Abstract W P248: miRNA Profiling of the Ischaemic Penumbra

MicroRNAs (miRNAs) are small non-coding RNA molecules (20 - 24 nucleotides) that inhibit mRNA translation. Demonstrated to have key roles in normal CNS development & function, they have also emerged to have effecter roles in the pathogenesis & endogenous repair mechanisms following stroke. To select 2 miRNAs to modulate therapeutically we profiled miRNA expression of the evolving (24h) & final (72h) peri-infarct tissue of adult spontaneously hypertensive stroke-prone rats (SHRSP) following 45 min transient middle cerebral artery occlusion (tMCAO). T2-weighted magnetic resonance imaging (MRI) was used for accurate dissection of the peri-infarct tissue, with equivalent brain regions taken from time-matched shams (n=6/group). Of the 754 miRNAs evaluated (TaqMan® human miRNA microarray card v3.0 Applied Biosystems) 89 were determined as differently regulated following tMCAO. 22 of these miRNAs were relevant in stroke & were thus validated by Taqman® qRT-PCR using specific probes (n=9 /group). 5 miRNAs were successfully validated; miR-34b & miR-520b were selected as miRNAs of interest due to their novelty, time of endogenous regulation & targets. An in vitro study to determine whether upregulation/knock-down of these miRNAs would demonstrate functional effects in classical hypoxic pathways was performed. A rat neuronal cell line (B50) & glial cell line (B92) were subjected to 9hr hypoxia (1% O2 -serum) & 24h reoxygenation (+serum) +/- miR-34b or miR-520b regulation. Upregulation of either miRNA in B50 cells demonstrated a reduction in apoptosis, assessed qualitatively by Caspase-3 immunocytochemistry & quantitatively by cell death detection ELISA (p<0.01 vs hypoxic non-treated cells (NTC)). Upregulation of either miRNA in B92 cells significantly reduced superoxide production, assessed by electron paramagnetic resonance (p<0.001 vs hypoxic NTC). MiR-520b significantly lowered levels of lipid peroxidation in B92 cells, assessed by malondialdehyde assay, & both were significantly effective in B50 cells (p<0.01 vs hypoxic NTC). These data suggest miR-34b & -520b upregulation ameliorates damage following hypoxia/reperfusion in cerebral cell lines. Future studies will assess the effect of modulating these miRNAs in vivo.

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Cell-Driven Angiogenesis and Neurogenesis after Stroke Is Regulated by Platelet’s Microparticles.

Blood ◽

10.1182/blood.v112.11.1899.1899 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1899-1899 ◽

Cited By ~ 1

Author(s):

David Varon ◽

Yael Haion ◽

Alexander Brill ◽

Ronen Leker

Keyword(s):

Survival Rates ◽

Brain Regions ◽

Artery Occlusion ◽

Survival Times ◽

Differentiation Potential ◽

Ischemic Brain ◽

Spontaneously Hypertensive ◽

Repair Mechanisms

Abstract Repair mechanisms based on the proliferation of endogenous cells have been identified in ischemic brain regions suggesting that precursors at the ventricular surface proliferate, migrate to sites of injury and differentiate into neurons. However, this response is significantly limited by short survival times and poor differentiation capacity of these cells. External factors can boost endogenous neural stem cells (eNSC)-dependent repair mechanisms in the cerebral cortex. Upon activation, platelets shed platelet derived microparticles (PMP) which contain a variety of growth factors central to angiogenesis and neurogenesis. We have previously reported a pro angiogenic effect of PMPs, both in vitro and in vivo. We thus hypothesized that the functional benefits obtained from the presence of newborn cells can be further enhanced by increasing the survival rates, migration and differentiation potential of these cells, as well as by promoting angiogenic response, applying PMPs at the site of a stroke. Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO) and were given BrdU to label newborn cells. Animals were treated with vehicle or PMP delivered via a biodegradable polymer applied to the brain surface. Rats were tested with a neurological severity score and infarct volumes were measured at 90 days post –PMCAO. We used immunohistochemistry to determine the fate of newborn cells and to count blood vessels in the ischemic brain. The results show that PMP treatment led to a dose dependent increase in neurogenesis and angiogenesis at the infarct boundary zone. PMP also significantly improved behavioral deficits without affecting infarct volumes.

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Analysis on Homoeopathic Preparation of Asterias Rubens for Evaluating Anti Breast Cancer Cell Line using Similia Principle

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11ispl4.4075 ◽

2020 ◽

Vol 11 (SPL4) ◽

pp. 805-808

Author(s):

Ravikumar Raju ◽

Teja ◽

Sravanathi P ◽

Muthu Babu K

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Cancer Cell ◽

Cancer Cell Line ◽

In Vitro Study ◽

In Vivo Studies ◽

Asterias Rubens ◽

Mcf 7

Breast cancer is the subsequent foremost reason of cancer death in a woman and ranks as the primary foremost reason of death in India. In its conduct, several measures and recommendation are considered. Homoeopathic medicines are one of the part of a corresponding, and another medicine is utilized for the treatment of cancer. The main purpose of the investigation is to evaluate the anticancer action of homoeopathic arrangements of Asterias rubens on the basis of the similia principle. We directed an in vitro study using MTT assay to control the result of ultra diluted homoeopathic preparation in contradiction of two human breast glandular cancer cell lines(MCF-7 and MDA-MD- 231), frequently used for the breast cancer treatment, by testing the feasibility of breast cancer (MCF-7 and MDA-MD-231) cell line, with various attenuations of Asterias rubens at 24 hrs. Multiple comparisons between tested reagents at different concentrations confirmed the significance of the said results. At a dilution of 1:25 6CH and 30CH potency shown superior activity on MCF-7 and no such significant changes on MDA-MD-231 at any dilutions As it fails to offer estrogen receptor(ER) Also progesterone receptor (PR) expression, and also HER2 (human epidermal development variable receptor2) so continuously a triple-negative breast cancer it will be a hostility manifestation for breast cancer with restricted medicine choices. However, further potency needs to be tested. These preliminary significant results warrant further in vitro and in vivo studies to estimate the possible of Asterias rubens a medicine to treat breast cancer.

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MPTP-Induced Modulation of Neurotransmitters in SH-SY5Y Human Neuroblastoma Cells

International Journal of Toxicology ◽

10.1080/109158198225919 ◽

1998 ◽

Vol 17 (6) ◽

pp. 677-701 ◽

Cited By ~ 1

Author(s):

Xiaoou Song ◽

Marion Ehrich

Keyword(s):

Cell Line ◽

Neuroblastoma Cells ◽

Neuronal Cell ◽

Cholinergic Antagonists ◽

Mao Inhibitor ◽

Human Neuroblastoma ◽

Hydroxyindoleacetic Acid ◽

Mao Activity

Neurotoxic effects of MPTP(1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) were evaluated in vitro using a human neuronal cell line, SH-SY5Y, that contained features contributing to expression of MPTP toxicity in vivo, namely, a transport system for dopam ine (DA) and monam ine oxidase (MAO) activity. In this model system, MPTP was found to reduce levels of catecholamines (DA, norepinephrine, epinephrine), serotonin (5-HT), and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA). MPTP enhanced 3H-DA release, which could contribute to the reduction in DA concentrations seen in these cells. In addition, MPTP inhibited MAO activity (Ki 2.26 X 10-5 M). Pretreatment with the MAO inhibitor pargy-line protected the cells from MPTP-induced alterations of catecholamines and the decrease in 5-HT. In this in vitro model, the cholinergic antagonists atro-pine and A-tubocurarine also protected cells from MPTP-induced alterations of catecholamines. The capability of cholinergic antagonists to prevent the MPTP-induced alterations of catecholamine concentrations suggests a possible cholinergic contribution to MPTP neurotoxicity in this cell line.

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Expression of trkA cDNA in neuroblastomas mediates differentiation in vitro and in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7447-7456.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7447-7456

Author(s):

H Matsushima ◽

E Bogenmann

Keyword(s):

Cell Differentiation ◽

Cell Line ◽

Neuronal Cell ◽

Human Neuroblastoma ◽

Myc Oncogene ◽

Ngf Receptor ◽

Neuronal Cell Differentiation ◽

Mature Neurons

The human trkA cDNA was transfected into a malignant human neuroblastoma (NB) cell line (HTLA230) to investigate its role in NB growth and differentiation. This cell line lacks expression of both endogenous trkA and gp75NGFR genes. Transfectants expressing the trkA mRNA and surface-bound receptors transcriptionally activate immediate-early genes (c-fos, c-jun, and jun-B) following nerve growth factor (NGF) stimulation. NGF treatment induces growth arrest as well as down-regulation of the amplified N-myc oncogene. Genes selectively expressed in mature neurons (SCG-10, ret proto-oncogene, GAP-43, etc.) are transcriptionally activated, and neurite outgrowth further demonstrates differentiation of transfectants following NGF stimulation. trkA-expressing NB cells remain tumorigenic in nude mice; however, subcutaneous treatment of tumor-bearing mice with NGF induces Schwannian and neuronal cell differentiation similar to the induction seen in human ganglioneuroblastomas. Thus, trkA expression in HTLA230 cells is sufficient to generate a functional NGF receptor complex that leads to growth-arrested and differentiated NB cells in vitro and in vivo in the presence of NGF. Hence, NGF may play a crucial role in NB cell differentiation and regression in vivo.

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Neuroprotective effect of Danshensu derivatives as anti-ischaemia agents on SH-SY5Y cells and rat brain

Bioscience Reports ◽

10.1042/bsr20130032 ◽

2013 ◽

Vol 33 (4) ◽

Cited By ~ 14

Author(s):

Sunisa Seetapun ◽

Jia Yaoling ◽

Yang Wang ◽

Yi Zhun Zhu

Keyword(s):

Rat Brain ◽

Infarct Volume ◽

Neuroprotective Effect ◽

In Vitro Study ◽

Artery Occlusion ◽

Neuroprotective Effects ◽

Inhibit Lipid Peroxidation ◽

Cysteine Derivative

Novel Danshensu derivatives (3–8) were designed and synthesized to improve bioactivity based on the strategy of ‘medicinal chemical hybridization’. Our previous studies indicated that these compounds exhibited noticeable cardioprotective activities. Here, we investigate whether these novel Danshensu derivatives exert neuroprotective activities. An in vitro study revealed that these compounds could increase cell viability and reduce LDH (lactate dehydrogenase) leakage. Moreover, Danshensu-cysteine derivative compounds 6 and 8 could significantly inhibit lipid peroxidation of cell membrane and regulate the expression of apoptosis-related protein (Bcl-2, Bax and caspase 3). An in vivo study demonstrated that treatment with compound 6 at 30 mg/kg markedly decreased the infarct volume of MCAO (middle cerebral artery occlusion) insulted rat brain. Furthermore, treatment with compound 6 showed the antioxidant capacity by increasing the activity of SOD (superoxide dismutase) and GPx (glutathione peroxidase) and decreasing the level of MDA (malondialdehyde) and the ROS (reactive oxygen species) production significantly. These results suggested that these novel conjugates exert significant neuroprotective effects as anti-ischaemia agents and those with high potential merit further investigation.

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In Vitro Labeling Strategies for Identifying Primary Neural Tissue and a Neuronal Cell Line after Transplantation in the Cns

Cell Transplantation ◽

10.1177/096368979300200207 ◽

1993 ◽

Vol 2 (2) ◽

pp. 131-149 ◽

Cited By ~ 51

Author(s):

Stephen M. Onifer ◽

Linda A. White ◽

Scott R. Whittemore ◽

Vicky R. Holets

Keyword(s):

Escherichia Coli ◽

Cell Line ◽

Neuronal Cell ◽

Fluorescein Isothiocyanate ◽

Initial Step ◽

Host Cells ◽

Lacz Gene ◽

Raphe Neurons

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS transplantation studies were tested by first characterizing the labels in vitro. The labels that were tested included 4,6-diamidino-2-phenylindole hydrochloride, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate-conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3′-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or tetanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in vivo after CNS transplantation. In vitro, 1,1-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neurons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Most labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retained in cellular debris. In vivo, DiI was visualized in transplanted, DiI-labeled raphe neurons, but not in astrocytes up to 1 mo posttransplant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. β-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No β-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels for use in CNS transplantation studies should be optimized for the specific population of donor cells under study, with the initial step being characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be assessed.

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Activation of p47phox as a Mechanism of Bupivacaine-Induced Burst Production of Reactive Oxygen Species and Neural Toxicity

Oxidative Medicine and Cellular Longevity ◽

10.1155/2017/8539026 ◽

2017 ◽

Vol 2017 ◽

pp. 1-14 ◽

Cited By ~ 5

Author(s):

Yu-jie Li ◽

Wei Zhao ◽

Xu-jiao Yu ◽

Feng-xian Li ◽

Zi-ting Liu ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Injury ◽

Neuroblastoma Cells ◽

Neuronal Cell ◽

Reactive Oxygen ◽

In Vitro Study ◽

Oxygen Species ◽

Membrane Translocation

Bupivacaine has been shown to induce neurotoxicity through inducing excessive reactive oxygen species (ROS), but the underlying mechanism remains unclear. NOX2 is one of the most important sources of ROS in the nervous system, and its activation requires the membrane translocation of subunit p47phox. However, the role of p47phox in bupivacaine-induced neurotoxicity has not been explored. In our in vitro study, cultured human SH-SY5Y neuroblastoma cells were treated with 1.5 mM bupivacaine to induce neurotoxicity. Membrane translocation of p47phox was assessed by measuring the cytosol/membrane ratio of p47phox. The effects of the NOX inhibitor VAS2870 and p47phox-siRNA on bupivacaine-induced neurotoxicity were investigated. Furthermore, the effect of VAS2870 on bupivacaine-induced neurotoxicity was assessed in vivo in rats. All these changes were reversed by pretreatment with VAS2870 or transfection with p47phox-siRNA in SH-SY5Y cells. Similarly, pretreatment with VAS2870 attenuated bupivacaine-induced neuronal toxicity in rats. It is concluded that enhancing p47phox membrane translocation is a major mechanism whereby bupivacaine induced neurotoxicity and that pretreatment with VAS2870 or local p47phox gene knockdown attenuated bupivacaine-induced neuronal cell injury.

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The Toxicity of Universal Dental Adhesives: An In Vitro Study

Polymers ◽

10.3390/polym13162653 ◽

2021 ◽

Vol 13 (16) ◽

pp. 2653

Author(s):

Adam Wawrzynkiewicz ◽

Wioletta Rozpedek-Kaminska ◽

Grzegorz Galita ◽

Monika Lukomska-Szymanska ◽

Barbara Lapinska ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Line ◽

Colorimetric Assay ◽

In Vitro Study ◽

In Vitro Toxicity ◽

Single Bond ◽

Dental Adhesives ◽

Apoptosis Detection

There is no consensus in the literature regarding the potential toxicity of universal dental adhesives (UDA). Being used in close proximity to the pulp, their biocompatibility should be an important factor in dental research. The aim of the present study was to evaluate the biocompatibility of UDA in an in vitro model. The study was performed using a monocyte/macrophage peripheral blood SC cell line (ATCC CRL-9855) on four specific UDA, namely: All-Bond Universal (Bisco); CLEARFIL Universal Bond Quick (Kuraray); G-Premio BOND (GC); Single Bond Universal (3M ESPE). The cytotoxicity of the investigated UDA was measured using the XTT colorimetric assay. The genotoxicity of the analyzed compounds was evaluated using an alkaline version of the comet assay. Furthermore, flow cytometry (FC) apoptosis detection was performed using the FITC Annexin V Apoptosis Detection Kit I. FC cell-cycle arrest assessment was performed using propidium iodide staining. The study observed significant differences in the toxicity of the UDA that were tested, as G-Premio BOND showed significant in vitro toxicity in all of the tests performed, while All-Bond Universal, CLEARFIL Universal Bond Quick and Single Bond Universal did not present any significant toxic effects toward SC cell line. The in vitro toxicity of UDA should be taken into consideration prior to in vivo and clinical studies. The flow cytometry could improve the accuracy of dental materials research and should be incorporated into the standardization criteria.

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