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During the past 10 years, much has been learned about homotransplantation of the whole liver using either a replacement (orthotopic) homograft or an auxiliary organ which is inserted at an ectopic site without removal of the recipient’s own liver. The available information concerning these operations has been reviewed from animal experiments, and from the handful of attempts at clinical application.
In this report, attention will be focused upon new or contraversial aspects of homotransplantation of the liver. Although interest in liver transplantation dates from Welch’s first reports almost 10 years ago (1, 2), there was at first little justification from laboratory experimentation for hope that such a procedure had a therapeutic clinical potential. In the recent past, the outlook has markedly changed.
Transplantation of the liver can be carried out in two general ways. First, the liver of the recipient may be removed and the homograft placed in its natural right subphrenic position (3–5). Survival after such an orthotopic homotransplantation is dependent upon immediate and continued function of the foreign hepatic tissue. Alternatively, recipient hepatectomy may be omitted in which case the homograft is inserted as an auxiliary organ in some ectopic site such as the pelvis, paravertebral gutter, or left subphrenic space (1, 2, 6–11). With the latter method, there is not total dependence upon the homograft; but as will be described, important physiologic problems are introduced relating to substrate competition between the two livers.
All experiments with whole organ liver transplantation have been carried out in dogs. This animal is not expensive, has suitably large structures to permit standard anastomotic techniques, and is easy to care for. There are, however, specific disadvantages. The canine liver is peculiarly susceptible to anoxic injury as a result of a vascular response to ischemia in which there is apparently constriction of intra-hepatic venous sphincters. Blood is entrapped in the rapidly engorgin parenchyma and the liver becomes swollen and cyanotic in this syndrome of « outflow Blood is entrapped in the rapidly engorging parenchyma and the intestinal tract results. These highly lethal events can largely be avoided if the homograft is cooled and if transfer can be carried out quickly. In the laboratory, the most effective means of using hypothermia is to perfuse cold electrolyte solution through the portal vein while the donor animal is being sacrificed by exsanguination (Fig. 1). Livers prepared in this way can usually tolerate 60 to 120 minutes of devascularization.
During removal of the host liver and insertion of the homograft, diversion of the temporarily-occluded inferior vena caval and spanchnic systems is necessary. In our experience, this can be done most easily by first performing a temporary portacaval anastomosis after which the combined venous pools can be decompressed through a single external bypass (Fig. 2). After the homograft has been placed and revascularized, the portacaval shunt is then removed (Fig. 3).
Once the technical steps of the operation are mastered, orthotopic homotransplantation can be carried out with an acceptable risk. In the pioneer studies of Moore (3, 4) and in our own laboratories (5, 12, 13), the operative mortality was staggering, being well over 50 %. At the present time, it is less than 5 %. In a recent series of 23 control animals in which no effort was made to prevent rejection (14), all but one animal survived operation. Twenty-two of the 23 dogs lived for at least 2 days and 19 of these (86 %) lived six days or more (Fig. 4).
In the untreated animal, the fate of the orthotopic homograft is not dissimilar to that of other homotransplanted tissues and organs. Since the liver is a vital organ without which life can be sustained for only a few hours, the functional end-point of rejection is very precisely defined by the death of the animal. Excluding the operative death, the mean survival in the above-described series of 23 animals was 7.1 days with a maximum of 10 days. In an occasional dog, protracted survival has been observed (12) as has also been noted with renal homografts.
The alterations in liver chemistries which follow orthotopic homotransplantation are quite predictable and are well correlated with the clinical course of the individual dog. After operation, there are usually 3 or 4 days of acceptable health, during which time the animals frequently resume alimentation. Progressive rises are then observed in the serum alkaline phosphatase, SGOT and SGPT, and a day or so later the serum bilirubin begins to increase. The biochemical alterations of rejection are progressive and inexorable until the time of death (Fig. 5). The animals develop dark urine and clay-colored stools, become listless and anorexic, and ultimately have unremittant terminal emesis.
Figure 6 shows the characteristic lesions of the rejecting orthotopic liver homograft—mononuclear infiltration of the portal tracts and the area in and around the central vein, accompanied by centrilobular necrosis. Early, many of these infiltrating cells are the pyronine-positive « large lymphoid » cells of Scothorne (15) but after the sixth day these are replaced by mature plasma cells. The changing character of cells in the homograft is reflected by similar alterations in the host’s own lymphoid organs. Lesions in the portal tract vessels are not prominent, but with electronmicroscopy mononuclear cells are found adhering to and apparently injuring the central sinusoidal endothelium. If hemodynamic factors are important in liver rejection they apparently occur at this level, a localization comparable to the tubular capillary lesion of Kountz in the rejecting renal homograft (16).
To date, azathioprine has been the most effective immunosuppressive agent for prolonging hepatic homograft survival. The use of this drug complicates the care of the animals in several ways. The attenuation of responsiveness to environmental antigens renders the kennel dog extremely susceptible to a variety of septic complications, the most common and lethal being pneumonitis. Anemia and weight loss develop (Fig. 7). In addition, azathioprine has a specific hepatotoxicity which tends to injure the homograft at the same time it acts to protect it from rejection (14). The influence of this agent upon the liver function of normal dogs is seen in Figure 7. After its administration is begun, sharp rises in SGOT, SGPT and alkaline phosphatase are observed within a few days, usually without jaundice. After 15 to 30 days of continuous therapy, there is partial but usually incomplete recovery. The histologic lesion caused by azathioprine has some resemblance to that of rejection in that centrilobular hepatocyte injury of frank necrosis (Fig. 8) occurred in more than two-thirds of the animals, always, however, without cellular infiltration.
Despite the handicap imposed by the use of a liver poison to prevent liver rejection, it has been possible after orthotopic homotransplantation to obtain a large number of chronic survivors. The operative mortality was increased over that of the nontreated controls, mainly due to pulmonary sepsis. Thirty-two of the 116 dogs died during the first week. Of the 84 remaining definitive test animals, 44 lived for 25 days or longer, and 24 lived for 50 days or more (Fig. 9). Fourteen are still alive from more than 2 to slightly less than 11 months. The best results were in those animals which also received methionine or its radioactive isotope, but the variability of survival was so great in all series that statistically significant advantage of these adjuvant agents could not be proved.
After homotransplantation to the treated animal, there was a great variability in the vigor of the rejection subsequently encountered. About one-fifth of the animals never had any clinically-detectable rejection. An example is shown in Figure 10. The dog received a homograft in March, 1964, had only minor early abnormalities in liver function, had all therapy discontinued in 4 months, and has been in good health since. In 6 such animals (Fig. 11) which had all therapy stopped at 4 months, only I has had a subsequent rejection and this a non-lethal one. The incidence and timing of successful discontinuation of therapy far exceeds that reported after renal homotransplantation (17–20). Moreover, none of these dogs had evidence of a late graft-versus-host reaction. In some of the dogs, red cell half-life was shortened in the early post-operative period but this did not recur after cessation of therapy (Fig. 12).
The biopsy of the dog whose course is depicted in Figures 10 & 12 was normal by light and electronmicroscopy after 4 months (Fig. 13A) and remained so 6 months later when re-biopsied (Fig. 13B). In this case, a virtually complete state of host-graft nonreactivity existed between the homograft and its host.
At the other end of the spectrum, observed in about one-third of the cases, was an inexorable rejection characterized by relentless deterioration of the liver chemistries, progressive jaundice, and death in all cases in 41 days or less (Fig. 14). The histologic features in these homograft were very similar to those of non-treated controls.
Finally, almost exactly one-half of the 84 definitive test animals underwent an obvious rejection which was often of great severity but which was reversible to a greater or lesser extent. Figure 15 depicts an example. After operation, this dog developed extremely poor liver function with a bilirubin that exceeded 6 mg % and collateral rises in the serum enzymes. He lost weight rapidly but as liver function improved, he began eating. The animal is still alive.
Perhaps the most interesting feature of these more than 40 examples was that intensification of immunosuppressive therapy was not used in any. This, we believe, delineates a general feature of transplantation biology which has been incompletely appreciated that rejection is subject to spontaneous remissions. This fact will make caution necessary in ascribing reversal to any preceding changes in therapy.
The course of the animal shown in Figure 16 is an even more striking example of spontaneous reversal of rejection. Jaundice did not occur in the first 120 days after operation at which time therapy was stopped. Subsequently rejection developed, reached its functional zenith, and reversed—all in the absence of all treatment.
The pathologic correlation with the foregoing clinical observations is extremely good and permits tentative reconstruction of the serial events after homotransplantation to the treated host Early after operation there is a more or less serious attack on the graft. If the animal dies at this time, the histologic picture is indistinguishable from that in the unmodified host (Fig. 17A). If the animals survive, many or even most of the infiltrating cells retreat from the graft, leaving large areas of necrosis.
Secondary to the necrosis there are many areas of reticulin collapse and condensation most heavily concentrated in the central areas (Fig. 17B). From this point onward, the dominant features are those of repair in which the most affected areas acquire fresh connective tissue (Fig. 18A). Some pyronine-positive cells often remain but rapid destruction of hepatocytes has ceased. A pseudolobular pattern often results with much connective tissue but with relatively few infiltrating cells (Fig. 18A).
Repair may continue even after cessation of therapy. In Figure 18A is a biopsy obtained after 4 months, at which time azathioprine was stopped. The abnormalities in this liver are evident. A substantial further improvement continued for the next 11 weeks when the biopsy in Figure 18B was obtained of a greatly improved liver.
Historically, the first whole organ liver transplants were carried out by Welch (1, 2) without removal of the host’s own liver and with insertion of the homograft into the pelvis by a technique similar to that shown in Figure 19A. With this preparation, the arterial supply is physiologically normal, but the ectopic liver receives its portal inflow from the inferior vena cava instead of the spanchnic venous system. Welch (1), Goodrich (2), Mehrez (7), Hallenbeck (21), Sicular (6), Paronetto (11) and Hagihara (9) all studied variants of this preparation in animals not treated with immunosuppression. The period of bile excretion which was obtained from such auxiliary homografts in unmodified hosts was 3 or 4 days.
More recently, auxiliary transplantation has been tested in dogs treated with azathioprine (8, 21). Such auxiliary homografts were quickly found to be much more severely damaged than had been observed after orthotopic homotransplantation. The pelvic liver underwent a rapid and drastic reduction in size, usually beginning within 2 weeks after operation (Fig. 20). Histologically, the diminutive liver had marked centrizonal hepatocyte loss, but with relatively good preservation of the blood vessels and the duct system. With the dissolution of hepatocytes, there was reticulin collapse.
It was subsequently shown that this acute atrophic process resulted at least in part from the abnormal way in which the ectopic livers had been revascularized (10). If auxiliary transplantation to the treated host is carried out with a comparable technique but with portal revascularization from the recipient splanchnic venous system (Fig. 19B), the homograft shrinkage is no longer observed (Table 1). Instead, there is a similar atrophic process in the recipient animal’s own liver (Fig. 21). Under these circumstances, the auxiliary homograft was found to be able to sustain life inasmuch as several animals had protracted survival after removal of their own shrunken autologous liver at a second-stage operation (Fig. 22).
These experiments have done much to clarify the physiologic requirements for homotransplantation of an auxiliary liver. Apparently there is a competition between the co-existing livers for some metabolite or other substance in the portal venous blood. That organ which has first access to the portal flow retains its functional and morphologic integrity. The other organ, whether it be the homograft of the autologous liver, undergoes atrophy predominantly affecting the centrizonular area.
Whether this substrate competition will prove to be of important clinical significance is not known. In the benign diseases for which liver transplantation might be contemplated, there would be pre-existing failure of the recipient patient’s liver, so that it might be incapable of metabolite extraction. Should this prove to bet he case, the exact method of auxiliary homograft revascularization will be less critical.
Eight attempts have been made at human liver homotransplantation, 7 in the United States and 1 in France. In 7 of these cases, the patient’s diseased liver was removed and an orthotopic transplant performed; but in Absolon’s case (22), the liver was rearterialized from the iliac artery and placed in the left paravertebral gutter without recipient hepatectomy. The summaries of these eight cases are given in Table 2. The indication for operation was cancer of the liver in six instances and biliary atresia in the other two.
The technical features of orthotopic clinical transplantation are similar to those in the dog, with a few important differences. First, the use of cadaveric homografts is mandatory, a fact which increases the difficulty of obtaining well-functioning and minimally-damaged tissue. In order to provide the donor liver with hypothermia and with oxygen from the time of death until its removal, a pump oxygenator has been used (23) to provide the corpse with an artificial circulation (Fig. 23). As soon as possible after death of the donor, cannulas are inserted into the abdominal aorta and inferior vena cava and perfusion begun from a glucose-or electrolyte-primed circuit into which a heat exchanger is incorporated. After dissection of the homograft is completed, it is removed and further flushed through the portal vein with chilled lactated Ringer’s solution (Fig. 24D).
In the recipient patient, the problems of hepatectomy are usually greatly complicated by the presence of the disease for which the operation is performed. Hepatomegaly or portal hypertension were present in all cases thus far treated, making removal an extremely formidable procedure. The recipient operation usually requires a thoracoabdominal incision. The restraining ligaments of the liver are incised (Fig. 24A) and the principal structures leaving and entering the liver are skeletonized (Fig. 24A-C). This phase of the operation may be done at a separate stage from the definitive transplant.
As mentioned earlier, failure in the dog to decompress the occluded portal vein during removal of the recipient liver and placement of the homograft rapidly leads to irreversible intestinal wall damage. In the human, occlusion of the portal vein has been found to be well tolerated in such cases, presumably due to the much richer collateral connections with the systemic venous system. Decompression of the occluded inferior vena cava is, however, probably important (Fig. 25A).
After the external bypass from the inferior to the superior vena cava is inserted, the diseased liver is removed (Fig. 25B) and the homograft inserted in the normal anatomic position. Anastomosis of the supra- and infrahepatic inferior vena cava, the portal vein, and the hepatic artery is carried out with standard vascular technique (Fig. 25C-D). Internal biliary drainage is provided by choledochocholedocostomy (Fig. 25D) or by cholecystenterostomy.
During the actual homotransplantation, high levels of fibrinolysins have been observed in all of the cases treated in Denver, leading to fatal hemorrhage in Case 1. Within a few hours after revascularization of the homograft this abnormal situation is spontaneously alleviated and may followed by a period of hypercoagulability. If clot-promoting drugs such as epsilon-amino-caproic acid (EACA) or purified fibrinogen are given during the fibrinolytic phase, the penalty has been found to be subsequent intravascular thrombosis, a complication which led to or contributed to the death of 3 patients in the Denver series.
Despite the provision for preservation of the cadaveric liver, varying degrees of acute ischemic injury were detected in all of the Colorado cases, with acute rises in SGOT, SGPT, and LDH, and temporarily deepening jaundice (Figs. 26, ,27).27). This immediate damage did not preclude early survival since it proved to be partially reversible in 4 of the 5 patients.
The immunosuppressive regimen employed differed from that described earlier for the dog in that large doses of prednisone and intermittent doses of actinomycin C were given for the clinical cases (Figs. 26, ,27).27). Clear evidence of uncontrolled rejection was not present in the cases treated at the University of Colorado and poor terminal function was present in only one (Fig. 27). In the last case, there was disruption of the common duct anastomosis which was probably due to inadequate blood supply of the donor portion of the reconstructed common duct.
The function obtained after Absolon’s auxiliary hepatic homotransplantation (22) is of special interest since this is the only example of auxiliary human liver transplantation to date. The homograft was subjected to a minimum of anoxia since the donor patient died while on cardiopulmonary bypass. During the first 10 postoperative days, the recipient patient, who had biliary atresia, had a fall of serum bilirubin from 22 mg % to 5 mg %. Death occurred at 13 days from septicemia and disruption of the cholecystenterostomy.
The pathologic features of the 5 human homografts treated at the University of Colorado have been previously reported (8, 13, 25) but were recently reviewed and re-interpreted by one of us (K.A.P.) in light of the vast amount of animal pathologic material which had subsequently been analyzed. One homograft was unusual (Case 1) in that the whole liver had undergone autolysis, probably due to the 7 hours of partial or complete anoxia which preceded restoration of circulation through the liver.
The other 4 cases all showed some cellular infiltration around the portal veins (Figs. 28–31). Although varying in density from one portal tract to another, it was never severe and was often mild. The larger portal tracts were rarely more than lightly infiltrated. Eighty-five to 90 % of the cells were small lymphocytes, between 5 to 10 % were pyroninophilic cells, and the remainder were neutrophiles and the occasional eosinophile. Of the pyroninophilic cells, some were larger lymphoid types while a few were plasma cells. Only in one case were these cells present around the central veins (Fig. 29B). There were foci of fibrinoid necrosis in the walls of several of the small branches of the hepatic artery in 2 of the homografts (Fig. 29C). Centrilobular and midzonal necrosis of hepatocytes was the outstanding feature of the longest lived case (Fig. 31), but the others only showed atrophy and disappearance of the liver cells immediately adjacent to the central veins (Fig. 29B, 30A). Various degrees of collapse of the central part of the lobular reticulin framework were present in 3 of the 4 cases and were accompanied by centrilobular cholestasis.
It is interesting that of the 4 cases that survived the immediate postoperative period, there was relatively good preservation of the general lobular architecture and cell structure in 3, that only one case showed the extensive centrilobular liver cell necrosis that is so common in treated canine hepatic homografts, and that cellular infiltration was not severe in any. These histological findings support the contention that the homograft reaction played no decisive role in the death of 4 of the patients. The centrizonal and midzonal necrosis in the homograft from the patient who lived 23 days may have been due to a homograft reaction, but other factors could have accounted partly or entirely for these changes.
Aided by Grants AM 062283, AM 06344, HE 07735, AM 07772, Al 04152 and FR 00051 from the United States Public Health Service; and by a grant from the Medical Research Council of Great Britain.
**Presented at the International Congress of Hepatology, Lyon, France, June 2–5, 1965.