Haibin Dai

A Rat Model of Studying Tissue-Type Plasminogen Activator Thrombolysis in Ischemic Stroke With Diabetes

Background and Purpose— Poststroke hyperglycemia and diabetes mellitus are associated with lower thrombolytic efficacy and an increased risk of postischemic cerebral hemorrhage. We aimed to develop a rodent model of thrombolysis in diabetic stroke that mimics the clinical situation. Method— Male 6-week Type I diabetic rats (14 weeks old) were subjected to embolic focal stroke and treated with tissue-type plasminogen activator at 1.5 hours. Reperfusion and 24-hour neurological outcomes were measured and compared with nondiabetic control rats. Results— Diabetic rats exhibited resistance to thrombolytic reperfusion, larger infarction volumes, and increased intracerebral hemorrhage. Conclusions— This animal model would be relevant to future studies investigating pathophysiological mechanisms and in developing new therapeutic approaches to enhance the efficacy of tissue-type plasminogen activator thrombolysis in stroke patients with diabetes or poststroke hyperglycemia.

Activation of the Central Histaminergic System is Involved in Hypoxia-Induced Stroke Tolerance in Adult Mice

We hypothesized that activation of the central histaminergic system is required for neuroprotection induced by hypoxic preconditioning. Wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice were preconditioned by 3 hours of hypoxia (8% O2) and, 48 hours later, subjected to 30 minutes of middle cerebral artery (MCA) occlusion, followed by 24 hours of reperfusion. Hypoxic preconditioning improved neurologic function and decreased infarct volume in WT or HDC-KO mice treated with histamine, but not in HDC-KO or WT mice treated with α-fluoromethylhistidine (α-FMH, an inhibitor of HDC). Laser-Doppler flowmetry analysis showed that hypoxic preconditioning ameliorated cerebral blood flow (CBF) in the periphery of the MCA territory during ischemia in WT mice but not in HDC-KO mice. Histamine decreased in the cortex of WT mice after 2, 3, and 4 hours of hypoxia, and HDC activity increased after 3 hours of hypoxia. Vascular endothelial growth factor (VEGF) mRNA and protein expressions showed a greater increase after hypoxia than those in HDC-KO or α-FMH-treated WT mice. In addition, the VEGF receptor-2 antagonist SU1498 prevented the protective effect of hypoxic preconditioning in infarct volume and reversed increased peripheral CBF in WT mice. Therefore, endogenous histamine is an essential mediator of hypoxic preconditioning. It may function by enhancing hypoxia-induced VEGF expression.

Histamine protects against NMDA-induced necrosis in cultured cortical neurons through H receptor/cyclic AMP/protein kinase A and H receptor/GABA release pathways

Using histamine and the H3 receptor antagonist thioperamide, the roles of histamine receptors in NMDA‐induced necrosis were investigated in rat cultured cortical neurons. Within 3 h of intense NMDA insult, most neurons died by necrosis. Histamine reversed the neurotoxicity in a concentration‐dependent manner and showed peak protection at a concentration of 10−7 m. This protection was antagonized by the H2 receptor antagonists cimetidine and zolantidine but not by the H1 receptor antagonists pyrilamine and diphenhydramine. In addition, the selective H2 receptor agonist amthamine mimicked the protection by histamine. This action was prevented by cimetidine but not by pyrilamine. 8‐Bromo‐cAMP also mimicked the effect of histamine. In contrast, both the adenylyl cyclase inhibitor 9‐(tetrahydro‐2‐furanyl)‐9H‐purine‐6‐amine and the cAMP‐dependent protein kinase inhibitor N‐[2‐(p‐bromocinnamylamino) ethyl]‐5‐isoquinolinesulfonamide reversed the protection by histamine. Thioperamide also attenuated NMDA‐induced excitotoxicity, which was reversed by the H3 receptor agonist (R)‐α‐methylhistamine but not by pyrilamine and cimetidine. In addition, the protection by thioperamide was inhibited by the GABAA receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by thioperamide was due to increased GABA release in NMDA‐stimulated samples. These results indicate that not only the H2 receptor/cAMP/cAMP‐dependent protein kinase pathway but also the H3 receptor/GABA release pathway can attenuate NMDA‐induced neurotoxicity.

Combination antibiotic therapy versus monotherapy for Pseudomonas aeruginosa bacteraemia: a meta-analysis of retrospective and prospective studies.

The choice of antibiotic monotherapy or combination therapy to treat Pseudomonas aeruginosa bacteraemia is controversial. The aim of this review was to compare both types of therapy to determine which delivers the best outcome for P. aeruginosa bacteraemia. We systematically searched electronic bibliographic databases, including PubMed, Ovid EMBASE and The Cochrane Library, for clinical studies that compared combination therapy with monotherapy in the treatment of P. aeruginosa bacteraemia. Eligible articles were analysed using Stata(®)/SE software v.12.0. Stratification analysis was conducted by study design and treatment type. Publication bias was assessed using Beggs funnel plot and Eggers test. Ten studies (eight retrospective and two prospective) involving 1239 patients were analysed. We found no difference between combination therapy and monotherapy when the data were combined (odds ratio = 0.89, 95% confidence interval 0.57-1.40; P = 0.614) or when data were analysed in subgroups. Neither combination therapy nor monotherapy treatment appears to have a significant effect on mortality rates in patients with P. aeruginosa bacteraemia. Further studies evaluating the effects of combination therapy or monotherapy in more specialised cases, such as when encountering a multidrug-resistant organism, are necessary.

Dexmedetomidine protects against oxygen–glucose deprivation-induced injury through the I2 imidazoline receptor-PI3K/AKT pathway in rat C6 glioma cells

Objectives  To explore the protection and the mechanism of dexmedetomidine on the oxygen–glucose deprivation (OGD) insults in rat C6 glioma cells.

Effects of dexmedetomidine on the release of glial cell line-derived neurotrophic factor from rat astrocyte cells

Dexmedetomidine (DEX) has been found to improve neuronal survival after transient global or focal cerebral ischemia in rats. Astrocyte cells may possess beneficial properties that promote neuronal recovery by secreting neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF). The purpose of this study was to investigate the effects of DEX on GDNF release from astrocytes and the possible mechanisms involved. Astrocyte cells were treated with DEX, and GDNF level in the conditioned media was determined by ELISA assay. The expression of CREB, p-CREB and PKCα was analyzed by Western blotting to explore the mechanisms involved in GDNF release. Our results showed that DEX stimulated GDNF release in a time- and dose-dependent manner; and this stimulation was blocked by the α2-adrenoreceptor antagonist yohimbine, but not by α1-adrenoreceptor antagonist prasozin, demonstrating that DEX induced GDNF release likely acts via activating the α2A adrenoreceptor. In addition, DEX-stimulated GDNF release was also blocked by the universal PKC inhibitor Ro-318220 and PKCα/β inhibitor Gö 6976, but not by PKCδ inhibitor rottlerin and PKCβ inhibitor LY333531. Interestingly, DEX also activated CREB phosphorylation, which was inhibited by Ro-318220, Gö 697 and ERK kinase inhibitor PD98059. Silencing CREB by siRNA decreased the DEX-stimulated GDNF release. In addition, the membrane translocation of PKCα was enhanced following DEX treatment. Furthermore, we found that DEX stimulated GDNF release rescued neurons against OGD-induced neurotoxicity; this effect was partly abolished by GDNF antibody. Thus, through α2A adrenergic receptors, DEX may activate astrocytes, and promote GDNF release to protect neurons after stroke, and this signaling is possibly dependent on PKCα and CREB activation.

Neuroprotective effect of carnosine on necrotic cell death in PC12 cells

The nervous tissue of many vertebrates, including humans, can synthesize beta-alanyl-L-histidine (carnosine). The biological functions of carnosine are still open to question, although several theories supported by strong experimental data have been proposed. The objective of this study was to examine the effects of carnosine on neurotoxicity in differentiated rat pheochromocytoma (PC12) cells. Neurotoxicity was induced by N-methyl-D-aspartate (NMDA), which caused time- and concentration-dependent cell death as measured by MTT and LDH assays. Pretreatment with carnosine significantly prevented the neurotoxicity in a concentration-dependent manner. The protective effect of carnosine was antagonized by the H1 receptor antagonist pyrilamine, but not by the H2 receptor antagonist cimetidine. In addition, alpha-fluoromethylhistidine, a histidine decarboxylase inhibitor, slightly reversed the protective action of carnosine. These results indicate that carnosine can effectively protect against NMDA-induced necrosis in PC12 cells, and its protection may in part be due to the activation of the postsynaptic histamine H1 receptor. The study suggests that carnosine may be an endogenous protective factor and calls for its further study as a new anti-excitotoxic agent.

Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats

Cerebral microvascular endothelial cells (ECs) are crucial for brain vascular repair and maintenance, but their physiological function may be impaired during ischemic stroke and diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, could exacerbate ischemia‐induced EC injury and dysfunction. We investigated the protective effect of autophagy on cultured human brain microvascular endothelial cells (HBMEC) that underwent MGO treatment. A further study was conducted to explore the underlying mechanisms of the protective effect. Autophagic activity was assessed by evaluating protein levels, using western blot. 3‐methyladenine (3‐MA), bafilomycin A1, ammonium chloride (AC), Beclin 1 siRNA, and chloroquine (CQ) were used to cause autophagy inhibition. Alarmar blue assay and lactate dehydrogenase release assay were used to evaluate cell viability. Streptozotocin was administered to induce type I diabetes in rats and post‐permanent middle cerebral artery occlusion was performed to elicit cerebral ischemia. Blood–brain barrier permeability was also assessed. Our study found that MGO reduced HBMEC cell viability in a concentration‐ and time‐dependent manner, and triggered the responsive autophagy activation. Autophagy inhibitors bafilomycin A1, AC, 3‐MA, and BECN1 siRNA exacerbated MGO‐induced HBMEC injury. FAK phosphorylation inhibitor PF573228 inhibited MGO‐triggered autophagy and enhanced lactate dehydrogenase release. Meanwhile, similar autophagy activation in brain vascular ECs was observed during permanent middle cerebral artery occlusion‐induced cerebral ischemia in diabetic rats, while chloroquine‐induced autophagy inhibition enhanced blood–brain barrier permeability. Taken together, our study indicates that autophagy triggered by MGO defends HBMEC against injuries.

Edaravone Protected Human Brain Microvascular Endothelial Cells from Methylglyoxal-Induced Injury by Inhibiting AGEs/RAGE/Oxidative Stress

Subjects with diabetes experience an increased risk of cerebrovascular disease and stroke compared with nondiabetic age-matched individuals. Increased formation of reactive physiological dicarbonyl compound methylglyoxal (MGO) seems to be implicated in the development of diabetic vascular complication due to its protein glycation and oxidative stress effect. Edaravone, a novel radical scavenger, has been reported to display the advantageous effects on ischemic stroke both in animals and clinical trials; however, little is known about whether edaravone has protective effects on diabetic cerebrovascular injury. Using cultured human brain microvascular endothelial cells (HBMEC), protective effects of edaravone on MGO and MGO enhancing oxygen-glucose deprivation (OGD) induced injury were investigated. Cell injury was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, cell account, lactate dehydrogenase (LDH) release and Rhodamine 123 staining. Advanced glycation end-products (AGEs) formation and receptor for advanced glycation end-products (RAGE) expression were measured by western blotting. Cellular oxidative stress was measured by reactive oxygen species (ROS) release. Treatment of MGO for 24 h significantly induced HBMEC injury, which was inhibited by pretreatment of edaravone from 10–100 µmol/l. What’s more, treatment of MGO enhanced AGEs accumulation, RAGE expression and ROS release in the cultured HBMEC, which were inhibited by 100 µmol/l edaravone. Finally, treatment of MGO for 24 h and then followed by 3 h OGD insult significantly enhanced cell injury when compared with OGD insult only, which was also protected by 100 µmol/l edaravone. Thus, edaravone protected HBMEC from MGO and MGO enhancing OGD-induced injury by inhibiting AGEs/RAGE/oxidative stress.

Hydroxysafflor yellow A protects methylglyoxal-induced injury in the cultured human brain microvascular endothelial cells

Individuals with diabetes have high concentration of methylglyoxal (MGO) and have advanced glycation end-products (AGEs) which play an important role in vascular complications, such as stroke. Our previous data demonstrated that hydroxysafflor yellow A (HSYA), a major active chemical component of the safflower yellow pigment, had antiglycation effect on the AGEs formation in vitro. It is not known whether HSYA can protect against MGO-induced injury in cultured human brain microvascular endothelial cells (HBMEC). Using cultured HBMEC, cell injury was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, lactate dehydrogenase (LDH) release and AnnexinV/PI staining. Advanced glycogen end-products and caspase-3 formation were measured by Western blotting. Incubation of MGO for 24h concentration-dependently induced HBMEC injury, which was protected by HSYA from 10 to 100 μmol/l. Caspase-3 expression and AnnexinV/PI staining illustrated that the protection of HSYA was probably associated with inhibiting cell apoptosis. Whats more, MGO promoted AGEs accumulation in the cultured HBMEC, which was also inhibited by 100 μmol/l HSYA. Thus, our results proved that HSYA could inhibit MGO-induced injury in the cultured HBMEC, which was associated with its antiglycation effect.