Experimental Studies On The Liver Immune Injury And Its Protection In Brain-dead Pigs

Background and objective:Liver transplantation has been regarded as the best way to cure terminal stage diseases of the liver. However, the shortage of donating livers leads to many patients died during the period of waiting for the donator livers. The huge amount of population in our country, the patients waiting for liver transplantation are increased day by day. As a result, the organ shortage phenomenon is very serious. Therefore, studying the changes of liver morphology and function in the brain-dead state may provide experimental foundation and theory basement for utilizing BDD.Many researches demonstrated that the brain-dead state injuries the liver. Olinga found that IL-10, IL-1βmRNA in the liver tissues were up-regulated in the brain-dead state of rats, and believed that the mechanism is related with the nonparenchymal cells, especially the activation of Kupffer's cell and autro-endothelial cell. Van Der Hoeven reported that ICAM-1, VCAM-1, CD45~+T cell obviously in brain-dead group in the same animal model, but the cell infiltration was less in control group, presuming that the expression of the mediation of inflammation and inflammation cell infiltrate were related to the brain-dead state. The correlation of transplantation organ and the immune system of receptor was induced by the expression of the mediation of inflammation and inflammation cell infiltrate, and then generated the immune response which affect the function state of post-transplantation donation organ in the near future and long-term periods. Though many studies indicated that the form and function of liver, inflammation injury, and immunity lesion were resulted by brain-dead state, the specific mechanism is still not clear.Nuclear factorκB(NF-κB) is closely related to inflammation and immune response. NF-KB regulates the expression of cytokine, growth factor and cellular adhesion molecule. At present, the mechanism of liver injury in brain-dead state is not clear; the excess activation of NF-KB might be one of the mechanisms that lead to liver injury in brain-dead state. Therefore, applying the NF-κB inhibitor in time may be the effective way to protect the liver in brain-dead state. Pyrrolidine dithiocarbamate (PDTC) is the inhibitor of NF-κB. In this study, we planed to establish the stable brain-dead model for pigs and hoped to provide the theory of utilizing brain-dead donor for clinic usage.This study containsⅢpartsPartⅠThe changes of BP, HR in the establishment of brain-dead model for pigsObjective: In this study, we planned to establish the brain-dead model by increasing intracranial pressure in a modified matched as slow, and intermittent way to observe the changes of BP, HR during establishment, the animal model for studying how the brain-dead state affects the modality, function and the immunogenic effects of liver.Methods: A total of 12 healthy pigs, female or male, weight (25±4kg), were randomized to control group (Group C) and brain-dead group (Group B), with 6 pigs in each group. The animals of group C were placed Foley balloon catheter incranially only, thus the non brain-dead model established. Brain-dead model was established in group B by increasing intracranial pressure in a modified matched as slow, the intermittent way, and brain-dead state was maintained for 24 h by respiration and circulation supports. MAP, heart rate, and central venous pressure of both groups were detected, and electroencephalogram was recorded in group B. The results were dealed with one-factor analysis of variance by SPSS 11.5 version. Results:1 Brain-dead model was established successfully in Group B (6 pigs).2 The homodynamic changes during the process2.1 After increasing intracranial pressure in a slow and intermittent way by Foley balloon catheter, the HR, BP in group B decreased slowly, and then increased sharply; breathing rate was stepped down, breath extent was deepened, the mean peak BP was (220.33±16.28)mmHg, and the mean peak HR was 266.33±21.83 per minute. These MAP and HR have significant difference compared with MAP(106.50±7.26mmHg) and HR (102.00±4.77 per minute) before intracranial pressure increased. Compared with pre-intracranial pressure MAP (106.50±7.26 mmHg), HR (102.00±4.77 per minute), there was significant difference (P＜0.05).2.2 There was significant difference between group B and C on MAP and HR after brain-dead for 6h (P＜0.05).3 Temporary and disorderly general tic were observed, HR and BP rose sharply, and disorderly and disturbance in brain wave could be observed.4 The vital signs of Group B in brain-dead states were consistent with the brain-dead donor criterion. The brain-dead states of group B was maintained for 24 h by respiration and circulation support, and then withdrawn the respiration and circulation supports, the brain-dead pigs went in "the heart and lung death" suddenly.PartⅡThe affections of Brain-dead state to the liver morphology and function of pigsObjective: In this study, we planned to explore the brain-dead state's affections to the liver morphology and function, and the roles NF-κB played in the process, as well as the protection of PTDC to it's lesions, utilizing the brain-dead model in partⅠ.Methods: A total of 18 healthy pigs, female or male, weight (25±4kg), were randomized to three groups, brain-dead group (group B), control group (group C) and PTDC group(group P) with 6 pigs in each group. According to the methods in PartⅠ, the brain-dead model was established in group B, with no drug intervention; also the brain-dead model were established in group P, applying PTDC 15mg/kg(added to Sodium chloride 100 ml) instantly after brain-dead, iv slowly; the group C were placed Foley balloon catheter intracranially and open/close abdomen operation only, no brain-dead model established. After the brain-dead model were established, the serum and liver tissues in the same locus were taken at 6h, 12h, and 24h after the conformation of brain-dead, the ALT, AST were determined by using automatic biochemistry analyzer, the IL-1β, IL-6, TNF-αlevel were determined by using ELISA method. The changes of NF-κB in gene level were determined by using real-time fluorescence quantitative polymerase chain reaction (real-time PCR), the expression of NF-κB in liver tissues were determined by immunity tissues chemistry method, and the liver tissues changes were observed by using HE staining. The results were dealed with one-factor analysis of variance by SPSS 11.5 version.Results:1 At 6h, 12h, 24h after the conformation of brain-dead, the changes of group C among the different time points had no statistic significance (P＞0.05). The levels of AST and ALT in group B and group P increased at 12h, and compared with the closed together time points, there was statistic significance (P＜0.05).The levels of group B and group P were higher than that of group C at 12 and 24 h (P＜0.05), but there were no statistic significance at 6h (P＞0.05).2 At 6h, 12h, 24h after the conformation of brain-dead, the serum IL-1β, IL-6, TNF-αlevel of group B and group P were higher than that of group C (P＜0.05); compared with the closer together time points of group B and group P, there was statistic significance (P＜0.05), but no statistic significance in group C(P＞0.05).3 At 6h, 12h, 24h after the conformation of brain-dead, there was no statistic significance in the transcriptional level of NF-κB mRNA and the expression level of NF-κB in liver tissues for group C(P＞0.05); compared with the closer together time points of group B and group P, there was statistic significance (P＜0.05); the transcription level in group B and group P was higher than that of group C (P＜0.05).4 The liver tissues were determined in normal at 12h after brain-dead. Under light microscope, within 12 h after the brain-dead, there showed slight claudy swelling, then became cell edema, and liver sinus to be pressed. The changes at 24 h were more obviously, the liver cell showed sparsity, but no liver cell death could be observed. In group P, the animals at 12 h after brain death the lesion of liver was lighter that of group B.PartⅢThe affections of Brain-dead state to the liver immunogenic changes in pigsObjective: In this study, we planned to explore the brain-dead state's affections to the liver immunogenic changes, and the roles NF-κB played in the process,as well as the affections of PTDC to the liver immunogenic, utilizing the brain-dead model in partⅠ.Methods: A total of 18 healthy pigs were randomized to three groups, brain-dead group (group B), PTDC group(group P), and control group (group C), with 6 pigs in each group. The establishment and management of the model are as same as partⅡ.The liver tissues in the same locus were taken at 6h, 12h, and 24h after the conformation of brain-dead of the animals.The changes of NF-κB, intercellular adhesion molecule-1(ICAM-1), monocyte chemotactic protein 1(MCP-1) in gene level were determined by Real-time fluorescence quantitative polymerase chain reaction (real-time PCR), the expression of NF-κB in liver tissues were determined by immunity tissues chemistry method, and the liver tissues changes were observed by using HE staining. The results were dealed with one-factor analysis of variance by SPSS 11.5 version.Results:1 At 6h, 12h, 24h after the conformation of brain-dead, there was no statistic significance in the transcriptional level of NF-κB, ICAM-1, MCP-1mRNA in liver tissues for group C(P＞0.05) ; compared with the closer together time points of group B and group P, there was statistic significance (P＜0.05); the transcription level in group B and group P was much higher than group C (P＜0.05)2. At 6 h, 12 h, 24 h after the conformation of brain-dead, there was no statistic significance in the expression level of NF-κB, ICAM-1, MCP-1 mRNA in liver tissues for group C(P＞0.05); compared with the closer together time points of group B and group P, there was statistic significance (P＜0.05); the transcription level in group B and group P was much higher than group C (P＜0.05).Conclusion1 In conclusion, this experiment applied an improved method of slow and intermittent encephalic pressure increase to establish pig BD model, which could simulate clinical BD state effectively. Porcine BD state could be maintained stably for more than 24 hours via effective respiration and circulation sustaining, which is helpful to the further observation of organs configuration and function changes in brain-dead state.2 "Tic reaction" appeared in all pigs before BD, so the disappearance of this phenomenon could be preliminarily considered as one of the BD verification indexes.3 The brain-dead state leads to the lesion changes of liver in function and morphology, the mediators of inflammation increases. The mechanism may be that the brain-dead state provoke the transcription level of NF-κB mRNA and protein translation increasing, then leads to the changes of mediators of inflammation, we presume that this may be one of the mechanisms, which results in the changes of liver in function and morphology.4 The brain-dead state leads to the level of NF-κB, ICAM-1, MCP-1 mRNA transcription and protein translation increasing. Liver tissues MCH-Ⅱexpresses increasing, leads to the immunogenicity enhanced. The mechanism may be the brain-dead state leads NF-κB activated, and then leads to the high expression of mediation of inflammation and the liver immunogenicity enhanced.5 In brain-dead state, the pigs was dealed with PDTC, liver tissues NF-κB mRNA transcription and protein translation were depressed, then inhibit the releasing of mediation of inflammation, with that it protect the liver damage both in function and morphology in brain-dead state of animals.6 The brain-dead state leads to the level of NF-κB, ICAM-1, MCP-1 mRNA transcription and protein translation decrease. Liver tissues MCH-Ⅱexpresses decreasing, and leads the immunogenicity depressed.7 The mechanism may be that PDTC depressed the degradation of I-κB, thus inhibited the NF-κB activation, decreased the releasing of mediation of inflammation, cytokine, immune factor, which protect the liver damages both in mophology and function in brain-dead state.