Dynamic Recording of Cell Death in the In Vitro Dorsal Vagal Nucleus of Rats in Response to Metabolic Arrest

2003 ◽  
Vol 89 (1) ◽  
pp. 551-561 ◽  
Author(s):  
Michael Müller ◽  
Klaus Ballanyi
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Neuronal Death ◽  
Time Course ◽  
Time Window ◽  
Membrane Integrity ◽  
Brain Slices ◽  
Limited Information ◽  
Histological Techniques ◽  
Metabolic Arrest

Anoxic/ischemic neuronal death is usually assessed in cell cultures or in vivo within a time window of 24 h to several days using the nucleic acid stain propidium iodide or histological techniques. Accordingly, there is limited information on the time course of such neuronal death. We loaded acute rat brain stem slices with propidium iodide for dynamic fluorometric recording of metabolic arrest-related cell death in the dorsal vagal nucleus. This model was chosen because dorsal vagal neurons show a graded response to metabolic inhibition: anoxia and aglycemia cause a sustained hyperpolarization, whereas ischemia induces a glutamate-mediated, irreversible depolarization. We found that the number of propidium iodide–labeled cells increased from 27% to 43% of total cell count within 1–7 h after preparation of slices. Compared with these untreated control slices, cyanide-induced anoxia (30 min) or aglycemia (1 h) did not cause further cell death, whereas 3-h aglycemia destroyed an additional 13% of cells. Ischemia (1 h) due to cyanide plus iodoacetate immediately labeled an additional 20% of cells, and an additional 48% of cells were destroyed within the following 3 h of postischemia. Continuous recording of propidium iodide fluorescence showed that loss of membrane integrity started within 25 min after onset of the ischemic depolarization and the concomitant intracellular Ca2+ rise. The results show that propidium iodide can be used to monitor cell death in acute brain slices. Our findings suggest that pronounced cell death occurs within a period of 1–4 h after onset of metabolic arrest and is apparently due to necrotic/oncotic mechanisms.

scholarly journals Acute Plasmalemma Permeability and Protracted Clearance of Injured Cells after Controlled Cortical Impact in Mice

2007 ◽  
Vol 28 (3) ◽  
pp. 490-505 ◽  
Author(s):  
Michael J Whalen ◽  
Turgay Dalkara ◽  
Zerong You ◽  
Jianhua Qiu ◽  
Daniela Bermpohl ◽  
...  
Keyword(s):  
Cell Death ◽  
Propidium Iodide ◽  
Time Course ◽  
Injury Severity ◽  
Cell Injury ◽  
Membrane Damage ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cellular Injury ◽  
Controlled Cortical Impact ◽  
Cortical Impact

Cell death after traumatic brain injury (TBI) evolves over days to weeks. Despite advances in understanding biochemical mechanisms that contribute to posttraumatic brain cell death, the time course of cell injury, death, and removal remains incompletely characterized in experimental TBI models. In a mouse controlled cortical impact (CCI) model, plasmalemma permeability to propidium iodide (PI) was an early and persistent feature of posttraumatic cellular injury in cortex and hippocampus. In cortical and hippocampal brain regions known to be vulnerable to traumatic cell death, the number of PI + cells peaked early after CCI, and increased with increasing injury severity in hippocampus but not cortex ( P < 0.05). Propidium iodide labeling correlated strongly with hematoxylin and eosin staining in injured cells ( r = 0.99, P < 0.001), suggesting that plasmalemma damage portends fatal cellular injury. Using PI pulse labeling to identify and follow the fate of a cohort of injured cells, we found that many PI+ cells recovered plasmalemma integrity by 24 h and were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling negative, but nonetheless disappeared from injured brain by 7 days. Propidium iodide-positive cells in dentate gyrus showed significant ultrastructural damage, including plasmalemma and nuclear membrane damage or overt membrane loss, in all cells when examined by laser capture microdissection and transmission electron microscopy 1 to 24 h after CCI. The data suggest that plasmalemma damage is a fundamental marker of cellular injury after CCI; some injured cells might have an extended window for potential rescue by neuroprotective strategies.

scholarly journals Evidence for the Involvement of Par-4 in Ischemic Neuron Cell Death

2001 ◽  
Vol 21 (4) ◽  
pp. 334-343 ◽  
Author(s):  
Carsten Culmsee ◽  
Yuan Zhu ◽  
Josef Krieglstein ◽  
Mark P. Mattson
Keyword(s):  
Cell Death ◽  
Neuronal Death ◽  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Current Data ◽  
Striatal Neurons ◽  
Similar Time ◽  
Chemical Hypoxia ◽  
Cultured Hippocampal Neurons

After a stroke many neurons in the ischemic brain tissue die by a process called apoptosis, a form of cell death that may be preventable. The specific molecular cascades that mediate ischemic neuronal death are not well understood. The authors recently identified prostate apoptosis response-4 (Par-4) as a protein that participates in the death of cultured hippocampal neurons induced by trophic factor withdrawal and exposure to glutamate. Here, the authors show that Par-4 levels increase in vulnerable populations of hippocampal and striatal neurons in rats after transient forebrain ischemia; Par-4 levels increased within 6 hours of reperfusion and remained elevated in neurons undergoing apoptosis 3 days later. After transient focal ischemia in mice, Par-4 levels were increased 6 to12 hours after reperfusion in the infarcted cortex and the striatum, and activation of caspase-8 occurred with a similar time course. Par-4 immunoreactivity was localized predominantly in cortical neurons at the border of the infarct area. A Par-4 antisense oligonucleotide protected cultured hippocampal neurons against apoptosis induced by chemical hypoxia and significantly reduced focal ischemic damage in mice. The current data suggest that early up-regulation of Par-4 plays a pivotal role in ischemic neuronal death in animal models of stroke and cardiac arrest.

Rapid Loss of Membrane Integrity and Irreversible Commitment to Cell Death of Primary AML Stem Cells: A Novel Activity for 4-benzyl, 2-methyl,1,2,4-Thiadiazolidine,3,5 Dione (TDZD-8).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2535-2535
Author(s):  
Monica L. Guzman ◽  
Xiaojie Li ◽  
Cheryl Corbett ◽  
Randall M. Rossi ◽  
Timothy Bushnell ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Time Course ◽  
Drug Exposure ◽  
Apoptotic Cell ◽  
Membrane Integrity ◽  
Ppar Gamma ◽  
Myelogenous Leukemia ◽  
Rapid Loss ◽  
Rapid Kinetics

Abstract Leukemia stem cells (LSCs) have been shown to play a crucial role in the pathogenesis of acute myelogenous leukemia (AML). However, current chemotherapy agents have not demonstrated effective targeting of the LSC population. Thus, failure to eradicate LSCs may lead to disease relapse and progression. Consequently, the identification and characterization of drugs that can efficiently eradicate the LSC population is required. We have identified a compound, 4-benzyl, 2-methyl,1,2,4-thiadiazolidine,3,5 dione (TDZD-8), with the novel characteristic of rapidly inducing cell death of different types of leukemia cells, including AML progenitor and stem cell populations. After 18–24 hours of treatment with 20 uM TDZD-8, the mean survival for primary AML cells was 7% (n=30), blast crisis CML 5% (n= 5), CLL 3% (n=13) and ALL 6% (n= 4). In contrast, normal specimens treated in the same fashion showed 80% mean survival (n=10). Functional assays were performed to determine the effect of TDZD-8 on primitive populations. NOD/SCID xenotransplant analysis showed a 93% decrease of engraftment for AML specimens, while normal cell engraftment was only reduced by 11%. Furthermore, colony-forming assays demonstrated a 93% decrease in CFU formation for AML cells (n=11, where 6 of the specimens showed no CFUs), in contrast to a 30% decrease in normal cells. Strikingly, cell death occurred with extremely rapid kinetics. Time course studies from the different primary AML specimens demonstrated that most of the cell death can be observed within 2 hours after exposure to TDZD-8 (where 10% of the samples tested appear dead with as little as 15–30 minutes of drug exposure). Furthermore, a 30 minute exposure to the drug was sufficient to irreversibly induce cell death of primitive CD34+CD38- cells and CFUs. Interestingly, TDZD-8 was originally described as a GSK-3b inhibitor and analogs to this molecule have also been found to activate PPAR-gamma. However, when we tested different known GSK-3b inhibitors (n=12) and PPAR-gamma activators (containing a thioazolidine ring) we did not observe the toxicity, specificity, or kinetics observed with TDZD-8. Molecular studies suggest that the mechanism by which TDZD-8 induces cell death involves a rapid thiol depletion, Nrf2 activation, and NF-kappaB inhibition. Moreover, a rapid loss of membrane integrity was observed using imaging flow cytometry (Amnis ImageStream) to determine permeability of different viability dyes. Furthermore, TDZD-8 induced cytochrome c release, but cell death was not blocked by pan-caspase inhibitors, suggesting a non-apoptotic cell death mechanism. Finally, preliminary in vivo experiments using mice transplanted with primary AML cells showed that Nrf2 was induced as soon as 1h after dosing animals with 4.4 mg/kg of TDZD-8 (i.p.). Together, these data suggest that TDZD-8 represents a novel class of anti-leukemia specific molecules capable of destroying leukemia progenitor and stem cells with extremely rapid kinetics.

Quercetin in attenuation of ischemic/reperfusion injury: A review

2020 ◽  
Vol 13 ◽  
Author(s):  
Milad Ashrafizadeh ◽  
Saeed Samarghandian ◽  
Kiavash Hushmandi ◽  
Amirhossein Zabolian ◽  
Md Shahinozzaman ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Membrane ◽  
Blood Supply ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Membrane Integrity ◽  
Therapeutic Effects ◽  
Molecular Pathways ◽  
Cell Membrane Integrity ◽  
Cell Membrane Disruption

Background: Ischemia/reperfusion (I/R) injury is a serious pathologic event that occurs due to restriction in blood supply to an organ, followed by hypoxia. This condition leads to enhanced levels of pro-inflammatory cytokines such as IL-6 and TNF-, and stimulation of oxidative stress via enhancing reactive oxygen species (ROS) levels. Upon reperfusion, blood supply increases, but it deteriorates condition, and leads to generation of ROS, cell membrane disruption and finally, cell death. Plant derived-natural compounds are well-known due to their excellent antioxidant and anti-inflammatory activities. Quercetin is a flavonoid exclusively found in different vegetables, herbs, and fruits. This naturally occurring compound possesses different pharmacological activities making it appropriate option in disease therapy. Quercetin can also demonstrate therapeutic effects via affecting molecular pathways such as NF-B, PI3K/Akt and so on. Methods: In the present review, we demonstrate that quercetin administration is beneficial in ameliorating I/R injury via reducing ROS levels, inhibition of inflammation, and affecting molecular pathways such as TLR4/NF-B, MAPK and so on. Results and conclusion: Quercetin can improve cell membrane integrity via decreasing lipid peroxidation. Apoptotic cell death is inhibited by quercetin via down-regulation of Bax, and caspases, and upregulation of Bcl-2. Quercetin is able to modulate autophagy (inhibition/induction) in decreasing I/R injury. Nanoparticles have been applied for delivery of quercetin, enhancing its bioavailability and efficacy in alleviation of I/R injury. Noteworthy, clinical trials have also confirmed the capability of quercetin in reducing I/R injury.

scholarly journals Moderately Inducing Autophagy Reduces Tertiary Brain Injury after Perinatal Hypoxia-Ischemia

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 898
Author(s):  
Brian H. Kim ◽  
Maciej Jeziorek ◽  
Hur Dolunay Kanal ◽  
Viorica Raluca Contu ◽  
Radek Dobrowolski ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Integrity ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Brain Slices ◽  
Brain Structures ◽  
Improved Outcomes ◽  
Hypoxia Ischemia ◽  
Tgfβ Receptor ◽  
Progressive Neurodegeneration

Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)β1 is highly elevated following HI and that delivering an antagonist for TGFβ receptor activin-like kinase 5 (ALK5)—SB505124—three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.

scholarly journals Neurotoxic Effect of Flavonol Myricetin in the Presence of Excess Copper

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 845
Author(s):  
Anja Sadžak ◽  
Ignacija Vlašić ◽  
Zoran Kiralj ◽  
Marijana Batarelo ◽  
Nada Oršolić ◽  
...  
Keyword(s):  
Cell Death ◽  
Intracellular Signaling ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Toxic Effects ◽  
Trypan Blue Exclusion ◽  
Atp Production ◽  
Mtt Method ◽  
Biophysical Approach ◽  
Induced Changes

Oxidative stress (OS) induced by the disturbed homeostasis of metal ions is one of the pivotal factors contributing to neurodegeneration. The aim of the present study was to investigate the effects of flavonoid myricetin on copper-induced toxicity in neuroblastoma SH-SY5Y cells. As determined by the MTT method, trypan blue exclusion assay and measurement of ATP production, myricetin heightened the toxic effects of copper and exacerbated cell death. It also increased copper-induced generation of reactive oxygen species, indicating the prooxidative nature of its action. Furthermore, myricetin provoked chromatin condensation and loss of membrane integrity without caspase-3 activation, suggesting the activation of both caspase-independent programmed cell death and necrosis. At the protein level, myricetin-induced upregulation of PARP-1 and decreased expression of Bcl-2, whereas copper-induced changes in the expression of p53, p73, Bax and NME1 were not further affected by myricetin. Inhibitors of ERK1/2 and JNK kinases, protein kinase A and L-type calcium channels exacerbated the toxic effects of myricetin, indicating the involvement of intracellular signaling pathways in cell death. We also employed atomic force microscopy (AFM) to evaluate the morphological and mechanical properties of SH-SY5Y cells at the nanoscale. Consistent with the cellular and molecular methods, this biophysical approach also revealed a myricetin-induced increase in cell surface roughness and reduced elasticity. Taken together, we demonstrated the adverse effects of myricetin, pointing out that caution is required when considering powerful antioxidants for adjuvant therapy in copper-related neurodegeneration.

scholarly journals Comparative Time-Course Physiological Responses and Proteomic Analysis of Melatonin Priming on Promoting Germination in Aged Oat (Avena sativa L.) Seeds

2021 ◽  
Vol 22 (2) ◽  
pp. 811
Author(s):  
Huifang Yan ◽  
Peisheng Mao
Keyword(s):  
Antioxidant Capacity ◽  
Avena Sativa ◽  
Proteomic Analysis ◽  
Time Course ◽  
Intact Cell ◽  
Membrane Integrity ◽  
Acid Synthesis ◽  
Ultrastructural Changes ◽  
Oat Seeds ◽  
Aged Seeds

Melatonin priming is an effective strategy to improve the germination of aged oat (Avena sativa L.) seeds, but the mechanism involved in its time-course responses has remained largely unknown. In the present study, the phenotypic differences, ultrastructural changes, physiological characteristics, and proteomic profiles were examined in aged and melatonin-primed seed (with 10 μM melatonin treatment for 12, 24, and 36 h). Thus, 36 h priming (T36) had a better remediation effect on aged seeds, reflecting in the improved germinability and seedlings, relatively intact cell ultrastructures, and enhanced antioxidant capacity. Proteomic analysis revealed 201 differentially abundant proteins between aged and T36 seeds, of which 96 were up-accumulated. In melatonin-primed seeds, the restoration of membrane integrity by improved antioxidant capacity, which was affected by the stimulation of jasmonic acid synthesis via up-accumulation of 12-oxo-phytodienoic acid reductase, might be a candidate mechanism. Moreover, the relatively intact ultrastructures enabled amino acid metabolism and phenylpropanoid biosynthesis, which were closely associated with energy generation through intermediates of pyruvate, phosphoenolpyruvate, fumarate, and α-ketoglutarate, thus providing energy, active amino acids, and secondary metabolites necessary for germination improvement of aged seeds. These findings clarify the time-course related pathways associated with melatonin priming on promoting the germination of aged oat seeds.

scholarly journals Neuroprotection by Histone Deacetylase-Related Protein

2006 ◽  
Vol 26 (9) ◽  
pp. 3550-3564 ◽  
Author(s):  
Brad E. Morrison ◽  
Nazanin Majdzadeh ◽  
Xiaoguang Zhang ◽  
Aaron Lyles ◽  
Rhonda Bassel-Duby ◽  
...  
Keyword(s):  
Cell Death ◽  
Histone Deacetylase ◽  
Neuronal Death ◽  
Histone Deacetylases ◽  
Essential Feature ◽  
Gene Promoter ◽  
Related Protein ◽  
Phosphatidylinositol 3 Kinase ◽  
Histone Deacetylase 1 ◽  
H3 Acetylation

ABSTRACT The expression of histone deacetylase-related protein (HDRP) is reduced in neurons undergoing apoptosis. Forced reduction of HDRP expression in healthy neurons by treatment with antisense oligonucleotides also induces cell death. Likewise, neurons cultured from mice lacking HDRP are more vulnerable to cell death. Adenovirally mediated expression of HDRP prevents neuronal death, showing that HDRP is a neuroprotective protein. Neuroprotection by forced expression of HDRP is not accompanied by activation of the phosphatidylinositol 3-kinase-Akt or Raf-MEK-ERK signaling pathway, and treatment with pharmacological inhibitors of these pathways fails to inhibit the neuroprotection by HDRP. Stimulation of c-Jun phosphorylation and expression, an essential feature of neuronal death, is prevented by HDRP. We found that HDRP associates with c-Jun N-terminal kinase (JNK) and inhibits its activity, thus explaining the inhibition of c-Jun phosphorylation by HDRP. HDRP also interacts with histone deacetylase 1 (HDAC1) and recruits it to the c-Jun gene promoter, resulting in an inhibition of histone H3 acetylation at the c-Jun promoter. Although HDRP lacks intrinsic deacetylase activity, treatment with pharmacological inhibitors of histone deacetylases induces apoptosis even in the presence of ectopically expressed HDRP, underscoring the importance of c-Jun promoter deacetylation by HDRP-HDAC1 in HDRP-mediated neuroprotection. Our results suggest that neuroprotection by HDRP is mediated by the inhibition of c-Jun through its interaction with JNK and HDAC1.

scholarly journals Hydrocortisone decreases apoptosis in jejunum of horses subjected to experimental ischemia and reperfusion

2011 ◽  
Vol 31 (6) ◽  
pp. 471-476 ◽  
Author(s):  
Geraldo Eleno S. Alves ◽  
Heloisa M.F. Mendes ◽  
Tiago G.S. Alves ◽  
Rafael R. Faleiros ◽  
Anilton C. Vasconcelos ◽  
...  
Keyword(s):  
Cell Death ◽  
Time Course ◽  
Apoptotic Cell ◽  
Treated Group ◽  
Apoptotic Cell Death ◽  
Apoptotic Cells ◽  
Ischemia And Reperfusion ◽  
Beneficial Effects ◽  
Time Zero ◽  
Venous Ischemia

In order to evaluate the effect of hydrocortisone on apoptosis in the jejunum of horses subjected to ischemia and reperfusion, ten horses were paired and grouped into two groups - treated (n=5) and non treated (n=5). Segments of the jejunum were used as controls (C), or as venous ischemia (VIsc), which were subjected to 2h of ischemia followed by 2 or 12h of reperfusion. C samples were collected at time zero (prior to ischemia) and VIsc samples were collected at 2h of ischemia and at 2 and 12h of reperfusion. TUNEL positive apoptotic cells were counted in 10 microscopical fields in deep mucosa from each horse throughout the time course. After 12h of reperfusion, the number of apoptotic cells in treated group were significantly lower than in untreated animals, indicating that hydrocortisone inhibits apoptosis. These results indicate that hydrocortisone has a beneficial effects favoring the maintenance of jejunal integrity in horses with ischemia and reperfusion injuries by preventing apoptotic cell death.

scholarly journals Ceramide and Related-Sphingolipid Levels Are Not Altered in Disease-Associated Brain Regions of APPSLand APPSL/PS1M146LMouse Models of Alzheimer's Disease: Relationship with the Lack of Neurodegeneration?

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Laurence Barrier ◽  
Bernard Fauconneau ◽  
Anastasia Noël ◽  
Sabrina Ingrand
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Animal Models ◽  
Neuronal Death ◽  
Time Course ◽  
Neuronal Loss ◽  
Brain Regions ◽  
App Processing ◽  
Ceramide Accumulation ◽  
The Brain

There is evidence linking sphingolipid abnormalities, APP processing, and neuronal death in Alzheimer's disease (AD). We previously reported a strong elevation of ceramide levels in the brain of the APPSL/PS1Ki mouse model of AD, preceding the neuronal death. To extend these findings, we analyzed ceramide and related-sphingolipid contents in brain from two other mouse models (i.e., APPSLand APPSL/PS1M146L) in which the time-course of pathology is closer to that seen in most currently available models. Conversely to our previous work, ceramides did not accumulate in disease-associated brain regions (cortex and hippocampus) from both models. However, the APPSL/PS1Ki model is unique for its drastic neuronal loss coinciding with strong accumulation of neurotoxic Aβisoforms, not observed in other animal models of AD. Since there are neither neuronal loss nor toxic Aβspecies accumulation in APPSLmice, we hypothesized that it might explain the lack of ceramide accumulation, at least in this model.

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