Transferase-mediated Biotinylated Deoxyuridine Triphosphate Nick End Labeling Research Articles

Neuroprotective Properties of Picroside II in a Rat Model of Focal Cerebral Ischemia

The aim of this study was to explore the effect of picroside II on neuronal apoptosis and the expression of caspase-3 and poly ADP-ribose polymerase (PARP) following middle cerebral artery occlusion/reperfusion in male Wistar rats. Picroside II (10 mg/kg) was administered intravenously into the tail vein of the animals. The neurological function deficits were evaluated with the Bederson’s test and the cerebral infarction volume was visualized with tetrazolium chloride (TTC) staining. The apoptotic cells were counted by in situ terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The immunohistochemistry stain and enzyme linked immunosorbent assay (ELISA) was used to determine the expressions of caspase-3 and PARP in brain tissue. The results indicated that rats in the control group showed neurological function deficit and cerebral infarction in ischemic hemisphere after two hours ischemia followed by 22 hours reperfusion. Caspase-3 and PARP expressions were also profound in the cortex, the striatum and the hippocampus, along with increased apoptotic cells in this group. Bederson’s score, infarction volume, and expressions of caspase-3 and PARP, as well as apoptosis in the treatment group were, however, significantly decreased compared to those in the control group indicating that intravenous treatment with picroside II might be beneficial to inhibit neuronal apoptosis and, thus, to improve the neurological function of rats upon cerebral ischemia reperfusion injury.

Anti-inflammation effects of picroside 2 in cerebral ischemic injury rats

BackgroundExcitatory amino acid toxicity, oxidative stress, intracellular calcium overload, as well as inflammation and apoptosis are involved in the pathological process after cerebral ischemic reperfusion injury. Picrodide 2 could inhibit neuronal apoptosis and play anti-oxidant and anti-inflammation role in cerebral ischemia/reperfusion injuries, but the exact mechanism is not very clear. This study aims to explore the anti-inflammation mechanism of picroside 2 in cerebral ischemic reperfusion injury in rats.MethodsThe middle cerebral artery occlusion reperfusion models were established with intraluminal thread methods in 90 adult healthy female Wistar rats. Picroside 2 and salvianic acid A sodium were respectively injected from tail vein at the dosage of 10 mg/kg for treatment. The neurobehavioral function was evaluated with Bederson's test and the cerebral infarction volume was observed with tetrazolium chloride (TTC) staining. The apoptotic cells were counted by in situ terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The immunohistochemistry stain was used to determine the expressions of toll-like receptor 4 (TLR4), nuclear transcription factor κB (NFκB) and tumor necrosis factor α (TNFα). The concentrations of TLR4, NFκB and TNFα in brain tissue were determined by enzyme linked immunosorbent assay (ELISA).ResultsAfter cerebral ischemic reperfusion, the rats showed neurobehavioral function deficit and cerebral infarction in the ischemic hemisphere. The number of apoptotic cells, the expressions and the concentrations in brain tissue of TLR4, NFκB and TNFα in ischemia control group increased significantly than those in the sham operative group (P < 0.01). Compared with the ischemia control group, the neurobehavioral scores, the infarction volumes, the apoptotic cells, the expressions and concentrations in brain tissue of TLR4, NFκB and TNFα were obviously decreased both in the picroside 2 and salvianic acid A sodium groups (P < 0.01). There was no statistical difference between the two treatment groups in above indexes (P > 0.05).ConclusionsPicroside 2 could down-regulate the expressions of TLR4, NFκB and TNFα to inhibit apoptosis and inflammation induced by cerebral ischemic reperfusion injury and improve the neurobehavioral function of rats.

Apoptosis in the Porcine Uterine Endometrium During the Estrous Cycle, Early Pregnancy and Post Partum

The mammalian uterus changes dramatically during the estrous cycle, pregnancy, and involution post partum. Dynamic changes in the uterine endometrium are a type of homeostasis and proceed with proliferation and exclusion of cells. Homeostasis of the uterus is closely related to apoptosis involving various hormones and cytokines. The objective of the present study was to determine the morphological features and occurrence of apoptosis in the porcine endometrium during the estrous cycle, early pregnancy, and post partum. Cyclic changes in the morphology of the surface epithelium were observed during the estrous cycle. The heights of surface epithelia were significantly high on day 4 of the estrous cycle and the early pregnancy. The heights of the surface epithelium remained low from days 1 to 31 post partum. We then used terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) of the 3'-terminal of fragmented DNA, which is effective for detection of apoptosis in various tissues. We found that apoptosis in the porcine endometrium contributed to homeostasis of the endometrium during the estrous cycle through control of cell proliferation and exclusion. Conversely, apoptosis on days 4 and 8 of gestation before the implantation window depended on the plasma estrogen and progesterone levels; however, suppressive homeostasis of apoptosis occurred at the time of implantation on days 15, 18 and 21 of gestation. Our study is the first to demonstrate apoptotic cell death in the porcine endometrium directly by TUNEL method. The results strongly suggest that uterine homeostasis is mainly controlled by apoptosis during the estrous cycle and early pregnancy.

Phosphatidylinositol 3-Kinase Activity Is Critical for Glucose Metabolism and Embryo Survival in Murine Blastocysts

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.

Changes in Expression and Localization of Connexin 43 mRNA and Protein in Porcine Ovary Granulosa Cells during Follicular Atresia

Gap junctions contain channels that connect neighboring cells by allowing the movement of molecules smaller than 1,200 Da. They are formed by connexins and may play a crucial role in the regulation of apoptotic cell death. To determine the role of connexin 43 (Cx43), which is dominantly expressed in granulosa cells, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression and localization of Cx43 mRNA and protein in granulosa cells during atresia using the quantitative real-time revese transcription-polymerase chain reaction, in situ hybridization, Western blot, and immunohistochemistry. Stages of follicular atresia were assessed based on histochemical terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) and/or the ratio of progesterone and 17beta-estradiol levels in follicular fluid measured by radioimmunoassay. Cx43 mRNA was detected in granulosa cells of secondary follicles and of healthy, early and progressed atretic tertiary follicles, but not in those of primordial or primary follicles. Both phosphorylated/activated and non-phosphorylated/native Cx43 proteins were detected in granulosa cells of secondary and tertiary follicles, but not in those of primordial or primary follicles. Moreover, in tertiary follicles, these Cx43 proteins were expressed most strongly in granulosa cells of healthy follicles, but only trace levels were noted in cells of early atretic and progressed atretic follicles, an indication that the expression levels of Cx43 protein decrease during follicular atresia. These findings indicate that Cx43 is involved in the apoptosis of granulosa cells during atresia in porcine ovaries.

"Anoikis" of oligodendrocytes induced by Wallerian degeneration: ultrastructural observations.

Oligodendrocytes undergo apoptosis in the white matter tracts remote from the experimental cord injury, although its significance is not understood. Our ultrastructural study, however, enabled us to speculate on its neurobiological implications. The spinal cords of male Wistar rats (4 week-old) were transected at Th11 level. At 4, 5, and 7 days after surgery the animals were transcardially perfusion-fixed. The removed cord was embedded in epoxy resin and examined by electron microscopy. Post-embedding terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) method was also performed. In the degenerative dorsal column above the transection, profiles of apoptotic oligodendrocytes were always found, embedded in a space formed by dilated degenerative myelin lamellae. Often, the dilated space in the myelin sheath lacked any apparent background proteinaceous matrix. In the electron microscopic TUNEL method, these apoptotic cells were electron dense in accordance with nuclear heterochromatinization. In conclusion, in the process of Wallerian degeneration, we observed the apoptosis of oligodendrocytes in a space formed by the split myelin sheath. These degenerative cells, which were enclosed in an ultrafiltrate-filled space formed by split myelin lamellae, were reminiscent of "anoikis."

Expression of matrix metalloproteinases and their tissue inhibitors in ovaries of senescence accelerated mice

We have shown that some corpora lutea (CLs) were abnormally accumulated in the ovaries of female SAMP1 mice. No significant differences in peripheral blood levels of progesterone or 17β-estradiol during estrus cycle were noted between SAMP1 and control-SAMR1 mice, and high activity of 20α-hydroxysteroid dehydrogenase was detected only in abnormally accumulated CLs (aaCLs) of SAMP1 mice. In the ovaries of SAMP1 mice, structural regression is inhibited in aaCLs, but functional regression does occur. In this study, we focused on the matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs), regulators of angiogenesis and angiodegeneration. In situ zymography (FIZ), terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) staining and histochemistry for hydroxysteroid dehydrogenases revealed that many TUNEL-positive apoptotic cells in regressing CLs of both SAMR1 and SAMP1 mice, but no apoptotic cells, were demonstrated in aaCLs of SAMP1 mice. MMPs and their mRNAs were detected in all ovaries of both SAMR1 and SAMP1 mice. In the murine ovaries, negative/trace activity of non-MMP proteinases was detected by FIZ techniques. Moderate/strong activities of MMPs were demonstrated in CLs of SAMR1 mice and in regressing CLs of SAMP1 mice, but trace MMP activity was noted in aaCLs of SAMP1 mice.

Cell Proliferation and Apoptosis in the Anterior Pituitary of Chicken during Inhibition and Resumption of Laying

The goal of this study was to determine whether tissue rejuvenation of the anterior pituitary with cell proliferation and apoptosis occurs during inhibition and resumption of egg-laying. White Leghorn laying hens were subjected to inhibition of laying by feed withdrawal. Feeding was resumed on the fourth day of egg-laying cessation. All birds were injected ip with bromodeoxyuridine (BrdU) 1 h before tissue collection. The anterior pituitary glands were collected from hens of the following groups: pretreatment (PT), 3 and 5 days after starvation (3DS and 5DS, respectively), 3 days after cessation of laying (3DC), 10 days after cessation of laying (10DC, 6 days after resumption of feeding), and the day of and 1 week after resumption of laying (RL and 1WRL, respectively). They were processed for the detection of proliferating cells and apoptotic cells by BrdU immunostaining and terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick-end-labeling (TUNEL). Immunostaining for the anterior pituitary hormones was also conducted. In the cephalic lobe the BrdU-positive cells showed a higher frequency in RL than in PT, 3DC, and 1WRL. BrdU-positive cell frequency in the caudal lobe was greater in RL than in PT. TUNEL-positive cells in both cephalic and caudal lobes were increased markedly in the RL group. Their frequency in the cephalic lobe was greater in RL than in PT, and that in the caudal lobe of RL was higher than in any other group of birds. The areas of FSH-like cells in 10DC and RL were greater than those in PT to 3DC, and those of LH-like cells in RL were greater than those of 3DS to 3DC. PRL-like cells were decreased until 3DC and then gradually increased until 1WRL. GH-, TSH-, and ACTH-like cell areas showed tendencies to increase until 3DC, with decreasing thereafter. The sizes of FSH-like cells in 10DC to 1WRL and LH-like cells in RL were larger than those around cessation of laying. These results suggest that during inhibition and resumption of laying cell proliferation and apoptosis occur in the anterior pituitary tissue, which may cause a rejuvenation of tissue to improve the function of this organ.

Apoptosis and expression of p53 response proteins and cyclin D1 after cortical impact in rat brain

We measured the temporal profile and cellular identification of apoptosis in rat brain after cortical contusion injury. Double staining immunohistochemistry was also used to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd45, PCNA) and cell cycle protein, Cyclin D1, in male Wistar rats 48 h after injury. Cortical contusion was induced in male Wistar rats with a pneumatic impactor device. The animals were sacrificed at different times after trauma (1, 2, and 14 h and 1, 2, 4, 7 and 14 days; n=4 per time point). Sham-operated rats ( n=4) and normal rats not subjected to any surgical procedure ( n=4) were used as controls for temporal profile determination. Additional 11 rats were used for study of protein expression. Coronal brain sections were analyzed using an in situ terminal deoxynucleotdyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL), hematoxylin, and immunohistochemical double staining methods. Apoptotic cells were observed as early as 2 h after the impact. Apoptotic cell death peaked at 2 days, gradually tapering off afterward, although scattered apoptotic cells were detected at 2 weeks after the impact. The number of apoptotic cells at 2 days far exceeded their number at other times ( p=0.009). Apoptotic cells were observed primarily in the cortex adjacent to the site of injury. In addition, apoptotic cells in conjunction with few injured cells were present in the ipsilateral hippocampus and localized to the granule layer of dentate gyrus. Our data indicate that DNA fragmentation is present in nearly all neurons subacutely after cortical contusion and persists for at least 2 weeks thereafter. Apoptosis is also present in neurons localized to the hilus of the dentate gyrus at a site remote from the area of injury suggesting a selective role for apoptosis in promoting secondary brain damage and dysfunction after traumatic brain injury. Using double staining, we were able to show that a great majority of apoptotic cells (>95%) were neurons and the rest were astrocytes and endothelial cells. Proteins associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd 45, PCNA) were expressed in the cytoplasm of normal cells of naive and sham rats. These proteins were translocated to the nuclei of apoptotic and injured cells at 48 h after cortical contusion. Cyclin D1 was not present in apoptotic cells. The differential expression of proteins associated with DNA damage, repair and the cell cycle protein Cyclin D1 in the contused brain suggest a potential role for these proteins in cell survival and apoptosis after cortical contusion.

Cell death, phagocytosis, and neurogenesis in mouse olfactory epithelium and vomeronasal organ after colchicine treatment.

The cytotoxic agent colchicine induced apoptotic cell death in the mouse olfactory epithelium and vomeronasal organ. The terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) method revealed the presence of many apoptotic bodies in the middle to basal region of the septal olfactory epithelium and vomeronasal organ near the boundary of the respiratory epithelium at 1 day after intraperitoneal (i.p.) injection of colchicine (4 mg/kg). In some areas of the nasal turbinates, massive apoptosis was observed in the olfactory epithelium. Electron micrographs showed that immature olfactory cells and globose basal cells were killed by the colchicine and had been phagocytized by the supporting cells and macrophages. In some areas of the nasal turbinates, mature olfactory cells were also killed, and thus only the supporting cells and horizontal basal cells remained in the epithelium. Bromodeoxyuridine (BrdU) labeling showed that regeneration occurred in the septum and vomeronasal organ at 4-6 days after colchicine treatment; however, there were no regenerated olfactory cells in some areas of the turbinates up to 30 days after colchicine treatment.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside

Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promotors of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM of cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase--mediated biotinylated--deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Apoptotic photoreceptor cell death after traumatic retinal detachment in humans.

To determine the mechanism of photoreceptor cell death after traumatic retinal detachment in humans. Clinical records from 1975 to 1993 of 75 patients, whose eyes were enucleated after traumatic retinal detachment, were reviewed for age, sex, previous ocular or systemic medical history, interval from initial trauma to enucleation, visual acuity, and types of trauma. The patients were divided into five groups of 15 cases each, based on the interval from initial trauma to enucleation. The retinal tissue was examined for two markers of apoptosis: (1) nicked nuclear DNA in situ by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) technique and (2) apoptotic bodies by light and electron microscopy. Of the 75 cases of ruptured globe and traumatic retinal detachment that were evaluated, 19 eyes (25.3%) showed TUNEL-positive labeling of photoreceptor cells. Nicked nuclear DNA was detected in photoreceptor cells of detached retinas as early as 8 hours after trauma. The detached retinas in seven of 15 eyes enucleated within 2 days after ocular trauma showed TUNEL-positive photoreceptor nuclei. The number of cases showing TUNEL-positive photoreceptor nuclei decreased as the interval between initial trauma and enucleation increased. The TUNEL-positive photoreceptor cells could still be seen in the detached retinas of two eyes enucleated 22 days after trauma. Light microscopy disclosed condensation and fragmentation of photoreceptor nuclei in the detached retinas. Electron microscopy showed structures resembling apoptotic bodies phagocytosed by neighboring cells in the TUNEL-positive retinas. Apoptosis is an important mechanism of photoreceptor cell degeneration in the early stage after traumatic retinal detachment in humans.

The experience of a clinical pharmacology unit based in the U.S.A.

At present, apoptosis is considered as a result of intracellular genetic program independently of the reason, regular or pathological, which launched it. Apoptosis is essentially different from necrosis, when swelling and rupture of cells with the effusion of their contents take place, inevitably followed by the development of inflammation. In phagocytosis of apoptotic cells and bodies, the macrophages do not produce proinflammatory cytokines, but increase the production of antiinflammatory cytokines. A late sign of apoptosis is internucleosomal decomposition of nuclear DNA, which can be exploited to reveal apoptotic cells in situ by means of TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling) reaction. Caspases are the key enzymes involved in the intracellular cascade pathway of apoptosis. The damage of mitochondria results in the release of a potent activator of the caspases—cytochrome C—into the cytoplasm. This phenomenon is connected with a change in the expression of Bcl-2 family genes which are under the control of the p53 gene; the activity of the latter determines the start of a cell's advance to apoptosis. The completion of many immunological processes ends with apoptosis, which involves the interaction of Fas (CD95/APO-1) receptor and its FasL ligand both widely expressed in the cells of the immune system. This latter either independently or together with the perphorin–granzyme system represents the main mechanism of cytotoxic killing by T and NK cells. Elimination of immune effector cells which have already fulfilled their function is also carried out by FAS-mediated apoptosis. Clonal deletion of immature T cells in the thymus takes place without the assistance of the Fas/FasL system. Apoptosis is realized during such immunological reactions as deletion of B lymphocytes in germinal centers post antigen activation. Adequately regulated apoptosis maintains cellular balance in the immune system. Both decreases and increases in apoptosis rates lead to pathology of the immune system.