Study of the Role of Transforming Growth Factor b-1 in Organ Damage Protection in Porcine Model of Brain Death L. Chena, X. Fengb, Y. Wangc, X. Xuc, C. Wanb, J. Wangd, and H. Mua,* a Department of Clinical Laboratory, bDepartment of Neurosurgery, The First Central Hospital of Tianjin, cKey Laboratory for Critical Care Medicine of the Ministry of Health; and dDepartment of Transplantation Surgery, The First Central Hospital of Tianjin, Tianjin, China
ABSTRACT Background. From the medical and ethical points of view, donation after brain death is a more acceptable organ source than that from a living donor because it has the advantage of providing multiple organs from a single donor source. Hence, it has become a more promising field of research which focuses on the protection of organs at brain death Here we investigated the role of transforming growth factor (TGF)-b1 in a porcine model of brain death. Methods. A porcine model of brain death was established by increasing the intracranial pressure (ICP) after which TGF-b1 was monitored by immunofluorescence at the following time points: before ICP was performed (t1), at brain death (t2), and at 3 (t3), 6 (t4), 9 (t5), and 18 (t6) hours after brain death. The data were analyzed using the fixed effect regression method and the correlation between the results was determined by Pearson analysis. Results. Our results showed that there was a significant increase in the levels of TGF-b1 (P < .05), urea (P < .01), creatinine (P < .01), and aspartate aminotransferase (AST; P < .01) during the 18-hour brain death process. There were negative correlations between TGF-b1 and urea, creatinine, alanine aminotransferase, AST, and total bilirubin. The negative correlations between TGF-b1 and creatinine and AST achieved statistical significance (P < .05). Conclusions. These findings taken together confirm that significant damages are caused to the myocardial fiber cell and kidney glomerulus during brain death process, and that TGF-b1 is associated with the protection of these organs.
O
RGAN TRANSPLANTATION is more available than ever before to a majority number of patients with end-stage organ failure as a curative treatment owing to the development of medicine and technology. As a result, donor shortage has become a more pressing problem in recent years as a large number of patients die every year while awaiting transplantation. From the medical and ethical point of view, organ donation after brain death (DBD) is more acceptable than that from a living donor for organ transplantation. In addition, DBD has the advantage of providing multiple organs from a single donor source. Increasingly, DBD has drawn great attention as a more reliable donor source. Brain death is a complex pathophysiologic process. Recent studies showed that brain death can cause a series of disturbances of normal homeostatic systems, which include
ª 2016 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 48, 205e209 (2016)
hemodynamic instability, hormonal impairment, significant changes in the internal environment, and inflammation. All these changes compromise the function of harvested organs [1,2]. To date, the technology to preserve DBD organs has been less than ideal. It is reported that about 25% of the potential DBD organs lose function gradually during the maintenance process, to some extent. Therefore, L.C., X.F., and Y.W. contributed equally to this work. This work was supported by National Clinical Key Specialty Project Foundation of the Ministry of Health, P.R. China (2013544) and National Natural Science Foundation of China (81101911). *Address correspondence to Hong Mu, Department of Clinical Laboratory, The First Central Hospital of Tianjin, Tianjin 300192, China. E-mail: [email protected] 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.01.007
205
.7464 (0.6)
.0003** (6.86)
.1285 (1.87)
.0000** (10.05)
.0000** (54.47)
.021* (3.616)
1.77 0.45
37.81 7.05
41.93 3.98
77.23 7.02
4.01 0.45
Note: *P < .05; **P < .01 Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BD, brain death; TBIL, total bilirubin; TGF, transforming growth factor.
260.93 85.06 .373 (0.91) 9.53 0.95 .000** (12.68) 114.33 20.07 .001** (3.86) 38.43 2.48 .810 (0.24) 286.43 16.87 .000** (5.18) 1.77 0.51 .677 (0.42) 536.80 152.23 .07 (1.91) 6.36 0.53 .000** (7.65) 103.75 21.36 .138 (1.55) 29.65 4.64 .553 (0.6) 118.38 38.61 .000** (4.79) 1.38 0.17 .76 (0.31) 594.24 129.54 .236 (1.22) 5.99 0.36 .000** (7.98) 95.45 11.94 .007** (3.03) 33.70 3.81 .087 (1.8) 107.85 24.27 .002** (3.52) 2.72 1.02 .867 (0.17) 1224.62 419.63 .83 (0.22) 4.49 0.54 .000** (6.94) 69.31 6.02 .028* (2.37) 33.49 5.24 .013* (2.71) 86.23 20.03 .009** (2.89) 2.82 0.94 .487 (0.71) 2174.10 638.66 .026* (2.4) 4.15 0.39 .047* (2.12) 70.81 7.24 .288 (1.09) 36.06 6.01 .017* (2.61) 70.57 18.33 .031* (2.31) 2.26 0.45 .276 (1.12) 1059.82 278.14
1583.28 162.93 .396 (0.87) 4.17 0.51 .546 (0.61) 74.82 6.81 .387 (0.89) 40.752 4.39 .084 (1.82) 54.19 12.11 .133 (1.57) 2.22 0.34 .61 (0.52)
BD 18h BD 9h BD 6h BD 3h BD
TGF-b1 (pg/mL) P (t) Urea (mmol/L) P (t) Creatinine (mmol/L) P (t) ALT (IU/L) P (t) AST (IU/L) P (t) TBIL (mmol/L) P (t)
Animals were anesthetized with general anesthesia. Cannula was inserted into the pig’s femoral artery and vein, separately, for continuous mean arterial pressure (MAP) monitoring and administration of fluids after weighing, anesthesia, and skin preparation. Another cannula was opened and connected to a microrespirator for respiratory wave monitoring; electrodes were inserted to monitor electroencephalographic and electrocardiographic activity. Then, cystostomy was performed, and the parietal central area of the skull was perforated and normal saline was injected by transcatheter progressively at the speed of 0.5 mL/min; ICP and MAP were monitored at the same time. Pressurization was started when MAP > ICP and then stopped when MAP < ICP. The pressure adjustment was continued until MAP was no longer increasing with the increasing ICP. Brain death was confirmed 30 minutes after anesthesia was terminated when the model had met the following criteria: (1) reflex to the light of the pupil disappeared, (2) corneal reflex disappeared, (3) no facial and head stimulation reactions,
BBD
Brain Death Model
Table 1. Levels of TGF-b1 and Biochemistry Markers in Brain Death Model During the BD Process
Seven healthy adult Landrace pigs weighing 33.6 4.7 kg, 4 male and 3 female, were purchased from the Experimental Animal Center in Tianjin, China. The experimental pigs were housed in climatecontrolled quarters (25 1 C at 50% humidity) with a 12-hour light/12-hour dark cycle and fed with standard food. They were kept fasting for 12 hours with no water for 6 hours before the experiment started. This study was approved ethically by Department of Clinical Laboratory, The First Central Hospital of Tianjin.
Reference time
MATERIAL AND METHODS Animals
n ¼ 34
understanding how the injury occurs and how it can be ameliorated has become an important research area for the preservation of DBD donor organs [3,4]. To explore and expand the organ source, the research in DBD will or has become an important hot topic with its huge clinical potential. Transforming growth factor (TGF)-b1, a gene located at 19q13, is a peptide with 25-kD molecular weight. It is secreted by many cell types. TGF-b1 is a kind of multifunctional cytokine that acts through its signaling pathway, stops the cell cycle at the G1 stage to stop proliferation, induces differentiation, or promotes apoptosis. It causes immunosuppression and angiogenesis. TGF-b also converts effector T cells that attack abnormal cells with an inflammatory (immune) reaction into regulatory (suppressor) T cells. TGF-b1 is proven to be associated with the arteriosclerosis, cancer, fibrosis, and inflammatory responses by the clinical research over the past decade [5e9]. However, the role of TGF-b1 in brain death process is rarely reported. We hypothesized that TGF-b1 could also trigger immunosuppression to lessen inflammatory injury of tissues induced by an immunologic reaction in the brain death process. In this study, we established a pig model of brain death by using intracranial pressure (ICP) method and then monitoring the concentration of TGF-b1 and other organ biochemistry markers during the continuous 18-hour brain death period. Moreover, we explored the TGF-b1 pathophysiology in DBD model and revealed the roles it might have played in the DBD organ damage process.
CHEN, FENG, WANG ET AL
P (F)
206
TGF-b1 IN ORGAN DAMAGE PROTECTION
Fig 1. Transforming growth factor (TGF)b-1 trend in the brain death model. The overall discrepancy was significant (F ¼ 3.616; P < .05). It increased from t1 (baseline 1059.82) to t3 (2174.10; t ¼ 2; P < .05) and then decreased to a final value of 260.93 at t7. It showed the process of stress response in TGFb-1. (*P < .05) (4) no independent breathing, (5) transcranial Doppler showed that the front and posterior cycles were oscillatory wave, sharp and small contraction wave, or the blood flow signal disappeared, (6) the electroencephalograph display resting potential, and (7) the atropine test was negative [10e12].
Blood Sampling Time Points Blood samples were collected from the internal jugular vein into a tube containing the coagulative agent. One hour later, blood was centrifuged for 10 minutes at 960 g and serum was isolated and stored at 80 C in polypropylene tubes until they were assayed. The time points of blood sample collected were as follow (time groups): (1) the time after the routine surgical operation was performed and before intracranial compression was set for the reference time group (reference time, t1); (2) the time of intracranial compression at which blood pressure was showing fluctuations wave was set as before brain death time group (BBD, t2), (3) the time that the experimental pigs just met the brain death standard was designed as brain death group (BD, t3), (4) 3 hours after brain death
207
Fig 3. Creatinine (CR) trend in the brain death model. The overall discrepancy was significant (F ¼ 10.05; P < .05). CR decreased from 77.23 to 69.31 (t ¼ 2.37; P < .05) and then increased to 114.33 at t7 (t ¼ 3.03; P < .01). (*P < .05; **P < .01) was designed as 3-hour post brain death group (3h PBD, t4), (5) 6 hours after brain death was designed as 6-hour post brain death group (6h PBD, t5), (6) 9 hours after brain death was designed as 9-hour post brain death group (9h PBD, t6), and (7) 18 hours after brain death was designed as 18-hour post brain death group (18h PBD, t7).
Organ Biochemistry Marker Detection The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and concentrations of urea, creatinine, and total bilirubin (TBIL) were measured. ALT, AST, and urea were analyzed using enzymatic rate assay and creatinine and TBIL were analyzed using colorimetric assay. All analyses were performed on a Modular DPP Roche Diagnostics (Tokyo, Japan) using Roche diagnostics’ reagents.
TGFb-1 Detection The concentration of TGF-b1 was determined by immunofluorescence method on Axon Gene Pix using a Ray Biotech regent. The measurement process was according to the manufacturer’s instructions.
Statistical Analysis The concentrations or activities of TGF-b1 and organ biochemistry marker are expressed as mean values standard error. The statistical significance of every marker at the successive brain death time points was analyzed by the method of fixed-effects regression (Stats IC v.12); correlations between the results of TGF-b1 and the organ biochemistry markers at the same brain death time point were analyzed by using Pearson analysis method (SPSS v.20.0). P < .05 was considered significant.
RESULTS
Fig 2. Urea trend in brain-death model. The overall discrepancy was significant (F ¼ 54.47, P < .01). It increased from t1 to t7 (4.01 to 9.53; t ¼ 2.12 to 12.68, P < .01). It showed the irreversible changes of urea in the brain death model. (*P < .05; **P < .01)
Values and changes of serum TGF-b1, and organ biochemistry markers in brain death model during t1et7 time-points are shown in Table 1. From the F test analysis, significant differences were observed in the concentration or activity of TGF-b1, urea, creatinine, and AST at consecutive
208
Fig 4. Aspartate aminotransferase (AST) trend in the brain death model. The overall discrepancy was significant (F ¼ 6.86; P < .01). AST increased from 37.81 to 286.43, that showed an irreversible damage of myocardial cells in the brain death models. (*P < .05; **P < .01)
time points, but not in that of ALT and TBIL. The significant point was further analyzed by t test, and a significant difference in TGF-b1 was observed at the time of brain death. Urea and AST showed significant differences from the time brain death occurred, so did creatinine after a delay of 1 time point. ALT showed 2 significant differences at both the time of brain death and 3 hours after brain death. TBIL presented no difference during the whole 18-hour brain death process. The general information tendency of TGF-b1 and biochemistry markers at continuous brain death process in Porcine Brain Dead Model are shown in Figs 1e6. The significant increase in urea and creatinine (P < .01) indicated that the glomerular filtration rate was attenuated during the process as though creatinine was 1 time point delayed. The sharp increase in AST (P < .01) activity showed that irreversible damage was caused to myocardial cells. From our observation of each curve, it seems that urea, creatinine, and AST changed in the opposite direction to that of TGF-b1.
CHEN, FENG, WANG ET AL
Fig 6. Total bilirubin (TBIL) trend in the brain death model. The overall discrepancy had no significance (F ¼ 0.6; P > .05), indicating that the hepatobiliary metabolism was mildly changed throughout the brain death process.
Results of the correlation analysis between TGF-b1 and other organ biochemistry markers at same time point are shown in Table 2. A significant negative correlation was observed between TGF-b1 and creatinine and AST. The negative correlation between TGF-b1 and urea just reached statistical significance at the .05 level. This could mean that TGF-b1 had a beneficial effect on the myocardium and renal glomerulus. DISCUSSION
Based on previous studies [1e4,13e15], significant complex pathophysiologic changes occurred at the time of brain death. These changes involve (1) sympathetic nerves excitement, (2) unbalance of hypothalamusepituitary and adrenocortical hormones, (3) hemodynamic instability, and (4) metabolic disorder and cytokine release, which reach their peak separately depending on the type of reaction and the seriousness of the injury during the brain death process. No matter how brain death is achieved, a proinflammatory environment always occurs. Essentially, these changes collectively induce irreversible damage to the organs. Cytokines play an important role in immunity, apoptosis, angiogenesis, and cell growth and differentiation in physiologic processes. They are involved in interactions between different cells; in particular, they could cross a disrupted bloodebrain barrier, activate complement and increase tissue permeability as cells respond to the brain death
Table 2. Correlation Between TGF-b1 and Organ Biochemistry Marker at Corresponding Time Point
Fig 5. Alanine aminotransferase (ALT) trend in the brain death model. ALT changed mildly (F ¼ 1.87; P > .05). It showed that there was little damage caused to the hepatocytes during brain death process. (*P < .05)
R P
Urea
Creatinine
ALT
AST
TBIL
0.360 .051
0.372 .043*
0.005 .980
0.404 .027*
0.198 .294
*P < .05. Abbreviation: BBD, before brain death time group.
TGF-b1 IN ORGAN DAMAGE PROTECTION
[1,2,15e17]. According to Ryan et al. [15], TGF-b1 may regulate T-cell survival and function; it works as a suppressor of interferon-geinduced macrophage upregulation and chemokine and cytokine generation. In our gradual induction of brain death in the pig model, the significantly increased concentration of urea and creatinine suggest that there was a decrease in the glomerular filtration rate throughout the brain death process. The sharp increase in urea could also be attributed to an increased protein degradation owing to tissue damage; a decrease in creatinine at t4 time point could be owing to a reduction in antidiuretic hormone while maintaining the adequate function of glomerulus at that time [1e3]. As to the 2 transferases, it seems to be somewhat paradoxical at first sight that there was a significant increase in aspartate transferase but not alanine transfer enzyme. However, it does suggest that there was only a mild damage to the hepatocytes. The increased activity of aspartate transferase could come from myocardial cell because the heart is another important source of aspartate transferase. In our study, TGF-b1 showed a significant negative correlation with the levels of AST and creatinine, suggesting that TGF-b1 is associated with the protection of myocardial fiber cells and kidney glomerulus. This result supports the findings of the previous studies that TGF-b1 may play a role as beneficial molecular media in brain death process [1,15]. This means that the protective mediator could be released massively when brain death occurs. Also, it demonstrates the multidimensional nature of stress response. Hence, it is better to choose a more mediator-specific measure over the antiinflammatory therapy to avoid the adsorption of both proinflammatory and antiinflammatory mediators when hemoadsorption is used indiscriminately. For example, using the monoclonal antibody of the proinflammatory cytokine mediator or its receptor to specifically pretreat brain death donor organ, unless measures are taken to avoid the occurrence of inflammation from the very beginning. Furthermore, our data show the significant probability that correlations between TGF-b1 and organ biochemistry indexes were modest at P < .05 level. This could imply that TGF-b1 was not the only contributing factor to the protection in brain death process. Thus, further research is needed to discover other contributing factors that are beneficial to organs during the process of brain death. In conclusion, TGF-b1 is associated with the protection of myocardium and kidney from injury at an early stage of brain death and may play an important role in the organ damage protection during brain death process. Thus, it is pertinent that cytokine-specific measure is taken to eliminate the harmful mediators and avoid TGF-b1 loss when protecting DBD organs. Successful implementation of this
209
will present a suitable pretreatment method that will improve the quality of DBD organs. ACKNOWLEDGMENTS The authors thank Xiang Zhu (Florida Hospital Orlando) who helped us with excellent data handling and statistical analysis.
REFERENCES [1] Ranasinghe AM, Bonser RS. Endocrine changes in brain death and transplantation. Best Pract Res Clin Endocrinol Metab 2011;25:799e812. [2] Auraen H, Mollnes TE, Bjortuft O, et al. Multiorgan procurement increases systemic inflammation in brain dead donors. Clin Transplant 2013;27:613e8. [3] Floerchinger B, Oberhuber R, Tullius SG. Effects of brain death on organ quality and transplant outcome. Transplant Rev (Orlando) 2012;26:54e9. [4] Lim KB, Schiano TD. Long-term outcome after liver transplantation. Mt Sinai J Med 2012;79:169e89. [5] Yoshimura A, Wakabayashi Y, Mori T. Cellular and molecular basis for the regulation of inflammation by TGF-beta. J Biochem 2010;147:781e92. [6] Itoh S, Itoh F. Implication of TGF-beta as a survival factor during tumour development. J Biochem 2012;151:559e62. [7] Vuong MT, Lundberg S, Gunnarsson I, et al. Genetic variation in the transforming growth factor-beta1 gene is associated with susceptibility to IgA nephropathy. Nephrol Dial Transplant 2009;24:3061e7. [8] Wang X, Nie J, Jia Z, et al. Impaired TGF-beta signalling enhances peritoneal inflammation induced by E. coli in rats. Nephrol Dial Transplant 2010;25:399e412. [9] Yanagita M. Inhibitors/antagonists of TGF-beta system in kidney fibrosis. Nephrol Dial Transplant 2012;27:3686e91. [10] Pratschke J, Wilhelm MJ, Kusaka M, et al. A model of gradual onset brain death for transplant-associated studies in rats. Transplantation 2000;69:427e30. [11] Ling F. Brain death criteria (adult). Chinese J Cerebrovasc Dis 2009;4:220e4. [12] Wan C, Feng X, Li M. Establishment of modified brain death model in pig. Tianjin Med 2014;42:318e20. [13] Rech TH, Moraes RB, Crispim D, et al. Management of the brain-dead organ donor: a systematic review and meta-analysis. Transplantation 2013;95:966e74. [14] Oltean S, Pullerits R, Floden A, et al. Increased resistin in brain dead organ donors is associated with delayed graft function after kidney transplantation. J Transl Med 2013;11:233. [15] Watts RP, Thom O, Fraser JF. Inflammatory signalling associated with brain dead organ donation: from brain injury to brain stem death and posttransplant ischaemia reperfusion injury. J Transplant 2013;2013:521369. [16] Tornatore L, Thotakura AK, Bennett J, et al. The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation. Trends Cell Biol 2012;22:557e66. [17] Gasparini C, Feldmann M. NF-kappaB as a target for modulating inflammatory responses. Curr Pharm Des 2012;18: 5735e45.
ABSTRACT Background. From the medical and ethical points of view, donation after brain death is a more acceptable organ source than that from a living donor because it has the advantage of providing multiple organs from a single donor source. Hence, it has become a more promising field of research which focuses on the protection of organs at brain death Here we investigated the role of transforming growth factor (TGF)-b1 in a porcine model of brain death. Methods. A porcine model of brain death was established by increasing the intracranial pressure (ICP) after which TGF-b1 was monitored by immunofluorescence at the following time points: before ICP was performed (t1), at brain death (t2), and at 3 (t3), 6 (t4), 9 (t5), and 18 (t6) hours after brain death. The data were analyzed using the fixed effect regression method and the correlation between the results was determined by Pearson analysis. Results. Our results showed that there was a significant increase in the levels of TGF-b1 (P < .05), urea (P < .01), creatinine (P < .01), and aspartate aminotransferase (AST; P < .01) during the 18-hour brain death process. There were negative correlations between TGF-b1 and urea, creatinine, alanine aminotransferase, AST, and total bilirubin. The negative correlations between TGF-b1 and creatinine and AST achieved statistical significance (P < .05). Conclusions. These findings taken together confirm that significant damages are caused to the myocardial fiber cell and kidney glomerulus during brain death process, and that TGF-b1 is associated with the protection of these organs.
O
RGAN TRANSPLANTATION is more available than ever before to a majority number of patients with end-stage organ failure as a curative treatment owing to the development of medicine and technology. As a result, donor shortage has become a more pressing problem in recent years as a large number of patients die every year while awaiting transplantation. From the medical and ethical point of view, organ donation after brain death (DBD) is more acceptable than that from a living donor for organ transplantation. In addition, DBD has the advantage of providing multiple organs from a single donor source. Increasingly, DBD has drawn great attention as a more reliable donor source. Brain death is a complex pathophysiologic process. Recent studies showed that brain death can cause a series of disturbances of normal homeostatic systems, which include
ª 2016 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
Transplantation Proceedings, 48, 205e209 (2016)
hemodynamic instability, hormonal impairment, significant changes in the internal environment, and inflammation. All these changes compromise the function of harvested organs [1,2]. To date, the technology to preserve DBD organs has been less than ideal. It is reported that about 25% of the potential DBD organs lose function gradually during the maintenance process, to some extent. Therefore, L.C., X.F., and Y.W. contributed equally to this work. This work was supported by National Clinical Key Specialty Project Foundation of the Ministry of Health, P.R. China (2013544) and National Natural Science Foundation of China (81101911). *Address correspondence to Hong Mu, Department of Clinical Laboratory, The First Central Hospital of Tianjin, Tianjin 300192, China. E-mail: [email protected] 0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.01.007
205
.7464 (0.6)
.0003** (6.86)
.1285 (1.87)
.0000** (10.05)
.0000** (54.47)
.021* (3.616)
1.77 0.45
37.81 7.05
41.93 3.98
77.23 7.02
4.01 0.45
Note: *P < .05; **P < .01 Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BD, brain death; TBIL, total bilirubin; TGF, transforming growth factor.
260.93 85.06 .373 (0.91) 9.53 0.95 .000** (12.68) 114.33 20.07 .001** (3.86) 38.43 2.48 .810 (0.24) 286.43 16.87 .000** (5.18) 1.77 0.51 .677 (0.42) 536.80 152.23 .07 (1.91) 6.36 0.53 .000** (7.65) 103.75 21.36 .138 (1.55) 29.65 4.64 .553 (0.6) 118.38 38.61 .000** (4.79) 1.38 0.17 .76 (0.31) 594.24 129.54 .236 (1.22) 5.99 0.36 .000** (7.98) 95.45 11.94 .007** (3.03) 33.70 3.81 .087 (1.8) 107.85 24.27 .002** (3.52) 2.72 1.02 .867 (0.17) 1224.62 419.63 .83 (0.22) 4.49 0.54 .000** (6.94) 69.31 6.02 .028* (2.37) 33.49 5.24 .013* (2.71) 86.23 20.03 .009** (2.89) 2.82 0.94 .487 (0.71) 2174.10 638.66 .026* (2.4) 4.15 0.39 .047* (2.12) 70.81 7.24 .288 (1.09) 36.06 6.01 .017* (2.61) 70.57 18.33 .031* (2.31) 2.26 0.45 .276 (1.12) 1059.82 278.14
1583.28 162.93 .396 (0.87) 4.17 0.51 .546 (0.61) 74.82 6.81 .387 (0.89) 40.752 4.39 .084 (1.82) 54.19 12.11 .133 (1.57) 2.22 0.34 .61 (0.52)
BD 18h BD 9h BD 6h BD 3h BD
TGF-b1 (pg/mL) P (t) Urea (mmol/L) P (t) Creatinine (mmol/L) P (t) ALT (IU/L) P (t) AST (IU/L) P (t) TBIL (mmol/L) P (t)
Animals were anesthetized with general anesthesia. Cannula was inserted into the pig’s femoral artery and vein, separately, for continuous mean arterial pressure (MAP) monitoring and administration of fluids after weighing, anesthesia, and skin preparation. Another cannula was opened and connected to a microrespirator for respiratory wave monitoring; electrodes were inserted to monitor electroencephalographic and electrocardiographic activity. Then, cystostomy was performed, and the parietal central area of the skull was perforated and normal saline was injected by transcatheter progressively at the speed of 0.5 mL/min; ICP and MAP were monitored at the same time. Pressurization was started when MAP > ICP and then stopped when MAP < ICP. The pressure adjustment was continued until MAP was no longer increasing with the increasing ICP. Brain death was confirmed 30 minutes after anesthesia was terminated when the model had met the following criteria: (1) reflex to the light of the pupil disappeared, (2) corneal reflex disappeared, (3) no facial and head stimulation reactions,
BBD
Brain Death Model
Table 1. Levels of TGF-b1 and Biochemistry Markers in Brain Death Model During the BD Process
Seven healthy adult Landrace pigs weighing 33.6 4.7 kg, 4 male and 3 female, were purchased from the Experimental Animal Center in Tianjin, China. The experimental pigs were housed in climatecontrolled quarters (25 1 C at 50% humidity) with a 12-hour light/12-hour dark cycle and fed with standard food. They were kept fasting for 12 hours with no water for 6 hours before the experiment started. This study was approved ethically by Department of Clinical Laboratory, The First Central Hospital of Tianjin.
Reference time
MATERIAL AND METHODS Animals
n ¼ 34
understanding how the injury occurs and how it can be ameliorated has become an important research area for the preservation of DBD donor organs [3,4]. To explore and expand the organ source, the research in DBD will or has become an important hot topic with its huge clinical potential. Transforming growth factor (TGF)-b1, a gene located at 19q13, is a peptide with 25-kD molecular weight. It is secreted by many cell types. TGF-b1 is a kind of multifunctional cytokine that acts through its signaling pathway, stops the cell cycle at the G1 stage to stop proliferation, induces differentiation, or promotes apoptosis. It causes immunosuppression and angiogenesis. TGF-b also converts effector T cells that attack abnormal cells with an inflammatory (immune) reaction into regulatory (suppressor) T cells. TGF-b1 is proven to be associated with the arteriosclerosis, cancer, fibrosis, and inflammatory responses by the clinical research over the past decade [5e9]. However, the role of TGF-b1 in brain death process is rarely reported. We hypothesized that TGF-b1 could also trigger immunosuppression to lessen inflammatory injury of tissues induced by an immunologic reaction in the brain death process. In this study, we established a pig model of brain death by using intracranial pressure (ICP) method and then monitoring the concentration of TGF-b1 and other organ biochemistry markers during the continuous 18-hour brain death period. Moreover, we explored the TGF-b1 pathophysiology in DBD model and revealed the roles it might have played in the DBD organ damage process.
CHEN, FENG, WANG ET AL
P (F)
206
TGF-b1 IN ORGAN DAMAGE PROTECTION
Fig 1. Transforming growth factor (TGF)b-1 trend in the brain death model. The overall discrepancy was significant (F ¼ 3.616; P < .05). It increased from t1 (baseline 1059.82) to t3 (2174.10; t ¼ 2; P < .05) and then decreased to a final value of 260.93 at t7. It showed the process of stress response in TGFb-1. (*P < .05) (4) no independent breathing, (5) transcranial Doppler showed that the front and posterior cycles were oscillatory wave, sharp and small contraction wave, or the blood flow signal disappeared, (6) the electroencephalograph display resting potential, and (7) the atropine test was negative [10e12].
Blood Sampling Time Points Blood samples were collected from the internal jugular vein into a tube containing the coagulative agent. One hour later, blood was centrifuged for 10 minutes at 960 g and serum was isolated and stored at 80 C in polypropylene tubes until they were assayed. The time points of blood sample collected were as follow (time groups): (1) the time after the routine surgical operation was performed and before intracranial compression was set for the reference time group (reference time, t1); (2) the time of intracranial compression at which blood pressure was showing fluctuations wave was set as before brain death time group (BBD, t2), (3) the time that the experimental pigs just met the brain death standard was designed as brain death group (BD, t3), (4) 3 hours after brain death
207
Fig 3. Creatinine (CR) trend in the brain death model. The overall discrepancy was significant (F ¼ 10.05; P < .05). CR decreased from 77.23 to 69.31 (t ¼ 2.37; P < .05) and then increased to 114.33 at t7 (t ¼ 3.03; P < .01). (*P < .05; **P < .01) was designed as 3-hour post brain death group (3h PBD, t4), (5) 6 hours after brain death was designed as 6-hour post brain death group (6h PBD, t5), (6) 9 hours after brain death was designed as 9-hour post brain death group (9h PBD, t6), and (7) 18 hours after brain death was designed as 18-hour post brain death group (18h PBD, t7).
Organ Biochemistry Marker Detection The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and concentrations of urea, creatinine, and total bilirubin (TBIL) were measured. ALT, AST, and urea were analyzed using enzymatic rate assay and creatinine and TBIL were analyzed using colorimetric assay. All analyses were performed on a Modular DPP Roche Diagnostics (Tokyo, Japan) using Roche diagnostics’ reagents.
TGFb-1 Detection The concentration of TGF-b1 was determined by immunofluorescence method on Axon Gene Pix using a Ray Biotech regent. The measurement process was according to the manufacturer’s instructions.
Statistical Analysis The concentrations or activities of TGF-b1 and organ biochemistry marker are expressed as mean values standard error. The statistical significance of every marker at the successive brain death time points was analyzed by the method of fixed-effects regression (Stats IC v.12); correlations between the results of TGF-b1 and the organ biochemistry markers at the same brain death time point were analyzed by using Pearson analysis method (SPSS v.20.0). P < .05 was considered significant.
RESULTS
Fig 2. Urea trend in brain-death model. The overall discrepancy was significant (F ¼ 54.47, P < .01). It increased from t1 to t7 (4.01 to 9.53; t ¼ 2.12 to 12.68, P < .01). It showed the irreversible changes of urea in the brain death model. (*P < .05; **P < .01)
Values and changes of serum TGF-b1, and organ biochemistry markers in brain death model during t1et7 time-points are shown in Table 1. From the F test analysis, significant differences were observed in the concentration or activity of TGF-b1, urea, creatinine, and AST at consecutive
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Fig 4. Aspartate aminotransferase (AST) trend in the brain death model. The overall discrepancy was significant (F ¼ 6.86; P < .01). AST increased from 37.81 to 286.43, that showed an irreversible damage of myocardial cells in the brain death models. (*P < .05; **P < .01)
time points, but not in that of ALT and TBIL. The significant point was further analyzed by t test, and a significant difference in TGF-b1 was observed at the time of brain death. Urea and AST showed significant differences from the time brain death occurred, so did creatinine after a delay of 1 time point. ALT showed 2 significant differences at both the time of brain death and 3 hours after brain death. TBIL presented no difference during the whole 18-hour brain death process. The general information tendency of TGF-b1 and biochemistry markers at continuous brain death process in Porcine Brain Dead Model are shown in Figs 1e6. The significant increase in urea and creatinine (P < .01) indicated that the glomerular filtration rate was attenuated during the process as though creatinine was 1 time point delayed. The sharp increase in AST (P < .01) activity showed that irreversible damage was caused to myocardial cells. From our observation of each curve, it seems that urea, creatinine, and AST changed in the opposite direction to that of TGF-b1.
CHEN, FENG, WANG ET AL
Fig 6. Total bilirubin (TBIL) trend in the brain death model. The overall discrepancy had no significance (F ¼ 0.6; P > .05), indicating that the hepatobiliary metabolism was mildly changed throughout the brain death process.
Results of the correlation analysis between TGF-b1 and other organ biochemistry markers at same time point are shown in Table 2. A significant negative correlation was observed between TGF-b1 and creatinine and AST. The negative correlation between TGF-b1 and urea just reached statistical significance at the .05 level. This could mean that TGF-b1 had a beneficial effect on the myocardium and renal glomerulus. DISCUSSION
Based on previous studies [1e4,13e15], significant complex pathophysiologic changes occurred at the time of brain death. These changes involve (1) sympathetic nerves excitement, (2) unbalance of hypothalamusepituitary and adrenocortical hormones, (3) hemodynamic instability, and (4) metabolic disorder and cytokine release, which reach their peak separately depending on the type of reaction and the seriousness of the injury during the brain death process. No matter how brain death is achieved, a proinflammatory environment always occurs. Essentially, these changes collectively induce irreversible damage to the organs. Cytokines play an important role in immunity, apoptosis, angiogenesis, and cell growth and differentiation in physiologic processes. They are involved in interactions between different cells; in particular, they could cross a disrupted bloodebrain barrier, activate complement and increase tissue permeability as cells respond to the brain death
Table 2. Correlation Between TGF-b1 and Organ Biochemistry Marker at Corresponding Time Point
Fig 5. Alanine aminotransferase (ALT) trend in the brain death model. ALT changed mildly (F ¼ 1.87; P > .05). It showed that there was little damage caused to the hepatocytes during brain death process. (*P < .05)
R P
Urea
Creatinine
ALT
AST
TBIL
0.360 .051
0.372 .043*
0.005 .980
0.404 .027*
0.198 .294
*P < .05. Abbreviation: BBD, before brain death time group.
TGF-b1 IN ORGAN DAMAGE PROTECTION
[1,2,15e17]. According to Ryan et al. [15], TGF-b1 may regulate T-cell survival and function; it works as a suppressor of interferon-geinduced macrophage upregulation and chemokine and cytokine generation. In our gradual induction of brain death in the pig model, the significantly increased concentration of urea and creatinine suggest that there was a decrease in the glomerular filtration rate throughout the brain death process. The sharp increase in urea could also be attributed to an increased protein degradation owing to tissue damage; a decrease in creatinine at t4 time point could be owing to a reduction in antidiuretic hormone while maintaining the adequate function of glomerulus at that time [1e3]. As to the 2 transferases, it seems to be somewhat paradoxical at first sight that there was a significant increase in aspartate transferase but not alanine transfer enzyme. However, it does suggest that there was only a mild damage to the hepatocytes. The increased activity of aspartate transferase could come from myocardial cell because the heart is another important source of aspartate transferase. In our study, TGF-b1 showed a significant negative correlation with the levels of AST and creatinine, suggesting that TGF-b1 is associated with the protection of myocardial fiber cells and kidney glomerulus. This result supports the findings of the previous studies that TGF-b1 may play a role as beneficial molecular media in brain death process [1,15]. This means that the protective mediator could be released massively when brain death occurs. Also, it demonstrates the multidimensional nature of stress response. Hence, it is better to choose a more mediator-specific measure over the antiinflammatory therapy to avoid the adsorption of both proinflammatory and antiinflammatory mediators when hemoadsorption is used indiscriminately. For example, using the monoclonal antibody of the proinflammatory cytokine mediator or its receptor to specifically pretreat brain death donor organ, unless measures are taken to avoid the occurrence of inflammation from the very beginning. Furthermore, our data show the significant probability that correlations between TGF-b1 and organ biochemistry indexes were modest at P < .05 level. This could imply that TGF-b1 was not the only contributing factor to the protection in brain death process. Thus, further research is needed to discover other contributing factors that are beneficial to organs during the process of brain death. In conclusion, TGF-b1 is associated with the protection of myocardium and kidney from injury at an early stage of brain death and may play an important role in the organ damage protection during brain death process. Thus, it is pertinent that cytokine-specific measure is taken to eliminate the harmful mediators and avoid TGF-b1 loss when protecting DBD organs. Successful implementation of this
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will present a suitable pretreatment method that will improve the quality of DBD organs. ACKNOWLEDGMENTS The authors thank Xiang Zhu (Florida Hospital Orlando) who helped us with excellent data handling and statistical analysis.
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