Survival Rates and Identification Results of the CLP Model of Sepsis
The survival rate of the mice after CLP surgery was closely related to the length of cecal ligation. The four-day survival rates of the mice in the 3/4 CLP, 1/2CLP, 1/3CLP, and Sham groups were 10%, 40%, 70%, and 100%, respectively. As the mortality rates of the 3/4CLP and 1/2CLP group were too high and non-conducive to subsequent experiments, 1/3CLP was selected as the experimental model for all subsequent experiments (Fig.2a).
The routine blood tests on day 1 post-CLP showed that the levels of white blood cells, neutrophils, lymphocytes, and monocytes in the CLP group were 2.25 ± 1.05, 0.47 ± 0.38, 1.56 ± 0.74, and 0.21 ± 0.139 (*103/μL), respectively, which were significantly lower than those in the Sham group (P < 0.05). However, there was no significant difference in routine blood tests between the two groups on day 4 post-CLP (P > 0.05, Table1).
On day 1 post-CLP, the level of serum ALT in the CLP group was 66.70 ± 36.33 Karu, which was significantly higher than that of Sham group (11.63 ± 2.23 karu, P < 0.05); however, there was no significant difference between the two groups on day 4 post-CLP (P > 0.05, Fig.2b).
The level of serum PCT in the CLP group was 958.4 ± 535.2 pg/mL and 607.1 ± 247.5 pg/mL on days 1 and 4 post-surgery, compared to only 124.1 ± 40.9 pg/mL and 156.4 ± 10.0 pg/mL in the Sham group. The level of serum PCT in the CLP group was significantly higher than that of the Sham group (P < 0.05; Fig.2c), indicating a severe infection in the CLP group.
A large number of colonies with neat white round edges and smooth surfaces were observed in the blood culture dish and peritoneal lavage fluid culture dish of the CLP group on day 1 post-CLP; however, no such results were observed in the blood culture dish or peritoneal lavage fluid culture dish of the Sham group, and the blood agar medium was bright red (Fig.2d). Therefore, these results demonstrated that the CLP model of sepsis had been successfully established.
Survival Rates and Identification Results of the "two-hit" Model of Sepsis
According to the survival curve of the "two hit" model, on day 11 following CLP, four mice in the CLP + Pa group died, one mouse in the Sham + Pa group died, and all mice in the CLP + NS and Sham + NS groups survived (Fig.3b).
Routines blood tests on day 5 post-CLP showed that the level of white blood cells, neutrophils, lymphocytes, and monocytes were 14.79± 1.28, 8.26± 0.57, 5.46± 0.74, and 1.03± 0.60 (*103/μL) in the CLP + Pa group; 7.02± 1.5, 1.72± 0.55, 4.74± 1.79, and 0.35± 0.05 (*103/μL) in the CLP+NS group; 10.80± 1.67, 2.25± 2.01, 7.14± 1.53, and 1.56± 1.02 (*103/μL) in the Sham + Pa group; and 8.76 ± 0.68, 1.71 ± 0.61,6.20 ± 0.95, and 0.60 ± 0.29 (*103/μL) in the Sham + NS group (Table.2). Compared with the CLP + Pa group, the white blood cells and neutrophils were significantly decreased in the three groups (P < 0.05).
On day 5 post-surgery, the bronchoalveolar lavage fluids of the four groups were cultured. Green colonies were observed in the NAC solid medium in the CLP + Pa group and Sham + Pa group, and the number of colonies in the CLP + Pa group was greater than that in the Sham + Pa group. However, no colonies were observed in either the CLP + NS group or Sham + NS group (Fig.3c).
We further observed the appearance of the lung tissue. On day 5 post-surgery, there were obvious bleeding points in the lung tissues of the CLP + Pa and Sham + Pa groups, especially in the CLP + Pa group. On day 11 post-surgery, the lung tissues of the surviving mice in the CLP+Pa group were consolidated dark red in color, and the majority of the lung lobes were affected, whereas there was only partial lung tissue damage in the Sham+Pa group. The lung tissue of the mice in the CLP+NS and Sham + NS groups was normal at the both time points (Fig.3d).
HE slices of the lungs in the CLP + Pa and Sham + Pa groups showed that the lung tissues exhibited widened alveolar septum, damaged alveolar structure, hemorrhage, serous exudation, and a large amount of inflammatory cell infiltration on day 5 after the operation. The degree of lung tissue injury was more severe in the CLP + Pa group. On day 11 post-surgery, there was greater consolidation of the alveolar cavity, focal pulmonary interstitial hemorrhage, and a large number of inflammatory cell infiltration in the connective tissues of the alveoli and alveolar septum in the CLP + Pa group. In the Sham + Pa group, only part of the inflammatory cells had infiltrated into the lung tissue. In the CLP + NS and Sham + NS groups, the lung tissue HE staining showed no significant change (Fig.3e).
On day 5 post-CLP, the lung indexes of the CLP + Pa, CLP + NS, Sham + Pa, and Sham + NS groups were calculated respectively: 0.72 ± 0.05%, 0.606 ± 0.02%, 0.70 ± 0.04%, and 0.51 ± 0.02%.The lung index of CLP + Pa group was significantly higher than that of the CLP + NS group and Sham + NS groups (P < 0.01). On day 11 post-CLP, the lung index of the four aforementioned groups were 0.74 ± 0.04%, 0.56 ± 0.02%, 0.66 ± 0.01%, and 0.51 ± 0.02%, respectively. The lung index of the CLP + Pa group was significantly higher than that of the other three groups (P < 0.01), indicating a serious lung infection in the CLP + Pa group (Fig.3f). Together these results prove that the "two-hit" model of sepsis was completely constructed.
The Body Weight Results and Abdominal Organ Changes of the "two-hit" Model
The body weight changes of the "two-hit" mice were continuously recorded within 11 days post-CLP. The results showed that the weight of the mice in the CLP operation group decreased continuously within 5 days after the operation, and increased slowly. The weight of the mice in the Sham operation group temporarily decreased on the first day after operation and subsequently underwent a gradual increase. The difference between the two groups was statistically significant (P < 0.01). After an intranasal instillation of P. aeruginosa, the weight of the mice in the Sham + Pa group decreased for a short time and gradually increased. On day 11 post-CLP, the weight gain of the mice in the CLP + Pa group was significantly lower than that in the other three groups(P < 0.01), which suggests that the infection in the CLP + Pa group was more serious than that of the other three groups (Fig.4a).
In both the CLP + Pa and CLP + NS groups, pale necrosis occurred at the distal end of cecal ligation on day 1 post-CLP; white purulent necrosis, adhesion with surrounding tissues, and edema appeared on day 4; both purulent secretion and the area of abdominal adhesion increased on day 5; and the necrotic area was wrapped and could not be completely separated on day 11. However, in both of the Sham + Pa and Sham + NS groups, the shape and structure of the cecum were intact, and no abnormalities were present throughout the experiment (Fig.4b).
The changes in the spleen size of each of the four groups were observed throughout the experimental cycle. From day 4 post-CLP, the spleens in the CLP + Pa and CLP + NS groups were significantly enlarged. On day 11 post-CLP, the size of the spleen in the CLP + Pa group was substantially larger than that of the other three groups, suggesting the inflammatory reaction in the CLP + Pa group was the most severe (Fig.4c).
On day 1, 4, 5 and 11 post-CLP, the spleen capsule in the CLP + Pa group gradually thickened, multinucleated macrophages gradually increased, and the number of white pulp decreased significantly, and the white pulp and red pulp boundary in the CLP + Pa group gradually blurred. The number of white pulp in the CLP + NS group was slightly reduced, and the boundary between the white pulp and the red pulp was still distinguishable. The fibrous capsule in the Sham + Pa group was thickened, and the ratio of white pulp to red pulp was normal. The capsule of the spleen tissue in the Sham + NS group was smooth, the Shape and size of the white pulp was regular, the boundary between the white and red pulp was clear and there was no abnormality (Fig.4d).
The spleen index was calculated at four different time points. The results showed that the spleen index was 0.26 ± 0.03%, 0.524 ± 0.06%, 0.70 ± 0.04%, and 1.36 ± 0.26%, respectively, in the CLP + Pa group; 0.25 ± 0.04%, 0.50 ± 0.03%, 0.67 ± 0.11%, and 0.89 ± 0.06%, respectively, in the CLP + NS group; 0.23 ± 0.02%, 0.29 ± 0.03%, 0.32 ± 0.03%, and 0.45 ± 0.03%, respectively, in the Sham + Pa group; and 0.24 ± 0.05%, 0.27 ± 0.05%, 0.29 ± 0.05%, and 0.28 ± 0.04%, respectively, in the Sham + NS group. The spleen index of the mice in the CLP operation group was significantly higher than that in Sham operation group(P < 0.01). However, on day 11 after CLP, the spleen index of the mice in the CLP + Pa group also significantly differed from that in the CLP + NS group(P < 0.01), which further highlighted the severity of the infection in the CLP + Pa group (Fig.4e). Our results showed that the spleens were in a state of hypersplenism due to infections and the immune cells were continuously consumed.
Immunization Results of the "two-hit" Model
On day 11 post-CLP, the white blood cells in the CLP + Pa group were 31.74 ± 7.14*103/μL, which was significantly higher than that in the CLP + NS group , Sham + Pa group and Sham + NS group (P < 0.05 or P < 0.01); the monocytes were 3.11 ± 1.28*103/μL, which was significantly higher than that in the CLP + NS and Sham + NS groups (P < 0.05). However, since the number of neutrophils and lymphocytes in the CLP + Pa group were exceeded the detection range, statistical analysis could not be performed (Table 3).
We further observed the changes in the level of TNF-α and IL-6. On day 1 post-CLP, the level of serum TNF-α was 167.00 ± 42.68 pg/mL in the CLP group and 45.90 ± 0.38pg/mL in the Sham group (P < 0.01), indicating that TNF-α was activated following CLP. On day 5 post-CLP, the level of serum TNF-α in the CLP + Pa and Sham + Pa groups were 110.69 ± 9.18pg/mL and 148.43 ± 13.42pg/mL, respectively. These findings were significantly different from those in the CLP + NS group (45.90 ± 0.38pg/mL) and Sham + NS group (45.81 ± 0.77pg/mL) (P < 0.0001), which suggests that serum TNF-α was produced after an intranasal instillation of P. aeruginosa. The level of serum TNF-α in the CLP + Pa group was significantly lower than that in the Sham + Pa group (P < 0.01), suggesting that the CLP operation may inhibit TNF-α reactivation. However, no significant difference was observed regarding the level of serum TNF-α between the four groups on days 4 and 11 post-CLP (Fig.5a). Moreover, the change trend of IL-6 was similar to that of TNF-α (Fig.5b).
The ratio of CD4+/CD8+ in whole blood was also detected at four different time points by flow cytometry to observe the cellular immune function. The results showed that the ratio of CD4+/CD8+ in the CLP + Pa group was 0.57 ± 0.10 on day 11 post-CLP, which was significantly lower than that of the other three groups (the ratio of CD4+/CD8+ in the CLP + NS group, Sham + Pa group, and Sham + NS group was 0.91 ± 0.04, 1.17 ± 0.03, and 1.48 ± 0.04, respectively) (P < 0.05 or P < 0.01, Fig.5c,d). These findings indicate that the "two-hit" model of sepsis may lead to the the decreased presence of immune cells . The above results indicate that the "two- hit" model of sepsis could lead to immunoparalysis.