Recent technical innovations in the field of liver transplantation have been driven by the critical shortage of cadaveric organs.1–3 It is hoped that the increasing number of adult living donor liver transplants (LDLT) and split liver transplants being performed annually will significantly reduce the number of deaths on the liver transplant waiting list. However, split liver transplantation for adults is hindered by the logistics of dividing a perfect donor liver to provide grafts of sufficient size for two small adult recipients of the same blood group. The split technique has the additional problem of prolonged cold ischemia times unless the liver is split in-situ. Currently LDLT is the technique with the greatest potential to address the organ shortage crisis but its widespread application is limited by the volume of liver that can be safely resected from a living donor, while at the same time providing a graft of sufficient size for the recipient. The size of graft required for successful liver transplantation is 30–40% of the expected liver volume for the recipient or 0.8–1.0% of the body weight.4 For most adult recipients of an LDLT graft, this will require a right lobe graft from another adult. Given the smaller volume of the left lobe graft it is most suitable for small adults or larger pediatric recipients, even when the caudate process is included.5, 6 Although grafts of 30% liver volume (0.8% body weight) or less have been transplanted successfully they are considered marginal or “small-for-size” and are associated with an increased incidence of complications and graft failure.7, 8 In addition, it is clear that even grafts greater than 40% liver volume or greater than 1% body weight can fail in recipients with severe portal hypertension or relative impedance to hepatic venous drainage.9–12 In both situations, the term small-for-size syndrome applies. This syndrome was first described by Starzl and colleagues in the laboratory13, 14 and then clinically,15 and is characterized by coagulopathy, ascites, and prolonged cholestasis. A better understanding of the pathophysiology of the small-for-size graft may lead to logical approaches to improving subsequent allograft function. Thus, successful transplantation of marginal size liver grafts would improve outcome for recipients and increase the margin of safety for living donors. The current study was undertaken to establish a large animal model that would simulate our clinical experience of the small-for-size liver graft and to evaluate the pathological findings associated with transplantation of these grafts.
Increasing shortage of cadaveric grafts demands the utilization of living donor and split liver grafts. The purpose of this study was to 1) define the “small-for-size” graft in a pig liver transplant model 2) evaluate pathological changes associated with small-for-size liver transplantation. Pigs were divided into four groups based on the volume of transplanted liver: (a) control group (n=4), 100% liver volume (LV) (b) group I (n=8), 60% LV (c) group II (n=8), 30% LV (d) group III (n=15), 20% LV. Tacrolimus and methyl prednisone were administered as immunosuppression. Animals were followed for 5 days with daily serum biochemistry, liver biopsies on day 3 and 5 for light microscopy, and tissue levels of thymidine kinase (TK) and ornithine decarboxylase (ODC). Liver grafts were weighed pretransplant and at sacrifice. All the recipients of 100%, 60%, and 30% grafts survived. Transplantation of 20% grafts (group III) resulted in a 47% mortality rate. Group III animals showed significantly prolonged prothrombin times (p<0.05), elevated bilirubin levels (p<0.05), and ascites. The rate of regeneration, as indicated by TK activity and graft weight was inversely proportional to the size of the transplanted graft. The severity of the microvascular injury was inversely proportional to graft size and appeared to be the survival-limiting injury. Frank rupture of the sinusoidal lining, parenchymal hemorrhage, and portal vein injury were prominent in group III animals 1 hour following reperfusion. This study established a reproducible large animal model of partial liver grafting; it defined the small-for-size syndrome in this model and described the associated microvascular injury. (Liver Transpl 2004;10:253–263.)
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Materials and Methods
Seventy outbred female pigs weighing between 17 to 34 kg were used as donors and recipients. An additional animal was utilized as a blood donor for each donor and recipient pair. The experiment was conducted in accordance with the NIH guidelines for the Care and Use of Laboratory Animals and approved by the University of Pittsburgh IACUC. After 12 hours of fasting, anesthesia was induced by intramuscular administration of thiopental sodium (25mg/kg) followed by endotracheal intubation and maintenance with oxygen, nitrous oxide, and isoflurane by positive pressure mechanical ventilation. An intravenous line was placed in the internal jugular vein for fluid administration and central venous pressure (CVP) monitoring and was tunneled subcutaneously to exit at the back of the neck for postoperative venous sampling. An additional internal jugular line was placed as part of the venovenous bypass (VVB) circuit and a carotid arterial line was used for intraoperative blood sampling.
Animals were divided into four groups: a control group (n=4) receiving whole liver transplants (100% LV), and three groups of partial liver transplants. Group I (n=8) received grafts of 60% LV; group II (n=8) received liver grafts of 30% LV and group III (n=15) received grafts of 20% LV.
Animals were followed for five days, at which time they were sacrificed. Postoperatively they were permitted food and water ad libitum starting the morning after surgery. Antibiotic prophylaxis, cefamandole 1g, was administered intraoperatively and then daily for three days. Analgesia, ketoprofen 1mg/kg intramuscularly every 12 hours, was administered for the first three days and then as needed to relieve signs of pain or distress. Animals underwent intensive clinical observation for the first 48 hours and intravenous fluids were administered as required based on clinical and biochemical parameters.
Immunosuppression was maintained using tacrolimus (0.04 mg/kg/d, IV) and methyl prednisone (125, 100, 75, 50, and 25 mg/d, IV) on postoperative day (POD) 1 to 5.
Hemodynamic monitoring of blood pressure, heart rate, and CVP was carried out continuously. Baseline blood samples were obtained preoperatively for electrolytes, liver function tests (serum aspartate aminotransferase (AST), total bilirubin, and serum albumin), and arterial blood gases. Following reperfusion hourly blood gas analysis was carried out for 12 hours and venous samples at 3, 6, and 12 hours following reperfusion were analyzed for liver function tests, prothrombin time (PT), and lactate.
Venous sampling for estimation of AST, total bilirubin, PT, hematocrit, creatinine, and electrolytes was continued on a daily basis until the end of the study at 5 days. Tacrolimus trough levels were drawn daily prior to the administration of the morning dose of the drug. Serum lactate measurement was discontinued at three days when the arterial line was removed.
All animals were scheduled for laparotomy with open liver biopsy on POD 3 and a repeat of the procedure on POD 5, prior to euthanasia. Recipients of 20% liver grafts (group III) did not tolerate a survival laparotomy on POD 3 and the schedule was modified for this group. Five of fifteen pigs underwent laparotomy with liver biopsy and were sacrificed on POD 3, and the remaining ten were scheduled for sacrifice on POD 5 without the laparotomy on POD 3. Animals in the remaining groups (I, II, and control) underwent laparotomy with open liver biopsy on POD 3 and again on POD 5 prior to sacrifice. All liver grafts were weighed at the time of sacrifice (GSW).
Tru-cut liver biopsies were obtained pretransplant, 5 and 60 minutes following reperfusion and on POD 3 and POD 5. Specimens were fixed with neutral buffered formalin, paraffin embedded, stained with hematoxylin and eosin (H+E) and examined by light microscopy. Sections were reviewed blindly by two independent pathologists without knowledge of the groups or timing of samples. Histological assessment was based on degree of hemorrhage and congestion, portal and septal edema, endothelial cell detachment, necrosis, apoptosis, and mitoses. All sections were reviewed and scoring was based on the severity of the findings as none, minimal, moderate, and significant.
Liver biopsies were also taken to evaluate liver regeneration. Ornithine decarboxylase (ODC) and thymidine kinase (TK) activity were measured preoperatively and on POD 3 and POD 5. ODC activity was determined utilizing the method of McGowan and Fausto and expressed as dpm/mg of protein16 and TK activity determined utilizing the method of Kahn with minor modification and expressed as dpm/ml of protein.17
After a mid-line laparotomy, the liver was mobilized, by dissecting all ligamentous attachments. The portal vein, hepatic artery, and bile duct in the hepatoduodenal ligament were isolated up to the bifurcation to the right and left lobes and the upper vena mobilized. The parenchymal transection outlined below was performed based on the pig liver anatomy.18, 19
The pig liver is comprised of three lobes, right, median, and left (Fig. 1, 2). These lobes are independent of each other along most of their surface and join together along their dorsal surface close to the vena cava. The median lobe is larger than both lateral lobes and is incompletely divided into right and left paramedian lobes along the umbilical fissure. The right paramedian lobe surrounds the gallbladder. The inferior vena cava (IVC) is entirely intrahepatic in its upper abdominal course being encircled by the right lobe of the liver. The left lobe and left paramedian division of the median lobe represent approximately 40% of the LV. The plane of transection in group I was between the right and left paramedian divisions of the median lobe as represented by line A in Figures 1 and 2. In groups II and III, the plane of transection was through the center of the right paramedian lobe to the right of the gall bladder and the middle hepatic vein. In addition, the lingular projection of the remaining part of the right paramedian lobe was resected representing a liver resection of approximately 70% of the liver volume. To achieve a liver volume of 20% in the group III recipients the grafts described above in group II were transplanted from a small donor (19 kg) into a larger recipient (30 kg).
The hepatic pedicles to the left lobe and left paramedian division of the median lobe were divided outside the liver in group I animals. In groups II and III, all pedicles to the right paramedian lobes were preserved until the parenchymal dissection was complete and sacrificed as required. Parenchymal transection was performed using the Cavitron Ultrasonic Surgical Aspirator (CUSA®: Valley Lab, Boulder, Colorado, USA) along the transection planes described above and outlined in Figures 1 and 2. Parenchymal vessels were individually ligated using 4-0 silk sutures. The hepatic veins, due to their intrahepatic location were isolated and clamped during parenchymal transection and over sown using 5-0 Prolene sutures. When parenchymal transection was complete the abdominal aorta and inferior mesenteric vein were cannulated. A thoracotomy, performed via a diaphragmatic incision, allowed clamping of the thoracic aorta and incision of the thoracic inferior vena cava. The liver was then perfused with lactated Ringers' solution and the abdominal cavity filled with crushed ice. On the back table the liver was flushed with cold lactated Ringers' solution via the portal vein, hepatic artery, and bile duct. Cholangiography was performed to confirm the integrity of the biliary tree. The liver graft was then weighed, implantation weight (GW). The graft weight at the time of implantation (GW) expressed as a percentage of the recipient's native liver weight (GWRLW) and as a percentage of total body weight (GWBW) was calculated as follows:
In the control group the liver was mobilized and the major vascular pedicles and bile duct were dissected free prior to perfusion. The operation was then completed as described above.
After midline laparotomy and mobilization of the liver the portal triad was dissected. Total hepatectomy was performed utilizing VVB by a centrifugal pump (Bio-Medicus Inc. Eden Prairie, USA), interconnecting the left internal jugular, splenic vein, and femoral vein via Tygon tubing. (Norton Industrial Plastics, Akron, OH; 16 or 18 Fr for the jugular vein, and 22 or 24 Fr for the splenic vein). The bypass blood flow was maintained over 30 ml/kg/min, under conditions of systemic heparinization (50 U/kg). The hepatic artery and common bile duct were divided close to the liver and the portal vein was clamped and divided. After clamping both the upper and lower IVC, the liver was excised. The transplant grafts (whole liver and partial liver grafts) were orthotopically transplanted with vascular anastomoses performed in standard sequence: upper IVC (5-0 Prolene), lower IVC (5-0 Prolene) and portal vein (7-0 Prolene) followed by reperfusion and discontinuation of VVB. Before reperfusion, methyl prednisone (500 mg) and tacrolimus (0.04 mg/kg) were given intravenously. The main hepatic artery of the graft liver was sutured to the recipient's common hepatic artery (8-0 Prolene) at the level of the gastroduodenal artery. The bile duct was reconstructed end-to-end using interrupted 6-0 PDS. A splenectomy was performed on all recipients following removal of the splenic limb of the VVB circuit. Allogeneic blood transfusion was given as required.
The results are expressed as the mean values ± SD One-factor ANOVA or t-test was performed and a p value less than 0.05 was considered significant.
All the recipients of 100%, 60%, and 30% liver grafts survived. Transplantation of 20% liver grafts resulted in a mortality rate of 47%. Seven pigs in group III died before schedule sacrifice, five as a result of liver failure and three died from unknown causes (Table 1).
|Scheduled Sacrifice||Day of Death||Cause of Death|
|Day 3 (n = 5)||Day 1 (n = 1)||Liver failure|
|Day 3 (n = 4)||Scheduled|
|Day 5 (n = 10)||Day 1 (n = 2)||Unknown|
|Day 2 (n = 2)||Liver failure|
|Day 3 (n = 1)||Liver failure|
|Day 4 (n = 1)||Unknown|
|Day 5 (n = 4)||Scheduled|
Two major complications were observed: 1) Injury of the bile duct supplying the right lobe: two animals in group II sustained an accidental injury to the bile duct during liver resection. This was detected by cholangiography and repaired on the back table. 2) Venous outflow obstruction: this occurred in one recipient in each of the groups II and III and was recognized intraoperatively. These animals were sacrificed during the procedure and excluded from the study. Gastric dilatation was observed at the time of sacrifice in two animals in group II and five animals in group III. Moderate amounts of ascites were observed in one animal in each group I and II, and five animals in group III.
There was no difference in the cold or warm ischemia times among the four groups. The mean cold ischemia times for the control group and groups I, II, and III were 138.0±38, 122.9±18.6, 151.0±21.7, and 139.0±20.6 minutes and the warm ischemia times were 31.5±1.9, 35.4±2, 32.6±3.5, and 32.4±3.0 minutes respectively.
Liver Function and Metabolism
Arterial blood gases normalized within 12 hours of reperfusion in all animals in the control group, and groups I and II. Two pigs in group III developed progressively worsening acidosis following reperfusion and died within 24 hours. A further two pigs in group III developed base deficits on POD 2 and died despite medical intervention. These findings were mirrored by elevated serum lactate levels. Lactate concentrations peaked 3 to 6 hours after reperfusion in all groups (Table 2). The levels of lactate in group III were significantly higher than in the control group and group I at 12 hours and on POD 1 (p<0.05). The higher serum lactate values were observed in pigs that died.
|5 min||60 min||3 hours||6 hours||12 hours||24 hours||48 hours||72 hours|
|Group I||6.3 ± 1.7||6.4 ± 1.9||9.5 ± 2.8||9.3 ± 4.2||1.3 ± 0.6||1.7 ± 1.1||1.9 ± 0.8||1.0 ± 0.2|
|Group II||4.7 ± 1.7||5.2 ± 2.4||7.1 ± 4.3||9.1 ± 4.7||3.4 ± 3.7||3.2 ± 1.5||1.9 ± 1.1||1.4 ± 0.9|
|Group III||5.9 ± 1.1||6.6 ± 1.5||8.4 ± 2.1||8.2 ± 4.0||5.3 ± 6.7||5.2 ± 4.3||3.6 ± 2.4||1.7 ± 0.7|
Experimental groups II and III, at 24 hours, demonstrated a significantly prolonged PT compared to the control group and group I (p<0.05). On the third POD, the PT in groups I, II, and III was prolonged compared to the control group. The PT remained prolonged in groups II and III to the fifth POD, which was the end of the study period. (p<0.05) (Table 3).
|12 hours||24 hours||2 days||3 days||4 days||5 days|
|Group I||13.5 ± 1.5||14.2 ± 1.7||14.0 ± 5.0||13.2 ± 1.6||13.3 ± 3.9||14.2 ± 6.0|
|Group II||14.4 ± 2.0||17.0 ± 1.9||14.7 ± 2.1||14.2 ± 2.1||16.6 ± 3.6||18.6 ± 6.0|
|Group III||15.0 ± 1.5||17.2 ± 1.9||15.5 ± 2.0||16.7 ± 4.7||16.2 ± 6.3||18.6 ± 5.3|
Serum AST values in control and group I peaked in the first 24 hours, being significantly higher than the other groups and then decreased rapidly (Table 4). On POD 3 and POD 4 the serum AST level of group III was significantly higher than the control. Serum albumin concentrations did not differ between groups.
|Preoperative||3 hrs||6 hrs||12 hrs||24 hrs||2 days||3 days||4 days||5 days|
|Serum AST U/L|
|Group I||47 ± 9.7||295 ± 204||400 ± 192||593 ± 239||654 ± 281||439 ± 197||293 ± 253||113 ± 65||75 ± 45|
|Group II||42 ± 8.5||119 ± 44||239 ± 42||303 ± 50||466 ± 69||355 ± 89||277 ± 139||185 ± 143||146 ± 109|
|Group III||46 ± 23||145 ± 44||257 ± 71||419 ± 115||409 ± 130||380 ± 106||268 ± 159||213 ± 166||254 ± 201|
|Serum Bilirubin mg/dl|
|Group I||0.2 ± 0.1||0.4 ± 0.2||0.3 ± 0.1||0.2 ± 0.07||0.2 ± 0.1||0.2 ± 0.1||0.2 ± 0.1||0.1 ± 0.05||0.2 ± 0.1|
|Group II||0.2 ± 0.1||0.3 ± 0.2||0.5 ± 0.2||0.6 ± 0.5||0.6 ± 0.4||0.5 ± 0.4||0.4 ± 0.3||0.2 ± 0.1||0.3 ± 0.3|
|Group III||0.2 ± 0.3||0.4 ± 0.2||0.7 ± 0.3||1.2 ± 0.5||1.3 ± 0.6||1.5 ± 0.9||1.0 ± 0.9||1.2 ± 1.8||1.9 ± 2.8|
Throughout the entire postoperative period, higher values of bilirubin were observed in group III, this reached statistical significance in the immediate postoperative period and on POD 1 and 2 (p<0.05) (Table 4).
Tacrolimus trough levels (Table 5) were consistently higher in recipients of the smaller grafts (groups II and III) compared to the control animals and group I despite the same dosage regimen, suggesting a deficiency of the liver in metabolizing the drug.
|Group||POD 1||POD 2||POD 3||POD 4||POD 5|
|Control||13.3 ± 3.0||10.2 ± 3.0||8.0 ± 1.9||9.1 ± 4.5||8.7 ± 4.3|
|Group I||6.4 ± 1.3||7.8 ± 2.2||7.9 ± 1.7||8.7 ± 1.7||6.0 ± 1.4|
|Group II||12.3 ± 4.2||13.1 ± 2.8||13.0 ± 3.2||12.0 ± 2.2||11.8 ± 3.6|
|Group III||11.8 ± 5.1||12.3 ± 2.7||11.1 ± 1.2||11.1 ± 1.2||10.2 ± 1.4|
Parameters of Regeneration
The rate of growth of the transplanted partial liver grafts was inversely proportional to the size of the graft as shown by the increase in graft volume at the time of sacrifice, with group III achieving the greatest increase in graft weight. The percentage increase in graft weight (GSW-GW × 100/GW) at the time of sacrifice is included in Table 6 and depicted in Figure. 3. Graft weights on POD3 are shown only for group III as no other animals were sacrificed prior to the end of the study. Survivors in group III increased their graft weight by 190% over the period of the study compared to an increase of 120% in group II and an increase of 61.8% in group III. Interestingly, the whole liver grafts in the control group increased in size by 32.8% over the period of the study. The percentage increase in graft weights were significantly different between groups I and II, and groups I and III (p<0.05). The difference in graft size between groups II and III approached but did not reach statistical significance (p=0.0514). When compared to the native liver volume of the recipient the 60% grafts achieved 96% of the expected liver volume (ELV) for the recipient by day 5, recipients of 30% grafts achieved 73% of the ELV and recipients of 20% grafts achieved 63% of the ELV for that animal. (Fig. 4). On average the rate of growth was 42.1g/day in the control group, 52.3g/day in the 60% group, 52.4g/day in the 30% group, and 60.8g/day in the 20% group.
|Control (100%)||Group-I (60%)||Group-II (30%)||Group-III (20%)|
|Donor body weight (kg)||25.9 ± 1.1||30.9 ± 5.5||26.5 ± 2.7||19.1 ± 1.7|
|Recipient body weight (kg)||27.2 ± 0.8||29.9 ± 4.1||26.9 ± 2.8||30.3 ± 2.9|
|GW (g)||641.5 ± 121.1||422.9 ± 73.8||216.9 ± 30.6||159.2 ± 22.1|
|GW/RLW (%)||87.4 ± 8.3||59.5 ± 5.0||33.6 ± 4.9||22.1 ± 3.4|
|GW/BW (%)||2.4 ± 0.4||1.41 ± 0.17||0.81 ± 0.09||0.53 ± 0.06|
|GSW (g)||852.3 ± 176.1||684.4 ± 118.9||478.8 ± 48.5||463.2 ± 122.0*|
|394.6 ± 22.5†|
|GW % increase||32.8%||61.8%||121%||190%*|
|Weight gain (mean g/day)||42.1||52.3||52.4||60.8|
Tissue levels of TK and ODC are outlined in Tables 7 and 8 respectively. Peak levels were recorded on POD 3 in all experimental groups with a decline in levels by POD 5. In group I, II, and III the TK activity was increased by a factor of 2.3, 3.9, and 6.7, respectively on day 3 compared to pretransplant activity. On day 5 levels had declined but in group III animals but were 3.5 times higher the pretransplant control values. Only in group III animals who were sacrificed on day 3, it is possible to compare graft weight and activity of TK and ODC; although graft weight continued to increase at the same rate the level of TK and ODC had already started to return towards normal. Levels of ODC mirrored those of TK, with a peak on POD 3 and a decrease in levels by POD 5, although these levels were still elevated compared to control values.
|Liver Volume||Pretransplant (Mean ± SD)||POD 3 (Mean ± SD)||POD 5 (Mean ± SD)|
|Control (100%)||1286 ± 1129||2717 ± 2354||1736 ± 1760|
|Group I (60%)||1494 ± 783||3510 ± 1734||2240 ± 1473|
|Group II (30%)||1545 ± 561||6141 ± 6696||2712 ± 1383|
|Group III (20%)||2435 ± 167||16543 ± 5553||10363 ± 3574|
|Liver Volume||Pretransplant (Mean ± SD)||POD 3 (Mean ± SD)||POD 5 (Mean ± SD)|
|Control (100%)||3 ± 7||198 ± 144||128 ± 191|
|Group I (60%)||27 ± 22||237 ± 138||156 ± 219|
|Group II (30%)||27 ± 22||210 ± 130||129 ± 93|
|Group III (20%)||7 ± 18||430 ± 589||100 ± 110|
Detailed pathological characteristics identified in each group are outlined in Table 9. Light microscopy of H+E stained sections demonstrated pathological changes in all grafts as early as 5 minutes after reperfusion (Table 9). In the control group and group I these changes were minimal congestion, hemorrhage, and periportal and septal edema. However, in the smaller grafts pathological findings were more severe. Transplantation of 30% and 20% liver grafts was associated with moderate to severe congestion and hemorrhage, and periportal/septal edema at 5 minutes. In addition there was evidence of periportal sinusoidal and portal vein endothelial cell detachment occurring as early as 5 minutes and peaking at 1 hour. The findings were most severe in the recipients of 20% liver grafts with the endothelial damage progressing to frank rupture and thrombosis of the periportal sinusoids at 1 hour associated with hepatocyte necrosis and apoptosis in zones 1 and 2 (Fig. 5). By POD 3 and 5, the liver morphology was almost completely recovered in the control group and groups I and II, with both liver parenchyma and nonparenchymal cells showing proliferation. On POD 3 group III had persistent portal endothelial and sinusoidal cell detachment and portal vein stretching. The findings persisted on POD 5 but were less severe; there was minimal necrosis and evidence of repair of the microvascular injury. Recipients of 30% and 20% grafts did show a high rate of hepatocyte and nonparenchymal cell proliferation on POD 3 and 5 demonstrating that these surviving marginal grafts did maintain the ability to regenerate (Fig. 6) All partial allografts showed evidence of a ductular reaction and nodular regenerative hyperplasia-like change. A cellular infiltrate associated with rejection was seen on POD 5 in all groups but this was minimal.
|Liver Volume||Pathology||5 min||1 hour||3 days||5 days|
|Disruption of Architecture||0||0||0||0|
|Disruption of Architecture||0||0||0||0|
|Disruption of Architecture||0||0||0||+|
|Disruption of Architecture||0||+++||++||+|
Overall results for adult LDLT are not equivalent to cadaveric transplantation or pediatric LDLT. Recent results from Japan report a 5 year survival of 81.5% in pediatric LDLT and 69.7% in adult LDLT.20 A similar study from the US reports a one year graft survival of 65% in adult LDLT recipients compared to 91% in children.21 These poorer outcomes in adults are largely attributed to the problems encountered following transplantation of marginal or small-for-size grafts, an uncommon problem in pediatric LDLT. Survival rates in adults are influenced not only by the size of the graft but also by the severity of illness of the recipient, with Child's class B and C recipients having poorer outcomes.9 The problem of increased portal vein blood flow through a reduced hepatic microvascular bed is regarded as central to the problems encountered following transplantation of small-for-size grafts. It is well described clinically and often referred to as the syndrome of portal hyperperfusion.22–24 This problem is accentuated the smaller the size of the graft and the greater the portal hypertension.
In order to study marginal grafts we set out to establish a reproducible large animal model of small-for-size liver transplantation that would provide clinically relevant data and allow us to pursue further in-depth studies. We chose the pig model because the anatomy, physiology, and size of the pig liver closely resembles that of humans. Several large animal models of partial liver transplantation have been reported but they have not defined a survival model of the small-for-size liver graft.25–32 Our approach in setting up this model was to reduce the size of the transplanted graft until the syndrome of the small-for-size graft was duplicated. The 20% liver graft fulfilled all of the criteria with a 47% mortality rate and surviving animals demonstrating prolonged PT, cholestasis, and ascites. Interestingly, the surviving recipients of 20% grafts, although they showed delayed recovery of synthetic liver function, did continue to regenerate and maintained a rate of regeneration that was inversely proportional to the size of the graft. Significant apoptosis was demonstrated in the smaller grafts, which started within one hour of transplantation and was still present at day 5. This may be related to the increased rate of proliferation in these grafts as demonstrated by Sakamoto,33 but in contrast to the cited study the apoptosis is unlikely to be the result of the liver disposing of superfluous hepatocytes. The 20% grafts, which demonstrated the greatest number of apoptotic bodies, achieved only 67% of their expected liver volume. The significance of apoptosis in the small-for-size graft is unknown. We postulate that the increased rate of regeneration is associated with an increased number of defective hepatocytes, which subsequently undergo apoptosis.
The most striking finding in this study was the severity of the microvascular injury in the smallest grafts. All reduced grafts demonstrated evidence of traumatic injury to the portal vein and periportal sinusoidal endothelium within one hour of transplantation. In groups II and III severe congestion was evident as early as 5 minutes and in group III this injury progressed to frank rupture and thrombosis of the periportal sinusoids. Although the microvascular injury in the 20% group was severe, animals surviving 5 days demonstrated evidence of repair and regeneration with a large number of proliferating hepatocytes and nonparenchymal cells. Documentation of the early pathological injury following transplantation of the small-for-size liver graft is scanty. Asakura, transplanting porcine grafts of 25% liver volume, documented disruption of the sinusoidal lining and hemorrhage in the periportal triads.34 However the timing and progression of this injury was not documented. Man studied the pathological changes in the microcirculation in a rat nonarterialized model of partial liver transplantation.35 In grafts of 28% LV they demonstrated sinusoidal congestion, vacuolar change in hepatocytes at 30 minutes, and electron microscopic evidence of damage to the sinusoidal lining cells at 30 minutes and 3 hours. A subsequent study from this group in human LDLT recipients also demonstrated electron microscopic evidence of injury to the sinusoidal lining cells in grafts less than 40% LV.36 One clinical case of hemorrhagic necrosis secondary to excessive portal blood flow in an adult LDLT was reported.37 This injury was recognized when the graft was removed at the time of retransplantation.
In our study the impact of preservation injury was minimal and similar in all groups. It is an unlikely contributing factor to the different pathological findings in various groups. It is not unreasonable to speculate that the microvascular changes demonstrated, with severe disruption of the sinusoidal lining in the 20% grafts, are at least in part a result of excessive portal blood flow, although blood flow rates were not estimated. Excessive portal venous flows of 373.4 ml/min/100g tissue have been described following transplantation of liver grafts less than 40% liver volume,22 compared to earlier reports of portal vein flow rates of 130ml/min/100g liver tissue in cadaveric whole liver transplants.38 When blood flow rates were studied in right lobe living donor grafts portal flow rates increased by a factor of 2 following transplantation compared to the rate of flow in the donor, and arterial flow rates were significantly reduced.39 The precise role of increased portal vein blood flow and reduced hepatic arterial flow in inducing injury following transplantation of partial liver grafts is complex and poorly understood. Shear stress in the portal vein induced by acute portal hypertension may play an important role in priming hepatocytes for regeneration and excessive flow is cited as a cause of liver allograft dysfunction.40–42 Reduced portal vein flow, on the other hand, following liver resection, results in liver atrophy and failure.43 The optimal rate of portal blood flow required to support liver regeneration and function in partial liver allografts, without causing a traumatic liver injury, is unknown.
In conclusion, this study has outlined a significant microvascular injury following the transplantation of partial liver grafts. The severity of the injury increased with reduction in graft size with the most severe injury in the small-for-size liver graft. It has also defined a reproducible model of the small-for-size graft, which will allow us to pursue further in-depth studies.