Transferase deoxy-UTP-nick End Labeling Research Articles

The effects of overwintering temperature on the survival of female adult mud crab, Scylla paramamosain, under recirculating aquaculture systems as examined by histological analysis of the hepatopancreas and expression of apoptosis-related genes

The effects of the overwintering temperature (8, 12, 16, and 20 °C) on survival, antioxidant capacity, and heat shock protein (HSP) gene expression in Scylla paramamosain were examined during an experimental period of eight weeks. The effects of the temperature on survival were parabolic, with the highest and lowest survival rates occurring at 16 °C and 8 °C, respectively. Histological examination of the hepatopancreas revealed that the hepatopancreas tissue structure remained intact and that cell boundaries were clearly observed at 16 °C and 20 °C. However, hepatopancreas cells in the 12 °C and 8 °C groups appeared to be vacuolated and indistinct, respectively. Furthermore, high levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and low levels of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in the hepatopancreas were observed in the 8 °C group. Hsp60 and hsp70 gene expression were dramatically upregulated, but hsp10, hsp40, hsp90, and glucose regulatory protein 78 (grp78) gene expression were downregulated at 8 °C. The terminal deoxynucleotidyl transferase deoxy-UTP nick end labeling (TUNEL) method showed that there was more DNA damage at 8 °C and 12 °C than at 16 °C. Moreover, apoptosis was significantly induced with decreasing temperature, and the 8 °C group had the highest apoptosis index. Overall, these results suggest that the overwintering temperature greatly impacts the survival of mud crabs and that these effects may be mediated through antioxidant, HSP response, and apoptosis pathways. Significantly, the optimal overwintering temperature for S. paramamosain was extrapolated to be 14.2–16.1 °C. This suggests that temperature control should be considered in the process of mud crab overwintering.

Neuroprotective effect of breviscapine on traumatic brain injury in rats associated with the inhibition of GSK3β signaling pathway

Breviscapine, a standardized Chinese herbal medicine extracted from Erigeron breviscapine, has been widely used to treat cerebrovascular diseases. However, there are no reports about the neuroprotective effects and underlying molecular mechanisms of breviscapine on traumatic brain injury (TBI). Therefore, this study was aimed to investigate the effects of breviscapine on rats with TBI insult and illuminate the underlying mechanism. We created a traumatic brain-injured model with breviscapine lateral ventricle injection and evaluated the expressional changes of glycogen synthase kinase 3 beta (GSK3β) as well as the GSK3β-involved signaling pathways including apoptosis and axonal growth. At 7, 14, 21days after injection, we found a great reduction of motor disability in TBI rats following breviscapine treatment, which was accompanied with a notably increased expression of phospho-Ser9-GSK3β (p-Ser9-GSK3β) and decreased expression of phosphor-Try216-GSK3β (p-Try216-GSK3β) at 7days after injection. Concomitantly, an enhanced expression of synaptic marker synaptophysin (SYP) together with a weakened expression of pro-apoptotic caspase3 was observed after TBI rats were treated with breviscapine. Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) immunohistochemical assay and SYP immunofluorescence staining also confirmed the result. This study suggests that breviscapine inhibits the GSK3β signaling pathway to promote neurobehavioral function following neurotrauma. These events may provide a new insight into the mechanism of breviscapine treating brain injury.

HIF-1α Involves in Neuronal Apoptosis after Traumatic Brain Injury in Adult Rats

Hypoxia-inducible factor-1α (HIF-1α), a well-identified hypoxia-related protein, is involved in regulating the biological functions of various cell types including neurons. The traditional biological function of HIF-1α is promoting the transcription of some pro-survival genes when exposing to low oxygen conditions. Meanwhile, some studies also point out that HIF-1α shows the detrimental role in several central nervous system (CNS) disorders. Up to now, the knowledge of HIF-1α function in CNS is still limited. To investigate whether HIF-1α is involved in CNS impairment and repair, we employed a traumatic brain injury model in adult rats. Upregulation of HIF-1α was observed in the injured brain cortex by western blot analysis and immunohistochemistry staining. Terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining suggested that HIF-1α was relevant to neuronal apoptosis after brain injury. In addition, glutamate excitotoxic model of primary cortex neurons was introduced to further investigate the role of HIF-1α in neuronal apoptosis; the result implied HIF-1α was associated with the regulation of p53 and BNIP3 in the apoptotic neurons. Based on our data, we suggested that HIF-1α might play an important role in neuronal apoptosis after traumatic brain injury in rat, which might also provide a basis for the further study on its role in regulating the transcription of target genes in apoptotic neurons, and might gain a novel strategy for the clinical therapy for traumatic brain injury.

Upregulation of CRM1 Relates to Neuronal Apoptosis after Traumatic Brain Injury in Adult Rats

Traumatic brain injury (TBI) initiates a complex series of neurochemical and signaling changes that leads to neuronal dysfunction and over-reactive astrocytes. There is increasing evidence that CRM1 mediated P27(Kip1), which is a potent inhibitor of G1 cyclin-dependent kinases complexes, nuclear export-dependent or -independent Jab1/CSN5, and cytoplasmic degradation in cells. Up to now, the function of CRM1 in central nervous system (CNS) is still with limited acquaintance. In our study, to investigate whether CRM1 is involved in CNS lesion, we performed a TBI model in adult rats. Western blot and RT-PCR analysis revealed that the level of protein and mRNA of CRM1 increased in ipsilateral brain cortex in comparison to the contralateral. Immunohistochemistry and immunofluorescence double labeling indicated that CRM1 was shutting into nucleus around the wound, and increased CRM1 co-localized with P27(Kip1). Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) staining suggested that CRM1 was involved in neuronal apoptosis after brain injury. We also investigated co-localization of CRM1 and active-caspase-3 in the ipsilateral brain cortex. In addition, the expression patterns of Bax and active-caspase-3 were parallel with that of CRM1. Based on our data, we suggested that CRM1 might play an important role in neuronal apoptosis following TBI, and might provide a basis for the further study on its role in regulating the expression of P27(Kip1) and cell cycle re-entry in TBI.

Phosphorylated retinoblastoma protein (p-Rb) is involved in neuronal apoptosis after traumatic brain injury in adult rats

Phosphorylated retinoblastoma protein (p-Rb), a well identified cell cycle related protein, is involved in regulating the biological functions of various cell types including neurons. One attractive biological function of p-Rb is releasing E2F transcription factor to induce S-phase entry and cellular proliferation of mitotic cells. However, some studies point out that the role of p-Rb in post-mitotic cells such as mature neurons is unique; it may induce cellular apoptosis rather than proliferation via regulating cell cycle reactivation. Up to now, the knowledge of p-Rb function in CNS is still limited. To investigate whether p-Rb is involved in CNS injury and repair, we performed a traumatic brain injury model in adult rats. Up-regulation of p-Rb was observed in the injured brain cortex by western blot analysis and immunohistochemistry staining. Terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining suggested that p-Rb was relevant to neuronal apoptosis after brain injury. In addition, glutamate excitotoxic model of primary cortex neurons was introduced to further investigate the role of p-Rb in neuronal apoptosis; the result implied p-Rb was associated with cell cycle activation in the apoptotic neurons. Based on our data, we suggested that p-Rb might play an important role in neuronal apoptosis after traumatic brain injury in rat; which might also provide a basis for the further study on its role in regulating cell cycle re-entry in apoptotic neurons, and might gain a novel strategy for the clinical therapy for traumatic brain injury.

Up-regulation of Che-1 Relates to Neuronal Apoptosis After Traumatic Brain Injury in Adult Rats

Che-1, a recently identified apoptosis related protein, affects the fate of various cell types when under stress. One attractive biological function of Che-1 is promoting the transcription of p53 after DNA damage; besides, it can also regulate cell cycle via interacting with retinoblastoma protein. Although previous evidence has showed its anti-apoptotic role in cancer cells, some studies point out that Che-1 might play an opposite role in central nervous system (CNS). However, the function of Che-1 in CNS is still with limited acquaintance. To investigate whether Che-1 is involved in CNS lesion, we performed a traumatic brain injury model in adult rats. Up-regulation of Che-1 was observed in the peritrauma brain cortex by performing western blotting and immunohistochemistry. Terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling and 4',6-diamidino-2-phenylindole staining suggested that Che-1 was involved in neuronal apoptosis after brain injury. We also investigated co-localization of Che-1 and active-caspase-3 in the ipsilateral brain cortex. In addition, the expression patterns of p53, Bax and PCNA were parallel with that of Che-1. Besides this, neurotrophin receptor-interacting MAGE homolog was found to be associated with Che-1 after brain trauma. Based on our data, we suggested that Che-1 might play an important role in neuronal apoptosis following TBI; and might provide a basis for the further study on its role in regulating the expression of p53 and cell cycle re-entry in traumatic brain injury.

Corneal collagen crosslinking in vitro: Inhibited regeneration of human limbal epithelial cells after riboflavin–ultraviolet-A exposure

To assess the hypothesis that during corneal crosslinking (CXL) treatment, riboflavin and ultraviolet-A (UVA) may have a toxic effect on human limbal epithelial cells. Center for Eye Research, Department of Ophthalmology, Oslo University Hospital Ullevål, Oslo, Norway. Experimental study. In this vitro study, limbal biopsies from corneoscleral rims collected after corneal transplantation were treated with the following combinations: riboflavin-UVA, riboflavin only, or UVA only; a control group received no treatment. After 3 weeks of cell culture, outgrowth of epithelium from the biopsies was evaluated by measuring the area of cell expansion and the number of cell layers. The explanted biopsies were analyzed for proliferation using immunohistochemistry marker Ki-67 and for apoptosis using the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay. The mean outgrowth from the biopsies was 2.25 mm(2) ± 6.90 (SD) in the riboflavin-UVA group, 181.4 ± 94.8 mm(2) in the riboflavin-only group, 128.5 ± 129.5 mm(2) in the UVA-only group, and 176.2 ± 114.0 mm(2) in the control group. There were no statistically significant between-group differences in the number of cell layers except in the riboflavin-UVA group, in which no cells were found. Detection of apoptosis with the TUNEL-assay was found in the riboflavin-UVA group only (4/5 sections). The proliferation marker Ki-67 was positive in some sections in all groups. Cytotoxicity and reduced cell expansion of human limbal epithelial cells occurred after riboflavin-UVA treatment in vitro, emphasizing the importance of avoiding riboflavin-UVA on the limbus during CXL.

Increased Apoptosis in Chorionic Trophoblasts of Human Fetal Membranes with Labor at Term.

To determine the association of apoptosis in the layers of human fetal membranes with labor at term. Human fetal membranes were collected from elective cesarean sections (n = 8) and spontaneous vaginal deliveries (n = 8) at term. The extent of apoptosis within the layers of fetal membranes was determined using terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) immunohistochemical assay and western blots. For TUNEL assays, 5-μm sections of formalin fixed membranes were used and the apoptotic index (number of apoptotic nuclei per total nuclei ×100) was determined in 5 independent microscopic fields. For Western blotting, proteins isolated from the amnion and choriodecidua layers were blotted against pro-apoptotic active caspase-3 and anti-apoptotic and Bcl-2. Data were expressed as the means ± SD and Student's t-test was used for statistical analysis. There was no statistical difference in maternal age, gestational age, gravidity, parity, race, and smoking between patients who delivered at term via either elective cesarean or vaginally. Apoptotic index in chorionic trophoblasts of membranes obtained after vaginal delivery was higher than those obtained from elective cesarean (11.57 ± 4.98 % and 4.05 ± 2.4 % respectively, p = 0.012). The choriodecidua layers after vaginal deliveries had higher expression of the pro-apoptotic active caspase-3 and less expression of the anti-apoptotic BcL-2 than those obtained from elective cesarean sections. Labor at term is associated with increased apoptosis in chorionic trophoblast cells of human fetal membranes. The cause-effect relation between apoptosis in fetal membranes and labor warrants further investigations.

Hypoxia‐inducible factor‐2α is involved in enhanced apoptosis in the placenta from pregnancies with fetal growth restriction

The aim of the present study is to investigate whether hypoxia-inducible factors (HIF-1alpha, HIF-2alpha, HIF-1beta) are involved in enhanced apoptosis in the human placenta from pregnancies with fetal growth restriction (FGR). Placental samples were obtained from women with normal term pregnancy (n = 18) or from pregnancy with FGR (n = 12). Placenta apoptosis was assessed by the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) staining. The expressions of HIF-1alpha, HIF-2alpha, and HIF-1beta were examined by immunohistochemical analysis. Enhanced apoptosis was observed in the placenta from pregnancies with FGR compared with normal term placenta. The apoptosis index in FGR group (1.45 +/- 1.26%) was significantly higher than that in the normal control group (0.18 +/- 0.16%; P < 0.01). There were no significant differences in the intensity of the staining for HIF-1alpha and HIF-1beta expressions between two groups, while HIF-2alpha was overexpressed in the placenta from pregnancies with FGR group (P < 0.05). The upregulation of HIF-2alpha protein expression in the placenta from pregnancies with FGR may, at least in part, be involved in the increased placental apoptosis.

Endothelial cell damage after riboflavin–ultraviolet-A treatment in the rabbit

Purpose To evaluate the possible cytotoxic effect of combined riboflavin–ultraviolet-A (UVA) treatment on the corneal endothelium. Setting Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. Methods The right eyes of 34 New Zealand White rabbits were treated with riboflavin and various endothelial UVA doses ranging from 0.16 to 0.9 J/cm 2 (0.09 to 0.5 mW/cm 2, 370 nm) and postoperative enucleation times of 4 hours and 24 hours. The endothelial cells were evaluated in histological sections. The terminal deoxynulceotidyl transferase deoxy-UTP-nick-end labeling (TUNEL) technique and transmission electron microscopy were used to detect apoptosis. Results There was no endothelial damage in the 6 rabbit eyes enucleated at 4 hours. In those enucleated at 24 hours, there was significant necrosis and apoptosis of endothelial cells in the corneas treated with an endothelial dose of ≥0.65 J/cm 2 (0.36 mW/cm 2), which is about twice the endothelial UVA dose used in the treatment of keratoconus patients. Conclusions In rabbit corneas with a corneal thickness less than 400 μm, the endothelial UVA dose reached a cytotoxic level of ≥0.65 J/cm 2 (0.36 mW/cm 2) using the standard surface UVA dose of 5.4 J/cm 2 (3 mW/cm 2). Pachymetry should be routinely performed before riboflavin–UVA treatment; in thinner corneas, irradiation should not be done because of the cytotoxic risk to the endothelium.

Lens epithelial cell death after cataract surgery

Purpose To determine whether lens epithelial cells (LECs) undergo apoptosis during healing after cataract surgery to further characterize the healing process of the postoperative lens capsule. Setting Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. Methods Apoptotic cells were detected in human postoperative lens capsules using the terminal deoxynucleotidyl transferase deoxy-UTP nick-end labeling (TUNEL) method. The effect of exogenous transforming growth factor-β2 (TGF-β2) on mouse LEC apoptosis was also examined in an organ-culture system. Results Three of 17 postoperative specimens contained TUNEL-positive cells. In 2 lens capsules obtained earlier than 10 days, many TUNEL-positive cells, presumably apoptotic LECs, were observed beneath the residual anterior capsule. In cell multilayers in capsule opacification extracted later than 10 days, a few TUNEL-positive cells were seen in 1 specimen; most cells remained negative. In mouse lenses organ-cultured with 1.0 ng/mL TGF-β2 for 48 hours, TUNEL-positive cells were detected beneath the lens capsule. Conclusions Lens epithelial cells undergo apoptosis during healing after cataract surgery, especially in the early phase. Transforming growth factor-β2 may be a factor inducing apoptosis in in vivo LECs.

Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation

Objective: This study was undertaken to determine whether preeclampsia and intrauterine growth retardation are associated with an increase in placental apoptosis. Study Design: Tissue specimens from 7 normal term placentas and each of 7 term placentas complicated by severe preeclampsia or intrauterine growth retardation were analyzed. Fas antigen and Bcl-2 protein expression were examined by the avidin/biotin immunoperoxidase method, whereas apoptosis was assessed by the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) method and transmission electron microscopy. Results: Fas antigen was immunolocalized in syncytiotrophoblasts in all placentas examined. No changes in the intensity of Fas antigen immunostaining in syncytiotrophoblasts were apparent among those placentas. Bcl-2 protein was abundantly immunolocalized in syncytiotrophoblasts in normal term placentas, but least abundant in term placentas complicated by severe preeclampsia or intrauterine growth retardation. Apoptosis was apparent in the nuclei of both cytotrophoblasts and syncytiotrophoblasts. The apoptosis positive rate of syncytiotrophoblast nuclei in severe preeclamptic and intrauterine growth retardation term placentas was significantly higher than that in normal term placentas (severe preeclampsia, P <.001; intrauterine growth retardation, P <.01). Transmission electron microscopy revealed the appearance of apoptotic nuclei in trophoblasts in severe preeclamptic term placenta. Conclusion: Decreased expression of Bcl-2 protein in syncytiotrophoblasts in severe preeclamptic and intrauterine growth retardation placentas may result in the increase in apoptosis in syncytiotrophoblasts in those placentas.(Am J Obstet Gynecol 2002;186:158-66.)

Expression of Fas ligand on bile ductule epithelium in biliary atresia—A poor prognostic factor

Purpose: The aim of this study was to investigate the possible role of Fas and Fas ligand system in biliary atresia. Methods: Immunohistochemical stains of Fas and Fas ligand (FasL) and in situ hybridization of Fas ligand messenger RNA (mRNA) were performed on paraffin-embedded liver specimens of 36 biliary atresia, 6 choledochal cysts, and 14 nontumorous parts of pediatric liver tumors. Apoptosis was detected by terminal deoxynucleotidyl transferase deoxy-UTP nick end labeling (TUNEL). The grade of liver fibrosis and results of bile drainage on the patients with biliary atresia were compared with the results of FasL expression. Results: Fas protein was positive on the hepatocytes and bile ductule epithelia of all the livers examined and also positive on some monocytes around the portal area in all the biliary atresia patients. FasL protein was positive on bile ductule epithelia in 10 biliary atresia patients and also positive on some monocytes in most of the biliary atresia patients. Positive signals of FasL mRNA were noted on hepatocytes in 4 biliary atresia, bile ductule epithelia in 19 biliary atresia patients, and some monocytes in most of the biliary atresia patients. Apoptotic nuclei were present among monocytes in all the biliary atresia livers but present among bile ductule epithelia only on the BA with positive FasL mRNA signals on ductule epithelium. The fibrosis grade was similar between biliary atresia with positive FasL mRNA signals and negative signals. The bile drainage was better in the biliary atresia without positive FasL mRNA signals. Conclusions: Fas ligand expression on bile ductule epithelia in biliary atresia may be induced to counterattack the infiltrating lymphocytes. Although the factors for post-Kasai bile drainage are multiple, the authors suggest Fas ligand expression on bile ductule epithelia may be a poor prognostic factor by playing a role in the continuous damage and obliteration of intrahepatic bile ducts after Kasai operation. J Pediatr Surg 35:1591-1596. Copyright © 2000 by W.B. Saunders Company.