dUTP-biotin Nick End Labeling-positive Cells Research Articles

Diacylglycerol O-acyltransferase 1 inhibitor increases plasma alanine aminotransferase and aspartate aminotransferase activities via a shedding of the intestinal villi and an increase in intestinal permeability in rats

Diacylglycerol O-acyltransferase 1 (DGAT1) is a key enzyme for fat absorption step in the enterocytes. We previously reported that DGAT1 inhibition increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in corn oil-loaded rats via protein kinase C (PKC) activation. In the present study, we investigated the mechanism with respect to the morphology and permeability of the small intestine, focusing on PKC function, and found that shortening of the intestinal villi and a decrease in the number of tdT-mediated dUTP-biotin nick-end labeling-positive cells in the tips of the villi were observed in the jejunum of DGAT1 inhibitor-treated rats loaded with corn oil. These results suggested that the tips of the villi were shed into the intestinal lumen. Next, fluorescein isothiocyanate-dextran, 110 kDa (FD-110) was administered intraduodenally to DGAT1 inhibitor-treated rats loaded with corn oil and we found that plasma FD-110 concentrations increased, indicating that the intestinal permeability to molecules with a molecular weight of approximately 110,000 (e.g., ALT and AST) increased. Taken together, the present results suggested that DGAT1 inhibitor-treatment in combination with corn oil causes ALT and AST to leak from the enterocytes into the blood by shedding the tips of the intestinal villi and increasing intestinal permeability.

Naringin protects against inflammation and apoptosis induced by intestinal ischemia-reperfusion injury through deactivation of cGAS-STING signaling pathway.

Effective amelioration of ischemia/reperfusion (I/R)-induced intestinal injury and revealing its mechanisms remain the challenges in both preclinic and clinic. Potential mechanisms of naringin in ameliorating I/R-induced intestinal injury remain unknown. Based on pre-experiments, I/R-injured rat intestine in vivo and hypoxia-reoxygenation (H/R)-injured IEC-6 cells in vitro were used to verify that naringin-alleviated I/R-induced intestinal injury was mediated via deactivating cGAS-STING signaling pathway. Naringin improved intestinal damage using hematoxylin and eosin staining and decreased alanine aminotransferase and aspartate aminotransferase contents in plasma. Naringin decreased inflammation characterized by reducing IL-6, IL-1β, TNF-α, and IFN-β contents in both plasma and IEC-6 cells. Naringin mitigated oxidative stress via recovering superoxide dismutase, glutathione, and malondialdehyde levels in the I/R-injured intestine. Naringin reduced the expression of apoptotic proteins, including Bax, caspase-3, and Bcl-2, and reduced terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling-positive cells both in vivo and in vitro, and decreased Hoechst 33342 signals in vitro. cGAS, STING, p-TBK1, p-IRF3, and NF-κB expressions were up-regulated both in vivo and in vitro respectively and the up-regulated indexes were reversed by naringin. Transfection of cGAS-siRNA and cGAS-cDNA significantly down-regulated and up-regulated cGAS-STING signaling-related protein expressions, respectively, and partially weakened naringin-induced amelioration on these indexes, suggesting that deactivation of cGAS-STING signaling is the crucial target for naringin-induced amelioration on I/R-injured intestine.

Mitochonic acid 5 regulates mitofusin 2 to protect microglia

Microglial apoptosis is associated with neuroinflammation and no effective strategies are currently available to protect microglia against inflammation-induced apoptosis. Mouse microglial BV-2 cells (5 × 106) were incubated with 10 μg/mL lipopolysaccharides for 12 hours to mimic an inflammatory environment. Then the cells were co-cultured with mitochonic acid 5 (MA-5) for another 12 hours. MA-5 improved the survival of lipopolysaccharide-exposed cells. MA-5 decreased the activity of caspase-3, which is associated with apoptosis. MA-5 reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells, and increased adenosine triphosphate levels in cells. MA-5 decreased the open state of the mitochondrial permeability transition pore and reduced calcium overload and diffusion of second mitochondria-derived activator of caspase (Smac). MA-5 decreased the expression of apoptosis-related proteins (mitochondrial Smac, cytoplasmic Smac, pro-caspase-3, cleaved-caspase-3, and caspase-9), and increased the levels of anti-apoptotic proteins (Bcl2 and X-linked inhibitor of apoptosis protein), mitochondria-related proteins (mitochondrial fusion protein 2, mitochondrial microtubule-associated proteins 1A/1B light chain 3B II), and autophagy-related proteins (Beclin1, p62 and autophagy related 5). However, MA-5 did not promote mitochondrial homeostasis or decrease microglial apoptosis when Mitofusin 2 expression was silenced. This shows that MA-5 increased Mitofusin 2-related mitophagy, reversed cellular energy production and maintained energy metabolism in BV-2 cells in response to lipopolysaccharide-induced inflammation. These findings indicate that MA-5 may promote the survival of microglial cells via Mitofusin 2-related mitophagy in response to lipopolysaccharide-induced inflammation.

MicroRNA let-7g regulates mouse granulosa cell autophagy by targeting insulin-like growth factor 1 receptor

As an important type of somatic cell, granulosa cells play a major role in deciding the fate of follicles. Therefore, analyses of granulosa cell apoptosis and follicular atresia have become hotspots of animal research. Autophagy is a cellular catabolic mechanism that protects cells from stress conditions, including starvation, hypoxia, and accumulation of misfolded proteins. However, the relationship between autophagy and apoptosis in granulosa cells is not well known. Here, we demonstrate that let-7g regulates the mouse granulosa cell autophagy signaling pathway by inhibiting insulin-like growth factor 1 receptor expression and affecting the phosphorylation of protein kinase B/mammalian target of rapamycin. Small interference-mediated knockdown of insulin-like growth factor 1 receptor significantly promoted autophagy signaling of mouse granulosa cells. In contrast, overexpression of insulin-like growth factor 1 receptor in mouse granulosa cells attenuated autophagy activity in the presence of let-7g. In addition, overexpression of let-7g increased the apoptosis rate, as indicated by an increased number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. Finally, 3-methyladenine as well as the lysosomal enzyme inhibitor chloroquine partially blocked apoptosis. In summary, this study demonstrates that let-7g regulates autophagy in mouse granulosa cells by targeting insulin-like growth factor 1 receptor and downregulating protein kinase B/mammalian target of rapamycin signaling, and that mouse granulosa cell autophagy induced by let-7g participates in apoptosis.

A Novel and Sensitive Approach for the Evaluation of Liver Ischemia-Reperfusion Injury After Liver Transplantation.

The purpose of our study was to evaluate the potential of x-ray propagation-based phase-contrast imaging (PCI) computed tomography (CT) for the detection and characterization of early changes after ischemia-reperfusion (IR) in a standardized rat liver transplantation (LTx) model. Syngeneic orthotopic liver transplantation was performed in male Lewis rats. Ischemia-reperfusion injury (IRI)-induced changes of liver parenchyma were investigated in a time-dependent manner (2, 16, 24, and 32 hours). X-ray phase-contrast images of formalin-fixated liver specimens were acquired in CT mode by using a voxel size of 8 × 8 × 8 μm. Necrapoptotic cell death was visualized with the TdT-mediated dUTP-biotin nick end labeling technique, and alterations of liver graft microhemodynamics, that is, acinar and sinusoidal perfusion failure, were evaluated by in vivo fluorescence microscopy. Acquired and reconstructed PCI-CT images showed an increase in necrotic liver parenchyma dependent on cold storage time, measuring 5.7% ± 1.6% after 2 hours (comparable to 2.6% ± 0.4% for sham livers), 11.5% ± 2.1% (16 hours; P < 0.05 vs control), 23.0% ± 0.5% (24 hours; P < 0.001 vs control), and 31.3% ± 2.2% (32 hours; P < 0.001 vs control). There were a significant lower number of perfused acini in dependence on increasing cold storage time. The acinar perfusion index reached 0.970 ± 0.006 after 2 hours of cold ischemia (comparable to 0.960 ± 0.009 for sham livers) and declined continuously after 16, 24, and 32 hours cold ischemia (0.58 ± 0.03, 0.49 ± 0.02, 0.41 ± 0.03, each P < 0.0001 vs controls). Comparable results were found for sinusoidal perfusion, reaching 1.8% ± 0.4% of nonperfused sinusoids for 2 hours of cold ischemia and 8.2% ± 0.8% after 16 hours, 18.8% ± 1.4% after 24 hours, and 39.0% ± 2.4% after 32 hours (each P < 0.0001 vs controls). Prolonged cold ischemia was associated with an increasing number of TdT-mediated dUTP-biotin nick end labeling-positive cells (hepatocytes and sinusoidal lining cells), reaching 0.4 ± 0.1 (sham), 0.7 ± 0.4 (2 hours), 6.4 ± 1.1 (16 hours), 2.1 ± 0.3 (24 hours), and 14.7 ± 3.5 (32 hours; P = 0.002) for hepatocytes. X-ray PCI of histological liver specimens can detect IR-induced tissue necrosis and can provide detailed complementary 3-dimensional information to standard histopathologic findings.

Dipeptidyl Peptidase IV Inhibitor Protects Against Tacrolimus-Induced Pancreatic and Renal Injury.

Background: Dipeptidyl peptidase IV (DPP IV) inhibitors had a protective effect in various disease models. This study was performed to evaluate whether sitagliptin, a DPP IV inhibitor, is effective against tacrolimus-induced pancreatic and renal injury. Method: Tacrolimus (TAC, 1.5mg/kg/day, s.c.) with or without sitagliptin (10, 20 mg/kg/day, gavage) were administered to rats for 4 weeks. We evaluated the effect of sitagliptin on DPP IV activity and GLP-1 level in tacrolimus-treated rats, and assessed the protective effect of sitagliptin in an experimental model of chronic tacrolimus-induced nephropathy and pancreatic injury. Oxidative stress and oxidative resistance were evaluated by measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG) and heme oxygenase-1 (HO-1). The protective mechanism of sitagliptin was also tested based on the glucagon like peptide-1 (GLP-1)/ (forkhead box O) FoxO pathway. Results: Tacrolimus treatment for 4 weeks caused a 2-fold increase in plasma DPP4 activity and a 5-fold decrease in serum active GLP-1 levels; however, concomitant treatment with sitagliptin reversed these effects. Sitagliptin treatment attenuated tacrolimus-induced pancreatic islet and renal dysfunction, with improvement of islet size and renal tubulointerstitial fibrosis. Tacrolimus treatment increased oxidative stress marker, 8-OHdG and decreased oxidative resistance gene (reduced HO-1), and its underlying pathways (FoxO1 activation and FoxO3 deactivation), whereas sitagliptin treatment reversed these changes. Treatment of primary rat islet and HK-2 cells with GLP-1 also decreased the oxidative injury caused by tacrolimus. Consequently, sitagliptin upregulated antioxidant enzymes e.g. manganese superoxide dismutase, and downregulated proapoptotic proteins e.g. B-cell lymphoma 2-interacting mediator of cell death and apoptotic cell death assessed by the number of TDT-mediated dUTP-biotin nick end labeling-positive cells. Conclusion:DPP IV inhibition by sitagliptin attenuated TAC-induced pancreatic islet and renal dysfunction and enhanced the GLP-1 signaling pathway, which may be associated with a decrease in oxidative injury and apoptotic cell death. These beneficial effects of the DPP IV inhibitor may be helpful in delaying the onset of TAC-induced diabetes and nephropathy in renal transplant patients.

The Retrograde Delivery of Adenovirus Vector Carrying the Gene for Brain-Derived Neurotrophic Factor Protects Neurons and Oligodendrocytes From Apoptosis in the Chronically Compressed Spinal Cord of twy/twy Mice

The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

Functional recovery in rat spinal cord injury induced by hyperbaric oxygen preconditioning

Background: It is a common belief that neurosurgical interventions can cause inevitable damage resulting from the procedure itself in surgery especially for intramedullary spinal cord tumors. The present study was designed to examine if hyperbaric oxygen preconditioning (HBO-PC) was neuroprotective against surgical injuries using a rat model of spinal cord injury (SCI).Methods: Sprague–Dawley rats were randomly divided into three groups: HBO-PC group, hypobaric hypoxic preconditioning (HH-PC) control group, and normobaric control group. All groups were subjected to SCI by weight drop device. Rats from each group were examined for neurological behavior and electrophysiological function. Tissue sections were analyzed by using immunohistochemistry, TdT-mediated dUTP-biotin nick end labeling, and axonal tract tracing.Results: Significant neurological deficits were observed after SCI and HBO-PC and HH-PC improved neurological deficits 1 week post-injury. The latencies of motor-evoked potential and somatosensory-evoked potential were significantly delayed after SCI, which was attenuated by HBO-PC and HH-PC. Compared with normobaric control group, pretreatment with HBO and hypobaric hypoxia significantly reduced the number of TdT-mediated dUTP-biotin nick end labeling-positive cells, and increased nestin-positive cells. HBO-PC and HH-PC enhanced axonal growth after SCI.Conclusions: In conclusion, preconditioning with HBO and hypobaric hypoxia can facilitate functional recovery and suppress cell apoptosis after SCI and may prove to be a useful preventive strategy to neurosurgical SCI.

Infusion of 4°C normal saline can improve the neurological outcome in a porcine model of cardiac arrest

This study sought to investigate induction of therapeutic hypothermia using ice-cold intravenous fluid after cardiopulmonary resuscitation (CPR). The effects on temperature, hemodynamics, cognitive performance and the accompanying neurohistopathological changes, and apoptosis were assessed. Fourteen piglets had 4 minutes of untreated ventricular fibrillation, followed by CPR. The animals in which spontaneous circulation was restored were randomly assigned to two groups: the hypothermia group (n = 7) was given an infusion of 4°C cold normal saline solution 30 mL/kg at an infusion rate of 1.33 mL/kg/min, followed by 10 mL/kg/h to 4 hours after restoration of spontaneous circulation; the control group (n = 7) was given the same infusion at room temperature. Variables were measured repeatedly until 4 hours after restoration of spontaneous circulation. Neurocognitive performance was evaluated 24 hours after CPR. Then animals were killed and the brains were removed for histopathology at 24 hours after restoration of spontaneous circulation. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method was used for apoptosis evaluation. Compared with the control group, the core temperature of the hypothermia group was significantly decreased (p < 0.01). The cerebral performance categories at 24 hours after restoration of spontaneous circulation in the hypothermia group were better than that in the control group (p < 0.05). The percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells in the cortex and dentate gyrus of the hippocampus were significantly reduced in the hypothermia group compared with the control group at 24 hours after restoration of spontaneous circulation. By observation of transmission electron microscopy, the neuron damages were significantly reduced in hypothermia group. 4°C normal saline solution is a safe and effective method to reduce brain damages and prevent apoptotic cell death after cardiac arrest.

Transient Protective Effect of B-Vitamins in Experimental Epilepsy in the Mouse Brain

The regulation of programmed cell death in the nervous system of vertebrates is a complex mechanism aimed to remove superfluous or damaged cells. Epileptic seizures can lead to an activation of pathways resulting in neuronal cell death. B-vitamins might have a neuroprotective potential reducing cell death following appropriate stimulation. Here, the role of the B-vitamins B(1) (thiamine), B(6) (pyridoxine), and B(12) (cobalamine) was investigated in a mouse model of experimental epilepsy induced by kainate. B-vitamin pre-treated animals showed a significantly reduced epileptic score during the first 15 min after kainate injection. The molecular response to kainate showed a bi-phased time course with early induction of Bcl-2 expression within 12 h and a second induction after 7 days of kainate exposure. B-vitamin pre-treatment resulted in significant higher Bcl-2 expression in control animals (no kainate) and at 12 h within the early phase. Bcl-2 expression was not affected by B-vitamins within the second phase. BAX expression was not significantly influenced during the whole experiment. Three days after kainate stimulation, the number of TdT-mediated dUTP-biotin nick end labeling-positive cells in the hippocampal region was lower in B-vitamin-treated animals. Therefore, B-vitamin pre-treatment may attenuate the response to epileptic stimulation.

Role of Nitric Oxide in the Pathogenesis of Encephalomyocarditis Virus-Induced Diabetes in Mice

The D variant of encephalomyocarditis virus (EMC-D virus) causes diabetes in mice by destroying pancreatic beta cells. In mice infected with a low dose of EMC-D virus, macrophages play an important role in beta-cell destruction by producing soluble mediators such as interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). To investigate the role of NO and inducible NO synthase (iNOS) in the development of diabetes in EMC-D virus-infected mice, we infected iNOS-deficient DBA/2 mice with EMC-D virus (2 x 10(2) PFU/mouse). Mean blood glucose levels in EMC-D virus-infected iNOS-deficient mice and wild-type mice were 205.5 and 466.7 mg/dl, respectively. Insulitis and macrophage infiltration were reduced in islets of iNOS-deficient mice compared with wild-type mice at 3 days after EMC-D virus infection. Apoptosis of beta cells was decreased in iNOS-deficient mice, as evidenced by reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. There were no differences in mRNA expression of antiapoptotic molecules Bcl-2, Bcl-xL, Bcl-w, Mcl-1, cIAP-1, and cIAP-2 between wild-type and iNOS-deficient mice, whereas expression of proapoptotic Bax and Bak mRNAs was significantly decreased in iNOS-deficient mice. Expression of IL-1beta and TNF-alpha mRNAs was significantly decreased in both islets and macrophages of iNOS-deficient mice compared with wild-type mice after EMC-D virus infection. Nuclear factor kappaB was less activated in macrophages of iNOS-deficient mice after virus infection. We conclude that NO plays an important role in the activation of macrophages and apoptosis of pancreatic beta cells in EMC-D virus-infected mice and that deficient iNOS gene expression inhibits macrophage activation and beta-cell apoptosis, contributing to prevention of EMC-D virus-induced diabetes.

Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver #

Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury, and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and antiinflammatory molecule with antiapoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days with 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production, and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate-buffered saline (PBS). Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal protein adducts, expression of hepatic tumor necrosis factor alpha, and resulted in hepatic steatosis and increased plasma aspartate aminotransferase and alanine aminotransferase. In ethanol-fed mice, treatment with rhTrx reduced 4-hydroxynonenal adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride, and improved liver enzyme profiles. Ethanol feeding also increased transferase-mediated dUTP-biotin nick-end labeling-positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis.

H5N1 Avian Influenza Virus Induces Apoptotic Cell Death in Mammalian Airway Epithelial Cells

In recent years, the highly pathogenic avian influenza virus H5N1 has raised serious worldwide concern about an influenza pandemic; however, the biology of H5N1 pathogenesis is largely unknown. To elucidate the mechanism of H5N1 pathogenesis, we prepared primary airway epithelial cells from alveolar tissues from 1-year-old pigs and measured the growth kinetics of three avian H5 influenza viruses (A/Crow/Kyoto/53/2004 [H5N1], A/Duck/Hong Kong/342/78 [H5N2], and A/Duck/Hong Kong/820/80 [H5N3]), the resultant cytopathicity, and possible associated mechanisms. H5N1, but not the other H5 viruses, strongly induced cell death in porcine alveolar epithelial cells (pAEpC), although all three viruses induced similar degrees of cytopathicity in chicken embryonic fibroblasts. Intracellular viral growth and the production of progeny viruses were comparable in pAEpC infected with each H5 virus. In contrast, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were detected only in H5N1-infected pAEpC, and the activities of caspases 3, 8, and 9 were significantly elevated in pAEpC infected with H5N1, but not with H5N2 and H5N3. These results suggest that only H5N1 induces apoptosis in pAEpC. H5N1 cytopathicity was inhibited by adding the caspase inhibitor z-VAD-FMK; however, there were no significant differences in viral growth or release of progeny viruses. Further investigations using reverse genetics demonstrated that H5N1 hemagglutinin protein plays a critical role in inducing caspase-dependent apoptosis in infected pAEpC. H5N1-specific cytopathicity was also observed in human primary airway epithelial cells. Taken together, these data suggest that avian H5N1 influenza virus leads to substantial cell death in mammalian airway epithelial cells due to the induction of apoptosis.

Acid sphingomyelinase deficiency increases susceptibility to fatal alphavirus encephalomyelitis.

Sindbis virus (SV), an enveloped virus with a single-stranded, plus-sense RNA genome, is the prototype alphavirus in the Togaviridae family. In mice, SV infects neurons and can cause apoptosis of immature neurons. Sphingomyelin (SM) is the most prevalent cellular sphingolipid, is particularly abundant in the nervous systems of mammals, and is required for alphavirus fusion and entry. The level of SM is tightly regulated by sphingomyelinases. A defect in acid sphingomyelinase (ASMase) results in SM storage and subsequent intracellular accumulation of SM. To better understand the role of the SM pathway in SV pathogenesis, we have characterized SV infection of transgenic mice deficient in the ASMase gene. ASMase knockout (ASM-KO) mice were more susceptible to SV infection than wild-type (WT) or heterozygous (Het) animals. Titers of SV were higher in the brains of ASM-KO mice than in the brains of WT mice. More SV RNA was detected by in situ hybridization, more SV protein was detected by immunohistochemistry, and more terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were present in the cortex and hippocampus of ASM-KO mice than in those of WT or Het mice. Interleukin-6 (IL-6), but not IL-1beta or tumor necrosis factor alpha, was elevated in infected ASM-KO mice compared to levels in WT or Het mice, but studies with IL-6-KO mice and recombinant SV expressing IL-6 showed no role for IL-6 in fatal disease. Together these data indicate that the increase in susceptibility of ASM-KO mice to SV infection was the result of more-rapid replication and spread of SV in the nervous system and increased neuronal death.

Early signs of neuronal apoptosis in the substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson’s disease

Parkinson’s disease is associated with a progressive loss of substantia nigra pars compacta dopaminergic neurons. The cellular and molecular mechanisms underlying Parkinson’s disease neurodegeneration have not been fully determined. Clinical investigations and subacute in vivo studies using the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have generated some observations suggesting that apoptosis is involved in neurodegeneration; however, this view remains equivocal. In this study, the substantia nigra pars compacta neurodegenerative process was examined in the chronic mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson’s disease treated with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) and probenecid (250 mg/kg) over five weeks. One day after chronic treatment, numerous terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were detected specifically in the substantia nigra pars compacta displaying shrunken volume, chromatin condensation, and DNA fragmentation. The number of apoptotic cells declined over time. No terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were found in untreated or probenecid-treated control animals. Cytomorphometric analysis of substantia nigra pars compacta nuclear loci revealed eccentric nucleoli dislocation and vesicular degranulation in all of the apoptotic neurons for the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson’s disease. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells phenotypically showed neuronal origin (NeuN-positive) with a loss of tyrosine hydroxylase immunoreactivity. While the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were not co-localized with astroglial (GFAP-positive) cells, some apoptotic cells were clearly associated with the activated microglial (macrophage antigen complex-1 and isolectin B 4-positive) cells suggesting an active process of dead cell removal. In the one-day and seven-day post-treated mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson’s disease, marked depression of tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and striatum was observed, which was correlated with significant reductions of striatal dopamine content and uptake. These results suggest that initial neuronal apoptosis and morphological changes are involved, at least in part, in the chronic neurodegeneration of mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson’s disease.

Age-related neural degeneration in nuclear-factor κB p50 knockout mice

Nuclear factor-κB is a transcription factor that regulates a variety of genes involved not only with immune and inflammatory responses, but also in cell survival. Nuclear-factor κB in the CNS is an area of current research interest; however, its role in age-related neural degeneration is obscure. The present study examines developmental degeneration changes in wild type and nuclear factor-κB p50 subunit knockout mice (p50 −/−) using various morphological methodologies. P50 −/− mice appeared normal at birth. At 6 and 10 months old, the body weight of p50 −/− mice was significantly less than that of wild type mice and they started to die from aging. Consistently, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling positive cells in the cortex were significantly more in p50 −/− mice than that in wild type mice, and neuronal cells in the cortex, hippocampus and caudate nucleus-putamen decreased in p50 −/− mice. Fewer myelinated axons of the optic nerve were found in p50 −/− mice than in wild type mice at 6 months. In p50 −/− mice, morphological examinations showed: 1) aging and degenerative changes in the cortex and hippocampus including increased lipofuscin granules in neural cytoplasm, 2) abnormal capillaries, 3) dark and watery alterations and organelle accumulations, 4) apoptotic glia cells, and 5) terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling and caspase-3 positive neurons. These results suggest that nuclear-factor κB may play an important role in neurovascular development, cell survival, and the aging process in the CNS. This new evidence linking nuclear-factor κB to myelination and aging may be of considerable importance for several areas of basic and clinical neuroscience.

Massive cell death of cerebellar granule neurons accompanied with caspase-3-like protease activation and subsequent motor discoordination after intracerebroventricular injection of vincristine in mice

Vincristine, a microtubule-depolymerizing agent, is known to induce neuronal cell damage. Biochemical, histological and behavioral alterations were investigated after intracerebroventricular injection of vincristine in mice. Intracerebroventricular injection of vincristine caused caspase-3-like protease activation followed by nucleosomal release in the cerebellum. Histological examinations showed that vincristine-induced damage was relatively specific to granule cells in the cerebellum, and terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick-end labeling-positive cells were observed among these cells. Chromatin condensation, one of the criteria for apoptosis, was seen on electron microscopy. Behavioral changes, namely head movements, pivoting and backward walking, were observed in parallel with the increase of caspase-3-like protease activity and nucleosomal release. Furthermore, motor function tests (bulb balance test and rotating rod test) showed deficits of motor coordination ability. These observations suggest that intracerebroventricular vincristine causes massive apoptosis of cerebellar granule cells accompanied with caspase-3-like protease activation, leading to motor dysfunction, in this model. These vincristine-treated mice should be a useful in vivo model for examination of neuronal apoptosis, which might be involved in a variety of neurodegenerative diseases.

Blockade of Smooth Muscle Cell Migration and Proliferation in Baboon Aortic Explants by Interleukin-1β and Tumor Necrosis Factor-α Is Nitric Oxide-Dependent and Nitric Oxide-Independent

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) are found in injured and atherosclerotic vessels and have been shown to influence smooth muscle cell (SMC) function in vitro. We have investigated the effects of IL-1β and TNFα on SMC migration and proliferation in baboon aortic explants, an in vitro model of arterial injury. Because platelet-derived growth factor (PDGF) is also present in the vessel wall, we have studied the interaction of PDGF with the cytokines. IL-1β and TNFα inhibited migration of SMCs and synthesis of DNA by SMCs. Cell death (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells and total DNA) was not altered by the cytokines. The cytokines increased levels of nitrite in the medium and L-nitroarginine partly reversed the inhibitory effects of the cytokines indicating a role for nitric oxide in these inhibitory effects. Treatment with indomethacin partially reversed the inhibition of migration, but not DNA synthesis by IL-1β suggesting cyclooxygenase products play an inhibitory role in migration. PDGF-BB reversed the inhibitory effect of the cytokines on SMC migration, but not mitogenesis, without changing levels of nitrite in the medium. These data show that IL-1β and TNFα decrease primate SMC migration and proliferation in arterial tissue partly through production of NO, and that PDGF antagonizes the effect of the cytokines. IL-1β and TNFα may act directly to limit injury-induced intimal hyperplasia by decreasing SMC migration and proliferation.

Oligodendrocyte Programmed Cell Death and Central Myelination Deficiency Induced in Transgenic Mice by Synergism between c-Myc and Oct-6

The basic helix-loop-helix transcription factor c-Myc is a potent trigger of programmed cell death when overexpressed during late oligodendrocyte development in transgenic mice. Here we provide evidence that c-Myc can act synergistically with the Pit, Oct, Unc homeodomain transcription factor Oct-6 to produce myelin disease pathogenesis in transgenic mice. More than 70% of c-myc/Oct-6 bitransgenic mice, obtained from crosses between phenotypically normal heterozygous mice of various My (c-Myc) and Oc (Oct-6) transgenic strains that express c-myc and oct-6 transgenes under transcriptional control of the myelin basic protein gene, developed severe neurological disturbances characterized by action tremors, recurrent seizures, and premature death. Affected bitransgenic mice exhibited multiple hypomyelinated lesions in the white matter that did not stain with myelin-specific antibodies against myelin basic protein, proteolipid protein, CNPase, and myelin-associated glycoprotein. The mice also exhibited a larger number of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling positive cells in the white matter as well as ultrastructural evidence of glial cell death and astrogliosis. These observations indicate that the myelin lesions observed in the c-myc/oct-6 bitransgenic mice result from the untimely programmed cell death of oligodendroglia and that the c-myc and oct-6 transgenes act synergistically in producing the lesions.

Apoptosis and proliferation of human hepatocellular carcinoma.

To investigate the contribution of apoptosis, a major mechanism of cell death, in the growth of hepatocellular carcinoma, we analyzed both apoptosis and cell proliferation in human hepatocellular carcinoma. We used the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method and proliferative cell nuclear antigen staining, respectively. Among 21 hepatocellular carcinoma specimens examined, four were well, ten were moderately, and seven were poorly differentiated hepatocellular carcinoma. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells in hepatocellular carcinoma were scattered individually or were sometimes clustered in the tumors. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling indices were 0.35 +/- 0.09, 0.81 +/- 0.29, and 1.9 +/- 0.94 in well, moderately, and poorly differentiated hepatocellular carcinoma, respectively. The proliferative cell nuclear antigen labeling indices were 6.6 +/- 0.9, 13.1 +/- 3.5, and 26.7 +/- 6.3 in hepatocellular carcinoma in the same respective order of differentiation. The differences in both terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling indices and proliferative cell nuclear antigen labeling indices (p < 0.05) were significant between well, moderately and poorly differentiated hepatocellular carcinoma. There was a positive correlation between the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen labeling indices in hepatocellular carcinoma (r = 0.84, p < 0.001). This study showed that the proliferation rate and the incidence of apoptosis increased as the differentiation grade of hepatocellular carcinoma was lowered, suggesting a rapid turnover of cancer cells in the lower differentiation grades. Apoptosis may thus play an important role in the growth of hepatocellular carcinoma.