| BackgroundSeptic shock caused by severe infections is the first death factor related to severe diseases at present, and the medical fees are staggering. Although there has been a rapid development in the usage of antibiotics, the intensive care, the supportive treatment, etc., however, in recent10years, the mortality rate has not appeared a decreasing trend. In case of serious infection or septic shock, micro-circulation stasis, blood distribution abnormalities and effective circulating blood volume reduction are caused by hypoxia and acidosis, plasma extravasation, and inflammatory metabolites, injury directly induced by endotoxin, etc. may jointly cause blood pressure dropped and tissue and organ perfusion reduced, of which the above-mentioned pathological changes become more obvious due to hypoxia or acidosis. Microcirculatory disturbance and inflammatory reaction are of its important pathophysiological basis, and to improve microcirculation and to alleviate inflammatory reaction is the key to the prognosis for corresponding effects. Continuously deteriorated microcirculation caused multiple-organ failure, and finally caused death. In the clinical treatment of septic shock, fluid resuscitation is the primary therapeutic measures for hemodynamics and tissue/organ perfusion, however, after the early goal-directed therapy, even if the capacity has been restored, microcirculatory disturbance may still continue to exist. Therefore, to find a more effective therapeutic method to improve microcirculation is imminent. It’s found in previous studies that, the poorer the quality of microcirculation image was, the worse the prognosis was; and, the survival rate of the patient with septic shock and having the microcirculation improved faster is higher than that of the patient with microcirculation improved slowly. To improve the microcirculation after septic shock as soon as possible would significantly improve the mortality rate.Toms, et al in1950s found that, after a small quantity of high molecular polymers is added to fluid, the flow resistance is significantly reduced, and on the condition that the driving pressure is not changed, the flow velocity is increased. This phenomenon is known as Toms effect, and this kind of high molecular polymers are referred as drag-reducing polymers). A number of domestic and international studies reported that, the drag-reducing polymers could prevent from the formation of delayed atherosclerosis, and the polymers have the functions of delaying indiabetic microangiopathy, diuresis and improving renal function, and reduce mechanical damage to red blood cells, etc. These effects may be related to the following mechanisms.1. To reduce the "plasma skimming effect", while the erythrocyte suspension is flowing inside microvessels, an area where is located near the proximal vessel wall and may produce a relatively small amount of cells. This phenomenon is referred as "plasma skimming". To add the drag-reducing polymers may significantly reduce the thickness of the plasma layer with relatively small red blood cells near the proximal vessel wall, causing the red blood cells to be redistributed near the proximal vessel wall, and facilitating the blood-gas exchange inside small arteries and blood capillaries.2. The red cell deformability is increased, so that the red cells could more easily flow through microcirculation.3. The Reynolds coefficient of plasma flow is reduced, so that the plasma flow turbulence and the vascular resistance could be reduced.4. To alleviate the critical radius of reversing the "Farhaeus-Lindqvist effect" may lower the resistance of red blood cell while passing a smaller microcirculation, thus increasing the oxygen supply from red blood cells for local tissue.since the70s of last century, drag-reducing polymers started being adopted in medical research, and its main functions include hemodynamic improvements; and, in recent years, it’s gradually found that, drag-reducing polymers play certain roles in improving microcirculatory disturbance of diabetic anima, prognosis for animal with acute myocardialischemia, reperfusion and fluid resuscitation from hemorrhagic shock, etc. Its effects on the microcirculation in septic shock has not been reported so far. The current study is proposed to establish a model of rat with septic shock, to observe directly microcirculation changes in spinotrapezius muscle under microscope, and to investigate the effects of drag-reducing polymers on the fluid resuscitation from septic shock.Materials and methods1.1Drugs and reagentsPolyethylene oxide (PEO5000; Sigma, Inc.),0.9%sodium chloride injection (Shandong Lukang Cisen Pharmaceutical Co., Ltd.), and,3%pentobarbital sodium (Laboratory Animal Center, Southern Medical University, Guangzhou) were used in this study.1.2InstrumentsAn IntelliVue MP60monitor (Philips Healthcare), a micropump (B. Braun), cellulose membrane tubing (Spectrum Laboratories, Inc.), a camrecord high-speed camera (OPTRONIS, Inc.) a prosilica-GE HD camera (ALLIED Vision Technologies, Inc) and axio microscope (Carl Zeiss, Inc.) were employed and the NIS-Elements software was used in this study. 1.3AnimalsA total of48male SPF (Specific pathogen Free) Wistar rats(weight range,180g to200g) were used for all the esperiments. the animals were provided by the Animal Experiment Center of Southern Medical University. The study was approved by the animal research ethics committee of the nanfang hospital, Southern Medical University. All animals were housed singly in standard cages. The environment was temperature and humidity controlled, with lights on and off at6:30AM and6:30PM.4Methods4.1Preparation of drag-reducing polymersPrecisely weigh and take10mg PEO, add10ml physiological saline, and mix into a solution with the concentration of1000ppm. Pour the prepared PEO solution into cellulose membrane tubing with the intercepted molecular weight of40000Da, and dialyze for24hours in physiological saline. Dilute the1000ppm solution after hemodialysis into the solutions respectively with the concentrations of10ppm and50ppm with a right amount of physiological saline, and then, place them into a refrigerator at4℃for alternate use.4.2Preparation of animal modelIn the research on sepsis, people usually establish a model of animals with sepsis by injecting a lethal dose of escherichia coli, endotoxin lipopolysaccharide(LPS) and CLP, etc. The experimental infection model is established by adopting the cecal ligation and puncture(CLP), and the CLP model is the most commonly used model for the study of infection and related diseases, and the gold standard for the experimental model of sepsis, which enables to compare the realistic simulation of the pathophysiological process of infection). This model adopted the cecal ligation and puncture, transferring intestinal contents and bacterial flora into blood and causing the organism to form inflammatory reactions. Methods: 180-200g male rat is anesthetized with an intraperitoneal injection of3%pentobarbital sodium for aseptic operation,40mg/kg, after the fixation, skin preparation and disinfection. Locate the cap end from a2cm lower abdominal median incision, and ligate at1/3of the middle-lower part of caecum(see Fig.l), avoiding from vascular injury. Penetrate with a14-gauge needle, and squeeze out a few intestinal contents, ensuring a through hole.(see Fig.2) Never tear mesenterium or blood vessel. Push back intestinal canal, and keep it from any obstruction caused by intestinal twist; and then, disinfect and suture the incision. Push back and observe after the preoperational resuscitation, and if any symptom of septic shock, it is recommended to do the experiment. The surgical procedures and operation time of each animal should be consistent as much as possible, so as to ensure the consistency of the model in infection gradient during the experiments. In the current study, the rats after CLP gradually produce symptoms of depression, dysphoria, pilo-erection, diarrhea, etc.; after the animals are sacrificed, their abdominal parts are dissected, with fetid bloody or purulent exudate inside abdominal cavity, intestinal canal flatulence and viscera losing its original color because of congestion and edema; and, it’s shown that the animal model is successfully reproduced.4.3Surgical preparation of laboratory animalThe animals exhibited signs of shock14hours after CLP operation. The animals were then anaesthetized with pentobarbital sodium (40mg/kg) by intraperitoneal injection. polyethylene catheters were implanted surgically into the femoral artery and femoral vein. The femoral artery catheter was used for monitor the mean arterial blood pressure and collect blood samples. And the femoral venous catheter was used for fluid infusion. The skin on the animals back was exposed. The Spinotrapezius muscle of the rat was isolated and placed under the axio microscope which is connected with the high-speed comera and the HD camera, the blood flow of the microcirculation was observed. The isolated Spinotrapezius muscle was treated with a continuous drip of equilibrium liquid ensure its surface was continuously moistened and wormly. The local microcirculation was photographed during the resuscitation.4.4Model grouping and data acquisitionThe models were randomized into four groups:The PEO1group or PEO2group was continuously infused with physiologic saline+10ppm polyethylene oxide respectively with physiologic saline+50ppm polyethylene oxide; the NS group was continuously infused with pure physiologic saline; The resuscitation was lasted for4hours, with an infusion rate of15ml/kg/h, and the NT group was not treated with fluid resuscitation. At the end of the resuscitation, the wound was sutured, and the survival time was observed. The microcirculation is recorded before resuscitation (TO) and at10,30,60,120, and240min after the beginning of resuscitation (T10, T30, T60, T120, and T240). blood samples were collected at the time before resuscitation、 at the end of resuscitation and72hours after the end of resuscitation for measurement of the lactic acid concentration. For serum preservation, after the plasma was allowed to coagulate, the blood was centrifuge for10min at3,000rpm. The supernatant liquid was collected and stored in a refrigerator at-80℃.4.4Statistical treatmentAll the data adopted SPSS13.0software for statistical analysis, and all measurement data is expressed as x±s; and, the intragroup pre-and-post comparison adopted the Repeated Measures analysis, while the intergroup comparison adopted the One-Way ANOVA measure analysis of variance(P<0.05, with significant difference). The preparation of charts and graphs adopted SPSS13.0and office2007.Results1Effects on heart rate and blood pressure Except that the NT group has blood pressure dropped, and the blood pressure at the end of resuscitation is lower than those of the rest(p<0.05), however, there is no significant difference between the rest three groups in blood pressure or heart rate.(p>0.05)2Effects on diameter and red cell velocity of small artery in spinotrapezius muscle of ratsThere is no Significant difference between the four groups in the diameter of blood vessel before and after the resuscitation, and the measured diameter of blood vessel is37.20±7.75(μm). The RBC flow velocity of the PEO1group after the administration is quickly increased, and reached the peak value (2228.9±292.7vs3115.7±376.0p<0.001) within10minutes, and the continuously sustained delivery could maintain stable, and although the velocity dropped, it is still insignificant, and the velocity during the resuscitation is obviously faster than that before the resuscitation (2228.9±292.9vs2759.7±215.7p=0.001). The RBC flow velocity of the PEO2group after the administration is significantly increased as well, and then it quickly dropped to the original level before the resuscitation, instead of being maintained. Both the RBC flow velocity of the NS group and that of the NT group are slowed, however, there is no significant difference (p>0.05). In the intergroup comparison, the RBC flow velocities of the PEO1and PEO2groups at the10th minute of the administration are respectively faster than those of the NS and NT groups (p<0.05); and, the velocity of the PEO2group after the administration for60minutes dropped to the level of the NS/NT group (p>0.05), while that of the PEO1group remained faster until the end of resuscitation. There is no significant difference between the NS group in the RBC flow velocity at different time point. The velocity of RBC of The NT group decreased to a lower level than that of T0(p<0.05).3Changes in rat plasma IL-6, TNF-a and lactic acid concentration and lactate clearance rate in the4th hour of resuscitation.Except that there is no noticeable change in the lactic acid concentration of the NT group before and after the resuscitation, the lactic acid concentrations of the rest three groups after the resuscitation are significantly lower than those before the resuscitation(p<0.01); and, in the intergroup comparison, the lactic acid concentrations of the PEO1, PEO2and NS groups after the resuscitation are significantly lower than that of the NT group(p<0.05), and their lactate clearance rates are also significantly higher than that of the NT group)(p<0.05). The lactic acid concentration of the PEO1group after the resuscitation is slightly lower than those of the NS and PEO2groups, without statistical significance, however, the lactate clearance rate of the group is obviously higher than those of the NS and PEO2groups(p<0.05). In the intergroup or intragroup comparison, there is no noticeable change in plasma IL-6or TNF-α concentration (p>0.05)4Changes in the survival time of ratsEach group has12rats enrolled for experimental purpose, and the PEO1group has5rats survived after72hours, with the survival rate of41.7%. The NS group only has2rats survived after72hours, with the survival rate of16.7%. The PEO2group only has one rat survived, with the survival rate of8.3%. However, none of the NT group could survive over48hours. The average survival time of the PEO1group(53±5.7hours) is significantly longer than that of the NT group(26±4.1hours) and that of the NS group(34±5.8hours). The survival time of the PEO2group is37±4.8hours, with no significant difference from that of the NS group.Conclusions1. A low-dose of polyethylene oxide (10ppm) can accelerate the removal of blood lactate, reduce lactate concentration, and improve the prognosis and survival time of septic shock rats. 2. A low-dose of polyethylene oxide (lOppm) can significantly improve the red cell velocity of small artery in spinotrapezius muscle of septic shock rats while it have no effect on diametes.3. A low-dose polyethylene oxide (lOppm) have no effect on blood pressure and heart rate under this experimental conditions. |
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