Poster Presentations

PS_1 Pegylation of Hemoglobin Cancels the Inherent Nitric Oxide Scavenging by Hemoglbin

P Cabrales¹, AG Tsai^1,2, JM Friedman³, M. Intaglietta²

¹La Jolla Bioengineering Institute, ²Department of Bioengineering, University of Californiaf, San Diego, La Jolla, CA, ³Department of Physiology and Biophysics Albert Einstein College of Medicine, New York, USA; [email protected]

Vasoactivity, i.e., vasoconstriction, presumably caused by nitric oxide (NO) scavenging or oxygen autoregulation has been defined as the principal problem associated with small hemodynamic radius hemoglobin (Hb) based blood substitutes. Conversely, Hb based materials with very large hydrodynamics radii, achieved by producing hyper-polymers of water trapping via polyethylene glycol (PEG) conjugation have been found to be vasoinactive, and may have vasodilator properties. These effects may be related to mechanisms of NO regulation by Hb in the erythrocyte, namely: i) NO transport in an allosterically controlled reversible chemical reaction with the Cys Betha 93 (SNO-Hb); and, ii) through nitrite reductase activity of deoxy Hb. Principal concerns with these mechanisms are: i) how do these reactions generate bioactive NO? ii) how does the free NO produced arrive to the site of action without reacting or been sequestered?; and, iii) if free NO is not capable of forming nitrosothiols, how are these being generated? A potential answers to these questions is that Hb can produce intermediates that can extract an electron from nitrite ultimately leading to the formation of reaction residues which can effectively generate nitrosothiols. This NO generating mechanism attributed to Hb is a possible pathway for PEGylated Hbs to remain vasoinactive, or may in some conditions cause them to act as vasodilators, even though it has been shown that PEG-Hb is as an effective NO scavenger as other vasoactive Hbs.

Supported by NIH BRP R24-HL64395, PPG HL71064, grants R01-HL62354, R01-HL62318 and R01-HL76182 and ARMY PR023075.

PS_2 Anesthesia Stability Associated with the Use of Perfluorocarbons During Cardiopulmonary Bypass Surgery

A Chávez-Negrete¹, R Verdin¹, BY SalazarVázquez^2,5, P Cabrales³, F Contreras⁴, J Barroso-Arranda⁴ M Intaglietta³

¹Specailities Hospital La Raza, Mexican Social Security Institute (IMSS), Mexico D.F., Mexico, ²Universidad Juárez del Estado de Durango, Dgo., Mexico, ³La Jolla Bioengineering Institute, La Jolla, CA, ⁴OASIS Hospital, Tijuana, Baja California, Mexico, ⁵Department of Bioengineering, Universtity of California, San Diego, La Jolla, CA; [email protected]

Blood substitutes are presently under development to alleviate blood shortages and reduce the use of allogeneic blood. Cardiopulmonary bypass (CPB) is a target for the deployment of these materials since the procedure utilizes blood on and beyond the blood losses associated with routine surgical interventions because of priming volume requirements in the extracorporeal circuit. Volatile anesthetic agents are routinely used to provide anesthesia in CPB. Volatile anesthetics also have a higher solubility in perfluorodecalin the main component of perfluorocarbon (Perftec Tijuana BC); However perfluorocarbons are oils that are immiscible in water and need to be emulsified before IV use. That is why the solubility of volatile anesthetics decreases in Perftec than in plain perflurocarbons. The aim of this study was investigate the effectiveness in terms of duration of the effect and uniformity of responses of the sevofluorane in cardiopulmonary bypass. Phase II, randomized clinical trial was carried out at Specialties Hospital, La Raza Mexican Social Security Institute, after obtain institutional review board approval, 30 patients (14 treatment, 16 controls) aged 53–68 undergoing elective rheumatic or degenerative valve replacement with hypotermic CPB and randomized to received either PFC 5mL/kg or allogeneic blood transfusion 300 mL. After tracheal intubation all patients were ventilated at an FIO2 of 1.0 and sevofluorane anesthetic. The results were compared with unpaired t-test, and p < 0.05 was significant. While oxygen transport efficacy of the PFC was published elsewhere, time under anesthesia PFC was 5.032 ± 0.1655 hr, (n = 14), control 3.996 ± 0.3156 hr (n = 13) (p = 0.003). The correlation to a Gaussian distribution have R² = 0.32 for PFC and R² = 0.02 for allogeneic blood transfusion. It is apparent that the increased duration of anesthesia with PFC is accompanied by a more uniform response.

PS_3 Microvascular Perspective of Current Polyethylene Glycol Surface Decorated Hemoglobins: Expanding Their Boundaries with New Designs

AG Tsai¹, P Cabrales², SA Acharya³, JM Friedman³, M Intaglietta¹

¹Dept. of Bioengineering, University of California, San Diego, CA; ²La Jolla Bioengineering Institute, La Jolla, CA; ³Dept. of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY/USA; [email protected]

Currently most polyethylene glycol hemoglobins (PEG-Hb) have: 1) high oxygen affinity (P50 = 4–8 mmHg), 2) Hb concentration of ∼ 4 g/dl, 3) colloid osmotic pressure higher than plasma (COP = 30 - 60 mmHg), and, 4) viscosity higher than plasma. High oxygen affinity has been shown to target oxygen release downstream from the resistance vessels circumventing autoregulatory microvascular vasoconstriction. Low Hb concentration lowers economic costs and extends resources; however, in combination with high COP provides for a maximal plasma concentration of 2–3 g/dl. Furthermore, the combination of high oxygen affinity and limited plasma Hb concentration may not provide sufficient oxygen once oxygen carrying capacity falls below the transfusion trigger. In this situation increasing infusion fluid Hb concentration could lead to a concomitant increase of COP which dilutes plasma Hb in vivo. To exploit the nonvasoactive properties of PEG-Hb oxygen carrying and delivery capacity, a new class of PEG-Hb molecules with lower oxygen affinity, increased Hb concentration with acceptable COP < 40 mmHg is currently being designed and studied in experimental models. Different PEG-linker chemistry, PEG chains molecular weight, and patterns of PEGylation are used to increase cooperativity, solution viscosity and COP, factors that lead to improved oxygen delivery capacity via higher microvascular perfusion which also correlates with improved survival.

Supported by NIH PPG HL71064, ARMY PR023075 and HL064395.

PS_4 Rheological Properties of Hemoglobin Vesicles Suspended in A Series of Plasma Substitute Solutions

A Sato, H Sakai, S Takeoka, E Tsuchida

Research Institute for Science and Engineering and Graduate School of Science and Engineering, Waseda University, Tokyo 169-8555, Japan; [email protected]

Hemoglobin vesicles (HbVs) are developed as artificial red cells. The HbV suspension does not possess a colloid osmotic pressure. Therefore, HbV must be suspended in or co-injected with an aqueous solution of a plasma substitute (water soluble polymer), which might interact with HbV. We clarified the unique rheological properties of HbVs suspended in plasma substitute solutions (e.g. recombinant human serum albumin (rHSA, Mw. 67 kDa), dextran (DEX, Mw. 40 kDa), modified fluid gelatin (MFG, Mw. 30 kDa), and hydroxyethyl starch (HES70, Mw. 70 kDa; HES130, Mw. 130 kDa; HES200, Mw. 200 kDa; HES670, Mw. 670 kDa)). In this paper, we further studied the rheological properties of the mixtures of HbV, plasma substitute solutions, and blood. The HbV suspended in rHSA was nearly Newtonian and that mixtures with blood exhibited very low viscoelasticity. On the other hand, DEX, MFG and HES670 induced flocculation of HbV possibly by depletion interaction and rendered the suspensions non-Newtonian with the shear thinning profile. Their mixtures with blood exhibited high viscosities. However, flocculation formation was reduced with increasing the mixing ratio of blood. Microscopically, the flow pattern of the flocculated HbV perfused through microchannels (4.5m wide, 20 cmH2O applied pressure) showed no plugging. Furthermore, the time required for passing was simply proportional to the viscosity. Collectively, the viscosity of HbV suspension was influenced by the presence of plasma substitutes. HbV suspension would provide unique opportunities to manipulate the rheological properties for various clinical applidations in addition to its use as a transfusion alternative.

PS_5 PEG-conjugated Hemoglobin Combination with Cisplatin Enforced the Antiangiogeic Effect in a Cervical Tumor Xenograft Model

Dai Min¹, Yu Minghua¹, Han Jianqun¹, Li Hongwei¹, Zhang Jian¹, Liu Qian², Xiu Ruijuan¹

¹Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China ²Peking Union Medical College Hospital (PUMCH), Beijing, China; [email protected]

Angiogenesis, an essential event involved in a tumor's progression and metastasis, is regulated by hypoxia. Hypoxia widely exists in solid tumor due to the abnormal vasculature of tumor tissue and insufficiency of tissue oxygenation. We speculate oxygen carrier can attenuated tissue hypoxia, thereby suppressed the antiangiogenic effect in solid tumor in the context that oxygen carriers have the ability to increase tissue oxygenation. In the present study, PEG-conjugated Hemoglobin solution (0.3 mg/kg i.v. or 0.6 mg/kg i.v.) intravenous administrated to BALB/c nude mice bearing the cervical tumor twice a week with or without the treatment of cisplatin (5 mg/kg i.p.) to investigate whether PEG-conjugated Hemoglobin has a chemo-sensitization effect though antiangiogenesis pathway. Tumor volume was measured every three days and intertumor hypoxia was detected by immunocytochemistry for Hypoxyprobe™ -1. Microvessel density and VEGF mRNA and protein levels were measured to test the antiagiogenesis effect. Our results showed that high concentration of PEG-conjugated Hemoglobin solution impede the growth of tumor compared with the control group significantly. Moreover, VEGF expression was declined when treated with PEG-conjugated Hemoglobin, maybe though the HIF regulation system. Collectively, treatment of PEG-conjugated Hemoglobin combination with cisplatin has an antiangiogeic effect, but the underline mechanism should be further studied.

PS_6 The Study on a Rat Exchange Transfusion Model by Artificial Red Blood Cells (Hemoglobin Vesicle)

Jing Fan, Xueqiao Wang

Tianjin Blood Center Tianjin, P.R. China; [email protected]

Objective: In a rat exchange transfusion model, as an artificial oxygen carrier, the security and validity with the artificial red blood cells (hemoglobin vesicle, HbV) has been verified. Methods The first, to construct a best rat exchange transfusion model. The second, using the model to transfuse blood with the artificial red blood cells. The finally, to evaluate the capabilities of the artificial red blood cells by some methods (such as: Blood routine test, Blood gas test, Flow cytometry and Pathology), and to compare the therapeutic effect of the different product in the rat acute hemorrhagic shock and resuscitation. Results: The artificial red blood cells (HbV) has been developed to provide the oxygen-carrying ability. The Studies have shown that after approximately 70% of hemoglobin is lost from circulation, then supply the artificial red blood cells suspension, the rats survive rate is 100%. The Pathology results indicate that no significant change in the viscera (heart, liver kidney lung). Conclusion: The artificial red blood cells (HbV) are the artificial oxygen carrier with bionics meaning. It can be helpful the acute hemorrhagic shock rats pass the first hour after the severe trauma, that is golden hour.

Key words: artificial red blood cells, hemoglobin vesicle, a rat exchange transfusion model

PS_7 EPO/EPOR System Changes During PEG-Conjugated Hemoglobin Solution with Chemotherapy Treatment in Cancer: Primary Investigation

Han Jianqun¹, Dai Min ¹, Yu Minghua ¹, Li Hongwei¹, Zhang Jian ¹, Liu Qian² and Xiu Ruijuan¹

¹Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China, ²Peking Union Medical College Hospital (PUMCH) Beijing, China; [email protected]

In solid neoplasias, adaptive responses to hypoxia are correlated with angiogenesis, enhanced metastatic spread and poor responses to therapy. Animal experiments and clinical reports have shown that EPO/EPOR the crucial pro-angiogenesis factor increased in cancer hypoxia adaption. Administration of blood substitutes has been suggested as a means of improving tumor oxygenation, so we postulated that EPO/EPOR system may be involved in the effect of PEG-conjugated hemoglobin with chemotherapy treatment. Methods: 50 male golden hamsters were divided into five groups and innoculated tumor cells to the submucosa of golden hamster's cheek pouch. Group served as controls. Group ? received intraperitoneal cisplatin (5 mg/kg). Group received both cisplatin and intravenous PEG-Hb(0.3 g/kg). Group received both cisplatin and intravenous PEG-Hb (0.6 g/kg). Group received intravenous PEG-Hb(0.3 g/kg). All agents administrated twice weekly. One week later hamsters were sacrificed, hypoxia marker pimonidazole staining was employed to detect tumor tissue oxygenation status. EPO/EPOR CD31 and HIF-1α was assayed using immunohistochemistry staining. Expression of EPO in serum detected by ELISA method. Results: Tumor hypoxia region was significantly in group compared to group. Expression of CD31, EPO and EPOR were decreased in group which was positive correlation to HIF-1α expression. Serum EPO concentration did not significantly changed among groups. Conclusion: Higher dose of PEG-conjugated hemoglobin solution with chemotherapy treatment is benefit to tumor tissue oxygenation, meanwhile EPO/EPOR pathway possible invovled. It is suggested that an antiangiogenic effect maybe participated in PEG-conjugated hemoglobin adjuvant to chemotherapy.

PS_8 Effects of Hemotech Blood Substitute on Platelets of Percutaneous Coronary Intervention Patients Ex Vivo

J Simoni, C Roongsritong, G Simoni, JF Moeller, A Kumar, M Pierce, GE Meyerrose

Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A. and HemoBioTech, Inc., Dallas, Texas, U.S.A; [email protected]

Coronary Artery Disease (CAD) is the single largest killer of Americans, both males and females. Every 26 seconds an American will suffer a coronary event such as a heart attack, and every minute someone will die from one. Percutaneous coronary intervention (PCI), with balloon dilatation or stent implantation has revolutionized the treatment of CAD. In recent years on average, more than 1 million coronary angioplasty procedures are performed annually at a cost of more than $30 billion. Although PCI has improved clinical outcomes, two major complications inherent to this procedure have become evident; thrombosis and restenosis. Restenosis that is a multifactorial process mainly involving remodeling following angioplasty and neointimal hyperplasia after stents, occurs in 33–50% of angioplasties and 15–20% of stents. In an attempt to reduce the rate of restenosis, emphasis has been placed on the development of drug-eluting stents (DES), which significantly improved short-term outcomes of PCI vis-à-vis bare metal stents (BMS). However, many current studies question their long-term effectiveness. Delayed healing and late thrombotic risk may supersede the DES reduced in-stent restenosis as compared with BMS. Our recent study found a link between thrombotic and restenotic events via PCI procedure-mediated endothelial injury with subsequent exposure of underlying collagen and accelerated synthesis of 8-isoprostane, both of which represent a strong platelet pro-aggregatory effect that triggers release of vasoconstrictive serotonin (ASAIO J 2007; 53(2):43). The aim of this present study was to investigate ex vivo effect of HemoTech, a free hemoglobin-based blood substitute with pharmacologic properties of adenosine and reduced glutathione (GSH) that was developed at Texas Tech University and licensed to HemoBioTech, Inc. for commercial development. HemoTech was previously found to have a platelet anti-aggregatory activity (ASAIO J 2000; 46(6):679–92). In this study, platelets from PCI patients obtained before, during and 6 hours after the procedure, were incubated with HemoTech (0.15 mM) and challenged with the platelet aggregation agonists (arachidonic acid, ADP, epinephrine and collagen). Results indicate that HemoTech markedly reduces the pro-aggregatory effects of collagen and ADP. The release of platelet serotonin in the presence of HemoTech was also reduced. This data suggests that adenosine and GSH that provide HemoTech with pharmacological properties are the main forces behind its platelet anti-aggregatory effects in PCI.

PS_9 Control of Peroxide-Mediated Hemoglobin Oxidative Reactions: Effects of Reduced Glutathione and Vitamin C

J Villanueva-Meyer, J Simoni, JF Moeller, G Simoni

Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A; [email protected]

Heme oxidation is still the central problem for free hemoglobin (Hb)-based blood substitute developers. The control of Hb oxidative reactions outside of red cells is difficult, since the plasma environment lacks the enzymatic antioxidant system that maintains heme in its functional reduced ferrous form. Spontaneous autoxidation of acellular Hb generates superoxide anion. The rate of this oxidation is augmented by hydrogen ions. Superoxide anion acts as a catalyst and promotes further Hb autoxidation and spontaneously or enzymatically dismutates to form hydrogen peroxide. Hydrogen peroxide reacts with ferric-Hb to produce two toxic intermediates, a ferryl heme iron and a globin tyrosyl radical. Ferryl heme acts as a radical and initiates lipid peroxidation to the same extent as hydroxyl radical. This reaction also produces lipid peroxyl radicals and lipid hydroperoxides that respectively accelerate lipid peroxidation and influence Hb redox cycling. A globin tyrosyl radical was found to be a potent oxidizer and crosslinker of low-density lipoproteins. It is believed that this reactive species may be formed after administration of free-Hb based oxygen carriers, especially in ischemic patients with a diminished ability to control oxidative reactions. The aim of the present study was to further investigate Hb radical chemistry following exposure to inorganic (hydrogen peroxide) and organic peroxides (lipid hydroperoxides). In particular, this research focused on preventing heme oxidation and formation of ferryl-Hb species by using naturally occurring antioxidants: ascorbates (vitamin C) and reduced glutathione (GSH). The results of this study suggest that while ascorbates are effective in reducing the formation of ferryl-Hb, GSH protects heme against excessive oxidation, thus augmenting ascorbate's protective effect. It seems that GSH diminishes the formation of ascorbyl radicals, responsible for the overall oxidation of heme at a late reaction stage. GSH, and to a lesser extent ascorbates, offers protection to the cellular membranes against oxidation induced by Hb activated with hydrogen peroxide and lipid hydroperoxides. The present study provides new evidence that combined treatment, using ascorbates and GSH, is a better option than either one alone.

PS_10 Novel Contributing Factor to Intrinsic Toxicity of Hemoglobin: Serotonin

J Simoni, G Simoni, C Roongsritong, JF Moeller, P Simoni, JA Griswold, DE Wesson

Texas Tech University Health Sciences Center, Lubbock, Texas, U.S.A; [email protected]

Decades of investigating hemoglobin (Hb) intrinsic toxicity significantly improved our knowledge on this intriguing molecule; however, many pathological reactions associated with its presence are not fully elucidated yet. In particular, there is a lack of understanding of some fundamental biological events that trigger the induction of genes upon Hb treatment. While the link between Hb-mediated changes in the cellular redox state and systemic inflammation has been established, it is still unknown to what extent its pro-oxidative potential contributes to other still unresolved toxic events. The vasoconstrictive effects of Hb need more investigation. Hb-mediated vasoconstriction is an early event that occurs within minutes after Hb infusion. Currently accepted mechanisms to explain this phenomenon (i.e., nitric oxide scavenging, calcium influx, eicosanoid, 8-isoprostane, 20-HETE and endothelin-1 formation, as well as others) cannot justify such a quick response. It is still unknown what triggers such a rapid hemodynamic response and to what extent the rise in blood pressure affects the perfusion rate of the vital organs. Our recently published study emphasized that the pseudo-peroxidase activity of Hb may contribute to activation of the renin-angiotensin-aldosterone system that may cause these early vasoconstrictive responses, and even via the aldosterone pathway, cardiac lesions observed after Hb administration (Artif Cells Blood Substit Immobil Biotechnol 2007;35(2):191–210). This present study investigated the possible role of Hb in release of serotonin (5-HT), using the human platelet aggregation model. Platelets are the source of many vasoactive factors, including 5-HT that is known to impact vascular tone through direct activation of Rho-kinase in vascular smooth muscle. Therefore, 5-HT can constrict normal vessels and those injured with compromised endothelium. In this ex vivo study, human platelets were exposed to different concentrations of Hb (.04 to .2 mM) at various time intervals (minutes to days). In an additional experiment, platelets exposed to Hb were challenged with collagen to mimic vascular injury. The results of this study suggest that while Hb can significantly accelerate the release of 5-HT from unstimulated platelets, it can slightly increase platelet aggregation in response to collagen. The observed effects were dose and time dependent. Collagen in the presence of the lower doses of Hb showed a stimulatory effect on 5-HT release, whereas Hb at higher concentrations had a less significant effect. It can be concluded that Hb itself can be a stimulus for 5-HT release, which may affect normal vessel tone. Also Hb at a low level, in the presence of collagen, can stimulate 5-HT release, which can influence the tone of injured arteries. 5-HT could be another factor in Hb-mediated vasoconstriction.

PS_11 Formulation and Characterization of New Perfluorocarbon Based Oxygen Carriers

CI Castro, SE Cuervo, LF Ponce, JC Briceño

Blood Substitutes Laboratory, Fundacion Cardioinfantil and U. of Los Andes. Bogota, DC, Colombia; [email protected]

Perfluorocarbon emulsions are being investigated as intravascular oxygen carriers (PFCOCs). However, the ideal formulation with the required safety, stability and shelf life for clinical use has not been devised yet. The objective of this study was to design, prepare and characterize PFCOCs in which PFC content, emulsion viscosity and osmolality can be varied. In this study formulations were established according to a factorial experimental design with the following factors and levels: PFC concentration (20 and 30 % v/v), viscosity (normal and high) and osmolality (normal and high). The emulsions contained perfluorooctylbromide (Exfluor Corp.) and soybean lecithin as surfactant (Degussa GmbH). Alginic acid sodium salt (Sigma-Aldrich Co.) and dextrose (Baxter Healthcare) were used as viscosity and osmolality modifiers, respectively. In order to characterise the emulsions viscosity, osmolality and stability (droplet size and macro) were determined after preparation. The effect of PFC concentration, viscosity, osmolality and time on emulsion stability (in terms of mean diameter and polydispersity of droplet size distributions) was as follows: polydispersity increases with PFC content; an increase in viscosity increases mean droplet diameter and polydispersity; an increase in osmolality reduces mean droplet diameter; Time does not affect mean droplet size but reduces polydispersity. All formulations were stable by both droplet size and macro analysis up to 80 days, when phase separation occurred. In conclusion, PFCOCs with controlled viscosity and osmolality have been prepared. Emulsion stability has to be improved. Initial evaluation in a rat model of normovolemic hemodilution appears promising.

PS_12 Evaluation of a New Perfluorocarbon Based Oxygen Carriers in a Rat Model of Normovolemic Hemodilution: Effect of Altitude

T Gardeazabal¹, P Cabrales², M Intaglietta², JC Briceño¹

¹Blood Substitutes Laboratory, Fundación Cardioinfantil and U. of Los Andes, Bogotá, DC, COLOMBIA; ²Department of Bioengineering, University of California-San Diego, La Jolla, CA, USA; [email protected]

The oxygen carrying capacity of perfluorocarbon based oxygen carriers (PFCOCs) has been extensively documented. Unlike hemoglobin, PFCs transport oxygen dissolved, their carrying capacity depending on oxygen's partial pressure, which in turn depends on barometric pressure. We decided to investigate if oxygen delivery and consumption in a rat model of hemodilution with PFCOCs was impeded by high altitude. Anesthetized rats were submitted to a two-exchange normovolemic hemodilution of 40% of volemia. First exchange was performed with 10%-HES. Second exchange with 80% PFCOC and 20% 10%-HES. The PFCOC contained 20%(v/v) of perfluorooctylbromide. Animals were then ventilated with 100% oxygen. Blood gasimetry, hematocrit and hemoglobin content were measured at baseline and 15 min after each exchange and arterio-venous oxygen content (CavO₂) was calculated. For the sea level group (La Jolla) barometric pressure was 1.0 atm and n = 9, while for the high altitude group (Bogotá, 2600 m above sea level) p = 0.74 atm and n = 6. Arterial variables showed a slight elevation in baseline values in the sea level group, but no significant differences were found between groups. As compared to baseline, CavO₂ decreased after the first exchange and recovered after the second exchange, but there were not significant differences between the groups. The PFCOC evaluated improved oxygen transport and delivery during experimental normovolemic hemodilution in rats. This effect was not affected by the lower PaO₂ obtained at 2600 m above sea level.

PS_13 The Study of Polymerized Human Hemoglobin on Modified Process Optimizing

FJ Li, HH Zhang, JF Wang, CM Yang

Institute of TianJin Union Biotechnology, Tianjin, China [email protected]

Purified hemoglobin was modified with PLP and polymerized with GDA to get the products. Comparing physical, chemical and biological properties of different procedure between modification before and after polymerization, there isn't significant difference in the molecular distribution, methemoglobin concentration, oxygen carrier capacity, P50 and spectral analysis. Furthermore the procedure of modification after polymerization can save PLP greatly, which can decrease cost greatly. So the procedure of modification after polymerization is a better way in research and production. Adding GSH could control the increasing of MetHb. Comparing physical, chemical and biological properties of different procedure, there isn't significant difference in the molecular distribution, methemoglobin concentration, oxygen carrier capacity and spectral analysis between adding GSH before and after PLP. But the P50 of adding GSH before is much lower than that of adding GSH after PLP. So procedure of adding GSH after PLP is a better way.

PS_14 Cross-Linked Inulin Polysaccaride—a Plant-Derived Plasma Substitute Candidate

SP Li, CY Zheng, T Liu GH Ma, ZG Su

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering. Chinese Academy of Sciences, Beijing, China [email protected]

Inulin, a polysaccharide from plant source, has a potential to become a safe and effective plasma substitute. In this study, cross-linked inulin was prepared and tested as a volume expander of blood vessels in situations where plasma substitute is required. Three cross-linked inulin polysaccharides, differing in molecular weight, were evaluated in vitro for their physical and chemical properties using commercially available plasma substitutes for comparison. Furthermore, these substitutes were evaluated in vivo by animal experiments. The results indicated that all these three products could efficiently improve the blood pressure of bleeding animals. These animals survived for the observation duration (14 days). Moreover, the histology investigation showed that the side effects on liver and kidney were molecular weight-dependant. The cross-linked inulin polysaccharide with low molecular weight does not cause detectable damages to liver and kidney.

PS_15 Cardioprotective Effect of Polymerized Human Placenta Hemoglobin (Polyphb) on Rat Heart In Vitro

T Li¹, J Li¹, WS Zhang¹, HH Zhang², CM Yang², J Liu¹

¹Laboratory of Anesthesiology & Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, P.R. China 610041. ²Institute of Tianjin Union Biotechnology, Tianjin, P.R. China 300457; [email protected]

Polymerized human placenta hemoglobin (PolyPHb), one of hemoglobin-based oxygen carriers, may have the potential to improve oxygen supply to the ischemia myocardium during heart arrest and storage for its nm-size and oxygen-carrying ability at low temperature. Therefore, this study was designed to investigate whether St. Thomas solution (STS) mixed with PolyPHb would provide more protection against myocardial ischemia/reperfusion injury for heart cold storage in vitro. Thirty male Sprague-Dawley rats weighing from 250 to 350 g were randomly divided into three groups (n = 10, in each group): STS group (STS), STS+Self-blood group (STS with 0.5g/dl rat self-blood), and STS+PolyPHb group (STS with 0.5g/dl PolyPHb). Rat hearts were isolated and perfused with Krebs-Henseleit solution (KHS) at 37°C on the langendorff apparatus. After 30 min of equilibration, they were arrested and stored at 4°C for 8 hours, then reperfused for 2 hours. The results indicate that there were no differences among the three groups in HR and CF; however, the percentage recovery of rate-pressure product (RPP, HR × LVDP) and dp/dtmax of hearts in STS+PolyPHb group were significantly higher than those in STS and STS+Self-blood groups during reperfusion (RPP: 62.33± 1.61% vs 22.39± 1.21% in STS group and 41.42± 1.19% in STS+Self-blood group, p < 0.001; dp/dtmax: 61.88± 5.66% vs 23.90± 5.95% in STS group and 40.23± 3.88% in STS+Self-blood group, p < 0.001), LDH and CK release were also significantly decreased (p < 0.001). Meanwhile, the infarct area in STS group (41.99± 2.11%) and STS+Self-blood group (24.62± 2.01%) was significantly larger than that in STS+PolyPHb group (16.55± 1.16%) (p < 0.05). Thus, those finding indicated that PolyPHb in STS could significantly protect the heart from ischemia/reperfusion injury and improve the function of rat hearts after prolonged cold storage in vitro.

PS_16 Resuscitative Effect of HES40 from Hemorrhagic Shock in Rats

Li Tao^1,2, Liu Liang-ming², Yang Chengmin¹

¹Tianjin Union Biotech Development Research Institute; ²State Key Laboratory of Trauma, Burns and Combined Injury, Department 2, Research Institute of Surgery, Daping Hospital, The Third Military Medial University, Chongqing 400042, China; [email protected]

Objective:To observed the resuscitative effect of 6% hydroxyethyl starch 40 (HES40) on hemorrhagic shock. Methods: Sixty SD rats subjected to hemorrhagic shock by 45% hemorrhage were divided into HES40 group and Lactated Ringer's(LR) group, which were further divided to 20 ml/kg, 30 ml/kg and 40 ml/kg group. The parameter observed included mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP), the maximal change rate of left intraventricular pressure ± dp/dt_max), artery blood gas and survival time and survival rate. Results: HES40 could significantly increase the MAP, the hemodynamic parameters of shock rats. The survival time and 24 hour survival rats of shock rats in HES40 group were significantly longer than in LR group. Meanwhile, HES40 had a better effect to improve blood gas than LR. Conclusion: HES40 has good beneficial effect to resuscitate hemorrhagic shock.

Key Words: Hemorrhagic shock; HES; LR

PS_17 Equivalent Efficiency Study of HES130 and Voluven on Hemorrhagic Shock in Rats

Li Tao^1,2, Liu Liang-ming², Yang Chengmin¹

¹Tianjin Union Biotech Development Research Institute; ²State Key Laboratory of Trauma, Burns and Combined Injury, Department ², Research Institute of Surgery, Daping Hospital, The Third Military Medial University, Chongqing 400042, China [email protected]

Objective:To compare the resuscitation effect of 6% hydroxyethyl starch 130 (HES130) and Voluven on hemorrhagic shock in rats. Methods: 60 SD rats subjected to 45% hemorrhage were divided into HES 130 group and Voluven group. Each of these two groups was further divided to 20 ml/kg, 30 ml/kg and 40 ml/kg group. The parameters observed included mean arterial blood pressure (MAP), hemodynamic parameters such as left intraventricular systolic pressure (LVSP), the maximal change rate of left intraventricular pressure ± dp/dt_max), artery blood gas and survival time and survival rate. Results: Effects of different volume of HES130 improving hemodynamic parameters were equivalent to Voluven except 20 ml/kg of HES130 elevated MAP slightly weaker than HES130 30 ml/kg and 40 ml/kg group and 40 ml/kg of Voluven increased LVSP higher than 40 ml/kg HES130. Artery blood gas had no significant differences among all groups after HES130 and Voluven infusion. Survial time and survival rate of shock rats after HES130 and Voluven infusion were almost same, except Voluven 40 ml/kg group was slightly better than other groups. Conclusion: HES130 has good beneficial effect on hemorrhagic shock. Middle and lower volume of HES130 has a equivalent antishock effect as compared to the same volume of Voluven.

Key Words: Hemorrhagic shock; HES; Voluven

PS_18 Equivalent Efficiency Study of HES200 and Haes on Hemorrhagic Shock in Rats

Li Tao^1,2, Liu Liang-ming², Yang Chengmin¹

¹Tianjin Union Biotech Development Research Institute ²State Key Laboratory of Trauma, Burns and Combined Injury, Department ², Research Institute of Surgery, Daping Hospital, The Third Military Medial University, Chongqing 400042, China; [email protected]

Objective: To compare the resuscitative effect of 6% hydroxyethyl starch 200 (HES200) and Haes on hemorrhagic shock in rats. Methods: 60 SD rats subjected to 45% hemorrhage were divided into HES200 group and Haes group. Each of these two groups was further divided to 20 ml/kg, 30 ml/kg and 40 ml/kg group. The observed variables included mean arterial blood pressure (MAP), hemodynamic parameters artery blood gas and survival time and survival rate. Results: HES200 and Haes significantly improved the hemodynamics of shock rats including MAP, left intraventricular systolic pressure (LVSP), the maximal change rate of left intraventricular pressure ± dp/dt_max). Different group between HES200 and Haes had no significant difference, except 40 ml/kg of Haes increased LVSP and ± dp/dt_max slightly higher than the same volume of HES200. But HES200 had a little better effect on survival time and survival rate than Haes. Conclusion: HES200 has good beneficial effect on hemorrhagic shock. There is no significant difference between HES200 and Haes in the effect of resuscitation shock.

Key Words: Hemorrhagic shock, HES, Haes

PS_19 Preparation and Characterization of Genipin Crosslinked Gelatin as a New Plasma Substitute

T Liu, GF Zhang, CY Zheng, SP Li, ZG Su

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering. Chinese Academy of Sciences, Beijing, China; [email protected]

Genipin a natural crosslinking agent with low cytotoxicity; is an aglucone of geniposide extracted from traditional Chinese medicine. In this study, genipin was used to prepare gelatin plasma substitute. The preparation conditions such as molecular weight range of gelatin, initiator concentration and ratio, reaction time, pH and temperature were optimized. The effects of product on blood volume and metabolism in blood were evaluated by in-vivo studies with male Sprague Dawley rats and chinchilla rabbits. Changes of blood pressure during blood exchanging process indicated that genipin-crosslinked gelatin was efficient on blood volume expand. Blood samples were collected in the first 48 hours and analyzed, which demonstrated that metabolic kinetics of product met well with the two-compartment model and its half life was about 6 hours in rabbit blood. All experimental animals survived and they were sacrificed after 2 weeks observation. tissues (including heart, liver, spleen, kidney and brain) and blood samples were collected for tissue sections and blood analysis. These investigations indicated that genipin-crosslinked gelatin has a potential to become a safe, efficient and thus promising candidate plasma substitute.

PS_20 Characterization of Hemospan® by Reverse-Phase High Performance Liquid Chromatography, Capillary Zone Electrophoresis, and SDS-PAGE

A Malavalli, G Mkrtchyan, S Spann, KD Vandegriff, RM Winslow

Sangart, Inc., 6175 Lusk Blvd., San Diego, CA USA; [email protected]

Hemospan® is a hemoglobin-based oxygen carrier currently undergoing clinical trials. Hemospan is produced by conjugation of human oxyhemoglobin to 5-kD maleimide-activated polyethylene glycol (PEG). Molecular properties of this product include increased O₂ affinity, molecular size, and colloidal osmotic pressure, leading to prolonged vascular persistence and blood volume expansion. In this study we present chemical analyses of the Hemospan drug product for product characterization. Studies were performed to identify the number of PEG chains conjugated to globins based on C₃ reverse-phase liquid chromatography (RP-HPLC), capillary zone electrophoresis (CZE) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). RP-HPLC is used to identify individual unmodified and PEG-modified globins, based on hydrophobicity and size. SDS-PAGE, with iodine staining specific for PEG, and short-capillary zone electrophoresis (CZE) are used to provide product profiles based on the number of PEGs conjugated to the globins (not based on α and β subunit separation). These methods can be used to determine the degree of PEGylation of Hemospan or any PEGylated hemoglobin sample. Thus, we have demonstrated that Hemospan contains PEGylated α and β globins with up to 4 PEG molecules conjugated to each globin, results that are consistent with the sites of maleimidation identified by tryptic-digestion and mass analysis using N-ethylmaleimide in place of MalPEG, revealing the following modified residues: α Lys7, 16 and 40; β Lys8, 17, 59, 66, 95 and 132; β Cys93. These biochemical methods provide simple, fast and accurate analytical tools for the characterization of PEGylated hemoglobin molecules and are helpful tools in continued drug development, release and stability testing.

PS_21 Hemospan® Dissociation and Haptoglobin Binding

A Malavalli, H Jan, G Mkrtchyan, KD Vandegriff, RM Winslow

Sangart, Inc., 6175 Lusk Blvd., San Diego, CA USA 92121 [email protected]

Hemospan® is a hemoglobin-based oxygen carrier currently undergoing clinical trials. Hemospan is produced by conjugation of human oxyhemoglobin to 5-kD maleimide-activated polyethylene glycol (PEG). The product has an average of 7 PEG polymers conjugated per hemoglobin tetramer. Molecular properties include increased oxygen affinity, size, and colloidal osmotic pressure. Since Hemospan is not chemically cross-linked, we analyzed dimer formation in Hemospan, as either PEGylated or free α β dimers. Dimer formation was first evaluated by dilution studies using size-exclusion chromatography (SEC) with unmodified hemoglobin or Hemospan at decreasing concentrations from 2.6, 0.06, to 0.015 mM (in heme). While unmodified hemoglobin showed clear α β dimer formation upon dilution, < 2% of free, non-PEGylated α β dimer was detected in the diluted Hemospan sample (at the limit of detection). Forced dissociation studies were then performed using SEC with 0.9 M MgCl₂. In this case, unmodified hemoglobin showed total conversion to α β dimers, while, again, Hemospan showed a profile with < 2% free, non-PEGylated α β dimers and > 98% PEGylated α β dimer species with 2-4 PEGs per dimer. To determine the ability of Hemospan to complex with human haptoglobin, we performed binding studies using equimolar mixtures of unmodified hemoglobin or Hemospan and haptoglobin (type 1-1). Mixed samples were evaluated by SEC and electrophoresis. These studies demonstrate that Hemospan binds to haptoglobin, which explains the transient disappearance of serum haptoglobin after dosing in humans. The studies also show that the dissociation of Hemospan tetramers to dimers is much less than for unmodified hemoglobin, but they do not distinguish whether Hemospan binds to haptoglobin as a PEGylated hemoglobin tetramer, dimer, or both.

PS_22 Characterization of Human Serum Albumin Complex with Carboxyfullerene

X Qu¹, T Komatsu^1,2, E Tsuchida¹

¹Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan; ² PRESTO, Japan Science and Technology Agency (JST) 332-0012, Saitama, Japan; [email protected]

Human serum albumin (HSA) is a versatile plasma protein that can bind a wide range of endogenous and exogenous compounds. The carboxyfullerene (CF), acting as a drug for neurodegenerative diseases, can also be trapped by HSA with a high binding constant (> 107 M-1) after being injected into the blood stream. Herein, we characterize for the first time the structure and photophysical properties of HSA complex with CF (HSA-CF). The CF is easily bound to HSA, yielding a stable orange-colored solution. The UV-vis. absorption spectrum is dominated by protein absorption at 280 nm and CF absorption at 490 nm. The spectroscopic pattern is similar to that of 1:1 mixture of HSA and CF; indicating that the CF is monomolecularly incorporated into HSA and no specific interaction between the two molecules at the ground state. Laser flash photolysis (Nd:YAG, SHG, 532 nm) to the rHSA-CF solution under N2 showed a triplet-triplet absorption of the CF chromophore (max: 734 nm). The lifetime of the triplet state was 46 s. In the presence of O2, energy transfer occurred from the triplet state of rHSA-CF to the O2 molecule to produce a singlet O2. The quenching rate constant (kq) was 1.8 × 108 M-1s-1, which is 8-fold lower than that of the CF monomer solution. The slower kq of the protein system suggests that the quenching reaction is diffusion controlled.

PS_23 Multiwavelength Pulse Spectrophotometry Applicable for Hemoglobin Vesicles

H Suzaki^1,2, H Sakai¹, N Kobayashi², T Ikeda³, H Horinouchi³, K Kobayashi³, S Takeda^1,2, T Togawa¹, E Tsuchida¹

¹Research Institute for Sci. & Eng., Waseda University, Tokyo; ²Ogino Memorial Lab., Nihon Kohden Co., Tokto; ³Dept. of Surgery, School of Medicine, Keio University, Tokyo, Japan; [email protected]

A pulse oxymeter (POM) is indispensable in a clinical setting to monitor non-invasively arterial blood O₂-saturation (SpO₂) and heart rate of a patient. POM is designed to analyze spectrum of Hb on the artery pulse with two wavelengths under the assumption that there are only two components of Hb, namely, deoxy- and oxy-Hb. Hb-vesicles (HbV, 250 nm) are artificial O₂ carriers, and the safety and efficacy as a transfusion alternative have been clarified. HbV encapsulates human Hb, however, the particle dispersion induces a strong light scattering in comparison with RBC, and gradual metHb formation is inevitable because of the lack of the metHb-reducing system in HbV. It is speculated that such peculiar characteristics of HbV would interfere with SpO₂-monitoring. Under such background, we aimed to clarify the interference effect of HbV on the commercially available POM, and to establish a method to avoid such effect using a multiwavelength system. Using the in vitro blood circulation system comprised of a pulsatory pump, an artificial lung, and the POM (Nihon Koden, DDG-3300; detection wavelengths: 660, 940 nm), we clarified that the stepwise addition of 60 vol% HbV to a pig blood tended to decrease the SpO₂ to 70%, and interference effect of HbV was evident. We introduced two detection wavelengths (620, 730 nm) in addition to 660 and 940 nm to a probe (TL-301), and tested its effectiveness in the 40%-blood exchange experiments using 5 beagles. FiO₂ was set 100, 20, and 10%, and the accurate SaO₂ and metHb content were monitored for 4 hrs. Equations to calculate SpO₂ and metHb content was established by a multiple regression analysis. Using the modified POM with 4 wavelengths, the deviation of SpO₂ is minimized from –7.2 ± 3.3% to 1.1 ± 1.9%, and metHb level can be measured with in the deviation 0.7 ± 1.3%. Optimization of wavelengths and more experiments will improve the accuracy of POM for HbV.

PS_24 Fluid Resuscitation with Hb-Vesicles in Hemorrhaged Rats and Profiles of Recovery for 14 Days

H Sakai¹, H Horinouichi², E Tsuchida¹, K Kobayashi²

¹Research Institute for Science and Engineering, Waseda University, Tokyo; ²Dept. of Surgery, School of Medicine, Keio University, Tokyo, Japan; [email protected]

Hb-vesicles (HbVs) are artificial O₂ carriers that encapsulate concentrated Hb solution in phospholipid vesicles. The efficacy of HbV for fluid resuscitation from hemorrhagic shock has been characterized for only several hrs in animal models, and subsequent profiles of recovery involving the degradation of HbV in RES and hematopoiesis remain unknown. This study undertakes, for the first time, 14 days observation after resuscitation with HbV suspended in recombinant human serum albumin (rHSA) solution. Wistar rats were anesthetized with 1.5% sevoflurane inhalation, while spontaneous breathing was maintained. Shock was induced by 50% blood withdrawal from a femoral artery. The rats showed significant hypotension, metabolic acidosis, and hyperventilation. After 15 min, they received HbV suspend in rHSA (HbV/rHSA) (n = 20), or shed autologous blood (SAB) (n = 20) through a femoral vein. Both groups showed rapid recovery of hemodynamic and blood gas parameters and they were stable for 6 hrs. There was no significant difference between the groups. After removing the catheters and awakening, the rats were housed in cages for up to 14 days. The HbV/rHSA group gained the body weight and the reduced Hct returned to the original level in 7 days, indicating the elevated hematopoiesis. Both groups showed elevation of AST and ALT at 1 day due to the systemic ischemia reperfusion injury. Splenomegaly was significant in the HbV/rHSA group at 3 days due to the accumulation of HbV, however, it subsided within 14 days. Histopathological observation indicated significant amount of HbV accumulated in the spleen macrophages, and complete disappearance within 14 days. In conclusion, HbV showed sufficient resuscitative effect that was comparable with transfusion. The injected HbV phagocytized in RES within 14 days, and the elevated hematopoietic activity resulted in the complete recovery of Hct within 7 days.

PS_25 Intestinal Barrier Function is Preserved Using Hb Vesicle (Hbv) in a Model of Isolated Arterially Perfused Murine Intestine

P Bercik¹, H Sakai², J Lu¹, K. Kobayashi³, E Tsuchida², SM Collins¹

¹Intestinal Disease Research Programme, McMaster University, Hamilton, Canada; ²Research Institute for Science and Engineering, Waseda University, Tokyo, Japan; ³Department of Surgery, School of Medicine, Keio University, Tokyo, Japan; [email protected]

Earlier animal experiments using HBOCs suggested changes in intestinal microvilli architecture within minutes of perfusion. This raised concerns that intestinal epithelial barrier function may be impaired and result in increased bacterial translocation. In this study we investigated the structural and functional integrity of an isolated intestinal arterially perfused by HbV. Methods: A 3-4 cm long segment of mouse small intestine was selected and the terminal branch of mesenteric artery was cannulated. HbV suspended in modified Krebs' solution supplemented with 5% albumin or the modified Krebs' solution alone (K) was perfused arterially and the venous outflow was collected in 3 min fractions. The intestinal lumen was perfused with normal saline. Mixture of macromolecular probes (C¹⁴-mannitol and Cr⁵¹-EDTA) was administered twice for 9 min at 60 min interval and their concentration in the venous outflow was determined by scintigraphy. Arterial pressure was monitored throughout the experiment. At the end of experiment tissue samples were collected for histology. Results: Arterial pressure remained stable during HbV but not during K perfusion. This was associated with changes in morphology; mild to moderate damage of villi occurred in the segments perfused with K while HbV perfusion resulted in minimal structural changes, such as increased desquamation of villi tips. Macromolecular probes were detected in the venous outflow 4 minutes after administration, reaching a peak at 12 min, and becoming undetectable after 24 min. The concentration of C¹⁴-mannitol and Cr⁵¹-EDTA remained similar after 60 min of HbV perfusion (increase of 22 and 21%, respectively) but augmented significantly during K perfusion by 288 and 239%, respectively. Conclusion: Prolonged HbV administration maintains both structural and functional integrity of an isolated mouse intestine. Our results show that paracellular permeability during HbV perfusion is not significantly altered and suggest that bacterial translocation is unlikely.

PS_26 “Brush” Effect of Biodegradable Bab Copolymer on the Long-Circulation Hemoglobin-Nanoparticles

Xiao-qian Shan, Yuan Yuan, Chang-sheng Liu

East China University of Science and Technology, Shanghai 200237, PR China [email protected]

B-A-B(PEG-PLA-PEG)type triblock copolymer with different PEG concentration were formulated to hemoglobin-loaded nanoparticles for the purpose of prolonging the circulation time in vivo. Nanoparticles were formulated using a modified multiple emulsion-solvent evaporation technique. A whole new method was raised for determining drug loading by FTIR with internal standards using calibration curves method (R² = 0.9996). The results revealed that introduction of PEG had no responsible for the particles size (the mean diameter of the particles is still about 200 nm comparing to the polymeric nanoparticles without PEG chains) but the molecule weight of the copolymer did. The different PEG chains influence both encapsulation efficiency and drug loading weakly but decrease the PVA density on the surface obviously. The lyophilized nanoparticles re-solution had a nice stability for 5 days because of the hydrophilic segment PEG like the brush delayed formation of nano-aggregates in an aqueous solution. Otherwise the fluorescence experiments showed that the PEG segments helped to avoid uptake by MPS, the more PEG content, the less particles uptake and the longer half-life time (when PEG content was 30%, the half-life time was over 24hrs), which means the BAB type triblock nanoparticles could be ‘stealth’ along the circulation in vivo.

PS_27 Surface Characteristics of Hemoglobin-Vesicles: Electrostatic Properties and Interaction with Blood Components

K Sou, H Sakai, E Tsuchida

Advanced Research Institute for Science and Engineering, Waseda University, Tokyo Japan; [email protected]

Phospholipid vesicles encapsulating concentrated Hb (HbV) have been developed as artificial oxygen carriers. The safety and efficacy of HbV as a transfusion alternative have been demonstrated in detail. The present HbV formulation has an oxygen-carrying capacity equal to red blood cells because of the high encapsulation efficiency. Negative show superior blood compatibility though conventional anionic vesicles containing acidic phospholipids are known to be complement activators. In this study, we tested the electrostatic property of the vesicles composing HbV and conventional anionic vesicles containing acidic phospholipid. Electrophoresis mobility measurement revealed that the magnitude of negative charge and the electrostatic interactivity of these two anionic vesicles are equal at pH 7.4. This indicated that the negative charge on vesicles is not a common factor for complement activation, suggesting that structure of acidic groups is critical to interact with the blood components. This finding should be important to formulate cellular type artificial oxygen carriers.

PS_28 Solid Phase Adsorption Method for Pegylation of Hemoglobin

XY Suo, CY Zheng, ZG Su

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering. Chinese Academy of Sciences, Beijing, China; [email protected]

Modification of hemoglobin with polyethylene glycol (PEG) can be used to prolong the circulating half-lives and decrease immunogenic reactions. However, preparation of the pegylated hemoglobin is problematic. Multiple site reactions and heterogeneous product formation could hinder the development of the blood substitute research. The key issue is how to minimize the random side reactions and to provide a homogeneous reaction products. To solve this problem, a novel solid phase adsorption method was proposed to prepare PEGylated hemoglobin. The PEGylated hemoglobin was characterized by multiangle laser light-scattering (MALLS), HPLC and SDS-PAGE. The advantage of this method is that it makes it possible to PEGylate hemoglobin at specific areas to keep its bioactivity under the protection of solid phase support. This method can also improve the yield of the mono-PEGylated hemoglobin compared with that of liquid phase reaction. Furthermore, the simultaneous pegylation and separation with unmodified hemoglobin were achieved. In summary, mono-PEGylated hemoglobin was prepared and separated in one-step, with high yield and bioactivity, using this solid phase adsorption method.

PS_29 Hboc May Attenuate Inflammation By Constraining The Late-Acting Inflammatory Protein Hmgb-1 In An Acute Hemorrhage Model

Jianguo Wu¹, Anne Hutchings¹, Cilina Rodriguez¹, Jin He¹, Paula Moon-Massat², Loring Rue III¹, Jeffrey Kerby¹.

¹Trauma, Burns & Surgical Critical Care, University of Alabama at Birmingham, 1670 University Blvd, Birmingham, AL 35294, ²Biopure Corporation, 11 Hurley St, Cambridge, MA 02141; [email protected]

It has been well established that blood transfusions can increase the risk for developing post-hemorrhage multiple organ failure (MOF). Although avoidance of immune responses to damaged erythrocytes can be achieved with the use of acellular hemoglobin based oxygen carriers, they are still associated with some inflammatory responses. This study was undertaken to investigate the effects of HBOC-201 on early and late cytokine responses following trauma-hemorrhage. Anesthetized mice (n = 5/group) were hemorrhaged over 30 minutes to a mean arterial pressure (MAP) of 25 ± 5 mm Hg. After an additional 1h of sustained hypotension, animals were resuscitated with lactated Ringer's solution, shed autologous blood, or HBOC-201, and allowed to recover for 2 or 24 h. Peripheral blood and liver tissues were collected for analysis by cytokine bead array, or by RT-PCR normalized to GAPDH mRNA. Survival rates were equal in all the groups. HBOC-201 increased post-resuscitation MAP, but had little effect on serum IL6, IL10, IL12, IFN-, TNF or MIP1 levels compared to other groups at 2 or 24 h post-resuscitation. However, at 24 h post-resuscitation, mRNA levels for IL10 were significantly higher in the livers of HBOC-201 treated animals compared to the autologous blood or lactated ringer's groups (0.022 ± 0.01 vs. 0.002 ± 0.001 or 0.003 ± 0.0002, p = 0.007), with no differences between the other cytokine mRNA studied. Interestingly, the relative mRNA level of HMGB-1 was slightly reduced in the livers of those treated with HBOC-201, compared to the autologous blood or lactated ringer's groups (0.05 ± 0.01 vs. 0.08 ± 0.03, or 0.10 ± 0.02, p = 0.063). These results suggest that, in addition to improving reperfusion, resuscitation with HBOC-201 may help reduce later immune responses by increasing the production of the anti-inflammatory cytokine IL10, and attenuating the late acting stimulatory protein, HMGB-1. This work was supported by a grant from Biopure.

PS_30 PEG-hemoglobin Can not Attenuate the Leukocytes/Endothelium Interactions Provoked by Cisplatin

Yu Minghua¹, Han Jianqun¹, Dai Min¹, Li Hongwei¹, Zhang Jian¹, Liu Qian², Xiu Ruijuan¹

¹Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China, ²Peking Union Medical College Hospital (PUMCH) Beijing, China; [email protected]

Hemoglobin-based oxygen carrying (HBOC) solutions are currently being developed as promising tools for a wide range of applications, including their use as artificial oxygen carrier instead of allogenic transfusion and as oxygen transporting tools for oxygen therapeutics. Rencent studies have demonstrated that HBOC solution has antiinflammatory effect which would confer it an additionally favorable potency for application in a number of clinical settings including shock resuscitation and ischemia/reperfusion injury. Leukocytes rolling and adhesion is an indication of the inflammation response of the endothelial cells. Our intravital microscopy study revealed that cisplatin exposing (5 mg/kg body weight) could invoked significantly higher (P < 0.01) numbers of rolling and sticking leukocytes on the wall of postcapillary venules in male Golden Syrian Hamster's cheek pouch bearing a human cervical carcinoma. Whereas co-administration of PEG-conjugated hemoglobin (0.3 g/kg i.v. or 0.6 g/kg i.v.) with cisplatin could not attenuate the enhanced leukocytes/endothelium interactions. This suggested that the evaluated PEG-conjugated hemoglobin might have no positive efficacy to attenuate cisplatin's troublesome side effects in which inflammatory mechanisms may play an important role.

PS-31 HRC 101, A High Molecular Weight Hemoglobin-Based Oxygen Carrier, Improves Survival in Murine Sickle Cell Disease

MW Crawford,¹ T Shichor,¹ T Engelhardt,¹ G Adamson,² D Bell,² FJL Carmichael,³ PCW Kim⁴

The Departments of ¹Anesthesia, Pharmacology, and ⁴Surgery, The Hospital for Sick Children, ³University of Toronto, Toronto, and ²Hemosol BioPharma, Mississauga, Ontario, Canada; [email protected]

Red cell transfusion decreases morbidity in patients with sićkle cell disease (SCD) but is not without risk. HRC 101, a novel high molecular weight, low oxygen affinity hemoglobin-based oxygen carrier (HBOC), was evaluated as a transfusion alternative in the SAD mouse (₂^human, ₂^{S, Antilles, D - Punjab}), which reproduces the acute spontaneous vaso-occlusive events of human SCD and the chronic steady state disease. HRC 101 (Hemosol BioPharma) is a covalent conjugate of human hemoglobin (Hb) and modified hydroxyethyl starch (HES), intended to provide both volume expansion capability and efficient tissue oxygenation. It is essentially free of Hb components ≤ 128 kDa and is prepared as a 10 g (Hb)/dL solution at iso-oncotic strength. Oxygen binding is non-cooperative (n₅₀ = 1.1) and p50 is ∼ 70 mmHg. Given its large molecular size, high p50 and high oxygen carrying capacity, together with its potential to bypass SCD-related vaso-occlusion, HRC 101 should minimize nitric oxide binding and oxygen steal but enhance oxygen delivery to the microcirculation during vaso-occlusive crisis. SAD mouse survival was measured during exposure to acute, severe hypoxic stress following HRC 101 administration. Wild-type (n = 30) and SAD (n = 36) mice received 0.02 mL/g HRC 101 or 5% albumin, then either 30% or 6% oxygen under anesthesia over 60 minutes. All wild-type mice survived 60 min., all SAD mice given albumin and 6% oxygen died (median time 9.0 min., P < 0.0001). HRC 101 increased survival in SAD mice breathing 6% oxygen (median survival time 48 min., P < 0.0001 vs. albumin). HRC 101-treated SAD mice (6% oxygen) were significantly less acidotic than those given albumin (P < 0.001). The results show that HRC 101 protects sickle mice from the lethal effects of acute, severe hypoxia, warranting further evaluation of HRC 101 as a therapeutic modality in SCD.

PS-32 O₂-Binding Ability of Membrane of Peg-Modified Albumin-Heme

A Nakagawa¹, Teruyuki Komatsu^1,2, Lu Gang¹, Eishun TSUCHIDA¹

¹RISE, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan, ²PRESTO, Japan Science and Technology Agency, Tokyo, Japan; [email protected]

Human serum albumin (HSA) incorporating the synthetic heme (albumin-heme) is an artificial O₂-carrier which can bind and release O₂ reversibly like hemoglobin under physiological condition (pH 7.4, 37°C). The surface modification of the albumin-heme with poly(ethylene glycol) (PEG) significantly improved its O₂-transporting efficacy.¹ The animal experiment proved that the PEG modified albumin-heme [PEG(albumin-heme)] solution worked as a resuscitative fluid for hemorrhagic shock.² It would be a totally synthetic O₂ carrying plasma expander. We have recently found new properties of the PEG(albumin-heme). Its aqueous solution was cast on the glass plate and a red membrane formed after water evaporation.³ The O₂-binding was reversibly observed depending on the O₂-pressure and the O₂ affinity (P_1/2) was 61 Torr (37°C). Furthermore the free-standing flexible thin film was obtained by addition of hyaluronic acid (HA, M_w: 2 MDa) as a polymer support [HSA:HA = 10:1 (w:w)]. The film also kept the O₂-binding ability (P_1/2: 60 Torr (37°C)). The PEG(albumin-heme) membrane would be used as an O₂-transporting thin film for medical applications.

PS-33 O₂ Binding Nanotubes Made of Albumin-Heme Complex

Gang Lu¹, Teruyuki Komatsu^1,2, Eishun Tsuchidaa

¹Research Institute for Science and Engineering, Waseda University, Tokyo, Japan. ²PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan; [email protected]

Human serum albumin (HSA) is a versatile protein in our blood plasma (4∼ 5 gdL-1) and acts as a transporter for a range of endogenous and exogenous compounds. We previously demonstrated that a synthetic heme, 2-[8-[N-(2-methyl-imidazolyl)] octanoyloxymethyl]-5, 10, 15, 20-tetrakis [[α,α,α,α -o-(1-methylcyclohexanamido)]phenyl] porphinatoiron(II) (FeP) is incorporated into HSA, and the formed artificial hemoprotein (HSA-FeP) can reversibly coordinate O₂ under physiological conditions (pH 7.4, 37°C) in a fashion similar to hemoglobin. We report herein the nanotubes prepared by a layer-by-layer deposition technique with HSA-FeP using an anodic porous alumina template and highlight their O₂ binding properties. With the alternate adsorption of HSA-FeP with different charges by varying the pH value at each deposition, we fabricated the hemoprotein nanotubes through layer-by-layer assembly based on the template synthesis method. The liberated nanotubes have a very uniform outer/inner diameter and can reversibly bind O₂ at 25°C. The O₂ binding affinity of the nanotubes was 2-fold lower than that of the monomeric HSA-FeP, which was kinetically due to the low association rate constants. The alternative layer-by-layer deposition technique with the very common blood protein, HSA, hybridized with the desired functional molecule, will lead to the development of a new field of smart nanotubes which can provide enhanced biological properties.

PS-34 Effects of Blockade of Cerebral Lymphatic Drainage on Cerebral Injury Folowing Subarachnoid Hemorrhage and Protective Effects of Extract of Ginkgo Biloba

ZC Cheng, BL Sun, MF Yang

Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; [email protected]

This study was designed to investigate the Effects of blockade of cerebral lymphatic drainage on cerebral injury following subarachnoid hemorrhage and the protective effects of extract Ginkgo biloba. The healthy adult male Wistar rats were selected as experiment animals. The SAH model was induced with freshly autologous arterial blood injection into the cisterna magna twice, CLB model in rats was established by occlusion of cervical lymphatic tubes and removal of cervical lymphatic nodes. The rats were randomly divided into normal control, SAH group, SAH+CLB group, SAH+CLB+pyridoxo group, SAH +CLB+Solvent group, higher dose and lower dose of ginkgolide (20 mg/kg, 80 mg/kg), higher dose and lower dose of ginkgo flavone glycoside (50 mg/kg, 200 mg/kg) 3d after second SAH preparation approximately observes the bone window(a side candate nucleus), The regional cerebral essence blood flow (rCBF) was recorded with the laser Doppler. Brain superoxide dismutase (SOD) activity and maleic dialdehyde (MDA) were determined .3d after termination of cisternal injection The (rCBF) Obviously reduces, brain SOD activity decreased while MDA content increased. The above pathological alternations were more severe in SAH+CLB group and SAH+CLB+Solvent group than those in SAH group. The extract Ginkgo biloba partly attenuated above pathological alternations. Cerebral lymphatic drainage pathway might play important role in the pathophysiology of cerebral ischemic injury after SAH. The extract Ginkgo biloba may relieve the deterioration of the cerebral ischemic injury by CLB after SAH.

PS-35 The Effect and Mechanism of Intranasal Administration of Recombined Colony-Stimulating Factor to Promote Neovascularization Inrats with Mcao

XY Han, BL Sun

Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000 [email protected]

This study was designed to investigate the reliability and feasibility of intranasal; (IN) pathway bypassing the blood-brain barrier (BBB) and observe the effect of intranasal administration of rhG-CSF to promote neovascularization on therapeutic angiogenesis in rats with MCAO. A blinded, vehicle-controlled study of IN rhG-CSF and IV rhG-CSF administration was performed by intraluminal middle cerebral artery occlusion (MCAO) model. Experiment 1: Ratswere randomly assigned into 3 groups: IN vehicle, IN rhG-CSF, IV rhG-CSF. The infarct volume in IN rhG-CSF group was reduced significantly by 35.1% as compared with IN vehicle. The vestibulomotor function of IN rhG-CSF improved significantly at 24 and 72 h (P = 0.02 and P = 0.04, respective-ly). Experiment 2: Rats were randomly divided into IN rhG-CSF, IV rhG-CSF and untreated group. The concentration of rhG-CSF in different brain regions was measured by ELISA. Olfactory bulb in IN rhG-CSF group obtained the highest concentration among all tissues, followed by hippocampus. The rhG-CSF concentrations of olfactory bulb and hippocampus in IN rhG-CSF group were significantly higher than that in IV rhG-CSF and control group. Experiment 3:Ratswere randomly assigned into 3 groups: IN vehicle, IN rhG-CSF, IV rhG-CSF. The expression of vascular endothelial growth factor (VEGF) in the brain were determined by immunohistochemistry respectively. The expression of VEGF in the rat brain of IN rhG-CSF group were significantly more than control group. It was concluded that Intranasal rhG-CSF could bypass BBB, reach the CNS, reduce infarct volume and promoted neovascularization and held back gliosis after ischemia and reperfusion lesion in rats following MCAO. Intranasal delivery of rhG-CSF holds a promising treatment for stroke and other CNS disorders.

PS_36 Evaluation of the Clearance of Macromolecular Tracers from CSF by Lymphatic Pathway in a Rat Model of SAH *

L Jia¹, BL Sun¹, MF Yang², H Yuan¹, YB Zhang¹

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Institute of Cerebral Microcirculation, Taishan Medical College; Taian, Shandong 271000, China; [email protected]

In In this experiment, lymphatic pathway blockade was made and subarachnoid hemorrhage (SAH) models were replicated. 100 μ g 125I labeled human albumin as macromolecular tracer was injected in to rat's lateral cerebral ventricle. Plasma recovery of 125I labeled human albumin was detected. Concentration-time curve was obtained in the experiment. Some pharmacokinetic parameters, including peak concentration [(Cmax) (μ g/ml)], time of maximum concentration [(Tmax)(h)], and elimination rate constant [(Ka) (h-1)] were calculated. The clearance of macromolecular tracers in CSF by lymphtic pathway was evaluated. It was found that in SAH rat, the ymphatic pathway blockade led to an decrease of 68.82% in Cmax. The ymphatic pathway blockade delayed Tmax by 5.47 hours. Ka was also decreased by 43.24% after lymphatic pathway blockade. The results suggest that in the condition of SAH, a good proportion of macromolecular substances in CSF was removed by lymphtic pathway, indicating improving the lymphatic dainage pathway of CSF may be a new approach for the therapeutic study on SAH and related cerebral injury.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS-37 Effects of Blockade of Cerebral Lymphatic Drainage on Cerebral Ischemic Injury Secondary to Subarachnoid Hemorrhage

L Jia¹, BL Sun¹, MF Yang², CB Zheng², YB Zhang¹, H Yuan¹

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China [email protected]

This study was designed to investigate the effects of cervical lymphatic blockade (CLB) on cerebral ischemic injury secondary to subarachnoid hemorrhage (SAH) and the influence of pyridoxol. Wistar rats were divided into control, SAH, SAH+CLB and pyridoxol groups. The expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhension molecule-1 (PECAM-1) in the brain were determined by immunohistochemistry respectively at the third day after SAH. It was found that the expression of PECAM-1 and VEGF in the brain of SAH group were significantly more than control group, SAH+CLB group increased further more than SAH group, but pyridoxol group decreased obviously compared with SAH+CLB group. It was conclusions that CLB could aggravate the cerebral ischemic injury secondary to SAH, but pyridoxol may relieve the aggravation of the cerebral ischemic injury by CLB after SAH.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS-38 The Effect of CSF on Pc12 Cells after Subarachnoid Hemorrhage and Cerebral Lymphatic Blockage

LL Jia¹, BL Sun¹, X Wang¹, MF Yang¹, CB Zheng¹, YB Zhang¹

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China [email protected]

This experiment was carried out to investigate the effect of cerebrospinal fluid (CSF) of subarachnoid hemorrhage (SAH) after cerebral lymphatic blockage (CLB) on nerve growth factor (NGF)-treated PC12 cells. The PC12 cells were randomly divided into blank control group (F-12 Ham's), normal CSF groups, SAH CSF groups and SAH+CLB CSF groups. At different points of time, the survival rate of PC12 was measured by MTT assay. Apoptosis protein of Bax and heat-shock protein 70 (Hsp70) were determined by immunohistochemical staining.Internal pH and [Ca2+] was also determined by laser scanning confocal fluorescence microscope. The survival rate of PC12 cells was obviously inhibited by SAH group and SAH+CLB group CSF. The expression of Bax in SAH+CLB group was higher than SAH group in a time dependent manner. It was concluded that cerebral lymphatic drainage pathway blockage aggravates SAH CSF-induced PC12 cells injury.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS-39 The Effect of CSF on Cultured Tat Hippocampal Neurons after Subarachnoid Hemorrhage and Cerebral Lymphatic Blockage

LL Jia¹, BL Sun¹, X Wang¹, MF Yang², CB Zheng², H Yuan¹

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; [email protected]

This study was to investigate the effect of cerebrospinal fluid (CSF) of subarachnoid hemorrhage (SAH) after cerebral lymphatic blockage (CLB) on hippocampal neurons. The hippocampal neurons were randomly divided into blank control group (DMEM), normal CSF groups, SAH CSF groups and SAH+CLB CSF groups. At different points of time, the survival rate of hippocampal neurons was measured by MTT assay. Apoptosis protein of Bax and heat-shock protein 70(Hsp70) were determined by immunohistochemical staining. Internal pH and [Ca2+] was also determined by laser scanning confocal fluorescence microscope. The survival rate of hippocampal neurons was obviously inhibited by SAH group and SAH+CLB group CSF. The expression of Bax in SAH+CLB group was higher than SAH group in a time dependent manner. It was concluded that cerebral lymphatic drainage pathway blockage aggravates hippocampal neurons injury.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724).

PS-40 Cerebral Lymphatic Blocking Increase the Production of Endothelin-1 in CSF after Experimental Subarachnoid Hemorrhage*

LL Jia¹, BL Sun¹, X Wang¹, MF Yang², H Yuan¹, YB Zhang¹

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; [email protected]

This experiment was carried out to investigate the effect of cerebral lymphatic blockage (CLB) on endothelin-1(ET-1) in CSF after subarachnoid hemorrhage (SAH). The New Zealand rabbits were randomly divided into sham operated(SO), SAH, SAH+cervical lymphatic blockade (CLB) groups. Endothelin-1(ET-1) was determined by radio-immunity method. There were a lot of Bax positive cells in SAH group. The number of Bax positive cells increased in SAH+CLB group. By Apoptotic changes of some cells in SAH goup were found. More apoptotic cells were seen in SAH+CLB group. Above resultes indicated that CLB deteriorates SAH-induced CVS and related cerebral ischemic injury. Above resultes indicated that cerebral lymphatic pathway facilitate the clearance of macromo and CLB aggrevite CVS after SAH.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS-41 Pharmacological Effect of Vasoactive Drug Modified by Polymeric Micelles on Hamster Cheek Pouch Microcirculation

Ailing Li, Hongwei Li, Jing Zhang, Songdan Gao, Ruijuan Xiu

Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China; [email protected]

Aims: to observe the pharmacological effect of vasoactive material A modified by polymeric micelles(PM-A) on the microcirculation of hamster cheek pouch under normal physiological condition, compared with that of enveloped by Lipid microspheres(Lipo-A). Methods: After successful anaesthetization and cannulation of jugular arterial to monitor blood pressure (BP) and jugular vein for the drug injection, the A3∼ A4 grade microvessel in hamster pouch was prepared. Then the microvascular diameters, blood velocity and vasomotion at points of before injection and 0, 5, 10, 15, 20, 25, 30 mins after injection were recorded and measured by MCIP. Results: After injection of drugs, BP decreased significantly at 0 point immediately (P < 0.05) in both groups. Diameter didn't change significantly during the 30mins (P > 0.05) except PM-A group showed an increased tendency, and interestingly appeared an alternative relaxation or constriction change. Blood velocity slowed a little, except the velocity at point of 20 mins in PM-A group which became faster, but there are no any statistic significance. Although PM-A showed a relative longer and stronger effect on the above three aspects, no significant difference between them. For vasomotion, during the 15mins after injection, PM-A group showed vasomotion characteristic of higher amplitude (P < 0.05). Conclusion: PM-A and Lipo-A could decrease BP significantly, maintain the diameter and velocity in a similar model. PM-A seems to be stronger that may be due to its ability to activate vasomotion which need to be studied in the near future.

PS-42 Effect of Cerebral Lymphatic Blockade, Ginkgolides and Ginkgoflavones against Brain Hydrops and Neuron Ultrastructural Injury on SAH Rat

XC Liu¹, BL Sun¹, MF Yang², QM Yu³

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; ³Institute of histology and Embryology, Shandong University School of Medicine, Jinan, Shandong, 250012, China; [email protected]

This study was designed to determine the effect of cerebral lymphatic blockade (CLB), Ginkgolides and Ginkgoflavones on brain water content and neuronic ultrastructure. Use 90 marure male Wister rats, the animals were divided into: negatived control group (a); SAH group (b); SAH+CLB group (c); SAH+CLB+pyridoxol group(d); SAH+CLB+ Normal Sodium group (e); SAH+CLB+20 mg/kg Ginkgolides group (f₂₀); SAH+CLB+80mg/kg Ginkgolides group (f₈₀); SAH+CLB+50 mg/kg Ginkgoflavones grsup (g₅₀); SAH+CLB+200 mg/kg Ginkgoflavones grsup (g₂₀₀). 24 h and 72 h after induction of SAH, brian water content was detected. 72h after induction of SAH, the rats were sacrificed and the hippocamre removed, the neuronal ultrastructure was observed under an elextron microscope. It was found that SAH led to increaded brain water content, c group were more severe than others group, f and g groups were a remardable drop to contrast c group. The neuronal ultrastructures of the rats from b to g group were different extent destruction. It was concluded that Cerebral lymphatic drainage pathway might play important role in cerebral dropsy and neuronal ultrastructure injury after SAH, Ginkgolides and Ginkgoflavones may relieve cerebral dropsy and neuronal ultrastructure injury by CLB after SAH.

PS-43 Ginkgo Biloba Extract Increases the Production of Heme Oxygenase-1 and Carbon Monoxide after Experimental Subarachnoid Hemorrhage*

BL Suna, CB Zhengb, MF Yangb, H Yuana

Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; Taishan Medical College, Taian, Shandong 271000, China; [email protected]

This study was designed to investigate the expression of Oxygenase-1 (HO-1) and production of carbon monoxide (CO) after subarachnoid hemorrhage (SAH) and the influence of Ginkgo biloba extract (EGb). Wistar rats were divided into non-SAH, SAH, vehicle, EGb1, and EGb2 groups. At different time points, the animals underwent detection of the expression of HO-1mRNA and HO-1 protein in the brain. Brain tissue CO and cGMP content were also determined. Increased expression of HO-1mRNA in cerebral cortex was found 24 and 72 hours after induction of SAH. HO-1 expression was increased in the brain section. CO content was increased after SAH. cGMP showed a tendency to increase in brain tissue. EGb dose-dependently promotes the expression of HO-1mRNA and HO-1 protein in the brain. EGb also increased the production of CO and cGMP. It was concluded that SAH induces the activation of HO-1 and production of CO. EGb exerts the protective effects on SAH-induced cerebral ischemic injury via the promotion of HO expression and CO production.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724).

PS-44 Alterations of Intracranial Pressure, Cerebral Blood Perfusion, Oxygen Free Radicals, and the Influence of Pyridoxine Following Experimental Subarachnoid Hemorrhage after Cerebral Lymphatic Drainage Blockage*

BL Sun¹, MF Yang², CB Zheng³, YS Chen¹, ZJ Wang², ZX Wang²

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; [email protected]

In this experiment, Wistar rats were divided into non Subarachnoid hemorrhage (SAH), SAH, SAH+ cerebral lymphatic drainage blockade (CLDB), SAH+ CLDB +pyridoxine groups. The results revealved that intracranial pressure (ICP) increased and cerebral perfusion pressure (CPP) decreased markedly during and 12 hours after induction of SAH. Meanwhile, regional cerebral blood flow (CBF) decreased significantly. 24 hours and 72 hours after SAH, brain superoxide dismutase (SOD) activity decreased and malonaldehyde (MDA) content increased remarkably. CLDB deteriorated above pathological alterations after SAH. Pyridoxine provented the changes of CBF, ICP, CPP after CLDB and SAH to a certain extent. It also alleviated the alterations of brain SOD activity and MDA content. It was concluded that cerebral lymphatic drainage pathway play an important role in the pathogenesis of secondary cerebral ischemic injury following SAH.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS_45 Study on Transdifferentiation of Adult Human Myoblasts into Neural Precursor Cells and its Transplantation in Cerebral Ischemic Rats

Renzhi Wang, Zhenxing Zhang, Junji Wei, Guilin Li, Wanchen Dou, Yukui Wei, Ming Feng, Ziheng Zhang, Zhaojian Li and Jun Kang

Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, 100730, P.R. China; [email protected]

As a promising therapeutic method, stem cell transplantation aroused much attention in treatment of ischemic cerebralvascular diseases. We have investigated the feasibility of inducing adult human myoblasts into neural precursor cells in vitro and delivered the neural precursor cells into the brain of cerebral ischemic rats. The myoblasts were harvested and isolated from minced adult human temporal muscle samples (volunteer). After purified clonally, they were incubated in vitro. Immunofluorescence cytochemistry and RT-PCR were used to analysis the expression of nestin, NF68, GFAP and GalC in order to identify the neural differentiation of the myoblasts. Then cerebral stroke model in rats with photochemically induced thrombosis of proximal cerebral middle artery (MCA) was introduced for cell transplantation. Neural precursor cells derived from myoblasts and myoblasts were transplanted into the brain cortex near the ischemic lesion 7 days after stroke in different groups. Each rat was subjected to a series of behavioral tests to assess neurologic function before and 7, 14, 21 and 28 days after stroke. We found that the human cells can became non-adherent aggregates as neurospheres which can express nestin in vitro after induction. The neural precursor cells also can express phenotypes of neural cells after induction with bFGF and EGF. Human cells survived near the ischemic regions after being transplanted. Some myoblast-derived neural precursor cells were found in corpus callosum area and the boundary of infarct lesion. Partial recovery of neurologic function was present in the tactile stimulation test, limb placement test and equilibrium test in rats of each group 2 weeks after cell transplantation. There were significant differences in recovery of neurological function in rats treated with myoblast-derived neural precursor cells compared with myoblasts and PBS injection (P < 0.05). No significant difference of lesion volume was observed in ischemic rats among the groups (P > 0.05). So our conclusions are follows: (1) Adult human myoblasts could be inducted into neural precursor cells in vitro. (2) After transplanted, myoblasts-derived neural precursor cells survived near the ischemic regions and could migrate primarily toward the lesion. They can express cell-specific antigens of neural cells in vivo. Transplantation of neural precursor cells derived from adult human myoblasts could enhance recovery of neurologic function of cerebral ischemia.

PS-46 Role of Lymphatic Drainage From CNS in the Development Of Cerebral Vasospasm And Related Cerebral Ischemic Injury*

X Wang¹, BL Sun¹, LL Jia¹, MF Yang², CB Zheng², YB Zhang¹

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; [email protected]

This experiment was carried out to investigate the possible role of cerebral lymphatic drainage pathway in the development of cerebral vasospasm (CVS) and related cerebral ischemic injury after subarachnoid hemorrhage (SAH). Wistar rats were divided into normal control, SAH, SAH+ cervical lymphatic blockade (CLB) groups. It was found that diameters of BA decreased remarkably after SAH. The increased thickness of BA wall and decread cavity were also observed after SAH. There were a lot of caspase-3 positive cells in SAH group. The number of aspase-3 positive cells increased in SAH+CLB group increased. By Apoptotic changes of some cells in SAH goup were found. More apoptotic cells were seen in SAH+CLB group. Above resultes indicated that CLB deteriorates SAH-induced CVS and related cerebral ischemic injury. It was concluded that cerebral lymphatic drainage pathway exerts intrinsic protective effects against CVS and related cerebral ischemic injury after SAH.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS-47 Cerebral Lymphatic Blockage Aggravates Cerebral Vasospasm after ASH and the Protection of Pyridoxol*

X Wang¹, BL Sun¹, LL Jia¹, WX Li¹, MF Yang², CB Zheng²

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; [email protected]

This experiment was carried out to investigate the effect of cerebral lymphatic drainage pathway to CVS after SAH and the protection of pyridoxol (PN). Wister rats were randomly divided into normal controls, SAH, SAH+cervical lymphatic blockade (CLB), SAH+CLB+Pyridoxol groups. In vivo basilar artery (BA) measurement and its reactivity to Ach was performed. It was found that diameter of BA and the reactivity to Ach are decreased remarkably after SAH. The index above-mentioned in SAH+CLB groups were reduced than that of SAH groups. Those in SAH+CLB+Pyridoxol groups were increased than that of SAH+CLB groups. Above resultes indicated that the disorder of cervical lymphatic drain could aggravate the vasaspasm of cerebral vessels after SAH, and cut down the reactivity to Ach of cerebral vessels, while pyridoxol could improve aboved symptom.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724)

PS-48 Study of Autotransfusion and Hemodilution of Perio-Peration in High Altitude

Wang zu-qian, zhao shijun, jia zhen et al.

Department of Anesthesiology, Qinghai University Affiliated Hospital.

Objective: To study the safe and effective in autotransfusion and hemodilution in high altitude (sea level 2260m) Methods Eighty patients were randomly divided two groups, ASAI-II?, age ranged from 23-68 ys, Hb ≥ 130 g/L, HCT ≥ 0.4 L/L before operation. Estimated volume of bleeding more than 800 mL during operation for one case. Group A (study), the blood was letting from vein are 400–800 mL preoperation or after general Anesthesia and transferring back during surgery, also transferring Hydroxyethyl stanch and Ringer,s lactate in order to hemodilute and controlled the banked blood transfusion by examination of Hb, HCT. Group B transfusion blood by routine treated. The changes of Hb, HCT, PLT, PT, APTT, FIB, TT, arterial blood gas and Bp (SBp, MBp, DBp) Were observed for two groups in perioperation. Result The volume of transfusion blood in group A (462 ± 220mL) decreased 60% than group B (1297 ± 640mL). The cost of transfusion blood in group A (46636.2 yuan) decreased 49.9% than group B (93379.08 yuan). Conclusion Herein hemoglobin and blood viscosity increased for influence of hypoxia in high altitude. So bleeding volume estimated more than that in major surgery, letting autologous blood preoperation and controlling hemodilution are safe and effective. It can save allogeniec blood in order to reduce the risk of transfusion blood.

PS-49 Effect of Different Molecular Weight of HBOCs with Low P50 on the Pressure in Rats

Kun-ping Yan^1,2, Chao Luo², Hong-li Zhao¹, Chao Chen^1,2,3

¹College of Life Science, Northwest University, Xi'an, P. R. China, 710069; ²Shaanxi Lifegen Co., Ltd., Xi'an, P. R. China, 710069; ³National Engineering Research Center for Miniaturized Detection Systems, Xi'an, P. R. China, 710069; [email protected]

Purpose: this study assessed the effects of different molecular weight of HBOCs with low P50 on the mean arterial blood pressure (MAP) following 50% isovolemic exchange transfusion in rats. Methods: rats were anesthetized with pentobarbital sodium, 6∼ 8 Sprague-Dawley rats per group, then an isovolemic exchange transfusion to 50% of total blood volume was performed with one of five different test HBOCs solution:1) Low MW glutaraldehyde-polymerized porcine hemoglobin (64 kDa = 80∼ 85%, P50 = 8.2 mmHg); 2) Medium MW glutaraldehyde-polymerized porcine hemoglobin (MW = 500 kDa, P50 = 7.3 mmHg); 3) Medium MW glutaraldehyde-polymerized porcine hemoglobin (MW = 500 kDa, P50 = 6.5 mmHg); 4) High MW glutaraldehyde-polymerized porcine hemoglobin (MW = 800 kDa, P50 = 6.1mmHg); 5) High MW glutaraldehyde-polymerized porcine hemoglobin (MW = 1100 kDa, P50 = 5.8 mmHg). MAP, total hemoglobin concentration and arterial blood gases were measured. Results: MAP responses with low MW Polyhemoglobin increased and sustained significantly above 10∼ 25 mmHg to the baseline through the exchange transfusion processes. MAP responses with medium MW Polyhemoglobin were stable and kept up unchanging to the baseline. MAP responses with high MW Polyhemoglobin weren't increased, but some parts of rats' MAP were decreased in different some extent in the middle of exchange transfusion, and then can be restored on the whole after 15∼ 60 min finishing the exchange transfusion. Conclusion: The low MW 64 kDa Polyhemoglobin with low P50 can cause blood pressure increasing. To the contrary, high MW Polyhemoglobin can cause blood pressure decreasing. Luckily, the medium MW Polyhemoglobin have no effects on the blood pressure.

PS-50 Influence of Smoking and Passive Smoking on Human Microcirculatory Function

Zhang Jian¹, Liu Shuying¹, Yang Xiaojie¹, Ingemar Bjorkhem², Xiu Ruijuan

Institute of Microcirculation, Chinese Academy of Medical Sciences and Peking¹ Union Medical College, 5, Dong Dan San Tiao, 100005, ²Department of Clinical Chemistry, Huddinge University Hospital, Karolinska Institute, S141 68, Stockholm, Sweden; [email protected]

To investigate the influence of smoking and passive smoking on human microcirculatory function, the nailfold, conjunctiva, tongue and lip were selected for intravital microscopic observation and recorded for off-line analysis. According to their contextualization, in this study, 102 subjects were enrolled and were assigned to smoking group (SM group, 34 cases), passive smoking group (PSM group, 34 cases) and control group (Ctrl group, 34 cases). Variations of heteromorphosis (Hm), venule dilation (Vd), aggregation (Ag), thrombus formation (Thb), segmental dilation (SD) and blood stasis (ST) were evaluated by using MCIP image processing system. It was shown that all parameters especial aggregation and blood stasis were significantly different between PSM group and Ctrl group (p < 0.01) in observed positions, among which conjunctiva was predominant. Compared with the Ctrl group, SM group presented significant aggravation of all above parameters (p < 0.01), which is striking in tongue and lip. However, there is no notable difference between SM group and PSM group (p > 0.05). According to our observation, it is suggested that compared with that of smoking condition, the passive smoking causes a very similar adverse effect on human microcirculatory function. A positive correlation could exist between passive smoking condition and RBC aggregation, thrombus formation, injury of endothelium and stasis of blood flow. These changes are possible participated in the process of cardiovascular events.

PS-51 Preliminary Characterization of Glutaraldehyde Cross-Linked Human Placenta Hemoglobin by Laser Doppler Electrophoresis

WB Zhang^1,2, HH Zhang¹, WG Liang¹, CM Yang¹

¹Tianjin Union Biotech Developing Co. Ltd., Tianjin, P.R. China; ²Postdoctoral Program at Tianjin Union Biotech Institution, jointly trained in Postdoctoral Station at Beijing Union Medicinal College; [email protected]

In this preliminary study laser Doppler electrophoresis (LDE) was used to analyze the surface electrostatic properties of glutaraldehyde cross-linked human placenta hemoglobin (G-PHb) to predict the thermodynamic stability of G-PHb solution. Zeta-potential was taken as the index of surface electrostatic properties of G-PHb. The results show that zeta-potential of G-PHb is less than 30 mV (People usually take 30 mV as threshold value for stable colloidal system) in the studied range of pH, which means that electrostatic properties would contribute to the physical instability of G-PHb solution; meanwhile, it also reflects that the stability of G-PHb solution should be improved by modifying pH, decreasing ion strength and/or adding some anti-flocculant. As a simple and fast method, LDE is suitable to monitor the electrostatic properties of G-PHb in solution and predict its thermodynamic stability.

PS-52 Is Antioxidant Improves the Damage to Gut Mucosal Barrier Occurred in The Process of Acute Brain Trauma and Severe Acute Pancreatitis: A Prospective Study from Basic to Clinic?

Peilin Cui^1,2, Dong Lv¹, Jun Zhang¹, Minghua Yu², Jianqun Han², Ming Dai², Hongwei Li², Zhaoxu Yang², Ruijuan Xiu²

¹Gastroenterology Department, Beijing Tiantan Hospital, Capital university of Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China; ²Institute of Microcirculation, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China [email protected]

The gut barrier is susceptible to be injured during the critical illness process, thus leading to the failure of intestinal defense mechanisms. It puts the patients at risk for infection and multiple organ dysfunction syndrome. So the specific prophylactic therapeutic is needed to prevent the gut barrier disruption during these severe stress conditions. Recently evidences are appearing that oxidative stress is involved in the injury of gut mucosal barrier. From these, a study was designed in an attempt to observed the effect of antioxidant on the protection of gut mucosal barrier and explore the putative mechanisms. In the animal models, the histology and the microcirculation of the indicated segment gut mucosa were observed; the bacteria translocation and gut mucosal permeability were detected; the gut mobility was monitored by a special instrument; and the immune globulins and some key components in the oxidant-reduction system in the mucosa were assayed. In the clinic patients, the ascites was aspirated to detect the bacteria translocation; the serum D-galactose and endotoxin and some specified inflammatory cytokines were assayed to evaluate the gut barrier permeability and damage; the biopsy mucosal tissue obtained from endoscopy was assessed the damage extent result from oxidative stress reaction and ischemia and/or hypoxia. Taken together, the results showed the gut mucosal barrier was disrupt during the process of acute brain trauma and severe acute pancreatitis; and oxygen free radicals (OFRs) played a very important role in the damage of gut mucosal barrier; antioxidant used in this study partially prevented and attenuated the damage.

PS-53 Anti-angiogenic Effects of Cisplatin Combined with Ginsenoside Rg3 in Nude Mice Cervical Cancer

Duan Ying-chun, Hu Ping, Huang Guang-rong, Yang Bo, Ren Qing, Zhou Yu-ping

The People's Hospital of Yunyang Medical College, Shiyan, 442000, China; [email protected]

Objective: To study the anti-angiogenic effects and anti-tumor effect of cisplatin combined with ginsenoside Rg3 in nude mice cervical cancer. Methods: 28 female nude mice were randomly divided into 4 groups to receive Saline (0.5 ml, qd), Rg3 (5 mg/kg) alone, Cisplatin (5 mg/kg) alone and Rg3 (5 mg/kg) combined with Cisplatin (5 mg/kg) respectively. Rg3 was given by gastrogavage once a day for 5 weeks, Cisplatin 3 was injected introperitoneally once 4 days for 5 weeks. And after treatmeat being stopping, mice were killed and the sizes of solid tumors were measured, the intratumor MVD was examined by immunohistochemical staining. Results: The tumor weight of treated group was significantly lower than that of control group. The tumor weight of Rg3 combined with Cisplatin group was lower than other groups. The MVD value of Rg3 group was significantly lower than that of Cisplatin group and control group. Conclusion: Rg3 combined with Cisplatin can significantly inhibit the growth of transplanted human Cervical Cancer in nude mice. Rg3 can obviously reduce the intratumoral MVD through inhibiting angiogenesis of malignant tumor.

Key words: ginsenoside Rg3; Cisplatin; angiogenesis; microvessel density

PS-54 Tracing Ferumoxide Labeled Flk1+ CD31- CD34- Human Bone Marrow Mesenchymal Stem Cells by Magnetic Resonance Imaging after Transplantation into Cynomolgus Monkey Brain

Ming Feng, RZ Wang, JJ Wei, CJ Wang

Department of Neurosurgery, Peking Union Medical College Hospital, CAMS&PUMC, Beijing, P.R. China; [email protected]

In this study Ferumoxide-PLL was used to lable Flk1+ CD31- CD34- human bone marrow mesenchymal stem cells (hBMSCs) and to observe the survival, the change of relax rate of MRI, and migration of hBMSCs after transplantation into the brains of cynomolgus monkeys. The results show that Ferumoxide-PLL can label hBMSCs effectively. Iron particles were found intracytoplamic of the hBMSCs by Prussian blue stain and transmission electron microscope. The relax rate of labled cells in MRI are 4.4 times and 4.2 times higher than that of the unlabled cells. Hypointensity area was found by MIR three weeks after transplantation. Large quantity of hBMSCs and new vessels were found in the tansplantation zone by pathology and immunofluo-rescence method.

PS-55 Akt Mediates Cardiomyocyte Hypertrophy Induced by Hydrogen Peroxide

Gao Songdan, Li Hongwei, Li Ailing, Zhang Qiuju, Xiu Ruijuan

Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China; [email protected]

Objective: Cellular mechanisms that mediate reactive oxygen species-induced cardiac hypertrophy have not been fully characterized. Akt signaling pathway has been implicated in cardiomyocyte hypertrophy. The aim of the study is to investigate the effect of Akt signaling on cardiomyocyte hypertrophy induced by hydrogen peroxide (H₂O₂). Methods: The neonatal rat cardiomyocytes cultured in primary generation were treated with low concentrations of H₂O₂. The cardiomyocyte hypertrophy was evaluated by the determination of average cell volume and protein content. The effcct of Akt inhibitor on cardiomyocyte hypertrophy induced by H₂O₂ was observed. Western blot was performed to detect the phosphorylation of AKT by H₂O₂ in cardiomyocytes. The effects of the inhibitors of PI3K on H₂O₂-induced AKT activation in cardiomyocytes were also determined. Results: H₂O₂ at 10 or 50 μ mol/L stimulated cardiomyocyte enlargement as measured by cell volume and the protein content per cell. After cardiomyocytes exposed to 50 μ mol/L H₂O₂, Akt phosphorylation increased within 5 to 10 minutes and decreased within 15 to 30 minutes. PI3K(phosphoinositide 3-kinase) inhibitor LY294002 and Wortmannin suppressed AKT phosphorylation induced by H₂O₂. AKT inhibitor significantly decreased the volume enlargement and protein content increase of cardiomyoctes induced by H₂O₂. Conclusion: Akt signaling is involved in cardiomyocyte hypertrophy induced by H₂O₂.

Key words: H₂O₂; Akt; hypertrophy; cardiomyocyte

PS-56 Differential Regulation of Protein Kinase C Isoforms in ROS-induced Cardiomyocyte Hypertrophy

Gao Songdan, Zhang Qiuju, Li Hongwei, Li Ailing, Xiu Ruijuan

Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China; [email protected]

Objective: Protein kinase C (PKC) plays a key role in myocardial hypertrophy. Whether its isoforms are involved differentially in reactive oxygen species (ROS)-induced cardiomyocyte hypertrophy are poorly defined. We examined whether PKC regulates cardiomyocyte hypertrophy induced by ROS. Methods: Cardiomyocytes were treated with low concentrations of H₂O₂ or DDC (inhibitor of superoxide dismutase). The cardiomyocyte hypertrophy was evaluated by the determination of average cell volume and protein content. The translocation of protein kinase C (PKC) isoforms, PKCα, PKCδ and PKCε from soluble to particulate fractions was determined in cardiomyocytes after treatment with H₂O₂ by Western blot analysis. The phosphorylation of PKCα, PKCδ and PKCε was also determined in cardiomyocytes after treatment with H₂O₂. The effects of inhibition of PKC by GF109203X on the hypertrophic response of cardiomyocytes induced by H₂O₂ or DDC were studied by the determination of average cell volume and protein content. Results: H₂O₂ at 10 or 50μ mol/L increased cardiomyocyte volum and protein content. Likewise, DDC at 1 and 5 μ mol/L stimulated cardiomyocyte hypertrophy as measured by cell volume and the protein content per cell. After cardiomyocytes stimulated with 50μ mol/L H₂O₂, the levels of PKCα and PKCε protein in cytosolic fractions began to decrease at 5 min, and the decrease was most significant at 10 and 30 min. Accordingly, the levels of PKCα and PKCε protein in membranous fractions increased at corresponding time. There was no effect of H₂O₂ on the levels of PKCδ protein in cytosolic and membranous fractions of cardiomyocytes. H₂O₂ induced the phosphorylation of PKCα, PKCδ and PKCε protein within 5 min treatmen, and the phosphorylation reached its highest level within 5 to 10 min. PKC inhibitor GF109203X significantly decreased the volume enlargement and protein content increase of cardiomyoctes induced by H₂O₂ or DDC. Conclusions: Reactive oxygen species (ROS) mediate the regulation of cardiomyocyte hypertyophy through different isoforms of PKC.

Key words: ROS; PKC; hypertrophy; cardiomyocyte

PS-57 Preparation of Phenothiazine Dye Modified Magnetic Particles for Blood Viruses Inactivation

TX Zhang, J Gao, WY Gao, C Chen

National Engineering Research Center for Miniaturized Detection Systems, Northwest University, Xi'an; Shaanxi Lifegen Ltd, Xi'an, P. R. China; [email protected]

Phenothiazinium photosensitizers, e.g. azures A and B, were respectively coupled to micro-superparamagnetic Fe₃O₄ particles by straightforward reactions to prepare dye modified magnetic particles with virus-inactivation activity. UV analysis of the products showed that the contents of the photosensitizers on the magnetic particles were in the range of 0.10∼ 0.15 μ mol/mg and photoinduced virus inactivation experiments indicated that the material exhibited potent activity on pseudorabies virus (PRV) which is used as the model of envelop viruses.

PS-58 P38-Mapk Signaling Pathway Plays an Important Role in the Acute Negative Inotropic Effect of β-Blockers in Human Ventricular Myocytes

HB Gong, ZL Zheng

Xuzhou Cardiovascular Disease Institute, Jiangsu, China; SE Harding; National Heart and Lung Institute, Imperial College, London, UK [email protected]

β-adrenoceptor (β AR) blockers have been currently shown to improve survival rate and heart function. However, the mechanism of initial decline of cardiac function after β -blockers treatment is still unclear. Our previous studies have shown that β AR-blockers themselves could directly and acutely depress the contraction of failing, but not non-failing, human myocytes and that the effect was mediated primarily through the inhibitory guanine nucleotide binding protein, Gi. The present work continued to investigate the mechanism of this effect using enzymatically isolated human myocytes or β₂AR-overexpressing rat myocytes (β₂AR/rat). Adenovirally-mediated overexpression of the human β₂AR in rat myocytes for 48h reduced basal contraction (% shortening: Control (Ad.GFP) 6.4 ± 0.3%; Ad. β₂AR.GFP 5.1 ± 0.3%, n = 51, P < 0.05), despite an increase in cyclic AMP. In addition, a further negative inotropic effect of the specific β₂AR blocker ICI 118,551, 1-3 μ M (ICI) was revealed in β₂AR/rat cells that was absent in control rat myocytes. The negative inotropic effect of ICI in human or β₂AR/rat could be prevented by pre-treatment with SB 203580 (5-10μ M), a specific p38-MAPK inhibitor: (HUMAN: basal 100%; ICI 64.7 ± 3.3%, p < 0.01 vs. basal; SB 104.3 ± 3.0%; SB+ICI 99.6 ± 5.0%; n = 15, 15, 7, 7 myocytes respectively, 6 - 8mM Ca^{2 +)} (RAT: basal 100%; ICI 59.2 ± 6.2%, p < 0.05 vs. basal; SB 79.9 ± 12.7%; SB+ICI 80.4 ± 13.1%; n = 6 myocytes, 2-4mM Ca^{2 +}), which indicates that the p38 MAPK signalling pathway plays an essential role in this negative inotropic effect. In contrast, the depressed basal contractility in human or β₂AR/rat myocytes was not affected by SB203580. Western Blot analysis showed that the phosphorylated p38-MAPK protein levels increased significantly in ICI-treated myocytes from β₂AR/rat or tissue from human CABG patients (HUMAN: arbitrary units: -ICI: 10.2 ± 2.4; +ICI: 17.8 ± 2.5, p < 0.01; n = 6) (RAT: arbitrary units: -ICI: 4.8 ± 0.9; +ICI: 9.1 ± 1.2, p < 0.05; n = 7). We have therefore demonstrated that the p38-MAPK signalling pathway plays an important role in the specific negative inotropic effect of β -blockers in rat and human myocardium.

PS-59 Alteration of Cardiac B-Adrenoceptor and Its Subtypes in Hypertensive-Diabetic Rats

HB Gong, L Wang, Xuzhou

Cardiovascular Disease Institute, Jiangsu, P.R. China; [email protected]

The alteration of cardiac β -adrenoceptor(β -AR) and its subtypes in hypertensive-diabetic rats were studied by radioligand binding assays, functional determination of isolated elective field driven left atria. The results showed that the β -AR density was increased by 35% (p < 0.01) and the K_D value was not changed in hypertensive-diabetic rats. The two sites analysis of CGP20712 competitive inhibition curves indicated that the ratio of two β -AR subtypes was not significantly changed. The maximal positive inotropic response induced by isoproterenol (Rmax) was decreased by 48% (P < 0.01), and the pD₂ value was not altered in hypertensive-diabetic rats. After blocking β₁-AR with CGP20712, the Rmax was not changed, and the pD₂ value was significantly decreased in hypertensive-diabetic rats. While after blocking β₂-AR with ICI118551, the Rmax and the pD₂ value were all decreased significantly. These results suggest that in hypertensive- diabetic rats cardiac β -AR was significantly upregulated, with the same degree for the β₁- and β₂-AR. The β -AR mediated Rmax is decreased, which is induced by alteration of β₁-AR. The sensitivity of β -AR to isoproterenol is unchanged, but the desensitization of β₁- and β₂-AR, respectively.

PS-60 Proliferation of Endogenous Neural Stem Cells in Rats after Cerebral Infarct Given Recombinent Human Granulocyt Colony-Stimulating Factor (Rhg-Csf) Through Intranasal Approach

MQ He¹, BL Sun¹, MF Yang²

¹Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College, Taian, Shandong 271000, China; [email protected]

In this study, cerebral infarct model was established in rats by thread-ligation method (reperfuse 2 hours later); Subcutaneous and intranasal delivery of rhG-CSF was chosen separately. Bromodeoxyuridine (BrdU) labeling method was used to identify the proliferation of neuronal stem cells (NSC) in the SGL and SVZ. The objective is to investigate the expression of Brdu in the brain of the rats after cerebral infarct and the influence of rhG-CSF. Our results show that there were BrdU-positive cells in ischemic group while seldom in sham-operation group. The number of BrdU-positive cells increased in the SGL and SVZ after Being applied rhG-CSF, and compared to subcutaneous approach, intranasal approach had a more effective function. There were significant differences between the divided groups. These indicate that ischemic stress stimulated the proliferation of NSC, rhG-CSF promoted the proliferation of inherent neural stem cells, and intranasal approach had a superiority than subcutaneous approach. So we consider rhG-CSF has the protective function on adult rats after cerebral ischemia. Intranasal approach would be a new and more effective way. For its convenience and effect, it is possible to apply into human in near future.

PS-61 Effect of Tumor Conditioned Medium on Endothelial Biological Behavior of HUVEC

Ailing Li, Hongwei Li, Ruijuan Xiu

Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China; [email protected]

Objective: to investigate the effect of tumor conditioned medium (TCM) on proliferation, cell cycle distribution, mitosis, apoptosis and in vitroangiogenesis of human umbilical vein endothelial cells (HUVECs) Methods: After having cultured and exposed the HUVEC to TCM from breast cancer cell MDA-MB-231, then measured its proliferation by MTT, observed its mitosis process by time-lapse recorder, test its apoptosis induced by H₂O₂ and cell cycle distribution by Hoechest33258 staining and flow cytometery (FCM), analyzed its in vitro angiogenesis via matrigel three-dimensional culture. Results: Following the stimulation of TCM, HUVECs showed higher pro-mitogenic ability with a higher rate of S and G2/M phase cell number and lower rate of G1 phase (P < 0.05), and showed higher anti-apoptotic ability than that of negative control group (ECGF-free, medium with 20% FBS), but similar to positive control group (ECGF-M, medium with ECGF and 20% FBS). TCM promoted the stretch speed of HUVEC in telophase and the formation of capillary like tube featured with large volume and lower density which is different with the situation formed in ECGF-M group (small size and higher density). Conclusion: Breast carcinoma cell line MDA-MB-231 could secret soluble pro-angiogenic factors that could induce angiogenic switch including cell cycle progression, proliferation and anti-apoptosis. The confirmative role and character of these factors remain to be further studied.

PS-62 Molecular Microcirculatory Study on Function of Cardiomyocytes

Li Hongwei, Li Ailing, Jia Shidong, Xiu Ruijuan

Institute of Microcirculation, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China; [email protected]

The cardiomyocytes in our experiments were divided into two groups, ie. those from whole heart and those from five parts of the heart (part 1: atrium sinistrum & dextrum, part 2: ventriculus dexter, Part 3: septum interventriculare, part 4: ventriculus sinister and part 5: cardiac apex). Cardiomyocytes were chosen for recording their spontaneous vivid pulsation under Time-Lapse Video Microscopy and analyzed with MCIP system (developed by our Institute). Meaningfully in our observation, cardiomyocytes recorded in each part displayed different pulsation frequency. In group I, the pulsation frequency of 190 primarily cultured cardiomyocytes is 106.54 ± 44.00 per min with range of 29-202 per min. And the pulsation frequency of 123 cardiomyocytes from 5 parts of whole heart in group II is listed as 75.30 ± 15.33 per min., 84.11 ± 23.46 per min., 91.15 ± 38.92 per min., 101.57 ± 12.94 per min., 111.53 ± 13.93 per min. for part 1, 2, 3, 4, 5 respectively, with average frequency 90.53 ± 25.56 per min at range of 29-175 per min. Ongoing functional study of cultured myocardial cells are followed by laser confocal microscopy; detection of apoptosis, DNA synthesis, and karyokinesis; receptor expression level and related signal transduction, which accumulate physiological basis in normal status for more advanced experimental trails and to be characterized for further investigation in the effect of certain Chinese medicines. Based on the above observations, we propose the following hypothesis: To strengthen the microvascular vasomotion in myocardium and pulsation function of cardiomyocytes is a promising means for improving myocardial microcirculation and metabolism. The expected results suggests us a new way to treat cardiovascular diseases by the enhancement of microvascular vasomotion hence to improve microcirculation in heart tissue, evaluate the effect of various treatments in ischemic heart diseases as well as select new drugs for cardiovascular diseases by testing their effects on microvascular vasomotion and pulsation of cardiomyocytes.

PS-63 Intraventricular Infusion of Raav1-Egfp Resulted In Transduction in Multiple Regions of Adult Rat Brain: A Comparative Study with Raav2 and Raav5 Vectors

Shi-fang Li, Ren-zhi Wang, Gui-lin Li, Wan-chen Dou, Zhao-jian Li, Zhen-xing Zhang

Department of Neurosurgery, Peking Union Medical College Hospital, Beijing, China; [email protected]

Most gene transfer studies conducted in the central nervous system (CNS) with recombinant adeno-associated virus (rAAV) vectors have been carried out by direct intraparenchymal injection. However, this delivery method usually results in transduction of cells in only a limited region and is quite invasive, which may hamper its potential clinical application. Injection of viral vectors into the cerebrospinal fluid (CSF) may provide an alternative strategy for widespread gene delivery to the CNS via the subarachnoid space. In this study we compared the transduction abilities of rAAV types 1, 2, and 5 when infused directly into the right lateral cerebral ventricle of adult rats. Multiple structures in the vicinity of the lateral ventricle were transduced by rAAV1, but not by rAAV2 or rAAV5 vectors. Double immunolabeling showed that the transduced cells included not only neurons, but also glia. Real-time quantitative RT-PCR experiments demonstrated that rAAV1-mediated EGFP mRNA expression was significantly higher than that induced by either rAAV2 or 5. Our data suggest that intraventricular infusion of rAAV1 vectors provides a useful method for broad gene delivery to cells in the adult rat CNS.

PS-64 Safety of Intraoperative Blood Salvage During Resection for Meningioma

Hui Liang, Bao-Guo Wang, Wen-Min Liu

Departments of Anesthesiology, Beijing Tian Tan Hospital, Capital Medical University, Beijing, People's Republic of China; [email protected]

In this study, a total of 134 patients who had undergone resection for meningioma in Beijing Tian Tan hospital during 1999 to 2006 were followed up to evaluate the risk of extracranial metastases of meningioma induced by intraoperative blood salvage (IBS). Among the patients, 89 (group1) received salvaged blood and 45 (group2) did not. There were no significant intergroup differences in clinical background, including age, sex, pathological classification, tumor growth pattern (whether invasion of venous sinus, whether invasive growth) and Simpson resection grades. Although in group 1, intraoperative blood loss was significantly higher than that of group 2, only 7.9% patients in group 1 were transfused with banked blood, which was much lower than that of group 1 (24.4%). The median follow-up for group 1 and 2 was 25 and 30 months, respectively. The recurrent rate was 7.6% and 11.8% in the two groups, respectively (p = 0.75). No extracranial metastases case was found. There is no increased risk of recurrence or metastases of meningioma for those who were transfused with salvaged blood. IBS may be safely used in resection for meningioma. However, only a prospective, large sample, multicenter, longer term, randomized trial would provide the most valid assessment of the safety of IBS used in resection for meningioma.

PS-65 The Relationship Between blood lipid and hemorrhelolgy in Hypertension patients

Liu Jiubo

Department of Blood Transfusion, the affiliated Taihe Hospital of Yunyang Medical College, Shiyan 442000 China; [email protected]

Objective: To explore the relationship between blood liquid and hemorrheology and their effect on hepertension. Method: SBP, DBP, Cho-c, TG, apoA, apoB, LDL-c, HDL-c and hemorrhelolgy were measured in 482 cases with hepertension, and were compared with those of 100 healthy subjects. Result The group Hepertension was positively correlated with blood viscosity of low shear rate (η bL), ESR, Equation K value of ESR, the time of erythrocyte electrophoresis. TG was positively correlated with η bL, blood viscosity of middle shear rate (η bM), ESR, Equation K value of ESR, SBP, DBP. BSV, EAI and the time of Erythrocyte Electrophoresis were positively correlated with DBP (P < 0.05∼ 0.01). η bM, η bL were positively correlated with SBP (P < 0.01). Conclusions hyperlipemia and hyperblood viscosity have close relation with hepertension. Blood lipid and blood viscosity interact with hypertension and are cause and effect each other. It produces vicious circle.

Key words: Blood lipid; Hemorrheology; blood pressure; Hypertension

PS-66 Construction and Identification of the Lentiviral Vector Transfered Akt Plasmid Gene

LI Dong-ye, Luo Yuan-yuan, Zhu Hong, Xia Yong, PAN Deng-feng

Department of Cardiology, Affiliated Hospital of Xuzhou Medical College, China; [email protected]

The direct protective effect of activated Akt gene in isolated cardiac myocytes and the whole heart has been well documented, due to the reduction of cell apoptosis both in vitro and in vivo. At the same time, Akt enhances the cardiac function through inducing cardiomyocytes hypertrophy. So the establishment of a stable transgenic Akt overexpression model is in strong desire. In this study, we generated a stable overexpression model of Akt plasmid gene in in vitroly cultured cardiomyocytes with lentiviral vector, so as to further study the mechanism of enhanced cardiac function through the hypertrophic effect in cardiomyocytes in vivo. The restriction enzymes, namely, Bgl II and Sal I were used to digest the plasmid pMSCV-Akt and lentiviral transfer vector PTK208. Akt gene was then subcloned into the lentiviral transfer vector to generate the resultant transfer vector PTK208-Akt. The recombinant plasmid was confirmed by the restriction enzymes and successful sequencing. The plasmid PTK208-Akt was then transformed into competent cells to get a high-yield and stable expression. Here the task of Akt gene cloning into the transfer plasmid of lentiviral vector PTK208 has been completed. It is concluded that the successful construction of PTK208-Akt lentiviral transfer vector provides a useful tool for further investigate the role of Akt gene and its possible mechanism in enhancing the cardiac function through cardiomyocytes hypertrophy.

PS-67 Application of Optical Proteinchip in Detecting Hba1c

Jixiao Niu¹, Honggan Zhang¹, Yu Niu², Gang Jin², Ruijuan Xiu¹

Institute of Microcircilation, Peking Union Medical College & Chinese Academy of Medical Science, Beijing, China, ²Institute of Mechanics, Chinese Academy of Sciences, Beijing, China; [email protected]

Our objective is to detect HbA1c in diabetic patients by optical proteinchip system and initially evaluate the potential for further application in clinical detection and general survey. Anti-human HbA1c monoclonal antibody was bound on the substrate surface of silicon wafer with aldehyde. The chip surface was incubated in a sample solution containng HbA1c, which was trapped by the antibody HbA1c with the special interaction between the HbA1c and antibody HbA1c. An immune complex formed by the antibody-antigen interaction, resulting in a variation of layer thickness on the silicon surface that was observed by imaging ellipsometry to investigate the content of HbA1c. Bio-Rad Varient ?ion exchange HPLC system was used as control assay methods at the same time. Results indicated that the gray was increased by 19.3 ± 6.1(gray value) after bound with antibody, and each variation of layer thickness corresponded to a concentration of HbA1c. The measure results of optical proteinchip system were markedly correlated with HPLC (P < 0.01). We initially set up an efficient assay method for HbA1c detection by optical proteinchip system. The optical proteinchip has advantages such as short measurement time, label free, high-throughput and has visualized results. It is more accurate to detect HbA1c through the special antibody-antigen interactions and may be applicable in clinical detection and general survey of diabetes.

(The study was supported by the Ministry of Science & thchnology of the People's Republic of China, No.2005DIB1J086)

PS-68 Enhanced Expression of Avβ3 Promote Endothelial-Tumor Cells Adhesion

Jixiao Niu, Honggang Zhang, Jing Zhang, Ruijuan Xiu

Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China; [email protected]

Our aim is to investigate the role of adhesion molecule α vβ 3 in the adhesion of tumor-endothelial cells in vitro. The human lung endothelial cells (HLEC) were isolated and identified by expression of vWF and uptake of Dil-Ac-LDL. The expression and regulation of α vβ 3 by vascular endothelial growth factor (VEGF) in HLEC were analyzed by RT-PCR and immune cytochemistry. The adhesion of human lung carcinoma cells A549 in HLEC was measured by a fluorescence plate reader after co-culture of dye-labeled tumor cells and endothelial cells. Results indicated that HLEC were positive expression for vWF and took up high levels of DiI-Ac-LDL. The expression of α vβ 3 in HLEC was up-regulated by VEGF. The adhesion of tumor cells was significantly increased under VEGF conditions. Furthermore, the tumor cell adherence could be inhibited by antibodies against α vβ 3. In a conclusion, adhesion molecule α vβ 3 may play an important role in the process of endothelial-tumor cells adhesion.

PS-69 CTGF Promotes the Proliferation of Human Umbilical Vein Endothelial Cell through ERK/Akt Signal Pathway

Su Yan¹, Luo Zhaohua¹, Li Hongwei¹, Li Ailing¹, Wang Chunling³, Cui Peilin¹, Jing Zhang¹ and Xiu Ruijuan¹

¹Department of biochemistry and molecular biology, Baotou Medical College, Baotou 014010, China; ²Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China Heart center, Meitan general hospital, Beijing, 100028, China; [email protected] [email protected]

Connective tissue growth factor (CTGF), a novel angiogenic factor, plays a role in angiogenesis. One recent study has shown that CTGF has a proliferative effect on fibroblast cell and vascular smooth muscle cells. However, there is no information on whether CTGF could also affect human umbilical vein endothelial cell (HUVECs). Thus, the purpose of this study was to assess whether CTGF could induce HUVECs proliferation and to study the mechanisms whereby CTGF signals in HUVECs. HUVECs were stimulated with increasing concentrations of CTGF for 48 hours. Cell proliferation was induced by CTGF as assessed by MTT. To gain more insights into the mechanism of action of CTGF, we investigated the extracellular signal-regulated kinase (ERK) and Akt signaling pathways. Transient phosphyorylation of the p42/44 mitogen-activated protein kinase (ERK1/2) and Akt occurred after addition CTGF to HUVECs. LY294002, a specific PI3K inhibitor, significantly inhibited HUVECs proliferation after CTGF stimulation. Our results demonstrate that CTGF induces HUVECs proliferation through both ERK1/2 and Akt signaling pathways. The proliferative action exerted by CTGF on HUVECs may account in part for the role of CTGF in angiogenesis.

PS-70 Different Transcriptional Regulation of Human Transforming Growth Factor-β 2 by Hypoxia Inducible Factor-1

Su Yan¹, Li Hongwei², Zhang Jing², Luo Zhaohua², Xiu Ruijuan²

¹Department of biochemistry and molecular biology, Baotou Medical College, Baotou 014010, China; ²Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China; [email protected] [email protected]

In this study, the putative hypoxia responsive element (HRE) in human transforming growth factor beta2 (TGF-β2) promoter was determined. HIF-1α protein was differentially expressed in Hela cells and HEK293 cells: a reasonable amount of HIF-1 was detected in Hela cells while little protein detected in HEK293 cells by western blot. Additionally, HIF-1α overexpression could dramatically increase its activity in HEK293 cells but only a little in Hela cells by a HRE reporter assay. Realtime PCR results show TGF-β 2 mRNA level were induced about 1.4-fold (P < 0.05) by HIF-1 overexpression in HEK293 cells but decreased significantly in Hela cells. Site-directed mutagenesis study of TGF-β 2 promoter was performed to map its HREs. Two HIF-1 responsive elements located in –73bp∼ –69bp and –49bp∼ -45bp which overlap with CRE/ATF(-74bp∼ -67bp) and E-box(-50bp∼ -45bp) were responsible for the hypoxia induction. This observation was further confirmed by a chromatin immuno-precipitation assay. These findings would provide theoretical evidences to the hypoxia related diseases.

Keywords: TGF-β 2, hypoxia, hypoxia responsive element, hypoxia inducible factor-1, transactivation

PS-71 Study on Injection Methods of Macromolecular Tracer's being administered To Rat's Caudate-Putamen*

TG Sun¹, BL Sun¹, L Jia¹, MF Yang²

¹Department of Neurology, Affiliated Hospital, Taishan Medical College; Taian, Shandong 271000, China; ²Taishan Medical College; Taian, Shandong 271000, China; [email protected]

This experiment was to discuss injection methods that macromolecular tracers entered into rat caudate-putament (CPu) in order to find a better way suitable for studying the drainage pathway from the parenchyma of the brain. Adult Sprague-Dawley rats were randomly divided into cutting needles group, not cutting needles 4ul group and not cutting needles 1ul group. Evans Blue-labeled albumin(EBA) solution was injected into the rat CPu with microsyringe under stereotaxic apparatus'control. Frozen sections of rat brain samples were made and observed by naked eye and fluorescence microscope respectively. The cutting needles way by which tracer's dose injected into CPu was inexact and unstable, however, not cutting needles way was exact and stable. Way of not cutting needles by which tracers were injected into rat CPu was economical, exact, stable and suitable for researching drainage of macromolecules from the parenchyma of the brain.

*This work was supported by the National Natural Science Foundation of China (30570651, 30670724); Science Development Plan of Tai'an(14-22)

PS-72 Soluble Angiopoietin Receptor Tie-2 in Patients with Acute Myocardial Infarction and its Detection by Optical Protein-chip

Wang Chunling¹, Li Hongwei², Wang Zhanhui³, Meng Yonghong³, Jin Gang³, Xiu Ruijuan²

¹Heart Centre, Mei-tan General Hospital, Beijing 100028, China ²Institute of Microcircilation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100005, China ³National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100080, China; [email protected]

Tie-2 receptor has been shown to play a role in the angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the Optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 27 patients with AMI were measured on admission (day1), day2, day3 and day7 after onset of chest pain and 28 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significantly correlation in sTie-2 results between two methods (r = 0.923, P < 0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum. Keywords: soluble Tie-2; acute myocardial infarction; ELISA; optical protein-chip; imaging ellipsometry

PS-73 The Benefits of Cell Transplantation with Human Bone Marrow Mesenchymal Stem Cells to Cerebral Ischemic Rats

Junji Wei, Renzhi Wang, Guilin Li, Wanchen Dou, Ming Feng, Jun Kang, Ziheng Zhang, Zhaojian Li

Department of neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, 100730, P.R. China; [email protected]

Ischemic cerebralvascular diseases (ICD) are a common disease in the world, which ranked the 3ed reason of death. Up to now, few therapeutic measures were present to ICD. Bone marrow mesenchymal stem cells could be used to treat ICD. But the distinct mechanism is still unknown. We treat the cerebral ischemic rats with human bone marrow mesenchymal stem cells (hBMSCs) to assess the effects of cell transplantation and investigate dynamically the survival, migration, differentiation of hBMSCs and the influence to cytokines secreting. Bone marrows of volunteers were collected and hBMSCs were isolated and cultivated until the third passage. 52 adult male Sprague-Dawley rats performed transient(2 hours) MCAO were divided randomly into the treated group (n = 32) and the control group (n = 20). All the rats received corresponding behavioral training before MCAO. hBMSCs were injected into the brain cortex of the treated rats while saline to the control rats after 24h of MCAO. Morris water maze test, Rotarod test and adhesive-removal test were performed orderly at different time points after transplantation. hBMSCs were identified with immunofluorescence. BDNF, NGF, NT-3 and VEGF of rats' brain were measured with real-time RT-qPCR. We found that a large number of hBMSCs survived 2 weeks after transplantation. However, the number of hBMSCs decreased at the 3rd week after transplantation and few cells could be detected at the end of 1 month. No definite evidence supported the differentiation of neural cell derived from hBMSCs. Our results also showed hBMSCs can migrate to the border of ischemia at the 2nd week after transplantation. The mRNA of neurotrophic factors and VEGF were increased more significantly in hBMSCs treanted group than control group at week 2 s and week 3 after transplantation, nevertheless, all the cytokines decreased at 4th week. The neurological function of rats treated with hBMSCs recovered dramatically compared with the control group. In summary, our experiment showed that the cerebral ischemic rats can benefit from cell transplantation with hBMSCs, but the survival time of hBMSCs was limited in vivo.

PS-74 Analysis of Differential Expression Profiles Provides New Insights into Cold-induced Mitochondrial Changes in Brown Adipose Tissue of Rat

Yang Yue, Hu Songnian, Yu Jun

Beijing Genomics Institute of the Chinese Academy of Sciences, Beijing, China 101300; [email protected]

Exposure to cold is a classical inducer of adaptive thermogenesis in brown adipose tissue (BAT). Mitochondria, the organelles that convert food to carbon dioxide, water and ATP, play fundamental roles in mediating effects on energy dissipation. To investigate this phenomenon, differential expression was studied by using combined proteomic and genomic strategies. Mitochondrial proteins of brown adipose tissue were compared between control and cold treated rats using two-dimensional gel electrophoresis. We observed significant changes in the abundance of 21 proteins among approximately 510 protein spots reproducibly detected on each gel. Among them, 6 protein spots were down-regulated, and 15 were up-regulated. Differentially expressed protein spots were identified by mass spectrometry. The expression level of proteins involved in respiratory chain and lipid metabolism were more abundant after cold exposure. Myosin light chain protein, which may be involved in the function of heat production of BAT, was down-regulated after cold exposure. Coenzyme Q (COQ) which may functions as a cofactor for uncoupling protein, however, COQ7 and COQ9 was unexpectativelly found to be down-regulated in BAT of cold-treated rats. By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of COQ that are differentially regulated in response to cold in BAT. As such, our work is expected to lead to new insights into the energy mechanism of BAT.

PS-75 Optical Protein Chip: A No Labeling Technique for Detecting Living Protein Maker In Breast Patient's Blood Sample

HG Zhang, C Qi, G Jin, RJ Xiu

Institute of Microcirculation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, P.R.China; [email protected]

In this paper, a novel type of biosensor system, optical protein chip was applied to detect protein marker CA153 in the serum from patients with breast cancer, this technique allows label-free samples and crude samples to be used directly without previous purification. The principle of this system is that the complex formed by interaction between an antibody molecule and its corresponding antigen can be detected on a silicon substrate by an optical sensor. The experimental step included 1, processing and modification of the silicon substrate surface with acidic peroxide solution; 2, washing and reacting with glutaraldehyde; 3, through the reaction of glutaraldehyde with 3-aminopropyltriethoxysilane, Fc regions of the anti-CA153 antibody molecules were covalently immobilized on the chip surface; 4, detection with crude serum samples. The results were analyzed that the thicknesses of layers in the analytical areas were measured with biosensor imaging ellipsometry, which produced an ellipsometric image of a surface of each chip with a lateral resolution of 2 μ m. In this paper, we studied on 60 serum samples from women with breast cancer and other breast diseases. The median patients age was 48.5 years (range, 22–75 years). From the results obtained by optical protein chip, the with-in CV values were 5.2%, 2.5% and 4.6% at 5, 10, and 18KIU/L, respectively (n = 10), and the interassay CVs were 7.5%, 3.8%, and 6.3%. The lower limit of detection was 1 KIU/L at a signal-to-noise ratio of 3. The limit of quantification, defined as the lowest amount detectable with CV < 20% (n = 10), was 4 KIU/L. The optical protein system provides a new technique for monitoring of biomolecular interaction events for the fields of proteomics. (This study was supported by the Ministry of Science & Technology of Chine, No.2005DIB1J086)

PS-76 Risk Assessment in Haematopoietic Stem Cell Transplantation: Hla Matching in Chinese

HG Zhang

Institute of Microcirculation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, P.R.China; [email protected]

HLA matching plays a critical preventive role in lowering risks of graft failure and graft- versus-host disease in haematopoietic stem cells transplantation. Consideration of potential donors for transplantation includes a rigorous assessment and identification of suitable unrelated donors when related donors are not available. In this paper, the probability of finding HLA-matched unrelated haematopoietic stem cells donors was studied. The present paper attempted to calculate the probability of finding HLA-A,B,DR matched unrelated stem cells donors based on the data of 156 Han population's HLA-A,B,DR gene frequencies and families haplotypes in China according the method described previously by Takahashi (Transfusion, 1987:5;394–398) and Beatty (Transpantation, 1988:4;714-718). Results of the studies showed that a haematopoietic stem cells bank with registries containing 1000, 10000 or 100000 donors, the probability were approximately 10.00%, 46.82%, 79.78%, respectively, the above results approximately represents the average likelihood of finding a HLA-A,B,DR matching without a “blank” allele from donors of Han population. Comparing with the results based on the data of Japanese, North American and Europe population, the results in this paper showed that the HLA heterogeneity in Chinese Han population is higher than Japanese, but it is lower than both of North American population and Europe population, i.e. in the haematopoietic stem cells bank with registries containing the same numerary donors, the probability of finding HLA-A, B, DR matched unrelated donor in Chinese Han population is lower than Japanese, but is higher than both of North American population and Europe population.

PS-77 Influence of Breast Carcinoma Cells on Normal Endothelial Cells in Vitro

HG Zhang, J Zhang, RJ Xiu

Institute of Microcirculation, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, 10005. China [email protected]

The present study, it was performed to evaluate the influence of breast carcinoma cells on normal endothelial cells (ECs). For this aim, ECs were isolated from human umbilical cord blood. Co-culture medium Z-MCF-7-EC and Z-MDA-MB-231-EC were established to co-culture the normal ECs and human breast carcinoma cell lines MCF-7 and MDA-MB-231, respectively. Real time observation was carried out during the co-culture of the breast carcinoma cells and normal ECs for more than 72 hours. After 5 days, it was found that some new vessel tubes between ECs on the medium were formatted. The behavior of these ECs was similar as endothelial progenitor cells (EPC). The new vessel formation was like fvasculogenesis by EPC. The results also showed that the shape and the microstructure of the ECs after co-culture were remodeling, and the expression levels of the genes related with the EC's behaviors, such as ESM, IGFBP-3, α vβ 3, VE-C, and Tie-2 in the ECs co-cultured with MCF-7 and MDA-MB-231 were all up-regulated.

In conclusion, the characters of ECs after co-culture with the breast carcinoma cell lines were different from normal ECs. The new vessels formation induced by breast carcinoma cells was vasculogenesis but not angiogenesis. (This study was supported by Grant of the Ministry of Science & Technology of China, No.2005DIB1J086)

PS-78 Hydrogen peroxide regulates cardiomyocyte hypertrophy via activation of nuclear factor-κ B

Zhang-Qiuju, Li-Hongwi, Gao-Songdan, Xiu-Ruijuan

Institute of Microcirculation, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100005, China; [email protected]

Objective: Reactive oxygen species (ROS) can act as signaling molecules to stimulate cardiomyocyte hypertrophy. Nuclear factor-κ B (NF-κ B) has been implicated in the signaling of cardiac hypertrophy. We tested whether NF-κ B activation is involved in ROS-induced signaling pathways of cardiomyocyte hypertrophy. Methods: The neonatal rat cardiomyocytes cultured in primary generation were treated with low concentrations of H2O2. Electrophoretic mobility shift assay(EMSA)was performed to detect the activation of NF-κ B induced by H2O2 in cardiomyocytes. The effects of the inhibition of PKC and AKT on NF-κ B activation induced by H2O2 were determined by EMSA. The phosphorylation and degradation of Iκ B-α stimulate by H2O2 were determined by Western blot. The effects of the inhibition of NF-κ B on the hypertrophic response of cardiomyocytes induced by H2O2 were studied by the determination of average cell volume and protein content. Results: The level of NF-κ B activation increased obviously within 30 min treatment with 50 μ mol/L H2O2, which activation was maximum at 60 min treatment and gradually reduced within 120 min. H2O2 increased the phosphorylation of Iκ B-α and promoted its degradation, indicated of NF-κ B activation induced by H2O2 through a Iκ B degradation-dependent way. The inhibitors of PKC and AKT significantly inhibited the activation of NF-κ B induced by H2O2, which indicated that PKC and PI3K/AKT pathway mediated NF-κ B activation induced by H2O2. Moreover, NF-κ B inhibitor significantly decreased the volume enlargement and protein content increase of cardiomyoctes induced by H2O2. Conclusion: Our date suggest that ROS may induce cardiomyocyte hypertyophy through NF-κ B activation dependent of PKC and PI3K/AKT pathway.

PS-79 Conjugate of Hemoglobin and Human Serum Albumin as a Candidate Blood Substitute

CY Zheng, XY Suo, T Liu, SP Li, ZG Su

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering. Chinese Academy of Sciences, Beijing, China, [email protected]

Hemoglobin (Hb) is the main protein of red blood cell and human serum albumin (HSA) is the main protein of human serum. Therefore, the conjugate containing both Hb and HSA was hoped to act as the real red blood cells and plasma to carry oxygen and expand blood vessels simultaneously. However, it is methodologically not easy to prepare well-defined conjugate of Hb and HSA to meet the need of biopharmaceutical research. The ineffective cross-linked polymers of Hb or HSA are liable to form due to the large amount of derivable lysines on these two molecules. The difficulty was circumvented by controlling the reaction environment under the adjustment of polyethylene glycol or the coordinating system of mannite and borate buffer. Moreover, when the conjugate of Hb and HSA was subjected to the exchange transfusion experiments with male Sprague Dawley rats, no significant side effect was observed. The conjugate was even overwhelmingly efficient in term of rescuing rats with 60% loss of the total blood, compared with other existing candidates, such as intra-molecular and inter-molecular cross-linked hemoglobins.