Terminal dUTP Nick-end Labeling-positive Cells Research Articles

Protective Effects of MiR-129-5p on Acute Spinal Cord Injury Rats.

BackgroundSpinal cord injury (SCI) is a severe devastating condition associated with serious disability and neurologic deficits. Aberrant micro RNA (miRNA) expression has been related to a variety of central nervous system diseases including SCI. In the present study, we aimed to discover the role of miR-129-5p on SCI.Material/MethodsAn acute SCI rat model was induced, following the modified Allen method. A total of 36 rats were randomly assigned into 4 groups (n=9 in every group): Sham group; Model group (SCI+saline); SCI+NC group; and SCI+miR-129-5p group (100 nm solution, every 2 days). Basso-Beattie-Bresnahan (BBB) locomotor rating score was carried out to determine functional recovery. TUNEL (terminal dUTP nick-end labeling) staining was used to evaluate cell apoptosis. Hematoxylin and eosin staining was performed to assess the pathological state of spinal cord. Furthermore, western blot assay was conducted to measure the calpain1 and calpain2 expression.ResultsOur data suggested that the expression level of miR-129-5p was markedly reduced in rats after SCI. Then miR-129-5p mimic was injected into the vertebral canal. We found that the SCI+miR-129-5p group had a high score in the BBB test compared with the SCI+NC group and the Model group. The overexpression of miR-129-5p obviously reduced tissue loss, damaged cells, and the number of TUNEL positive cells. Moreover, western blot assay exhibited that overexpression of miR-129-5p decreased calpain1, calpain2, and cleaved caspase-3 expression.ConclusionsOur findings suggested that overexpression of miR-129-5p improved neurological function by promoting functional recovery, reducing tissue loss and cell apoptosis in rats in an SCI model, possibly through downregulation of calpain1 and calpain2.

Berbamine ameliorates isoproterenol-induced myocardial infarction by inhibiting mitochondrial dysfunction and apoptosis in rats.

Berbamine (BBM), a bisbenzylisoquinoline alkaloid from roots, bark, and stem of Berberis plant such as Berberis aristata has a wide range of pharmacological activities. However, the evidence for the cardioprotective effect of BBM is inadequate and the molecular mechanism of BBM remains unclear. This study investigated the underlying molecular mechanism of BBM-mediated cardioprotection on isoproterenol (ISO)-induced mitochondrial dysfunction and apoptosis in rats. The assays of mitochondria antioxidant status, mitochondrial marker enzymes, and electron microscopic analysis of mitochondria revealed BBM significantly prevented the mitochondrial dysfunction induced by ISO. The ISO-induced elevation of mitochondrial oxidative stress was also curbed by BBM. Furthermore, pretreatment with BBM protected the heart tissue from ISO-induced apoptosis as evident from decreased terminal dUTP nickend-labeling positive cells and decreased expression of Bax, cytochrome c, cleaved caspase-9, and caspase-3, and poly (ADP-ribose) polymerase and increased expression of Bcl-2 in ISO-induced rats. These current findings suggest that BBM exerts a significant cardioprotective effect on ISO-induced myocardial infarction in rats.

Sildenafil Mediates Blood-Flow Redistribution and Neuroprotection After Neonatal Hypoxia-Ischemia

The best conceivable treatment for hypoxia-ischemia (HI) is the restoration of blood flow to the hypoxic-ischemic region(s). Our objective was to examine whether boosting NO-cGMP signaling using sildenafil citrate, a phosphodiesterase-type 5 inhibitor, could modify cerebral blood flow and reduce lesions in the developing brain. HI was induced in P7 Sprague-Dawley rats by unilateral carotid artery occlusion and hypoxia, and followed by either PBS or sildenafil. Blood-flow velocities were measured by ultrasound imaging with sequential Doppler recordings to evaluate collateral recruitment. Cell death, blood-brain barrier integrity, and glial activation were analyzed by immunohistochemistry. Motor behavior was evaluated using an open-field device adapted to neonatal animals. Sildenafil citrate (10 mg/kg) induced collateral patency, reduced terminal dUTP nick-end labeling-positive cells, reactive astrogliosis, and macrophage/microglial activation at 72 hours and 7 days post-HI. Sildenafil also reduced the number of terminal dUTP nick-end labeling-positive endothelial cells within lesion site. Seven days after HI and sildenafil treatment, tissue loss was significantly reduced, and animals recovered motor coordination. Our findings strongly indicate that sildenafil citrate treatment, associated with a significant increase in cerebral blood flow, reduces HI damage and improves motor locomotion in neonatal rats. Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.

Neuroprotective effects of diallyl sulfide against transient focal cerebral ischemia via anti-apoptosis in rats

Objectives: Diallyl sulfide (DAS) is the main organosulfur component of garlic and it is known for multiple pharmacological actions. Recent studies have demonstrated that DAS has neuroprotective effects against ischemia/reperfusion injury. While some of the possible mechanisms behind this protection have been explored, its ability to inhibit apoptosis has yet to be fully explained. In the present study, the effects of DAS on focal cerebral ischemia in rats were tested and its anti-apoptotic action was explored.Methods: To examine the protective effects of DAS, focal cerebral ischemia/reperfusion was induced in rats by transient middle cerebral artery occlusion for 2 hours followed by reperfusion for 24 hours. The animals received DAS in quantities of 100, 150, and 200 mg/kg (intraperitoneal; every day), for 7 days before transient middle cerebral artery occlusion. The neurological score and infarct volume were measured at 24 hours after the end of reperfusion. Apoptotic cells were counted by terminal dUTP nick end labeling staining and apoptotic mechanisms were studied by fluorescence immunohistochemistry staining and western blot analysis.Results: For animals with induced ischemia/reperfusion, those pretreated with 200 mg/kg DAS showed an infarct volume (22·36±0·67%) significantly lower than that of the non-treated ischemia/reperfusion group (38·23±0·72%), and the percentage of terminal dUTP nick-end labeling-positive cells (23·46±1·02%) of the DAS-pretreated group was also significantly decreased compared to non-treated (36·41±1·58%). Fluorescence immunohistochemistry staining and western blot analysis indicated that DAS reduced caspase-3 expression and increased Bcl-2 expression.Conclusion: These results suggest that the mechanism by which DAS protects the brain from ischemia/reperfusion injury is related to its anti-apoptotic effects in part.

3,3′-diindolylmethane inhibits prostate cancer development in the transgenic adenocarcinoma mouse prostate model

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol, which is present in cruciferous vegetables and has been reported to possess anti-carcinogenic properties. In the present study, we examined whether DIM inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen and proliferating cell nuclear antigen, and increased the number of terminal dUTP nick-end labeling-positive cells in the dorsolateral lobes of the prostate. Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate cancer cells were cultured with various concentrations of DIM. DIM induced a substantial reduction in the numbers of viable cells and induced apoptosis in LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly (ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability and the translocation of cytochrome c and Smac/Diablo from the mitochondria. Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid. These results indicate that DIM inhibits prostate carcinogenesis via induction of apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

Glyoxalase I Is Critical for Human Retinal Capillary Pericyte Survival under Hyperglycemic Conditions

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

Changes of apoptosis-related proteins in hippocampus of SAM mouse in development and aging

Expression of Caspase and Bcl-2 proteins was examined in the hippocampus of senescence-accelerated mice (SAM, P8 and R1 strain) from E19 to 16 months of age. Immunoblotting analysis showed no upregulation of pro-apoptotic proteins (caspase-2L, -3, -6, -8, -9, and Bax) with age while all the anti-apoptotic proteins (caspase-2S, Bcl-2, and Bcl-XL) remained unchanged during aging. Terminal dUTP nick end labeling (TUNEL) and electron microscopy on the hippocampus of 3- and 16-month-old SAM revealed very few TUNEL positive cells in both groups. Morphometric study further showed neuronal loss in the hippocampus was not age-related. Our results suggest apoptosis and cell loss are minor events in the hippocampus of SAM mice and are unlikely to be the cause of functional decline during aging in SAM.

Lung Alveolar Septation Defects in Ltbp-3-Null Mice

Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) modulate the secretion and activation of latent TGF-beta. To explore LTBP function in vivo, we created an Ltbp-3(-/-) mouse that has developmental emphysema with decreased septation in terminal alveoli. Differences in distal airspace enlargement were obvious at day 6 after birth. Secondary septation was inhibited, so by days 21 to 28 the mean linear intercept was approximately twofold greater in mutant versus control lungs. There were no differences in lung collagen and elastin, visualized by immunohistochemistry, or in myofibroblast numbers, determined by alpha-smooth muscle actin-positive cells, between mutant or wild-type lungs as the animals aged, other than differences associated with altered lung structure in mutant animals. However, from day 10 there was twice the number of alveolar type II cells in mutant alveoli compared to controls. At days 6 and 10, a transient enhancement in cell proliferation in the mutant lungs was observed by both 5-bromo-2'-deoxy-uridine and proliferating cell nuclear antigen labeling, accompanied by enhanced numbers of terminal dUTP nick-end labeling-positive cells at days 4, 6, and 10. Finally, there was a transient decrease in TGF-beta signaling at days 4 to 6 in Ltbp-3(-/-) lungs. These results indicate that in the absence of Ltbp-3, a temporary decrease in TGF-beta signaling in the lungs at days 4 to 6 alters cell proliferation, correlating with inhibition of septation and developmental emphysema.

Superoxide Dismutase Inactivation in Pathophysiology of Asthmatic Airway Remodeling and Reactivity

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Furthermore, oxidation and nitration of MnSOD were identified in the asthmatic airway, correlating with physiological parameters of asthma severity. These findings link oxidative and nitrative stress to loss of SOD activity and downstream events that typify asthma, including apoptosis and shedding of the airway epithelium and hyperresponsiveness.

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, ΔC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.

Protection from Lethal Apoptosis in Lipopolysaccharide-Induced Acute Lung Injury in Mice by a Caspase Inhibitor

LPS (lipopolysaccharide) is one of the major factors that induce acute lung injury. Recently, it was reported that LPS induced disseminated endothelial apoptosis, preceding nonendothelial tissue damage. Caspases play important roles in apoptosis, including tumor necrosis factor-alpha-induced apoptosis, in several systems. We therefore investigated whether the injection of a caspase inhibitor prevents LPS-induced apoptosis and acute lung injury in mice. LPS (30 mg/kg) was administered intravenously to Institute for Cancer Research mice. Electron microscopic findings demonstrated characteristic features of apoptosis in endothelial cells and alveolar epithelial cells. The caspase-3 activity and the number of terminal dUTP nick-end labeling-positive cells in lung tissues were significantly increased after LPS administration. Benzyloxycarbonil-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), which is a broad-spectrum caspase inhibitor, was injected before and after the administration of LPS. The injection of Z-VAD.fmk suppressed the caspase-3 activity in lung tissues, and significantly decreased the number of terminal dUTP nick-end labeling-positive cells. Furthermore, the survival rate of mice was prolonged significantly by the injection of Z-VAD.fmk. These results indicate that apoptosis may play an important role in acute lung injury, and thus that inhibition of caspase activity may constitute a new therapeutic approach for treatment of this disease.