Number Of Apoptotic Nuclei Research Articles

Preconditioning donor with a combination of tacrolimus and rapamacyn to decrease ischaemia–reperfusion injury in a rat syngenic kidney transplantation model

Reperfusion injury remains one of the major problems in transplantation. Repair from ischaemic acute renal failure (ARF) involves stimulation of tubular epithelial cell proliferation. The aim of this exploratory study was to evaluate the effects of preconditioning donor animals with rapamycin and tacrolimus to prevent ischaemia-reperfusion (I/R) injury. Twelve hours before nephrectomy, the donor animals received immunosuppressive drugs. The animals were divided into four groups, as follows: group 1 control: no treatment; group 2: rapamycin (2 mg/kg); group 3 FK506 (0, 3 mg/kg); and group 4: FK506 (0, 3 mg/kg) plus rapamycin (2 mg/kg). The left kidney was removed and after 3 h of cold ischaemia, the graft was transplanted. Twenty-four hours after transplant, the kidney was recovered for histological analysis and cytokine expression. Preconditioning treatment with rapamycin or tacrolimus significantly reduced blood urea nitrogen and creatinine compared with control [blood urea nitrogen (BUN): P < 0·001 versus control and creatinine: P < 0·001 versus control]. A further decrease was observed when rapamycin was combined with tacrolimus. Acute tubular necrosis was decreased significantly in donors treated with immunosuppressants compared with the control group (P < 0·001 versus control). Moreover, the number of apoptotic nuclei in the control group was higher compared with the treated groups (P < 0·001 versus control). Surprisingly, only rapamycin preconditioning treatment increased anti-apoptotic Bcl2 levels (P < 0·001). Finally, inflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, showed lower levels in the graft of those animals that had been pretreated with rapamycin or tacrolimus. This exploratory study demonstrates that preconditioning donor animals with rapamycin or tacrolimus improves clinical outcomes and reduce necrosis and apoptosis in kidney I/R injury.

Abstract 8534: Combination of Cyclosporine and Erythropoietin Improves Brain Infarct Size and Neurological Function in Rats after Ischemic Stroke

Objectives: This study tested the superiority of combined cyclosporine (CsA)-erythropoietin (EPO) therapy compared with either one in limiting brain infarction area (BIA) and preserving neurological function in rat after ischemic stroke (IS). Methods: Fifty adult-male SD rats were equally divided into sham control (group 1), IS plus intra-peritoneal physiological saline (at 0.5/24/48h after IS) (group 2), IS plus CsA (20.0 mg/kg at 0.5/24h, intra-peritoneal) (group 3), IS plus EPO (5,000IU/kg at 0.5/24/48h, subcutaneous) (group 4), combined CsA and EPO treatment (group 5) after occlusion of distal left internal carotid artery. Results: BIA on day 21 after acute IS was highest in group 2 and lowest in group 5 (all p&lt;0.01). The sensorimotor functional test showed highest frequency of left turning in group 2 and lowest in group 5 (all p&lt;0.05). mRNA and protein expressions of apoptotic markers and number of apoptotic nuclei on TUNEL were highest in group 2 and lowest in group 1 and 5, whereas the anti-apoptotic markers exhibited an opposite trend (all p&lt;0.05). The expressions of inflammatory and oxidized protein were highest in group 2 and lowest in group 1 and 5, whereas anti-inflammatory markers showed reversed changes in group 1 and 5 (all p&lt;0.05). The number of aquaporin - 4+ and glial fibrillary acid protein+ stained cells were highest in group 2 and lowest in groups 1 and 5 (all p&lt;0.01). Conclusion: Combined treatment with CsA and EPO was superior to either one alone in protecting rat brain from ischemic damage after IS (the proposed mechanisms of potential therapeutic impact of combined therapy with CsA and EPO on acute IS have been summarized in the figure)

Combination of cyclosporine and erythropoietin improves brain infarct size and neurological function in rats after ischemic stroke

BackgroundThis study tested the superiority of combined cyclosporine A (CsA)-erythropoietin (EPO) therapy compared with either one in limiting brain infarction area (BIA) and preserving neurological function in rat after ischemic stroke (IS).MethodsFifty adult-male SD rats were equally divided into sham control (group 1), IS plus intra-peritoneal physiological saline (at 0.5/24/48 h after IS) (group 2), IS plus CsA (20.0 mg/kg at 0.5/24h, intra-peritoneal) (group 3), IS plus EPO (5,000IU/kg at 0.5/24/48h, subcutaneous) (group 4), combined CsA and EPO (same route and dosage as groups 3 and 4) treatment (group 5) after occlusion of distal left internal carotid artery.ResultsBIA on day 21 after acute IS was higher in group 2 than in other groups and lowest in group 5 (all p < 0.01). The sensorimotor functional test showed higher frequency of left turning in group 2 than in other groups and lowest in group 5 (all p < 0.05). mRNA and protein expressions of apoptotic markers and number of apoptotic nuclei on TUNEL were higher in group 2 than in other groups and lowest in group 1 and 5, whereas the anti-apoptotic markers exhibited an opposite trend (all p < 0.05). The expressions of inflammatory and oxidized protein were higher in group 2 than in other groups and lowest in group 1 and 5, whereas anti-inflammatory markers showed reversed changes in group 1 and other groups (all p < 0.05). The number of aquaporin-4+ and glial fibrillary acid protein+ stained cells were higher in group 2 as compared to other groups and lowest in groups 1 and 5 (all p < 0.01).Conclusioncombined treatment with CsA and EPO was superior to either one alone in protecting rat brain from ischemic damage after IS.

Ovine Oocytes Vitrified at Germinal Vesicle Stage as Cytoplast Recipients for Somatic Cell Nuclear Transfer (SCNT)

The development of embryos produced by somatic cell nuclear transfer (SCNT) using vitrified oocytes as cytoplast recipients has been reported in cattle but not in sheep. This study investigated the parthenogenetic development of ovine oocytes vitrified and thawed at the germinal vesicle (GV) stage, matured in vitro, and then activated using two activation protocols. The optimal activation protocol was then used to assess development when vitrified oocytes were used as cytoplast recipients for SCNT. No blastocysts were obtained from vitrified oocytes activated by CA+CHX/CB (calcium ionophore A23187 + cycloheximide, and cytochalasin B); in contrast, vitrified oocytes activated by Sr/CB (strontium chloride (SrCl(2)) + cytochalasin B) developed to blastocyst, although the number was significantly lower (p < 0.05) than in toxicity and control groups (3.8 vs. 20.0 and 27.3%, respectively). In SCNT embryos, cleavage at both 24 and 48 h postactivation (31.0 vs. 55.1% and 48.0 vs. 85.0%) was significantly lower (p < 0.05) in vitrified oocytes compared to controls. However, no significant differences were observed in the frequency of development to blastocyst (13.0 vs. 23.4%), the number of hatched blastocysts (7.0 vs. 10.3%), total cell numbers (90.3 ± 4.9 vs. 97.6 ± 4.6), number of apoptotic nuclei (13.1 ± 0.9 vs. 13.2 ± 1.4), or the proportion of diploid embryos (60.0 vs. 75.0%). This study demonstrates for the first time that ovine oocytes vitrified at the GV stage can be used successfully as recipient cytoplasts for SCNT.

Vascular endothelial growth factor expression up-regulated by endometrial ischemia in secretory phase plays an important role in endometriosis

Primary human endometrial cells were exposed to hypoxia preconditioning (HPC), HPC-hypoxia, and hypoxia conditions, and then endometrial tissue treated with ischemia preconditioning (IPC) was transplanted onto the chick embryo chorioallantoic membrane to investigate the role of slight ischemia of endometrium in the pathologic process of endometriosis. IPC up-regulated vascular endothelial growth factor expression and decreased apoptosis of endometrial cells, thus facilitating the endometrial fragments' ectopic implantation.

Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.

Effects of intracellular zinc depletion on the expression of VDAC in cultured hippocampal neurons

An experiment was conducted to investigate whether intracellular zinc depletion can actually change expression of voltage-dependent anion channel 1 (VDAC1) and VPAC2 in cultured hippocampal neurons as well as their significance. Hippocampal neurons were obtained by primary culture from hippocampus of newborn Wistar rats. Cultured hippocampal neurons were exposed to a cell membrane-permeable zinc chelator N,N,N′,N′-tetrakis (2-pyridyl methyl) ethylenediamine (TPEN) (2 µM), and to TPEN plus zinc sulfate (5 µM) for 1 or 24 hours. Cultures were then processed to detect neuronal injury by lactate dehydrogenase (LDH) assay, intracellular Ca2+ with the fluorescent probe fluo-3/AM, reactive oxygen species (ROS) generation using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay, nuclear morphology by Hoechst 33342, VDAC1, and VDAC2 protein levels by western blot, and VDAC1 and VDAC2 mRNA levels by RT–PCR. The results demonstrated that exposure of hippocampal neurons to TPEN (2 µM) for 24 hours induced notably neuronal injury, significantly increased the number of apoptotic nuclei, up-regulated the expression of VDAC1 protein level and down-regulated the expression of VDAC2 protein level. Significant down-regulation of mRNA levels for VDAC1 and VDAC2 were observed in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced neuronal injury and above alterations in VDAC1 and VDAC2 protein levels and mRNA levels. Present results implicate a possibility that up-regulation of VDAC1 and down-regulation of VDAC2 may participate in hippocampal neuron injury induced by zinc deficiency.

PKCδ Is Activated in a Dietary Model of Steatohepatitis and Regulates Endoplasmic Reticulum Stress and Cell Death

Hepatic steatosis can progress to the clinical condition of non-alcoholic steatohepatitis (NASH), which is a precursor of more serious liver diseases. The novel PKC isoforms δ and ε are activated by lipid metabolites and have been implicated in lipid-induced hepatic disease. Using a methionine- and choline-deficient (MCD) dietary model of NASH, we addressed the question of whether hepatic PKCδ and PKCε are activated. With progression from steatosis to steatohepatitis, there was activation and increased PKCδ protein content coincident with hepatic endoplasmic reticulum (ER) stress parameters. To examine whether similar changes could be induced in vitro, McA-RH 7777 (McA) hepatoma cells were used. We observed that McA cells stored triglyceride and released alanine aminotransferase (ALT) when treated with MCD medium in the presence of fatty acids. Further, MCD medium with palmitic acid, but not oleic or linoleic acids, maximally activated PKCδ and stimulated ER stress. In PKCδ knockdown McA cells, MCD/fatty acid medium-induced ALT release and ER stress induction were completely blocked, but triglyceride storage was not. In addition, a reduction in the uptake of propidium iodide and the number of apoptotic nuclei and a significant increase in cell viability and DNA content were observed in PKCδ knockdown McA cells incubated in MCD medium with palmitic acid. Our studies show that PKCδ activation and protein levels are elevated in an animal model of steatohepatitis, which was recapitulated in a cell model, supporting the conclusion that PKCδ plays a role in ALT release, the ER stress signal, and cell death.

Protective effect of immunosuppressive treatment before orthotopic kidney autotransplantation

BackgroundIschemia reperfusion injury (IRI) is one of the risk factors for delayed graft function, acute rejection and long term allograft survival after kidney transplantation. IRI is an independent antigen inflammatory process that produces tissue damage. Our objective was to study the impact of immunosuppressive treatment (IS) on IRI applying only one dose of IS before orthotopic kidney autotransplantation. MethodsTwenty-four rats allocated in four groups were studied. One group served as control (G1: autotransplanted rats without IS) and the rest received IS 12h before kidney autotransplantation (G2: Rapamycin, G3: Mycophenolate mofetil and G4: Tacrolimus). ResultsImproved renal function and systemic inflammatory response were found among IS groups compared to the control group (Delta Urea p<0.0001; Delta Creatinine p<0.0001; Delta C3 p<0.001). The number of apoptotic nuclei in renal medulla in G1 was higher than in IS groups (p<0.0001). Tubular damage was less severe in IS groups respecting G1 (p<0.001). C3, TNF-α and IL-6 expression in kidney samples was reduced when IS was used compared to the control group. No differences were observed among the different immunosuppressive drugs tested. However, Heme oxygenase-1(HO-1) was increased only in Rapamycin treatment. ConclusionsThese data suggest that the use of IS administered before transplant attenuates the IRI process after kidney transplantation in an animal model.

POD09 Using the chick embryo model system to study the neurotoxicity of TDP43

We have successfully developed the chick embryo model of ALS. We carried out overexpression of HA and Myc tagged TDP-43WT, TDP-43Q331K and TDP-43M337V constructs (Sreedharan et al, 2008). We also...

Adipose-derived mesenchymal stem cells markedly attenuate brain infarct size and improve neurological function in rats

BackgroundThe therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on brain infarction area (BIA) and neurological status in a rat model of acute ischemic stroke (IS) was investigated.MethodsAdult male Sprague-Dawley (SD) rats (n = 30) were divided into IS plus intra-venous 1 mL saline (at 0, 12 and 24 h after IS induction) (control group) and IS plus intra-venous ADMSCs (2.0 × 106) (treated interval as controls) (treatment group) after occlusion of distal left internal carotid artery. The rats were sacrificed and brain tissues were harvested on day 21 after the procedure.ResultsThe results showed that BIA was larger in control group than in treatment group (p < 0.001). The sensorimotor functional test (Corner test) identified a higher frequency of turning movement to left in control group than in treatment group (p < 0.05). mRNA expressions of Bax, caspase 3, interleukin (IL)-18, toll-like receptor-4 and plasminogen activator inhibitor-1 were higher, whereas Bcl-2 and IL-8/Gro were lower in control group than in treatment group (all p < 0.05). Western blot demonstrated a lower CXCR4 and stromal-cell derived factor-1 (SDF-1) in control group than in treatment group (all p < 0.01). Immunohistofluorescent staining showed lower expressions of CXCR4, SDF-1, von Willebran factor and doublecortin, whereas the number of apoptotic nuclei on TUNEL assay was higher in control group than in treatment group (all p < 0.001). Immunohistochemical staining showed that cellular proliferation and number of small vessels were lower but glial fibrillary acid protein was higher in control group than in treatment group (all p < 0.01).ConclusionsADMSC therapy significantly limited BIA and improved sensorimotor dysfunction after acute IS.

Autologous bone marrow cell implantation attenuates left ventricular remodeling and improves heart function in porcine myocardial infarction: An echocardiographic, six-month angiographic, and molecular–cellular study

Background We investigated the potential benefits and the underlying mechanisms of autologous bone marrow-derived mononuclear cell (BMDMNC) implantation in a porcine model of acute anterior wall myocardial infarction (AAWMI) by studying 6-month left ventricular (LV) function and LV remodeling. Methods After being aspirated from the iliac crest and cultured for 1 week, BMDMNCs were implanted immediately after AAWMI induction through the left anterior descending artery ligation. Thirty male mini-pigs (16–18 kg) were equally divided into group 1 [AAWMI plus saline injection into infarct-ischemia area (IA)], group 2 (AAWMI plus 3.0 × 10 7 BMDMNC transplantation into non-IA), group 3 (AAWMI plus 3.0 × 10 7 BMDMNC transplantation into IA), group 4 (sham control plus 3.0 × 10 7 BMDMNC transplantation into LV myocardium), and group 5 (normal control). Results By day 90, echocardiography demonstrated an increased LV end-diastolic and end-systolic dimensions but reduced LV ejection fraction (LVEF) in groups 1 and 2 than in other groups (all p < 0.01). Six-month angiographic study showed a lower LVEF and wall motion score but a higher mitral regurgitation in groups 1 and 2 than in other groups (all p < 0.01). In IA and peri-infarct area, the number of small vessels and mRNA expressions of endothelial nitric oxide synthase, Bcl-2, interleukin (IL)-10, and peroxisome proliferator-activated receptor-γ coactivator-1α were lower, whereas the number of apoptotic nuclei, caspase-3, Bax, endothelin-1, IL-8, and matrix metalloproteinase was higher in groups 1 and 2 than in other groups (all p < 0.01). Conclusions Autologous BMDMNC transplantation into IA rather non-IA improves LV function and reduces LV remodeling via eliciting a broad-spectrum of molecular–cellular defensive mechanisms.

Microcebus murinus retina: A new model to assess prion-related neurotoxicity in primates

No effective treatment currently exists for prion diseases and therefore the development of experimental non-human primate models of prion neurotoxicity, to better understand the underlying mechanism and to test new treatments relevant to humans, represents an urgent medical need. However, the establishment of such models is challenging due to animal welfare and cost considerations. We describe here the use of Microcebus murinus retina, in primary cultures and in vivo, as a new experimental primate model to rapidly examine the effects in the central nervous system of PrP 106–126, a neurotoxic fragment of the human prion protein. We demonstrate that PrP 106–126 triggered rod photoreceptor cell loss by apoptosis and a change in morphology of microglial cells in mixed neuronal–glial cultures of retinal cells. In addition, 2 days after intravitreal injection of PrP 106–126, retinas showed a significant increase in the number of apoptotic nuclei, mainly in the ganglion cell layer.

Female sex and estrogen receptor-β attenuate cardiac remodeling and apoptosis in pressure overload

We investigated sex differences and the role of estrogen receptor-beta (ERbeta) on myocardial hypertrophy in a mouse model of pressure overload. We performed transverse aortic constriction (TAC) or sham surgery in male and female wild-type (WT) and ERbeta knockout (ERbeta(-/-)) mice. All mice were characterized by echocardiography and hemodynamic measurements and were killed 9 wk after surgery. Left ventricular (LV) samples were analyzed by microarray profiling, real-time RT-PCR, and histology. After 9 wk, WT males showed more hypertrophy and heart failure signs than WT females. Notably, WT females developed a concentric form of hypertrophy, while males developed eccentric hypertrophy. ERbeta deletion augmented the TAC-induced increase in cardiomyocyte diameter in both sexes. Gene expression profiling revealed that WT male hearts had a stronger induction of matrix-related genes and a stronger repression of mitochondrial genes than WT female hearts. ERbeta(-/-) mice exhibited a different transcriptional response. ERbeta(-/-)/TAC mice of both sexes exhibited induction of proapoptotic genes with a stronger expression in ERbeta(-/-) males. Cardiac fibrosis was more pronounced in male WT/TAC than in female mice. This difference was abolished in ERbeta(-/-) mice. The number of apoptotic nuclei was increased in both sexes of ERbeta(-/-)/TAC mice, most prominent in males. Female sex offers protection against ventricular chamber dilation in the TAC model. Both female sex and ERbeta attenuate the development of fibrosis and apoptosis, thus slowing the progression to heart failure.

Microgravity-induced apoptosis in cultured glial cells.

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.

Gamma-Ray Susceptibility of Immature and Mature Hippocampal Cultured Cells

Ionizing radiation suppresses neurogenesis in the mammalian brain. This in vitro study compared the detrimental effect of acute gamma-irradiation on immature hippocampal cells with mature cells. Both rat immature (0.5 day in vitro (DIV)) and mature hippocampal cells (14 DIV) were irradiated with 0-4 Gy gamma-rays. Cell viability was analyzed by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA fragmentation study was performed by extracting intracellular DNA. Morphological features of apoptosis were characterized by 4',6-diamidine-2'-phenylindole, dihydrochloride (DAPI) staining. MTT assay revealed that the survival rate of immature hippocampal cells declined in a dose-dependent manner within the range of irradiation applied, but was not changed in mature cells. Intranucleosomal DNA fragmentation in a ladder like pattern was dose-dependently increased in immature cells, but not in mature cells. The number of apoptotic nuclei in immature cells increased significantly in a dose-dependent manner within the range of irradiation applied. Active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expressions in immature hippocampal cells at 6 hr after 2 Gy exposure were markedly higher than control levels. The significantly greater radiosensitivity of immature hippocampal cells than that of the mature cells, indicates that the susceptibility of such hippocampal cells depends on their maturation. In addition, gamma-irradiation may induce caspase-dependent apoptosis in immature hippocampal cells.

Sirolimus in Kidney Transplant Donors and Clinical and Histologic Improvement in Recipients: Rat Model

Background Ischemia-reperfusion (I/R) injury is one of the risk factors for delayed graft function, acute rejection episodes, and impaired long-term allograft survival after kidney transplantation. This antigen-independent inflammatory process produces tissue damage. Isogeneic transplantation in a rat model is a useful method for study of nonimmunologic risk factors for kidney damage. Objective To study the effect of sirolimus on I/R injury using only 1 dose of the drug in the donor. Materials and Methods Eighteen rats were allocated to 3 groups of 6 rats each: sham group, control group, and rapamycin group. Results Improved renal function and systemic inflammatory response were observed in the rapamycin group compared with the control group (Δurea, Δcreatinine, and ΔC3, all P < .01). The number of apoptotic nuclei in the renal medulla in the control group was higher than in the rapamycin group ( P < .01). Tubular damage was less severe in the rapamycin group compared with the control group ( P < .01). Complement 3 and tumor necrosis factor-α expression in the kidney samples were significantly decreased when rapamycin was given to the donor rats ( P > .01). Bcl-2 protein was upregulated in the rapamycin group compared with the control group ( P < .01). Conclusion Administration of rapamycin in donors attenuates the I/R injury process after kidney transplantation in a rat model.

60 EFFECTS OF REPETITIVE IONOMYCIN TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

In bovine somatic cell nuclear transfer (SCNT), oocyte activation is an essential element for the successful development of cloned embryos. Chemical treatment with ionomycin induces transient levels of Ca2+, and this has been used in the activation of reconstructed embryos. In vitro-fertilized oocytes are naturally activated by sperm following several Ca2+ transients known collectively as Ca2+ oscillations. We aimed to improve the developmental efficiency of embryos produced by SCNT by mimicking this Ca2+ oscillation artificially. SCNT was performed as follows; bovine calf skin fibroblast cells were transferred into the perivitelline space of IVM oocytes, and then these NT couplets were treated with electrical fusion (2 pulses, 1.75 kV cm-1, 15 μs). Reconstructed embryos were subsequently cultured in SOF medium (5% CO2, 5% O2, and 38°C). To mimic Ca2+ oscillation, we carried out 3 different repetitive ionomycin (10 μM) treatments at 1-h intervals. There were 3 groups: group 1 (4 min, 1 time), group 2 (30 s, 4 times), and group 3 (1 min, 4 times). The difference in embryo development amongst these experimental groups was then analyzed using the one-way ANOVA test after arcsine transformation to maintain homogeneity of variance. All analyses were performed using SAS (version 8.1, SAS Institute, Cary, NC, USA). Significant differences among the treatments were determined when the P-value was &lt;0.05. In experiment 1, to assess developmental efficiency, the cleavage rate was investigated on Day 2 and the formation rate of blastocysts (BL) was examined on Day 7. In group 3, a significant increase in BL formation was observed [47/263 (17.8%), 50/259 (19.3%), and 67/258 (26.0%), respectively]. In experiment 2, culturing each group of embryos with different ionomycin treatments caused no significant differences among the groups in terms of the total number of BL (164.3, 158.5, and 145.1, respectively). In experiment 3, expression of apoptosis-related genes in each group was evaluated by real-time PCR and the TUNEL assay. The 3 BL within each group were analyzed for the expression of apotosis-related genes. Expression of the anti-apoptotic Bcl-2 gene was increased in group 3, whereas the expression of the pro-apoptotic Bax was decreased. A decrease in the number of apoptotic nuclei was also observed in group 3. In conclusion, the present study demonstrated that repetitive ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.

High concentration of magnolol induces hepatotoxicity under serum-reduced conditions

Although magnolol is cytoprotective against warm ischemia/reperfusion injury, its effect on cold preservation has not been fully investigated. This study aimed at examining whether magnolol maintains the liver graft integrity after cold preservation and elucidating the underlying mechanisms in terms of apoptotic signaling under both normothermic and hypothermic conditions. After being preserved in Ringer's lactate (RL) at 4 degrees C for 6h ex vivo, the magnolol-treated grafts demonstrated significantly higher AST, ALT, and LDH levels in perfusates than those from negative controls. TUNEL staining showed no difference in the number of apoptotic nuclei in both groups, whereas a more intense apoptotic signal in magnolol-treated grafts was shown as compared with the controls. In vitro data showed no significant difference in viability of RL-preserved clone-9 hepatocytes between the magnolol-treated and control groups, while magnolol pretreatment at 30min before cold preservation prominently induced hepatocyte cell death. RT-PCR and Western blotting analyses revealed a suppression in Bcl-2, but an up-regulation in Bax expression in clone-9 cells after magnolol treatment. Magnolol suppressed the ratios of NF-kappaB to I-kappaBalpha protein contents and I-kappaBalpha phosphorylation induced by TNF-alpha, and potentiated mitochondrial cytochrome c release and subsequent caspase-3 cleavage. Conversely, caspase-3 inhibitor attenuated magnolol-induced hepatotoxicity. We concluded that magnolol could not protect liver grafts from cold ischemia/reperfusion injury. High concentration of magnolol under serum-reduced conditions attenuates NF-kappaB-mediated signaling and induces intrinsic apoptotic pathway, thereby inducing in vitro hepatotoxicity.

Apoptosis and macrophage infiltration occur simultaneously and present a potential sign of muscle injury in skeletal muscle of nutritionally compromised, early post-hatch turkeys

Physical stress and malnutrition may cause elimination of myonuclei and produce inflammatory response in muscle. The objective of this study was to histochemically determine the association of apoptosis and/or macrophage infiltration with changes in muscle satellite cell mitotic activity in pectoralis thoracicus muscle of early post-hatch turkey toms. Feed-deprived birds and birds provided with three different levels of crude protein and amino acids (0.88 NRC, 1.00 NRC, and 1.12 NRC) were used in this model. The number of apoptotic nuclei was significantly elevated (P<0.05) and presence of macrophage infiltration was readily detectable in feed-deprived and 0.88 NRC treatment groups 72 h and 96 h post-hatch suggesting potential muscle injury and/or muscle remodeling. The number of apoptotic nuclei was the same (P>0.05), and there was no detectable macrophage infiltration present in birds placed on 1.00 NRC and 1.12 NRC diet 72 h, 96 h, and 120 h post-hatch. At 120 h post-hatch, feed-deprived and 0.88 NRC birds were characterized by no detectable levels of macrophage infiltration and a significant drop (P<0.05) in apoptotic nuclei. Understanding mechanisms that correlate early nutrition with skeletal muscle growth and development may present a useful tool in optimizing muscle health and improving meat quality and yield.