- Email: [email protected]
Influence of Ischemia Time in Injury of Deep Peribiliary Glands of the Bile Ducts Graft: A Prospective Study
Influence of Ischemia Time in Injury of Deep Peribiliary Glands of the Bile Ducts Graft: A Prospective Study D. Diogoa,*, C. Pachecob, R. Oliveirac, R. Martinsa, P. Oliveiraa, M.A. Ciprianoc, J.G. Tralhãod, and E. Furtadoa a Adult and Paediatric Liver Transplantation Unit, Coimbra Hospital and University Centre, Coimbra, Portugal; bFaculty of Medicine, University of Coimbra, Coimbra, Portugal; cDepartment of Pathologic Anatomy, Coimbra Hospital and University Centre, Coimbra, Portugal; and dDepartment of Surgery, Coimbra Hospital and University Centre, Coimbra, Portugal
ABSTRACT The deep peribiliary glands (DPBG) are a niche of progenitor cells in the wall of the biliary duct (BD) and are the second line of multiplication when severe lesion of the epithelium occurs. Previous studies have identified DPBG injury as a cause of post-liver transplant (LT) biliary stenosis; this complication is a major cause of post-LT morbidity. The incidence of biliary stenosis in our center is high (38.1%). This study evaluates the lesion of DPBG in response to ischemia. Graft BD was collected in adult LT between August 2016-July 2017, from donation after brain death. Samples of 45 grafts were collected at 2 moments: BD1during graft preparation and BD2-before biliary anastomosis. Histological analysis of the samples was performed and then classified according to degree of lesion (0, 50%, and >50%). A comparison was made between the degree of lesion and graft ischemia, graft histology, donor, and procurement variables. The DPBG lesion was more frequent in BD2 (20.9% vs 7%, P ¼ .079). BD2 lesions with DPBG lesions had higher medians and means at all times of ischemia. The difference was greater in the warm ischemia time (0: 43.3 12.53 minutes vs 50%: 52.4 14.38 minutes, P ¼ .068). The group of BD1 with DPBG lesion presented superior median cold ischemia time (CIT). In the analysis of the remaining variables there were also no statistically significant differences. We concluded that during the period of CIT there is already lesion of the DPBG, which increases after reperfusion of the graft, in greater association with longer warm ischemia time.
B
ILIARY complications are the main cause of post-liver transplant (LT) morbidity and mortality, with a negative impact on graft survival, leading to significant costs and decreased quality of life for patients [1,2]. The high frequency and severity of this pathology led some authors to call it the “Achilles heel” of liver transplantation [3,4]. The etiology of non-anastomotic strictures (NAS) postLT is multifactorial [1,2,5]. We know that cholangiocytes are cells sensitive to ischemia and that their death leads to the loss of bile duct epithelium [4,6,7]. For its regeneration, in addition to oxygen and nutrients, a proliferative reserve, consisting of deep peribiliary glands (DPBG), is required. These glands are a niche of progenitor and pluripotential cells, which exist in the fibromuscular layer of biliary duct (BD) and are the second line of multiplication when severe lesion of the epithelium occurs [8,9].
ª 2019 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1545e1548 (2019)
We consider that our unit has a high rate of biliary complications after LT, with 16.7% of our patients developing NAS. AIM
The purpose of this study is to evaluate the influence of ischemia on the injury of the DPBG of the BD of the liver grafts, through an exploratory and transversal study included in a larger prospective study.
*Address correspondence to Dulce Diogo, Coimbra Hospital and University Centre, Praceta Prof. Mota Pinto, 3000-075 Coimbra, Portugal. E-mail: [email protected]. pt 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.01.041
1545
1546
DIOGO, PACHECO, OLIVEIRA ET AL
MATERIAL AND METHODS
Table 2. Graft Ischemia and Reperfusion Times (Minutes)
This is an unicentric study, performed in the Pediatric and Adult Liver Transplantation Unit with collaboration of the Department of Pathologic Anatomy of Coimbra Hospital and University Centre. During one year (August 1, 2016-July 31,2017), a systematic collection of BD tissue samples in liver grafts for transplantation was performed in adult recipients, exclusively from donation after brain death. It was only performed in cases where it was possible to do it, without causing graft damage (45 of a total of 60 grafts). The same preservation solution was used in all cases (Celsior). Samples were collected sequentially in 2 periods: the first sample we designated as BD1 and was collected during the preparation of the graft in the back table; the second sample (BD2) after re-perfusion of the graft immediately before making the bile anastomosis. The collection was performed without cauterization and the time of the procedure was recorded. Each sample was placed in a 4% formaldehyde container and sent to the Department of Pathologic Anatomy.
Histological Evaluation of Bile Duct Samples Total inclusion of each sample was performed and observed in 4 mm sections stained with hematoxylin and eosin using a Nikon Eclipse 50i optical microscope. Images were taken with a Nikon-Digital Sight DS-Fi1 camera. The microscopic evaluation included a categorization of the quality of the material in good, acceptable, and bad quality, excluding the last from the study. Subsequently, histological parameters were analyzed, categorized, and adapted to those described by Op den Dries et al [10] (Table 1).
Ischemia Times and Reperfusion Times We calculated the cold ischemia time (CIT) and the warm ischemia time of the graft and 2 additional times were conceptualized: the CIT0 (since the time of beginning of CIT to the time of collection of BD1) and reperfusion time (RT) 1 and RT2 (since the time of reperfusion of the first and second vessels in LT to the time of collection of BD2). The search and the analysis of the donor and procurement variables were also performed: age, ABO and Rh group, cause of death, hemodynamic stability, perfusion volume of the preservation solution; graft histology of after reperfusion: steatosis, fibrosis and hemosiderosis, and ischemia/reperfusion injury (IRI). These variables were stratified by groups.
Statistical Analysis We used IBM SPSS Statistics 24 software (IBM, Armonk, NY, United States) for the statistical treatment of data. It was applied as significance level or P value of .05, with 95% confidence interval. Qualitative variables were characterized according to the absolute and relative frequency. Using the Shapiro-Wilk test, the distribution of the quantitative variables was classified as normal or asymmetric, respectively, by the mean and standard deviation or the Table 1. Deep Peribiliary Glands Injury Classification Injury Grade
0 1 2
Deep Peribiliary Glands Injury
0% 50% >50% (Adapted from Op den Dries et al [10].)
CIT0 (n ¼ 39) CIT (n ¼ 44) WIT1 (n ¼ 44) TIT (n ¼ 44) WIT2 (n ¼ 44) RT1 (n ¼ 32) RT2 (n ¼ 32)
Median (IQR)/Mean (SD)
Min.eMax.
225 (190e263) 320.5 (290.5e366.0) 49.5 (38.5e61) 382.5 (335.3e426) 44.9 12.42 140 (125.0e164.0) 91 (76.3e111.75)
144e525 138e670 30e181 178e707 13e77 64e350 37e297
Abbreviations: CIT, cold ischemia time; CIT0, cold ischemia time until colleting the first bile duct sample; IQR, interquartile range; RT1, reperfusion time since reperfusion of the first vessel until colleting the second bile duct sample; RT2, reperfusion time since reperfusion of the second vessel until colleting the second bile duct sample; SD, standard deviation; TIT, total ischemia time (CIT þ WIT1); WIT1, warm ischemia time until reperfusion of the first vessel; WIT2, warm ischemia time since reperfusion of the first vessel until reperfusion of the second vessel.
median and interquartile range (P25-P75). Parametric and nonparametric tests were used to compare the variables. Qualitative variables were compared according to the c2 test or Fisher’s exact test. To compare dichotomous variables, the Student t test or the Mann-Whitney test were used, and in the case of variables with three or more categories, the Kruskal-Wallis test was used.
RESULTS
Collections were performed on 45 grafts: 45 BD1 and 44 BD2, and a BD2 was excluded due to poor sample quality. The median age of the donors was 61 years (52e70) and 62.2% were male. 46.7% of the donors were of group A and 86.6% presented hemodynamic instability with need for amines. 72.8% of the grafts presented IRI on postreperfusion biopsy. The graft ischemia and reperfusion times are described in Table 2. The BD1 group with grade 1 DPBG injury presented a median time of CIT superior to the group without injury (255 vs 220.5 minutes), with only 3 cases presenting injury and statistical significance not being obtained (P ¼ .20). The BD2 samples had a higher percentage of DPBG injury than BD1, BD1: 7% vs BD2: 20.9%, with P ¼ .079 (Table 3). The BD2 samples with DPBG injury had higher medians and means in all ischemia and reperfusion times, the most relevant difference being in warm ischemia time 2 (grade 0: 43.3 12.53 minutes vs 50%: 52.4 14.38 minutes), with P ¼ .068 (Table 4). For the remaining variables, the study groups were homogeneous. DISCUSSION AND CONCLUSIONS
In this series, we observed that lesions of DPBG already occur during the CIT period (the first sample of BD to present Table 3. Comparison of the Degree of DPBG Injury Between BD1 and BD2
DPBG injury
Injury Grade
BD1 (n ¼ 43)
BD2 (n ¼ 43)
P Value
0 1 2
93% (n ¼ 40) 4.7% (n ¼ 2) 2.3% (n ¼ 1)
79.1% (n ¼ 34) 20.9% (n ¼ 9)
.079*
Abbreviations: BD, bile duct sample; DPBG, deep peribiliary glands. *c2 test.
ISCHEMIA TIME AND DPBG INJURY
1547
Table 4. Comparison of the Ischemic and Reperfusion Times and the Lesion of the Deep Peribiliary Glands in BD2 Deep Peribiliary Glands Injury
CIT, median (IQR) WIT1, median (IQR) WIT2, mean (SD) TIT, median (IQR) RT1, median (IQR) RT2, median (IQR)
0
1
P Value
320 (290.5e365.8) (n ¼ 33) 51.5 (39e61.8) (n ¼ 33) 43.3 12.53 (n ¼ 34) 373.5 (335.3e426) (n ¼ 33)
346.5 (290.3e365.3) (n ¼ 9) 42 (39.5e66) (n ¼ 9) 52.4 14.38 (n ¼ 8) 387 (347e407.3) (n ¼ 9) 141.5 (123.8e160.5) (n ¼ 6) 91 (80.8e109.5) (n ¼ 6)
.713* .443*
139 (117.5e164) (n ¼ 25) 87.5 (75.3e109.3) (n ¼ 25)
.068† .550* 1* .920*
Abbreviations: CIT, cold ischemia time; IQR, interquartile range; RT1, reperfusion time since reperfusion of the first vessel until colleting the second bile duct sample; RT2, reperfusion time since reperfusion of the second vessel until colleting the second bile duct sample; TIT, total ischemia time (CIT þ WIT1); WIT1, warm ischemia time until reperfusion of the first vessel; WIT2, warm ischemia time since reperfusion of the first vessel until reperfusion of the second vessel. *Mann-Whitney test. † Student t test.
damage was collected and fixed at 225 minutes CIT) and that the percentage of samples with glandular injury increased after graft reperfusion. Cholangiocytes are cells sensitive to anoxia, but are even more sensitive to re-oxygenation/reperfusion [4], by the exponential increase in the production of reactive oxygen species that damages the membranes, the deoxyribonucleic acid, and causes cell death by apoptosis [7,11]. We obtained a difference between BD1 and BD2 with respect to the volume of lesion of the DPBG, with P value approaching statistical significance (P ¼ .079). Comparing these results with those of the study we considered the reference in this field, we found that Op den Dries et al [10], did not obtain differences between the BD group collected at the end of the CIT and the BD group collected after reperfusion of the graft (P ¼ .493). In addition, the injury of DPBG in both samples is considerably higher than those identified in the present study. The explanation may be related to the CIT difference between the 2 works; our BD1 sample has been subjected to a considerably lower CIT (225 vs 406 minutes) and also because Op den Dries et al also used BD samples collected in grafts also from donation after cardiac death [10]. Our series, although smaller than that presented by Op den Dries et al, is more homogeneous; it is a unicentric study, all the grafts were from donation after brain death, the same preservation solution was used in all cases and in 43 of the 45 BD analyzed, we obtained 2 samples collected sequentially, and it was possible to evaluate with lower bias the evolution of the DPBG injury since CIT to the postreperfusion period of the graft. Previous studies have demonstrated a relationship between the histological injury of the BD of the graft in response to the IRI and the development of NAS after liver transplantation [12,13]. Op den Dries et al, were the first to demonstrate this relationship, through the analysis of the injury of the DPBG, and found that the increase of the degree of damage of the DPBG corresponds to an increase in the incidence of NAS [10,14].
We can conclude from the analysis of the present series that, after the CIT period, there is already a lesion of the DPBG and that aggravates after the re-perfusion of the graft and that the lesion of the DPBG is greater in association with longer times of ischemia, mainly with warm ischemia time 2, with the difference between groups approaching statistical significance. This study is ongoing in our unit, so the size of the sample keeps increasing. Taking into account the results of the previous studies mentioned, the next step will be to compare the lesion of the DPBG of our series with the development of NAS. REFERENCES [1] De Vries Y, von Meijenfeldt FA, Porte RJ. Post-transplant cholangiopathy: classification, pathogenesis, and preventive strategies. Biochim Biophys Acta Mol Basis Dis 2018;1864(4 Pt B): 1507e15. [2] Mourad MM, Algarni A, Liossis C, Bramhall SR. Aetiology and risk factors of ischaemic cholangiopathy after liver transplantation. World J Gastroenterol 2014;20:6159e69. [3] Koneru B, Sterling MJ, Bahramipour PF. Bile duct strictures after liver transplantation: a changing landscape of the Achilles’ heel. Liver Transpl 2006;12:702e4. [4] Ryu CH, Lee SK. Biliary strictures after liver transplantation. Gut Liver 2011;5:133e42. [5] Cursio R, Gugenheim J. Ischemia-reperfusion injury and ischemic-type biliary lesions following liver transplantation. J Transplant 2012:164329. [6] Yoo KS, Lim WT, Choi HS. Biology of cholangiocytes: from bench to bedside. Gut Liver 2016;10:687e98. [7] Zhai Y, Petrowsky H, Hong JC, Busutil RW, KupiecWeglinski JW. Ischaemia-reperfusion injury in liver transplantationfrom bench to bedside. Nat Rev Gastroenterol Hepatol 2013;10: 79e89. [8] Carpino G, Cardinale V, Onori P, Franchitto A, Berloco PB, Rossi M, et al. Biliary tree stem/progenitor cells in glands of extrahepatic and intrahepatic bile ducts: an anatomical in situ study yielding evidence of maturation lineages. J Anat 2012;220:186e99. [9] Cardinale V, Wang Y, Carpino G, Mendel G, Alpini G, Gaudio E, et al. The biliary tree - a reservoir of multipotent stem cells. Nat Rev Gastroenterol Hepatol 2012;9:231e40.
1548 [10] Op den Dries S, Westerkamp AC, Karimian N, Gouw AS, Bruinsma BG, Markmann JF, et al. Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures. J Hepatol 2014;60:1172e9. [11] Martins RM, Teodoro JS, Furtado E, Rolo AP, Palmeira CM, Tralhão JG. Recent insights into mitochondrial targeting strategies in liver transplantation. Int J Med Sci 2018;15:248e56. [12] Hansen T, Hollemann D, Pitton MB, Heise M, HoppeLotichius M, Schuchmann M, et al. Histological examination and evaluation of donor bile ducts received during orthotopic liver
DIOGO, PACHECO, OLIVEIRA ET AL transplantation–a morphological clue to ischemic-type biliary lesion? Virchows Arch 2012;461:41e8. [13] Brunner SM, Junger H, Ruemmele P, Schnitzbauer AA, Doenecke A, Kirchner GI, et al. Bile duct damage after cold storage of deceased donor livers predicts biliary complications after liver transplantation. J Hepatol 2013;58:1133e9. [14] Karimian N, Op den Dries S, Porte RJ. The origin of biliary stricures after liver transplantation: is it the amount of epithelial injury or insufficient regeneration that counts? J Hepatol 2013;58: 1065e7.
ABSTRACT The deep peribiliary glands (DPBG) are a niche of progenitor cells in the wall of the biliary duct (BD) and are the second line of multiplication when severe lesion of the epithelium occurs. Previous studies have identified DPBG injury as a cause of post-liver transplant (LT) biliary stenosis; this complication is a major cause of post-LT morbidity. The incidence of biliary stenosis in our center is high (38.1%). This study evaluates the lesion of DPBG in response to ischemia. Graft BD was collected in adult LT between August 2016-July 2017, from donation after brain death. Samples of 45 grafts were collected at 2 moments: BD1during graft preparation and BD2-before biliary anastomosis. Histological analysis of the samples was performed and then classified according to degree of lesion (0, 50%, and >50%). A comparison was made between the degree of lesion and graft ischemia, graft histology, donor, and procurement variables. The DPBG lesion was more frequent in BD2 (20.9% vs 7%, P ¼ .079). BD2 lesions with DPBG lesions had higher medians and means at all times of ischemia. The difference was greater in the warm ischemia time (0: 43.3 12.53 minutes vs 50%: 52.4 14.38 minutes, P ¼ .068). The group of BD1 with DPBG lesion presented superior median cold ischemia time (CIT). In the analysis of the remaining variables there were also no statistically significant differences. We concluded that during the period of CIT there is already lesion of the DPBG, which increases after reperfusion of the graft, in greater association with longer warm ischemia time.
B
ILIARY complications are the main cause of post-liver transplant (LT) morbidity and mortality, with a negative impact on graft survival, leading to significant costs and decreased quality of life for patients [1,2]. The high frequency and severity of this pathology led some authors to call it the “Achilles heel” of liver transplantation [3,4]. The etiology of non-anastomotic strictures (NAS) postLT is multifactorial [1,2,5]. We know that cholangiocytes are cells sensitive to ischemia and that their death leads to the loss of bile duct epithelium [4,6,7]. For its regeneration, in addition to oxygen and nutrients, a proliferative reserve, consisting of deep peribiliary glands (DPBG), is required. These glands are a niche of progenitor and pluripotential cells, which exist in the fibromuscular layer of biliary duct (BD) and are the second line of multiplication when severe lesion of the epithelium occurs [8,9].
ª 2019 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1545e1548 (2019)
We consider that our unit has a high rate of biliary complications after LT, with 16.7% of our patients developing NAS. AIM
The purpose of this study is to evaluate the influence of ischemia on the injury of the DPBG of the BD of the liver grafts, through an exploratory and transversal study included in a larger prospective study.
*Address correspondence to Dulce Diogo, Coimbra Hospital and University Centre, Praceta Prof. Mota Pinto, 3000-075 Coimbra, Portugal. E-mail: [email protected]. pt 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.01.041
1545
1546
DIOGO, PACHECO, OLIVEIRA ET AL
MATERIAL AND METHODS
Table 2. Graft Ischemia and Reperfusion Times (Minutes)
This is an unicentric study, performed in the Pediatric and Adult Liver Transplantation Unit with collaboration of the Department of Pathologic Anatomy of Coimbra Hospital and University Centre. During one year (August 1, 2016-July 31,2017), a systematic collection of BD tissue samples in liver grafts for transplantation was performed in adult recipients, exclusively from donation after brain death. It was only performed in cases where it was possible to do it, without causing graft damage (45 of a total of 60 grafts). The same preservation solution was used in all cases (Celsior). Samples were collected sequentially in 2 periods: the first sample we designated as BD1 and was collected during the preparation of the graft in the back table; the second sample (BD2) after re-perfusion of the graft immediately before making the bile anastomosis. The collection was performed without cauterization and the time of the procedure was recorded. Each sample was placed in a 4% formaldehyde container and sent to the Department of Pathologic Anatomy.
Histological Evaluation of Bile Duct Samples Total inclusion of each sample was performed and observed in 4 mm sections stained with hematoxylin and eosin using a Nikon Eclipse 50i optical microscope. Images were taken with a Nikon-Digital Sight DS-Fi1 camera. The microscopic evaluation included a categorization of the quality of the material in good, acceptable, and bad quality, excluding the last from the study. Subsequently, histological parameters were analyzed, categorized, and adapted to those described by Op den Dries et al [10] (Table 1).
Ischemia Times and Reperfusion Times We calculated the cold ischemia time (CIT) and the warm ischemia time of the graft and 2 additional times were conceptualized: the CIT0 (since the time of beginning of CIT to the time of collection of BD1) and reperfusion time (RT) 1 and RT2 (since the time of reperfusion of the first and second vessels in LT to the time of collection of BD2). The search and the analysis of the donor and procurement variables were also performed: age, ABO and Rh group, cause of death, hemodynamic stability, perfusion volume of the preservation solution; graft histology of after reperfusion: steatosis, fibrosis and hemosiderosis, and ischemia/reperfusion injury (IRI). These variables were stratified by groups.
Statistical Analysis We used IBM SPSS Statistics 24 software (IBM, Armonk, NY, United States) for the statistical treatment of data. It was applied as significance level or P value of .05, with 95% confidence interval. Qualitative variables were characterized according to the absolute and relative frequency. Using the Shapiro-Wilk test, the distribution of the quantitative variables was classified as normal or asymmetric, respectively, by the mean and standard deviation or the Table 1. Deep Peribiliary Glands Injury Classification Injury Grade
0 1 2
Deep Peribiliary Glands Injury
0% 50% >50% (Adapted from Op den Dries et al [10].)
CIT0 (n ¼ 39) CIT (n ¼ 44) WIT1 (n ¼ 44) TIT (n ¼ 44) WIT2 (n ¼ 44) RT1 (n ¼ 32) RT2 (n ¼ 32)
Median (IQR)/Mean (SD)
Min.eMax.
225 (190e263) 320.5 (290.5e366.0) 49.5 (38.5e61) 382.5 (335.3e426) 44.9 12.42 140 (125.0e164.0) 91 (76.3e111.75)
144e525 138e670 30e181 178e707 13e77 64e350 37e297
Abbreviations: CIT, cold ischemia time; CIT0, cold ischemia time until colleting the first bile duct sample; IQR, interquartile range; RT1, reperfusion time since reperfusion of the first vessel until colleting the second bile duct sample; RT2, reperfusion time since reperfusion of the second vessel until colleting the second bile duct sample; SD, standard deviation; TIT, total ischemia time (CIT þ WIT1); WIT1, warm ischemia time until reperfusion of the first vessel; WIT2, warm ischemia time since reperfusion of the first vessel until reperfusion of the second vessel.
median and interquartile range (P25-P75). Parametric and nonparametric tests were used to compare the variables. Qualitative variables were compared according to the c2 test or Fisher’s exact test. To compare dichotomous variables, the Student t test or the Mann-Whitney test were used, and in the case of variables with three or more categories, the Kruskal-Wallis test was used.
RESULTS
Collections were performed on 45 grafts: 45 BD1 and 44 BD2, and a BD2 was excluded due to poor sample quality. The median age of the donors was 61 years (52e70) and 62.2% were male. 46.7% of the donors were of group A and 86.6% presented hemodynamic instability with need for amines. 72.8% of the grafts presented IRI on postreperfusion biopsy. The graft ischemia and reperfusion times are described in Table 2. The BD1 group with grade 1 DPBG injury presented a median time of CIT superior to the group without injury (255 vs 220.5 minutes), with only 3 cases presenting injury and statistical significance not being obtained (P ¼ .20). The BD2 samples had a higher percentage of DPBG injury than BD1, BD1: 7% vs BD2: 20.9%, with P ¼ .079 (Table 3). The BD2 samples with DPBG injury had higher medians and means in all ischemia and reperfusion times, the most relevant difference being in warm ischemia time 2 (grade 0: 43.3 12.53 minutes vs 50%: 52.4 14.38 minutes), with P ¼ .068 (Table 4). For the remaining variables, the study groups were homogeneous. DISCUSSION AND CONCLUSIONS
In this series, we observed that lesions of DPBG already occur during the CIT period (the first sample of BD to present Table 3. Comparison of the Degree of DPBG Injury Between BD1 and BD2
DPBG injury
Injury Grade
BD1 (n ¼ 43)
BD2 (n ¼ 43)
P Value
0 1 2
93% (n ¼ 40) 4.7% (n ¼ 2) 2.3% (n ¼ 1)
79.1% (n ¼ 34) 20.9% (n ¼ 9)
.079*
Abbreviations: BD, bile duct sample; DPBG, deep peribiliary glands. *c2 test.
ISCHEMIA TIME AND DPBG INJURY
1547
Table 4. Comparison of the Ischemic and Reperfusion Times and the Lesion of the Deep Peribiliary Glands in BD2 Deep Peribiliary Glands Injury
CIT, median (IQR) WIT1, median (IQR) WIT2, mean (SD) TIT, median (IQR) RT1, median (IQR) RT2, median (IQR)
0
1
P Value
320 (290.5e365.8) (n ¼ 33) 51.5 (39e61.8) (n ¼ 33) 43.3 12.53 (n ¼ 34) 373.5 (335.3e426) (n ¼ 33)
346.5 (290.3e365.3) (n ¼ 9) 42 (39.5e66) (n ¼ 9) 52.4 14.38 (n ¼ 8) 387 (347e407.3) (n ¼ 9) 141.5 (123.8e160.5) (n ¼ 6) 91 (80.8e109.5) (n ¼ 6)
.713* .443*
139 (117.5e164) (n ¼ 25) 87.5 (75.3e109.3) (n ¼ 25)
.068† .550* 1* .920*
Abbreviations: CIT, cold ischemia time; IQR, interquartile range; RT1, reperfusion time since reperfusion of the first vessel until colleting the second bile duct sample; RT2, reperfusion time since reperfusion of the second vessel until colleting the second bile duct sample; TIT, total ischemia time (CIT þ WIT1); WIT1, warm ischemia time until reperfusion of the first vessel; WIT2, warm ischemia time since reperfusion of the first vessel until reperfusion of the second vessel. *Mann-Whitney test. † Student t test.
damage was collected and fixed at 225 minutes CIT) and that the percentage of samples with glandular injury increased after graft reperfusion. Cholangiocytes are cells sensitive to anoxia, but are even more sensitive to re-oxygenation/reperfusion [4], by the exponential increase in the production of reactive oxygen species that damages the membranes, the deoxyribonucleic acid, and causes cell death by apoptosis [7,11]. We obtained a difference between BD1 and BD2 with respect to the volume of lesion of the DPBG, with P value approaching statistical significance (P ¼ .079). Comparing these results with those of the study we considered the reference in this field, we found that Op den Dries et al [10], did not obtain differences between the BD group collected at the end of the CIT and the BD group collected after reperfusion of the graft (P ¼ .493). In addition, the injury of DPBG in both samples is considerably higher than those identified in the present study. The explanation may be related to the CIT difference between the 2 works; our BD1 sample has been subjected to a considerably lower CIT (225 vs 406 minutes) and also because Op den Dries et al also used BD samples collected in grafts also from donation after cardiac death [10]. Our series, although smaller than that presented by Op den Dries et al, is more homogeneous; it is a unicentric study, all the grafts were from donation after brain death, the same preservation solution was used in all cases and in 43 of the 45 BD analyzed, we obtained 2 samples collected sequentially, and it was possible to evaluate with lower bias the evolution of the DPBG injury since CIT to the postreperfusion period of the graft. Previous studies have demonstrated a relationship between the histological injury of the BD of the graft in response to the IRI and the development of NAS after liver transplantation [12,13]. Op den Dries et al, were the first to demonstrate this relationship, through the analysis of the injury of the DPBG, and found that the increase of the degree of damage of the DPBG corresponds to an increase in the incidence of NAS [10,14].
We can conclude from the analysis of the present series that, after the CIT period, there is already a lesion of the DPBG and that aggravates after the re-perfusion of the graft and that the lesion of the DPBG is greater in association with longer times of ischemia, mainly with warm ischemia time 2, with the difference between groups approaching statistical significance. This study is ongoing in our unit, so the size of the sample keeps increasing. Taking into account the results of the previous studies mentioned, the next step will be to compare the lesion of the DPBG of our series with the development of NAS. REFERENCES [1] De Vries Y, von Meijenfeldt FA, Porte RJ. Post-transplant cholangiopathy: classification, pathogenesis, and preventive strategies. Biochim Biophys Acta Mol Basis Dis 2018;1864(4 Pt B): 1507e15. [2] Mourad MM, Algarni A, Liossis C, Bramhall SR. Aetiology and risk factors of ischaemic cholangiopathy after liver transplantation. World J Gastroenterol 2014;20:6159e69. [3] Koneru B, Sterling MJ, Bahramipour PF. Bile duct strictures after liver transplantation: a changing landscape of the Achilles’ heel. Liver Transpl 2006;12:702e4. [4] Ryu CH, Lee SK. Biliary strictures after liver transplantation. Gut Liver 2011;5:133e42. [5] Cursio R, Gugenheim J. Ischemia-reperfusion injury and ischemic-type biliary lesions following liver transplantation. J Transplant 2012:164329. [6] Yoo KS, Lim WT, Choi HS. Biology of cholangiocytes: from bench to bedside. Gut Liver 2016;10:687e98. [7] Zhai Y, Petrowsky H, Hong JC, Busutil RW, KupiecWeglinski JW. Ischaemia-reperfusion injury in liver transplantationfrom bench to bedside. Nat Rev Gastroenterol Hepatol 2013;10: 79e89. [8] Carpino G, Cardinale V, Onori P, Franchitto A, Berloco PB, Rossi M, et al. Biliary tree stem/progenitor cells in glands of extrahepatic and intrahepatic bile ducts: an anatomical in situ study yielding evidence of maturation lineages. J Anat 2012;220:186e99. [9] Cardinale V, Wang Y, Carpino G, Mendel G, Alpini G, Gaudio E, et al. The biliary tree - a reservoir of multipotent stem cells. Nat Rev Gastroenterol Hepatol 2012;9:231e40.
1548 [10] Op den Dries S, Westerkamp AC, Karimian N, Gouw AS, Bruinsma BG, Markmann JF, et al. Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures. J Hepatol 2014;60:1172e9. [11] Martins RM, Teodoro JS, Furtado E, Rolo AP, Palmeira CM, Tralhão JG. Recent insights into mitochondrial targeting strategies in liver transplantation. Int J Med Sci 2018;15:248e56. [12] Hansen T, Hollemann D, Pitton MB, Heise M, HoppeLotichius M, Schuchmann M, et al. Histological examination and evaluation of donor bile ducts received during orthotopic liver
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