An automatic patient-specific seizure onset detection method using intracranial electroencephalography.

OBJECTIVE: This study presents a multichannel patient-specific seizure detection method based on the empirical mode decomposition (EMD) and support vector machine (SVM) classifier. MATERIALS AND METHODS: The EMD is used to extract features from intracranial electroencephalography (EEG). A machine-learning algorithm is used as a classifier to discriminate between seizure and nonseizure intracranial EEG epochs. A postprocessing algorithm is proposed to reject artifacts and increase the robustness of the method. The proposed method was evaluated using 463 hours of intracranial EEG recordings from 17 patients with a total of 51 seizures in the Freiburg EEG database. RESULTS: The proposed method had better performance than most of the existing seizure detection systems, including an average sensitivity of 92%, false detection rate (FDR) of 0.17/hour, and time delay (TD) of 12 sec. Moreover, the FDR could be further reduced by a TD extension. CONCLUSIONS: Given its high sensitivity and low FDR, the proposed patient-specific seizure detection method can greatly assist clinical staff with automatically marking seizures in long-term EEG or detecting seizure onset online with high performance. Early and accurate seizure detection using this method may serve as a practical tool for planning epilepsy interventions.

Application of microscopical techniques in the study of autolysosome dynamics in PC12 neurites.

Autophagy is a principal degradation pathway for the turnover of intracellular proteins or cytoplasmic organelles in response to starvation. During autophagic activation, autophagosomes fuse with lysosomes to form autolysosomes where incorporated materials are degraded. However, the dynamics of autolysosomes in neurites of live cells was still poorly known. In this study, various subsets of microscope were applied to analyse the autophagy induction and highly dynamic transport of autolysosomes in rat PC12 neurites. Beading formation was found in degenerating PC12 neurites under phase contrast light microscope after serum deprivation. The monomeric red fluorescence protein-green fluorescence protein-light chain 3-labelled autolysosomes accumulated throughout PC12 neurites after 18 h of serum deprivation as revealed by fluorescence microscope analysis. The single-membrane autolysosomes were also visualized in PC12 cells under transmission electron microscope. Moreover, fluorescence recovery after photobleaching experiment, which was conducted by confocal laser scanning microscope, demonstrated that autolysosomes were motile vesicles and moved along PC12 neurites during starvation. The directional transport of monomeric red fluorescence protein -labelled autolysosomes in neurites was further monitored by a motorized video microscope. Both anterograde and retrograde transport of autolysosomes were observed. In addition, the autolysosomes were precisely mapped by using 2D Gaussian fitting and then their highly dynamic movement was robustly tracked by using multidimensional assignment. Collectively, by using different microscopical techniques, our results confirmed the dynamic transport of autolysosomes in starved PC12 neurites and may provide valuable insight into understanding the biophysical characteristics of autolysosomes in neurites under physiological and pathological conditions.

Salidroside stimulated glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase.

In the present study, we reported the metabolic effects of salidroside, one of the active components of Rhodiola Rosea, on skeletal muscle cells. Salidroside dose-dependently stimulated glucose uptake in differentiated L6 rat myoblast cells. Inhibitor of AMP-activated protein kinase (AMPK) by pretreating the cells with compound C potently reduced salidroside-stimulated glucose uptake, while inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin exhibited no significant inhibitory effect on salidroside-mediated glucose transport activation. Western blotting analyses revealed that salidroside increased the phosphorylation level of AMPK and acetyl-CoA carboxylase (ACC). In addition, salidroside enhanced insulin-mediated Akt activation and glucose uptake, and such enhancement can be specifically inhibited by compound C. In summary, AMPK activation was involved in the effects of salidroside on glucose transport activation and insulin sensitivity. Salidroside can be further developed as potential compound for the anti-diabetic therapy.

[Three-dimensional motion analysis for GLUT4 vesicles in TIRF microscopy].

In this paper, GLUT4 vesicles are observed in real-time under TIRF microscopy and a new three-dimensional single particle tracking algorithm according to the unique features of TIRF is put forward. Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time and mobile vesicles were segmented by an adaptive background subtraction method. Kalman filtering was then introduced to track the granules so as to reduce the searching range and to avoid the disturbance of background noise and false targets. In the experiments the algorithm was applied in analyzing the long-distance movement of GLUT4 vesicles. The experimental results indicate that the algorithm has achieved robust tracking of the vesicles in the imaging plane and has effectively calculated the position in the direction orthogonal to the imaging plane.

Rapid Decoding of Hand Gestures in Electrocorticography Using Recurrent Neural Networks.

Brain-computer interface (BCI) is a direct communication pathway between brain and external devices, and BCI-based prosthetic devices are promising to provide new rehabilitation options for people with motor disabilities. Electrocorticography (ECoG) signals contain rich information correlated with motor activities, and have great potential in hand gesture decoding. However, most existing decoders use long time windows, thus ignore the temporal dynamics within the period. In this study, we propose to use recurrent neural networks (RNNs) to exploit the temporal information in ECoG signals for robust hand gesture decoding. With RNN's high nonlinearity modeling ability, our method can effectively capture the temporal information in ECoG time series for robust gesture recognition. In the experiments, we decode three hand gestures using ECoG signals of two participants, and achieve an accuracy of 90%. Specially, we investigate the possibility of recognizing the gestures in a time interval as short as possible after motion onsets. Our method rapidly recognizes gestures within 0.5 s after motion onsets with an accuracy of about 80%. Experimental results also indicate that the temporal dynamics is especially informative for effective and rapid decoding of hand gestures.

Inhibition of excitatory amino acid efflux contributes to protective effects of puerarin against cerebral ischemia in rats.

OBJECTIVE: To investigate whether the protective effects of puerarine (Pur) against cerebral ischemia is associated with depressing the extracellular levels of amino acid transmitters in brain of rats. METHODS: Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) for 60 min followed by 24 h reperfusion. Pur (50, 100 mg/kg, i.p.) was administered at the onset of MCAO. The infarct rate and edema rate were detected on TTC (2,3,5-triphenyltetrazolium chloride)-stained coronal sections. The extracellular levels of amino acid transmitters were monitored in striatum of rats with ischemic/reperfusion injury using in vivo microdialysis technique. Furthermore, the protective effects of Pur against glutamate-induced neurotoxicity were detected. Glutamate-induced apoptotic and necrotic cells in hippocampus were estimated by flow cytometric analysis of Annexin-V and PI labeling cells. RESULTS: Pur (100 mg/kg) significantly decreased infarct size by 31.6% (P<0.05), reduced edema volume (P<0.05), and improved neurological functions (P<0.05) following MCAO. In these rats, the ischemia-induced extracellular levels of aspartate (Asp), glutamate (Glu), y-aminobutyric acid (GABA), and taurine (Tau) were significantly reduced in striatum of vehicle-treated animals by 54.7%, 56.7%, 75.8%, and 68.1% (P<0.01 and P<0.05). Pur reduced the peak values of Glu and Asp more obviously than those of GABA and Tau, and the rate of Glu/GABA during MCAO markedly decreased in Pur-treated MCAO rats, compared with the vehicle-treated MCAO rats. Meanwhile, apoptosis and necrosis induced by Glu in cultured hippocampal neurons were significantly reduced after Pur treatment. CONCLUSION: Acute treatment with Pur at the onset of occlusion significantly depresses ischemia-induced efflux of amino acids, especially, excitotoxicity in the striatum, a mechanism underlying the neuroprotective effect on cellular survival.

[Protective effects of breviscapine against cultured rat hippocampal neuronal toxicity induced by glutamate].

The aim of this study was to investigate the effects of breviscapine on cultured rat hippocampal neuronal toxicity induced by glutamate. Primary hippocampal neurons were prepared from 2 day-old SD rats. After 8 days cultured in vitro, the cultures subjected to 30 min treatment of 0.1, 0.5 and 1.0 mmol x L(-1) L-glutamate, separately. Breviscapine (10, 20 and 40 micromol x L(-1)) was added into the cultures during 30 min treatment of L-glutamate and for the following 24 h respectively. After 24 h of L-glutamate treatment, flow cytometric analysis of Annexin V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that L-glutamate dose-dependently induced hippocampal neuronal apoptosis and necrosis. In agreement with these results, RT-PCR experiments indicated a biphasic regulation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA after L-glutamate treatment, i. e up-regulation by 0.1 mmol x L(-1) L-glutamate and down-regulation by 0.5 and 1.0 mmol x L(-1) L-glutamate. However, breviscapine markedly reduced apoptosis and necrosis due to toxicity of 0.5 mmol L(-1) L-glutamate. Compared with the vehicle-treated L-glutamate group, the apoptosis was reduced by 30.4% and 40.1%, and necrosis was reduced by 32.5% and 38.8%, after treatment by breviscapine of 20 and 40 micromol x L(-1). Meanwhile, breviscapine obviously reversed the down-regulation of XIAP expression induced by L-glutamate (up-regulation by 45.1% and 54.9% when compared with that of the vehicle-treated glutamate group). The results from the detection of confocal laser scanning microscopy with Fluo-3, a Ca2+ probe showed an obvious increase in intracellular Ca2+ during L-glutamate treatment; and breviscapine of 20 or 40 micromol x L(-1) significantly slowed down glutamate-induced Ca2+ influx and lowered the intracellular Ca2+ peak in hippocampal neurons (P < 0.01). These results suggest that neuroprotective effect of breviscapine against glutamate excitotoxicity was associated with inhibition of the accumulation of intracellular Ca2+ and up-regulation of XIAP expression in hippocampal neurons.

Neurochemical effects of exercise and neuromuscular electrical stimulation on brain after stroke: a microdialysis study using rat model.

Treadmill exercise and neuromuscular electrical stimulation are common clinical approaches for stroke rehabilitation. Both animal and clinical studies have shown the functional improvements after these interventions. However, the neurochemical effects on the ischemic brain had not been well studied. This study aimed at evaluating the effects of treadmill exercise and neuromuscular electrical stimulation (NMES), and studying their effects during a 2-week training, on the levels of common neurotransmitters (aspartate, glutamate, taurine and gamma-aminobutyric acid (GABA)) in the hippocampus following transient focal cerebral ischemia. Either treadmill exercise or neuromuscular electrical stimulation was prescribed to the rats 24 h after cerebral ischemia whereas Control group remained in cages for 2 weeks. Microdialysis technique was used to collect dialysates from ipsilesional hippocampus in vivo. It was found that the glutamate level was increased significantly during treadmill exercise and then returned to baseline level. Both interventions did not trigger significant effects on aspartate and glutamate basal levels during the 2 weeks. The relatively high taurine level in Control groups may suggest that the interventions might suppress the taurine release in hippocampus. GABA and aspartate levels did not showed significant changes over the 2 weeks in all groups. These results provide insights to explain the neurochemical effects on the ischemic injured brain during the course of rehabilitation.

Astragaloside IV improved barrier dysfunction induced by acute high glucose in human umbilical vein endothelial cells.

The purpose of the present study was to examine the effects of astragaloside IV, a saponin isolated from Astragalus membranaceus (Fisch) Bge, on the impairment of barrier function induced by acute high glucose in cultured human vein endothelial cells. High glucose (27.8 mM) induced a decrease in transendothelial electrical impedance and an increase in cell monolayer permeability in human umbilical vein endothelial cells. Endothelial barrier dysfunction stimulated by high glucose was accompanied by translocation and activation of protein kinase C (PKC), the redistribution of F-actin and formation of intercellular gaps, suggesting that increases in PKC activity and rearrangement of F-actin could be associated with endothelial barrier dysfunction induced by acute high glucose. Application of astragaloside IV inhibited high glucose-induced endothelial barrier dysfunction in a dose-dependent manner, which is compatible with inhibition of PKC translocation and improvement of F-actin rearrangements. Western blot analysis revealed that high glucose-induced PKC alpha and beta2 overexpression in the membrane fraction were significantly reduced by astragaloside IV. These findings indicate that astragaloside IV protected endothelial cells from high glucose-induced barrier impairment by inhibiting PKC activation, as well as improving cytoskeleton remodeling.

Development of cerebral infarction, apoptotic cell death and expression of X-chromosome-linked inhibitor of apoptosis protein following focal cerebral ischemia in rats.

The aim of this study was to investigate the role of apoptosis or necrosis in the development of delayed infarct, and the relationship between the level of XIAP gene, caspase-3 activation and ischemic cell death following transient focal cerebral ischemia. Adult male Sprague-Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 50 min and reperfusion for 0.5, 4, 8, 24 h, 3, 7, 14 days. On TTC-stained coronal sections, delayed infarct was observed to develop in the whole MCA territory, especially in frontoparietal cortex after ischemia. Near total infarct was shown in striatum 24 h after MCAo, while delayed infarct was evident in the cortex. By day 3, the infarct had progressively expanded to the nearly whole area of the frontoparietal cortex. Flow cytometric analysis of Annexin-V (marks apoptosis) and PI (propidium iodide, marks necrosis) labeling cells showed that MCAo dominantly induced necrosis in ischemic core, striatum. Apoptosis contributed to delayed infarct and cell death in the border zone, dorsolateral cortex and hippocampus. The time-course of caspase-3 activation was consistent with the changes of apoptosis and infarct following MCAo. Further RT-PCR experiments indicated that there was a biphasic regulation of XIAP in time- and region-dependent manner after ischemia. In the infarct core (striatum), following a transient and slight increase during 0.5 h to 4 h post-MCAo, expression of XIAP mRNA markedly decreased. On the other hand, a longer and larger upregulation of XIAP was observed at early time points in border zone (0.5 to 8 h, in dorsolateral cortex; 0.5 to 24 h in hippocampus), then the level of XIAP reduced. A negative correlation was observed between apoptosis and regulation of XIAP gene in these regions. Our findings suggest a possible association between expression of XIAP gene, apoptosis and delayed infarct following ischemia.

Activity of Ginkgo biloba extract and quercetin on thrombomodulin expression and tissue-type plasminogen activator secretion by human umbilical vein endothelial cells.

OBJECTIVE: In order to investigate the pharmacological properties of Ginkgo biloba extract (GBE) on improving blood circulation, the regulating action of GBE and quercetin (a main flavonoid ingredient in GBE) on thrombomodulin (TM) expression and tissue-type plasminogen activator (t-PA) secretion was studied. METHODS: Using flow cytometer and gel image system respectively, we evaluated the TM expression and the t-PA secretion by human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: The increase of TM expression on HUVECs surface was induced by GBE rather than quercetin in a dose- and time-dependent manner. Both GBE and quercetin increased the t-PA release significantly. CONCLUSION: The effect of GBE on improving blood circulation may be partly attributed to its promoting TM expression and t-PA secretion by endothelial cells, and quercetin participated in the effect of GBE on t-PA secretion. However, the action of GBE on increasing TM expression needs further study.

[Effect of dialysis time in vivo on recovery of amino acids for micro-dialysis probe].

OBJECTIVE: To investigate the variety of vitro recovery of amino acids for microdialysis probe after different dialysis time in vivo. METHODS: Probes were dialyzed in the amino acids standard solutions with microdialysis system,amino acid standard solutions and the microdialysate of probe were detected by the method of precolumn derivation with HPLC-RF. RESULT: After using different time of probe made by regenerated cellulose membrane, the vitro recoveries of Asp, Glu and GABA were not completely same (Asp: F=19.669, P=0.000; Glu: F=103.955, P=0.000; GABA: F=3.454, P=0.040); while the vitro recovery of Tau had no obvious difference(F=2.001, P=0.152). After using 6 h in vivo, recovery remain percentage (RRP) of Asp, Glu,Tau and GABA was 64.34 %, 67.36%, 103.11 % and 98.23 %, respectively, the recoveries of Asp, Glu decreased obviously (Asp: P < 0.01,Glu: P <0.05). After using 12 h in vivo, the RRP of Asp, Glu, Tau and GABA was 43.44 %, 24.42%, 77.45 % and 67.36 %, respectively, the recoveries of Asp, Glu and GABA decreased obviously (Asp: P < 0.001, Glu: P < 0.001, GABA: P < 0.05). After using 24 h in vivo, the RRP of Asp, Glu,Tau and GABA was 36.26 %, 12.24 %, 89.48 % and 71.35 %, respectively, the recoveries of Asp, Glu, GABA decreased obviously (Asp: P < 0.0001, Glu: P < 0.0001, GABA: P < 0.01). CONCLUSION: Dialysis in vivo could lead to the decline of recovery of probe, the decline is more obvious after longer dialysis. So when making brain dialysis experiments, the use time of probe should not be too long. To improve the validity of data, some calibration should be made on the recoveries of probe.

The study of anti-metabolic syndrome effect of puerarin in vitro.

Puerarin is an isoflavone extracted from Chinese plant, Pueraria lobata (Wild.) Ohwi. It has been reported to have comprehensive pharmacological action in treatment of diabetes and cardiovascular diseases. The purpose of this study was to link the scattered effects of puerarin and to find the common mechanisms underlying. We investigated the effect of puerarin on the pivotal common pathogenic factors of metabolic syndrome, which includes obesity, Type II diabetes and cardiovascular diseases. Recently, a large body of evidence indicates that there is a complicated interplay among insulin resistance, adipocytes and endothelial dysfunction that links the abnormalities of metabolic syndrome. Results of present study showed that puerarin could potentiate insulin-induced preadipocyte differentiation, promote glucose-uptake of adipocytes that have been induced insulin resistance by high glucose, and prevent TNF-a-induced apoptosis and viability loss of endothelial cells. Furthermore, we found that these effects are probably due to promote PPARgamma expression and partly through inhibiting abnormal TNF-a-induced intracellular-free Ca(2+) accumulation of endothelial cells. Overall, our synthetical study links the comprehensive pharmacological actions of puerarin to the recognized common pathogenesis of metabolic syndrome, and provides a new insight into the mechanism of puerarin effect.

Inhibitory effects of jujuboside A on EEG and hippocampal glutamate in hyperactive rat.

In this study, the inhibitory effect of jujuboside A (JuA) on a penicillin sodium (Na-PCN) induced hyperactivity model was investigated. Cortical EEG (electroencephalogram) and the concentration of hippocampal Glutamate (Glu) were monitored simultaneously in vivo as indicators of rat's excitatory state. Power spectral density (PSD) and gravity frequency of PSD were calculated. JuA (0.05 g/L and 0.1 g/L) inhibited the EEG excitation effect caused by Na-PCN by increasing the power of delta1 and delta2 bands (P<0.01 vs model) and lowering the gravity frequency of PSD (P<0.01 vs model). JuA also remarkably reduced the Glu elevation induced by Na-PCN (P<0.05 vs model). Diazepam also depressed Glu concentration and lowered the gravity frequency, but it showed a different EEG pattern in increased beta2-activity (P<0.01 vs model). EEG excitation caused by Na-PCN correlated with Glu elevation during the first hour. Neurophysiological inhibitory effects of JuA and diazepam were more persistent than their Glu inhibitory effects.

[Effect of l-tetrahydropalmatine on expression of adhesion molecules induced by lipopolysaccharides in human umbilical vein endothelium cell].

OBJECTIVE: To investigate the effect of l-tetrahydropalmatine (l-THP) on adhesion molecule expression induced by LPS in human umbilical vein endothelium cell (HUVEC). METHOD: HUVEC were obtained from the human umbilical cord vein and treated by LPS or LPS plus different concentration of l-THP. Expression of ICAM-1 and E-selectin was assayed by flow cytometer. RESULT: l-THP(250 mg x L(-1)) markedly attenuated ICAM-1 and E-selectin expression induced by LPS(P <0.05). l-THP(50 mg x L(-1) inhibited E-selectin expression (P < 0.05) but had no effect on ICAM-1 (P > 0.05). l-THP (10 mg x L(-1)) had no effect on expression of both adhesion molecules. DXM (50 mg x L(-1)) completely inhibited both responses induced by LPS (P < 0.05). CONCLUSION: l-THP depresses expression of ICAM-1 and E-selectin induced by LPS, which suggests that I-THP represent a promising candidate to be developed into therapies for the treatment of inflammation.

Regulative effects of hawthorn leave flavonoids on cytotoxicity, NO and Ca2+ in hypoxia-treated human umbilical vein endothelial cells.

OBJECTIVE: To evaluate the potential effect of HLF (Hawthorn leave flavonoids, w/w, 80% flavonoids) against thrombus formation, effect of HLF on hypoxia-treated human umbilical vein endothelial cell (HUVECs) was studied. METHOD: The levels of cytotoxicity and NO upon HUVECs were studied by flow cytometry. Moreover, the level of calcium ion in HUVECs was examined through laser scanning confocal microscopy. RESULT: Data from this study showed that HLF at concentrations of 5 micrograms/ml and 10 micrograms/ml decreased the cytotoxicity of hypoxia to HUVECs (P<0.05, P<0.01). The intracellular levels of NO and calcium ion were downregulated by HLF at concentrations of 5 micrograms/ml (P<0.01; P<0.01) and 10 micrograms/ml (vs control, P<0.01; P<0.01) too. CONCLUSION: Results observed suggest that HLF protect HUVECs from hypoxia partly through its regulative effect on NO and calcium ion levels.

A flow cytometry-based assay for quantitative analysis of cellular proliferation and cytotoxicity in vitro.

A novel method based on flow cytometry (FCM), which can count the number of detected cells, has been developed for the evaluation of cellular proliferation and cytotoxicity in vitro. It provides a tool that directly counts cell number without being influenced by the metabolic state of the cells, discriminates target cells from effector cells in cell-mediated cytotoxicity assay, and with less treatment step and free radioactivity. In this paper, we have prepared the PG cells (a highly metastatic human lung cancer cell line) and peripheral blood lymphocytes (PBL) with various concentrations and ratios of concentration to validate the method. The results were compared with MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) assay and the regression analysis results showed that this method worked very well. We have also used this method to evaluate mitogen-induced proliferation and cytotoxicity. The results indicated that this method might yield high sensitivity and reliability.

Novel human endothelial cell-engineered polyurethane biomaterials for cardiovascular biomedical applications.

A tri-block coupling-polymer composed of 4,4'-methylenediphenyl diisocyanate and poly (ethylene oxide) (PEO), abbreviated MPEO, was used as the template surface-modifying additive (SMA), based on which selected amino acids (lysine, arginine, glycin, and aspartic acid) and RGD peptide were respectively conjugated as functional endgroups of the PEO spacer-arms through sulfonyl chloride-activation routes. After the immobilization of biofunctional factors, the SMA-MPEO derivatives were noncovalently introduced onto the biomedical poly(ether urethane) (PEU) surfaces by physical blending methods. The SMA synthesis and PEU surface modification were monitored and analyzed by nuclear magnetic resonance spectroscopy, attenuated total reflection-infrared spectroscopy, and X-ray photoelectron spectroscopy. The human umbilical vein endothelial cells (HUVECs) were collected and harvested manually by collagenase digestion. The cell culture was performed respectively on the MPEO derivative-modified PEU surfaces and also on the surfaces of the commercially available polystyrene cell-culture plates (TCPS) for control. The cell adhesion rates and cell proliferation rates of the in vitro cultivated HUVEC were measured using flow cytometry. The individual cell viability rates were determined with MTT assay. The cell morphologies of the living HUVECs were investigated by optical inverted microscopy, and more detailed information was acquired from scanning electrical microscopy. The results indicated that the efficacy of SMA functional endgroups was the dominant factor for HUVEC compatibility; the proper-sized PEO spacers (M(w) 2 k) could support and mobilize the functional endgroups, optimizing the surface (interface) environment for the cell growth. As the endgroups of the SMA-MPEO derivatives and the bio-functional factors, the basic amino acids (lysine and arginine) demonstrated similar performances to that of the widely acknowledged cell growth-promoter, RGD peptide, which were superior to TCPS. Therefore, these MPEO derivative-modified PEU materials are promising to serve as novel polymeric permanent implants or interventional devices for cardiovascular biomedical applications.

Dynamic investigation of leukocyte-endothelial cell adhesion interaction under fluid shear stress in vitro.

To establish a method to investigate the dynamic adhesion between leukocytes and human umbilical vein endothelial cells (HUVECs) under definite shear stress. A parallel plate flow chamber system was developed to produce the definite shear stress in vitro. After the cultured HUVECs were loaded in the flow chamber, the circulation solution containing acridine orange (AO)-labeled leukocytes was perfused to flow through chamber at 0.71 dynes/cm(2). In this case, leukocyte-endothelial cell adhesion process was induced. Lipopolysaccharide(LPS) was used as the chemical stimulus and dexamethasone(DXM) was used as the anti-inflammatory reagent. The adhesion process was recorded in videotape by Olympus IX70 fluorescence microscope and CCD-camera. Then the number of adhesion leukocyte, slow and fast rolling velocities of leukocytes on the surface of HUVECs were measured based on the captured images. The number of static adhering and slow rolling leukocytes on the HUVECs treated with LPS was significantly increased by 23.7-fold and 4.1-fold compared with that of the control group. Meanwhile, both the slow and fast rolling velocities of the leukocytes on HUVECs treated with LPS were significantly decreased by 25.6% and 26.1%. When HUVECs were treated with both LPS and DXM, the effect of LPS was inhibited obviously. This developed method can be used in studying ECs adhesion function affected by different chemical and physical stimulus and evaluating the various compounds interfering with cell adhesion.

Inhibitive Effect of N-nitro-L-arginine on Hippocampal Neurons Excitation.

The inhibitive effect of N-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor) on the excitation of cultured rat hippocampal CA1 neurons, stimulated by penicillin G(PG), was investigated. A rapid intracellular NO production induced by PG (4 000 IU/ml) was disclosed when monitored with laser scanningconfocal microscopy (LSCM). Pretreatment of L-NNA (0--10 &mgr;mol/L) dose-dependently inhibited the NO production, and the intercellular level of glutamate (15 min after PG stimulation) as well. Meanwhile, L-NNA(1 and 10 &mgr;mol/L) also significantly inhibited the immunoreactive methionine enkephalin (ir-M-ENK) level. Furthermore, the immunoreactive dynorphin B (ir-DYN-B) level was increased significantly by L-NNA (10 &mgr;mol/L). Finally, beta-FNA (100 &mgr;mol/L, an M-ENK receptor inhibitor) facilitated the inhibitive effect of L-NNA on the Glu level, while nor-BIN (100 &mgr;mol/L, a DYN receptor inhibitor) suppressed that effect. In conclusion,L-NNA could inhibit NO production induced by PG (4 000 IU/ml) stimulation, thuslowering the M-ENK level and increasing the DYN-B level, and resulted in a down-regulation of the Glu level and the neuron excitation.