Terminal dUTP Nick-end Labeling Staining Research Articles

Transfer of microRNA-486-5p from human endothelial colony forming cell–derived exosomes reduces ischemic kidney injury

Administration of human cord blood endothelial colony-forming cells (ECFCs) or their exosomes protects mice against kidney ischemia/reperfusion injury. Here we studied the microRNA (miRNA) content of ECFC exosomes and the role of miRNA transfer in kidney and endothelial cell protection. ECFC exosomes were enriched in miR-486-5p, which targets the phosphatase and tensin homolog (PTEN) and the Akt pathway. In cultured endothelial cells exposed to hypoxia, incubation with ECFC exosomes increased miR-486-5p, decreased PTEN, and stimulated Aktphosphorylation. Exposure of hypoxic endothelial cellsto conditioned medium from ECFCs pretreated with anti-miR-486-5p blocked increases in miR-486-5p and phosphorylated Akt, restored expression of PTEN, and enhanced apoptosis. Coculture of endothelial cells with ECFCs enhanced endothelial miR-486-5p levels. Targeting of PTEN by miR-486-5p was observed in endothelial cells, and PTEN knockdown blocked apoptosis. In mice with ischemic kidney injury, infusion of ECFC exosomes induced potent functional and histologic protection, associated with increased kidney miR-486-5p levels, decreased PTEN, and activation of Akt. Infusion of exosomes from ECFCs transfected with anti-miR-486-5p had no protective effect. Thus, delivery of ECFC exosomes reduces ischemic kidney injury via transfer of miR-486-5p targeting PTEN. Exosomes enriched in miR-486-5p could represent a therapeutic tool in acute kidney injury.

Hydrogen Gas Inhalation Attenuates Seawater Instillation-Induced Acute Lung Injury via the Nrf2 Pathway in Rabbits.

Seawater instillation-induced acute lung injury involves oxidative stress and apoptosis. Although hydrogen gas inhalation is reportedly protective in multiple types of lung injury, the effect of hydrogen gas inhalation on seawater instillation-induced acute lung injury remains unknown. This study investigated the effect of hydrogen gas on seawater instillation-induced acute lung injury and explored the mechanisms involved. Rabbits were randomly assigned to control, hydrogen (2% hydrogen gas inhalation), seawater (3mL/kg seawater instillation), and seawater + hydrogen (3mL/kg seawater instillation + 2% hydrogen gas inhalation) groups. Arterial partial oxygen pressure and lung wet/dry weight ratio were detected. Protein content in bronchoalveolar lavage fluid (BALF) and serum as well as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 levels were determined. Hematoxylin-eosin staining was used to monitor changes in lung specimens, and malondialdehyde (MDA) content and myeloperoxidase (MPO) activity were assayed. In addition, NF-E2-related factor (Nrf) 2 and heme oxygenase (HO)-1 mRNA and protein expression were measured, and apoptosis was assessed by measuring caspase-3 expression and using terminal deoxy-nucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. Hydrogen gas inhalation markedly improved lung endothelial permeability and decreased both MDA content and MPO activity in lung tissue; these changes were associated with decreases in TNF-α, IL-1β, and IL-6 in BALF. Hydrogen gas also alleviated histopathological changes and cell apoptosis. Moreover, Nrf2 and HO-1 expressions were significantly activated and caspase-3 expression was inhibited. These results demonstrate that hydrogen gas inhalation attenuates seawater instillation-induced acute lung injury in rabbits and that the protective effects observed may be related to the activation of the Nrf2 pathway.

Total flavonoid extract from Coreopsis tinctoria Nutt. protects rats against myocardial ischemia/reperfusion injury.

This study aimed to evaluate the protective effects of total flavonoid extract from Coreopsis tinctoria Nutt. (CTF) against myocardial ischemia/reperfusion injury (MIRI) using an isolated Langendorff rat heart model. Left ventricular developed pressure (LVDP) and the maximum rate of rise and fall of LV pressure (±dp/dtmax) were recorded. Cardiac injury was assessed by analyzing lactate dehydrogenase (LDH) and creatine kinase (CK) released in the coronary effluent. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels were determined. Myocardial inflammation was assessed by monitoring tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), interleukin-8 (IL-8), and interleukin-6 (IL-6) levels. Myocardial infarct size was estimated. Cell morphology was assessed by 2,3,5-triphenyltetrazolium chloride and hematoxylin and eosin (HE) staining. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Pretreatment with CTF significantly increased the heart rate and increased LVDP, as well as SOD and GSH-Px levels. In addition, CTF pretreatment decreased the TUNEL-positive cell ratio, infarct size, and levels of CK, LDH, MDA, TNF-α, CRP, IL-6, and IL-8. These results suggest that CTF exerts cardio-protective effects against MIRI via anti-oxidant, anti-inflammatory, and anti-apoptotic activities.

Allicin improves cardiac function by protecting against apoptosis in rat model of myocardial infarction.

To study the effects of allicin on cardiac function and underlying mechanism in rat model of myocardial infarction (MI). Ninety-four Wistar rats were randomly assigned to 6 groups (n=14-16 per group): sham control group [underwent thoracotomy without left anterior descending (LAD) occlusion and only received an injection of the same amount of citrate buffer], MI control group (subjected to LAD occlusion and only received an injection of same amount of citrate buffer), positive control group (subjected to LAD occlusion and received an injection of diltiazem hydrochloride at the dose of 1.5 mg/kg), and MI + allicin groups (subjected to LAD occlusion and received an injection of allicin at the doses of 1.2, 1.8, and 3.6 mg/kg). All of the drugs were administered intraperitoneally daily for 21 days. The infarct area was measured by myocardial staining. Hematoxylin-eosin staining was used to observe the pathological changes. Cardiac function parameters were assessed by echocardiography. The myocardial apoptotic index was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. The expression of Bax and Bcl-2 were detected by quantificational real-time polymerase chain reaction and Western blot. Treatment with allicin could attenuate the myocardial infarct area (P<0.05) and relieve the changes of the myocardium. The left ventricular anterior wall diastolic and systolic thicknesses were increased in the allicin-treated groups (P<0.05), while there was no signifificant difference in the left ventricular posterior wall diastolic and systolic thickness (P>0.05). The left ventricular internal diameter in systole, ejection fraction, fractional shortening, and stroke volume were dramatically elevated in allicin-treated rats (P<0.05). Allicin dose-dependently reduced creatine kinase and lactate dehydrogenase levels (P<0.05). The myocardial apoptotic index was also markedly lowered, and Bax expression was signifificantly decreased, whereas Bcl-2 expression exhibited an opposite trend in allicin-treated rats (P<0.05). Allicin appears to exert a cardioprotective effect that may be linked to blocking Bcl-2/Bax signaling pathway-denpendent apoptosis, further improving cardiac function.

Effect of baicalin on hippocampal damage in kainic acid-induced epileptic mice.

The aim of the present study was to determine the effect of baicalin on the expression of miR-497 and its target B-cell lymphoma-2 (Bcl-2) in the hippocampus of kainic acid (KA)-induced epileptic mice. To establish status epilepticus (SE), 0.1 µg/5 µl KA was injected into the lateral cerebral ventricle in mice, which then received an intraperitoneal injection of baicalin (100 mg/kg) after 1 and 8 h. Hematoxylin and eosin staining was used to observe the pathological changes in morphology and neuronal apoptosis was determined by terminal transferase-mediated dUTP nick end-labeling staining. Western blot analysis was used to detect the expression of Bcl-2 and cleaved caspase-3 proteins in the hippocampus, while reverse transcription-quantitative polymerase chain reaction was used to quantify hippocampal miR-497 expression. The results showed that baicalin significantly attenuated neuronal damage and apoptosis in the hippocampus 72 h after SE. In addition, baicalin decreased SE-induced expression of miR-497 and cleaved caspase-3 protein, while upregulating the expression of Bcl-2 protein. In conclusion, the present results suggest that baicalin possesses potent antiapoptotic properties and attenuates hippocampal injury in mice after SE, which may be associated with the downregulation of miR-497 and cleaved caspase-3 and the upregulation of Bcl-2.

Subclinical concentrations of sevoflurane reduce oxidative stress but do not prevent hippocampal apoptosis.

Sevoflurane is generally considered a pro-apoptotic agent in the neonatal brain. However, recent studies have suggested that low levels of sevoflurane anesthesia may be neuroprotective and have a memory enhancing effect. The present study aimed to investigate whether sevoflurane exerts a neuroprotective effect at subclinical concentrations, with regard to oxidative state. In the current study, postnatal day 7 (P7) Sprague-Dawley rats were continuously exposed to 0.3, 1.3, or 2.3% sevoflurane for 6 h. ELISA was used to quantify the levels of superoxide dismutase, glutathione peroxidase (GSH-px) and malondialdehyde (MDA) in the plasma and the hippocampus. Terminal deoxynucleotidyl-transferase dUTP nick-end labeling staining was used to observe hippocampal neuronal apoptosis. Altered object exploration tests for recognition memory were employed to investigate long-term behavioral effects at postnatal day 28. The results demonstrated that a single 6 h exposure to a subclinical concentration (1.3%) of sevoflurane at P7 reduces MDA and GPH-px production in rats. Sevoflurane induced hippocampal apoptosis in a dose-dependent manner and altered recognition memory testing indicated no differences among the groups. Although early exposure to a subclinical concentration of sevoflurane reduced oxidative stress, it did not prevent the process of sevoflurane-induced hippocampal apoptosis. These changes did not affect subsequent recognition memory in juvenile rats.

House dust mite–induced asthma causes oxidative damage and DNA double-strand breaks in the lungs

Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) invivo and invitro. We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX)foci in bronchial epithelium. BEAS-2Bcells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology.

Effects of selenium on ischaemia-reperfusion injury in a rat testis model.

Selenium is shown to have beneficial effects on ischaemia-reperfusion (IR) injury. Our aim was to assess the effects of selenium on IR-induced testicular damage in terms of biochemical and histopathological evaluation. A total of 32 rats were randomised into four groups: control, IR, IR+selenium (IR+S) and S. Detorsion was applied after 3h of torsion. Testicular tissue superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), total antioxidant capacity (TAC) and DNA fragmentation levels were determined. Testicular tissue samples were examined by histopathological examination and terminal deoxynucleotidyl transferase dUTP nick end-labelling staining. The control, IR and IR+S groups had higher SOD values compared with the S group; SOD levels of the control and IR+S groups were higher than those of the IR group (P<0.05). Further, MDA levels of the IR group were higher than those in the other three groups (P<0.05). The IR group revealed lower TAC levels than the three groups (P<0.05 for all). GSH levels of the IR group were significantly lower than those in the other three groups (P<0.05 for all). In contrast, GSH levels of the IR+S group increased compared with those of the S group. The IR group had more DNA fragmentation than the control and S groups (P<0.05). It is concluded that selenium possibly reduces oxidative stress and apoptosis caused by testicular IR injury in rats. The testicular protective effect of selenium appears to be mediated through its anti-apoptotic and antioxidative effects. However, selenium does not affect DNA fragmentation.

Ferulic acid prevents liver injury induced by Diosbulbin B and its mechanism.

The rhizome of Dioscorea bulbifera Linn, traditionally used to treat thyroid disease and cancer in China, is reported to induce serious liver injury during clinical practice. Diosbulbin B (DB), a diterpene lactone, has been found to be the main toxic compound in D. bulbifera. The present study aims to investigate the protection of ferulic acid (FA) against DB-induced acute liver injury and its engaged mechanism. Mice were orally administered FA (20, 40, 80 mg/kg) once daily for 6 consecutive days; and then orally given DB (250 mg/kg) on the last day. Daily FA (40, 80 mg/kg) decreased DB (250 mg/kg)-induced increase in serum levels of alanine/aspartate aminotransferase (ALT/AST) and alkaline phosphatase (ALP). Histological evaluation showed that FA (80 mg/kg) ameliorated DB-induced hepatocellular degeneration and lymphocyte infiltration. Results of terminal dUTP nick-end labeling (TUNEL) staining assay showed that FA (80 mg/kg) decreased the DB-increased number of apoptotic hepatocytes. FA (40, 80 mg/kg) reduced DB-increased liver malondialdehyde (MDA) amount. FA (40, 80 mg/kg) decreased DB-increased serum levels of tumor necrosis factor alpha (TNF-α) and interferon-γ (IFN-γ), and liver myeloperoxidase (MPO) activity. FA (80 mg/kg) reversed the DB-induced decrease in expression of inhibitor of kappa B (IκB) and the increase in nuclear translocation of the p65 subunit of nuclear factor kappa B (NFκBp65). Taken together, our results demonstrate that FA prevents DB-induced acute liver injury via inhibiting intrahepatic inflammation and liver apoptosis.

Atorvastatin activates autophagy and promotes neurological function recovery after spinal cord injury.

Atorvastatin, a lipid-lowering medication, provides neuroprotective effects, although the precise mechanisms of action remain unclear. Our previous studies confirmed activated autophagy following spinal cord injury, which was conducive to recovery of neurological functions. We hypothesized that atorvastatin could also activate autophagy after spinal cord injury, and subsequently improve recovery of neurological functions. A rat model of spinal cord injury was established based on the Allen method. Atorvastatin (5 mg/kg) was intraperitoneally injected at 1 and 2 days after spinal cord injury. At 7 days post-injury, western blot assay, reverse transcription-polymerase chain reaction, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining results showed increased Beclin-1 and light chain 3B gene and protein expressions in the spinal cord injury + atorvastatin group. Additionally, caspase-9 and caspase-3 expression was decreased, and the number of TUNEL-positive cells was reduced. Compared with the spinal cord injury + saline group, Basso, Beattie, and Bresnahan locomotor rating scale scores significantly increased in the spinal cord injury + atorvastatin group at 14-42 days post-injury. These findings suggest that atorvastatin activated autophagy after spinal cord injury, inhibited apoptosis, and promoted recovery of neurological function.

Treadmill exercise alleviates chronic mild stress-induced depression in rats.

Depression is a major cause of disability and one of the most common public health problems. In the present study, antidepressive effect of treadmill exercise on chronic mild stress (CMS)-induced depression in rats was investigated. For this, sucrose intake test, immunohistochemistry for 5-bromo-2′-deoxyuridine, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, and Western blot analysis for brain-derived neurotrophic factor, cyclic adenosine monophosphate response element binding protein, and endothelial nitric oxide synthase were conducted. Following adaptation to the animal vivarium and two baseline fluid intake tests, the animals were divided into four groups: the control group, the CMS-induced depression group, the CMS-induced depression and exercise group, and the CMS-induced depression and fluoxetine-treated group. The animals in the CMS groups were exposed to the CMS conditions for 8 weeks and those in the control group were exposed to the control conditions for 8 weeks. After 4 weeks of CMS, the rats in the CMS-induced depression and exercise group were made to run on a motorized treadmill for 30 min once a day for 4 weeks. In the present results, treadmill exercise alleviated CMS-induced depressive symptoms. Treadmill exercise restored sucrose consumption, increased cell proliferation, and decreased apoptotic cell death. The present results suggest the possibility that exercise may improve symptoms of depression.

Curcumin accelerates reendothelialization and ameliorates intimal hyperplasia in balloon-injured rat carotid artery via the upregulation of endothelial cell autophagy.

Delayed reendothelialization and intimal hyperplasia (IH) contribute to the failure of vascular interventions. Curcumin (Cur) has been used for various types of diseases with antioxidant, antiproliferative and anti-inflammatory effects. However, investigations involving the application of Cur in inhibiting IH are limited. The aim of the present study was to evaluate the potential therapeutic effects of Cur and its underlying mechanisms on a rat model of carotid artery (CA) intimal injury. In vitro, an endothelial cell (EC) migration assay was conducted using cultured primary human umbilical vein endothelial cells (HUVECs) that were exposed to Cur. In vivo, CA angioplasty injury was used to generate a rat model of intimal injury. CAs were collected at 3 days, and 1 and 4 weeks after injury, respectively, for western blot analysis and double-immunofluorescence analyses, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, oxidative stress indicator analysis and hematoxylin and eosin staining of the neointima. In vivo, Cur significantly enhanced the migration and healing of HUVECs and simultaneously promoted microtubule-associated protein light chain 3-II (LC3-II) expression when HUVECs were subjected to an artificial scratch. In vitro, endangium from the Cur-treated rats exhibited a significantly reduced number of apoptotic ECs and oxidative stress level compared to that of the sham group. In addition, Cur treatment markedly improved quantification of the LC3-II concomitant with the downregulation of p62 in the injured CA. At 1 week following injury, sizable neointimal lesions had developed, although prominent intima thickening was not observed. At 4 weeks, apparent hemadostenosis occurred resulting from the exorbitance IH. Cur treatment markedly reduced the thickness of the neointimal lesion. It is noteworthy that high-dose Cur may have exerted more significant effects than low-dose Cur. Cur can potentially become a therapeutic drug for angiostenosis by imparting a protective effect that accelerates reendothelialization and ameliorates IH and was mediated by its pro-autophagic effect.

Pharmacological preconditioning and postconditioning with nicorandil attenuates ischemia/reperfusion-induced myocardial necrosis and apoptosis in hypercholesterolemic rats.

Pharmacological preconditioning and postconditioning may reduce myocardial necrosis and apoptosis during ischemia/reperfusion (I/R), however, hypercholesterolemia interferes with the associated cardioprotective mechanisms. The present study investigated whether pharmacological preconditioning and postconditioning with nicorandil could attenuate myocardial necrosis and apoptosis induced by I/R in hypercholesterolemic rats, and explored the possible mechanisms involved. Male Wistar rats (n=160) were fed normal (normocholesterolemic group, n=10) or high-cholesterol (hypercholesterolemic group, n=150) diets for 8 weeks. Hearts harvested from the normal and hypercholesterolemic rats were subsequently placed on modified Langendorff perfusion apparatus and 30-min global ischemia was performed, followed by 120-min reperfusion. Nicorandil (1, 3, 10, 30, 100 µmol/l), and mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel blocker 5-hydroxydecanoic acid sodium salt (5-HD) (100 µmol/l) or soluble guanylyl cyclase (sGC) blocker 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µmol/l) were perfused for 10 min, prior to ischemia or at the onset of reperfusion. The myocardial infarct size was determined by triphenyltetrazolium chloride staining, and cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. In order to investigate the potential mechanisms, the expression levels of caspase-3, B-cell lymphoma-2 (Bcl-2) proteins and Bcl-2-associated X protein (Bax) were measured using western blot analysis. The present study demonstrated that, in hypercholesterolemic rats, pharmacological preconditioning and postconditioning with nicorandil decreased I/R-induced myocardial necrosis and apoptosis in a concentration-dependent manner. The optimal preconditioning and postconditioning concentration of nicorandil determined to have anti-infarct and anti-apoptosis effects was 30 µmol/l, which significantly (P<0.05) reduced the infarct size to 14.88±3.25% and 15.96±3.29%, and attenuated the percentage of cardiomyocyte apoptosis to 25.20±3.93% and 26.18±4.82%, respectively, compared with the I/R group. However, the cardioprotective effects of nicorandil were partially suppressed by cotreatment with 5-HD or ODQ. Western blot analysis demonstrated that pharmacological preconditioning and postconditioning with nicorandil significantly downregulated caspase-3 and Bax expression, and upregulated Bcl-2 expression compared with the I/R group (P<0.05). The results of the present study suggest that pharmacological preconditioning and postconditioning with nicorandil may protect hypercholesterolemic hearts against I/R-induced necrosis and apoptosis; and the cardioprotective effects of nicorandil may be due to the dual pharmacological mechanisms of opening the mitoKATP channels and a nitric oxide/sGC-dependent mechanism, and regulation of the expression of caspase-3, Bax and Bcl-2.

Insulin-Like Growth Factor-1 Contributes to Mucosal Repair by β-Arrestin2–Mediated Extracellular Signal-Related Kinase Signaling in Experimental Colitis

Insulin-like growth factor-1 (IGF-1) possesses the ability to attenuate intestinal damage and promote mucosal repair of colitis. β-Arrestins, as the scaffolding proteins of G protein-coupled receptors or non-G protein-coupled receptors signaling, can be involved in IGF-1-mediated signaling pathways. However, the interaction of IGF-1 and β-arrestin2 in the mucosal repair of experimental colitis remains unexplored. Ulcerative colitis was induced in β-arrestin2 wild-type mice and β-arrestin2 knockout littermates by using 3% dextran sulfate sodium for 5 days, followed by regular water consumption for 1, 2, 3, and 4 weeks to analyze the mucosal repair from experimental colitis. Disease activity index and histologic score analyses were performed. Apoptosis and proliferation were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Ki-67 staining, respectively. The expressions of β-arrestin2, phospho (p)-IGF-1R, and p-extracellular signal-regulated kinase (ERK)1/2 were examined. Furthermore, β-arrestin2 was overexpressed or altered in HCT116 cells by transfection before IGF-1 treatment in vitro. IGF-1 and β-arrestin2 expression was up-regulated in the repairing phase of experimental colitis. Targeted deletion of β-arrestin2 delayed the repair of colitis by inhibiting cell proliferation without affecting the levels of IGF-1 and p-IGF-1R. The β-arrestin2/ERK signaling pathway was involved in IGF-1-mediated mucosal repair through promoting epithelial cell and goblet cell regeneration from experimental colitis. These results indicate that IGF-1 contributes to the mucosal repair by β-arrestin2-mediated ERK signaling in experimental colitis.

Astaxanthin reduces matrix metalloproteinase-9 expression and activity in the brain after experimental subarachnoid hemorrhage in rats

We have previously shown that astaxanthin (ATX) reduces the blood–brain barrier (BBB) disruption and neurovascular dysfunction following subarachnoid hemorrhage (SAH) insults. However, the underlying mechanisms remain unclear. It is known that the matrix metalloproteinases (MMPs), especially matrix metalloproteinase-9 (MMP-9) plays a crucial role in the pathogenesis of secondary brain injury after SAH. And ATX has the ability to regulate MMP-9 in other models. Herein, we investigated whether ATX could ameliorate MMP-9 activation and expression in a rat model of SAH. A total of 144 rats were randomly divided into the following groups: control group (n=36), SAH group (n=36), SAH+vehicle group (n=36), and SAH+ATX group (n=36). The SAH model was induced by injection of 0.3ml autologous blood into the prechiasmatic cistern. ATX (20μl of 0.1mmol) or vehicle was administered intracerebroventricularly 30min after SAH induction. Mortality, neurological function, brain edema and blood–brain barrier (BBB) permeability were measured at 24 and 72h after SAH. Biochemical and zymographic methods were used to analyze MMP-9 expression and activity in brain samples. Immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were also evaluated at 24h. Our data indicated that ATX could significantly reduce the expression and activity of MMP-9, leading to the amelioration of brain edema, BBB impairment, neurological deficits and TUNEL-positive cells at 24h but not 72h after SAH. The ATX-mediated down-regulation of MMP-9 was correlated with the decreased levels of IL-1β, TNF-α, oxidative stress, activated microglia and infiltrating neutrophils. These results suggest that the neurovascular protection of ATX in SAH is partly associated with the inhibition of MMP-9 expression and activity.

Impact of the radius of the injector system on the cell viability in descemet membrane endothelial keratoplasty: an ex vivo experimental study.

To evaluate the impact of the radius of the injector system on the viability of the endothelial cells in Descemet membrane endothelial keratoplasty (DMEK). Descemet membranes (DMs) of 30 corneoscleral rims were used in a paired design and divided into three groups (A, B, C). The right corneas were used as control (without shooting through the injector after DM preparation) and the left cornea for simulation of the implantation step (shooting through a glass injector of different radii). The injector in Group A had a diameter of 0.5 mm, group B 0.9 mm and group C 1.4 mm. Prior to preparation, endothelial cell count was measured and endothelial cell quality assessed. Quantification of potential damage was achieved by staining with a viability bioassay (calcein acetoxymethyl and ethidium homodimer-1) and DAPI. Apoptosis was evaluated by TUNEL (terminal dUTP nick-end labelling) staining. The average percentage of the endothelial cell death ratios was 4.9 ± 3.3% in group A (n = 5), 3.7 ± 3.7% in group B (n = 5) and 3.9 ± 3.6% in group C (n = 5) with no significant difference compared to the control. The average percentage of apoptotic endothelial cells was 0.05 ± 0.05%, 0.60 ± 0.53% and 0.15 ± 0.26% in groups A, B and C, respectively, with no significant difference compared to the control. Prepared grafts for transplantation in DMEK procedures may be injected through a smaller sized injector system of up to 0.5 mm with no significant increase in cell loss. Clinical studies are necessary to draw a final conclusion.

Impacts of chronic sleep deprivation on learning and memory, autophagy and neuronal apoptosis in mice

Objective To establish chronic sleep deprivation mouse model, evaluate the learning and memory ability of mice and observe autophagy and apoptosis levels in mouse brain. Methods C57BL/6 mice (n=20) were randomly separated into sleep deprivation group and control group. After 2-month sleep deprivation by using an adapted multiple platform method, the behavioral performance of mice was measured by IntelliCage system. The expression of microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) and Beclin-1 was detected by Western blotting. Confocol microscopy was used to observe autophagosome. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to detect neuronal apoptosis level in mouse brain. Results The results of behavioral test showed that the incorrect visit ratio was much higher in sleep deprivation group than that in control group. Moreover, the expression of LC3-Ⅱ(sleep deprivation group 1.681±0.186, control group 1.125±0.048, t=2.892, P=0.027 6) and Beclin-1(sleep deprivation group 1.144±0.048, control group 1.006±0.017, t=2.721, P=0.018 6) in mouse hippocampus and cortex was significantly elevated in sleep deprivation group than those in control group. Accordingly, the confocal microscopy observation also revealed an increased nuclear LC3-positive puncta in hippocampus and cortex of sleep deprived mice (hippocampus in sleep deprivation group 1.665±0.153, in control group 0.819±0.072, t=5.024, P=0.002 4; cortex in sleep deprivation group 1.925±0.175, in control group 1.195±0.111, t=3.521, P=0.012 5). In addition, TUNEL staining showed a much higher percentage of TUNEL-positive nuclei in these brain regions (hippocampus in sleep deprivation group 47.24±4.15, in control group 19.26±3.72, t=5.025, P=0.007 4; cortex in sleep deprivation group 42.25±1.25, in control group 27.50±3.23, t=4.262, P=0.005 3). Conclusions Chronic sleep deprivation can impair the learning and memory, increase the expression of LC3-Ⅱ and Beclin-1, elevate the formation of autophagosome, and promote apoptosis in mouse brain. These findings suggest that autophagy and apoptosis might be involved in the cognitive impairment induced by chronic sleep deprivation. Key words: Sleep deprivation; Neurons; Autophagy; Apoptosis; Memory disorders; Disease models, animal

Hyperbaric oxygen preconditioning inhibits skin flap apoptosis in a rat ischemia–reperfusion model

BackgroundHyperbaric oxygen (HBO) improves skin flap function and inhibits partial necrosis induced by ischemia–reperfusion (I/R) injury. Our study aimed to evaluate the mechanism underlying HBO regulation of the antiapoptosis factors associated with I/R injury of skin flaps. MethodsThe rats were divided into sham surgery, I/R, and HBO groups. Rats from the HBO group received HBO preconditioning followed by I/R surgery. Blood perfusion of the skin flaps was measured with laser Doppler flowmeters. Tissue morphology and apoptosis were subsequently assessed based on hematoxylin-eosinhe and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Protein expression of phosphorylated apoptosis signal-regulating kinase 1 (pASK-1), phosphorylated c-Jun N-terminal kinase (pJNK), B-cell lymphoma-2 (Bcl-2), and Bcl2-associated X protein (Bax) was examined by immunodetection, and Bcl-2 messenger RNA expression was detected by quantitative polymerase chain reaction. In addition, caspase-3 activity was also measured. ResultsThe result of microcirculation analysis showed that the survival and blood perfusion rates significantly increased in the skin flap after HBO exposure. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining revealed that cell apoptosis was significantly attenuated in the HBO group. Furthermore, HBO preconditioning increased the expression of Bcl-2 and inhibited pASK-1, pJNK, and Bax expression as determined by both immunohistochemistry and Western blot. Caspase-3 activity and the Bax/Bcl-2 ratio declined in the HBO group. ConclusionsHBO preconditioning effectively ameliorates I/R injury by regulating the apoptosis signal-regulating kinase 1 and/or c-Jun N-terminal kinase pathway and anti- and proapoptosis factors.

Melatonin attenuates hepatic ischemia through mitogen-activated protein kinase signaling

BackgroundMelatonin exerts a protective effect during hepatic ischemia–reperfusion (I/R) injury through modulation of the apoptotic cell death program. Mitogen-activated protein kinases mediate various intracellular processes such as cell differentiation, survival, and death. This study investigated whether melatonin exerts a protective effect through the activation of Raf-MEK-ERK and its downstream targets, including 90 ribosomal S6 kinase (p90RSK) and Bad, during hepatic I/R damage. MethodsHepatic ischemia was induced in mice by occlusion of the hepatic artery, portal vein, and bile duct. Adult mice were subjected to 1 h of hepatic ischemia and 3 h of reperfusion. Vehicle or melatonin (10 mg/kg, intraperitoneal) was injected 15 min before ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were measured, and terminal deoxynucleotidyl transferase dUTP nick-end labeling histochemistry was performed. Moreover, Western blot and immunoprecipitation analyses were performed. ResultsMelatonin treatment attenuated hepatic I/R-induced increases in alanine aminotransferase and aspartate aminotransferase levels and also ameliorated hepatic injury-induced pathologic lesions and increases of positive terminal deoxynucleotidyl transferase dUTP nick-end labeling staining in hepatic tissues. Hepatic I/R injury induced decreases in the phosphorylation of Raf-1, MEK1/2, and extracellular-regulated kinase (ERK)1/2, whereas melatonin attenuated decreases in these phosphorylation levels. Moreover, melatonin prevented the injury-induced decreases in phosphorylation of downstream targets, p90RSK and Bad. Immunoprecipitation analysis showed that the interaction between phospho-Bad and 14-3-3 was decreased in vehicle-treated animals, while melatonin prevented this decrease. Melatonin also attenuated the injury-induced increase in cleaved caspase-3. In cultured hepatocytes, melatonin treatment prevented the hydrogen peroxide-induced cell death and decrease in phosphorylation of ERK1/2. Moreover, blocking MEK by PD98059 attenuated the effect of melatonin. ConclusionsThese data suggest that melatonin protects hepatic cells against hepatic I/R damage through the activation of the Raf-MEK-ERK cascade and phosphorylation of its downstream targets.

The effects of porcine pulmonary surfactant on smoke inhalation injury

BackgroundOur previous study, consistent with others, demonstrated that administering an exogenous surfactant was a potential therapy for acute lung injury and acute respiratory distress syndrome. However, the underlying mechanisms remain largely unknown. In the present study, we investigated the effect of instilled porcine pulmonary surfactant (PPS) on rat inhalation injury model induced by smoke and the possible mechanism. Materials and methodsFifteen Sprague–Dawley rats were equally randomized to three groups as follows (n = 5 in each group): sham control group (C group), inhalation injury group (II group), and inhalation injury + PPS treatment group (PPS group). Lung tissues were assayed for wet/dry ratio, histologic, terminal dUTP nick-end labeling staining, and Western blotting examinations. The myeloperoxidase activity was tested in lung tissues as well. Bronchoalveolar lavage fluid was collected to determine the total protein concentrations, inflammatory cytokines, surfactant protein A (SP-A), and SP-D. ResultsOur present work exhibited that PPS had therapeutic effects on smoke inhalation injury reflected by significant increase of PaO2 values, improved edema status, decreased vascular permeability, amelioration of lung histopathology, and reduction of inflammatory response. In addition, PPS treatment could increase endogenous SP-A levels both in lung tissue and bronchoalveolar lavage fluid. Further correlation analysis showed that SP-A was negatively correlated with both myeloperoxidase activity and interleukin 8 levels. ConclusionsThese results indicate that PPS can attenuate smoke-induced inhalation injury at least partly through stimulating production of endogenous SP-A and inhibiting the release of proinflammatory cytokines such as interleukin 8. The increasing production of endogenous SP-A may be due to the antioxidant effect of PPS, which contains no SP-A.