| Severe sepsis and septic shock are vital clinical issues in critical care settings.The incidence of sepsis is on the rise due to ageing populations,invasive medical procedures and the rising incidence of cancer annually.Of millions of new cases of sepsis worldwide each year,more than a quarter of them die eventually,despite tremendous efforts by clinicians.Although the mortality rate of sepsis has declined,the number of patients who die from severe sepsis and septic shock is increasing.The cost of sepsis treatment in the United States exceeds billions of dollars annually,which is still rising at a rate of 10 % a year.Successful management of such patients in intensive care unit(ICU)is based on the following three aspects: timely and reasonable application of antibiotics,hemodynamic support,and infection source control.Fluid resuscitation is a key hemodynamic support in the early stage of sepsis,however,there has been no international concensus concerning the optimal kind of fluid,including the appropriate amount.While rapid optimization of intravascular volume has been shown to improve the prognosis,a number of studies have reported that fluid overload often occurs following an early goal directed therapy(EGDT)in the management of severe sepsis and septic shock,which results in poor clinical prognosis.Therefore,more work is needed to investigate whether it is critical to administer timely and appropriate fluid resuscitation in terms of sepsis treatment.In recent years,critical attention has focused on the function of vascular endothelium and the glycocalyx as endothelial barrier has been studied gradually.As is well known,the luminal surface(the thickness of the glycocalyx was 0.2-2?m)of the endothelial cells that line the vasculature is coated with a glycocalyx of membrane-bound macromolecules comprised of glycoproteins,proteoglycans,glycosaminoglycan and plasma proteins.In 1966,Luft firstly obtained an electron microscope photograph of the glycocalyx.Glycocalyx plays a key role in maintaining endothelial permeability,preventing tissue edema,regulating leukocyte migration and inhibiting intravascular coagulation.In 2004,Adamson et al.suggested that the colloidal osmotic pressure between glycocalyx and the vascular endothelium was very low,which chanllenged the classical Starling mechanism.It has been long believed that there was no gradient of osmotic pressure across the blood vessels.Instead,the colloidal osmotic pressure gradient formed by both the inner and outer sides of the glycocalyx resists intravascular hydrostatic pressure.The glycocalyx plays an important role in various physiological conditions,which,however,is extremely vulnerable to physiological and pathophysiological changes,such as ischemia reperfusion injury,hypervolemia and inflammation.Specifically in case of sepsis which leads to damage to glycocalyx,fluid resuscitation is beneficial at early stages of sepsis.It is technically challenging and impractical to measure the thickness of glycocalyx directly.Syndecan-1 is an indirect marker of the degradation of glycocalyx,and its plasma levels can be easily measured,so these markers are often reused in numerous studies.The plasma concentrations of syndecan-1 and heparan sulfatate were proportionate to the degree of damage to the glycocalyx.Sepsis and severe coagulopathy caused by dysfunction of endothelial cells are associated with vascular response disorders and tissue edema.It has been found that damaged glycocalyx is central to organ failure during sepsis.In the present study,the alterations of endothelial glycocalyx in early septic shock rabbits induced by different pathogenicfactors were observed on the basis of clinical indexes,biochemical indexes,routine pathology indexes,immunohistochemistry and Western blot.All the five parts of studies were aimed to investigate the role of endothelial glycocalyx in fluid resuscitation in early septic shock,to pinpoint the mechanism of fluid resuscitation which affects septic shock,to explore the protection and function of glycocalyx in clinical treatment and to provide a novel approach for clinical understanding of liquid distribution and fluid therapy of septic shock.The study is composed of five parts.Part ? Rabbit Septic Shock Model Induced by LPS and Cecal Ligation and PunctureObjective: To investigate the approahces to establisha rabbit model of septic shock.Methods: Twenty-four New Zealand male rabbits were randomly divided into cecal ligation and puncture(CLP)group(model group1;n=6),lipopolysaccharide(LPS)group(model group 2;n=6),CLP control group(n=6),and LPS control group(n=6).After establishment of rabbit model of LPS-or CLP-induced septic shock using CLP or LPS methods,blood samples were collected to compare the clinical indicators of each group.The rabbits were sacrificed,and the heart,lung,liver and kidney were harvested and fixed in 4 % polyformaldehyde solution for HE staining.Results: The modeling time of CLP group and LPS group was significantly different(P<0.001).There were no statistical differencesin white blood cell count between CLP group and LPS group(t=2.215,P>0.05),as well as between CLP control group and LPS control group(t=0.900,P>0.05).However,significant differences were found between model group and control group.There was no statistical difference in the determination of lactic acid between CLP group and LPS group(t=1.218,P>0.05),as well as between CLP control group and LPS control group(t=0.560,P>0.05).Nevertheless,no significant differences were noted between model group and control group.In addition,model group and control group showed significant differences in central venous blood oxygen saturation(ScvO2)results(t=3.539,P<0.05),as well as between model group and control group(t=-3.700,P<0.05).There was no significant differences between the two control groups(t=-2.023,P>0.05).Escherichia coli and a small number of gram-positive cocci were detected in blood culture of CLP and LPS group,,which,however,were negative in the control group.HE staining revealed more severe damage to organs and tissues in CLP group.Summary:1.Septic shock model can be successfully eastablished using both CLP method and LPS method,and the latter is easier to implement.2.It is suitable to monitor blood pressure in the middle of the rabbit ears in physiological state,other than in septic shock.3.LPS causes severe inflammatory response in animals in a short period of time,which may be very different from the actual physiological changes in clinical settings.CLP,by contrast,is closer to the condition in clinical practice.Part ? Effects of different doses of resusciation fluid on endothelial glycocalyx in early septic shockObjective: To investigate the effects of a combination of norepinephrine and different doses of resuscitation fluid injected intravenously on endothelial glycocalyx of rabbits with early septic shock.Methods: Twenty-four New Zealand male rabbits were randomly divided into control group(n=6),sepsis group(n=6),sepsis+10 ml treatment group(10 ml group;n=6),and sepsis+30 ml treatment group(30 ml group;n=6).After establishment of rabbit model of LPS-induced septic shock,we compared the effect of a combination of norepinephrine with different doses of resuscitation fluid administered intravenously on lactic acid,ScvO2,and atrial natriuretic peptide(ANP).In addition,the concentrations of plasma syndecan-1 were compared after glycocalyx was damaged.Expressions of related proteins of various organs and tissues were measured by ELISA and those of related antibodies were detected by immunohistochemistry method.Results:1.The mean levels of lactic acid were lower in both 10 ml and 30 ml group after resuscitation(P<0.05),and the decrease in the mean value was greater in 10 ml group(3.29)than in 30 ml group(1.36).ScvO2 results showed that after resuscitation,the increase in the mean value was smaller in 10 ml group(10.52)than in 30 ml group(18.79).2.Concentration of ANP was the lowest in the control group,followed by LPS group,10 ml group and 30 ml group.Plasma syndecan-1 concentration was the lowest in control group,followed by 10 ml group,30 ml group and LPS group.3.Immunohistochemical results showed that the expression of syndencan-1 in renal tissues was decreased in the order of control group,10 ml group,30 ml group and LPS group.The expressions of syndecan-1 and angiotensin-1 in renal tissues was decreased in the order of control group,10 ml group,30 ml group and LPS group.The expressions of angiotensin-2,matrix metalloproteinase-2,7 and intercellular adhesion molecule-1 was increased in 10 ml group,followed by 30 ml group and LPS group.Univariate analysis demonstrated significant differences in indicators among the four groups(P<0.05),and between-group differences were found(P<0.05).In addition,mean values of angpt-1 were decreased gradually in both control group and LPS group,whereas the other indicators were increased.Summary:1.Appropriate and timely resuscitation in combination with norepinephrine exerted a protective effect on endothelial glycocalyx.2 The concentrations of ANP and syndecan-1 were remarkably increased due to 30ml/kg resuscitation fluid as opposed to 10ml/kg.3.The expression of angpt2 was increased,whereas that of angpt1 was reduced after 30ml/kg resuscitation,thereby increasing vascular instability and permeability.4.As compared with 10ml/kg resuscitation fluid,30ml/kg fluid contributed to activation of MMP-2,7,which accelerated the destruction of the glycocalyx,thus affecting the integrity of the glycocalyx.5.Excessive fluid(30ml/kg as opposed to 10ml/kg)aggravated glycocalyx damagePart ? The changes in endothelial glycocalyx in rabbits with septic shock induced by different pathogenic factorsObjective: To observe the alterations of endothelial glycocalyx in early septic shock rabbits induced by different pathogenic factors.Methods: Thirty-six New Zealand male rabbits were randomly divided into CLP group(n=6),CLP control group(n=6),CLP 30 ml treatment group(n=6),LPS group(n=6),LPS control group(n=6)and LPS 30 ml treatment group(n=6)according to a random number table.After establishment of rabbit model of LPS and CLP induced septic shock,they were managed accordingly and the clinical indicators and time for model establishment of each group were recorded and compared.Concentrations of syndecan-1 in plasma were measured by ELISA at different time points early after septic model was established.After six hours,the rabbits were sacrificed,and various organs and tissues were harvested.Western blot and immunohistochemistry were used to detect the expression of endothelial glycocalyx.Results: Results of plasma markers revealed that the concentration of plasma syndecan-1 was significantly higher in CLP group than in LPS group.HE staining revealed less renal damage in LPS group than in CLP group.Immunohistochemical results showed that the expression of syndencan-1 which was darker was higher in LPS group than in CLP group.Western blot results indicated that the expressions of syndecan-1 in renal tissues in LPS groups were higher than that in CLP groups.Summary:1.The endothelial glycocalyx was damaged by both LPS and CLP.2.LPS causes less damage to endothelial glycocalyx than CLP.3.After fluid resuscitation,endothelial glycocalyx will be more likely to be partially restored in LPS group in a short term.Part ? Effects of timely and delayed resuscitation on endothelial glycocalyx in rabbits with early septic shockObjective: To observe the alterations of endothelial glycocalyx in early septic shock rabbits treated with different doses and timing of resuscitation fluid combined with norepinephrine,and to explore the effects of different doses and timing of resuscitation fluid on pulmonary microcirculation.Methods: Thirty New Zealand male rabbits were randomly divided into control group(n=6),30 ml group(timely group;n=6),60 ml group(timely group;n=60),CLP group(n=6),and sepsis delayed treatment group(delayed group;n=6).After establishment of rabbit model of CLP-induced septic shock,we compared the effect of a combination of norepinephrine with different doses and timing of resuscitation fluid administered intravenously on lactic acid,ScvO2,and oxygenation index.Concentrations of plasma syndecan-1were measured by ELISA.Western blot and immunization group were used to detect the expression of related proteins in lung tissues.Results:1.Results of lactic acid,ScvO2 and oxygenation index after resuscitation at different time points and doses: In the timely resuscitation group and delayed resuscitation group,the mean ScvO2 values of lactic acid changed after the resuscitation,as compared with those before resuscitation(P < 0.05).After resuscitation,the mean values of lactic acid and ScvO2 in delayed group were higher than those in the timely group.The oxygenation index in the three groups(timely,delayed,and 60 ml groups)was not statistically different before resuscitation(P>0.05)which,however,differed significantly in these three groups after resuscitation(P < 0.0001).In addition,oxygenation index was improved in both timely and delayed groups,other than in 60 ml group.2.Results of plasma markers revealed that plasma syndecan-1 concentration was the lowest in the control group,followed by 30 ml group,delayed group,sepsis group and 60 ml group.The mean value of each group was increased in the order of 0h,3h and 6h in a time-dependent manner(all P<0.05).3.Immunohistochemical results showed that the expression of syndencan-1 in lung tissues was decreased in the order of control group,30 ml group,delayed group,CLP group and 60 ml group.4.After fluid resuscitation at different doses,Western blot results showed that there was a statistical difference in syndecan-1 index and ICAM-1 index between any two groups(P < 0.05).No significant differences were found in MMP-2 index between delayed group and CLP group(P>0.05),but MMP-2 index differed significantly between each other two groups(P<0.05).Summary:1.Compared with the timely group,the expressions of MMP-2,angpt2 and ICAM-1 were significantly higher in the tissues of delayed group,resulting in significant degradation of glycocalyx and destruction of endothelial barrier function.2.The expressions of MMP-2,angpt2 and ICAM-1 in 60 ml group were significantly higher.Excessive liquid resuscitation had a greater effect on the glycocalyx than delayed resuscitation.3.Appropirate liquid resuscitation combined with norepinephrine which keeps systemic circulation stable could protect the glycocalyx and reduce its shedding,which was beneficial for patients with septic shock.Part ? Changes in endothelial glycocalyx in severe sepsis and its clinical significanceObjective: To determine the relationship between fluid therapy and changes in glycocalyx in severe sepsis,and to explore the value of glycocalyx as a parktaker of microcirculation disturbances in predicting the prognosis of sepsisMethods: A total of 15 adult patients who were admitted to ICU due to severe sepsis after thoracotomy were enrolled in this study as the experimental group.Eleven patients who had recovered after thoracotomy were selected as a control group.Patients in the experimental group were given adequate fluid resuscitation and follow-up organ support treatment according to the 2012 Sepsis Guidelines.In the sepsis group,blood was collectedat baseline(upon ICU admission)and at 3,6,24,and 48 h after ICU admission.In the meantime,blood samples were also drawn from these 11 patients in the control group.Syndecan-1 concentrations were determined at each of the above time points;blood lactate levels were analyzed at the baseline and at 6h after admission to determine lactate clearance.Fluid balance at 3h,6h,24 h,and 48 h,Acute Physiology and Chronic Health Evaluation(APACHE)II score,Sequential Organ Failure Assessment(SOFA)score,6h lactate clearance and duration of mechanical ventilation,length of ICU stay,and ICU survival were recordedin the experimental group.Results: Plasma syndecan-1 concentrations were significantly higher in experimental group than in control group,and were correlated with fluid balance(at 3h,6h,24 h and 48h)in experimental group in a time-dependent manner(P< 0.05,r=0.974).Prognosis was not related to trends in plasma syndecan-1 concentrations(ascending/descending)in experimental group(=0.682,P>0.05).In terms of prognosis,fluid balance at 24 h was significantly different(Z=-2.481,P<0.05),whereas no significant differences were found at other time points.Changes in plasma syndecan-1 concentrations were not correlated with duration of mechanical ventilation,and length of ICU stay,SOFA scores and APACHE.As compared with plasma syndecan-1 concentrations,lactic acid clearance had a high diagnositic value in predicting patient outcome(AUC=0.591),with the optimal cut-off point of 0.40% and the correct index of 0.5.Summary:1.Vascular endothelial glycocalyx plays a pivotal role in the pathogenesis of severe sepsis.The plasma concentration of syndecan-1,a plasma marker,represents the severity of the patient's condition.The higher syndecan-1 concentration,the more severe the damaged glycocalyx is,and the more fluid required in fluid resuscitation.The fluid balance at 24 h after admission to the ICU is closely related to the survival rate.2.Despite a certain understanding of the structure and function changes of endothelial glycocalyx in inflammation,the specific changes and mechanisms of its action need further exploration.3.In spite of a significant increase in plasma syndecan-1 concentration in patients with severe sepsis,lactate clearance has a greater diagnostic value in predicting patient outcome..4.Future work is needed to explore how to inhibit or reduce glycocalyx shedding and how to regenerate it by manual intervention to fight the inflammatory responses,thereby protecting the normal structure and function of blood vessels.Conclusions:1.Both intravenous LPS method and CLP method can be used to establish septic shock model successfully.2.As compared with the LPS model,CLP brings about more serious damage to the endothelial glycocalyxl.3.The Appropriate fluid resuscitation is very important in septic shock treatment,which could protect the endothelial glycocalyx,whereasinappropriate fluid resuscitation will aggravate the damage to glycocalyx,triggering a vicious circle.4.The fluid resuscitation of septic shock is time-limited,and liquid resuscitation performed at the wrong time is detrimental to the patient,and aggravate the damage to the glycocalyx.5.In the treatment of sepsis and septic shock,positive protection of endothelial glycocalyx may improve the prognosis of such patients. |
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