Establishment Of Non-human Primate Acute Liver Failure Model

Liver, which has multiple functions such as synthesis, secretion, metabolization and detoxication, is the most important organism of human-being. Liver failure, induced by the necrosis of large amount of liver cells, leads to the accumulation of toxicity and metabolic disturbance which can worsen the damage of liver cells. Acute liver failure (ALF), whose development is rapid with dangerous status and poor prognosis, is the appearance of severe complications rapidly after the first signs of liver disease (such as jaundice), and indicates that the liver has sustained severe damage. The complications are hepatic encephalopathy and impaired protein synthesis (as measured by the levels of serum albumin and the prothrombin time in the blood). However, most patients who survived from ALF are on the stage of liver cirrhosis. ALFF includes fulminant liver failure and subacute liver failure. Fulminant hepatic failure (FHF) is usually defined as the severe impairment of hepatic functions with the occurrence of hepatic encephalopathy and coagulopathy within 8 weeks after onset of disease. Subacute liver failure is defined as the occurrence of hepatic encephalopathy within 26 weeks after onset. Liver transplant with the survival rate of 50%～70% is the only treatment reported to have preferable effect. However, the shortage of liver donation and ethics factor restrict its application. In this condition, artificial liver support system whose mechanism is based on the reversibility of hepatic injury and reproducibility of hepatic cells, is established to temporarily substitute for the damaged liver to create condition for hepatic transplant and regeneration of hepatic cells. It can eliminate toxicity while supplying the necessity to improve internal environment and create condition for recover of hepatic function and regeneration of hepatic cells and thus decrease the mortality.The effect of ALF treatment is hard to estimate because of the complex illness state, multiple influence factors, lack of control in clinical study. Therefore, the establishment of ideal ALF animal model is the basement of ALF study. The requirements for ideal ALF animal model raised by Terblanche in 1974 includes reversibility, reproducibility, death from liver failure, a therapeutic window, a large animal model, and minimal hazard to personnel. The first four requirements are most important while the latter two can promote the popularization of the model.During the past 3 decades, many animal model most of which are FHF model have been established. The methods include surgery, medicine and gene knock out. At present most animal model are rat, pig or dog models whose clinical manifestation, physiology and biochemistry index, pathology change are different from human being. The physiology feature as well as immune system of non-human primate whose gene has 98% same part with human is far more similar to human being than rodent or dog. The study of the establishment of non-human primate ALF model and its mechanism can supply a preferable foundation for the clinical practice of artificial liver support system.Background:Acute liver failure (ALF), whose mortality is 80%, is the appearance of severe complications without preferable treatment. Liver transplant with the survival rate of 50%～70% is the only treatment reported to have preferable effect. However, the shortage of liver donation and ethics factor restrict its application. The requirement for ideal ALF animal model raised by Terblanche in 1974 includes reversibility, reproducibility, death from liver failure, a therapeutic window, a large animal model, and minimal hazard to personnel.The first four requirements are most important while the latter two can promote the popularization of the model.1) Reversibility:Untreated experimental animals with induced ALF should have a high mortality of around 80% while those who have effective treatment should recover. Methods used now to establish the models, such as hepatic necrosis induced by fully blocking blood supply and hepatectomy, all lead to reversible biochemistry and histology change. Therefore, those methods could not be used to establish ideal ALF animal model.2) Reproducibility:Ideal ALF animal model should be reproducible and stable. However, basic indexes such as disease stage, survival time and coma duration time can't be predicted in methods at present used in establishing models. The death of all untreated animals could not be guaranteed in experiments.3) Death from liver failure:Mortality is the endpoint to evaluate curative effect. Death from liver failure but no other causes is very important for the collection of experiment data and assessment of curative effect.4) Therapeutic window:Time interval from treating to killing animals should be long enough to intervene and assess curative effect.5) Large animal model Large ALF animal model is convenient for blood collection or serial continual study. Treatments studied in experiments should be done in animals similar to human being for better clinical practice. Therefore large animal model is more suitable for artificial liver support system study.6) Minimal hazard to personnel:All chemicals used in experiments should be harmless to personnel. However, hepatotoxic agents used now such as CTC and nitrosamine are all harmful to human being in some ketamineObjective:To establish a large animal ALF model for study in artificial liver support system with stable reversibility and reproducibility.In our study, macaca fascicularis ALF model was set by intravenous injection of different doses of D-gal under ketamine (0.1 ml/kg) anesthesia condition. Clinical, biochemistry and histology changes were observed to explore suitable D-gal doses for macaca fascicularis ALF model and settle a solid foundation for further study and remedy for acute liver failure.Methods:Male macaca fascicularis were selected as model animals.15 macaca fasciculariss were randomly divided into 3 groups after preliminary experiment:the first one is large dose group (n=5,medicine dose:0.45g/kg), the second moderate dose group (n=5,medicine dose:0.3g/kg), the third low dose group (n=5,medicine dose:0.15g/kg). Blood ammonia, PT, AST and TBL were under dynamic monitoring. D-gal were injected into macaca fascicularis through vena jugularisexterna in 10 minutes under anesthesia state induced by combined intramuscular injection of sumianxinⅡ(0.1 mL/kg) and ketamine (0.1 ml/kg). ICP was monitored by the encephalic Codman micro sensor which was placed through the hole in the calvarium. Blood was drawn daily before and after surgery to monitor hepatic function, kidney function, blood sugar, blood ammonia, coagulation test, BCAA/AAA through laboratory dept. in Nanfang hospital while postoperation survival condition was under dynamic monitoring. Clinical manifestations as well as disease progression were also observed and the survival time was recorded. Thorough Autopsy was immediately done after the death to take liver, kidney, spleen, pancreas, heart, lung, gastric and brain. Specimens prepared for light microscope were fixed by 10% formaldehyde to be made into paraffin sections dyed by HE. Specimens prepared for electron microscope were fixed by glutaraldehyde and osmic acid to be made into resin sections dyed by uranyl acetate and lead citrate. Pathological changes were observed under light and electron microscope.Results:The mean survival time of the first group was (56.1±8.1)h with the occurrence of hepatic coma in 80% macaca fascicularis before death. The mean survival time of the second group was(109.8±11.2)h with the occurrence of hepatic coma in 60% macaca fascicularis before death. All is survival except one was death after 98h in third group after injection D-gal, they take a better turn after 4 days, all index return normal gradually. Among the 5 macaca fasciculariss induced by 0.3g/kg D-gal,2 macaca fascicularis was 105h and 120h separately. Blood pressure of all macaca fascicularis in the third group remained stable after being given medicine with ICP more than doubled. Serum aminotransferase of macaca fascicularis increased 12h after being given medicine and became higher and higher as the disease progressed with the continuous rise of bilirubin, cholic acid, ammonia and lactic acid, the.decrease of platelet,the prolonging of activated clotting time and normal blood gas analysis results. There was significant difference between the survival time of different groups. Linear correlation analysis showed that there was negative correlation between the D-gal dose and survival time. K-M survival analysis showed that survival time of the macaca fasciculariss receiving 0.45g/kg D-gal was significant shorter than the other two groups (x2=21.933,P=0.000).Autopsy showed that macaca fasciculariss receiving D-gal had smaller liver with sharp margin, mottled surface and scattered from pin-point like to rice-like bleeding points. There was kermesinus congestion on the cut surface of liver with yellow chyliform necrotic tissues. Intestinal gas could also been seen in the intestinal tract with congested and swelling wall and scattered bleeding points. The brain tissue had sever edema with the most obvious pathological change around stellate cells and perivascular space. The shape and size of gall bladder was normal. There was no abnormality of spleen, lung and kidney under naked eyes.The structure of hepatic lobule in the control group was normal with no hepatic necrosis under light microscope. However, there were hepatic patchy necrosis with significant bleeding and swelling hepatic cells as well as narrow hepatic sinus in macaca fasciculariss receiving D-gal. Cytoplasmic puffing and vacuolar degeneration could also be seen in hepatic cells. Some hepatic cells had fatvacuole and some had karyoclasis. Cellular infiltration of neutrophil and lymphocyte could also be seen in some portal area. Large patchy necrosis with obscure hepatic lobules and severe bleeding appeared in the hepatic tissue of macaca fasciculariss receiving 1.0 g/kg. The hepatic ultrastructure of macaca fascicularis without receiving D-gal under electron microscope showed rich glycogenosome in cytoplasma, abundant normal endoplasmic reticulum, plentiful normal mitochondria with clear mitochondrialcrista and membrane and nucleus with even chromatin and clear membrane as well as nucleolus. After given D-gal, the hepatic ultrastructure showed homogenized glycogenosome in cytoplasma, endoplasmic reticulum degranulation, swelling mitochondria with obscure mitochondrialcrista, some dissolved mitochondria,abnormal nucleus with congested chromatin and intranuclear pseudoinclusions and significant decrease of microvilli of intracellular bile canaliculi.Conclusion:1.Successful establishment of ALF induced by D-gal was done with the finding of optimum dose.2.Macaca fascicularis ALF model induced by D-gal has the similar clinical, biochemistry and pathological characteristic with human being.3. Macaca fascicularis ALF model,which is suitable for the assessment of artificial liver support system, has nice reversibility and reproductivity, appropriate time window with all animals dying from ALF.