Transferase-mediated Deoxyuridine Triphosphate Nick-end Labeling Research Articles

Hepatocellular Necrosis, Apoptosis, and Proliferation after Transcatheter Arterial Embolization or Chemoembolization in a Standardized Rabbit Model

Purpose To evaluate the effect of transcatheter arterial chemoembolization versus transcatheter arterial embolization on hepatocellular damage, apoptosis, proliferation, and proinflammatory response in a rabbit VX2 tumor model. Materials and Methods Rabbits implanted with VX2 tumors in left liver lobes were randomly divided into three groups: a control group (n = 9) that underwent infusion of distilled water into the left hepatic artery, an embolization group (n = 15) that underwent left hepatic artery embolization with polyvinyl alcohol (PVA) particles, and a chemoembolization group (n = 15) that underwent left hepatic artery infusion of a mixture of 10-hydroxycamptothecin and iodized oil followed by PVA embolization. Serum and liver samples were collected at 6 hours, 3 days, and 7 days postoperatively. Liver damage was measured by liver function tests and histologic analysis. Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling were performed to quantify proliferating and apoptotic cells. Serum tumor necrosis factor (TNF)–α levels were measured to assess proinflammatory response. Results Compared with embolization, chemoembolization caused liver injury with a greater increase in serum alanine aminotransferase and aspartate aminotransferase levels on days 3 and 7; histologic analysis showed increased hepatic necrosis in adjacent liver tissue beginning at day 3 and increased serum levels of TNF-α at 6 hours. By contrast, chemoembolization resulted in a slower increase in hepatocyte proliferation. Additionally, increased apoptotic hepatocytes were observed after embolization and chemoembolization. Conclusions In contrast to embolization, nonsuperselective transcatheter arterial chemoembolization increased hepatocellular damage and stimulated systemic proinflammatory cytokine release, but inhibited hepatocyte proliferation.

Redox factor 1 inhibits the apoptosis process after intracerebral hemorrhage

<title/>Objective: To determine the role of redox factor 1 (Ref-1) in the apoptotic process in perihematoma brain tissue from intracerebral hemorrhage (ICH) patients.Methods: Thirty ICH patients were selected, and normal brain tissue, inevitably lost during surgery, was used as the control group, while the brain tissue from 1 cm around the hematoma was used as the experimental group. Experimental tissues were further divided according to the time from syndrome onset to the time of operation as follows: <6 hours (n = 6); 6-12 hours (n = 7); 12-24 hours (n = 5); 24-72 hours (n = 6); and >72 hours (n = 6). Apoptotic cells were observed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) reaction. Protein and mRNA expression of Ref-1 and Bax were determined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) respectively.Results: Immunohistochemistry indicated that Ref-1 expression was the maximal in control tissue, while it slowly declined to a nadir between 12 and 72 hours in the experimental tissue. Bax expression was the lowest in the control tissue, and gradually increased from 12 to 72 hours in the experimental tissue. RT-PCR data showed that patterns of Ref-1 and Bax expression similar to the immunohistochemistry results. Correlation analysis demonstrated a negative correlation among Ref-1, apoptosis, and Bax.Conclusion: Ref-1 may play an important role in protecting brain cells and may be able to inhibit apoptosis following ICH.

5-Lipoxygenase contributes to the progression of hepatocellular carcinoma

5-Lipoxygenase (5-LOX) has been implicated in the development and progression of lung, pancreatic and esophageal cancers. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore the role of 5-LOX in the pathogenesis of HCC. The expression of 5-LOX was detected in human HCC, HepG2 cells and diethylnitrosamine (DEN)-induced rat HCC using immunohistochemistry (IHC) staining or reverse transcriptase-polymerase chain reaction. Apoptosis in rat HCC was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. Cell viability and apoptosis were determined in HepG2 cells by MTT assay and flow cytometry, respectively. IHC staining showed that the 5-LOX protein was highly expressed in human HCC, HepG2 cells and rat HCC, but not in the normal liver tissues. 5-LOX mRNA expression in human and rat HCC was also significantly increased compared to normal liver tissues. Zileuton, a 5-LOX inhibitor, reduced the nodule incidence and the mean number of nodules per nodule-bearing liver in DEN-induced rats. Further study using TUNEL assay showed that zileuton treatment induced apoptosis in the liver as the result of inhibition on 5-LOX levels. This result is consistent with our observation of significantly higher apoptotic indices in rats treated with DEN/zileuton, which were significantly higher compared to those from the control groups. In addition, zileuton reduced cell viability and induced apoptosis in a concentration- and time-dependent manner as detected using HepG2 cells in our in vitro analysis. In conclusion, 5-LOX is expressed in HCC, and the inhibition of 5-LOX blocks the development of HCC via the induction of apoptosis in tumor cells.

Saffron: A potential candidate for a novel anticancer drug against hepatocellular carcinoma

Saffron has been proposed as a promising candidate for cancer chemoprevention. The purpose of this investigation was to investigate the chemopreventive action and the possible mechanisms of saffron against diethylnitrosamine (DEN)-induced liver cancer in rats. Administration of saffron at doses of 75, 150, and 300 mg/kg/day was started 2 weeks prior to the DEN injection and was continued for 22 weeks. Saffron significantly reduced the DEN-induced increase in the number and the incidence of hepatic dyschromatic nodules. Saffron also decreased the number and the area of placental glutathione S-transferase-positive foci in livers of DEN-treated rats. Furthermore, saffron counteracted DEN-induced oxidative stress in rats as assessed by restoration of superoxide dismutase, catalase, and glutathione-S-transferase levels and diminishing of myeloperoxidase activity, malondialdehyde and protein carbonyl formation in liver. The results of immunohistochemical staining of rat liver showed that saffron inhibited the DEN-mediated elevations in numbers of cells positive for Ki-67, cyclooxygenase 2, inducible nitric oxide synthase, nuclear factor-kappa B p-65, and phosphorylated tumor necrosis factor receptor. Saffron also blocked the depletion in the number of cells positive for TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) and M30 CytoDeath in liver tissues of DEN-treated rats. In vitro experiments carried out using HepG2 cells also confirmed these findings and showed inhibition of nuclear factor-kappa B activation, increased cleavage of caspase-3, as well as DNA damage and cell cycle arrest upon saffron treatment. This study provides evidence that saffron exerts a significant chemopreventive effect against liver cancer through inhibition of cell proliferation and induction of apoptosis. This report also shows some evidence that saffron protects rat liver from cancer via modulating oxidative damage and suppressing inflammatory response.

Xenon augmented hypothermia reduces early lactate/N‐acetylaspartate and cell death in perinatal asphyxia

Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia-ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry. Thirty-six newborn piglets were randomized (all groups n = 9), with intervention from 2 to 26 hours, to: (1) normothermia; (2) normothermia + 24 hours 50% inhaled xenon; (3) 24 hours hypothermia (33.5°C); or (4) 24 hours hypothermia (33.5°C) + 24 hours 50% inhaled xenon. Serial MRS was acquired before, during, and up to 48 hours after hypoxia-ischemia. Mean arterial blood pressure was lower in all treatment groups compared with normothermia (p < 0.01) (although >40mmHg); the combined therapy group required more fluid boluses (p < 0.05) and inotropes (p < 0.001). Compared with no intervention, both hypothermia and xenon-augmented hypothermia reduced the temporal regression slope magnitudes for phosphorus-MRS inorganic phosphate/exchangeable phosphate pool (EPP) and phosphocreatine/EPP (both p < 0.05); for lactate/N-acetylaspartate (NAA), only xenon-augmented hypothermia reduced the slope (p < 0.01). Xenon-augmented hypothermia also reduced transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)(+) nuclei and caspase 3 immunoreactive cells in parasagittal cortex and putamen and increased microglial ramification in midtemporal cortex compared with the no treatment group (p < 0.05). Compared with hypothermia, however, combination treatment did not reach statistical significance for any measure. Lactate/NAA showed a strong positive correlation with TUNEL; nucleotide triphosphate/EPP showed a strong negative correlation with microglial ramification (both p < 0.01). Compared with no treatment, xenon-augmented hypothermia reduced cerebral MRS abnormalities and cell death markers in some brain regions. Compared with hypothermia, xenon-augmented hypothermia did not reach statistical significance for any measure. The safety and possible improved efficacy support phase II trials.

Colitogenic role of tumour necrosis factor (TNF) receptors in trinitrobenzene sulphonic acid colitis: TNF-R1 ablation does not affect systemic inflammatory response

Tumour necrosis factor (TNF)-α plays a critical role in the pathogenesis of T helper type 1-mediated colitis such as Crohn's disease. However, the roles of its two receptors in mediating pathology remain largely unknown. In this study, trinitrobenzene sulphonic acid (TNBS) was used to induce colitis in TNF-receptor single or double knock-out (DKO) BALB/c mice and in wild-type counterparts. TNF-R1(-/-) mice had significantly less weight loss, reduced mortality, colon shortening and oedema, colon histological damage and lower levels of colon myeloperoxidase compared with wild-type (WT) BALB/c mice. A similar manifestation was also observed in TNF-R2(-/-) and TNF-R1(-/-) TNF-R2(-/-) (TNF-R DKO) mice. Strikingly, systemic inflammatory response (including splenomegaly and monocyte expansion) was found in WT and TNF-R1(-/-) mice after TNBS, instead of TNF-R2(-/-) and TNF-R DKO mice. Attenuated pathology of colitis in TNF-R1(-/-) or TNF-R2(-/-) mice correlated with lower amounts of interleukin (IL)-6, IL-1β, monocyte chemotactic protein (MCP)-1, IL-12p70 and interferon (IFN)-γ production in the colons. Importantly, ablation of TNF-R1 or TNF-R2 reduced the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL)-positive apoptotic epithelial cells in the affected colons compared with WT TNBS-instilled controls, which might be due to the heightened ratio of Bcl-2/Bax and reduced activity of nuclear factor (NF)-κB. These findings suggest that either TNF-R1 or TNF-R2 plays a pathogenic role in the pathology of colitis and TNF signalling via TNF-R1 or TNF-R2 alone is not sufficient for inducing mucosal damage.

Alchornea glandulosaEthyl Acetate Fraction Exhibits Antiangiogenic Activity: Preliminary Findings fromIn VitroAssays Using Human Umbilical Vein Endothelial Cells

Alchornea glandulosa has traditionally been used in Brazilian folk medicine for the treatment of immune-inflammatory diseases and as an antiulcer agent to heal gastric ulcer and gastritis. Angiogenesis is a complex multistep process that consists of proliferation, migration, and anastomosis of endothelial cells and has a major role in the development of pathologic conditions, such as inflammatory diseases. To investigate a possible link between the anti-inflammatory activities and antiangiogenic effects of A. glandulosa ethyl acetate fraction (AGF), this study examined which features of the angiogenic process could be disturbed by this fraction. The antiangiogenic activity of AGF was determined in vitro by using human umbilical vein endothelial cells (HUVEC), and cell viability, proliferation, apoptosis, invasion, and capillary-like structures formation were addressed. To elucidate the mechanism of action, nuclear factor κB (NFκB), a transcription factor implicated in these processes, was also evaluated in HUVEC incubated with AGF. A significant decrease in proliferation, a relevant increase in apoptosis, and a strong reduction in invasion capacity (as assessed by bromodeoxyuridine, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and double-chamber assays, respectively) were observed. AGF also led to a drastic reduction in the number of capillary-like structures formed when HUVEC were cultured on growth factor-reduced Matrigel-coated plates. In addition, incubation of HUVEC with AGF resulted in reduced NFκB activity. These findings emphasize the antiangiogenic potential of AGF and support its therapeutic use for disorders that involve excessive angiogenesis, such as chronic inflammation and tumor growth.

Deleterious Effect of Hyperoxia at Birth on White Matter Damage in the Newborn Rat

White matter damage (WMD) remains the leading cause of cerebral palsy in children born prematurely. The release of an excessive amount of reactive oxygen species is recognized as a risk factor for WMD. We hypothesize that free radical injury during reoxygenation at birth may be harmful to the immature white matter and may underlie, at least in part, the pathogenesis of WMD. We tested this hypothesis in rat pups delivered from normoxic pregnant rats, and by investigating an animal model based on protracted antenatal hypoxia in the pregnant rat and mimicking the main features of human WMD in rat pups. From embryonic day (E)5 to E21, the pregnant rats were placed in a chamber supplied with a gas mixture that either induced hypoxia (FiO₂ = 10%) or maintained normoxia (FiO₂ = 21%). On E21, the dams were removed from the chamber and housed under either normoxia (FiO₂ = 21%), hyperoxia (FiO₂ = 60%) or slowly reoxygenated (FiO₂ from 15% at E21 to 21% at postnatal day 7). Postnatal hyperoxia was associated with a significantly increased density of activated microglial cells (+105%) and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling)-positive cells (+85%) within the developing white matter. Myelin content (–31%) and mature oligodendrocyte density (–37%) in the normal developing white matter were significantly decreased by postnatal hyperoxia. Postnatal hyperoxia significantly potentiated the myelination delay and oligodendroglial dysmaturation induced by antenatal hypoxia. In contrast, progressive reoxygenation at birth did not induce any change in white matter inflammation, myelination and cell death as compared with normoxic controls, and prevented most of the WMD observed following antenatal hypoxia. This study demonstrates a deleterious effect of hyperoxia at birth on the developing white matter in normal rat pups. Postnatal hyperoxia worsened the WMD induced by antenatal hypoxia. Hyperoxia at birth should be avoided in preterm infants at risk of WMD.

Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants

We describe here the isolation and cytogenetic characterization of three meiotic prophase I mutants, denoted ietsugu (its), iesada (isa), and iemochi (imo), isolated by a novel N-ethyl-N-nitrosourea mutagenesis screen for adult zebrafish gonadogenesis. Histological examination and flow cytometry analysis of testes from these mutants showed that each contained neither spermatids nor sperm. Staining for Sycp3 and cleaved Caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) assay further revealed that its had defects at the onset of meiosis, and that isa and imo spermatocytes failed to progress past the zygotene stage with apoptosis occurring in the testicular somatic cells. Staining for phosphorylated histone H2AX showed that foci formation in leptotene spermatocytes was disrupted in isa and imo. Furthermore, in vitro differentiation experiments revealed the possibility that the defects and sterility associated with mutations were germ line autonomous. Our results thus indicate that each responsible gene is necessary for meiotic progression during spermatogenesis and for male fertility.

Expression Level of Hand2 Affects Specification of Enteric Neurons and Gastrointestinal Function in Mice

Hand2 is a basic helix-loop-helix transcription factor required for terminal differentiation of enteric neurons. We studied Hand2 haploinsufficient mice, to determine whether reduced expression of Hand2 allows sufficient enteric neurogenesis for survival, but not for development of a normal enteric nervous system (ENS). Enteric transcripts that encode Hand2 and the neuron-specific embryonic lethal abnormal vision proteins HuB, HuC, and HuD were quantified. Immunocytochemistry was used to identify and quantify neurons. Apoptosis was analyzed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling procedure. Intracellular microelectrodes were used to record inhibitory junction potentials. Gastrointestinal transit and colonic motility were measured in vivo. Levels of of enteric Hand2 transcripts were associated with genotypes of mice, in the following order: Hand2(+/+) > Hand2(LoxP/+) > Hand2(+/-) > Hand2(LoxP/-). Parallel reductions were found in expression of HuD and in regional and phenotypic manners. Numbers of neurons, numbers of neuronal nitric oxide synthase(+) and calretinin(+), but not substance P(+) or vasoactive intestinal peptide(+) neurons, decreased. No effects were observed in stomach or cecum. Apoptosis was not detected, consistent with the concept that Hand2 inhibits neuronal differentiation, rather than regulates survival. The amplitude of inhibitory junction potentials in colonic circular muscle was similar in Hand2 wild-type and haploinsufficient mice, although in haploinsufficient mice, the purinergic component was reduced and a nitrergic component appeared. The abnormal ENS of haploinsufficient mice slowed gastrointestinal motility but protected mice against colitis. Reduced expression of factors required for development of the ENS can cause defects in the ENS that are subtle enough to escape detection yet cause significant abnormalities in bowel function.

Increased Cavernous Expression of Transforming Growth Factor-β1 and Activation of the Smad Signaling Pathway Affects Erectile Dysfunction in Men with Spinal Cord Injury

Transforming growth factor-β1 (TGF-β1) is implicated in bladder fibrosis after spinal cord injury (SCI) and in the fibrosis in the corpus cavernosum tissue after cavernous nerve injury. We investigated the differential expression of TGF-β1 and the Smad transcription factor, the key molecule for the initiation of TGF-β-mediated fibrosis, in cavernous tissue from SCI patients. After obtaining informed consent and approval from the patients and our institutional review board, we enrolled 5 patients with psychogenic erectile dysfunction (ED) (mean age 36.8 years; range 20-50 years) and 10 patients with neurogenic ED from SCI (mean age 38.8 years; range 18-50 years). Cavernous tissues were obtained by percutaneous biopsy and stained with Masson trichrome, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), or antibodies to TGF-β1 and phospho-Smad2. Semi-quantitative analysis of TGF-β1 and phospho-Smad2 was performed, and the numbers of apoptotic cells were counted. We also quantified the cavernous collagen area with the use of an image analyzer system. The expression of TGF-β1 and phospho-Smad2 protein was significantly higher in the SCI group than in the psychogenic group. The TUNEL assay revealed a higher apoptotic index in the SCI group than in the psychogenic group. Higher TGF-β1 and phospho-Smad2 expression and more apoptotic cells were noted mainly in endothelial cells, smooth muscle cells, and fibroblasts of the SCI group. Double labeling of cavernous tissue with TUNEL and antibody to phospho-Smad2 revealed that most TUNEL-positive cells showed immunoreactivity to phospho-Smad2 staining. Cavernous collagen content was significantly greater in the SCI group than in the psychogenic group. Upregulation of TGF-β1 and activation of the Smad signaling pathway may play important roles in SCI-induced cavernous fibrosis and deterioration of erectile function, which warrants early pharmacological intervention to protect erectile tissue from irreversible damage.

Neuroprotection With Intraventricular Brain-Derived Neurotrophic Factor in Rat Venous Occlusion Model

The increasing number of neurosurgical procedures for elderly patients and the development of skull base neurosurgery have increased interest in cerebral venous injury that might occur in a neurosurgical setting. Brain-derived neurotrophic factor (BDNF) may have neuroprotective effects against cerebral venous ischemia. To investigate the intraventricular effects of BDNF infusion on infarct size, suppression of apoptosis, and regional cerebral blood flow (rCBF) in cerebral venous ischemic lesions in a rat 2-vein occlusion model. Thirty-three male Wistar rats were randomly divided into BDNF-treated and vehicle control groups; each group was further randomly divided into 2-day and 7-day postocclusion groups. BDNF (2.1 μg/day) or vehicle was delivered continuously via intraventricular infusion pumps. Two adjacent contralateral cortical veins were then photochemically occluded. Two and 7 days after occlusion, we histologically measured ratios of infarct volume to contralateral hemisphere volume and counted (2-day group) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive apoptotic cells in the penumbra. rCBF was measured via full-field laser perfusion imaging. The mean infarct volume after venous occlusion was significantly smaller in BDNF-treated rats than in controls at 2 days (1.49 ± 1.44% vs 3.66 ± 1.51%; P < .05) and 7 days (0.93 ± 0.47% vs 1.69 ± 0.58%; P < .05). Two days after occlusion, there were significantly fewer TUNEL-positive apoptotic cells in the BDNF-treated rats (17.0 ± 15.1) than in the controls (39.0 ± 19.6; P < .05). There were no differences in rCBF. After 2-vein occlusion, continuous intraventricular administration of BDNF protected the cerebral cortex against apoptosis and reduced infarct size without affecting rCBF.

Cilostazol Minimizes Venous Ischemic Injury in Diabetic and Normal Rats

We evaluated the effects of cilostazol on venous infarction produced by a photothrombotic two-vein occlusion (2VO) model in diabetic and control rats. The cerebral blood flow (CBF) between the occluded veins was measured by laser Doppler flowmetry for 4 hours after 2VO. Infarct size and immunohistochemistry were evaluated 24, 48, 96, and 168 hours after 2VO. Cilostazol was administered 1 hour after 2VO, and thereafter at a continuous oral dose of 60 mg/kg per day. Cilostazol reduced the infarct size, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive apoptotic and B-cell lymphoma 2-associated X protein (Bax)-positive cells, and improved the CBF in control rats. In diabetic rats, cilostazol reduced the infarct size, and the number of TUNEL-positive apoptotic and Bax-positive cells, 96 and 168 hours after 2VO, but did not improve the CBF 4 hours after 2VO. Cilostazol increased the number of B-cell lymphoma 2 (Bcl-2)-positive cells in both strains 48, 96, and 168 hours after 2VO, but did not improve vessel wall thickness or collagen deposits. Cilostazol appeared to limit venous infarcts by improving the penumbral CBF in nondiabetic rats, and inhibited pro-apoptotic changes through Bcl-2 overexpression, without improving the CBF in diabetic rats.

Maitake (D Fraction) Mushroom Extract Induces Apoptosis in Breast Cancer Cells by BAK-1 Gene Activation

For many years mushrooms have been used empirically in traditional medicine to treat several diseases. Study of the maitake mushroom, with its immunomodulatory and antitumoral properties, has led to the isolation of several bioactive compounds. One of these, D fraction, is known to reduce tumor cell viability. This study examined the effect of isolated D fraction on viability and apoptosis of human breast cancer cells (MCF7). These cells were treated with maitake (D fraction) extract at 18 μg/mL, 36 μg/mL, 91 μg/mL, 183 μg/mL, or 367 μg/mL or were left untreated (control) for 24 hours. MCF7 incubation with the maitake extract resulted in decreased cell viability [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay] in a dose-dependent manner. Apoptosis was statistically significantly increased in a dose-dependent manner at every concentration tested (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay). Upon incubation with D fraction, a microarray assay revealed upregulation of BAK-1 and cytochrome c transcripts, 2 proteins directly involved in the apoptotic pathway. Reverse transcriptase polymerase chain reaction studies confirmed these findings; BAK-1 was one of most overexpressed gene, as observed by microarray assay. These findings confirm the apoptotic effect of maitake D fraction in breast cancer cells and further highlight the involvement of cytochrome c release to the cytoplasm. Cytoplasmic release of cytochrome c, another player in the apoptotic pathway, was also increased after incubation with D fraction in a dose-dependent manner. This finding indicates that the effect of this compound involves mitochondrial dysfunction. The identification of the molecular mechanisms by which D fraction exerts its effects is crucial for the development of preventive and therapeutic strategies for cancer.

Endothelium‐coated tumor clusters are associated with poor prognosis and micrometastasis of hepatocellular carcinoma after resection

Distinct morphologic features of microvascular endothelium exist in tumor tissues. The objective of this study was to investigate the prognostic value of endothelium-coated tumor clusters (ECTCs) in hepatocellular carcinoma (HCC). ECTCs were evaluated by immunohistochemical staining for cluster of differentiation 34 (CD34) (a cell surface glycoprotein which is expressed specifically on tumor microvascular endothelium in HCC) in 239 specimens from patients with primary HCC. Overall survival (OS) and time to recurrence (TTR) were determined using Kaplan-Meier analysis and a Cox proportional hazards regression model. Levels of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, and Ki-67 expression, and E-cadherin expression were assessed to determine tumor apoptosis, proliferation, and invasiveness, respectively. The presence of ECTCs was associated with a poor prognosis in all patients and in patient subgroups stratified by tumor size, TNM classification, and Barcelona Clinic Liver Cancer stage and tumor invasiveness. In a multivariate Cox proportional hazards analysis, the presence of ECTCs emerged as an independent prognostic indicator of both poor OS (P = .001; hazard ratio, 1.949) and shorter TTR (P < .001; hazard ratio, 2.085). Furthermore, the presence of ECTCs was associated with micrometastatic endothelium-coated emboli (P < .001; chi-square test) and early relapse after resection (P < .001; chi-square test). In addition, patients who had endothelium-coated emboli, in which tumor cells displayed high proliferation and low apoptosis, had poor OS and shorter TTR. The current results suggested that the presence of ECTCs was an efficient, simple, and convenient predictor of a poor prognosis in patients with HCC that potentially may serve as a novel target for the prevention and treatment of HCC metastasis.

Relationship between Cellular Apoptosis and the Expression of p75 Neurotrophin Receptor and Tyrosine Kinase A Receptor in Tissue Surrounding Haematoma in Intracerebral Haemorrhage

Cellular apoptosis and the expression of p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase A receptor (TrkA) were investigated in the tissues surrounding haematoma in patients with intracerebral haemorrhage. Specimens of tissue from near the haematoma (haemorrhagic samples) and tissue from a distant site (control samples) were collected from 14 patients with basal ganglia haemorrhage undergoing surgical intervention. Cellular apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL), and p75(NTR) and TrkA location, protein and gene expression were studied using immunohistochemistry, Western blot and real-time polymerase chain reaction, respectively. The percentage of apoptotic cells and expression of p75(NTR), but not of TrkA, were significantly higher in the haemorrhagic samples than in the control samples. There was a positive correlation between the percentage of TUNEL-positive cells and the percentage of p75(NTR)-positive cells. These results suggest that the p75(NTR)-dependent signal transduction pathway plays an important role in apoptosis after intracerebral haemorrhage.

In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility

BACKGROUND: Ovarian tissue cryopreservation, in combination with autotransplantation or long-term culture, has been proposed as a means of fertility preservation. However follicle density within ovarian cortex has a profound impact on the success of in vivo and in vitro systems designed to support follicle growth and restore fertility. The objective of this study was to investigate the dye neutral red (NR) as a tool to quantify follicle density in situ, without compromising follicle viability and developmental potential. METHODS: In the first experimental series thin slices of cryopreserved and fresh ovine cortical tissue were incubated in 50 μg/ml NR and assessed for the presence of red colouration. Slices were then used for follicular structure isolation and viability evaluation using 5-(and 6)-carboxyfluoresceindiacetate succinimidylester (CFDA-SE), or prepared histologically for follicle counting or evaluation of apoptosis via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL). An additional subset of slices were cultured for 8 days, followed by re-evaluation of follicle viability. NR staining was further assessed in a pilot study using thin slices of cryopreserved human ovarian tissue donated by 17 patients undergoing laparoscopic sterilisation or elective Caesarean section. RESULTS: In both ovine and human ovarian cortex NR concentrated in follicular structures within weakly stained stroma. NR colouration was observed in 41.7 ± 4.6% of cryopreserved and 49.3 ± 6.5% of the fresh ovine tissue slices, and NR staining was consistently predictive of the presence of follicles. Non-stained ovine slices contained highly apoptotic follicles, while lower levels of apoptosis were observed in NR positive slices, indicating preferential detection of viable follicles by NR. Following culture the majority of ovine slices re-stained with NR, no significant increases in the levels of apoptosis were observed and 94.6 ± 3. 1% of follicles were viable by CFDA-SE. In the human study, NR identified follicles in 19.3 ± 3.7% of tissue slices, and follicle density tended to decrease with advancing patient age. CONCLUSIONS: NR predicts viable follicle density in situ in slices of ovine and human ovarian cortex. Furthermore incubation of tissue in NR prior to culture does not compromise subsequent follicle survival in vitro, indicating the potential suitability of this approach in fertility preservation regimes.

Native macrophages genetically modified to express heme oxygenase 1 protect rat liver transplants from ischemia/reperfusion injury

We investigated whether native macrophages overexpressing heme oxygenase 1 (HO-1) could protect rat orthotopic liver transplant (OLT) against cold ischemia/reperfusion injury (IRI). Livers from Sprague-Dawley rats were stored at 4°C in University of Wisconsin solution for 24 hours, and then they were transplanted into syngeneic recipients. Bone marrow-derived macrophages (BMMs) that were transfected ex vivo with heme oxygenase 1 adenovirus (Ad-HO-1), β-galactosidase adenovirus (Ad-β-gal), or HO-1 small interfering RNA (siRNA) were infused directly into the OLT before reperfusion. Controls were OLT conditioned with unmodified or scrambled siRNA-transfected cells. The transfer of Ad-HO-1/BMMs increased the survival of OLT to 100% (versus 40%-50% for controls) and decreased serum alanine aminotransferase levels and histological features of hepatocellular damage. In contrast, an infusion of macrophages transfected with HO-1 siRNA/Ad-β-gal failed to affect IRI. Gene therapy-induced HO-1 suppressed toll-like receptor 4 expression, decreased expression of proinflammatory tumor necrosis factor α, interleukin-1β, monocyte chemoattractant protein 1, and chemokine (C-X-C motif) ligand 10, and attenuated endothelial intercellular cell adhesion molecule 1 expression with resultant diminished OLT leukocyte sequestration. Although Ad-HO-1/BMMs decreased the frequency of apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and ameliorated caspase-3 activity, the expression of interleukin-10 and antiapoptotic B cell lymphoma 2/B cell lymphoma extra large increased in well-functioning OLT. Thus, the transfer of native macrophages transfected ex vivo with HO-1 can rescue rat iso-OLT from IRI. Our study validates a novel and clinically attractive concept: native macrophages transfected ex vivo with the antioxidant HO-1 can be applied at the time of transplantation to mitigate otherwise damaging antigen-independent liver inflammation and injury resulting from the peritransplant harvesting insult. If this new, refined strategy is proven to be effective in allo-OLT recipients, it should be considered in clinical settings to increase the supply of usable donor organs and ultimately improve the overall success of liver transplantation.

Hydrogen sulphide protects mouse pancreatic β‐cells from cell death induced by oxidative stress, but not by endoplasmic reticulum stress

Hydrogen sulphide (H₂S), a potentially toxic gas, is also involved in the neuroprotection, neuromodulation, cardioprotection, vasodilatation and the regulation of inflammatory response and insulin secretion. We have recently reported that H₂S suppresses pancreatic β-cell apoptosis induced by long-term exposure to high glucose. Here we examined the protective effects of sodium hydrosulphide (NaHS), an H₂S donor, on various types of β-cell damage. Isolated islets from mice or the mouse insulinoma MIN6 cells were cultured with palmitate, cytokines (a mixture of tumour necrosis factor-α, interferon-γ and interleukin-1β), hydrogen peroxide, thapsigargin or tunicamycin with or without NaHS. We examined DNA fragmentation, caspase-3 and -7 activities and reactive oxygen species (ROS) production in the treated cells thereafter. Apoptotic cell death in isolated islets was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method. NaHS suppressed DNA fragmentation and the activities of caspase-3 and -7 induced by palmitate, the cytokines or hydrogen peroxide. In contrast, NaHS failed to protect islets and MIN6 cells from apoptosis induced by thapsigargin and tunicamycin, both of which cause endoplasmic reticulum stress. NaHS suppressed ROS production induced by cytokines or hydrogen peroxide but it had no effect on ROS production in thapsigargin-treated cells. NaHS increased Akt phosphorylation in MIN6 cells treated with cytokines but not in cells treated with thapsigargin. Treatment with NaHS decreased TUNEL-positive cells in cytokine-exposed islets. H₂S may prevent pancreatic β-cells from cell apoptosis via an anti-oxidative mechanism and the activation of Akt signalling.

Effect of Sildenafil Citrate on Penile Weight and Physiology of Cavernous Smooth Muscle in a Post–radical Prostatectomy Model of Erectile Dysfunction in Rats

To evaluate the gross morphometric changes and in vitro responses of the corpus cavernosus of rats treated with sildenafil citrate after cavernous neurotomy. The animals were divided into 3 groups. Group 1 consisted of sham-operated rats (n = 16); group 2 consisted of rats that underwent bilateral cavernous neurotomy (BCN) (n = 16); and group 3 consisted of rats that underwent unilateral cavernous neurotomy (UCN) (n = 16). Each group of rats was further classified into 2 subgroups according to whether or not they received sildenafil treatment. The rats were killed on postoperative day 14, and penectomy was performed. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), and organ-bath studies were evaluated by Phenylephrine (Phe), acetylcholine (Ach), sodium nitroprusside (SNP), and electrical field stimulation (EFS) responses. Penile weight in the BCN group was significantly lower than that of sham-treated group. UCN allowed much more preservation of penile weight compared with that in the sham-treated group. Sildenafil citrate treatment had positive effects on penile weight of both BCN (P = .003) and UCN (P = .004) groups. BCN increased smooth muscle apoptosis when compared with the sham or UCN group. Sildenafil citrate had a positive effect on the apoptotic index. In the BCN group, responses to Phe, Ach, SNP, and EFS decreased significantly, and sildenafil treatment corrected the responses to Phe, Ach, and SNP. Our experimental study results support that early and daily sildenafil citrate treatment has a protective affect on the adrenergic and cholinergic systems, which play a role in erectile function.