General suitability of techniques for in situ detection of apoptosis in small intestinal epithelium

The present study was designed to evaluate different techniques for the in situ detection of apoptosis in human and rat small intestinal epithelium. The techniques included light microscopy (LM) and transmission electron microscopy (TEM) observation of epoxy resin-embedded tissue, scanning electron microscopy (SEM), TUNEL assay, and antibodies directed against caspase cleavage products of caspase 3, cytokeratin 18 (CK 18), and apoptotic single-strand DNA (ssDNA). All techniques, if the labeling was positive, showed apoptotic cells exclusively at the villus tip. LM and TEM were the most reliable and revealed morphological signs typical of cells that have died via apoptosis. SEM indicated the extension of the process. The antibody recognizing cleaved caspase 3 could be considered an appropriate marker for apoptotic epithelial cells in human and rat small intestine. However, the majority of epithelial cells lining the proximal small intestinal villus contained only low levels of intact CK 18. Therefore, sufficient amounts of cleaved CK 18 for immunohistochemical detection were not generated during apoptosis, rendering the application of the antibody inappropriate. The antibody detecting formamide-denatured ssDNA in apoptotic cells was both suitable and reliable; however, the particular staining procedure used compromised the tissue preservation. In comparison to this, the TUNEL assay was less reliable. Although it was performed with a commercially available ready-to-use kit, its application conditions had to be adjusted for each specimen on the basis of the findings produced by other techniques.     

Immunohistochemical analysis of cleaved caspase-3 detects high level of apoptosis frequently in diffuse large B-cell lymphomas of the central nervous system

The purpose of the present paper was to examine the level of apoptosis and the relationships among apoptosis, apoptosis-associated proteins, and proliferating potential in lymphoma tissues to clarify the characteristics of apoptosis in diffuse large B-cell lymphomas (DLBCL) of the central nervous system (CNS). The formalin-fixed, paraffin-embedded tissues of CNS and non-CNS DLBCL (20 cases each) were studied by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) and immunohistochemistry, using antibodies against single-stranded DNA (ssDNA), cleaved caspase-3, bcl-2, bax, p53, Fas and Ki-67. The cleaved caspase-3 immunohistochemistry detected apoptosis of the lymphoma cells most sensitively compared to TUNEL and ssDNA immunohistochemistry. High expression (grade + + or + + +) of cleaved caspase-3 was found more frequently in CNS DLBCL (11 cases, 55%) than non-CNS DLBCL (three cases, 15%; P = 0.009). Bax-positivity of lymphoma cells was increased in six cases of CNS DLBCL, which also showed high positivity of cleaved caspase-3. There was no significant correlation between the cleaved caspase-3-positivity and the Ki-67 positivity. The present study indicates that the number of apoptotic cells and expression level of cleaved caspase-3 were significantly higher in CNS DLBCL than non-CNS DLBCL, and that the correlation of bax and cleaved caspase-3 expression was often present in CNS DLBCL.     

Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver

In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.     

Differential expression of apoptotic markers in jimpy and in Plp overexpressors: evidence for different apoptotic pathways

Point mutations and duplications of proteolipid protein (PLP) gene in mammals cause dysmyelination and oligodendrocyte cell death. The jimpy mouse, which has a lethal Plp point mutation, is the best characterized of the mutants; transgenic mice, which have additional copies of Plp gene, are less characterized. While oligodendrocyte death is a prominent feature in jimpy, the pathways leading to death have not been investigated in jimpy and Plp overexpressors. Using immunohistochemistry and immunobloting, we examined expression of cleaved caspase-3, Poly (ADP-ribose) polymerase (PARP), caspase-12, and mitochondrial apoptotic markers in spinal cord in jimpy males and Plp overexpressors. Compared to controls, cleaved caspase-3 is increased 10x in jimpy white matter spinal cord, and 3x in Plp overexpressor. In jimpy, the number of cleaved caspase-3 cells far exceeds the number of TUNEL(+) cells. The majority of cleaved caspase-3(+) cells were not TUNEL(+) and these cells exhibited staining in perikarya and in processes. Only 30% of the cleaved caspase-3(+) cells were TUNEL(+) and exhibited both nuclear and perinuclear staining. This observation suggests that activation of caspase-3 begins earlier and overlaps for a period of time with DNA fragmentation. In both Plp mutants, quantitative immunobloting of PARP showed a 45% increase in total as well as cleaved form, indicating that oligodendrocytes die via apoptosis. Most interestingly, cleavage of caspase-12, a caspase associated with unfolded protein response, is dramatically increased in jimpy but not at all in Plp overexpressors. Mitochondrial markers cytochrome c and Bcl-X(L) are upregulated in both Plp mutants but levels of expression are different between mutants, suggesting that apoptosis in these two Plp mutants follows different pathways. In jimpy, mitochondrial apoptotic markers may play a role in amplifying the apoptotic signal. Our data shows for the first time, in vivo, that mutations in Plp gene increase oligodendrocyte death by activating the caspase cascade but the trigger to upregulate this cascade follows different pathways.     

Apoptosis and proliferation in lungs of human fetuses exposed to chorioamnionitis

AIMS: To determine whether chorioamnionitis has an impact on the extent of apoptosis and proliferation in fetal lungs. Fetuses exposed to chorioamnionitis have an increased risk of aquiring lung tissue damage in utero. METHODS AND RESULTS: Lung tissue sections from 35 stillborn fetuses were used in this study. Chorioamnionitis-exposed fetuses were subdivided depending on whether pneumonia was diagnosed (n = 13) or not (n = 10); 12 unaffected fetuses served as controls. Apoptotic and proliferating cells were determined by in-situ terminal deoxytransferase-mediated dUTP nick end labelling (TUNEL) assay and by anti-Ki67 immunohistochemistry, and quantified. The median apoptotic index in lungs of chorioamnionitis-exposed fetuses increased 2.4-fold compared with chorioamnionitis-negative stillborn controls (P = 0.043) and rose 21.6-fold when chorioamnionitis-exposed fetuses additionally developed pneumonia (P < 0.001). Compared with the proliferation index of the control group (PI = 2.3), the median percentage of proliferating cells in the lungs of chorioamnionitis-exposed fetuses decreased (PI = 1.4) (P = 0.036), but increased 1.8-fold (P = 0.036) in fetal lungs of the chorioamnionitis/pneumonia group. By double labellings combining the TUNEL assay or the Ki67 antigen with cell marker proteins, we identified distal airway epithelial cells as the cell type undergoing apoptosis in chorioamnionitis-exposed fetal lungs, while epithelial, endothelial and smooth muscle cells proliferated. Immunolabellings of cleaved caspases -8 and -9 revealed that apoptosis is mediated via initiator caspase-8. CONCLUSION: Chorioamnionitis induces apoptosis of distal airway epithelial cells via the caspase-8 pathway and interferes with the normal proliferative activity of epithelial, endothelial, and smooth muscle cells in fetal lungs. Thus, apoptosis and proliferation are an important feature of chorioamnionitis-associated lung injury in utero.     

No evidence for apoptosis of decidual leucocytes in normal and molar pregnancy: implications for immune privilege

Complete hydatidiform moles are totally paternally derived and represent complete allografts that might be expected to provoke maternal immune rejection. Our previous and other studies have shown expression of Fas by increased numbers of activated decidual CD4(+) T cells in both complete and partial molar pregnancy as well as increased FasL(+) expression by molar trophoblasts compared with trophoblasts in normal pregnancies. As the Fas/FasL system represents a major apoptotic pathway that can play a role in immune privilege, the aim of this study was to investigate whether apoptosis of decidual immune cells, particularly T cells, could be responsible for maternal immune tolerance in molar pregnancy. Using terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labelling (TUNEL), a significant increase in TUNEL(+) cells was demonstrated in decidua associated with partial (P = 0.0052) and complete (P = 0.0096) hydatidiform mole compared with normal early pregnancy. Co-labelling immunoperoxidase studies showed that the TUNEL(+) cells in both normal and molar pregnancies were not activated CD45RO(+) immune cells, CD3(+) T cells, CD56(+) uterine natural killer (NK) cells or CD14(+) CD68(+) macrophages. Double immunohistochemical labelling with antiactive caspase-3 and leucocyte markers confirmed the lack of leucocyte apoptosis. Double immunostaining with anticytokeratin to detect trophoblast and M30 CytoDeath, which detects a neoepitope of cytokeratin 18 revealed after caspase-mediated cleavage, revealed apoptotic extravillous trophoblast cells within decidual tissue. We conclude that there is no evidence that apoptosis of decidual leucocytes plays a role in maintaining maternal tolerance in either normal or molar pregnancy.     

Mitochondria-mediated apoptosis of lung epithelial cells in idiopathic interstitial pneumonias

We previously demonstrated that the up-regulation of p53, Fas, and DNA damage are present in lung epithelial cells from patients with idiopathic interstitial pneumonias (IIP). Fas ligation induces apoptosis of lung epithelial cells predominantly through the direct activation of the caspase cascade via caspase-8 activation, whereas the up-regulation of p53 and other cellular stresses can induce mitochondria-mediated apoptosis. In this study, we investigated the incidence of mitochondria-mediated apoptosis of epithelial cells in IIP. We performed TUNEL staining to detect apoptotic cells and western blot analysis and immunohistochemistry to assess the expression and activation of caspases and the cytochrome c release from mitochondria in lung tissues from eight patients with usual interstitial pneumonia, five patients with nonspecific interstitial pneumonia, and eight patients with normal lung parenchyma. The expressions of pro- and cleaved caspase-8, 9, 3, and cytochrome c release from the mitochondria were all significantly increased in the lung tissues of IIP compared with normal lung parenchyma. The positive signals for caspases in epithelial cells were increased in IIP compared with normal lung parenchyma by immunohistochemistry. The results of TUNEL and electron microscopy suggested that apoptotic cells were predominantly epithelial cells. TUNEL-positive cells in % of epithelial cells were significantly increased in IIP compared with normal lung parenchyma, and significantly correlated with cytochrome c release from the mitochondria and with the expression of cleaved caspase-3 in epithelial cells. We conclude that mitochondria-mediated apoptosis may be involved in the pathophysiology of IIP.     

Aggregatibacter actinomycetemcomitans infection enhances apoptosis in vivo through a caspase-3-dependent mechanism in experimental periodontitis

The purpose of this study was to test the hypothesis that diabetes aggravates periodontal destruction induced by Aggregatibacter actinomycetemcomitans infection. Thirty-eight diabetic and 33 normal rats were inoculated with A. actinomycetemcomitans and euthanized at baseline and at 4, 5, and 6 weeks after inoculation. Bone loss and the infiltration of polymorphonuclear leukocytes (PMNs) in gingival epithelium were measured in hematoxylin-eosin-stained sections. The induction of tumor necrosis factor alpha (TNF-α) was evaluated by immunohistochemistry and of apoptotic cells by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. After A. actinomycetemcomitans infection, the bone loss in diabetic rats was 1.7-fold and the PMN infiltration 1.6-fold higher than in normoglycemic rats (P < 0.05). The induction of TNF-α was 1.5-fold higher and of apoptotic cells was up to 3-fold higher in diabetic versus normoglycemic rats (P < 0.05). Treatment with a caspase-3 inhibitor significantly blocked noninflammatory cell apoptosis induced by A. actinomycetemcomitans infection in gingival epithelium and connective tissue (P < 0.05). These results provide new insight into how diabetes aggravates A. actinomycetemcomitans-induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection.     

一氧化氮和caspase-3在多巴胺诱导PC12细胞

目的：探讨一氧化氮（NO）和半胱氨酸蛋白酶3（caspase-3）在多巴胺诱导PC12细胞凋亡中的可能作用。方法：流式细胞仪定量检测PC12细胞的凋亡率,原位末端标记法（TUNEL）观察凋亡细胞的形态,Griess法测定NO₂^-的浓度,荧光分光光度计法检测caspase-3的活力,半定量RT-PCR法检测诱导型一氧化氮合酶（iNOS）mRNA的表达水平。结果：多巴胺（0.15-0.60 mmol/L）可剂量依赖性地诱导PC12细胞凋亡,表现为凋亡细胞的TUNEL染色阳性;iNOS mRNA的表达、NO的合成及caspase-3的活力均有明显的增加（P<0.01）;特异性的iNOS抑制剂aminoguanidine和caspase-3抑制剂Ac-DEVD-CHO通过减少NO的生成和抑制caspase-3的激活阻断PC12细胞凋亡。结论：NO的生成可能是多巴胺诱导PC12细胞凋亡的触发因子,caspase-3的激活是其中的效应因子。     

Apoptosis and expression of bcl-2 protein in invasive ductal adenocarcinoma of the pancreas

Apoptosis plays a central role in the development and/or progression of cancer. There are several methods for detection of apoptotic cells in tissue sections including light and electron microscopy, in situ nick end-labeling (ISEL), TdT-mediated dUTP nick-end labeling (TUNEL) and immunohistochemical detection of proteins associated with apoptosis. Apoptosis was assessed by the monoclonal antibody M30 CytoDEATH (M30), which is specific for neo-epitope in cytokeratin 18 that becomes available at an early caspase cleavage during apoptosis. Expression of bcl-2 protein was evaluated, because bcl-2 protein plays an important role in the regulation of apoptosis. Twenty-six invasive ductal adenocarcinomas of the pancreas were studied immunohistochemically with antibodies M30 and bcl-2. The mean apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells number) was 2.75%. High AI (> 10%) was observed in 4 cases of the 26 pancreatic carcinomas (15%). Protein bcl-2 was expressed in 3 cases (11.5%). The AI did not correlate with the expression of protein bcl-2. In conclusion, the detection of neo-epitope in cytokeratin 18 by monoclonal antibody M30 can be used for quantification of apoptotic cells with immunohistochemical techniques in tissue sections. It is a new approach to evaluate apoptosis in pancreatic carcinomas. The low positivity of bcl-2 expression in pancreatic adenocarcinomas suggests that bcl-2 protein does not play a central role in pancreatic tumorigenesis and cancer progression.     

Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm². JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation.     

C5b-9 terminal complement complex assembly on apoptotic cells in human arterial wall with atherosclerosis

Apoptosis plays an important role in atherosclerosis. The factors regulating this process are not well defined. We examined the relation of apoptotic cells with the terminal complement complex C5b-9 in human atherosclerotic lesions. The extent of apoptosis was determined using TdT dUTP nick-end labeling (TUNEL) and immunohistochemistry of apoptosis regulators caspase-3, caspase-9, Bax, and Bcl-2. C5b-9 was localized by immunohistochemistry and immunoelectron microscopy. The apoptotic index was higher in fibrous plaques when compared with intimal fatty streaks and intimal thickenings. Bax expression was present in TUNEL+ apoptotic cells, and Bcl-2 was rarely present in the atherosclerotic wall. Active caspase 9 and caspase 3 deposits were present in the same areas, suggesting an involvement of the mitochondrial pathway. C5b-9 deposits colocalized with TUNEL+ cells, and the percent of double-positive cells was 2% in fatty streaks, 12% in intimal thickenings, and 35% in fibrous plaques. Colocalization of apoptotic cells with C5b-9 was also confirmed by immunoelectron microscopy. In conclusion, some apoptotic cells carry C5b-9 deposits, suggesting that complement might be activated by apoptotic cells and involved in the promotion of apoptosis, contributing to the progression of atherosclerotic lesions.     

Immunosuppressant prograf® (tacrolimus) induces histopathological disorders in the peritubular tissue of rat testes

Treatment with tacrolimus (FK-506) has been shown to induce a significant decrease in the number of spermatocytes, spermatids, and Sertoli cells. Regarding the importance of the peritubular tissue for the maintenance of Sertoli cells, the integrity of the cellular and extracellular components of the peritubular tissue was evaluated in adult rats that were treated with 1 mg/kg/day of FK-506 for 30 and 60 days. Testicular sections were used for a quantitative analysis of the peritubular cells (PCs) and were submitted to the PAS method. Paraffin sections were submitted to the TUNEL method and to immunohistochemistry for the detection of caspase-3. Several testicular fragments were analyzed under a transmission electron microscope (TEM). A weak PAS reaction was noted in the peritubular tissue of the tacrolimus-treated animals. Next to the damaged peritubular tissue, the Sertoli cell nuclei were absent or dislocated from the basement membrane. In the treated animals, the number of PCs decreased significantly compared to the control animals, and these cells showed apoptotic features, were TUNEL positive, and were caspase-3 immunolabeled. Using the TEM, apoptosis was confirmed in myoid cells; moreover, the thickness and undulation of the basal laminae and an enlargement of the collagen I layer adjacent to the myoid cells was observed. Long-term treatment with the immunosuppressor induced peritubular myoid cell death by apoptosis and disarrangement of the peritubular extracellular layers. Future studies are necessary to confirm whether the structural alterations in the seminiferous epithelium are related to the effect of FK-506 on peritubular tissue.     

Caspase-3在脑出血后神经细胞凋亡中的作用

目的:探讨Caspase-3表达与大鼠脑出血后神经元凋亡的关系。方法:应用立体定向技术,将自体不凝血注入大鼠尾状核制备脑出血模型,将动物随机分为假手术组、脑出血组,分别在不同时间点断头取脑,连续切片作TUNEL染色和Caspase-3免疫组化染色。结果:脑出血后6h血肿内部及周边组织出现TUNEL阳性细胞, 72h达高峰, 2w时仍有少量表达;Caspase-3在脑出血后6h表达明显增高, 24h达到高峰,各组与假手术组之间差异显著(P<0 05 )。脑出血后的TUNEL阳性细胞与Caspase-3的表达呈正相关(r=0. 547,P<0 01),且Caspase-3的高峰时间早于凋亡的发生。结论:脑出血后的细胞凋亡与Caspase-3的表达有关。     

Avian encephalomyelitis virus induces apoptosis via major structural protein VP3

Avian encephalomyelitis virus (AEV) strain L(2)Z was investigated for its apoptotic activity in specific-pathogen-free chick embryo brain tissue. DNA fragmentation analysis and electron microscopy observation demonstrated that AEV could induce apoptosis in chick embryo brain tissues characterized by chromatin condensation, plasma membrane blebbing, cell shrinkage, and nucleosomal DNA fragmentation after 4 days postinfection. AEV structural protein genes VP1, VP2, and VP3 were transfected into Cos-7 and chick embryo brain (CEB) cells, respectively. The results showed that only VP3 protein was an apoptotic inducer, as demonstrated by DNA fragmentation analysis and TUNEL assay at 24 and 48 h posttransfection. Furthermore, expression of VP3 protein resulted in the activation of caspase-3-like proteases in both cells, which could be inhibited by a caspase-3-like protease-specific inhibitor Ac-DEVD-CHO peptide, suggesting that AEV VP3 protein induces apoptosis through a caspase-3-like protease pathway. In addition, VP3 protein localized to mitochondria in the Cos-7 and CEB cells at 24 h posttransfection observed by confocal microscopy, indicating that mitochondria may play an important role in VP3-induced apoptosis. Taken together, our results show that AEV could induce apoptosis in chick embryo brain tissue, structural protein VP3 could serve as an apoptotic inducer resulting in apoptosis in cell culture through a caspase-3-like protease pathway, which may be related to its localization to mitochondria.     

糖尿病足伤口皮肤细胞凋亡情况及AGEs对人皮肤成纤维细胞凋亡的影响

 目的：检测糖尿病足伤口皮肤细胞凋亡情况,探讨晚期糖基化终产物（AGEs）对人皮肤成纤维细胞凋亡的影响。方法：选择36例足部伤口患者,糖尿病组18例,非糖尿病组18例。对2组临床和生化数据进行统计分析。采用免疫组化和TUNEL法检测伤口皮肤细胞凋亡情况。体外培养人原代皮肤成纤维细胞,分别给予正常浓度糖、持续高糖、波动高糖和AGEs干预72 h,采用蛋白质印迹法和流式细胞术测定细胞凋亡情况。结果：与非糖尿病组相比,糖尿病组血糖水平明显增高,伤口持续时间长（P<0.05）。Cleaved caspase-3在非糖尿病组和糖尿病组的免疫反应评分分别为1.04±0.23和3.04±0.31（P<0.05）;TUNEL检测非糖尿病组和糖尿病组的细胞凋亡指数分别为（3.8±0.8）%和（8.4±1.5）%（P<0.05）。人原代皮肤成纤维细胞在正常浓度糖、持续高糖、高糖波动、AGEs干预下,cleaved caspase-3蛋白表达水平分别为0.80±0.13、1.22±0.18、1.46±0.32和1.83±025,凋亡率分别为（2.43±0.19）%、（2.89±0.51）%、（3.99±0.24）%和（6.83±0.36）%。 AGEs组cleaved caspase-3蛋白表达水平和凋亡率均明显高于正常浓度糖组和持续高糖组（P<0.05）。结论：糖尿病皮肤创面细胞凋亡增加,可能是糖尿病皮肤伤口难愈的重要原因之一。与持续高糖、高糖波动组相比,AGEs促进人皮肤成纤维细胞凋亡的作用更强。     

The activation of caspase-3 and DNA fragmentation in B cells phagocytosed by macrophages

Apoptotic signaling of mammalian cells involves two pathways: the death receptor and mitochondrial pathways. In this in vivo study, we investigated apoptotic signaling of B cells in mouse germinal centers (GCs) of gut-associated lymphoid tissues (GALTs) using transmission electron microscopy (TEM), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), immunofluorescence of members of caspase family and cFLIP(L), and caspase activity assay. It was very difficult to ultrastructurally differentiate B cells undergoing apoptosis from B cells differentiating into memory cells or plasma cells among B cells constituting GCs. Isolated B cells in GCs showed no active form of caspase-3 or TUNEL immunoreactivity, but expressed cFLIP(L). Contrary to isolated B cells, apoptotic B cells phagocytosed by macrophages exhibited immunoreactivity of the active form of caspase-3 and TUNEL, but lacked the cFLIP(L) expression. The caspase activity assay in GALTs clearly showed intense activity of caspase-3, caspase-9, and caspace-8 that was high in order. Therefore, the death receptor pathway accompanying the increased activity of caspase-3 and caspase-8 may be blocked by the expression of cFLIP(L) in B cells of GALTs. Moreover, both the activation of caspase-3 and DNA fragmentation first occur only when B cells are phagocytosed by macrophages.     

Caspase-3, TUNEL and ultrastructural studies of small follicles in adult human ovarian biopsies

BACKGROUND: The aim of this study was to investigate evidence for cell death by apoptosis in small unilaminar ovarian follicles of adult humans. METHODS: Cortical biopsies from 13 healthy donors were either frozen and protein extracted for western blots or fixed for immunohistochemistry (IH) to localize procaspase-3 and active-caspase-3, to detect DNA fragmentation in situ and undertake routine transmission electron microscopy (TEM). RESULTS: Blots identified the presence of the inactive pro-form of caspase-3, and IH localized this in all follicles studied. In contrast, the active form of caspase-3, a major effector of apoptosis, was only detected in large antral follicles that also had microscopic signs of atresia. Active caspase-3 was not detected in primordial (n = 87), primary (n = 8) or secondary follicles. The atretic follicles were also the only ovarian structures with positive evidence of DNA fragmentation after terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) treatment. Confocal microscopy showed dual labelling for both active caspase-3 and TUNEL in individual granulosa cells in large atretic follicles, but no such labelling was evident in any other follicles. No apoptotic bodies were seen by TEM in sections of 39 small follicles from seven patients. CONCLUSION: This study found evidence for TUNEL and active caspase-3 only in human ovarian antral follicles.     

Subclinical concentration of sevoflurane potentiates neuronal apoptosis in the developing C57BL/6 mouse brain

The effect of general anesthetics on the developing brain is receiving growing attention. Nonetheless, there remains a paucity of evidence regarding the effect of sevoflurane, a widely used anesthetic in pediatric anesthesia. This study was designed to investigate the effect of sevoflurane on nerve cell apoptosis in the developing brain. Techniques to detect cell apoptosis included immunohistochemistry of cleaved caspase-3 and single-strand DNA, as well as electron microscopy. Elevated cleaved caspase-3 was also validated semi-quantitatively by immunoblotting assay. Mouse pups (day 7 postnatal) were subjected to sevoflurane inhalation. Twelve hours later, dramatically increased cleaved caspase-3 and single-strand DNA immunoreactivity were detected in the pup brains. Immunoblotting assay of cleaved caspase-3 revealed a significant increase after anesthetic exposure. Electron microscopy disclosed typical apoptotic morphology of the degenerative nerve cells. Blood glucose levels in the anesthetized group were not different from those of the control group, indicating that the neuronal apoptosis observed in the anesthetized group was not the result of hypoglycemia. Our results indicate that subanesthetic concentration of sevoflurane can trigger neuronal apoptosis in the postnatal mouse brain.     

人参皂苷Rh4诱导肝癌细胞HepG2的凋亡及分子机制

目的:探讨人参皂苷Rh4对人肝癌HepG2细胞凋亡的作用及机制。方法:采用MTT比色法测定不同浓度(10、20和40μmol/L)人参皂苷Rh4对人肝癌HepG2细胞活力的抑制作用;用流式细胞术检测定细胞凋亡率;通过Hoechst 33258和TUNEL染色观察人参皂苷Rh4诱导人肝癌HepG2细胞凋亡的形态学变化;Western blot法检测凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9的表达情况。结果:人参皂苷Rh4能够明显促进人肝癌HepG2细胞的凋亡,且呈剂量依赖性;TUNEL和Hoechst 33258染色实验结果表明,人参皂苷Rh4作用24 h后,细胞呈现明显皱缩、肿胀、破裂等凋亡形态;Western blot分析结果表明,随着人参皂苷Rh4给药浓度的增加,抗凋亡蛋白Bcl-2表达量逐渐下降,而促凋亡蛋白Bax、cleaved caspase-3和caspase-9的表达逐渐升高。结论:人参皂苷Rh4可诱导人肝癌HepG2细胞凋亡,其作用机制可能与下调Bcl-2以及上调Bax、cleaved caspase-3和caspase-9蛋白表达有关。