金黄色葡萄球菌败血症模型 z-bo 2020-12-06 348

　　Background: As a multifunctional organ in sepsis, the liver plays a central role in sepsis. In the human intensive care unit (ICU), cholestatic liver dysfunction is a common morbidity, with side effects such as infection, sepsis, major surgery, blood transfusion, and hepatotoxicity of antibiotics and other drugs. Diagnosis is usually based on an increase in serum bilirubin concentration, which occurs through the transaminase aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), increased lactate dehydrogenase and/or hypoproteinemia Come to support. Persistent hyperbilirubinemia is associated with increased mortality in patients with S. aureus sepsis. The pathogenesis of sepsis-related liver dysfunction is unclear. We have previously conducted two related sepsis models. Experimental pigs were inoculated with blood-borne Staphylococcus aureus, and the study time was up to 48 hours. During the observation period from inoculation to euthanasia, the pigs were awake. The clinical symptoms of sepsis include increased body temperature, increased neutrophils, C-reactive protein (CRP) and interleukin-6 (IL-6), and decreased serum iron. Increase the hypercoagulable state of the blood. The decrease in the number of platelets indicates coagulation system dysfunction, and it is particularly noteworthy that abnormal liver function is an indirect indicator of hyperbilirubinemia and normal creatine kinase levels, AST (CK), and fiber Protein leaks from small blood vessels in the liver. A small amount of bacteria was isolated from liver tissue, indicating that the liver's influence is part of the systemic inflammatory response, rather than the result of local liver infection. These observations led to the hypothesis of liver dysfunction in pigs with experimental Staphylococcus aureus sepsis. 48 hours after inoculation with blood-borne Staphylococcus aureus, the galactose elimination ability test (GEC) was introduced to confirm and quantify the presence of liver insufficiency. Animal Welfare and Model Robustness is a challenge designed by the division of research, some pigs have apnea and cardiac arrest during vaccination. Before other pigs were euthanized, they developed acute respiratory dysfunction and severe pneumonia due to severe clinical diseases. In this model, the inoculation concentration has a certain guiding effect on the severity of sepsis. We used this observation to form the second hypothesis of this study, that is, the infusion rate of Staphylococcus aureus is the degree of clinical disease induced. In short, the signs of liver dysfunction cannot be reconstructed within the scope of our pre-defined humanitarian endpoints. Therefore, it is difficult to establish a balance between scientific goals and animal welfare in a conscious porcine sepsis model.

　　Method: Animals: 6 females, hybrid pigs. Six pigs did not have Staphylococcus aureus. Based on standard hematology and biochemical results and a daily adaptation period of 8-19 days, pigs are considered clinically healthy. The body weight (BW) on the day of vaccination was 30‒36 kg. Establish permanent intravenous infusion access: 1 to 3 days before vaccination, intramuscular injection of the following drugs before surgery: 0.83 mg/kg body weight zolazepam anesthesia and 0.83 mg/kg body weight thiacycloethylamine, 0.83 mg/kg body weight toluene Thiazine, 0.83 mg/kg body weight ketamine, and 0.17 mg/kg body weight butorphanol (10 mg/ml). At the time of transfer, propofol was injected intravenously. A permanent central venous catheter is aseptically inserted into the auricular vein of each ear and guided into the internal jugular vein. Fix the catheter. To prevent the catheter from clotting, deposit 2.5-3.5 ml of saline with heparin (1000 ml/ml) as a locking solution. After resuming anesthesia, the pigs were allowed to be kept in cages with level 2 biosafety.

　　Vaccination and experimental design: Establish an intravenous infusion 1 to 3 days after the vaccination procedure, and use the same preoperative medication to anesthetize the pig again. Intubation of the pig’s trachea (100% oxygen, 0.5-1 liter/min) for echocardiography and pulse oximetry monitoring. The experiment was carried out three times in a row. In the first two rounds, one pig was inoculated with Staphylococcus aureus, one pig was inoculated with saline, and the third round was inoculated with two pigs. The specific swine strain of Staphylococcus aureus was injected into four pigs through the left ear vein catheter, suspended in 0.9% NaCl, at a dose of 1×108 cfu/kg BW. The 1, 2, and 3 rounds of infusion doses were 30, 10, and 20 ml, respectively, using a fixed infusion rate of 3 mL/min. The infusion time was 10, 3.33, and 6.66 minutes, corresponding to 1×107, 3×107 and 1.5×107 CFU/min/kg body weight respectively. Two control pigs were injected with volume saline (0.9% sodium chloride) corresponding to Parallel vaccination.

　　 Observation period after vaccination: After vaccination, the pig can recover from anesthesia. Blood collection and intravenous injection of analgesics can be repeated from the right ear venous catheter. Constant infusion of isotonic saline (0.9% sodium chloride, 3 ml/hour, interrupted only when blood sampling or analgesics infusion. Clinical examination (such as temperature measurement, auscultation, blood oxygen saturation measurement) is only judged as clinical symptoms Only carried out when worsening or painful. During the entire experimental study period, 0.01‒0.02 mg/kg body weight buprenorphine was injected intravenously in pigs and control pigs.

　　 Humane endpoint: The humane endpoint is based on the endpoint used in the previous sepsis study conducted by our group. It further elaborates on reducing the subjectivity of animal welfare assessment. In short, the endpoints include: (1) Severe dyspnea symptoms, defined as the inability of pigs to maintain blood oxygen saturation above 90%. (2) Cannot stand up and/or move. (3) Marked encephalitis, such as nystagmus, paralysis, or head tilt.

　　 Blood measurement: The baseline blood sample (B) is taken out of the intravenous catheter 1 to 3 days before vaccination. Start before vaccination (0 hour PI), and collect blood every 6 hours during the rest of the study. Perform clinical chemistry analysis on serum. Reactive protein was analyzed by sandwich enzyme-linked immunosorbent assay with dendrimer coupled with cytidine diphosphate choline, and serum amyloid A (SAA) was measured by sandwich ELISA. And at the following time points PI: 0, 6, 12, 24, 36 and 48 hours for bacterial culture.

　　 Galactose elimination ability test: Galactose (0.5 g/kg body weight, 12% solution) was infused through the right ear vein catheter for more than 5 minutes. 15-60 minutes after the start of the infusion, heparin-stabilized arterial blood samples were obtained at five-minute intervals. Within 30 minutes of sampling, centrifuge the blood (4°C, 2500 rpm, 10 minutes) and keep the plasma at −20°C until analysis. Urine was collected 4 hours after the injection of galactose.

　　 Euthanasia: After completing the test, inject an overdose of propofol (3-6 mg/kg body weight) and immediately euthanize the pig after bleeding from the axilla.

　　 Pathology and Bacteriology: liver obtained from liver lymph nodes and five pre-defined areas. The specimens were fixed with 10% formalin for 24 hours, and after routine treatment, they were cut into 3–4μm. The sections were stained with HE for evaluation.

　　Result: Inoculated pig case 1 (1×107 CFU/min/kg body weight; 10 minutes of infusion): There was only slight tachycardia during bacterial infusion (heart rate increased from 60 to 90 beats per minute). The pig was restless for the first 6 hours; after that, it lay down and did not want to stand up. Becomes anorexic, but still willing to drink water. Limbs get cold. This pig completed the study without reaching any humane endpoint. By increasing body temperature, it shows sepsis, bacteremia and neutrophils, while increasing IL-6, CRP, SAA, and reducing iron. There was no significant change in liver blood parameters, but the total bilirubin blood concentration increased between 24 and 42 hours.

　　 Case 2: (3×107 CFU/min/kg body weight; 3.33 minutes infusion): In addition to mild tachycardia, a short spontaneous arrhythmia occurred during bacterial infusion. Compared with other pigs, this pig showed earlier and severe clinical symptoms, that is, it became numb and muscle tremors. After 30 hours, the pig was no longer able to stand up and was euthanized. According to the humanized endpoint: "Inability to get up and/or move." An increase in body temperature indicates sepsis, bacteremia, and neutrophils , While increasing IL-6, CRP, SAA and reducing serum iron. In addition, the AST level reached a peak at 6 hours (5 times the baseline value) and remained elevated. The CK level has a similar pattern.

　　 Cases 3 and 4: (1.5×107 CFU/min/kg body weight; 6.66 minutes infusion): Clinically, the behavior of pigs is similar to Case 1. During the study period, both pigs received a small amount of feed and did not reach the humane endpoint. Increased body temperature of vaccinated pigs indicates sepsis and bacteremia and neutrophils, while increasing IL-6, CRP, SAA, and reducing serum iron. Except for a brief increase in liver bilirubin, other liver parameters are not affected.

　　 Conclusion: The signs of liver dysfunction cannot be reconstructed within our pre-defined humanitarian endpoint. The balance between scientific goals and animal welfare is not feasible, and we find it difficult to use the conscious swine sepsis model. We recommend that this model be re-established as a long-term anesthesia model in order to establish a translation model related to sepsis.