Transferase-mediated dUTP Nick-end Labeling Technique Research Articles

Apoptotic investigation of brain tissue cells in dogs naturally infected by canine distemper virus

BackgroundCanine distemper caused by canine distemper virus that belongs to the Morbillivirus genus of the Paramyxoviridae family is still a global epidemic significant infectious disease, especially in pet dogs in China and serious harm to the development of the dog industry. It has been known that apoptosis caused by the canine distemper virus can show in culture cells, lymphoid tissues, and the cerebellum. However, its occurrence in brain tissue cells remains unclear. To investigate the relationship among canine distemper infecting brain tissues, apoptosis in brain tissue cells, and demyelinating pathogenesis was investigated.Methods16 naturally infected dogs that exhibited clinical signs of CD and tested positive for the anti-CDV monoclonal antibody and six healthy dogs that served as the control, were used in the research. Brain specimens were divided into the cerebrum, brain stem, and cerebellum embedded in paraffin and made the sections respectively. Approximately 5 µm-thick sections were stained by hematoxylin–eosin, methyl green pyronin, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique, and immunohistochemistry. CDV nucleocapsid protein was detected by immune streptavidin-biotinylated peroxidase complex.ResultsAlterations in the brain tissues of CDV-infected dogs involved both various cells and nerve fibers. CDV had varying degrees of cytotropism to all brain tissue cells; apoptosis also occurred in all brain cells, especially in the endothelia of cerebral vessels, astrocytes, oligodendrocytes, and ependymal cells, the more serious infection, the more obvious apoptosis. Serious infections also involved the pyramidal and Purkinje cells. The nervous fibers exhibited demyelinating lesions (showed small multifocal vacuole), and some axonal neuron atrophy gradually disappeared (formed large vacuole).ConclusionsApoptosis in brain tissue cells was mainly related to the propagation path and cytotropism of CDV. The apoptosis of astrocytes, oligodendrocytes, and some neurons may play a significant role in the demyelinating pathogenesis in dogs with acute canine distemper. A lot of diverse nervous signs shown in the clinic may be related to different neuron apoptosis.

Boron Supplementation Promotes Osteogenesis of Tibia by Regulating the Bone Morphogenetic Protein-2 Expression in African Ostrich Chicks.

The present study aimed to explore the effects of supplemental boron on osteogenesis of tibia and to investigate the possible relationship between additional boron and the expression of bone morphogenetic protein-2 (BMP-2) in tibia of ostrich chicks. Therefore, forty-eight African ostrich chicks (15days old) were supplemented with 0mg/L, 40mg/L, 80mg/L, 160mg/L, 320mg/L, and 640mg/L of boron in drinking water for 75days. The paraffin sections of tibia used to measure histomorphometric parameters by hematoxylin and eosin (HE) staining, Masson's staining, and immunohistochemistry (IHC). Enzyme-linked immunosorbent assay was performed to assess the level of BMP-2, osteocalcin (BGP), glucocorticoids (GCs), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) in serum. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) technique was performed to detect the cell apoptosis. The results indicated that low dose of supplemental boron (40mg/L-160mg/L) in drinking water promotes bone development by increasing the mature ossein. The expression of BMP2 on 45days was higher than 90days. Serum level of BMP-2, BGP, and GCs changed significantly in groups with low dosage of boron, and OPG/RANKL ratio was upregulated from 0 to 160mg/L. Cell apoptosis was least in 40mg/L and 160mg/L groups. Taken together, low dose of boron supplemented in drinking water could promote osteogenesis and growth and development of tibia by regulating the expression and secretion of BMP-2 and providing a dynamically balanced environment for tibia growth, development, and reconstruction by regulating the concentrations of BGP, GCs, and OPG/RANKL ratio in serum.

Increased Th1 immune response in SERPINB3 transgenic mice during acute liver failure

Acute liver failure (ALF) is characterized by severe neurological complications, known as acute hepatic encephalopathy, where brain ammonia and inflammatory processes play a dominant role. In experimental models of acute liver failure SERPINB3 was found significantly increased in microglia, the intrinsic immune cells of the central nervous system. The aim of the present study was to investigate the extent of brain tissue damage and the inflammatory milieu in experimental acute liver failure using a SERPINB3-transgenic mouse model. C57BL/6J wild-type and transgenic mice were inoculated with acetaminophen or phosphate-buffered saline and sacrificed 20 h postinjection. Proliferation and apoptotic activity were analyzed in brain tissue by immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique. The expression of cytokines was analysed in brain and liver tissue by real time polymerase chain reaction and in the corresponding serum samples using a Bio-Plex system. Acetaminophen induced a significantly lower body temperature and shorter survival in transgenic than in wild-type mice, despite liver function was similar in both groups. The brain of transgenic mice, expressing SERPINB3 positivity in microglia, showed increased glial cell number, associated to significant lower apoptotic death events, compared with wild-type mice. In mice injected with acetaminophen, remarkably higher values of cytokines mRNA were observed in the liver of both groups, with a trend toward higher values in transgenic animals. In brain tissue similar increase of tumor necrosis factor-α was detected in transgenic and wild-type mice, while IL-10 mRNA increased only in the wild-type group. A remarkable increase of circulating Th1 cytokines was detected in serum of transgenic mice, while in the wild-type group they remained rather unchanged. These figures were associated with lower levels of granulocyte macropage colony-stimulating factor, despite similar increase of IL-10 values in both groups. In conclusion, in acute liver failure SERPINB3 determines an enhanced inflammatory background, mainly mediated by higher levels of Th1 proinflammatory cytokines.

Development of a novel synthetic oligopeptide for the detection of DNA damage in human spermatozoa

The integrity of DNA in spermatozoa is considered an additional parameter of semen quality and a potential fertility predictor. Significant progress has been made in recent years towards the development of reliable tests for sperm chromatin integrity and DNA damage assessment. However, most of the techniques available are labor intensive, require expensive instrumentation or utilize enzymes whose activity could be compromised by the highly condensed nature of sperm chromatin. In addition, all the methods currently available involve the destruction of the sperm tested; none is able to select intact spermatozoa that could then be used for fertilization. The aim of the present study was to create a peptide ligand-based stain, capable of binding specific DNA structures, thereby revealing the presence of DNA damage, preferably in living cells. The peptide was bioinformatically modelled on the critical region of the p53 protein associated with DNA binding and fluorescently labeled with a terminal rhodamine B dye. The ability of this 21 amino acid synthetic peptide (DW1) to detect DNA damage in intact and fixed human spermatozoa was assessed in detail. Human sperm samples (n=20) were treated with reagents that induce single- and/or double-stranded DNA breaks. The effect of these treatments on peptide-labelling was measured and compared with results obtained using established tests for the evaluation of DNA damage, such as comet assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and sperm chromatin dispersion test. The peptide had a high affinity for single-stranded DNA, and DNA lesions such as double- and single-stranded breaks. The proportion of spermatozoa with intense staining was found to be closely associated with the percentage of cells possessing DNA damage. The analysis of 10 sperm samples using DW1 staining and TUNEL technique showed a significant correlation between the extent of DNA fragmentation for the two methods (r=0.892, Pearson's correlation, P<0.05). We have produced a novel peptide-based stain capable of detecting DNA damage in individual sperm cells. Evaluation of sperm DNA fragmentation using this peptide may be an inexpensive and easier to use alternative to the tests in current use. Additionally, although DW1 currently requires removal of the membrane using a detergent, further research may allow this approach to be applied to the selection of viable spermatozoa with intact DNA for use in ICSI and/or intra-cytoplasmic morphologically selected sperm injection.

Influence of hormonal and nonhormonal estrus synchronization methods on follicular and oocyte quality in primiparous lactating does at early postpartum period

High-yield lactating does need effective estrus synchronization methods to improve their reproductive outcome by enhancing ovarian function. The aim of the current work was to analyze ovarian follicular and oocyte characteristics of hormonal and nonhormonal estrus synchronization regimes in primiparous lactating rabbit does (Oryctolagus cuniculus) in the early postpartum period (Day 11). Females were randomly treated with either (1) a hormonal standard treatment with 25 IU equine chorionic gonadotropin (eCG) 48h before artificial insemination (eCG group) or (2) an alternative nonhormonal treatment consisting of doe-litter separation 24h before artificial insemination (Bio group). No significant differences were found in serum estradiol and progesterone concentrations between experimental groups. During the histologic study, the Bio group presented a higher number of primordial (P<0.05) and primary follicles (P=0.07) compared with that of the eCG group, whereas secondary and antral follicular populations were similar. Rates of late atretic follicles assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique were not different between treatments, but the eCG group showed a significantly higher number of mid-atretic follicles compared with that of the Bio group. Nuclear in vitro oocyte maturation (IVM), measured as metaphase II rate, and in vitro steroidogenic response of cumulus-oocyte complexes, measured by ELISA, did not show significant differences between treatments. However, confocal study showed that cytoplasmic maturation of oocytes, in terms of cortical granule migration rate, was significantly higher in the Bio group compared with that after the eCG treatment. In conclusion, transient doe-litter separation seems to improve ovarian response in terms of follicular health and oocyte competence compared with that after the eCG treatment. Therefore, a 24-h-long transient weaning could be an alternative nonhormonal method for synchronizing estrus in primiparous lactating rabbit does inseminated in the early postpartum period.

Protective effect of erythropoietin pretreatment in testicular ischemia-reperfusion injury in rats

This study was designed to investigate the effects of recombinant erythropoietin (EPO), a hormone widely used for treatment of uremic anemia, in rats subjected to testicular ischemia and reperfusion (I/R). Thirty-five male rats were divided into the following: control, sham operated, ischemia (I), I/R, and I/R + EPO groups. In the I group, 2 hours of left unilateral testicular torsion were performed, and in the I/R and I/R + EPO groups, an additional 2 hours of testicular detorsions were performed. The I/R + EPO group was pretreated intraperitoneally with EPO (500 IU/kg) before reperfusion. Testicular tissue samples were examined for biochemical and histopathologic parameters. Apoptotic cells in all testes were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling technique and caspase 3 immunohistochemistry. At histopathologic examination, ischemic changes in primary spermatocytes were noted in all torted testes. Cellular damage and apoptosis were more severe in ischemic groups than the EPO-pretreated group. There were statistically significant differences in tissue biochemical parameters in the I and I/R groups compared with the I/R + EPO group. The results of the present study suggest that EPO exerts protective effects against I/R injury via the modulation of free radical scavenger's activities, which decreases lipid peroxidation levels and attenuation of apoptosis.

Apoptotic myocytes generate monocyte chemoattractant protein-1 and mediate macrophage recruitment

The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-1 (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2-1 mumol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 mumol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-1 mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-1. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a trans-well system. The culture medium of STS-treated myocytes recruited monocytes in a MCP-1-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 min of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 +/- 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody showed that 69.8 +/- 3.9% of TUNEL-positive myocytes expressed MCP-1 protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-1, which have a critical role in the active recruitment of macrophages.

Simultaneous detection of a cell surface antigen and apoptosis by microwave-sensitized TUNEL assay on paraffin sections

The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique has been described as a sensitive method for detection of apoptotic nuclei in tissues and preferential staining of apoptotic strand breaks. Short-term microwave pre-treatment, a non-enzymatic pre-treatment technique of antigen retrieval, has been demonstrated to optimize the TUNEL method for in situ detection of apoptotic cells in formalin-fixed paraffin-embedded tissue sections. In the present study, we sensitized internal mammary artery sections by short-term microwave pre-treatment and used a two-step indirect enzymatic method to gain as an end product differentially stained cells, namely TUNEL-positive cells and these positive for the surface marker von Willebrand factor (vWF). This technique enables to clearly distinguish between apoptotic, non-apoptotic and vWF-positive cells that are phenotypic for endothelial cells. Phenotypic identification of cells is simplified by double staining with cell surface markers. This rapid, sensitive and reproducible technique allows simultaneous detection of DNA fragmentation and phenotypic markers in the same paraffin-embedded human tissue section.

Renal vascular endothelial growth factor in neonatal obstructive nephropathy. II. Exogenous VEGF

Chronic unilateral ureteral obstruction (UUO) in the neonatal rat causes delayed renal maturation, tubular apoptosis, and interstitial inflammation. Vascular endothelial growth factor (VEGF) acts as a survival factor for tubular cells and reduces renal injury in several models of renal disease. To determine whether exogenous VEGF attenuates renal injury from UUO, rats were subjected within the first 48 h of life to sham operation, partial UUO, or complete UUO. Saline vehicle or VEGF(121) (50 mg/kg) was injected twice daily for 7 days, after which kidneys were harvested for histological study. The density of peritubular capillaries was measured with platelet-endothelial cell adhesion molecule-1 immunostaining, proliferating nuclei were detected by proliferating-cell nuclear antigen staining, apoptosis by the transferase-mediated dUTP nick end-labeling technique, macrophages by ED-1 immunostaining, and collagen by Sirius red staining. Glomerular number and maturation index were also determined in each group. Following chronic complete UUO in the neonatal rat, peritubular capillary density was significantly decreased. Cortical capillary density was further reduced by exogenous VEGF in the partially obstructed kidney. While UUO also decreased glomerular number and delayed glomerular maturation, exogenous VEGF exerted no additional effects. Cellular proliferation and tubular apoptosis increased in proportion to the severity of obstruction, but exogenous VEGF had no additional effects on proliferation, tubular apoptosis, or macrophage infiltration. However, VEGF reduced interstitial apoptosis in the kidney with partial UUO. We conclude that VEGF does not have salutary effects on the renal lesions caused by chronic UUO in the neonatal rat and may actually worsen obstructive nephropathy by aggravating the interstitial lesions.

The Absence of Delayed Graft Function Is Predicted by the Presence of Manganese-Superoxide Dismutase in Distal Tubules of Renal Allografts

Acute tubular necrosis (ATN) in renal allograft biopsies correlates poorly with delayed graft function (DGF). Factors involved in the pathogenesis of DGF were evaluated in biopsies in an attempt to refine the recognition of DGF. Anti-cubulin and anti-AE-1/AE-3 antibodies identified proximal and distal tubules, respectively. The terminal deoxynucleotide transferase-mediated dUTP nick-end labeling technique and active caspase-3 staining were used to demonstrate apoptosis. Antibodies against superoxide dismutase (SOD) were used as markers of the protective tubular response. Tubular regeneration was evaluated using anti-ki 67 and antivimentin antibodies. Of a total of 40 biopsies, 9 were associated with DGF. ATN was seen in 16 biopsies; 5 were associated with DGF. The finding of ATN in the biopsy of a graft predicted DGF in only 56% of cases. Absence of distal caspase-3 staining predicted the absence of ATN in 87% of cases. The presence of caspase-3 predicted ATN in 54% of cases. The detection of manganese-SOD in distal tubules predicts the absence of DGF in 76% of the cases. The use of immunohistochemical staining on posttransplant renal biopsies improved its predictive value with respect to ATN and DGF: The absence of active caspase-3 in distal tubular epithelium predicts the absence of ATN in 87% of cases, whereas its presence predicts ATN in 54% of cases. The presence of manganese-SOD in distal tubules predicts the absence of DGF in 76% of cases.

Smooth Muscle Cell Hypertrophy in Varicose Veins Is Associated with Expression of Estrogen Receptor-β

Varicose veins are characterized by dilated and thickened vein walls. This study examined whether the changes that occur in varicose veins are associated with smooth muscle cell (SMC) hypertrophy, cellular proliferation or apoptosis. Moreover, the association between SMC hypertrophy and the expression of the estrogen receptor-β (ERβ) was investigated. Varicose veins were obtained from male patients during vascular stripping surgery (n = 11) and nonvaricose veins during coronary bypass surgery, also from male subjects (n = 12). The cellular volume of the SMC in both the longitudinal and circular layer of the vessel wall was measured using stereological methods. Apoptosis was detected using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) technique. SMC proliferation and ERβ expression were investigated by immunohistochemistry. Neither in the longitudinal nor in the circular layer of the varicose vein wall were signs of apoptosis or proliferation present. However, the mean cellular volume of the SMC in the circular layer of the varicose veins was strongly increased (5,291 ± 363 µm³) as compared to non-varicose veins (2,812 ± 212 µm³, p < 0.001). Moreover, ERβ expression in the circular layer of varicose veins (63 ± 4%) significantly differed from nonvaricose veins (39 ± 4%; p = 0.001). Interestingly, the SMC volume correlated with ERβ expression (r = 0.71, p < 0.001). These data show that cell death or proliferation of SMC do not, or only rarely, occur in varicose veins. However, remodeling of varicose veins can mainly be attributed to increased volumes of the SMC of the circular layer and this increase correlates with ERβ expression.

Apoptosis and p63 expression in the pathogenesis of bullous lesions of endemic pemphigus foliaceus

The physiopathological mechanism involved in the formation of the intraepithelial bullae in pemphigus foliaceus (PF), which is endemic in Brazil, has not been elucidated [1, 2]. Apoptosis has been reported to occur in some dermatoses that may present a positive Nikolsky sign such as PF and graft-versus-host disease (GVHD) [3, 4, 5]. In the first disease, apoptotic keratinocytes are present in the acantholytic region and close to the bullae, but little is known about their role in this dermatosis. Recently, the p53 homologues, p63 and p73, which probably originate from a common ancestor gene, have been described [6]. The p63 gene is located in the 3q27-28 locus and is selectively expressed in the basement membrane of various epithelia, being considered as a stem cell marker for these tissues [7]. However, nothing is known about the role of p63 in bullous dermatoses. The aim of the present study was to determine apoptosis and p63 expression in skin samples from patients with PF. After approval by the Research Ethics Committee of the University Hospital, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, and after obtaining informed consent from each patient, skin biopsies were taken from ten patients with a diagnosis of PF. Clinical criteria were established and active lesions were confirmed by a positive Nikolsky sign and histology. For PF patients, biopsies of injured skin and of apparently healthy skin located at least 3 cm from the active lesion were obtained. The control group (n=5) consisted of skin samples from patients undergoing abdominal plastic surgery. A positive control group for apoptosis (n=5) was included using skin samples from GVHD patients [4]. The biopsies were fixed in formalin, embedded in paraffin and sectioned. For the apoptosis study, the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) technique was performed using an Apoptag kit (Intergen Company, Oxford, UK), developed with 3,3′diaminobenzidine (DAB) (Sigma, St. Louis, Mo.). For p63 expression, immunohistochemistry was performed with a p63 antibody (Santa Cruz Biotechnology, clone 4A4; Santa Cruz, Calif.) at 1:100 dilution, and p63 was identified using the streptavidin-biotin-peroxidase method (Novostain Universal kit, Novocastra, Newcastle upon Tyne, UK), developed with 3-amino-9-ethyl-carbazole (AEC) (Vector Laboratories, Burlingame, Calif.). In the control group, p63 expression was limited to the basal layer of the epidermis (Fig. 1b). Indeed, p63 is essential for the maintenance of a precursor population (stem cells) in various epithelial tissues and may be considered a marker of cell undifferentiation [6, 7]. In these samples, apoptotic keratinocytes were identified in some cells close to the horny layer (Fig. 1a). Apoptotic keratinocytes in Isabela Zuccolotto · Ana Maria Roselino · Leandra Naira Zambelli Ramalho · Sergio Zucoloto

Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.

Vasa vasorum hypoperfusion is responsible for medial hypoxia and anatomic remodeling in the newborn lamb ductus arteriosus.

Postnatal constriction of the full-term ductus arteriosus produces hypoxia of the muscle media. This is associated with anatomic remodeling (including smooth muscle death) that prevents subsequent reopening. We used late-gestation fetal and neonatal lambs to determine which factors are responsible for the postnatal hypoxia. Hypoxia [measured by 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide technique] and cell death (measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique) were observed in regions of the constricted ductus wall within 4 h after delivery. Although there was a decrease in ductus luminal flow during the first 6 h after delivery (measured by Doppler transducer), the amount of oxygen delivered to the ductus lumen (3070 +/- 1880 micromol O2 x min(-1) x g(-1)) far exceeded the amount of oxygen consumed by the constricted ductus (0.052 +/- 0.021 micromol O2 x min(-1) x g(-1), measured in vitro). Postnatal constriction increased the effective oxygen diffusion distance across the ductus wall to >3x the limit that can be tolerated for normal tissue homeostasis. This was owing to both an increase in the thickness of the ductus (fetus, 1.12 +/- 0.20 mm; newborn, 1.60 +/- 0.17 mm; p < 0.01) and a marked reduction in vasa vasorum flow (fetus, 0.99 +/- 0.44 mL x min(-1) x g(-1); newborn, 0.21 +/- 0.08 mL x min(-1) x g(-1); p < 0.01). These findings suggest that hypoxic cell death in the full-term ductus is caused primarily by changes in vasa vasorum flow and muscle media thickness and can occur before luminal flow has been eliminated. We speculate that in contrast with the full-term ductus, the preterm ductus is much less likely to develop the degree of hypoxia needed for vessel remodeling inasmuch as it only is capable of increasing its oxygen diffusion distance to 1.3x the maximally tolerated limit.

Ovarian follicle apoptosis at the onset of standing estrus in virgin and repeat-breeder dairy heifers

There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.

Apoptosis index is a predictor of metastatic phenotype in patients with early stage squamous carcinoma of the tongue

Patients with squamous carcinoma of the oral tongue in clinical stages TIN0M0 and T2N0M0 with a tumor thickness < or = 3 mm usually do not have lymph node (LN) metastasis. However, factors that are useful in predicting LN metastasis in thicker tumors (> 3 mm thick) need to be identified. The authors investigated the clinical relevance of the apoptotic index (AI), the proliferation index, and tumor grade in relation to LN metastasis in patients with early stage squamous carcinoma of the oral tongue. Twenty-three patients with squamous carcinoma of the anterior two-thirds of the tongue measuring < 2 cm in height and > 3 mm in thickness were evaluated for tumor grade, AI (by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique), and proliferation index (by proliferating cell nuclear antigen [PCNA] expression). The mean AI value was significantly higher in LN positive patients compared with LN negative patients (P = 0.012). The LN positive and LN negative subgroups did not differ in the mean PCNA index, and there was no significant difference in the distributions of tumor grade between LN positive and LN negative subsets. Four of 12 tumors with an AI < or = 5% and 10 of 11 tumors with an AI > 5% had LN metastasis (P = 0.009; risk ratio, 20). The AI maintained its significance with respect to LN metastasis in the multivariate analysis (P = 0.003). The 4-year recurrence free survival was significantly better in patients with tumors that had an AI value < or = 5% compared with patients with tumors that had an AI > 5% (92% vs. 32%) (P = 0.033). However, the AI lost its impact on recurrence free survival within a Cox proportional hazards model (P = 0.068). A higher AI value is a predictor of LN metastasis and may serve as a prognostic factor in patients with early stage squamous carcinoma of the oral tongue. The authors present a hypothesis to explain this rather surprising finding.

Nifedipine induces apoptosis in cultured vascular smooth muscle cells from spontaneously hypertensive rats

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7-10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca2+ channel antagonist, nifedipine (5 X 10(-5) M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 x 10(-7) M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.

Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.

We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)

Tumor necrosis factor alpha decreases the viability of mouse blastocysts in vitro and in vivo.

Mouse blastocysts were exposed for 24 h to various concentrations of recombinant mouse tumor necrosis factor alpha (TNFalpha) and observed for their capacity to implant in vitro on a fibronectin-coated substrate or to develop in vivo after their transfer into surrogate females. Compared with findings in control blastocysts, exposure to TNFalpha resulted in a significant reduction in the average number of cells in the inner cell mass (ICM) lineage. This effect was associated with a significant increase in the frequency of cells identified as engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling technique. No difference was found in the incidence of nuclear fragmentation between control and TNFalpha-exposed blastocysts. When TNFalpha-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to maintain a structured ICM cluster at the center of the trophectoderm outgrowth. Although no difference was found in the average surface area of the outgrowths, implants derived from TNFalpha-treated blastocysts contained significantly fewer nuclei than implants from control embryos. After transfer into recipient mice, TNFalpha-pretreated blastocysts implanted at about the same rate as control embryos, but a significantly higher rate of resorption was found among fetuses after exposure to the cytokine. In addition, the weight of the surviving fetuses was significantly lower than for control fetuses. These data indicate that the impact of TNFalpha on blastocysts is specifically aimed at the ICM lineage and that TNFalpha decreases the ability of embryos to differentiate into fetuses after implantation.

Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects.

Several laboratories have demonstrated the presence of apoptotic cell death in atherosclerotic plaques. Apoptosis occurs in at least 2 stages. The final "execution" phase, which includes DNA fragmentation, is brief ( approximately 6 hours) and irreversible and can be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. The TUNEL technique is only selective (rather than specific) for apoptotic nuclei, because these contain a far greater degree of DNA fragmentation than do nonapoptotic nuclei. Nonapoptotic cell nuclei that show high levels of RNA synthesis and splicing can also be labeled. This could explain the large variation in the reported percentages of TUNEL-positive nuclei in the plaques. Therefore, the TUNEL technique should be combined with additional techniques, such as markers of transcription and morphological criteria. Recent studies indicate that human fatty streaks differ from adaptive intimal thickenings by the presence of cells containing pro-apoptotic proteins. However, apoptotic cell death is present only in advanced atherosclerotic plaques that show a dense macrophage infiltration. This indicates that although both smooth muscle cells and macrophages within the human fatty streaks become susceptible to apoptosis, additional factors (mainly macrophage- and lipid-derived factors) are necessary to terminate the cell death pathway.