| Hemorrhagic shock remains a major cause of death and disability in battlefield injuries, as well as in civilian trauma. Fluid resuscitation is an essential component of therapy for hemorrhagic shock. The aim of fluid resuscitation is to expend effective blood volume, increase perfusion in microcirculation, and to alleviate injuries in tissue and organ. Plasma expanders used in fluid resuscitation can not only expend the blood volume, but also influence plasma viscosity and other rheological behaviors, which are important determinants in tissue perfusion, oxygen supply, microcirculation and improvement in function of tissue and organs. It was generally perceived that lowered blood viscosity in hemodilution and hemorrhagic shock could improve the tissue perfusion due to reduced flow resistance. But the studies began in 90th in the last century showed that plasma expander with high viscosity, which could increase perfusion in microcirculation and improve hemodynamic parameters, is beneficial in extreme hemodilution and severe hemorrhagic shock. Plasma expander with high viscosity is known to elvate plasma viscosity and wall shear stress in microvascular, and to improve function of endothelial cell in microvascular. But because of the limitation in animal study, the effects of hydro- viscosity on microvessel endothelial cell and the mechanisms are still to be investigated.Microvessel endothelial cells locate in circumstances with complex mechanical forces. Changes in mechanical properties, such as stiffness, elasticity, and adherent property are outcomes of mechanical stimulation, as well as maintenance of cellular function. Mechanical properties of microvessel endothelial cells are important in perfusion of microcirculation. Atomic force microscopy (AFM) is a novel method for high-resolution three-dimensional topography in living cell, and can detect local mechanical properties of cells. The present study will use in vitro perfusing system of luminar flow and function of detecting local mechanical properties by AFM. The aim of the study is to observe the influence of hydro- viscosity on the mechanics of microvessel endothelial cell, and to investigate the roles of cytoskeleton, NO (nitric oxide) and ET-1 (Endothelin-1). The regulatory mechanisms of hydro-viscosity on the mechanics of microvessel endothelial cell will give more insight in the treatment of hemorrhagic shock and the research in blood and plasma substitutes.Establishment of system of rat cerebral microvessel endothelial cells perfused with medium of different hydro-viscosities. To investigate he influence of hydro- viscosity on the mechanics of microvessel endothelial cell, it is necessary to establish an in vitro system to simulate shear stress of blood flow. We developed a parallel plate flow chamber according to the fundamental of distribution of shear stress, which showed equally in ease of use and performance compared with overseas commercial chambers. Based on the parallel plate flow chamber, in vitro perfusing system of luminar flow was developed. Rat cerebral microvessel endothelial cells (rCMECs) were isolated and cultured in vitro. The purity of the cells meets the demand in further study and can keep stable in the shear stress. Sodium alginate solutions show Newton property. Compared with Dextran 70, sodium alginate of a comparatively low concentration can result in high viscosity with a low colloid oncotic pressure and a low concentration. Sodium alginate solutions do not influence the growth of rCMECs. Thus, sodium alginate can act as'viscosity modifier'. The system of microvessel endothelial cells perfused with medium of different hydro-viscosities meet the demand in further study.Determination of parameters for local mechanical properties of rCMECs in quantitative analysis by AFM. Force volume is the commonly used working pattern for detection of local mechanical properties by AFM. The Force-Curve can be collected during the scanning of surface of the cells by contact mode. Young's modular is the commonly used parameter in quantitative analysis of local mechanical properties. Method to evaluate Young's modular suitable for rCMECs in the present was developed based on Hertz model. Meanwhile,'Deformation Consumed Work'calculated based on the Force-Curve was advanced for the first time in this study for analyzing the local mechanical properties. Deformation Consumed Work, which shows viscoelasticity of cells, describes the differences in Consumed Work between deformation of the cell and elastic homogeneous body. Deformation Consumed Work is suitable in quantitative analysis of nonlinear deformation of cells. Furthermore, it can eliminate the influence of hard base and show no changes with different indetations. As a novel parameter in quantitative analysis of local mechanical properties, Deformation Consumed Work may be an alternative or compensatory of Young's modular calculated by Hertz model. Deformation Consumed Work of endothelial cell can reflect the energy dissipation of blood flow in wall of vascular generated by heart.Effect of hydro-viscosity on morphology and local mechanical properties of rCMECs. Under the shear rate of 800s-1, hydro-viscosity of 0.93, 2.08 and 4.76 mPa s, shear stress of 7.4, 16.6 and 38.1 dyn/cm2 for 2h, the changes of distribution of angle of rCMECs reflect orientation caused by the shear stress. Higher viscosity causes some cells to become thinner and longer. As the hydro-viscosity and shear stress become higher, the height and the roughness of the cell decrease. Meanwhile, the stiffness elevates, but Deformation Consumed Work and non-specific adherent force decrease. The changes of local mechanical properties have threshold. When the hydro-viscosity reaches a certain level, local mechanical properties do not show more changes. The elevate stiffness is the result of reorganization of cytoskeleton. Decrease in Deformation Consumed Work reflects reduction in energy dissipation of blood flow in wall of vascular generated by heart. Decrease in non-specific adherent force reflects lower frequency of adhesion of white blood cell to endothelial cell. These results indicated that appropriately elevated hydro-viscosity can modify local mechanical properties of rCMECs, and can reduce energy dissipation in microcirculation, restrain the adhesion of white blood cell to endothelial cell. These mechanisms are beneficial in perfusion in microcirculation. But if the blood viscosity becomes too high in vivo, the local mechanical properties of endothelial cells cannot improve.Effect of hydro-viscosity on cytosleletal actin reorganization and production of NO and ET-1 in rCMECs. At a constant shear rate, higher viscosity causes changes in distribution of cytoskeleton F-actin, and stress fibers become dense in the central region of rCMECs. The dramatic increase ofβ-actin mRNA reflects response of rCMECs to shear stress, which includes a greater turnover of actin, and stronger adherent force to basement. ET-1 mRNA increases when hydro-viscosity and shear stress become higher. But if hydro-viscosity and shear stress reach a certain threshold, the ET-1 mRNA decreases. The increases of NO production show concomitance with the F-actin reorganization. These results indicated that cytosleletal actin is the base of changes in local mechanical properties, and NO production shows relationship with cytoskeletal reorganization.Taken together, system of microvessel endothelial cells perfused with medium of different hydro-viscosities was established. Parameters for local mechanical properties in quantitative analysis by AFM were determined. Three-dimensional topography and local mechanical properties of rCMECs changed significantly when hydro-viscosity became higher and the shear rate kept constant. The regulatory mechanisms of hydro-viscosity of certain degree on the mechanics of microvessel endothelial cell will be beneficial in the treatment of severe hemorrhagic shock and the viscosity of blood and plasma substitutes should be taken into consideration.
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