Evaluation Of The Quality Of Liver And Kidney Donor In The State Of Brain Death In Rabbits

China has become the second biggest country after the U.S on organ transplant. The survival rates of transplant recipients are also close to or reached the international advanced level. But the scarce sources of donors have become the bottleneck of human organ transplantation. In order to reach the international standards, the former Ministry of Health and the China Red Cross Society activated cardiac death organ donation and transplantation experiment in the country of16provinces and autonomous regions. Brain dead donor will gradually replace the death, in the next two years to become a major source of organ transplants. However, a large number of clinical studies have reported that there was a poor recent or long-term prognosis for brain dead donors compared with living relatives or cadaveric organ transplantations. Previous studies also reported that the injury of brain death organs may be related to hemodynamic changes, the release of inflammatory cytokines, apoptosis, consumption of coagulation factors, endocrine and hormonal changes, et al. And the relevant pathophysiological changes impacted on the quality of brain-dead organ donor, and would be the key issues constraining the widely use of brain death donor organs.Objective The aim of our study was to establish a high degree of simulated clinical, scientific and stable rabbit animal model of brain death and investigate the mechanism of brain death of rabbitts on liver and kidney damage and further find sensitive indicators to assess the qualities of brain-dead liver to provide direction for improving the quality of brain-dead organs.Methods (1)40twelve-week-old male New Zealand white rabbits were randomly divided into sham group and the group of brain death. Each group was further divided into4sub-groups according to2,4,6and8h after brain death (n=5). Rabbits in sham groups was performed with femoral artery catheterization, endotracheal intubation and burr holes catheterization but no intracranial pressure and kept the states till the end of the experiments; the ones in brain death group was performed in turn with femoral artery, trachea intubation, skull drilling tube and slow intracranial pressure till brain death and maintained the states of brain death till the point for sample conllection. With the intraoperative use of biological functional experimental system, animal ventilator, intelligent temperature control instrument to monitoring the heart rate, breathing wave, mean arterial pressure and brain waves in the process of establishing and maintaining the states of brain death in rabbits. Each group collected the serum samples, liver and kidney tissues in the2,4,6and8h after brain death.The liver function indicated by AST, ALT and renal function indicated by BUN and Cr in brain death groups and the sham groups were detected in the points of2,4,6and8h after brain death. Morphological changes were observed with light microscopy at different time points in liver and kidney tissues. The ELISA kit was used to detect the serum levels of IL-I?, IL-6, IL-8and TNF-? The liver and kidney inflammation-related factors of ICAM and HSP70were detected by immunohistochemistry. The numbers of apoptotic cells in the liver and kidney was calculated by TUNELWe used the proteomics techniques, composed by the two-dimensional gel electrophoresis technology, bio-mass spectrometry and bioinformatics database technology comparative analysis to screen and identify the different proteins in liver and kidney tissue between the brain death and the sham groups. The western blot was used to verify the different protein named PHB in kidney and the different expressive protein named RUNX1was analyzed by immunohistochemistry.Results (1) We proposed for the first time the standards of establishment of brain death model of New Zealand rabbits and established a new method of slow intracranial pressure till brain death. In the modeling process, the arterial blood pressure, shooted up to the peak was obviously higher than the sham groups (P>0.05) and then sharply declined to below the normal level during a time of brain death. But there was no significant difference in heart rate during process of the establishment of brain death but in the2h after brain death, the heart rate was significantly lower than the sham ones (P<0.01).(2) We found no obvious liver functional and morphological changes within6h after brain death in New Zealand rabbits. But after brain death after8h, the levels of ALT and AST increased significantly (P<0.05) and there were significant balloon degeneration, sinusoidal pressure, no significant hepatic cord structure in liver cells and plenty of periportal infiltration of lymphocytes accompanied with partial focal necrosis. Although BUN values were with no obviously change (P>0.05) within4h after brain death, there were significantly increased renal Cr levels (P<0.05) and the edema of renal tubular cell, the increased degeneration, the occlusion in part proximal tubule and significant inflammation were seen in the kidney issues. There were a gradual increase for the levels of IL-1?, IL-6, IL-8and TNF-? expression and in the8h after brain death group these indicators were significantly higher than the sham ones'(P<0.05). Gradual increase for ICAM and the numberds of apoptotic cells (P<0.05) and on the contrary a gradually downregulated for HSP70(P<0.05) were found in our study.(3) We used proteomics technology successfully to screen and identify the different proteins in the liver after brain death. They were as follows: dihydropyrimidinase related protein4, aldehyde dehydrogenase, peroxiredoxin-6,3-phosphoinositide dependent protein kinase-1, runt related transcription factor1,3-mercaptopyruvate sulfurtransferase, inorganic pyrophosphatase, alcohol dehydrogenase [NADP+], glutamate--cysteine ligase regulatory subunit and Cytochrome B5. These proteins were related to regulation of cell proliferation and differentiation, metabolism, detoxification, anti-oxidation and oxidation. And we found ten different proteins in the kidney after brain death. They were as follows:Prohibitin, PRP38pre-mRNA processing factor38(yeast) domain containing A, beta-1,3-N-acetylgalactosaminyltransferase1, Annexin A5, Calcineurin subunit B type1, Superoxide dismutase, V-type proton ATPase subunit G1, Cytochrome b-c1complex subunit1, NADH dehydrogenase1beta subcomplex subunit10and Peroxiredoxin-3. They were proliferation and differentiation, signal transduction, processing and modification of proteins, electron transport chain and redox-related proteins. Among them, the different protein named RUNX1in liver decreased in a time dependent manner (P<0.05). But PHB expression in kidneys on the contrary performed a gradual increase (P<0.05).Conlusions The brain death model in New Zealand rabbit we first established is a scientific, highly simulated clinical rabbit brain death model and was worthy of promoting and reference for other animal studies of brain death.In the early brain death of the rabbit, although significant changes in kidney function has not yet been found, but complex pathophysiological changes standed for inflammatory response and apoptosis have occurred and affected the qualities of livers and kidneys of brain dead rabbits.RUNX1expression in liver might be sensitive indicator for assessing the quality of brain dead rabbit liver, but its detail mechanism remains to be further studied. Kidney PHB expression may become sensitive molecular markers for kidneys quality assessment of brain dead rabbits and it was provide a new idea for clinic to improve the quality of brain death organ kidney.