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.

Apoptosis in polycystic kidney disease: involvement of caspases

2000 ◽  
Vol 278 (3) ◽  
pp. R763-R769 ◽  
Author(s):  
Shujath M. Ali ◽  
Victoria Y. Wong ◽  
Kristine Kikly ◽  
Todd A. Fredrickson ◽  
Paul M. Keller ◽  
...  
Keyword(s):  
Cell Death ◽  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Apoptotic Cell ◽  
Renal Cysts ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Homozygous Mutation ◽  
Polycystic Kidney ◽  
Apoptosis Pathway ◽  
Cystic Kidneys

Polycystic kidney disease (PKD) is characterized by the development of large renal cysts and progressive loss of renal function. Although the cause of the development of renal cysts is unknown, recent evidence suggests that excessive apoptosis occurs in PKD. With the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, we have confirmed the presence of apoptotic bodies in cystic kidneys of congenital polycystic kidney (cpk) disease mice carrying a homozygous mutation at 3 wk of age. Apoptosis was localized primarily to the interstitium with little evidence of cell death in cyst epithelium or noncystic tubules. In addition, we observed that the expression of various caspases, bax and bcl-2, was upregulated in cystic kidneys. With the use of various substrates in enzyme activity assays, we have demonstrated a greater than sevenfold increase in caspase 4 activity and a sixfold increase in caspase 3 activity. These data suggest that there is a caspase-dependent apoptosis pathway associated with PKD and support the hypothesis that apoptotic cell death contributes to cyst formation in PKD.

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 In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2234-2243 ◽  
Author(s):  
Giorgio Zauli ◽  
Marco Vitale ◽  
Elisabetta Falcieri ◽  
Davide Gibellini ◽  
Alessandra Bassini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Apoptotic Cell ◽  
High Surface ◽  
Suspension Cultures ◽  
Apoptotic Cell Death ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Terminal Phase ◽  
Serum Free

Abstract To investigate the fate of human megakaryocytes, CD34+ hematopoietic progenitor cells were purified from the peripheral blood or bone marrow of healthy donors and seeded in serum-free chemically defined suspension cultures. In the presence of thrombopoietin (TPO; 100 ng/mL), CD34-derived cells showed an eightfold numerical expansion and a progressive maturation along the megakaryocytic lineage. Megakaryocyte maturation was characterized ultrastructurally by the presence of a demarcation membrane system and phenotypically by a high surface expression of αIIbβ3 integrin. The number of mature megakaryocytes peaked at days 12 to 15 of culture. On the other hand, the number of platelets released in the culture supernatant by CD34-derived megakaryocytes peaked at days 18 to 21, when a high percentage of megakaryocytes showed the characteristic features of apoptosis, as evaluated by electron microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end-labeling technique (TUNEL) and uptake of propidium iodide. In other experiments, primary αIIbβ3+ megakaryocytic cells were directly purified from the bone marrow aspirates of normal donors and seeded in serum-free suspension cultures. In the absence of cytokines, αIIbβ3+ megakaryocytes progressively underwent apoptotic cell death. The addition of TPO but not interleukin-3 or erythropoietin showed some protection of αIIbβ3+ cells from apoptosis at early culture times (days 2 to 4), but it did not show any significant effect at later time points. These findings suggest that the terminal phase of the megakaryocyte life span is characterized by the onset of apoptosis, which can be modulated only to a certain extent by TPO.

Oligodendrocytes and their precursors require phosphatidylinositol 3-kinase signaling for survival

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2529-2537
Author(s):  
G.S. Vemuri ◽  
F.A. McMorris
Keyword(s):  
Cell Death ◽  
Enzyme Activity ◽  
Growth Factors ◽  
Growth Factor ◽  
Cell Survival ◽  
Nuclear Dna ◽  
Apoptotic Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Signal transduction in response to several growth factors that regulate oligodendrocyte development and survival involves the activation of phosphatidylinositol 3-kinase, which we detect in oligodendrocytes and their precursors. To investigate the role of this enzyme activity, we analyzed cell survival in cultures of oligodendrocytes treated with wortmannin or LY294002, two potent inhibitors of phosphatidylinositol 3-kinase. Cell survival was inhibited by 60–70% in these cultures within 24 hours, as quantitated by a tetrazolium staining assay for viable cells and by measurement of DNA content. Similar results were obtained with oligodendrocyte precursor cells. Nuclei of the dying cells contained fragmented DNA, as revealed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays, indicating that the cells were dying by apoptosis. Moreover, a significant increase in the number of cells with fragmented nuclear DNA was detected as early as 4 hours, well before any significant differences could be detected in glucose transport or cell viability. Exogenous addition of insulin-like growth factor-I, neurotrophin-3, platelet-derived growth factor, basic fibroblast growth factor, ciliary neurotrophic factor, N-acetyl cysteine, vitamin C, vitamin E, progesterone or serum did not prevent cell death in the presence of wortmannin or LY294002. These findings indicate that survival of oligodendrocytes and their precursors depends on a phosphatidylinositol 3-kinase mediated signaling pathway. Inhibition of this critical enzyme activity induces apoptotic cell death, even in the presence of exogenous growth factors or serum.

Coptidis rhizoma Induces Apoptosis in Human Colorectal Cancer Cells SNU-C4

2004 ◽  
Vol 32 (06) ◽  
pp. 873-882 ◽  
Author(s):  
Youn Jung Kim ◽  
Soon Ah Kang ◽  
Mee Suk Hong ◽  
Hae Jeong Park ◽  
Mi-Ja Kim ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Colorectal Cancer ◽  
Colorectal Cancer Cells ◽  
Coptidis Rhizoma ◽  
Human Colorectal Cancer Cells

Coptidis rhizoma has been used as traditional herb medicine in gastrointestinal disorders in the Eastern Asia. We investigated whether the anticancer effects of the C. rhizoma induced apoptosis on human colorectal cancer cells SNU-C4. The cytotoxic effect of C. rhizoma was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To determine apoptotic cell death, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR) and caspase-3 enzyme assay were performed. In this study, C. rhizoma treatment (100 μg/ml) revealed typical morphological apoptotic features. Additionally, C. rhizoma treatment (100 μg/ml) increased levels of BAX and CASPASE-3, and decreased levels of BCL-2. Caspase-3 enzyme activity by treatment of C. rhizoma (100 μg/ml) also significantly increased compared to the control (p<0.05). These data indicate that C. rhizoma caused cell death by apoptosis through caspase pathways on human colorectal cancer cells SNU-C4.

scholarly journals Alteration in Downstream Hypoxia Gene Signaling in Neonatal Glutathione Peroxidase Overexpressing Mouse Brain after Hypoxia-Ischemia

2015 ◽  
Vol 37 (4-5) ◽  
pp. 398-406 ◽  
Author(s):  
R. Ann Sheldon ◽  
Raha Sadjadi ◽  
Matthew Lam ◽  
Russell Fitzgerald ◽  
Donna M. Ferriero
Keyword(s):  
Cell Death ◽  
Brain Injury ◽  
Glutathione Peroxidase ◽  
Apoptotic Cell ◽  
Hypoxia Inducible Factor ◽  
Artery Ligation ◽  
Epo Receptor ◽  
Carotid Artery Ligation ◽  
Mouse Cortex ◽  
Hypoxia Ischemia

We have previously shown that glutathione peroxidase (GPx) overexpressing mice (hGPx-tg) have reduced brain injury after neonatal hypoxia-ischemia (HI) as a consequence of reduced hydrogen peroxide accumulation. However, this protection is reversed with hypoxia preconditioning, raising the question of the roles of the genes regulated by hypoxia-inducible factor-1α (HIF-1α) and their transcription products, such as erythropoietin (EPO), in both the initial protection and subsequent reversal of protection. hGPx-tg and their wild-type (WT) littermates underwent the Vannucci procedure of HI brain injury at postnatal day 9 - left carotid artery ligation followed by exposure to 10% oxygen for 50 min. Brain cortices and hippocampi were subsequently collected 0.5, 4 and 24 h later for the determination of protein expression by Western blot for GPx, HIF-1α, HIF-2α, EPO, EPO receptor, ERK1/2, phospho-ERK1/2, spectrin 145/150 (as a marker of calpain-specific necrotic cell death), and spectrin 120 (as a marker of apoptotic cell death mediated via caspase-3). As expected, the GPx overexpressing mouse cortex had approximately 3 times the GPx expression as WT naïve. Also, GPx expression remained higher in the GPx overexpressing brain than WT at all time points after HI (0.5, 4, 24 h). HIF-1α was not significantly changed in hGPx-tg as a consequence of HI but decreased in the WT cortex 4 h after HI. HIF-2α decreased in the WT hippocampus after HI. EPO was higher in the GPx overexpressing cortex and hippocampus 30 min after HI compared to WT, but the EPO receptor was unchanged by HI. ERK1/2 phosphorylation increased in the hippocampus at 4 h after HI and in the cortex at 24 h after HI in both WT and hGPx-tg. Spectrin 145/150 was increased in the WT cortex at 4 and 24 h after HI, and spectrin 120 increased 24 h after HI, perhaps reflecting greater injury in the WT brain, especially at 24 h when brain injury is more evident. The effect of GPx overexpression does not appear to upregulate the HIF pathway, yet EPO was upregulated, perhaps via ERK. This might explain, in part, why cell death takes a necrotic or apoptotic path. This may also be an explanation for why the GPx overexpressing brain cannot be preconditioned. This information may prove valuable in the development of therapies for neonatal HI brain injury.

scholarly journals Hypothermia is not therapeutic in a neonatal piglet model of inflammation-sensitized hypoxia–ischemia

2021 ◽  
Author(s):  
Kathryn A. Martinello ◽  
Christopher Meehan ◽  
Adnan Avdic-Belltheus ◽  
Ingran Lingam ◽  
Tatenda Mutshiya ◽  
...  
Keyword(s):  
Cell Death ◽  
Microglial Activation ◽  
Acute Infection ◽  
Brain Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Resonance Spectroscopy ◽  
List Type ◽  
Neonatal Encephalopathy ◽  
Perinatal Brain Injury ◽  
Hypoxia Ischemia

Abstract Background Perinatal inflammation combined with hypoxia–ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown. Methods Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1–25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured. Results Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT. Conclusions We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI. Impact Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models.

Verapamil Reduces Coronary Endothelium Damage and Cardiomyocyte Necrosis but not Apoptosis after Ischemia and Reperfusion: Ex Vivo Study in Rat Hearts

2002 ◽  
Vol 15 (3) ◽  
pp. 225-232 ◽  
Author(s):  
P. Di Napoli ◽  
A. A. Taccardi ◽  
A. Grilli ◽  
M. Felaco ◽  
L. Di Gioacchino ◽  
...  
Keyword(s):  
Cell Death ◽  
Ischemia Reperfusion Injury ◽  
Ex Vivo ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Cardiomyocyte Apoptosis ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Heart Weight ◽  
Acute Administration ◽  
Rat Hearts

We tested the hypothesis of beneficial effects of the calcium-blocker verapamil in a model of ischemia-reperfusion, and investigated its effects against coronary microcirculation and cardiomyocyte apoptosis. Isolated working rat hearts were subjected to 15 min global ischemia and 22–180 min reperfusion in the presence or absence of verapamil (0.25 μM). We evaluated creatinephosphokinase (CK) in coronary effluent, heart weight changes, microvascular permeability (extravasation of fluoresceinelabeled albumin), ultrastructural alterations, and cardiomyocyte apoptosis (by 1.5% agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling technique). In this model, 0.25 μM verapamil significantly reduced myocardial damage, CK release and vascular hyperpermeability, concomitant with a reduction in endothelial and cardiomyocyte lesions; on the contrary, 0.25 μM verapamil was unable to reduce cardiomyocyte apoptosis. In conclusion, in the absence of perfusing granulocytes, the acute administration of a pharmacologically relevant verapamil concentration reduces ischemia-reperfusion injury and prevents coronary endothelial cell and cardiomyocyte necrotic cell death but it is unable to reduce apoptotic cell death in isolated working rat hearts.

Differential Effects of Mixtures of Cholesterol Oxidation Products on Bovine Aortic Endothelial Cells and Human Monocytic U937 Cells

2005 ◽  
Vol 24 (3) ◽  
pp. 173-179 ◽  
Author(s):  
Aaron J. O’Sullivan ◽  
Yvonne C. O’Callaghan ◽  
Nora M. O’Brien
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Oxidation Products ◽  
Interactive Effects ◽  
Cholesterol Oxidation ◽  
U937 Cells ◽  
Cholesterol Oxidation Products ◽  
Aortic Endothelial

Cholesterol oxidation products or oxysterols are of interest due to their hypothesized role in the development of atherosclerosis. The objective of the present study was to assess the cytotoxic effects of mixtures of oxysterols: 25-hydroxycholesterol (25-OHC), 7 β-hydroxycholesterol (7 β-OHC), and cholesterol-5 β,6 β-epoxide ( β-epox) on two cell types associated with the atherosclerotic process, bovine aortic endothelial (BAE) cells and human monocytic U937 cells. Cells were exposed to 25-OHC, 7 β-OHC, or β-epox, or equimolar mixtures (30 μM) of 25-OHC and 7 β-OHC, 25-OHC and β-epox, or 7 β-OHC and β-epox for 48 h. Cell viability was assessed using the fluorescein diacetate/ethidium bromide (FDA/ EtBr) assay and nuclear morphology following staining with Hoechst 33342. 25-OHC was the least toxic of the oxysterols and did not induce apoptosis in either cell line. Both 7 β-OHC and β-epox treatments were cytotoxic and induced apoptosis in the cells. Cotreatment with 25-OHC did not alter the toxicity of 7 β-OHC and β-epox in U937 cells but did decrease the percentage apoptotic cell death. In contrast, in the BAE cells cotreatment with 25-OHC had a slight protective effect on 7 β-OHC and β-epox–induced toxicities and a marked decrease in apoptotic cell death. The 7 β-OHC and β-epox mixture induced a significant increase in apoptotic cell death in U937 cells but decreased this mode of cell death in the BAE cells. The effects of oxysterols on glutathione levels also differed between the cells with changes noted in U937 and not in BAE cells. Results demonstrate interactive effects when oxysterols are studied as mixtures rather than single compounds in vitro.

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