Transferase-mediated dUTP-digoxigenin Nick-end Labeling Assay Research Articles

Protective effect of anisodamine on bleomycin-induced acute lung injury in immature rats via modulating oxidative stress, inflammation, and cell apoptosis by inhibiting the JAK2/STAT3 pathway.

BackgroundPediatric acute lung injury (ALI) is one of the most common causes of infant mortality. Although lung-protective strategies have developed in recent years, no ALI treatment is currently available. Anisodamine (Ani) is a common drug used to treat gastrointestinal smooth muscle spasm. The protective effects of Ani against acute kidney injury and myocardial injury have been reported. However, the efficacy of Ani on bleomycin (BLM)-induced ALI has not been examined previously. In the present study, we aimed to examine the effects of Ani on bleomycin (BLM)-induced ALI on immature rats.MethodsThe ALI rat model was established by intratracheally administration of BLM. Ani treatment was performed by an intravenous injection at different concentrations. The lung function of each rat was measured, and then lung tissue structures, apoptosis, and collagen deposition were observed by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and Masson’s staining, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of inflammatory cytokines. The expression of apoptosis-related proteins and fibrosis-related markers was determined by reverse transcription-polymerase chain reaction and/or Western blot analysis. Finally, the expression levels of Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were determined.ResultsOur findings indicated that lung function was remarkably decreased in BLM-induced rats, which could be reversed by Ani. Ani treatment increased the levels of antioxidant enzymes, decreased the apoptotic rate and apoptosis-related proteins, and downregulated the expression of fibrosis-related markers. Additionally, Ani treatment also attenuated inflammatory response and suppressed the activation of the JAK2/STAT3 pathway.ConclusionsOur results demonstrated that Ani had potent activity against BLM-induced ALI in immature rats through inhibiting the JAK2/STAT3 signaling pathway. Our findings provide supporting evidence to further investigate the therapeutic effect of Ani against ALI in children.

Apoptosis of cardiomyocytes in diabetic cardiomyopathy involves overexpression of glycogen synthase kinase-3β.

To evaluate the role of glycogen synthase kinase-3β (GSK-3β) in the apoptosis of cardiomyocytes in diabetic cardiomyopathy (DCM). Diabetes mellitus (DM) in rats was induced by intraperitoneal injection of 1% streptozotocin (STZ), and lithium chloride (LiCl) was used to decrease the expression of GSK-3β. Hematoxylin/eosin (HE) staining and the terminal deoxyribonucleotide transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay was conducted to evaluate the pathological injury and apoptosis of cardiomyocytes respectively. Western blot was applied to detect the protein expressions of Cleaved-caspase 3, caspase 3, Bax and Bcl-2 in rat cardiomyocytes. Real-time polymerase chain reaction (RT-PCR) was applied to detect the gene expressions of phosphoinositide 3-kinases (PI3K), Akt, and GSK-3β in rat cardiomyocytes. DM-induced cardiomyocyte injuries, which were presented as capillary basement membrane thickening, interstitial fibrosis, cardiomyocyte hypertrophy and necrosis in HE staining and increased apoptosis detected by TUNEL assay. When comparing with the control group, the mRNA expression of PI3K and Akt in DM group obviously decreased but the mRNA expression of GSK-3β obviously elevated (P < 0.05). In addition, the ratio of Cleaved-caspase 3/caspase 3 and Bax/Bcl-2 were notably increased in DM group compared with control group (P < 0.05). LiCl, as an inhibitor of GSK-3 apparently reduced the expression of GSK-3β mRNA (P < 0.05) but not the PI3K and Akt comparing with the DM group. LiCl also attenuated the myocardial injury and apoptosis induced by DM. The myocardial injury induced by DM is associated with the up-regulation of GSK-3β. LiCl inhibited the expression of GSK-3β and myocardial apoptosis in diabetic myocardium.

In Vivo Study of the Effects of ERβ on Apoptosis and Proliferation of Hormone-Independent Prostate Cancer Cell Lines PC-3M.

Objective To evaluate the in vivo therapeutic effects of attenuated Salmonella carrying PCDNA3.1-ERβ plasmid in hormone-independent prostatic cancer in nude mice and to clarify the mechanism by which estrogen receptor β (ERβ) induces apoptosis and proliferation in prostatic cancer cells in mice. Methods The orthotopic prostatic cancer models of mice were randomly divided as follows: MOCK group, treated with PBS, PQ group, treated with attenuated Salmonella alone, PQ-PCDNA3.1 group, treated with attenuated Salmonella carrying PCDNA3.1 plasmid, and PQ-PCDNA3.1-ERβ group, treated with the attenuated Salmonella carrying PCDNA3.1-ERβ plasmid. Then, 10 μl of the plasmid-containing solution, comprising 1 × 107 cfu of the bacteria, was administered via intranasal delivery to each group except the MOCK group. The experimental methods included flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay, immunohistochemistry, and western blotting. Results Compared with the MOCK, PQ, and PQ-PCDNA3.1 groups, the weights of tumors in the PQ-PCDNA3.1-ERβ group were significantly reduced. The results of flow cytometry and TUNEL assay revealed that the number of apoptotic cells in the PQ-PCDNA3.1-ERβ group significantly increased. Compared with PQ-PCDNA3.1 group, the protein expression levels of ERβ, Bad, p-caspase 9, p-caspase 3, and cleaved PARP in the PQ-PCDNA3.1-ERβ group were significantly increased, while the expression levels of Akt, p-Akt, and Bcl-xl were decreased (P < 0.05). Conclusion The attenuated Salmonella carrying PCDNA3.1-ERβ plasmid could inhibit the growth of orthotopic prostatic cancer in mice by increasing the expression of ERβ.

Wound Healing, Inflammation, and Corneal Ultrastructure After SMILE and Femtosecond Laser–Assisted LASIK: A Human Ex Vivo Study

To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].

Ginsenoside Rg3 inhibits angiogenesis in a rat model of endometriosis through the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway.

ObjectiveThis study aimed to investigate the link between the inhibitory effect of ginsenoside Rg3 on the ectopic endometrium growth and the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway, a mechanism known to inhibit angiogenesis and induce ectopic endometrial cell apoptosis.Materials and methodsA model of endometriosis was established by allotransplantation in rats. The rats were randomly divided into 5 groups: the ginsenoside Rg3 low-dose group (group A,5mg/kgBW/d of ginsenoside Rg3), the ginsenoside Rg3 high-dose group (group B, 10mg/kgBW/d of ginsenoside Rg3), the gestrinone group (group C, 0.5mg/kgBW/d of gestrinone), the control group (groupD, 10ml/kg BW/d of 0.5%CMC-Na) and the ovariectomized group (group E, 10ml/kgBW/d of 0.5%CMC-Na). Rats were executed after 21 days of continuous administration. The ectopic endometrium volume was measured and the inhibitory rate was calculated. The levels of serum estradiol (E2) and progesterone (P) were detected by Electro-Chemiluminescence Immunoassay (ECLI). The protein expressionof VEGF, VEGFR-2, p-Akt, and p-mTOR inthe ectopic endometrium wastested by immunohistochemistry(IHC) and Western Blotting. The mRNA expression levels of VEGF, VEGFR-2, Akt, and mTOR were tested by Real-Time Polymerase Chain Reaction (PCR). The apoptosis rate of the ectopic endometrial cells was detected by Terminal Deoxynucleotidyl Transferase-mediated Digoxigenin-dUTP Nick-End Labeling Assay(TUNEL).Main resultsTissue measurements revealed a dose-dependent inhibition effect of ginsenoside Rg3 on the growth of the ectopic endometrium in treated rats compared to controls. Immunohistochemical and Western Blotting assays confirmed that the expression of VEGF, p-Akt, and p-mTOR was down-regulated in ginsenoside Rg3 -treated lesions. Real-time PCR results also showed that the mRNA expression levels of VEGF, Akt, and mTOR in the ectopic endometrium were reduced.ConclusionsThe present study demonstrates, for the first time, that ginsenoside Rg3 suppresses angiogenesis in developing endometrial lesions. The ginsenoside Rg3 inhibitory effect on the growth of the ectopic endometrium in EMs rats might occur through the blocking of the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway, thus halting angiogenesis and promoting the apoptosis of ectopic endometrial cells.

Sclerotic Effect of Oxytetracycline on the Submandibular Gland: An Experimental Model

Oxytetracycline has been suggested as an alternate therapy for chronic recurrent sialadenitis and sialorrhea. We conducted an experimental study to investigate the sclerotic effect of this drug on the submandibular gland by histopathologic methods. Our subjects were 20 New Zealand white rabbits, which were divided into two groups of 10. The right submandibular gland of the rabbits in the active-treatment group was injected with 0.3 ml of oxytetracycline (100 mg/ml), and that of the controls was injected with saline. Four weeks after the injections, all the glands were removed. Histopathologic studies, including hematoxylin and eosin and Masson trichrome staining, were carried out. The glands were evaluated for tissue inflammation, congestion, fibrosis, edema, lipomatosis, and atrophy. To investigate apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) immunohistochemical staining was used. In the study group, inflammation (n = 9), congestion (n = 9), fibrosis (n = 6), edema (n = 6), and lipomatosis (n = 4) were observed; in the sham group, only lipomatosis was seen (n = 5). The TUNEL assay results for acinar cells were 4.51 ± 1.41% in the oxytetracycline group and 2.08 ± 1.76% in the control group (p = 0.006); the corresponding figures for the duct cells were 7.05 ± 0.87% and 3.10 ± 2.26% (p = 0.001). Based on our findings, we conclude that oxytetracycline might be a viable alternative for the treatment of chronic recurrent sialadenitis and sialorrhea. However, more research in this area is needed.

The effect of 7 days of letrozole pretreatment combined with misoprostol on the expression of progesterone receptor and apoptotic factors of placental and decidual tissues from first-trimester abortion: a randomized controlled trial

ObjectiveTo evaluate if letrozole-induced suppression of estradiol reduces progesterone receptor expression and apoptosis in the first-trimester placenta. Study DesignWe performed a double-blinded, randomized, placebo-controlled trial. We randomized 20 women requesting first-trimester abortion with gestation up to 63 days to receive either letrozole 10 mg daily or placebo pretreatment for 7 days before administrating 400 mcg of vaginal misoprostol followed by suction abortion. We collected the placental and decidual tissues on which we performed immunohistochemical staining for progesterone receptor and apoptotic markers (active caspase 3, caspase 3, Bcl2, CD95, fas ligand) and determined H-scores of each based on the intensities of staining. We performed terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay for apoptosis in the samples of four women to confirm the findings from apoptotic markers. ResultsWe excluded one woman in the letrozole group from the analysis because she had passage of abortus after taking letrozole, leaving 19 women (9 in the letrozole group, 10 in the placebo group) for analysis. There was no significant difference in the H-scorings of progesterone receptor and apoptotic markers, as well as proportion of apoptotic cells on TUNEL assay between the two groups. The H-scores for the progesterone receptor were 8.17±2.67 (mean±SD) in the letrozole group and 9.01±2.82 in the placebo group (p=0.36). ConclusionWe did not detect a difference in the expression of progesterone receptor and apoptotic markers in placental and decidual tissues after letrozole pretreatment for 7 days in first-trimester abortion. ImplicationsWe did not confirm the hypothesis that letrozole reduces progesterone receptor expression and induces apoptosis in the first-trimester placenta. Further studies are required to allow better understanding of the mechanism by which estrogen suppression following the use of letrozole can lead to improved abortion rate in the first trimester.

Effects of KH-204 on the expression of heat shock protein 70 and germ cell apoptosis in infertility rat models.

BackgroundIdiopathic infertility is a significant number of causes of male infertility. Empirical treatments are used for idiopathic male infertility, and antioxidant supplementation is a kind of management of oxidative stress related infertility. We investigated the antioxidant effects of the modified Ojayeonjonghwan (KH-204) in a rat model of cryptorchidism.MethodMale rats were divided into four groups (n = 8 in each): a normal control group, a cryptorchidism-induced control group and two cryptorchidism-induced groups treated p.o. with either 200 or 400 mg/kg, KH-204 for 4 weeks. The testes and epididymides from rats in all groups were removed, weighed and subjected to histological examination and semen analysis after surgery. Oxidative stress was assessed by measuring 8-hydroxy-20-deoxyguanosine (8-OHdG), superoxide dismutase (SOD) and heat shock protein (HSP) levels. Apoptosis was determined using a terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling assay.ResultsTreatment with the multi-herbal medicine KH-204 (1) increased the mean weight of the cryptorchid testes; (2) restored sperm counts, motility and germinal cell layer thickness; (3) decreased levels of 8-OHdG and increased levels of SOD; and (4) decreased HSP70 levels and apoptosis.ConclusionsKH-204 reduces the oxidative stress in an experimental rat model of cryptorchidism, and it may alleviate HSP expression and germ cell apoptosis.

Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AX (γH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues.

Morphology of the developing muscularis externa in the mouse esophagus

Muscularis externa of mouse esophagus is composed of two skeletal muscle layers in the adult. But less attention is paid to the histogenesis of the muscularis externa of the esophagus, and controversies still exist about the developmental process and the spatio-temporal expression characteristics of muscle-specific proteins during the development of esophageal muscularis externa. To further probe into the developmental pattern of muscularis externa of the mouse esophagus and the expression characteristics of different muscle-specific proteins, immunohistochemical and terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick-end labeling apoptotic staining methods are used to investigate the expression patterns of different muscle-specific proteins and to elucidate the relationship of these protein expressions with the development of muscularis externa of the mouse esophagus. Thus, an understanding of the developing esophageal muscularis externa may be important for developing therapeutic strategies for the treatment of human esophagus diseases. Serial sections of mouse embryos from embryonic day (ED) 12 to ED18, and full-length esophagi from postnatal first to 5th day were stained with monoclonal antibodies against α-smooth muscle actin (α-SMA), α-sarcomerical actin (α-SCA), desmin, and monoclonal anti-skeletal myosin (MHC), while apoptosis was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The expression of α-SMA was started at ED12. During the development of ED14-ED15, α-SMA positive cells were seen extending from the walls of left three, four, and six arch arteries toward the dorsal wall of esophagus. Stronger expression of α-SCA and desmin could be detected at ED14 and ED15, expression intensity in caudal segment and inner layer was stained stronger than that of cranial segment and outer layer, but after ED16, strong expression of α-SCA and desmin was found in the outer layer of muscularis externa. Expression of MHC was first detected in the outer layer of cranial segment of muscularis externa at ED17. At ED18, MHC had extended to the level of thyroid gland, staining intensity in the outer layer and cranial segment was stronger than that of inner layer and caudal segment. One to five days after birth, the thickness of the esophageal muscle layer was obviously increased. Most of the muscle cells in the cranial segment of esophagus showed strong expression of α-SCA and clear cross striations at higher magnification. With progression toward the caudal segment, expression intensity of α-SCA became weaker, but the expression intensity of desmin was the same at different levels of esophagus. The muscle fibers were arranged densely with high expression of MHC in the cranial segment. During the development of esophageal muscularis externa, few apoptotic cells were observed. α-SMA, α-SCA, desmin, and MHC show different expression patterns. The differentiation of outer layer of esophageal muscularis externa is quicker than that of inner layer, and the caudal segment is quicker than that of the cranial segment. Besides, apoptosis may not participate in the development of esophageal muscularis externa. The smooth muscle cells from arch arteries may participate in the development of esophageal muscularis externa.

Hypoxia confers protection against apoptosis via the PI3K/Akt pathway in endothelial progenitor cells

The recruitment of endothelial progenitor cells (EPC) to ischemia has recently been suggested as an important mechanism of tissue repair. Although tissue ischemia can facilitate EPC mobilization, recruitment, and retention at the hypoxic site, the effects of hypoxia on EPC survival are not well known. In the present study, we examined whether hypoxia (2% O2) would suppress apoptosis induced by serum withdrawal and whether survival signals, such as the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinase (ERK) pathways, were involved in this process. After being serum-starved for 24 h, EPC were cultured under normoxic or hypoxic conditions (2% O2) for 24 h. Cell survival was assessed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, annexin V-propidium iodide dual-color flow cytometry, and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay. The expressions of signaling proteins were evaluated by Western blot analysis. Under hypoxic conditions, EPC were resistant to apoptosis induced by serum starvation. The inhibition of the PI3K/Akt pathway using the LY294002 inhibitor prevented hypoxia-inhibited apoptosis in EPC and altered the phosphorylation state of glycogen synthase kinase-3beta, an effector protein involved in regulation of EPC apoptosis. However, ERK inhibitor PD98059 had no significant effect on cell survival. Our data demonstrated that hypoxia inhibited serum withdrawal-induced apoptosis in EPC, which might be associated with the activation of the PI3K/Akt pathway.

Synergistic antitumoral activity and induction of apoptosis by novel pan Bcl-2 proteins inhibitor apogossypolone with adriamycin in human hepatocellular carcinoma

To investigate the in vitro and in vivo activities and related mechanism of apogossypolone (ApoG2) alone or in combination with adriamycin (ADM) against human hepatocellular carcinoma (HCC). The IC50 of ApoG2 in vitro was tested by WST assay, and the synergistic effect was analyzed using the CalcuSyn method. Cell apoptosis was determined using 4',6-diamidino-2- phenylindole staining and flow cytometric analysis. Western blotting was used to determine the expression of apoptosis-related proteins. In vivo activity was evaluated in the xenograft model in nude mice, and apoptosis in tumor tissues was determined by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. The IC50 of ApoG2 in HCC cells was 17.28-30.63 micromol/L. When ApoG2 was combined with ADM, increased cytotoxicity and apoptosis were observed in SMMC-7721 cells compared to treatment with ApoG2 alone. The Western blotting results indicated that the ApoG2 induced apoptosis in SMMC-7721 cells by downregulating anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL, up-regulating pro-apoptotic protein Noxa, and promoting the activities of caspases-9 and -3. The tumor growth of xenograft SMMC-7721 was inhibited in nude mice when ApoG2 was administered orally without causing damage to the normal tissues. The in vivo study also indicated an increasing anti-tumoral effect when ApoG2 at 100 or 200 mg/kg dosages were used together with ADM at 5.5 mg/kg, with relative tumor proliferation rate (T/C) values of 0.456 and 0.323, respectively. Apoptosis induced in vivo by ApoG2 alone or combined with ADM was confirmed by TUNEL assay in tumor tissues. ApoG2 is a potential non-toxic target agent that induces apoptosis by upregulating Noxa, while inhibiting anti-apoptotic proteins and promoting the effect of chemotherapy agent ADM in HCC.

Down-regulation of Sonic hedgehog signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells

The Sonic hedgehog (SHh) pathway plays a critical role in normal embryogenesis and carcinogenesis, but its function in cancer cells treated with 5-fluorouracil (5-FU) remains unknown. We examined the expression of a subset of SHh signaling pathway genes, including SHh, SMO, PTC1, Su(Fu) and HIP in human hepatocellular carcinoma (HCC) cell lines, Hep3B and HepG2, treated with 5-FU by reverse transcription-polymerase chain reaction. Using trypan blue analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, we also detected the apoptosis of Hep3B cells resulting from the transfection of pCS2-Gli1 expression vector combined with 5-FU treatment. The motility of the cells was detected by scratch wound closure assay. The expression and subcellular location of PTC1 protein in Hep3B cells treated by 5-FU were also investigated by Western blot analysis and immunofluorescent microscopy. The results indicated that the expression of SHh pathway target molecules at both messenger RNA and protein levels are evidently down-regulated in Hep3B cells treated with 5-FU. The overexpression of Gli1 restores cell viability and, to some extent, the migration abilities inhibited by 5-FU. Furthermore, 5-FU treatment affects the subcellular localization of PTC1 protein, a key member in SHh signaling pathway. Our data showed that the down-regulation of SHh signaling pathway activity was involved in 5-FU-induced apoptosis and the inhibition of motility in hedgehog-activated HCC cell lines. This implies that the combination of SHh signaling pathway inhibitor and 5-FU-based chemotherapy might represent a more promising strategy against HCC.

Cytotoxic effect of CdSe quantum dots on mouse embryonic development

The aim of this study was to examine the cytotoxic effect of quantum dots (QD), a novel luminescent material, on early post-implantation embryonic development. Mouse blastocysts were incubated in medium with or without CdSe-core QD (250 or 500 nmol/L) for 24 h. Cell apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay and Annexin V/propidium iodide staining, and proliferation was investigated by dual differential staining. Pre-implantation and post-implantation development was assessed by in vitro and in vivo analyses, respectively. The apoptotic staining analysis showed that CdSe-core QD induced apoptosis in mouse blastocysts in a dose-dependent manner. Pretreatment of blastocysts with CdSe-core QD inhibited cell proliferation, primarily in the inner cell mass. CdSe-core QD also inhibited post-implantation embryonic development; fewer CdSe-core QD-pretreated blastocysts reached the later stages of development compared to the controls. The pre-implantation development of morulas into blastocysts was also inhibited by CdSe-core QD. Furthermore, CdSe-core QD at 500 nmol/L were associated with resorption of post-implantation blastocysts and a decrease in fetal weight. The cytotoxicity of CdSe QD in embryonic development was significantly reduced by the addition of a ZnS coating. Our results show that CdSe-core QD induce apoptosis in mouse blastocysts, inhibit cell proliferation, retard early post-implantation blastocyst development, and increase early-stage blastocyst death in vitro and in vivo.

Silencing livin gene by siRNA leads to apoptosis induction, cell cycle arrest, and proliferation inhibition in malignant melanoma LiBr cells

The aim of the present study was to investigate the effects of silencing the livin gene by small interfering RNA (siRNA) on the expression of livin and the effects on apoptosis, cell cycle, and proliferation in human malignant melanoma LiBr cells. Three chemically-synthetic siRNA duplexes targeting livin were transiently transfected into the LiBr cells, and the effects on livin expression were detected both at the mRNA level by real-time RT-PCR and at the protein level by Western blotting. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, flow cytometric analysis, and the expression of procaspase-3 and activated caspase-3 analysis by Western blotting. Cell cycle was analyzed by flow cytometry. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. One of the 3 designed siRNA could effectively knock down the livin expression both at the mRNA and protein levels in dose- and time-dependent manners; 100 nmol/L with maximum downregulation on mRNA at 48 h, and on the protein at 72 h after transfection. Silencing livin could significantly induce apoptosis, arrest cell cycle at the G0/G1 phase, and inhibit proliferation in LiBr cells. Meanwhile, caspase-3 was activated. The livin gene could serve as a potential molecular target for gene therapy by siRNA for malignant melanoma.

Cell proliferation and apoptosis in stromal corneal dystrophies.

The aim of our study was to evaluate corneal cell proliferation and apoptosis in cases of granular, macular and lattice dystrophy, and to provide evidence which may help to clarify whether apoptosis is a pathogenic factor in any of these dystrophies. The study group comprised 39 eyes (from 33 patients) which had undergone penetrating keratoplasty (PK) for stromal dystrophies: these comprised 12 eyes (from 9 patients, 55.5% males) with granular dystrophy, 13 eyes (12 patients, 33.3% males) with macular dystrophy, and 14 eyes (13 patients, 61.5% males) with lattice type I dystrophy. A further 4 corneal buttons from enucleated eyes of 4 patients with choroideal melanoma served as controls. Immunocytochemical analysis of Ki67 (DNAcon Kit, DakoCytomation A/S, Glostrup, Denmark) was used for evaluation of cell proliferation. Apoptosis was detected by use of the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling) assay method (Apoptag reagent, Q-Biogene, Strasbourg, France). Statistical comparisons were made using the Mann-Whitney test. No Ki67-positive cells were detected in the study-group or control corneas. In control corneas no apoptotic activity was found. In the study group the mean (normalised) apoptotic keratocyte number was 1.1+/-1.7 in granular dystrophy and 0.5+/-1.1 in lattice type I dystrophy (p = 0.36, 0.63 respectively). Compared to the controls, the difference was statistically significant only for macular dystrophy (1.6+/-1.2; p = 0.01). Keratocyte apoptosis seems to be a concomitant or pathogenic factor in macular dystrophy. However, the pathways that are triggered to result in increased apoptotic cell death remain to be clarified.

Hepatocyte morphology and kinetics after portal vein embolization

Macroscopic volume changes after portal vein embolization (PVE) can be assessed accurately by computed tomography, but histological changes remain poorly understood. The aim of this study was to clarify hepatocyte morphology and kinetics after PVE. The resected livers from 25 patients who underwent extended hepatectomy after PVE and five normal livers were examined using hepatocyte paraffin 1 staining for histomorphometric analysis of hepatocytes. Cell kinetics were determined by Ki-67 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay. Kupffer cells were examined by CD68 immunostaining. The number of hepatocytes was similar in the embolized lobe, non-embolized lobe and normal liver, but hepatocyte volume was greater in the non-embolized lobe than in the embolized lobe (P = 0.017). The Ki-67 labelling index was higher in the non-embolized lobe (P < 0.001) whereas the apoptotic index was higher in the embolized lobe (P < 0.001). There were more Kupffer cells per unit area in the embolized lobe (P < 0.001). Hepatocyte hypertrophy and replication leads to volume enlargement of the non-embolized hepatic lobe, whereas hepatocyte atrophy and apoptosis causes a decrease in volume of the embolized lobe.

A Short Duration Transient Ischemia Induces Apoptosis in Retinal Layers: An Experimental Study in Rabbits

To investigate retinal cell apoptosis in an experimental transient, short duration ocular ischemia model. An experimental ischemia model, which simulates creating temporary high intraocular pressure to control intraocular bleeding during pars plana vitrectomy, was set up. Rabbits were randomly divided into three groups. Group 1 was the control group. In Group 2, intraocular pressure was increased to 97 mmHg for 5 minutes. In Group 3, intraocular pressure was increased to 97 mmHg for 10 minutes. After 24 hours, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay was used to detect retinal apoptosis in rabbit eyes. Only nuclear staining in retinal cells was counted. Groups with 5 minutes and 10 minutes of ischemia showed significantly higher amount of ganglion cell layer apoptosis when compared with the control group (p<0.05). Light microscopy and standard hematoxylin-eosin did not show any significant damage in the retina cells. Apoptotic cell death in the retinal cell layers occurs in temporary ischemia-reperfusion as early as 5 and 10 minutes duration.

Epithelial Cell, Keratocyte, and Endothelial Cell Apoptosis in Fuchs' Dystrophy and in Pseudophakic Bullous Keratopathy

To elucidate the pathomechanism of Fuchs' dystrophy and pseudophakic bullous keratopathy (PBK) by examining cell apoptosis in different corneal layers. The authors studied corneal buttons obtained from 21 eyes following central penetrating keratoplasty: 14 corneal buttons (13 patients, age 70.8+/-10.0 years) with Fuchs' dystrophy, and 7 buttons (7 patients, age 69.6+/-10.2 years) with PBK. Four buttons from enucleated eyes with choroidal melanoma served as controls. Histologic changes were examined using light microscopy with hematoxylin-eosin (HE) staining. The average numbers of apoptotic cells per field of view (125x magnification) in separate samples of the epithelial, stromal, and endothelial layers were determined using the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) assay. In 11 of the Fuchs' dystrophy corneas and 2 of the PBK corneas, apoptotic activity was detected. In the control corneas no apoptotic activity was found. Compared to the controls there was a statistically significant difference in the mean (normalized) apoptotic cell numbers for all three layers (p=0.01 in each case) in the Fuchs' dystrophy corneas, and for the stromal layer (p<0.01) in PBK corneas. The apoptotic cell numbers for the epithelial and endothelial layers of the latter were higher, but the difference was not statistically significant (p=0.07, 0.07). Apoptosis may play a role in the pathomechanism of Fuchs' dystrophy and in keratocyte death in corneas with PBK.

Olfactory Epithelial Lesions Induced by Various Cancer Chemotherapeutic Agents in Mice

In order to examine and compare the potential toxicity in the olfactory epithelium, the antitumor drug vincristine sulfate (VCR), vinblastine sulfate(VBL), vindesine sulfate (VDS), paclitaxel (PTX), mitomycin C (MMC), 5-fluorouracil, (5-FU) or cisplatin (CDDP) was intravenously injected once(designated as day 1) at an estimated 10% lethal dose (LD(10)) to male BALB/c mice. The animals were necropsied on days 2, 5 and 15, and nasal tissues were examined by light-microscopy, counting of epithelial cells positive for terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL), immunohistochemical staining with keratin antibody, and electron microscopy. Further, to delineate the drug disposition in the target organ, whole-body radioluminography was performed 1 hour and 24 hours after treatment with the LD(10) of PTX or 5-FU. Of the antitumor drugs employed, only the antimicrotubule agents, VCR, VBL, VDS, and PTX, induced single cell death in the olfactory epithelium, especially sensory cells on day 2, atrophy of the olfactory epithelium on day 5, and myelin fragmentation in the trigeminal nerve on day 15. PTX induced the strongest changes among the 4 antimicrotubule agents. The cell death was confirmed to be apoptosis by TUNEL assay and electron microscopy, whereas the change in horizontal basal cells of the olfactory epithelium was shown not to be apoptosis by keratin staining. In quantitative radioluminography,radioactivity of PTX in the nasal tissues both 1 hour and 24 hours after administration was about 4- or 5-fold higher than those of 5-FU. These results suggest that tubulin-targeting antitumour drugs could induce apoptosis in the olfactory epithelial cells of mice and that high drug distribution may effect the onset of the olfactory lesions.