Transferase-mediated Deoxyuridine Triphosphate Nick-end Labeling Research Articles

Combined liver-kidney perfusion enhances protective effects of normothermic perfusion on liver grafts from donation after cardiac death.

It has been shown that combined liver-kidney normothermic machine perfusion (NMP) is able to better maintain the circuit's biochemical milieu. Nevertheless, whether the combined perfusion is superior to liver perfusion alone in protecting livers from donation after circulatory death (DCD) is unclear. We aimed to test the hypothesis and explored the mechanisms. Livers from 15 DCD pig donors were subjected to either static cold storage (group A), liver-alone NMP (group B), or combined liver-kidney NMP (group C). Livers were preserved for 6 hours and reperfused ex vivo for 2 hours to simulate transplantation or were transplanted in situ. During perfusion, group C showed an improved acid-base and biochemical environment in the circuit over group B. After reperfusion, the architecture of the liver grafts was best preserved in group C, followed by group B, then group A, as shown by the histology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining of both hepatocytes and biliary epithelium. Ki-67 staining showed substantial hepatocyte proliferation and biliary epithelial regeneration after perfusion in group B and group C. Group C produced more bile in the reperfusion phase than those in group A and group B, with more physiological bile composition and less severe biliary epithelium injury. Von Willebrand factor-positive endothelial cells and E-selectin expression decreased in both group B and group C. Combined liver-kidney NMP not only produced more adenosine triphosphate, protected the nitric oxide signaling pathway, but also diminished oxidative stress (high mobility group box-1 protein and 8-hydroxy-2-deoxy guanosine levels) and inflammatory cytokine (IL6 and IL8) release when compared with liver-alone NMP and CS. In addition, the 7-day survival rate of liver transplant recipients was higher in group C than that in groups A and B. In conclusion, combined liver-kidney NMP can better protect DCD livers from warm ischemia and reperfusion injury probably by maintaining the stability of the internal environment and by abolishing oxidative stress injury. Liver Transplantation 24 67-79 2018 AASLD.

Intravesical Immunomodulatory Imiquimod Enhances Bacillus Calmette-Guérin Downregulation of Nonmuscle-invasive Bladder Cancer.

The Toll-like receptor (TLR)2/4 agonist bacillus Calmette-Guérin (BCG), although not failure proof, has been the most efficient immunomodulatory treatment of immunogenic nonmuscle-invasive bladder cancer (NMIBC) for > 40 years. We investigated the role of the immunomodulatory molecule TLR7 agonist imiquimod through the BCG key receptors TLR2/4 and the main downstream molecules of the mammalian target of rapamycin pathway in NMIBC treatment. A total of 40 Fischer-344 rats, 7 weeks old, received 4 doses of 1.5 mg/kg N-methyl-N-nitrosourea intravesically on weeks 0, 2, 4, and 6 for cancer induction. At week 8, the rats were randomized into 4 groups (10 per group) and treated intravesically once a week for 6 weeks: control (0.2 mL of vehicle); BCG (2× 106 colony-forming units Connaught strain in 0.2 mL); imiquimod (20 mg/kg in 0.2 mL), and associated treatment BCG plus imiquimod in 0.2 mL. The bladders were extracted and analyzed for histopathology, immunohistochemistry, cell proliferation (Ki-67), apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and immunoblotting for TLR2, TLR4, p-P70S6K, and p-4E-BP1 proteins. The histopathology results showed that BCG and imiquimod decreased bladder tumorigenesis compared with the control group, with a proliferation decrease (Ki-67) and an apoptosis increase (TUNEL). BCG upregulated TLR2/4, imiquimod upregulated TLR4, and both downregulated P70S6K1. Imiquimod is able to efficiently decrease bladder carcinogenesis through upregulation of TLR7/4 and downregulation of P70S6K1 protein, generating new perspectives to boost BCG effects in the future.

Alpha1-adrenergic receptor antagonist tamsulosin ameliorates aging-induced memory impairment by enhancing neurogenesis and suppressing apoptosis in the hippocampus of old-aged rats

ABSTRACTAge-related memory decline is closely associated with decreased neurogenesis and increased apoptosis in the hippocampus. Noradrenaline exerts its effect by selectively binding to and activating adrenergic receptors (ARs). Tamsulosin, α1-AR antagonist, is reported to have access to the brain and interact with α1-AR. In this study, the effects of tamsulosin on short-term and spatial learning memory in terms of neurogenesis and apoptosis were investigated using rats. Step-down avoidance test for short-term memory and radial 8-arm maze test for spatial learning memory were conducted. Neurogenesis was detected by 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry and apoptosis was evaluated by caspase-3 immunohistochemisty and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNE) staining. Western blot for protein kinase C (PKC), cAMP-responsive element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, Bcl-2, and Bax was conducted. In the aged rats, short-term and spatial learning memory was declined. Hippocampal nerogenesis was suppressed and hippocampal apoptosis was enhanced in the aged rats. In addition, phosphorylation of PKCα, CREB, PI-3 kinase, and Akt was decreased in the hippocampus of old-aged rats. Tamsulosin activated PKC/CREB and PI-3 kinase/Akt pathways. With these pathways, BDNF-TrkB signaling enhanced hippocampal neurogenesis and suppressed apoptosis in the old-aged rats. As the results, tamsulosin improved performance of short-term and spatial learning memory in the aged rats.

Effects of Rosiglitazone on the Outcome of Experimental Periapical Lesions in Mice

IntroductionThe purpose of this study was to evaluate a protocol for systemic administration of rosiglitazone in mice in order to stimulate apoptosis of osteocytes in the jaws and to evaluate the effect of osteocyte apoptosis induced by rosiglitazone in the progression of periapical lesions in mice at 7, 21, and 42 days. MethodsC57BL/6 mice at 4–5 weeks of age were used. In phase 1, mice (n = 24) were treated with rosiglitazone (gavage, 10 mg/kg dose) or without (phosphate-buffered saline + 10% dimethyl sulfoxide) for 1, 2, or 3 weeks. We used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and 4′-6-diamidino-2-phenylindole methods for quantification of apoptotic cells. In phase 2, mice (n = 30) received rosiglitazone for 2 weeks or just vehicle for 1 week (n = 30), and periapical lesions were induced for 7, 21, or 42 days. We performed the measurement of periapical lesions, tartrate-resistant acid phosphatase staining, dual-energy x-ray absorptiometry for the evaluation of bone mineral density (BMD) in long bone, and gene evaluation using real time quantitative polymerase chain reaction of osteocyte markers (Sost, Hyou1, and Dmp1) and receptor activator of nuclear factor kappa-B ligand (RANKL) (Tnfsf11). ResultsIt was observed that systemic administration of rosiglitazone for 2 weeks showed apoptosis of osteocytes in a more expressive manner. In phase 2, in the groups that received rosiglitazone, a trend toward larger periapical lesions was observed (P > .05). Rosiglitazone also induced a greater number of osteoclasts and a greater expression of Sost and Hyou1 at 21 days of lesions. Moreover, there were no statistically significant differences in RANKL and Dmp1 expression or in the BMD of femurs. ConclusionsRosiglitazone stimulated apoptosis of osteocytes, interfering in the progression of periapical lesions in mice.

Ginkgetin inhibits proliferation of human leukemia cells via the TNF-α signaling pathway.

Ginkgetin is known to be an anticancer agent in many studies. However, its effectiveness in treating chronic myeloid leukemia [corrected] remains unknown. The present study aimed to evaluate the effects of ginkgetin on the growth of the K562 cell line. The MTT assay was employed to examine the proliferation of K562, and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining was conducted to detect the apoptotic rates. Furthermore, changes of tumor necrosis factor-α (TNF-α) were detected by Western blot analysis. Ginkgetin inhibited the proliferation of K562 cells in a dose- and time-dependent manner. Concentrations of ginkgetin required to induce 50% death of K562 at 24, 48 and 72 h were 38.9, 31.3 and 19.2 μM, respectively. Moreover, treatment of ginkgetin increased K562 apoptosis in vitro along with increased levels of TNF-α. Interestingly, anti-TNF-α antibody prevented ginkgetin-induced K562 cell apoptosis and growth inhibition via deactivation of caspase-8, caspase-9 and caspase-3. Concomitantly, downregulation of TNF-α by etanercept in vivo attenuated ginkgetin-induced inhibitory effects on the tumor growth in an xenograft mouse model. Our results indicate that ginkgetin effectively inhibits K562 cell proliferation, and TNF-α plays a key role in ginkgetin-induced cell apoptosis.

The Differentiation-Inducing Effect of a Retinoic Acid Derivative on a Pulmonary Carcinoma Cell Line

Pulmonary carcinoma is a main cause of cancer death internationally. Chemotherapeutic regimens in most solid tumors have limited effect. And in the targeted inhibition of carcinogenic driver mutations with molecular therapies, in a large number of cases, patients acquire resistance to drugs. Therefore, the development of new therapeutic approaches is highly desirable. In the present study, we evaluated the applicability of 4[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl- 2-naphthalenyl)carbamoyl]benzoic acid (Am80), a synthetic retinoid, as a differentiation inducer in pulmonary carcinoma and examined its effect on the viability and differentiation of Calu-6 cells (a human nonsmall cell lung cancer cell line), with the final goal of establishing a new approach to pulmonary carcinoma therapy. The cell viability of Calu-6 cells after Am80 treatment was assessed by the CellTiter-Glo Luminescent Cell Viability Assay. The early apoptosis of cells was detected by Terminal deoxynucleotidyl Transferase-mediated deoxyuridine triphosphate Nick End Labeling (TUNEL) staining. The differentiation-inducing effect of Am80 on Calu-6 cells was investigated by immunostaining. Am80 induced the differentiation of Calu-6 to alveolar type I and II cells. However, Am80 reduced the viability of Calu-6 cells without inducing apoptosis. In addition, in order to investigate whether Am80 inhibits tumor growth in vivo, we conducted an in vivo experiment to determine he effect of the intratumoral administration of Am80 in mice harboring sc tumors composed of Calu-6 cells. Am80 significantly influenced the suppression of tumor growth.

Mechanism of apoptosis involved in gastric mucosal lesions in Tibetans with high-altitude polycythemia.

The Tibetan high plateau is a low-oxygen environment, which may cause the pathogenesis of high-altitude polycythemia (HAPC). Gastric mucosal lesions (GML) are a common complication of HAPC. The molecular mechanisms involved in HAPC-induced GML have remained to be fully elucidated and were therefore investigated in the present study. Gastric tissues of patients with heavy, HAPC-induced GML and healthy controls were assessed by ultrastructural and histopathological analysis. In addition, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis were used to detect cell apoptosis in the gastric mucosa tissues. Moreover, the expression of genes associated with the phosphoinositide-3 kinase (PI3K) pathway was assessed by RT-qPCR to investigate the mechanism of cell apoptosis in HAPC-induced GML. The results revealed a significant increase in the number of red blood cells, gastric vessels and the diameter of gastric mucosal vessels in HAPC-induced GML patients compared with those in healthy controls. In addition, more red blood cells were distributed in gastric tissue not only at the vascular level but also in the tissue space. The number of vacuoles was increased in the gastric mucosal cells. Furthermore, a significant increase in apoptosis of the gastric mucosal cells was identified. The expression of phosphatase and tensin homolog was significantly higher in gastric mucosa from patients with HAPC-induced GML compared with that in the healthy controls. All of the pathologic changes suggested that significant cell apoptosis occurred in the HAPC-induced GML tissues, which may be associated with the PI3K pathway. These findings may provide novel insight for the treatment of gastric lesions caused by HAPC in the future.

Apoptosis, Expression of PAX3 and P53, and Caspase Signal in Fetuses with Neural Tube Defects.

Neural tube defects (NTDs) are among the most common and severe congenital malformations of the central nervous system. Animal studies have shown that apoptosis is involved in the development of NTDs. However, little evidence is available from human studies. We aim to examine the level of apoptosis and expression of apoptosis-regulating proteins of human terminated fetuses. A total of 37 NTD cases and 21 controls from pregnancy terminations were recruited. Tissues of the central nervous system were obtained through autopsy. Apoptosis of neuroepithelial cells was examined by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay. Expression of PAX3, p53, and caspase 3/8/9 in central nervous tissue was measured using Western blotting. More TUNEL-positive apoptosis cells were observed in the central nervous tissue of NTD cases than those of controls (p < 0.05). In spinal cord tissue, lower PAX3 expression, higher p53 expression, and increased levels of cleaved caspase 3(17kD) and cleaved caspase 8 (18kD) were found in anencephaly cases but not in spina bifida cases when compared with controls. In brain tissue, levels of PAX3 were significantly reduced in both encephalocele and spina bifida subtypes; the expression levels of cleaved caspase 3(17 kD) of encephalocele cases and cleaved caspase 8(47/45 kD) in spina bifida cases were higher than in controls; no difference was found in the expression of p53 or caspase 9 between NTDs and controls. These findings provide some evidence that excessive apoptosis in fetal central nervous tissues may be associated with the development of human NTDs. Birth Defects Research 109:1596-1604, 2017. © 2017 Wiley Periodicals, Inc.

Pentamidine blocks hepatotoxic injury in mice.

VLX103 effectively decreases toxin-induced liver injury in mice and may be an effective therapy for this and other forms of human liver disease. (Hepatology 2017;66:922-935).

Anti-tumor effect of cisplatin in human oral squamous cell carcinoma was enhanced by andrographolide via upregulation of phospho-p53 in vitro and in vivo.

Oral squamous cell carcinoma is one of the most common neoplasm in the world. Despite the improvements in diagnosis and treatment, the outcome is still poor now. Thus, the development of novel therapeuticapproaches is needed. The aim of this study is to assess the synergistic anti-tumor effect of andrographolide with cisplatin (DDP) in oral squamous cell carcinoma CAL-27 cells in vitro and in vivo. We performed Cell Counting Kit-8 proliferation assay, apoptosis assay, and western blotting on CAL-27 cells treated with andrographolide, DDP or the combination in vitro. In vivo, we also treated CAL-27 xenografts with andrographolide or the combination, and performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis of Ki-67. The results showed the combination of andrographolide and DDP synergistically inhibited CAL-27 cell proliferation in vitro and caused tumor regression in vivo in the CAL-27 xenografts. In addition, the synergistic anti-tumor effect of andrographolide with synergistic was due to an enhanced apoptosis. Moreover, the combination therapy upregulated the expression level of p-p53 in vitro and decreased Ki-67 expression in vivo. Our data indicate that the combination treatment of andrographolide and DDP results in synergistic anti-tumor growth activity against oral squamous cell carcinoma CAL-27 in vitro and in vivo. These results demonstrated that combination of andrographolide with DDP was likely to represent a potential therapeutic strategy for oral squamous cell carcinoma.

Melatonin Inhibits Neural Cell Apoptosis and Promotes Locomotor Recovery via Activation of the Wnt/β-Catenin Signaling Pathway After Spinal Cord Injury.

The spinal cord is highly sensitive to spinal cord injury (SCI) by external mechanical damage, resulting in irreversible neurological damage. Activation of the Wnt/β-catenin signaling pathway can effectively reduce apoptosis and protect against SCI. Melatonin, an indoleamine originally isolated from bovine pineal tissue, exerts neuroprotective effects after SCI through activation of the Wnt/β-catenin signaling pathway. In this study, we demonstrated that melatonin exhibited neuroprotective effects on neuronal apoptosis and supported functional recovery in a rat SCI model by activating the Wnt/β-catenin signaling pathway. We found that melatonin administration after SCI significantly upregulated the expression of low-density lipoprotein receptor related protein 6 phosphorylation (p-LRP-6), lymphoid enhancer factor-1 (LEF-1) and β-catenin protein in the spinal cord. Melatonin enhanced motor neuronal survival in the spinal cord ventral horn and improved the locomotor functions of rats after SCI. Melatonin administration after SCI also reduced the expression levels of Bax and cleaved caspase-3 in the spinal cord and the proportion of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cells, but increased the expression level of Bcl-2. These results suggest that melatonin attenuated SCI by activating the Wnt/β-catenin signaling pathway.

Artesunate Suppresses the Growth of Prostatic Cancer Cells through Inhibiting Androgen Receptor.

Prostatic cancer (PCa) is a leading cause of cancer related death in males and is often regarded as a kind of androgen-sensitive cancer. Artesunate (ART), a semi-synthetic derivative of the Chinese herb Artemisia annua, is such an anti-cancer agent. However, the effects and mechanism of ART on PCa cells remains unclear. The study aims to elaborate the mechanism of the involvement of androgen receptor (AR) in anti-prostatic cancer (PCa) of artesunate (ART). PCa cells 22rvl were used in vivo and in vitro, and the viability and apoptosis were conducted using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, respectively. Ectopic expressions of AR and DNA methyltransferase (DNMT) were detected in cells in overexpression or interference of AR or DNMT3b. ART dose-dependently suppressed tumor growth, inhibited cell viability, enhanced apoptosis, decreased AR expression, and increased the expression and the catalytic activity of DNMT3b in 22rv1 cells either in transplanted mice or in vitro. Furthermore, AR downregulated DNMT3b expression, and overexpression of AR or interference of DNMT3b could reverse ART-induced cytotoxicity and apoptosis in 22rvl cells, whereas overexpression of DNMT3b could not change the effect profiles of ART on the cells. The results indicated that ART suppressed tumor growth of prostatic cancer cells through AR-DNMT3b pathway, underlying ART will allow for the utilization of this Chinese therapeutic agent for the potential treatment of prostate cancer.

Protection of Cultured Cortical Neurons by Luteolin against Oxidative Damage through Inhibition of Apoptosis and Induction of Heme Oxygenase-1.

Luteolin, one of the most common flavonoids present in many types of natural products, possesses diverse biological properties including anti-oxidant activity. In this study, we investigated neuroprotective effect of luteolin and its underlying signaling pathways using primary cultured rat cortical cells. Luteolin was demonstrated to attenuate H2O2- or xanthine/xanthine oxidase-induced oxidative damage and generation of intracellular reactive oxygen species (ROS). It enhanced the phosphorylation of Bad at Ser112 and attenuated H2O2-induced activation of caspase 3, indicating anti-apoptotic action. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay confirmed this finding, showing inhibition of H2O2-induced DNA fragmentation. We also found that luteolin significantly up-regulated the expression of anti-oxidant enzyme heme oxygenase (HO)-1. Treatment with tin protoporphyrin IX, a selective HO-1 inhibitor, abolished neuroprotective and anti-apoptotic effects of luteolin, suggesting a critical role of HO-1 up-regulation. It was also shown to increase the phosphorylation of mitogen-activated protein kinase (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinases (JNK) and Akt. Treatment of the cells with specific inhibitors including SB203580, SP600125, and LY294002 suppressed the luteolin-induced HO-1 expression, suggesting the involvement of p38 MAPK, JNK, and Akt in HO-1 induction. In contrast, HO-1 expression was not reduced by U0126, implying that ERK may not be directly involved in HO-1 induction. These results indicate that luteolin exhibits neuroprotective effect through the inhibition of ROS and apoptotic cell death. Furthermore, up-regulation of HO-1 expression via p38 MAPK, JNK and Akt may contribute, at least in part, to luteolin-mediated neuroprotection. Based on these findings, luteolin may serve as a potential intervention for neurodegenerative diseases associated with oxidative stress.

Is levetiracetam neuroprotective in neonatal rats with hypoxic ischemic brain injury?

The aim of this study was to determine if levetiracetam (LEV) is neuroprotective in neonatal rats with hypoxic-ischemic brain injury (HIBI). The study included 7-d-old male Wistar rats that were randomly divided into the LEV400, LEV800, control, and sham groups. All the rats, except those in the sham group, underwent ligation of the carotid artery and were then kept in a hypoxic chamber containing 8% oxygen for 2 h. At the end of the hypoxic period the rats in the control group were administered saline solution 0.5 mL, the rats in the LEV400 group were administered LEV 400 mg.kg-1, and rats in the LEV800 group were administered LEV 800 mg.kg-1 via the intraperitoneal route. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method was used to evaluate neuronal apoptosis in the rats. The Morris Water Maze (MWM) test was performed at age 14 weeks in order to evaluate cognitive function. The number of apoptotic neurons in the right hemispheres was significantly lower in the sham, LEV400, and LEV800 groups than in the control group (p < 0.001, p < 0.001, and p < 0.001, respectively). In addition, the number of apoptotic neurons in the right hemispheres was significantly lower in the LEV800 group than in the LEV400 group (p = 0.001). Platform finding time (PFT) during MWM testing was significantly shorter in the sham and LEV800 groups on d 4 than on d 1 (p = 0.001 and p = 0.006, respectively); however, PFT did not significantly change between d 1 and d 4 in the control or LEV400 groups (p = 0.91 and p = 0.096, respectively). Based on the present findings, LEV exhibited a dose-dependent neuroprotective effect in neonatal rats with HIBI (Ref. 27).

Impact of Pioglitazone on Macrophage Dynamics in Adipose Tissues of Cecal Ligation and Puncture-Treated Mice.

Pioglitazone improves sepsis-induced organ injury accompanied with anti-inflammatory effects on visceral adipose tissue. However, its action in adipose immune cells remains to be ascertained. We investigated the effects of pioglitazone on visceral adipose macrophage population and polarisation in cecal ligation and puncture (CLP)-induced sepsis mice. Eight-week-old male mice were assigned to 3 groups: 1) sham-operated group, 2) CLP group, or 3) pioglitazone-treated CLP group. Pioglitazone (10 mg/kg) was injected intraperitonally for 7 d and CLP surgery was performed. Visceral adipose tissues were collected 24 h after the surgery. mRNA expression of several macrophage markers (inducible nitric oxide synthase (iNOS) for M1, arginase1 (Arg1) and interleukin (IL)-10 for M2, CD163 and F4/80 for mature macrophages) and inflammatory adipokines (IL-6, monocyte chemoattractant protein-1: MCP-1) was quantified by real-time RT-PCR. Tissue sections were subjected to the immunohistochemical analysis and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. CLP significantly enhanced Arg1, IL-10 and iNOS mRNA expressions as compared with the sham group, and pioglitazone significantly increased the mRNA level of CD163 and F4/80 in CLP mice. Expression of IL-6 and MCP-1 stimulated by CLP was reduced by pioglitazone treatment. Increased CD11b/c- and CD163-positive cells as well as apoptotic cells were observed in the CLP group and the pioglitazone-treated group. The data indicate that M1/M2 macrophage activation of visceral adipose tissues is induced in CLP-induced mice, and the function of macrophages recruited from surrounding organs may be modulated by pioglitazone treatment.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

The role of inversely operating glutamate transporter in the paradoxical analgesia produced by glutamate transporter inhibitors

Controlling extracellular glutamate level in a physiological range is important to maintain normal sensory transmission. Here, we investigated the paradoxical action of glutamate transporters in the rat formalin test to elucidate a possible role of inversely operating transporters in its analgesic mechanism. The effects of glutamate transporter inhibitor on formalin-induced pain behavior were examined. Then we performed a microdialysis study to clarify the differential change in extracellular glutamate concentration by intrathecal administration of transportable and non-transportable blockers. And we further investigated the mechanism pharmacologically via pretreatment with antagonists of various receptors and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Intrathecally-injected glutamate transporter inhibitors, non-transportable DL-threo-β-benzyloxyaspartat (TBOA) and transportable trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC), produced paradoxical antinociception in the formalin test. In normal rats, inhibition of the glutamate transporter increased extracellular glutamate. In the formalin model rats, TBOA suppressed while t-PDC enhanced glutamate release. When tPDC was pretreated 30min prior to formalin injection, glutamate release was blocked. Blocking α-2 adrenergic receptors reversed the tPDC analgesia. Increased apoptosis was not apparent in the spinal dorsal horn of tPDC-treated rats compared to the control group. These data suggest that glutamate transporters in a formalin-induced pain state work in a reverse mode and can be blocked from releasing glutamate by TBOA and preloaded tPDC. The analgesic mechanism of TBOA may be related to the blockade of inversely operating transporter, and that of tPDC may be associated with the activation of noradrenergic neurotransmission but not with dorsal horn neurotoxicity.

Neuroprotective effect of Korea Red Ginseng extract on 1-methyl-4-phenylpyridinium-induced apoptosis in PC12 Cells

ABSTRACTBackground: Korean Red Ginseng (KRG) is a valuable herb in Asian countries that is used as a crude substance to inhibit inflammation and to enhance vitality, longevity and immunity. The protective effects of KRG against the toxicity of 1-methyl-4-phenylpyridinium (MPP+) were investigated in vitro in the present study.Methods: PC12 cells were pretreated with the water extract of KRG for 24 h, then incubated with MPP+ for 24 h. The growth of the cells was assessed using a live cell viability assay, the ratio of apoptotic cells was measured using flow cytometry and morphology of the apoptotic cells was detected using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The expressions of caspase-3 and -9 were measured by quantitative real-time PCR.Results: Pretreatment of the KRG extract increased cell viability significantly when compared with that of only MPP+-treated cells (p < .01). KRG-pretreated cells revealed reductions of Annexin V-FITC and propidium iodide double staining cells (p < .01), TUNEL-positive cells (p < .01), and caspase-3 and -9 activities (p < .01) when compared with only MPP+-treated cells.Conclusion: These results indicate that KRG treatment suppresses MPP+-induced apoptosis in PC12 cells by regulating caspase cascades, suggesting a possible role for KRG in the prevention and treatment of Parkinson’s disease.

Lithium posttreatment confers neuroprotection through glycogen synthase kinase-3β inhibition in intracerebral hemorrhage rats

OBJECTIVE Inflammation and apoptosis are two key factors contributing to secondary brain injury after intracerebral hemorrhage (ICH). The objective of this study was to evaluate the effects of lithium posttreatment on behavior, brain atrophy, inflammation, and perihematomal cell death. Furthermore, the authors aimed to determine the role of the pro-apoptotic glycogen synthase kinase-3β (GSK-3β) after experimental ICH. METHODS Male Sprague-Dawley rats (n = 108) were subjected to intracerebral infusion of semicoagulated autologous blood. Window of opportunity and dose optimization studies of lithium on ICH-induced injury were performed by measuring neurological deficits. Animals with ICH received vehicle administration or lithium posttreatment (60 mg/kg) for up to 21 days. Hemispheric atrophy was evaluated. Perihematomal cell death was quantified through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). The number of myeloperoxidase (MPO)-positive neutrophils and OX42-positive microglia in the perihematomal areas were calculated. Western blotting was used for the quantification of GSK-3β, heat shock protein 70 (HSP70), nuclear factor-κB p65 (NF-κB p65), and cy-clooxygenase-2 (COX-2). RESULTS Lithium, at a dose of 60 mg/kg initiated from 2 hours after injury, exhibited the best effects of improving neurological outcomes 3, 5, 7, 14, 21, and 28 days after ICH, reduced the hemispheric atrophy at 42 days after surgery, and reduced the number of TUNEL-positive cells, MPO-positive neutrophils, and OX42-positive microglia in the perihematomal areas. Furthermore, lithium posttreatment modulated GSK-3β, increased HSP70, and decreased NF-κB p65 and COX-2 expression in the ipsilateral hemisphere. CONCLUSIONS Lithium posttreatment at a dose of 60 mg/kg, initiated beginning 2 hours after injury, improves functional and morphological outcomes, and inhibits inflammation and perihematomal cell death in a rat model of semicoagulated autologous blood ICH through inactivation of GSK-3β.

Invited commentary

Investigations into ischemia and reperfusion injury in association with repair of thoracoabdominal aneurysms have commonly focused on the spinal cord and neurologic sequelae. However, visceral ischemia can also lead to significant morbidity. Clinicians have well appreciated this complication when faced with intestinal gangrene or coagulopathy from hepatic ischemia. Less advanced degrees of visceral ischemia or less specific manifestations, such as systemic inflammatory response syndrome or multiorgan failure, may not be recognized for their underlying etiology. Furthermore, we have been limited in both our preventative intraoperative options and in our understanding of the underlying pathophysiology. Keschenau et al have used a pig model of thoracic aortic clamping to investigate small-bowel mucosal and muscularis injury after 1 hour of ischemia and reperfusion. Terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) is a well-established method for detecting DNA fragmentation, which is characteristic of apoptosis, caspase enzymes initiate protein substrate cleavage during cell breakdown, the annexin V affinity assay quantifies the number of apoptotic cells, and proliferating cell nuclear antigen is a nuclear factor involved in DNA replication of proliferating cells. The results indicate that the most extensive histochemical damage occurred in the clamp-only group, which had no distal perfusion, supporting the common clinical practice of initiating some type of mesenteric perfusion when ischemia time is >1 hour. The results, however, are not as clear as one would have hoped. There were effectively no major differences between the groups in the amount of cellular apoptosis, including the sham group without any aortic clamping. It is further unclear why distal perfusion should increase mucosal proliferation but selective mesenteric perfusion does not—the hypothesis that the latter provides insufficient energy to initiate apoptosis is unconvincing. Finally, the number of animals in each group was small and potentially insufficient to demonstrate significant differences between the groups. This research has some inherent limitations. Reperfusion for 2 hours may not allow sufficient time for small-bowel injury to fully manifest or repair itself. How much of these effects are due to extracorporeal circulation compared with shunt-based distal perfusion is unclear. These findings in pigs may not translate directly to humans. Clinically, this study reinforces the usefulness of mesenteric perfusion with thoracic aortic clamping of >1 hour but cannot provide us with any specific recommendations on the type of distal perfusion or whether simultaneous visceral perfusion provides additional protection. Research in this area, however, should be encouraged and the authors commended on their efforts. Extracorporeal circulation increases proliferation in the intestinal mucosa in a large animal modelJournal of Vascular SurgeryVol. 64Issue 4PreviewExtracorporeal circulation induces ischemia/reperfusion injury in the small intestinal wall. One reason for this damage is a perfusion shift from the muscular toward the mucosal layer. This study investigated the effect of this perfusion shift on the small-intestinal apoptosis and proliferation. Full-Text PDF Open Archive