have great potential for the development of novel therapies for nervous system diseases.However,the proliferation of endogenous neural stem cells following brain ischemia is insufficient for central nervous system self-repair.Ginkgolide B has a robust neuroprotective effect.In this study,we investigated the cell and molecular mechanisms underlying the neuroprotective effect of ginkgolide B on focal cerebral ischemia/reperfusion injury in vitro and in vivo.Neural stem cells were treated with 20,40 and 60 mg/L ginkgolide B in vitro.Immunofluorescence staining was used to assess cellular expression of neuron-specific enolase,glial fibrillary acid protein and suppressor of cytokine signaling 2.After treatment with 40 and 60 mg/L ginkgolide B,cells were large,with long processes.Moreover,the proportions of neuron-specific enolase-,glial fibrillary acid protein-and suppressor of cytokine signaling 2-positive cells increased.A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion.Six hours after ischemia,ginkgolide B（20 mg/kg） was intraperitoneally injected,once a day.Zea Longa＇s method was used to assess neurological function.Immunohistochemistry was performed to evaluate the proportion of nestin-,neuron-specific enolase-and glial fibrillary acid protein-positive cells.Real-time quantitative polymerase chain reaction was used to measure m RNA expression of brain-derived neurotrophic factor and epidermal growth factor.Western blot assay was used to analyze the expression levels of brain-derived neurotrophic factor and suppressor of cytokine signaling 2.Ginkgolide B decreased the neurological deficit score,increased the proportion of nestin-,neuron-specific enolase-and glial fibrillary acid protein-positive cells,increased the m RNA expression of brain-derived neurotrophic factor and epidermal growth factor,and increased the expression levels of brain-derived neurotrophic factor and suppressor of cytokine signaling 2 in the ischemic penumbra.Together,the in vivo and in vitro findings suggest that ginkgolide B improves neurological function by promoting the proliferation and differentiation of neural stem cells in rats with cerebral ischemia/reperfusion injury.

Korean red ginseng promotes hippocampal neurogenesis in mice

Neurogenesis in the adult hippocampus plays a major role in cognitive ability of animals including learning and memory.Korean red ginseng (KRG) has long been known as a medicinal herb with the potential to improve learning and memory;however,the mechanisms are still elusive.Therefore,we evaluated whether KRG can promote cognitive function and enhance neurogenesis in the hippocampus.Eight-week-old male C57BL/6 mice received 50 mg/kg of 5-bromo-2′-deoxyuridine (BrdU) intraperitoneally and 100 mg/kg of KRG or vehicle orally once a day for 14 days.Pole,Rotarod and Morris water maze tests were performed and the brains were collected after the last behavioral test.Changes in the numbers of BrdU- and BrdU/ doublecortin (DCX;a marker for neuronal precursor cells and immature neurons)-positive cells in the dentate gyrus and the gene expression of proliferating cell nuclear antigen (a marker for cell differentiation),cerebral dopamine neurotrophic factor and ciliary neurotrophic factor in the hippocampus were then investigated.KRG-treated mice came down the pole significantly faster and stood on the rotarod longer than vehicle-treated mice.The Morris water maze test showed that KRG administration enhanced the learning and memory abilities significantly.KRG also significantly increased BrdU- and BrdU/DCX-positive cells in the dentate gyrus as well as the proliferating cell nuclear antigen,cerebral dopamine neurotrophic factor and ciliary neurotrophic factor mRNA expression levels in the hippocampus compared to vehicle.Administration of KRG promotes learning and memory abilities,possibly by enhancing hippocampal neurogenesis.This study was approved by the Pusan National University Institutional Animal Care and Use Committee (approval No.PNU-2016-1071) on January 19,2016.

Neural stem cell proliferation and differentiation in a neonatal rat model of hypoxia/ischemia injury Acupuncture at Ren,Du,and urinary bladder meridians

BACKGROUND： Acupuncture improves the prognosis of neonatal hypoxic-ischemic encephalopathy （HIE）. However, the cytological mechanism of acupuncture therapy remains poorly understood. In situ neural stem cell （NSC） proliferation theory proposes that the proliferation and differentiation of NSCs plays an important role in HIE treatment with acupuncture. OBJECTIVE： To investigate NSC proliferation and differentiation in the brain of a rat model of HIE during acupuncture at Ren, Du, and urinary bladder meridians. DESIGN, TIME AND SEB＇ING： A randomized, controlled animal experiment was performed at the Central Laboratory of Shantou University Medical College from July 2005 to June 2009. MATERIALS： A 32# 1-cun stainless steel acupuncture needle was purchased from Suzhou Acupuncture Supplies Co. Ltd., China.METHODS： A total of 90 Sprague-Dawley rats, aged 7 days, were randomly assigned to acupuncture, model and normal groups, with 30 animals in each group. Animals in acupuncture and model groups were subjected to left common carotid artery ligation followed by hypoxia for 2 hours to establish neonatal HIE models. Acupuncture group rats underwent acupuncture at Ren, Du, and urinary bladder meridians, once a day. MAIN OUTCOME MEASURES： The number, appearance, and distribution of bromodeoxyuridine （BrdU）-positive cells in the cerebral cortex and hippocampus of each group were compared. In addition, NSC differentiation in the occipital cortex and hippocampal dentate gyrus 40 days following model establishment was detected.RESULTS： BrdU-positive cells were dispersed in the cerebral cortex and hippocampus. The number of BrdU-positive cells in occipital cortex and hippocampal dentate gyrus of HIE rats remained unchanged following 3 and 7 days of acupuncture, but a significant increase was detected on days 14 and 28 （P 〈 0.01 or P 〈 0.05）. At 40 days, immunofluorescence showed that a majority of BrdU-positive cells were co-lableled with the neuron marker, and neuron specific enolase, and a few were co-labeled with the astrocyte marker, and glial fibrillary acidic protein. CONCLUSION： Acupuncture at Ren, Du, and urinary bladder meridians promoted NSC proliferation in the occipital cortex and hippocampal dentate gyrus of HIE rats. Moreover, acupuncture-induced neoformative NSCs mostly differentiated into neurons.

Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.

Mammalin cochlear supporting cells transdifferentiation into outer hair cells

Objective To study the recovery of the outer hair cells in the bat cochlea after gentamicin exposure. Methods Bats were injected with a daily dose of gentamicin for 15 consecutive days and bromodeoxyuridine (BrdU) was given from day 16 to day 40 of this recovery phase. Hearing was assessed by overt acoustic behavior and auditory brainstem responses analysis, which was performed one day prior to the first injection and a day after the last injection (day 16). On day 40 animals were sacrificed for detection of cells that could take up BrdU. Results After 15 days of gentamicin treatment, all of the animals were proved to be deafened with significant increases of ABR thresholds, compared with control group. The findings in immunocytochemical stained samples and scanning electron microscopy revealed that BrdU labeled nuclei were observed in the cochlea in all of the deafened animals most commonly in the regions of the first-row and second-row Deiter’s cells (DCs) and occasionally in the regions of the third-row DCs. Conclusion We suggest that, under sufficient drug and enough time, the bat cochlear supporting cells can directly transdifferentiate into the outer hair cells after aminoglycoside exposure. This transdifferentation process is essential for repair of outer hair cells and recovery of normal function after gentamicin exposure.

Probing the Cell Cycle with Flow Cytometry

Flow cytometry is a versatile technique to study different aspects of the cell cycle from subpopulations of cells to detailed cell kinetic information. In this paper a basic review of cell kinetic parameters is presented followed by detailed descriptions of the different flow cytometric methodologies that can be used to extract pertinent information for a particular study. The methodologies range from simple DNA profile analysis, the use of bromodeoxyuridine to cell cycle-associated proteins such as the cyclins.

Effects of Scutellaria baicalensis on neurogenesis in the hippocampal dentate gyrus and on spatial memory of adult rats

We investigated the effects of ethanol extracted Scutellaria baicalensis（EESB） on spatial memory function and neurogenesis in the hippocampal dentate gyrus.Adult Sprague-Dawley rats were orally administered 50,100,or 200 mg/kg of EESB for 6 successive days.The radial-arm maze test showed that 200 mg/kg of EESB improved the spatial memory of adult rats.Confocal microscopy results showed that 100 mg/kg of EESB increased the number of bromodeoxyuridine（BrdU）-and neuron-specific nuclear protein-positive cells in the granular cell layer,and that 100 and 200 mg/kg of EESB increased the number of BrdU-/neuron-specific nuclear protein-positive cells in the sub-granular zone.200 mg/kg of EESB increased the number of BrdU-/glial fibrillary acid protein-positive cells in the subgranular zone.These findings indicate that EESB can effectively promote neurogenesis in the hippocampal dentate gyrus and improve spatial memory function.

Psilocybin facilitates fear extinction in mice by promoting hippocampal neuroplasticity

Background:Posttraumatic stress disorder(PTSD)and depression are highly comorbid.Psilocybin exerts substantial therapeutic effects on depression by promoting neuroplasticity.Fear extinction is a key process in the mechanism of first-line exposure-based therapies for PTSD.We hypothesized that psilocybin would facilitate fear extinction by promoting hippocampal neuroplasticity.Methods:First,we assessed the effects of psilocybin on percentage of freezing time in an auditory cued fear conditioning(FC)and fear extinction paradigm in mice.Psilocybin was administered 30 min before extinction training.Fear extinction testing was performed on the first day;fear extinction retrieval and fear renewal were tested on the sixth and seventh days,respectively.Furthermore,we verified the effect of psilocybin on hippocampal neuroplasticity using Golgi staining for the dendritic complexity and spine density,Western blotting for the protein levels of brain derived neurotrophic factor(BDNF)and mechanistic target of rapamycin(mTOR),and immunofluorescence staining for the numbers of doublecortin(DCX)-and bromodeoxyuridine(BrdU)-positive cells.Results:A single dose of psilocybin(2.5 mg/kg,i.p.)reduced the increase in the percentage of freezing time induced by FC at 24 h,6th day and 7th day after administration.In terms of structural neuroplasticity,psilocybin rescued the decrease in hippocampal dendritic complexity and spine density induced by FC;in terms of neuroplasticity related proteins,psilocybin rescued the decrease in the protein levels of hippocampal BDNF and mTOR induced by FC;in terms of neurogenesis,psilocybin rescued the decrease in the numbers of DCX-and BrdU-positive cells in the hippocampal dentate gyrus induced by FC.Conclusions:A single dose of psilocybin facilitated rapid and sustained fear extinction;this effect might be partially mediated by the promotion of hippocampal neuroplasticity.This study indicates that psilocybin may be a useful adjunct to exposure-based therapies for PTSD and other mental disorders characterized by failure of fear extinction.

BrdU抗体染色技术检测昆虫器官的细胞增殖

BrdU(5-bromo-2'-deoxyuridine)是一种人工合成的核苷酸类似物,常用于标记活体组织的增殖细胞。它的结构类似于胸腺嘧啶,在细胞分裂的S期,可以取代胸腺嘧啶而插入正在复制的细胞DNA中。通过免疫组化手段,用BrdU抗体检测整合在细胞DNA中的BrdU分子,可以反映出细胞周期的活力,即细胞增殖的速率。本文介绍一种优化的BrdU染色流程,用来标记昆虫小器官的细胞增殖速率。通过这种技术,我们重新评估了Dpp信号通路中的转录抑制因子Brinker在翅芽发育过程中调控细胞增殖的作用,发现它并不是以前所认为的在翅囊区是一种生长抑制因子。

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn

BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. CONCLUSIONS: Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.

Relations of proliferative activities of gastric carcinoma cells to lymphatic involvement, venous invasion and prognosis

Background This study was to evaluate bivariate bromodeoxyuridine(BrdUrd)/DNA flow cytometric analysis in detection of gastric carcinoma and to study the relations of cellular BrdUrd labeling indices (LI), G_2/M-phase fraction(G_2/MPF) and DNA ploidy pattern to lymphatic involvement, venous invasion and prognosis.Methods Fresh tumor samples from 60 patients with gastric carcinoma were analyzed by bivariate BrdUrd/DNA flow cytometry. The results were correlated with lymphatic vessel invasion, lymphatic node metastasis, the number of matastatic lymphatic nodes, and venous invasion. Propidium iodide (PI) was used as a fluorescent probe for total cellular DNA, and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescent-labeled goat anti-mouse antibody was used as a second antibody. S-phase fractions were measured by in vitro BrdUrd labeling, and DNA ploidy and G_2/MPF were also measured. Comparison of survival was performed with the log-rank test, the Chi-square test for qualitative data, and Student’s t test for quantu data. Results BrdUrd LI and G_2/MPF values were significantly higher in tumors with lymphatic vessel invasion than in those without invasion respectively (P<0.01); the patients who had tumors with lymphatic vessel invasion showed a significantly poor prognosis (P<0.01). Both BrdUrd LI and G_2/MPF values were significantly higher in tumors with lymphatic node metastasis than in those without metastasis (P<0.01). A statistical significant difference was noted in the 5-year survival rates between the patients with lymph node metastasis and those without metastasis. Compared with diploid carcinoma, the incidence of lymph node metastasis was significantly higher in aneuploid carcinoma (P<0.05), and the patients with aneuploid carcinoma showed a significantly poor prognosis (P<0.05). BrdUrd LI was significantly higher in patients with more than 5 metastatic lymph nodes than those with 1-4 metastatic lymph nodes (P<0.05) and those without metastasis (P<0.01). G_2/MPF values in those patients either with more than 5 metastatic lymph nodes or 1-4 metastatic lymph nodes were higher than those without metastasis (P<0.01 and P<0.05). A statistical significance was seen in the 5-year survival rates among the patients with no metastatic lymph node, 1-4 metastatic nodes and more than 5 metastatic nodes (P<0.01). G_2/MPF values were significantly higher in patients with venous invasion than in those without invasion (P<0.01).Conclusions Positive correlations exist between cellular BrdUrd LI, G_2/MPF with lymphatic involvement and prognosis, and DNA aneuploid with lymphatic involvement and prognosis. The same was true between G_2/MPF value and venous invasion in gastric carcinoma.