270 APOPTOSIS AND ONCOSIS ASSESSMENT IN CRYOPRESERVED MOUSE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 242
Author(s):  
M. E. O. A. Assumpção ◽  
A. R. S. Coutinho ◽  
W. B. Feitosa ◽  
C. M. Mendes ◽  
R. Simões ◽  
...  
Keyword(s):  
Cell Death ◽  
Liquid Nitrogen ◽  
Caspase 3 ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Water Bath ◽  
Slow Freezing ◽  
Control Group ◽  
Embryo Viability ◽  
Cryopreserved Embryos

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis of cryopreserved embryos may be a negative factor for their viability. The aim of this study was to detect apoptosis and to characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were separated to be subjected to two cryopreservation protocols (slow freezing and vitrification) and a control group (fresh). In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. Straws were placed in a methanol bath at -7�C until it reached -31�C and then plunged and stored in liquid nitrogen. The embryos were thawed in air for 10 s and in a 25�C water bath for 20 s. In the vitrification method, embryos were exposed to 10% and 20% EG for 5 min, followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and then plunged and stored in liquid nitrogen. These embryos were thawed in a 25�C water bath for 20 s. For the cell death evaluation, cell membrane integrity from the fresh and cryopreserved embryos was assessed by Hoechst and propidium iodide (H/PI staining). Morphology and apoptosis were assessed by means of the haematoxylin-eosin staining (HE) and by electron microscopy (MET). To confirm apoptosis, 64 cryopreserved mouse morulae (34 submitted to slow freezing and 30 to vitrification) were used to evaluate Caspase-3 activity. The cryopreserved embryos were divided into experimental and control groups and incubated with Caspase-3 and buffer solution, respectively. Afterward, the embryos were incubated with rhodamine and the Caspase activity was determined under a fluorescence microscope. H/PI staining detected more membrane permeability in the vitrification (69.7%) than in the slow-freezing (48.4%) or fresh (13.8%) groups (P < 0.05; Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow freezing induced pyknosis and chromatin condensation. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), whereas in the slow-freezing the presence of cytoplasmic condensation and eosinophilic structures indicating apoptosis were observed. MET examination of the ultrastructure confirmed the HE results. The Caspase-3 activity showed a fluorescence increase in both experimental groups compared with the control group. In conclusion, staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. Regarding the cryopreservation techniques, both slow freezing and vitrification showed oncosis and apoptosis injuries. However, in this experiment vitrification caused more cellular injuries, with less embryo viability, than slow freezing. This work was supported by FAPESP 04/01252-4 and CAPES.

scholarly journals 82 EVALUATION OF CELL DEATH IN CRYOPRESERVED MOUSE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 191
Author(s):  
A.R.S. Coutinho ◽  
A.B. Nascimeto ◽  
C.M. Mendes ◽  
R. Simoes ◽  
C.F. Lucio ◽  
...  
Keyword(s):  
Cell Death ◽  
Liquid Nitrogen ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Embryonic Cell ◽  
Slow Freezing ◽  
Cell Membrane Integrity ◽  
Frozen Embryos ◽  
Mammalian Embryos ◽  
Cryopreserved Embryos

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis may be important in determining the viability of cryopreserved embryos. Our goal was to detect apoptosis and characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were collected, selected, and separated into three groups: fresh, slow-freezing, and vitrification. In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. After loading, the straws were placed into methanol at −7°C for 5 min, seeded and after 5 min cooled at 0.5°C/minute. After 10 minutes at −31°C, straws were plunged into and stored in liquid nitrogen. Slow-frozen straws were thawed in air for 10 s, and then immersed in a 25°C water bath for 20 s. Embryos were vitrified by exposing them to 10% and 20% EG for 5 min followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and the 0.25-mL straws then plunged into and stored in liquid nitrogen. The vitrified straws were warmed by immersing them in 25°C water for 20 s. Cell membrane integrity was assessed by Hoechst and propidium iodide double staining (H/PI). Fresh and thawed embryos were scored (following IETS recommendations) and then fixed after 30 min in PBS + 10% FCS. Morphology and apoptosis were assessed with Haematoxylin-Eosin staining (HE) and by electron microscopy (MET). The number of Grade I embryos recovered after thawing was higher for slow-frozen embryos (61.5%) than vitrified embryos (29.5%). H/PI detected more membrane permeability in the vitrified embryos (69.7%), than in the slow-frozen (48.4%) or non-frozen (13.8%) groups (P < 0,05, Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow-freezing induced pyknosis and chromatin condensation. Mitotic pattern was observed in the fresh and slow-frozen group, but not in vitrification group suggesting that the embryos were either not randomly allocated to the groups or not-treated and fixed at the same age, or that vitrification changed the nuclear status of the embryos. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), while in the slow-frozen group the presence of cytoplasmic condensation and eosinophilic structures indicated apoptosis. The ultrastructure examination confirmed the HE observations. In conclusion, the results demonstrated that staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. According to these data, vitrification caused more cellular injuries than slow-freezing, and oncosis was the predominant injury. It is important to point that specific molecular apoptosis tests must be performed to confirm these results. This work was supported by FAPESP 04/01252-4.

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.

Two steroidal saponins from Camassia cusickii induce L1210 cell death through the apoptotic mechanism

2001 ◽  
Vol 79 (11) ◽  
pp. 953-958 ◽  
Author(s):  
Ellyawati Candra ◽  
Kimihiro Matsunaga ◽  
Hironori Fujiwara ◽  
Yoshihiro Mimaki ◽  
Yutaka Sashida ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Flow Cytometric Analysis ◽  
Apoptotic Cell Death ◽  
Morphological Observation ◽  
Steroidal Saponin ◽  
Steroidal Saponins ◽  
L1210 Cells

Two steroidal saponins, tigogenin hexasaccharide-1 (TGHS-1, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside) and tigogenin hexasaccharide-2 (TGHS-2, (25R)-5α-spirostan-3β-yl 4-O-[2-O-[3-O- (β-D-glucopyranosyl)-β-D-glucopyranosyl]-3-O-[4-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranosyl]- β-D-galactopyranoside), were isolated from the fresh bulbs of Camassia cusickii. In murine leukemic L1210 cells, both compounds showed cytotoxicity with an EC50 value of 0.06 µM. The morphological observation revealed that TGHS-1 and TGHS-2 induced shrinkage in cell soma and chromatin condensation, suggesting apoptotic cell death. The cell death was confirmed to be apoptosis by Annexin V binding to phosphatidylserine in the cell membrane and excluding propidium iodide. A typical apoptotic DNA ladder and the cleavage of caspase-3 were observed after treatment with TGHS-1 and TGHS-2. In the presence of both the compounds, cells with sub-G1 DNA content were detected by flow cytometric analysis, indicating that TGHS-1 and TGHS-2 (each EC50 value of 0.1 µM) are the most powerful apoptotic saponins known. These results suggest that TGHS-1 and TGHS-2 induce apoptotic cell death through caspase-3 activation.Key words: steroidal saponin, tigogenin hexasaccharide, apoptosis, DNA fragmentation, murine leukemic L1210 cells.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

132 SUCCESSFUL EMBRYO TRANSFER OF IN VIVO-PRODUCED RED DEER (CERVUS ELAPHUS) EMBRYOS AFTER CRYOPRESERVATION BY SLOW FREEZING OR VITRIFICATION

2007 ◽  
Vol 19 (1) ◽  
pp. 183
Author(s):  
J. P. Soler ◽  
G. G. Kaiser ◽  
N. Mucci ◽  
L. B. Ferre ◽  
R. H. Alberio
Keyword(s):  
Liquid Nitrogen ◽  
Embryo Transfer ◽  
Cervus Elaphus ◽  
Red Deer ◽  
Slow Freezing ◽  
Alternative Methods ◽  
Embryo Cryopreservation ◽  
Chi Square ◽  
Cryopreserved Embryos

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P &lt; 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos. Table 1. Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos

217 QUALITY CHANGES IN POST-THAW SPRINGBOK (ANTIDORCAS MARSUPIALIS) EPIDIDYMAL SPERM MAINTAINED IN FERTILIZATION MEDIUM

2006 ◽  
Vol 18 (2) ◽  
pp. 216
Author(s):  
F. P. Chatiza ◽  
G. M. Pieterse ◽  
P. Bartels
Keyword(s):  
South Africa ◽  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Water Bath ◽  
Quality Changes ◽  
Epididymal Sperm ◽  
Plasma Membrane Integrity ◽  
Free Ranging ◽  
Over Time

The availability of gametes from the cropping of excess wildlife species provides the opportunity for the advancement of knowledge into assisted reproductive technology for possible future conservation measures. Little is known about the longevity of springbok (Antidorcas marsupialis) spermatozoa maintained in fertilization medium. The aim of this project was to determine the quality changes of post-thawed springbok spermatozoa incubated in fertilization medium by measuring plasma membrane integrity over time. Testes (n = 12) were obtained from two geographically distinct free-ranging springbok populations in South Africa. Spermatozoa were flushed from the cauda epididymides within three hours of the animals' death. Samples from an individual male were pooled, diluted to 400 × 106 sperm/mL with Biladyl (Minitüb, Tiefenbach, Germany) fraction A (no glycerol) and equilibrated in a water bath for 6 h at 4°C. An equal volume of Biladyl fraction B (containing 12% glycerol) was added to the sample to make a final concentration of 200 × 106 sperm/mL. Samples were loaded into 0.25-mL straws and frozen in liquid nitrogen vapor (5 cm above the liquid nitrogen level) for 20 min after which they were plunged into liquid nitrogen. Straws from each sample were thawed for 20 s at 36°C in a water bath. Thawed spermatozoa (100 μL) was added to 1 mL IVF-TALP medium containing heparin and PHE (Vajta et al. 1996 Theriogenology 45, 683–689) in 2-mL Nunc tubes (AEC, Amersham, South Africa) and incubated at 38.7°C, in humidified 5% CO2 balance air for 30 h. Aliquots were extracted from the incubating spermatozoa to determine plasma membrane integrity at 6-h intervals. Propidium iodide (Sigma, South Africa) at 50 ng/mL (10 min at RT) was used to evaluate membrane integrity under fluorescence microscopy at ×400, with a 450-nm excitation filter, a 510-nm dicroic beam splitter, and a 520-nm barrier filter. Cells with damaged plasma membrane have nuclei that fluorescence red. Eosin/nigrosin was also used to evaluate membrane integrity under ×400 bright-field microscopy. Cells with damaged plasma membrane stain purple-red, whereas the balance of cells remain translucent. The average post-thaw motility of spermatozoa in populations A and B was 69% (n = 6) and 68% (n = 6), respectively. Plasma membrane integrity of post-thawed springbok spermatozoa deceased steadily in IVF-TALP medium over the 30-h period (Table 1). Cryopreserved epididymal sperm derived from free-ranging springbok populations survive in IVF-TALP media and may be useful in future conservation activities where an isolated gene pool requires genetic supplementation through one or more assisted reproduction techniques such as IVF or AI. Further research is required to confirm and extend these findings. Table 1. Percentage plasma membrane integrity of post-thawed springbok sperm over time

Vascular Endothelium Is Severely Perturbed and Undergoes Apoptosis in Experimental Heatstroke in Primates.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3972-3972
Author(s):  
George T. Roberts ◽  
Muhammad A. Chishti ◽  
Fallah H. Al-Mohanna ◽  
Raafat M. El-Sayed ◽  
Abderezak Bouchama
Keyword(s):  
Cell Death ◽  
Endothelial Cell ◽  
Vascular Endothelium ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Apoptotic Cell Death ◽  
Control Group ◽  
Tissue Sections ◽  
Endothelial Cell Death

Abstract Introduction: Ultrastructural evidence of endothelial cell (EC) injury has been associated with diffuse microvascular thrombosis in human heatstroke (HS). In vitro studies have also shown that heat stress accelerates apoptotic cell death. Using a recently described baboon model of heatstroke, we sought to examine pathological changes in the vascular endothelium and whether apoptosis is a mechanism of endothelial cell death. Hypothesis: Major structural vascular endothelium alterations occur in HS and apoptosis is a mechanism of endothelial cell death in HS. Methods: Anesthetized baboons (Papio hamadyras) were heat-stressed in a neonatal incubator maintained at 44 1.5 °C, until rectal temperature attained 42.5°C (moderate heatstroke; n =4) or systolic blood pressure fell to < 90 mm Hg (severe heatstroke n =4). Animals were resuscitated with normal saline and allowed to cool at room temperature. Four sham-heated animals served as control group. Spleen, liver, heart, kidney, gut, lung and adrenal tissue were obtained either by immediate autopsy in non-survivors or after euthanasia at 72-h for survivors. Vascular endothelium ultrastructure was evaluated by transmission electron microscopy (TEM) of ultra-thin tissue sections. Biological activity of EC was determined by light microscopy (LM) using a polyclonal antibody targeting von Willebrand Factor (vWF). Apoptosis was assessed, also in tissue sections, by deoxyuridine triphosphate nick end-labeling (TUNEL) procedure. Results: In heatstroke animals, there were marked EC changes in lungs, spleen, jejunum, kidneys and liver, demonstrated by TEM, as increased cytoplasmic membrane convolutions that included formation of villi projecting into the vessel lumina, and increase in the width of the gaps between ECs. Migration of neutrophils, platelets and erythrocytes through these widened gaps was noted. Weibel-Palade bodies were increased both in size and number in EC of jejunum, lungs and kidneys. This increase correlated with increased endothelial expression of immunologically detectable vWF. TEM also showed that there was increased apoptosis manifested by nuclear chromatin condensation and karyorrhexis and formation of cytoplasmic myelin whorls. Increased EC apoptosis was also observed by TUNEL in the jejunum, lungs, liver and spleen. All these changes were greater in animals with severe HS than in animals with moderate HS, whereas sham heated control animals showed no significant changes. Conclusion: Widespread EC injury with apoptotic cell death is consistent with the hypothesis that the endothelium may play a pathogenic role in heatstroke.

scholarly journals The Efficacy of Cinnamomum burmanii Extract on the Protection of Neuronal Cell Death in Haloperidol Induced Male Wistar Rats

2018 ◽  
Vol 2 (4) ◽  
pp. 1-11
Author(s):  
Nita Parisa ◽  
MT Kamaluddin ◽  
Theodorus Theodorus
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Positive Control ◽  
Control Group ◽  
Cinnamon Extract ◽  
White Rats ◽  
Before And After ◽  
Mean Differences

Background Haloperidol is categorized as the first class antipsychotic drug. Long-term use of haloperidol may convey to increased Reactive Oxygen Species (ROS) that will yield oxidative damage which further leads to cell death. Several studies had identified the effects of cinnamon extract on cell death. This study aimed to determine the efficacy of cinnamon extract (Cinnamomum burmanii) on the protection of neuronal cell death in haloperidol-induced male Wistar white rats. Methods This study was experimental with pre and post-test design. Thirty male Wistar rats were divided into 5 groups, induced with haloperidol and followed by treatment. Caspase-3 and dopamine were assayed by ELISA sandwich method using ELISA kit. Mean difference of caspase expression and dopamine levels before and after induction were shown (p<0.05). Results There were mean differences of caspase-3 expression level in the positive control group, cinnamon extract of 100 and 200mg/kgBW before and after treatment (p<0.05). Whereas for dopamine levels, there were mean differences in positive control group, cinnamon extract of 50, 100 and 200mg/kgBW before and after treatment (p<0.05). With Post Hoc test, it was found that there were no mean differences of caspase-3 expression level between positive group with cinnamon extract group of 100 and 200mg/kgBW (p>0,05) and there were also no mean differences of positive group dopamine level with group of cinnamon extract of 100 and 200mg/kgBW (p>0.05). Conclussion Cinnamomum burmanii extract at dose of 100 and 200mg/kgBW were effective in the protection against neuronal cell death in haloperidol induced male Wistar white rats.

Caspase-3 Activity and Carbonyl Cyanide m-Chlorophenylhydrazone-induced Apoptosis in HL-60 cells

2001 ◽  
Vol 29 (3) ◽  
pp. 243-249 ◽  
Author(s):  
Petr Mlejnek
Keyword(s):  
Cell Death ◽  
Dna Cleavage ◽  
Caspase 3 ◽  
Chromatin Condensation ◽  
Experimental System ◽  
Apoptotic Bodies ◽  
Nucleosomal Dna ◽  
Carbonyl Cyanide ◽  
Induced Apoptosis ◽  
Fluoromethyl Ketone

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being “truly” necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as “truly” necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.

135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C

2007 ◽  
Vol 19 (1) ◽  
pp. 185
Author(s):  
Y. Takagi ◽  
M. Shimizu ◽  
M. Morimura ◽  
S. Yokomizo ◽  
K. Hara ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Viability ◽  
Chi Square ◽  
Significant Difference ◽  
Morula Stage ◽  
Chi Square Test ◽  
Student’S T

Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (&lt;−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P &gt; 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P &lt; 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P &lt; 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days. Table 1. Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C

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