| Part 1Pneumonia After Cecal Ligation and Puncture:A Clinically Relevant "two-hit" Model of sepsisBackgroundSepsis is systemic inflammatory response syndrome (SIRS) originated from infection. The study of sepsis is the hotspot of research because of its high morbility and mortality. Studies of patients and animal models have revealed that changes in the immune reponse during sepsis play a decisive role in the outcome. Sepsis has long been recognized that the early phase of sepsis caused by LPS attact is dominated by a hyperinflammatory state mediated by systemic production of inflammatory cytokines, and induced multiple organ failure. In most current animal models of sepsis, the hyperinflammatory " cytokine storm" may lead to deaths. In contrast, most patients with sepsis may eradicate their primary infection only to develop and succumb to secondary infection, and have a protracted hospital course, condition aggravation and the modest increase of mortality. It may be associated with immunosuppression caused by compensatory anti-inflammatory response syndrome (CARS).ObjectiveThis study was designed to construct a rat sepsis model and investigate the change of immune reponse in different stage of sepsis for the cognition of immunoparalysis timing. In addition, we modulate a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the pneumonia, and to explore the correlation between the secondary damage and immune state.MethodsA total of 36 rats weighted of 250g were enrolled in this study. Thirty rats were subjected to cecal ligation and puncture; six rats were enrolled in normal-control group. Randomly select 12 rats survived from CLP, and inject intranasally 200μ 1 of Streptococcus pneumonia suspension (1 X 1010 CFU/ml) into these rats in a "heads-up" position at 4 or 7days post-CLP. Observe the living condition and survival rate of each kind of models, and collect the blood and tissue samples of each rat including normal-group, 1、4、7days post-CLP and 24h after infusion of S. pneumonia.Detection included the examination of blood routin, bacterial count, hepatic and renal function. Use flow cytometry (FCM) to test the percentage of dendritic cells, CD4、CD8 T-cell and T-regulatory cells. The serum levels of pro- and anti-inflammatory cytokines were detected by ELISA. Use HE staining to evaluate the damage of rat spleen, and the apoptosis of spleen cells were detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining.ResultsCecal ligation and puncture alone resulted in 73.33% survival at 14 days, and the group of secondary infection at 4 days post-CLP resulted in 26.67% survival (compared to CLP, P<0.05), but with no increase in the group of secondary infection 7 days post-CLP.Blood leucocyte counts investigated in animals undergoing CLP peaked in the early-stage of sepsis (15.57±2.77×109/L), with suffering from bacterimia. but at the metaphase of "two-hit" models decreased ability of the host to clear bacteria (1.78 vs. 0.25 log CFU/ml, compared with). In addition, the hepatic and renal function of septic rats also increased, but the results between the two-hit and the same stage of sepsis without statistically significant difference.The propotion of dendritic cells of spleen peaked in the first day of sepsis(0.87±0.31%), but at the metaphase DC、CD4+ and CD8+ T-cells decreased significantly. T-regulatory cells have peaked in the 4 days post-CLP (3.14±0.74%), and the propotion of Treg cells have switched back in the late stage of injury. The results between the two-hit and the same stage of sepsis without statistically significant difference.The serum levels of TNF-α、IL-6 and IL-1β peaked in the 24h post-CLP, HMGB-1 peaked at 4 days post-CLP (1.76±0.71 ng/ml). The anti-inflammatory cytokines IL-10、TGF-β1 peaked in the middle stage of sepsis, and decreased later on, with the exception of sTNFR-Ⅰ maintained high levels anaphasis (1.56±0.39 pg/ml). The level of IL-1β andTGF-β1 have significantly rised in the 7 days "two-hit" rats, without difference of 4 days double injury.The results of HE staining and TUNEL test indicated that sepsis could cause the remarkable damage and apoptosis of spleen in the CLP-rats. In addition, the total number of apoptosis cells increased significantly 4 days postinjury compared with the same stage of sepsis(74.48 vs.52.99, P<0.05). Rats undergoing CLP 7 days followed by pneumonia have no statistically significant difference.ConclusionsThe septic model of rats was successfully constructed by cecal ligation and puncture (CLP), whose clinically symptoms and signs were consistent with classical septic model. It could result in bacteremic and liver and kidney dysfunction. At middle stage of sepsis, immune cells were decreased, while anti-inflammatory cytokines and spleen apoptosis were enhanced. The mortality of this clinically relevant "two-hit" model constructed by sepsis and pneumonia was significantly higher than single sepsis model, meanwhile, the ability of bacteria clearance was decreased. These changes could be associated with imbalance of immune response and exacerbation of apoptosis. These findings demonstrated the occurrence of immunoparalysis at middle stage of sepsis.Part 2Modulation and Characterization of Sepsis Model in Chronic Kidney Disease RatsBackgroundThe translation of sepsis treatment from animal models into humans has largely failed, partly because the less complex animal models do not mimic human sepsis. Most patients with sepsis have at least one underlying preexisting chronic illness. The recent studies have indicated that pre-existing renal injury may worsen sepsis and septic acute kidney injury (AKI), with the significantly increased mortality and deteriorated kidney dysfunction. The possible mechanism could attribute to uremia-induced leukocyte dysfunction, inflammatory cytokine accumulation from less renal clearance, or other coexisting illness and so on. The variation of immune function may be one of the reason of the high mortality in CKD (chronic kidney disease)-sepsis patients.ObjectiveThis study was designed to establish a chronic kidney disease rat model followed by cecal ligation and puncture to induce sepsis, and investigate the change of immune reponse in different stage of CKD-sepsis. By contrast with single sepsis model, this two-stage model can be used to recognize and analysis the change of immune state in each stage of CKD-sepsis and analysis the mechanism of CKD-sepsis induce immunosuppression.MethodsA total of 24 rats weighted of 250g were subjected to 5/6 nephrectomy (performed in two stage:resecting the upper and lower left kidney poles and using Avitene hemostasis, followed by right nephrectomy lweek later). These CKD rats were performed sub-lethal cecal ligation and puncture to induce sepsis subsequently. Observe the living condition and survival rate of each model, and collect the blood and tissue samples of each rat including normal-group,1、4、7days post-CLP of both models. Detection included the examination of blood routin, bacterial count, hepatic and renal function. Use flow cytometry (FCM) to test the percentage of dendritic cells, CD4、CD8 T-cell and T-regulatory cells. The serum levels of pro- and anti-inflammatory cytokines were detected by ELISA. Use HE staining to evaluate the damage of rat spleen, and the apoptosis of spleen cells were detected by using TUNEL test.ResultsThe survival rate of CKD-sepsis group decreases significantly compared to CLP alone (40% vs.73.33%, P=0.094). Blood leucocyte counts investigated in CKD rats were higher than normal group (10.21 ×109/L vs.4.04 X109/L, P<0.05), and peaked in the early-stage of sepsis (14.55±2.88X 109/L). These rats with sepsis suffered from bacterimia, and pre-existing CKD weaken the ability of bacteria clearance (1.17±0.68 log CFU/ml in the 4 days post-CLP of CKD rats). In addition, the hepatic and renal function of CKD-sepsis rats deteriorated compared to the single sepsis group. At the late stage of sepsis, Scr and BUN sustained hige level, implied with the delay recovery of renal function.The propotion of dendritic cells, CD4、CD8 T-cells and Treg cells of spleen increased post-CLP in the CKD rats, and decreased in the mid-stage more significantly than sepsis alone. The propotion of DC sustained to reduce, but without marked variation in propotion of CD4、CD8 T-cells. Treg cells of CKD-sepsis rats were higher than single-CLP group, and peaked in the lat-stage of sepsis.The serum levels of cytokines in CKD rats were higher than normal-group. Pro inflammatory cytokines were lower than the single sepsis rats post-CLP. The levels of anti-inflammatory cytokines increased in CKD-CLP rats, while TGF-β1 and sTNFR-I maintained high-level until the late-stage of sepsis, and kept exceeding the level of single CLP group in each timing. Principal component analysis (PCA) suggested that anti-inflammatory cytokines were superior at metaphase of CKD-sepsis, resulting in immunoparalysis and recovery delay.TUNEL test indicated that apoptosis cells increased in CKD-sepsis rats compared with CLP alone (67.40 vs.52.99/HP,in 4 days postinjury). These findings demonstrated that CKD may aggravate the apoptosis condition of spleen.ConclusionsThe two-stage model of rats was successfully constructed by CKD followed CLP, which duplicated the high mortality of clinical CKD patients. Pre-existing CKD may promote sepsis-induced immune dysfunction, with the exacerbation and delay of immunosuppression at the middle stage of sepsis, and ultimately increased the motality of CKD-sepsis rats. |
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