Triphosphate (dUTP)-biotin Nick End Labeling Research Articles

Streptococcus suis contributes to inguinal lymph node lesions in piglets after highly pathogenic porcine reproductive and respiratory syndrome virus infection.

The swine pathogens porcine reproductive and respiratory syndrome virus (PRRSV) and Streptococcus suis have both been reported to cause damage to the immune organs. Inguinal lymph node (ILN) injury has been reported in PRRSV-infected pigs with secondary S. suis infection, but not much is known about the mechanism. In this study, secondary S. suis infection after highly pathogenic (HP)-PRRSV infection caused more severe clinical symptoms, mortality, and ILN lesions. Histopathological lesions were seen in ILNs with a marked decrease in lymphocyte numbers. Terminal deoxynucleotidyl transferase (TdT)-mediated de-oxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) assays revealed that HP-PRRSV strain HuN4 alone induced ILN apoptosis, but dual-infection with S. suis strain BM0806 induced greater levels of apoptosis. Besides, we found that some HP-PRRSV-infected cells underwent apoptosis. Furthermore, anti-caspase-3 antibody staining confirmed that ILN apoptosis was mainly induced by a caspase-dependent pathway. Pyroptosis was also observed in HP-PRRSV-infected cells, and there was more pyroptosis in piglets infected with HP-PRRSV alone compared with those with secondary S. suis infection, and HP-PRRSV-infected cells underwent pyroptosis. Altogether, this is the first report to identify pyroptosis in ILNs and which signaling pathway is related to ILN apoptosis in single or dual-infected piglets. These results contribute to a better understanding of the pathogenic mechanisms during secondary S. suis infection.

Perindopril inhibits myocardial apoptosis in mice with acute myocardial infarction through TLR4/NF-κB pathway.

To explore the anti-apoptotic effect of perindopril on myocardial cells in mice with acute myocardial infarction (AMI). A total of 48 mice were randomly divided into 4 groups before intervention, namely sham operation group (Sham group, n=12), AMI group (n=12), 1.5 mg/kg perindopril treatment group (Perindopril group, n=12), and 1.5 mg/kg perindopril treatment and Toll-like receptor-4 (TLR4) knockout group (TLR4-/-Perindopril group, n=12). Mice in the control group and AMI group were gavaged with normal saline, and those in the Perindopril group and TLR4-/-Perindopril group were gavaged with perindopril for 7 d. On the 4th day after drug administration, mice in the AMI group, Perindopril group and TLR4-/-Perindopril group were subjected to the ligation of the anterior descending coronary artery to induce AMI, and those in the Sham group underwent the same operation, but had a loose knot at the anterior descending coronary artery. At 24 h after the above operation, color echocardiography was performed on mice to observe changes in cardiac function. Then, the mice were sacrificed. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay was carried out to determine myocardial apoptosis. Immunohistochemistry and Western blotting technique were employed to detect the protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50 in infarction zones. The messenger ribonucleic acid (mRNA) expression levels of TLR4 and NF-κB p50 in infarction zones were measured via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Perindopril could significantly reduce the number of apoptotic myocardial cells after AMI. Mouse echocardiography showed that ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) of AMI mice in the Perindopril groups were markedly superior to those in the AMI group. AMI mice in the Perindopril group had decreased expression levels of Bax protein and TLR4 and NF-κB p50 mRNA and protein, as well as the Bax/Bcl-2 ratio. Knockout of TLR4 attenuated the effect of perindopril in alleviating myocardial apoptosis after AMI. Perindopril inhibits myocardial apoptosis in mice with AMI through the TLR4/NF-κB pathway.

Therapeutic Efficacy of Intranasally Delivered Mesenchymal Stem Cells in a Rat Model of Parkinson Disease

Safe and effective cell delivery remains one of the main challenges in cell-based therapy of neurodegenerative disorders. Graft survival, sufficient enrichment of therapeutic cells in the brain, and avoidance of their distribution throughout the peripheral organs are greatly influenced by the method of delivery. Here we demonstrate for the first time noninvasive intranasal (IN) delivery of mesenchymal stem cells (MSCs) to the brains of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. IN application (INA) of MSCs resulted in the appearance of cells in the olfactory bulb, cortex, hippocampus, striatum, cerebellum, brainstem, and spinal cord. Out of 1 × 10⁶ MSCs applied intranasally, 24% survived for at least 4.5 months in the brains of 6-OHDA rats as assessed by quantification of enhanced green fluorescent protein (EGFP) DNA. Quantification of proliferating cell nuclear antigen-positive EGFP-MSCs showed that 3% of applied MSCs were proliferative 4.5 months after application. INA of MSCs increased the tyrosine hydroxylase level in the lesioned ipsilateral striatum and substantia nigra, and completely eliminated the 6-OHDA-induced increase in terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine, 5'-triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining of these areas. INA of EGFP-labeled MSCs prevented any decrease in the dopamine level in the lesioned hemisphere, whereas the lesioned side of the control animals revealed significantly lower levels of dopamine 4.5 months after 6-OHDA treatment. Behavioral analyses revealed significant and substantial improvement of motor function of the Parkinsonian forepaw to up to 68% of the normal value 40-110 days after INA of 1 × 10⁶ cells. MSC-INA decreased the concentrations of inflammatory cytokines-interleukin-1β (IL-1β), IL-2, -6, -12, tumor necrosis factor (TNF), interferon-γ (IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF)-in the lesioned side to their levels in the intact hemisphere. IN administration provides a highly promising noninvasive alternative to the traumatic surgical procedure of transplantation and allows targeted delivery of cells to the brain with the option of chronic application.

Saffron Supplement Maintains Morphology and Function after Exposure to Damaging Light in Mammalian Retina

To test whether the saffron extract (Crocus sativus L.) given as a dietary supplement counteracts the effects of continuous light exposure in the albino rat retina. Three experimental groups of Sprague-Dawley rats were used. Experimental animals were prefed either saffron or beta-carotene (1 mg extract/kg/d) before they were exposed to bright continuous light (BCL) for 24 hours. Flash electroretinograms (fERGs) were recorded in control and treated rats the day before and 1 week after light exposure. At the end of the second recording session, the animals were killed and the retinas were quickly removed, fixed, cryosectioned, and labeled so that the thickness of the outer nuclear layer (ONL) could be analyzed. Changes in protein level and cellular localization of fibroblast growth factor (FGF)2 were determined by Western blot analysis and retinal immunohistochemistry, respectively. In a second series of experiments, rats were killed at the end of light exposure, and the amount of apoptotic figures in the ONL was assessed by terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). BCL induced DNA fragmentation, characteristic of dying cells, almost exclusively in the photoreceptor layer. The rate of photoreceptor death induced by BCL is expressed as the frequency of TUNEL-positive profiles per millimeter. The photoreceptor layer was largely preserved in saffron-treated animals because it was the fERG response. In addition, the rate of photoreceptor death induced by BCL appeared drastically reduced in treated animals. In beta-carotene prefeeding experiments, morphologic analysis showed preservation of the ONL similar to that obtained with saffron prefeeding, whereas the fERG response was unrecordable. Western blot analysis showed that exposure to light induced a strong upregulation of FGF2 in control and beta-carotene-treated rats, but s no change was noted in saffron-treated rats. These results show that saffron may protect photoreceptors from retinal stress, maintaining both morphology and function and probably acting as a regulator of programmed cell death.

Effects of vascular endothelial growth factor on apoptosis of sinusoidal endothelial cells caused by ethanol: experiment with rats

To investigate the cell death pattern of sinusoidal endothelial cells (SECs) caused by ethanol and the effects of vascular endothelial growth factor (VEGF) on this cell death, as well as the underlying mechanism involving Ets-1 and Caspase-8. SECs were isolated from male Wistar rats and cultured in medium containing ethanol (25 - 100 mmol/L). VEGF (20 - 30 ng/ml) was added into the medium to be co-incubated for up to 6 h. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) technique. The protein expression of Ets-1, prototype of anti-apoptotic Ets family, was determined by Western blotting and the Caspase-8 was measured by FLICE/caspase-8 colorimetric protease assay kit. Three days after culture, the SECs showed spindle-like shapes and nearly confluent, however, the cells tended to shrink and die during the time course of ethanol incubation under phase contrast microscope. The control SECs contained only a few percent of TUNEL-positive cells; however, the TUNEL-positive cells started to increase 2 hours after the addition of ethanol (100 mmol/L), and about 75% of the cells were TUNEL-positive 6 hours after ethanol incubation (P < 0.05) under fluorescent microscope. Again, TUNEL-positive cells increased 6 hours after ethanol (25 - 100 mmol/L) incubation in a dose dependent manner (P < 0.05). Six hours after VEGF (20 - 30 ng/ml) was added into the medium with ethanol (100 mmol/L) the percentage of TUNEL positive SECs decreased in a dose dependent manner (P < 0.05), the level of ethanol-induced apoptotic cells in the presence of VEGF (30 ng/ml) being around 71% 6 hours after ethanol incubation alone. The Ets-1 protein level of the SECs decreased 6 hours after ethanol (100 mmol/L) incubation, which was prevented almost completely by VEGF (30 ng/ml). The Caspase-8 activity level was significantly increased to 44.9 +/- 14.3 2 hours after ethanol (100 mmol/L) incubation, and decrease to 30.4 +/- 2.0 and 25.2 +/- 2.2 respectively after the addition of VEGF (20 - 30 ng/ml) (both P < 0.05). VEGF prevents the apoptosis of primary cultured SECs induced by ethanol, through at least in part, inhibition of ethanol-induced down-regulation of Ets-1 protein expression and ethanol-induced up-regulation of Casepase-8 activity in SECs.

Granzyme B leakage-induced apoptosis is a crucial mechanism of cell death in nasal-type NK/T-cell lymphoma

This study aims to investigate the role of granzyme B in the apoptosis of nasal-type NK/T-cell lymphoma. Twenty-four nasal-type NK/T-cell lymphomas were examined by TdT-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) assay and immunohistochemical staining for active caspase 3, poly(ADP-ribose) polymerase (PARP-1/p85)/p85, and Bcl-2. In addition, HANK-1 and NKL cell lines were analyzed using Western blot analysis. Immunoprecipitation was performed to identify the binding of granzyme B and intrinsic serpin proteinase inhibitor 9 (PI-9). To localize granzyme B, immunogold labeling and immunofluorescence staining were performed. The expression level of granzyme B in tumor tissue was correlated with the apoptosis rate (P=0.015), degree of necrosis (P=0.002), and the levels of active caspase 3 (P=0.036) and poly ADP-ribose polymerase (PARP)-1/p85 (P=0.040). The granzyme B-positive HANK-1 cell line showed increased spontaneous cell death compared to the granzyme B-negative NKL cell line. The untreated HANK-1 cells released cytochrome c into the cytosol with cleavage of caspase 3 and PARP-1. Treatment with granzyme B inhibitor and caspase inhibitor decreased the cleavage of PARP-1. By performing immunogold labeling, granzyme B was identified within the cytolytic granules as well as in the cytosol. Confocal microscopy and immunoprecipitation assays confirmed the colocalization of PI-9 and granzyme B, which formed an SDS-resistant complex. These results suggested that granzyme B leakage induces cell death in NK/T-cell lymphomas via both caspase-dependent and -independent mechanisms, and this leads to the extensive necrosis that is commonly seen in NK/T-cell lymphoma.

Epithelial cell turnover in relation to ongoing damage of the gastric mucosa in patients with early gastric cancer: increase of cell proliferation in paramalignant lesions

Gastric cancer is typically an end result of Helicobacter pylori-associated chronic gastritis. The pathogenesis is thought to involve effects on gastric mucosal epithelial cell turnover. In this study, we aimed to compare apoptosis and proliferation in the noncancer-containing mucosa of H. pylori-positive patients with early gastric cancer with these phenomena in H. pylori-positive controls. Two specimens each were obtained from the greater and lesser curvatures of the corpus and from the greater curvature of the antrum. The histopathological grading used was the updated Sydney System. Apoptotic epithelial cells were detected using the terminal deoxy nucleotidyl transferase-mediated deoxy-uridine triphosphate (dUTP) biotin nick-end labeling (TUNEL) method. The expression of Ki 67 was evaluated by immunostaining. Forty-five H. pylori-positive patients with endoscopic mucosal resection for early gastric cancer and 52 H. pylori-positive controls were studied. Gastric cancer was associated with a higher frequency of incomplete intestinal metaplasia (IM; odds ratio [OR], 19.1; 95% confidence interval [CI], 6.9-53.2; P < 0.001). The apoptotic index (AI) in the greater curvature of the corpus and the proliferation index (PI) in each part were significantly higher in cancer patients than in the control group. The median PI in the antrum was significantly higher in the incomplete IM group than that in the complete IM group (17.6 vs 12.6; P = 0.009). The PI and the AI in the greater curvature of the corpus correlated with the activity score, and the PI correlated with the IM score. In the cancer patients, H. pylori-induced gastritis was associated with increased cell proliferation and apoptosis compared with mucosal findings in the controls. IM seems to be one of the most important factors affecting cell proliferation and may be one of the components of carcinogenesis that results in proliferation-dominant cell kinetics.

Spatial distribution of Bax and Bcl-2 in osteocytes after bone fatigue: complementary roles in bone remodeling regulation?

Osteocyte apoptosis appears to play a key role in the mechanism by which osteoclastic resorption activity targets bone for removal, because osteocyte apoptosis occurs in highly specific association with microdamage and subsequent remodeling after fatigue. However, beyond terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay, little is known about the mechanisms controlling osteocyte apoptosis in vivo. In the current studies, expression of Bax, a proapoptotic gene product, and Bcl-2, an antiapoptotic gene product, was determined in osteocytes of fatigued rat bone using immunocytochemical staining and compared with TUNEL staining patterns. Bax and Bcl-2 were evident in osteocytes by 6 h after loading. Moreover, Bax and Bcl-2 in osteocytes were expressed differently as a function of distance from microdamage sites. The peak of Bax expression and TUNEL+ staining in osteocytes was observed immediately at the microcrack locus, which is where bone resorption occurs in this system; in contrast, Bcl-2 expression, the antiapoptotic signal, reached its greatest level at some distance (1-2 mm) from microcracks. These data suggest that near sites of microinjury in bone, those osteocytes that do not undergo apoptosis are prevented from doing so by active protection mechanisms. Moreover, the zone of apoptotic osteocytes around microcracks was effectively "walled in" by a surrounding halo of surviving osteocytes actively expressing Bc1-2. Thus, the expression pattern of apoptosis-inhibiting gene products by osteocytes surrounding the apoptotic osteocyte at microdamage sites also may provide important signals in the guidance of resorption processes that occur in association with osteocyte apoptosis after fatigue.

Cell proliferation and apoptosis during fracture healing.

This study investigated the relation between cell proliferation and apoptosis during fracture healing in a mouse femoral fracture model. Left femoral osteotomies were performed in 30 mature male CFLP mice immobilized with uniplanar external fixators. Six animals were killed on days 2, 4, 8, 16, and 24 postfracture for examination. Localization of cell proliferation was examined using immunohistochemistry with proliferating cell nuclear antigen (PCNA) monoclonal antibody. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) method. Images of each time-specific specimen were captured. The total callus area, the positively labeled cells by PCNA, and TUNEL per high-power field were quantified. Cell proliferation and apoptosis were found coexisting during the entire period of study. In the early phases of fracture healing (days 2-8), PCNA-positive labeling was predominant and peaked at day 8 and the TUNEL-positive labeling was minimal. In later stages of fracture healing (days 16-24), PCNA expression declined at day 16 as callus ossification and remodeling spread within the fracture site and apoptosis was the dominant cell activity with the TUNEL-positive labeling peaking at day 16 and declining sharply at day 24. These cell activities were reflected by the change of fracture callus, where there was a continuous increase in total callus area to day 16 and subsequent decrease at day 24. This study indicated that cell proliferation and apoptosis are coupled events during fracture repair, cell proliferation is active at the early stages, and apoptosis is active during the phase of callus remodeling.

Acinar Cell Hypertrophy, Proliferation and Cell Death in Rat Submandibular Glands Induced by Long-Term Isoproterenol Administration.

The purpose of this study was to elucidate the relationship between cell proliferation and cell death of submandibular gland (SMG) acinar cells in rats treated with the β-adrenergic secretagogue isoproterenol (IPR) which elicits massive growth of salivary glands. We analyzed this process using computerized imaging analysis, 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining, the terminal deoxyribonucleotidyl transferase (TdT)-mediated-deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) method and ultrastructural observations for 10 days of IPR treatment and for 3 weeks thereafter. Proliferation of acinar cells occurred at days 2 and 3 of IPR administration, and acinar cell hypertrophy continued during the entire 10-day treatment period. Numerous TUNEL-positive cells and vesicular structures were found in the acinar area by day 3 post-IPR administration. Ultrastructurally, vacuolated structures that resembled degenerating apoptotic bodies were located in and between the acinar cells. A different type of cell death, which was associated with an electron-lucent and edematous cytoplasm but not with mitochondrial lysis or cell membrane destruction, was also recognized in the acinus. It is suggested that this other type of cell death may correspond to secondary necrosis.

Glycine Prevents Apoptosis of Rat Sinusoidal Endothelial Cells Caused by Deprivation of Vascular Endothelial Growth Factor

Apoptosis of sinusoidal endothelial cells (SECs) is one of the initial events in the development of ischemia-reperfusion injury of the liver. Glycine has been shown to diminish ischemia-reperfusion injury in the liver and improve graft survival in the rat liver transplantation model. Here, we investigated the effect of glycine on apoptosis of primary cultured rat SECs induced by vascular endothelial growth factor (VEGF) deprivation. Isolated rat SECs were cultured in EBM-2 medium supplemented with 10% fetal bovine serum (FBS) and growth factors including 20 ng/mL VEGF for 3 days. SECs at 3 days of culture showed spindle-like shapes; however, cells started shrinking and detaching from dishes by VEGF deprivation. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining in these conditions. Control SECs contained only a few percent of TUNEL-positive cells; however, they started increasing 4 hours after VEGF deprivation, and the percentage of TUNEL-positive cells reached about 50% at 8 hours and almost 100% at 16 hours after VEGF deprivation. Interestingly, this increase in TUNEL-positive cells after VEGF deprivation was prevented significantly when glycine (1-10 mmol/L) was added to the medium, the levels being around 60% of VEGF deprivation without glycine. Furthermore, strychnine (1 μmol/L), a glycine receptor antagonist, inhibited this effect of glycine, suggesting the possible involvement of the glycine receptor/chloride channel in the mechanism. Moreover, Bcl-2 protein levels in SECs were decreased 8 hours after VEGF deprivation, which was prevented almost completely by glycine. It is concluded that glycine prevents apoptosis of primary cultured SECs under VEGF deprivation.(Hepatology 2000;32:542-546.)

In situ apoptosis in the thyroid.

Recent evidence has emphasized the importance of programmed cell death, or apoptosis, in the maintenance of tissue homeostasis and pathogenesis of tumors. This study analyzed the significance of apoptosis in relation to the expression of p53 and bcl-2 proteins, tissue proliferation defined by Ki-67 expression, and tissue histology in thyroid tissue. Extent of apoptosis was defined by morphological criteria and the terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate (dUTP) biotin nick end labeling (TUNEL) assay. Immunocytochemistry was performed for p53, bcl-2, and Ki-67 expression. There was good correlation between TUNEL-reactive cells and morphological evaluation criteria for apoptosis. The extent of apoptosis was significantly associated with the type of thyroid lesion (r = 0.66990, p = 0.000012), both proliferative (namely multinodular goiter) and neoplastic (benign and malignant). A higher extent of apoptosis was evident in medullary and anaplastic carcinomas. Apoptosis also correlated to p53 protein accumulation (r = 0.485, p = 0.00041) and Ki-67 immunoreactivity (r = 0.435, p = 0.001). An inverse correlation was observed between bcl-2 expression and the extent of apoptosis (r = -0.33369, p = 0.01912). A direct correlation was also observed between p53 expression and Ki-67 immunoreactivity (r = 0.623, p = 0.0002). By inhibiting apoptosis, bcl-2, may cause a shift in tissue kinetics toward the preservation of genetically aberrant cells, thereby facilitating tumor progression. These results imply that rapidly proliferating tumors appear to have a high cell turnover state in which there may be increased chance of apoptosis among the proliferating cells. The ability of apoptosis to occur in the presence of a possibly mutant p53 protein suggest the existence of at least two p53 dependent apoptotic pathways, one requiring activation of specific target genes and the other independent of it. However, keeping in mind the limited number of subjects studied in each subgroup and the rather low correlation coefficients, these possibilities would have to be substantiated in a larger study population.

Apoptosis and growth inhibition of head and neck tumor cell line induced by epidermal growth factor receptor tyrosine kinase inhibitor.

Overexpression of the epidermal growth factor (EGF) receptor, a hallmark of aerodigestive squamous cell carcinoma of the head and neck (SCCHN), correlates with aggressive tumor behavior. There is evidence that SCCHN cells auto-activate their EGF receptors. The receptor has therefore attracted interest as a potential therapeutic target. We tested the in vitro therapeutic efficacy of PD153035--a potent, specific inhibitor of the tyrosine kinase intrinsic to the EGF receptor--by employing a well-characterized cell line derived from human gingival SCCHN. DNA-synthesis and cell number were assayed for growth-inhibitory effects, phosphorylation of the EGF receptor was quantitated by immunoblot, and cell apoptosis was detected by terminal deoxytransferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) in situ assay. PD153035, at nanomolar concentrations, inhibited autophosphorylation of the EGF receptor induced by EGF stimulation and the inhibition occurred in a dose-dependent manner. Under the same conditions, PD153035 inhibited cell growth, and induced apoptosis of SCCHN cells in vitro. We conclude that selective inhibition of the EGF receptor tyrosine kinase completely abolishes EGF receptor phosphorylation resulting from receptor stimulation, and results in growth inhibition and apoptosis of SCCHN cells in vitro. By inducing cytostasis and apoptosis, this new class of inhibitors may be of therapeutic value against SCCHN.

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis.

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.

Acute Testicular Ischemia Results in Germ Cell-Specific Apoptosis in the Rat

Testis torsion-induced aspermatogenesis is not necessarily due to permanent loss of blood flow nor to dysfunctional Leydig cells or Sertoli cells. This investigation was undertaken to gain further insight into the mechanism underlying torsion-induced germ cell loss. Male rats were subjected to 1-h or 2-h ischemiainducing torsion, and testes were examined at either 1, 2, 4, 24, or 48 h after torsion, depending on the study. Testes were examined for evidence of 1) in situ apoptosis by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) technique, 2) apoptosis by the DNA technique, 3) leukocyte margination and diapedesis in testicular vessels by immunocytochemical and histological techniques, and 4) testicular lipid peroxidation by the thiobarbituric acid reactive substances assay. The first TUNEL evidence for torsion-induced apoptosis was at 4 h after repair of 1-h torsion. Induction of apoptosis was confirmed by the electrophoretic laddering of DNA fragments. It was hypothesized that apoptosis was induced by reactive oxygen species arising from reperfusing leukocytes. A significant increase in both leukocyte margination and diapedesis occurred 4 h after torsion repair as did a significant increase in intratesticular lipid peroxidation products. These events were contemporaneous with the first appearance of apoptosis and consistent with the hypothesis that post-torsion, germ cell-specific apoptosis is induced by reactive oxygen species.

Developmental neuronal death is not a universal phenomenon among cell types in the chick embryo retina.

The goal of this study was to investigate whether all the cell types present in the chick embryo retina undergo developmental neuronal death. Apoptosis was investigated in retinal sections at different developmental stages, processed either with propidium iodide, which stains pyknotic nuclei intensely, or with terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). Internucleosomal DNA fragmentation was investigated in tissue extracts by agarose gel electrophoresis. TUNEL-positive (T+) cells and pyknotic nuclei were first detectable in the ganglion cell layer (GCL) around embryonic day (ED) 8 and peaked at ED 10. In the inner nuclear layer (INL), T+ and pyknotic cells first appeared on ED 8, reached maximum frequency on ED 11, and were largely absent after ED 14. DNA ladders were observed at all the stages, when T+ and pyknotic cells were abundant, but not on ED 4, when only scattered dead cells were observed histologically. Dying cells were virtually never detected in the outer nuclear layer (ONL) from ED 4 to postnatal day 2. After unilateral midbrain ablation on ED 5, there was a striking increase in the number of pyknotic and T+ cells in both the GCL and in the INL of the contralateral eye but not in the ONL. The absence of apoptotic cell death in the ONL during normal development and after tectal ablation shows that developmental death is not universal among the various cell populations present in the chick embryo retina and raises questions regarding mechanisms controlling both photoreceptor survival and the matching of pre- and postsynaptic elements in the outer plexiform layer of this species.

Detection of apoptotic cells in cytology specimens: an application of TdT-mediated dUTP-biotin nick end labeling to cell smears.

We applied TdT-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) to cytologic smears in order to detect the cells undergoing apoptosis. These smears were obtained by scraping the cut surface of 9 cases of carcinoma, including renal-cell carcinoma (3 cases), esophageal squamous-cell carcinoma (3 cases), and gastric adenocarcinoma (3 cases), and were fixed and prepared by different methods. The results were also compared with those of tissue sections. TUNEL in smears was generally associated with higher background nuclear stain than in tissue sections. Smears that were fixed in 4% or 8% paraformaldehyde or absolute methanol exhibited results comparable with those of tissue sections, with minimum background in all cases examined. There were no significant differences in TUNEL labeling index among tissue sections and smears fixed in 4% or 8% paraformaldehyde or in absolute methanol. Smears treated in Carnoy's fixative (3:1 methanol:acetic acid) and air-dried smears demonstrated a higher background. TUNEL positivity could not be detected in slides decolorized from May-Grünwald-Giemsa stain. Markedly high background, which may occur as a result of artifactural DNA breaks, was also observed in slides decolorized from Papanicolaou stain, in which TUNEL-positive cells could be evaluated only in 3/8 cases. Application of the TUNEL method to cytology specimens has disadvantages or limitations compared to its application to histological sections, but the method is considered the most suitable one for detecting cells undergoing apoptosis in cytology materials.

Acute testicular ischemia results in germ cell-specific apoptosis in the rat.

Testis torsion-induced aspermatogenesis is not necessarily due to permanent loss of blood flow nor to dysfunctional Leydig cells or Sertoli cells. This investigation was undertaken to gain further insight into the mechanism underlying torsion-induced germ cell loss. Male rats were subjected to 1-h or 2-h ischemia-inducing torsion, and testes were examined at either 1, 2, 4, 24, or 48 h after torsion, depending on the study. Testes were examined for evidence of 1) in situ apoptosis by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) technique, 2) apoptosis by the DNA "laddering" technique, 3) leukocyte margination and diapedesis in testicular vessels by immunocytochemical and histological techniques, and 4) testicular lipid peroxidation by the thiobarbituric acid reactive substances assay. The first TUNEL evidence for torsion-induced apoptosis was at 4 h after repair of 1-h torsion. Induction of apoptosis was confirmed by the electrophoretic laddering of DNA fragments. It was hypothesized that apoptosis was induced by reactive oxygen species arising from reperfusing leukocytes. A significant increase in both leukocyte margination and diapedesis occurred 4 h after torsion repair as did a significant increase in intratesticular lipid peroxidation products. These events were contemporaneous with the first appearance of apoptosis and consistent with the hypothesis that post-torsion, germ cell-specific apoptosis is induced by reactive oxygen species.

Deafferentation Causes Apoptosis in Cortical Sensory Neurons in the Adult Rat

The present study provides an experimental model of the apoptotic death of pyramidal neurons in rat olfactory cortex after total bulbectomy. Terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL), DNA electrophoresis, and neuronal ultrastructure were used to provide evidence of apoptosis; neurons in olfactory cortex were counted by stereology. Maximal TUNEL staining occurred in the piriform cortex between 18 and 26 hr postbulbectomy. Within the survival times used in the present study (up to 48 hr postlesion), cell death was observed exclusively in the piriform cortex; there was no evidence of cell death in any other areas connected with the olfactory bulb. Neurons undergoing apoptosis were pyramidal cells receiving inputs from, but not projecting to, the olfactory bulb. The apical dendrites of these neurons were contacted by large numbers of degenerating axonal terminals. Gel electrophoresis of DNA purified from lesioned olfactory cortex showed a ladder pattern of fragmentation. Inflammatory cells or phagocytes were absent in the environment of degenerating neurons in the early stages of the apoptotic process. The present model suggests that deafferentation injury in sensory systems can cause apoptosis. In addition, olfactory bulbectomy can be used for investigating molecular mechanisms that underlie apoptosis in mature mammalian cortical neurons and for evaluating strategies to prevent the degeneration of cortical neurons.

Apoptosis is inhibited early in the dysplasia-carcinoma sequence of Barrett esophagus.

To evaluate the alteration of apoptosis in the esophageal epithelium during the esophagitis-Barrett esophagus-adenocarcinoma sequence. Archival tissue samples of 85 lesions in 58 cases were used. The lesions represented 7 groups: normal esophagus (n = 10), reflux esophagitis (n = 12), Barrett metaplasia (n = 21), Barrett low-grade dysplasia (n = 17), Barrett high-grade dysplasia (n = 5), well- or moderately differentiated adenocarcinoma (n = 10), and poorly differentiated adenocarcinoma (n = 10). Apoptotic cells with fragmented DNA were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) method. Monoclonal antibodies against bcl-2 protein were applied using the avidin-biotin complex immunoperoxidase method. The esophagitis group showed many apoptotic cells on the epithelial surface; in the other groups, few apoptotic cells were seen. Weak bcl-2 expression was seen in the basal cells in normal subjects and those with esophagitis. There was overexpression of bcl-2 in 72% of Barrett metaplasia, 100% of Barrett low-grade dysplasia, 25% of Barrett high-grade dysplasia, 40% of well- or moderately differentiated adenocarcinoma, and 20% of poorly differentiated adenocarcinoma. Increased apoptosis in reflux esophagitis may be a protective mechanism counteracting increased proliferation. Inhibition of apoptosis by overexpression of bcl-2 protein occurs early in the dysplasia-carcinoma sequence of Barrett esophagus. The resulting prolongation of cell survival may promote neoplastic progression. Despite the absence of apoptosis, bcl-2 was not widely overexpressed in Barrett high-grade dysplasia and adenocarcinoma, suggesting that cells acquire other ways of avoiding apoptosis as malignancy appears.