"Reproduction of a model of ""two-hit"" sepsis model with complication of pneumonia in rat"
10.3760/cma.j.issn.2095-4352.2015.10.005
- VernacularTitle:“二次打击”脓毒症继发肺炎大鼠模型的构建与评价
- Author:
Yingying CHEN
;
Huixian LI
;
Shuai MA
;
Bo DENG
;
Jianxin LU
;
Feng DING
- Publication Type:Journal Article
- Keywords:
"Sepsis;
Animal model;
""Two-hit"";
Immunoparalysis"
- From:
Chinese Critical Care Medicine
2015;(10):805-810
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo reproduce a clinically relevant two-hit model of sepsis complicated by pneumonia and to explore the correlation between two-hit and immune state.Methods Eighty-one male Sprague-Dawley ( SD ) rats were divided into groups according to the random number table. Forty-five male rats were assigned respectively to sepsis-alone group, pneumonia 4 days and 7 days after sepsis groups, respectively. Survival rate of each group was observed. Another group of 36 male rats were divided into normal control group, sepsis-alone for 1, 4 and 7 days groups, and sepsis complicated by pneumonia for 4 days and 7 days after sepsis groups, each group consisted of 6 rats. Cecal ligation and puncture (CLP) was done in rats, andStreptococcus pneumoniae suspension (bacteria count 1×1010 cfu/mL) was injected via the nose on the 4th day or 7th day after CLP. Rats were sacrificed at corresponding time points, and 1 day after challenge ofStreptococcus pneumoniae on the 4 days or 7 days post CLP for the collection of blood and tissue samples to make bacterial count of the blood, splenocyte count, biochemical indices, cytokines concentration, pathological changes in spleen and apoptotic cells.Results① Compared with the rats of sepsis-alone group, the rats in pneumonia 4 days after CLP group had poor survival rate (4 vs. 11,χ2 = 6.533,P = 0.011), while no difference was found between pneumonia 7 days after CLP group and sepsis-alone group (9 vs. 11,χ2 = 0.600,P = 0.439).② The blood bacterial count and all the biochemical indexes were sharply increased on 1 day post-CLP in the rats of sepsis-alone group, and then they gradually lowered. Compared with the rats of 1 day post-CLP, the proportion of splenocytes were decreased on the 4th day post-CLP [dendritic cells (DC): (0.69±0.09)% vs. (0.87±0.31)%, CD4+T cells: (21.05±2.89)% vs. (24.84±4.59)%, CD8+ T cells: (10.62±1.79)% vs. (13.40±1.31)%, allP< 0.05], but T-regulatory cell (Treg) count was higher on the 4th day after CLP compared with sepsis-alone rats [(3.14±0.74 )% vs. (2.87±1.08)%,P< 0.05]. The biochemical indices, including alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (SCr) were obviously lowered on 7 days post-CLP compared with 1 day after CLP [ALT (U/L): 35.33±11.52 vs. 81.00±38.40, AST (U/L): 70.33±42.16 vs. 156.00±28.11, BUN (mmol/L): 5.30±2.27 vs. 9.13±4.04, SCr (μmol/L): 55.33±10.67 vs. 96.67±45.79, allP< 0.05]. The serum levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) peaked on the 1st day after CLP [TNF-α:(18.03±2.88) ng/L, IL-6: (10.37±4.20) ng/L, IL-1β: (102.44±51.46) ng/L], and high mobility group box-1 (HMGB1) peaked on the 4th day after CLP [(1.76±0.71)μg/L]. The levels of anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1 ) and soluble tumor necrosis factor receptor-Ⅰ (sTNFR-Ⅰ) maintained at high levels [7 days post-CLP: TGF-β1 was (0.90±0.56) ng/L, sTNFR-Ⅰ was (1.56±0.39) ng/L]. The spleen pathology became more marked with the time in the group of sepsis-alone, meanwhile the number of apoptotic spleencytes increased 4 days post-CLP as compared with that of the 1st day post-CLP (cells/HP: 52.99±20.79 vs. 16.05±3.28,P< 0.05).③ Compared with the same period of sepsis-alone group, the rats with pneumonia 4 days post-CLP group showed a higher blood bacterial count (log cfu/mL: 1.78±0.54 vs. 0.25±0.18,P< 0.05), while no difference was found between 7-day of post-CLP pneumonia group and sepsis-alone group (log cfu/mL: 0.57±0.46 vs. 0.13±0.12,P> 0.05). The same trend of changes, with slight reduction in splenocytes and biochemical indices were found between the groups of sepsis followed by pneumonia and sepsis-alone, but no significant difference was found. The level of HMGB1 in the 4-day group of sepsis with complication of pneumonia was further decreased compared with sepsis-alone group (μg/L:1.17±0.74 vs. 1.76±0.71,P< 0.05), and IL-1β in the 7-day group of sepsis complicated pneumonia was further higher than those of sepsis-alone group in the same period (ng/L: 105.73±25.06 vs. 61.04±31.29,P< 0.05), while there were no differences in levels of other cytokines between two-hit group and sepsis-alone group. Apoptosis of spleencytes in the 4-day group of sepsis complicated pneumonia was more marked than that of sepsis-alone group at the same period (cells/HP: 74.48±22.47 vs. 52.99±20.79,P< 0.05), while no difference was found between the 7-day groups of sepsis complicated pneumonia and the sepsis-alone group (cells/HP: 28.70±4.13 vs. 30.43±14.55, P> 0.05).Conclusions The mortality of this two-hit model with complication of pneumonia 4 days after CLP was significantly higher than that of single sepsis model. The ability of bacteria clearance was decreased, and immunocyte apoptosis was exacerbated. These findings may be with the result of the occurrence of immunoparalysis in the mid stage of sepsis. The two-hit model reproduced on 7 days after CLP might suggest reconstruction of host immune function, and maybe associated with the recovery of immune response.
