scholarly journals Potentiation of neurite outgrowth and reduction of apoptosis by immunosuppressive agents: implications for neuronal injury and transplantation

2006 ◽  
Vol 20 (5) ◽  
pp. 1-7 ◽  
Author(s):  
Jason Sheehan ◽  
Anne Eischeid ◽  
Randi Saunders ◽  
Nader Pouratian
Keyword(s):  
Cyclosporin A ◽  
Neurite Outgrowth ◽  
Apoptotic Cell ◽  
Immunosuppressive Agents ◽  
Neuronal Cell ◽  
Neuronal Injury ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Neuroblastoma ◽  
Cord Injury ◽  
Induced Apoptosis

Object Immunosuppressive agents are believed to play a role in recovery from spinal cord injury, but the underlying mechanisms by which neuronal function is improved by these agents are poorly understood. In this study, the authors evaluate the effect of immunosuppressive medications on neurite outgrowth and cell survival after a pharmacologically induced injury. Methods Differentiated human neuroblastoma SH-SY5Y cells were injured using the calcium agonist thapsigargin. After cellular injury, neurite outgrowth in the presence or absence of immunosuppressive agents was measured. Apoptosis was quantified with the aid of a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay. Neurite outgrowth was severely restricted following thapsigargin injury. Outgrowth was potentiated, however, by the addition of concentrations of 1 and 10 μM cyclosporin A in a dose-dependent fashion. Similarly, addition of 10 nM FK506 increased the percentage of neurites in the 20- to 40-micron range. A low dose (1 μM) of dexamethasone did not have a significant effect on neurite outgrowth, but a higher dose (10 μM) increased the percentage of neurites in the 10- to 45-micron range. These agents also lessened the degree of thapsigargin-induced apoptosis. Conclusions Immunosuppressive agents such as cyclosporin A, FK506, and dexamethasone can potentiate neurite outgrowth and protect against apoptotic cell death in a human postmitotic neuronal cell line. Such effects may have implications for lessening neuronal injury after neurotrauma, stroke, or neurodegeneration.

scholarly journals Mitomycin C Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via a Mitochondrial-Mediated Pathway

2015 ◽  
Vol 35 (3) ◽  
pp. 1125-1136 ◽  
Author(s):  
Chuqi Yan ◽  
Dechao Kong ◽  
Dong Ge ◽  
Yanming Zhang ◽  
Xishan Zhang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Viability ◽  
Mitomycin C ◽  
Apoptotic Cell ◽  
Chronic Inflammatory Disease ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Treatment ◽  
Induced Apoptosis ◽  
Fibroblast Like Synoviocytes

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.

scholarly journals Apoptosis in the Mouse Central Nervous System in Response to Infection with Mouse-Neurovirulent Dengue Viruses

1998 ◽  
Vol 72 (1) ◽  
pp. 823-829 ◽  
Author(s):  
Philippe Desprès ◽  
Marie-Pascale Frenkiel ◽  
Pierre-Emmanuel Ceccaldi ◽  
Claudia Duarte Dos Santos ◽  
Vincent Deubel
Keyword(s):  
Central Nervous System ◽  
Cell Death ◽  
Nervous System ◽  
Virus Infection ◽  
Virus Replication ◽  
Parental Strain ◽  
Apoptotic Cell ◽  
Neuronal Cell ◽  
Induced Apoptosis

ABSTRACT Apoptosis has been suggested as a mechanism by which dengue (DEN) virus infection may cause neuronal cell death (P. Desprès, M. Flamand, P.-E. Ceccaldi, and V. Deubel, J. Virol. 70:4090–4096, 1996). In this study, we investigated whether apoptotic cell death occurred in the central nervous system (CNS) of neonatal mice inoculated intracerebrally with DEN virus. We showed that serial passage of a wild-type human isolate of DEN virus in mouse brains selected highly neurovirulent variants which replicated more efficiently in the CNS. Infection of newborn mice with these neurovirulent variants produced fatal encephalitis within 10 days after inoculation. Virus-induced cell death and oligonucleosomal DNA fragmentation were observed in mouse brain tissue by day 9. Infected mouse brain tissue was assayed for apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and for virus replication by immunostaining of viral antigens and in situ hybridization. Apoptotic cell death and DEN virus replication were restricted to the neurons of the cortical and hippocampal regions. Thus, DEN virus-induced apoptosis in the CNS was a direct result of virus infection. In the murine neuronal cell line Neuro 2a, neuroadapted DEN virus variants showed infection patterns similar to those of the parental strain. However, DEN virus-induced apoptosis in these cells was more pronounced after infection with the neurovirulent variants than after infection with the parental strain.

scholarly journals A Comparison of High-Content Screening versus Manual Analysis to Assay the Effects of Mesenchymal Stem Cell–Conditioned Medium on Neurite Outgrowth In Vitro

2010 ◽  
Vol 15 (5) ◽  
pp. 576-582 ◽  
Author(s):  
Karina T. Wright ◽  
Gareth J. Griffiths ◽  
William E. B. Johnson
Keyword(s):  
Content Analysis ◽  
Neurite Outgrowth ◽  
Conditioned Medium ◽  
Neuroblastoma Cell ◽  
High Content Screening ◽  
Human Neuroblastoma Cell ◽  
Control Medium ◽  
Human Neuroblastoma ◽  
Cord Injury ◽  
High Content Analysis

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

scholarly journals In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

1996 ◽  
Vol 44 (9) ◽  
pp. 959-968 ◽  
Author(s):  
A Negoescu ◽  
P Lorimier ◽  
F Labat-Moleur ◽  
C Drouet ◽  
C Robert ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Rapid Identification ◽  
Cell Labeling ◽  
Cell Fraction ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Current Debate ◽  
Double Labeling ◽  
Apoptotic Morphology ◽  
Induced Apoptosis

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

scholarly journals Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis

2004 ◽  
Vol 379 (3) ◽  
pp. 805-813 ◽  
Author(s):  
Yan SHOU ◽  
Li LI ◽  
Krishnan PRABHAKARAN ◽  
Joseph L. BOROWITZ ◽  
Gary E. ISOM
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Cyclosporin A ◽  
Apoptotic Cell ◽  
Calcineurin Inhibitors ◽  
Apoptotic Response ◽  
Cytochrome C Release ◽  
Cortical Cells ◽  
Subsequent Activation ◽  
Induced Apoptosis

In cyanide-induced apoptosis, an increase in cytosolic free Ca2+ and generation of reactive oxygen species are initiation stimuli for apoptotic cell death. Previous studies have shown that cyanide-stimulated translocation of Bax (Bcl-associated X protein) to mitochondria is linked with release of cytochrome c and subsequent activation of a caspase cascade [Shou, Li, Prabhakaran, Borowitz and Isom (2003) Toxicol. Sci. 75, 99–107]. In the present study, the relationship of the cyanide-induced increase in cytosolic free Ca2+ to activation of Bad (Bcl-2/Bcl-XL-antagonist, causing cell death) was determined in cortical cells. Bad is a Ca2+-sensitive pro-apoptotic Bcl-2 protein, which on activation translocates from cytosol to mitochondria to initiate cytochrome c release. In cultured primary cortical cells, cyanide produced a concentration- and time-dependent translocation of Bad from cytosol to mitochondria. Translocation occurred early in the apoptotic response, since mitochondrial Bad was detected within 1 h of cyanide treatment. Mitochondrial levels of the protein continued to increase up to 12 h post-cyanide exposure. Concurrent with Bad translocation, a Ca2+-sensitive increase in cellular calcineurin activity was observed. Increased cytosolic Ca2+ and calcineurin activation stimulated Bad translocation since BAPTA [bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid], an intracellular Ca2+ chelator, and cyclosporin A, a calcineurin inhibitor, significantly reduced translocation. BAPTA also blocked release of cytochrome c from mitochondria as well as apoptosis. Furthermore, treatment of cells with the calcineurin inhibitors cyclosporin A or FK506 blocked the apoptotic response, linking calcineurin activation and the subsequent translocation of Bad to cell death. These observations show that by inducing a rapid increase in cytosolic free Ca2+, cyanide can partially initiate the apoptotic cascade through a calcineurin-mediated translocation of Bad to mitochondria.

scholarly journals Elevation of Cleaved p18 Bax Levels Associated with the Kinetics of Neuronal Cell Death during Japanese Encephalitis Virus Infection

2019 ◽  
Vol 20 (20) ◽  
pp. 5016
Author(s):  
Prapimpun Wongchitrat ◽  
Arisara Samutpong ◽  
Hatairat Lerdsamran ◽  
Jarunee Prasertsopon ◽  
Montri Yasawong ◽  
...  
Keyword(s):  
Cell Death ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cell Apoptosis ◽  
Neuronal Apoptosis ◽  
Apoptotic Cell ◽  
Neuronal Cell ◽  
Encephalitis Virus ◽  
Apoptotic Cell Death ◽  
Human Neuroblastoma

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

scholarly journals Hypoxia-Ischemia and Hypothermia Independently and Interactively Affect Neuronal Pathology in Neonatal Piglets with Short-Term Recovery

2019 ◽  
Vol 41 (1-2) ◽  
pp. 17-33 ◽  
Author(s):  
Caitlin E. O’Brien ◽  
Polan T. Santos ◽  
Ewa Kulikowicz ◽  
Michael Reyes ◽  
Raymond C. Koehler ◽  
...  
Keyword(s):  
Cell Death ◽  
Motor Cortex ◽  
Apoptotic Cell ◽  
Clinical Care ◽  
Neuronal Injury ◽  
Interactive Effects ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Neonatal Piglets ◽  
Sham Procedure ◽  
Hypoxia Ischemia

Therapeutic hypothermia is the standard of clinical care for moderate neonatal hypoxic-ischemic encephalopathy. We investigated the independent and interactive effects of hypoxia-ischemia (HI) and temperature on neuronal survival and injury in basal ganglia and cerebral cortex in neonatal piglets. Male piglets were randomized to receive HI injury or sham procedure followed by 29 h of normothermia, sustained hypothermia induced at 2 h, or hypothermia with rewarming during fentanyl-nitrous oxide anesthesia. Viable and injured neurons and apoptotic profiles were counted in the anterior putamen, posterior putamen, and motor cortex at 29 h after HI injury or sham procedure. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) identified genomic DNA fragmentation to confirm cell death. Though hypothermia after HI preserved viable neurons in the anterior and posterior putamen, hypothermia prevented neuronal injury in only the anterior putamen. Hypothermia initiated 2 h after injury did not protect against apoptotic cell death in either the putamen or motor cortex, and rewarming from hypothermia was associated with increased apoptosis in the motor cortex. In non-HI shams, sustained hypothermia during anesthesia was associated with neuronal injury and corresponding viable neuron loss in the anterior putamen and motor cortex. TUNEL confirmed increased neurodegeneration in the putamen of hypothermic shams. Anesthetized, normothermic shams did not show abnormal neuronal cytopathology in the putamen or motor cortex, thereby demonstrating minimal contribution of the anesthetic regimen to neuronal injury during normothermia. We conclude that the efficacy of hypothermic protection after HI is region specific and that hypothermia during anesthesia in the absence of HI may be associated with neuronal injury in the developing brain. Studies examining the potential interactions between hypothermia and anesthesia, as well as with longer durations of hypothermia, are needed.

scholarly journals Activation of Canonical Notch Signaling Pathway Is Involved in the Ischemic Tolerance Induced by Sevoflurane Preconditioning in Mice

2012 ◽  
Vol 117 (5) ◽  
pp. 996-1005 ◽  
Author(s):  
Qianzi Yang ◽  
Wenjun Yan ◽  
Xin Li ◽  
Lihong Hou ◽  
Hui Dong ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Signaling Pathway ◽  
Apoptotic Cell ◽  
Intraperitoneal Administration ◽  
Neuronal Cell ◽  
Notch Signaling Pathway ◽  
Ischemic Tolerance ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Brain Infarct ◽  
Sevoflurane Preconditioning

Background A wealth of evidence has demonstrated that sevoflurane preconditioning induces brain ischemic tolerance, but the mechanism remains poorly understood. This study was designed to investigate the role of canonical Notch signaling in the neuroprotection induced by sevoflurane preconditioning in a mouse model. Methods C57BL/6 mice were pretreated with 1-h sevoflurane exposure at a dose of 2.5% for 5 consecutive days. Twenty-four hours after the last exposure, all mice were subjected to focal cerebral ischemia by right middle cerebral artery occlusion for 60 min. Neurobehavioral scores, brain infarct volumes, and cellular apoptosis were determined at 72 h after reperfusion (n = 10 per group). The activation of Notch signaling was evaluated (n = 5 per group), and its role in ischemic tolerance was assessed by intraperitoneal administration of γ-secretase inhibitor DAPT (100 mg/kg, n = 10 per group) and conditional Notch-RBP-J knockout technique (n = 8 per group). Results Sevoflurane preconditioning reduced brain infarct volumes (42.5%), improved neurologic outcomes (P < 0.01 vs. control), and attenuated neuronal cell apoptosis (cells positive for terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling reduced to 21.2%). The expression of Notch1 intracellular domain (1.35 folds) and the transcriptions of Hes1 (1.95 times) and Hes5 (1.48 times) were up-regulated. DAPT augmented the brain infarcts (1.64-fold) and decreased neurologic scores (P = 0.43 vs. sevoflurane) in sevoflurane-preconditioned mice. Brain infarct volumes, neurobehavioral scores, and apoptotic cell numbers showed no significance between Notch knockout mice with sevoflurane preconditioning and wild-type mice without preconditioning. Conclusions Sevoflurane preconditioning-induced protective effects against transient cerebral ischemic injuries are mediated by the activation of canonical Notch signaling pathway in mice.

scholarly journals 1-Methyl-4-phenylpyridinium (MPP+)-induced apoptosis and mitochondrial oxidant generation: role of transferrin-receptor-dependent iron and hydrogen peroxide

2003 ◽  
Vol 371 (1) ◽  
pp. 151-164 ◽  
Author(s):  
Shasi V. KALIVENDI ◽  
Srigiridhar KOTAMRAJU ◽  
Sonya CUNNINGHAM ◽  
Tiesong SHANG ◽  
Cecilia J. HILLARD ◽  
...  
Keyword(s):  
Cell Death ◽  
Transferrin Receptor ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Neuroblastoma Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Ros Generation ◽  
Cellular Models ◽  
Pre Treatment ◽  
Induced Apoptosis

1-Methyl-4-phenylpyridinium (MPP+) is a neurotoxin used in cellular models of Parkinson's Disease. Although intracellular iron plays a crucial role in MPP+-induced apoptosis, the molecular signalling mechanisms linking iron, reactive oxygen species (ROS) and apoptosis are still unknown. We investigated these aspects using cerebellar granule neurons (CGNs) and human SH-SY5Y neuroblastoma cells. MPP+ enhanced caspase 3 activity after 24h with significant increases as early as 12h after treatment of cells. Pre-treatment of CGNs and neuroblastoma cells with the metalloporphyrin antioxidant enzyme mimic, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), completely prevented the MPP+-induced caspase 3 activity as did overexpression of glutathione peroxidase (GPx1) and pre-treatment with a lipophilic, cell-permeable iron chelator [N,N′-bis-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid, HBED]. MPP+ treatment increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling)-positive cells which was completely blocked by pre-treatment with FeTBAP. MPP+ treatment significantly decreased the aconitase and mitochondrial complex I activities; pre-treatment with FeTBAP, HBED and GPx1 overexpression reversed this effect. MPP+ treatment increased the intracellular oxidative stress by 2—3-fold, as determined by oxidation of dichlorodihydrofluorescein and dihydroethidium (hydroethidine). These effects were reversed by pre-treatment of cells with FeTBAP and HBED and by GPx1 overexpression. MPP+-treatment enhanced the cell-surface transferrin receptor (TfR) expression, suggesting a role for TfR-induced iron uptake in MPP+ toxicity. Treatment of cells with anti-TfR antibody (IgA class) inhibited MPP+-induced caspase activation. Inhibition of nitric oxide synthase activity did not affect caspase 3 activity, apoptotic cell death or ROS generation by MPP+. Overall, these results suggest that MPP+-induced cell death in CGNs and neuroblastoma cells proceeds via apoptosis and involves mitochondrial release of ROS and TfR-dependent iron.

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