Resveratrol attenuates high-fat diet-induced disruption of the blood-brain barrier and protects brain neurons from apoptotic insults.

The blood-brain barrier (BBB) maintains brain microenvironment. Our previous study showed that oxidized low-density lipoprotein (oxLDL) can damage the BBB by inducing apoptosis of cerebrovascular endothelial cells. This study was aimed at evaluating the effects of resveratrol on high-fat diet-induced insults to the BBB and brain neurons. Exposure of mice to a high-fat diet for 8 weeks increased levels of serum total cholesterol (146 ± 13) and LDL (68 ± 8), but resveratrol decreased such augmentations (119 ± 6; 45 ± 8). Permeability assays showed that a high-fat diet induced breakage of the BBB (88 ± 21). Meanwhile, resveratrol alleviated this interruption (16 ± 6). Neither resveratrol nor a high-fat diet caused the death of cerebrovascular endothelial cells. Instead, exposure to a high-fat diet disrupted the polymerization of occludin and zonula occludens (ZO)-1, but resveratrol significantly attenuated those injuries. Neither a high-fat diet nor resveratrol changed the levels of occludin or ZO-1 in brain tissues. Resveratrol protected brain neurons against high-fat diet-induced caspase-3 activation and genomic DNA fragmentation. This study shows that resveratrol can attenuate the high-fat diet-induced disruption of the BBB via interfering with occludin and ZO-1 tight junctions, and protects against apoptotic insults to brain neurons.

GATA-2 transduces LPS-induced il-1ß gene expression in macrophages via a toll-like receptor 4/MD88/MAPK-dependent mechanism.

Lipopolysaccharide (LPS) is a critical factor for inducing acute lung injury. GATA-2, a transcription factor, contributes to the control of cell activity and function. Exposure of RAW 264.7 cells to LPS induced interleukin (IL)-1ß mRNA and protein expression and GATA-2 translocation from the cytoplasm to nuclei in concentration- and time-dependent manners. A bioinformatic search revealed that GATA-2-specific binding elements exist in the 5'-promoter region of the il-1ß gene. LPS could enhance the transactivation activity of GATA-2 in macrophages. Knocking-down translation of GATA-2 mRNA using RNA interference significantly alleviated LPS-induced IL-1ß mRNA and protein expression. As to the mechanism, transfection of toll-like receptor (TLR) 4 small interfering (si)RNA into macrophages concurrently decreased LPS-caused increases in nuclear GATA-2 levels. Sequentially, treatment with myeloid differentiation factor 88 (MyD88) siRNA decreased LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) kinase 1/2 and subsequent translocation of GATA-2. Reducing MAPK activities using specific inhibitors simultaneously decreased GATA-2 activation. Furthermore, exposure of primary macrophages to LPS significantly increased the transactivation activities of GATA-2 and IL-1ß mRNA and protein expression. Transfection of GATA-2 siRNA inhibited LPS-induced IL-1ß mRNA expression. Results of this study show that LPS induction of il-1ß gene expression in macrophages is mediated by GATA-2 via activation of TLR4, MyD88, and MAPKs.

Propofol protects against nitrosative stress-induced apoptotic insults to cerebrovascular endothelial cells via an intrinsic mitochondrial mechanism.

BACKGROUND: Cerebrovascular endothelial cells (CECs), major component cells of the blood-brain barrier, can be injured by oxidative stress. Propofol can protect cells from oxidative injury. The aim of this study was to evaluate the effects of propofol on nitrosative stress-induced insults to CECs and its possible mechanisms. METHODS: Primary CECs isolated from mouse cerebral capillaries were exposed to2 nitric oxide (NO) donors: sodium nitroprusside (SNP) or S-nitrosoglutathione (GSNO). Cellular NO levels, cell morphologies, and cell viabilities were analyzed. DNA fragmentation and apoptotic cells were quantified using flow cytometry. Proapoptotic Bcl2-antagonist-killer (Bak) and cytochrome c were immunodetected. Bak translocation was analyzed using confocal microscopy. Caspases-9 and -3 activities were measured fluorometrically. Permeability of the CEC monolayer was assayed by measuring the transendothelial electrical resistance. RESULTS: Exposure of CECs to SNP increased cellular NO levels and simultaneously decreased cell viability (P < .01). Meanwhile, treatment of CECs with propofol at a therapeutic concentration (50 µM) decreased SNP-induced cell death (P < .01). SNP induced DNA fragmentation and cell apoptosis, but propofol decreased the cell injury (P < .01). Sequentially, propofol decreased SNP-enhanced Bak levels and translocation from the cytoplasm to mitochondria (P < .05). Exposure of CECs to propofol attenuated GSNO-induced cell death, apoptosis, and caspase-3 activation (P < .01). Additionally, propofol protected CECs against SNP-induced disruption of the CEC monolayer (P < .05). Consequently, SNP-enhanced cascade activation of caspases-9 and -3 was decreased by propofol (P < .01). CONCLUSION: This study suggested that propofol at a therapeutic concentration can protect against nitrosative stress-induced apoptosis of CECs due to downregulation of the intrinsic Bak-mitochondrion-cytochrome c-caspase protease pathway.

Socioeconomic deprivation and associated risk factors of traumatic brain injury in children.

BACKGROUND: This study aimed to investigate the relative risks of low income (family socioeconomic deprivation) and associated factors for traumatic brain injury (TBI) in children. METHODS: Using Taiwan National Health Insurance Research Database and adjusting the covariates, we conducted a population-based case-control study analyzing 8,291 pediatric patients, aged 0 year to 17 years, diagnosed with TBI, and 33,164 sex- and age-matched controls to study the association of low income and TBI. The relative risks of TBI for socioeconomically deprived children with various coexisted medical conditions were evaluated. RESULTS: After adjustment, pediatric population with low income were at increased risk of TBI (odds ratio [OR], 1.71; 95% confidence interval [CI], 1.52-1.92). Among the coexisting medical conditions, low-income pediatric population with mental disorders had significantly increased TBI risk when compared with matched controls (adjusted OR, 1.99; 95% CI, 1.51-2.63). Increased risk of TBI was also found in low-income children with epilepsy when compared with children of regular family income (adjusted OR, 3.11; 95% CI, 1.65-5.86). The adjusted OR of TBI for low-income children with mental disorders and epilepsy was as high as 4.45 (95% CI, 1.96-10.1). Among TBI patients, low-income children who had epilepsy were at significantly higher risk of post-TBI intracranial hemorrhage when compared with controls (OR, 10.6; 95% CI, 3.30-33.9). CONCLUSION: We found a significantly increased risk of TBI in socioeconomically deprived children, particularly among children with mental disorders, epilepsy, or both. Low-income children should be considered for special attention to reduce TBI risk and post-TBI morbidities. LEVEL OF EVIDENCE: Prognostic study, level III.

Risk and outcomes for traumatic brain injury in patients with mental disorders.

OBJECTIVE: To investigate the risk of traumatic brain injury (TBI) and post-injury mortality in patients with mental disorders. BACKGROUND: Patients with mental disorders are at higher risk of injuries. However, the association between mental disorders and TBI is still not understood. We conducted case-control studies to investigate whether people with pre-existing mental disorders are at higher risk of TBI and post-injury mortality. METHODS: Using reimbursement claims, we analysed 16,635 patients with TBI and 66,540 controls with adjustment of covariates to study the association of mental disorders and TBI. A nested case-control study was also conducted to analyse contributory factors for post-injury mortality. RESULTS: People with mental disorders were at increased risk of TBI (odds ratio (OR) 1.94, 95% confidence interval (CI) 1.86 to 2.02). Men, older age, living in highly urbanised areas and patients on low income had a higher risk of TBI and post-injury mortality. Psychiatric medication intensity and frequency of psychiatric visits was significantly correlated with TBI in a severity dependent relationship (p for trend <0.0001). Patients receiving advanced psychiatric healthcare had an increased risk of TBI (OR 2.98, 95% CI 2.67 to 3.33) and post-injury mortality (OR 1.92, 95% CI 1.34 to 2.77). A history of receiving psychiatric related outpatient care (OR 1.77, 95% CI 1.70 to 1.85), hospitalisation (OR 3.21, 95% CI 2.79 to 3.70) or emergency visits (OR 3.53, 95% CI 3.15 to 3.94) were highly associated with subsequent TBI. CONCLUSIONS: Patients with mental disorders have an increased risk of TBI. Intensity of psychiatric medication is associated with increased post-injury mortality. Special attention to prevent TBI among this disabled population is mandatory.

Anticancer agent 2-methoxyestradiol improves survival in septic mice by reducing the production of cytokines and nitric oxide.

Cytokine production is critical in sepsis. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, inhibits hypoxia-inducible factor 1α (HIF-1α) and is an antiangiogenic and antitumor agent. We investigated the effect of 2ME2 on cytokine production and survival in septic mice. Using i.p. LPS or cecal ligation and puncture (CLP), sepsis was induced in BALB/c mice that were simultaneously or later treated with 2ME2 or vehicle. Twelve hours after the LPS injection, serum and peritoneal fluid cytokine and nitric oxide (NO) levels were analyzed using enzyme-linked immunosorbent assay and the Griess reaction. Lung injuries were histologically analyzed, and liver and kidney injuries were biochemically analyzed. Survival was determined 7 days after LPS injection or CLP procedure. In vivo and in vitro effects of 2ME2 on LPS-induced macrophage inflammation were determined. The effect of 2ME2 on HIF-1α expression, nuclear factor κB (NF-κB), and inducible NO synthase (iNOS) in LPS-treated RAW264.7 cells, a murine macrophage cell line, was determined using Western blotting. 2-Methoxyestradiol treatment reduced LPS-induced lung, liver, and kidney injury. Both early and late 2ME2 treatment prolonged survival in LPS- and CLP-induced sepsis. 2-Methoxyestradiol significantly reduced IL-1ß, IL-6, TNF-α, and NO levels in septic mice as well as in LPS-stimulated peritoneal macrophages. 2-Methoxyestradiol treatment also reduced the LPS-induced expression of HIF-1α, iNOS, and pNF-κB in RAW264.7 cells, as well as iNOS and pNF-κB expression in siHIF-1α-RAW264.7 cells. 2-Methoxyestradiol prolongs survival and reduces lung, liver, and kidney injury in septic mice by inhibiting iNOS/NO and cytokines through HIF-1α and NF-κB signaling.

Resveratrol attenuates oxidized LDL-evoked Lox-1 signaling and consequently protects against apoptotic insults to cerebrovascular endothelial cells.

Cerebrovascular endothelial cells (CECs) are crucial components of the blood-brain barrier. Our previous study showed that oxidized low-density lipoprotein (oxLDL) induces apoptosis of CECs. This study was designed to further evaluate the effects of resveratrol on oxLDL-induced CEC insults and its possible molecular mechanisms. Resveratrol decreased the oxidation of LDL into oxLDL. Additionally, the oxLDL-caused oxidative stress and cell damage were attenuated by resveratrol. Exposure of CECs to oxLDL induced cell shrinkage, DNA fragmentation, and cell apoptosis, but resveratrol defended against such injuries. Application of Lox-1 small interference (si)RNA into CECs reduced the translation of this membrane receptor, and simultaneously increased resveratrol protection from oxLDL-induced cell apoptosis. By comparison, overexpression of Lox-1 attenuated resveratrol protection. Resveratrol inhibited oxLDL-induced Lox-1 mRNA and protein expressions. Both resveratrol and Lox-1 siRNA decreased oxLDL-enhanced translocation of proapoptotic Bcl-2-associated X protein (Bax) from the cytoplasm to mitochondria. Sequentially, oxLDL-induced alterations in the mitochondrial membrane potential, cytochrome c release, and activities of caspases-9, -3, and -6 were decreased by resveratrol. Pretreatment with Z-VEID-FMK (benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone) synergistically promoted resveratrol's protection against DNA fragmentation and cell apoptosis. Therefore, this study shows that resveratrol can protect CECs from oxLDL-induced apoptotic insults via downregulating Lox-1-mediated activation of the Bax-mitochondria-cytochrome c-caspase protease pathway.

Resveratrol protects against oxidized LDL-induced breakage of the blood-brain barrier by lessening disruption of tight junctions and apoptotic insults to mouse cerebrovascular endothelial cells.

Cerebrovascular endothelial cells (CEC) comprise the blood-brain barrier (BBB). In a previous study, we showed that oxidized LDL (oxLDL) can induce apoptosis of mouse CEC. Resveratrol possesses chemopreventive potential. This study aimed to evaluate the effects of resveratrol on oxLDL-induced insults to mouse CEC and its possible mechanisms. Exposure of mouse CEC to 200 µmol/L oxLDL for 1 h did not cause cell death but significantly altered the permeability and transendothelial electrical resistance of the cell monolayer. However, resveratrol completely normalized such injury. As for the mechanisms, resveratrol completely protected oxLDL-induced disruption of F-actin and microtubule cytoskeletons as well as occludin and zona occludens-1 (ZO-1) tight junctions. The oxLDL-induced decreases in the mitochondrial membrane potential and intracellular ATP levels were normalized by resveratrol. Exposure of mouse CEC to 200 µmol/L oxLDL for 24 h elevated oxidative stress and simultaneously induced cell apoptosis. However, resveratrol partially protected against oxLDL-induced CEC apoptosis. The oxLDL-induced alterations in levels of Bcl-2, Bax, and cytochrome c were completely normalized by resveratrol. Consequently, resveratrol partially decreased oxLDL-induced activation of caspases-9 and -3. Therefore, in this study, we show that resveratrol can protect against oxLDL-induced damage of the BBB through protecting disruption of the tight junction structure and apoptotic insults to CEC.

Lipoteichoic acid-induced TNF-α and IL-6 gene expressions and oxidative stress production in macrophages are suppressed by ketamine through downregulating Toll-like receptor 2-mediated activation oF ERK1/2 and NFκB.

Lipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that ketamine has anti-inflammatory and antioxidant effects on gram-negative LPS-induced macrophage activation. In this study, we further evaluated the effects of ketamine on the regulation of LTA-induced TNF-alpha and IL-6 gene expressions and oxidative stress production in macrophages and its possible mechanisms. Exposure of macrophages to a therapeutic concentration of ketamine (100 microM) inhibited LTA-induced TNF-alpha and IL-6 expressions at protein or mRNA levels. In parallel, ketamine at 100 microM reduced LTA-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Sequentially, ketamine reduced the LTA-triggered translocation of nuclear factor-kappaB (NFkappaB) from the cytoplasm to nuclei and its transactivation activity. Pretreatment with PD98059, an inhibitor of ERK, decreased LTA-enhanced NFkappaB activation and TNF-alpha and IL-6 mRNA syntheses. Cotreatment with ketamine and PD98059 synergistically suppressed the LTA-induced translocation and transactivation of NFkappaB and biosyntheses of TNF-alpha and IL-6 mRNA. Application of Toll-like receptor 2 (TLR2) small interfering RNA (si)RNA into macrophages decreased the levels of this receptor, and simultaneously ameliorated LTA-augmented NFkappaB transactivation and consequent production of TNF-alpha and IL-6 mRNA. Cotreatment with ketamine and TLR2 siRNA synergistically lowered TNF-alpha and IL-6 mRNA syntheses in LTA-activated macrophages. Ketamine and TLR2 siRNA could reduce the LTA-induced increases in production of nitrite and intracellular reactive oxygen species in macrophages, and their combination had better effects than a single exposure. Thus, this study shows that one possible mechanism involved in ketamine-induced inhibition of LTA-induced TNF-alpha and IL-6 gene expressions and oxidative stress production is through downregulating TLR2-mediated phosphorylation of ERK1/2 and the subsequent translocation and transactivation of NFkappaB.

Cytoskeleton interruption in human hepatoma HepG2 cells induced by ketamine occurs possibly through suppression of calcium mobilization and mitochondrial function.

Ketamine is an intravenous anesthetic agent often used for inducing and maintaining anesthesia. Cytoskeletons contribute to the regulation of hepatocyte activity of drug biotransformation. In this study, we attempted to evaluate the effects of ketamine on F-actin and microtubular cytoskeletons in human hepatoma HepG2 cells and its possible molecular mechanisms. Exposure of HepG2 cells to ketamine at

Aphonia and quadriplegia---a rare complication following epidural labor analgesia.

We report an obstetric patient who developed unusual neurological complications after an epidural injection of a local anesthetic and a narcotic in a seemingly faultless manner. Ten minutes after receiving a loading dose, the patient developed aphonia, quadriplegia, and facial palsy while retaining normal consciousness and stable hemodynamics. The episode spontaneously resolved 40 minutes later. We wish to draw attention to anesthesiologists of the possibility that even in the presence of a negative aspiration test or without noticeable dural puncture, the injected drugs may enter the intrathecal or subdural space instead of pooling entirely in the epidural compartment. Once an unexpected high block and unusual symptoms or signs are noted, a prompt differential diagnosis must be made and treatment initiated as soon as possible.

Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes.

During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-gamma in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism.

Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway.

Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, oxLDL can damage the blood-brain barrier through induction of CEC apoptosis via a Bax-mitochondria-caspase protease pathway.

Confocal laser scanning microscopy: I. An overview of principle and practice in biomedical research.

Evolving from conventional microscopic technologies, confocal microscopy has proved itself to play an important role in the biomedical research during the past decade. Confocal microscope has many advantages over traditional microscope including the ability to look deeply into inside cells with less photodamage and photobleach, reconstruct three-dimensional images, and chart intracellular dynamic events in the living cells. With these remarkable properties and the availability of fluorescent dyes for living cells, the confocal microscopy has been widely used in solving many unknown questions in biological and pharmacological fields. In clinics, confocal microscope has also served as an important tool to observe the living cells in skins and eyes. For anesthesiologists, confocal microscope has made possible novel experimental approaches for the effects of multiple anesthetic agents on cells. Furthermore, the technology of fibreoptical confocal endomicroscopy is now on its way of maturation. It will soon be the era for confocal microscopy to explore the "cell behavior" inside of intact living tissues.

Propofol suppresses macrophage functions and modulates mitochondrial membrane potential and cellular adenosine triphosphate synthesis.

BACKGROUND: Propofol is an intravenous anesthetic agent that may impair host defense system. The aim of this study was to evaluate the effects of propofol on macrophage functions and its possible mechanism. METHODS: Mouse macrophage-like Raw 264.7 cells were exposed to propofol, at 3, 30 (a clinically relevant concentration), and 300 microm. Cell viability, lactate dehydrogenase, and cell cycle were analyzed to determine the cellular toxicity of propofol to macrophages. After administration of propofol, chemotactic, phagocytic, and oxidative ability and interferon-gamma mRNA production were carried out to validate the potential effects of propofol on macrophage functions. Mitochondrial membrane potential and cellular adenosine triphosphate levels were also analyzed to evaluate the role of mitochondria in propofol-induced macrophage dysfunction. RESULTS: Exposure of macrophages to 3 and 30 microm propofol did not affect cell viability. When the administered concentration reached 300 microm, propofol would increase lactate dehydrogenase release, cause arrest of cell cycle in G1/S phase, and lead to cell death. In the 1-h-treated macrophages, propofol significantly reduced macrophage functions of chemotactic and oxidative ability in a concentration-dependent manner. However, the suppressive effects were partially or completely reversed after 6 and 24 h. Propofol could reduce phagocytic activities of macrophages in concentration- and time-dependent manners. Exposure of macrophages to lipopolysaccharide induced the mRNA of interferon-gamma, but the induction was significantly blocked by propofol. Propofol concentration-dependently decreased the membrane potential of macrophage mitochondria, but the effects were descended with time. The levels of cellular adenosine triphosphate in macrophages were also reduced by propofol. CONCLUSIONS: A clinically relevant concentration of propofol can suppress macrophage functions, possibly through inhibiting their mitochondrial membrane potential and adenosine triphosphate synthesis instead of direct cellular toxicity.