Research Of Blood Purification Technology In The Treatment Of Sepsis From Basic To Clinical

Sepsis is one of the main causes of death in critically ill patients. The pathophysiology of sepsis is complex and not completely understood till now. The pro-inflammatory and anti-inflammatory response leads to cell and organ dysfunction and, in severe cases, death. Thus, the goal of the intervention is to restore the homeostasis of circulating mediators rather than to inhibit selectively the pro-inflammatory or anti-inflammatory mediators. The blood purification has been reported to remove a wide array of inflammatory mediators. The effects are broad-spectrum and auto-regulating. It is also demonstrated to restore immune function through improving antigen-presenting capability, adjusting leukocyte recruitment, oxidative burst and phagocytosis, and improving leukocyte responsiveness. A great deal of work has to be done in order to find and optimize the best extracorporeal blood purification therapy for sepsis. New devices specifically target the pathophysiological mechanisms involved in these conditions. High-volume hemofiltration, hemoadsorption, coupled plasma filtration adsorption, high cut-off membrane are now being trialed in septic patients. Preliminary data indicate the feasibility of these modified techniques in sepsis. Their impact on patient prognosis, however, still needs proof by large randomized clinical trials. Finally, the emerging paradigm of sepsis-induced immune suppression provides additional rationale for the development of extracorporeal blood purification therapy for sepsis.To investigate the effect of blood purification on sepsis caused by abdominal infection, and to find the best blood purification methods and optimize treatment strategies, Our study was divided into the following sections:(1) establish a rat model of sepsis;(2) development of venovenous extracorporeal blood purification circuits in rodents for sepsis and Safety assessment;(3) Effects of hemoadsorption with a novel adsorbent in the treatment of sepsis:in vivo and in vitro study (4) Effect of high adsorption homofiltrationi in treatment of septic rats;(5) clinical studies of high-volume hemofiltration in treatment of sepsis. CRISMA Laboratory, University of Pittsburgh provided us a full range of technical support.The First Part Establishment of sepsis model of ratObjective:In order to access the effect of blood purification treatment in sepsis, we explored the approach to setup stable rat sepsis model of different severity for different blood purification therapy research purposes.Methods:Adult male Sprague-Dawley rats(n=30) were randomly divided into three groups(n=10each group). Sepsis in the experimental groups was induced by CLP, whereas the control group underwent a sham operation neither ligated nor punctured. In CLP group1(CLP1), the cecum was ligated at the position of1/4, then punctured twice with a21-gauge needle and a small amount of the bowel contents was extruded through the puncture holes; in the CLP group2(CLP2), cecum was ligated at the position of1/3, puncture3times. Survival time of rats will be observed in7days, the blood was dawnd at48hours after surgery to detect blood lactate and glucose levels.The pathological changes of kidney and liver would be observed.Results:All rats of control group survived in7day,50%rats survived in CLP1Group, only10%rats survived in CLP2group. The blood lactate level increased, blood glucose decreased at48hours after CLP. Liver histology slice after CLP showed swelling of hepatocytes with focal piecemeal necrosis. Kidney histology after CLP showed significant vacuolization in tubulesConclusion:By controlling the ligation length and puncture numbers of cecum we can create different severity of sepsis model induced by abdominal infection, it was silmilar with the pathophysiology characters of human sepsis, which can cause tissue hypoperfusion, metabolic disorders and organ damage. The Second Part Development of venovenous extracorporeal blood purification circuits in rodents for sepsis and safety assessmentBackground/Aim:Unlike pharmacologic interventions in sepsis, extracorporeal blood purification, which is widely used in septic patients, is not typically studied in experimental rodents. Most of the previous studies have performed extracorporeal blood purification in larger animals and typically use arterio-venous (AV) vascular access. In order to explore the possibility of blood purification technology applied in a rat model of sepsis, we developed a veno-venous (VV) purification model in the rat as an adjunct for the treatment of sepsis.Methods:Using adult male Sprague-Dawley rats, we cannulated the femoral artery or vein and the jugular vein with P50tubing and created an AV or VV circuit. Blood flow was maintained by arterial pressure in the AV circuit, whereas in the VV circuit the blood flow was regulated using a rotary pump. The safety of this circuit was evaluated using the changes of blood interleukin6, rectal temperature, and7-d survival with sham extracorporeal circulation (circuit connection without treatment) compared with the control (without circuit). The main side complications of this VV circuit were compared with those of the AV circuit.Results:The differences in interleukin6, body temperature, and cumulative survival were not statistically significant after extracorporeal circulation. The main complications of extracorporeal circulation occurred less often with VV compared with AV therapy:massive bleeding (2.5%versus15%, P=0.04); clot formation (2.5% versus15%, P=0.04). This VV circuit has been successfully used in different septic rodent models with different techniques (such as hemoadsorption and hemofiltration).Conclusions:VV blood purification in a rodent model appears to be effective and is safer than AV circuit. The Third Part Effects of hemoadsorption with a novel adsorbent on sepsis: in vivo and in vitro studyBackground/Aims:Sepsis is characterized by an overproduction of cytokines in the blood and tissues. Hemoadsorption may attenuate the dysfunctional inflammatory response and improve outcomes by removing cytokines from the circulating blood. We hypothesized that CTR, a new adsorbent, can remove cytokines and improve organ function.Methods:The sterile CTR sorbent beads were filled into columns of three sizes: small (0.5ml), medium (1.0ml) and large (2.0ml). Each size of column was tested using IL-6capture in vitro. In vivo, rats were subjected to cecal ligation and puncture (CLP) and18hours later were randomly assigned to receive treatment with either0.5ml, lml, or2.0ml of CTR beads (CTR0.5, CTR1and CTR2respectively) or sham treatment (n=10each) for four hours. Blood was drawn at18hours (hrs) after CLP, at0hrs after the4hr treatment, and then again at24hrs and48hrs after treatment. Plasma cytokines (TNF-a, IL-1β, IL-6, and IL-10), high mobility group boxl (HMGB-1), alanine aminotransferase (ALT), creatinine and cystatin C were measured. Survival time was recorded.Results:In vitro study showed that IL-6removal was accelerated with increasing bead mass. IL-6capture was faster than TNF-a capture. The mortality rates at7days after CLP were50%,63.64%,62.5%, and72.72%for the sham, CTR0.5, CTR1, CTR2respectively. At later time points (24hrs and48hrs) after intervention, the cytokine concentrations (TNF-α, IL-1β, IL-6, and IL-10) were significantly lower in the CTR0.5and CTR2bead treatment groups (p<0.05). CTR2groups showed a significant decrease in HMGB-1after two days (p<0.05). There were no statistically significant differences reached on ALT and creatinine, but the results show strong evidence for late renal protection. Additionally, cystatin C levels after24hr of treatments in CTR1and CTR2groups were significantly lower compared to the sham treatments.Conclusion:CTR appeared to have a favorable effect on survival despite no immediate effects on cytokine removal. However, CTR, especially CTR2beads, did result in a late decrease cytokines and HMGB-1. CTR1andCTR2beads also significantly reduced kidney injury at24hrs after treatments. Further study is needed. The Fouth Part Effect of high adsorption homofiltrationi in treatment of septic ratsBackground/Aim:The extracorporeal blood purification is proposed as immunomodulatory adjuvant septic shock. Recent advances in the field of renal replacement therapy membranes have allowed the development of new techniques. These therapies are very low compared with one another in the medical literature. The objective of this study was to compare the mortality of septic shock in rats after high adsorption hemofiltration versus after hemoperfusion.Materials and Methods:36rats were subjected to cecal ligation and puncture (CLP) Eighteen hours after induction of sepsis, they were randomized into three groups to receive a session4h high adsorption hemofiltration (adsorbent membrane hemofiltration endotoxins (Oxiris(?), Gambro)), or a session of4hours hemoperfusion (cartridge bearings adsorbent cytokines (CytoSorb(?), CytoSorbents)), or a session of4hours extracorporeal circulation without blood purification element (control group). The primary endpoint was survival to7days (log-rank test). Furthermore, assays of cytokines (TNF, GMCSF, IFN-y, IL-la, IL-1p, IL-2, IL-4, IL-6, IL-10, IL-12) on blood samples were carried out at the beginning,2hours and4hours after each session.Results:Twelve rats were included in the hemofiltration group,11in the hemoperfusion group and11in the control group (2rats died before randomization). Overall survival at day7was66%in group high adsorption hemofiltration (8/12),45%in the hemoperfusion group (5/11) and18%in the control group (2/11). Mortality was significantly reduced in the high adsorption hemofiltration group compared to control group (P=0.011). All cytokine assays showed no significant difference between the3groups.Conclusion:In this animal model of septic shock, high adsorption hemofitrtion seemed improve survival. But there were no significant difference in all cytokine level before and after treatment, no difference between the3group. The Fifth Part5.1Effect of Continuous high volume hemofiltration on patients with severe acute respiratory distress syndromeOBJECTIVE:To investigate the effect of continuous high-volume hemofiltration (HVHF) on patients with severe acute respiratory distress syndrome.METHODS:Sixty-five patients with severe ARDS from2007to2012were divided into control group (28patients) and HVHF treatment group (37patients). The patients in HVHF treatment group were treated with HVHF (45ml/kg/h) and the other normal treatment.28patients of control group was received the normal treatment except hemofiltration therapy. We compared the oxygenation index (PaO2/FiO2). extravascular lung water index (EVLWI), PaCO2, heart rate, mean arterial pressure between control group and treatment group before and after treatment. The duration of mechanical ventilation,ICU stay time and percentage of withdraw from ventilation also were compared.RESULTS:The oxygenation index (PaO2/FiO2),extravascular lung water index (EVLWI), PaCO2,we compared,were improved after the treatment in both the HVHF treatment group and the control group(p<0.05), but improvement in the HVHF treatment group is more obvious than control group(p<0.05). The heart rate and mean arterial pressure were improved after the treatment in both groups(p<0.05), but no obvious difference compared between the treatment group to the control group (p>0.05). The duration of mechanical ventilation,ICU stay time and percentage of withdraw from ventilation were showed obvious improvement in the HVHF treatment group(p<0.05).Conclusion:HVHF is an effective assistant treatment for severe ARDS. It can improve the lung function,shorten the duration of mechanical ventilation, percentage of withdraw from ventilation and the survival rate, but no obvious hemodynamics influence to patients.5.2Study the effects of high-volume hemofiltration on acute lung injury and acute kidney injury patients induced by sepsisObjective:To evaluate the effects of high-volume hemofiltration(HVHF) on the organ function in patients with acute lung injury(ALI) and acute kidney injury(AKI).Methods:108cases septic patients with ALI and AKI underwent HVHF were enrolled our Department between August2007and December2011. All the patients were randomly divided in two groups(routine treatment, group A,68cases) and (high-volume hemofiltration, group B,40cases).The artery lactate level were checked by arterial blood gas analysis(ABGA) at first and at72hour in two groups; serum high sensitivity reactive protein(hs-CRP) was detected by immunity-cross muddy method on admission and comparison before treatment and after72hour in two groups; Alveolar-arterial oxygen pressure difference P(A-a)DO2and oxygenation index(OI) was checked. The serum Cystatin C (Cyst c) and serum creatinine clearance rate (CCR) levels was checked by at first and after72hour in two groups. Data were expressed as mean±standard deviation and the analysis of variance was done with Spss12.0software. Two group average comparison was conducted with t-test and the ratio by chi-square test. The change were considered as statistically significant if P value was less than0.05.Results:(①The difference of artery lactate、hs-CRP in two groups[2.7±1.5vs1.7±0.7(mmol/L), P<0.05and99.5±20.4vs35.8±18.8(mg/L), P<0.05] in two groups after72hours was significant (P<0.01);②The levels of P(A-a)DO2in HVHF group were reduced more significantly than routine treatment [155.4±27.4vs115.5±23.1(mmHg), P<0.05], but the level of OI was increased [99.5±20.4vs295.2±38.8,P<0.01];③The Cystatin C (Cyst C) level in B groups was reduced gradually after72hour treatment [3.95±2.06vs2.06±1.12(mg/L), P<0.05], but the creatinine clearance rate (CCR) at72th hour in group B were higher than group A[90.21±30.35vs108.71±31.33, P<0.01].Conclusions:HVHF could improve the organ function in patients with ALI and AKI, at the same time take precaution multiple organ dysfunction syndrome(MODS), so it could improve the survival rate of patients. Our study from basic to clinical showed that blood purification technology is a feasible approach to the treatment of sepsis.(1) Sepsis model in rats were established by CLP, which was the most commonly sepsis model. By controlling the ligation length and puncture numbers of cecum we established two kind of different severity of sepsis model, it was silmilar with the pathophysiology characters of human sepsis, which can cause tissue hypoperfusion, metabolic disorders and organ damage.(2) We developed the VV extracorporeal blood purification in a rodent model IT appears to be effective and is safer than AV circuit.(3) CTR appeared to have a favorable effect on survival despite no immediate effects on cytokine removal. However, CTR, especially CTR2beads, did result in a late decrease cytokines and HMGB-1. CTR1and CTR2beads also significantly reduced kidney injury at24hrs after treatments. Further study is needed.(4) In this animal model of septic shock, high adsorption hemofitrtion seemed improve survival. But there were no significant difference in all cytokine level before and a fter treatment, no difference between the3group.(5) In clinical research, we observed the effect of high volume hemofiltration in treatment of sepsis, the results showed that the HVHF technology can improve organ function and improve the prognosis.The research provides much useful iinformation about blood purification technique for the treatment of sepsis and can help us to manage the treatment of blood purification.Various new techniques and new strategy still need large-scale randomized controlled clinical trials to test, the pathogenesis of sepsis also need further study.