their predictive value (Abouna et al., 1969; Drapanas, Zemel and Vang, 1966; van

Transcription

1 Br. J. exp. Path (1976) 57, 515 AN ELECTRON MICROSCOPIC STUDY OF PIG LIVER VIABILITY AFTER EXTRACORPOREAL STORAGE R. F. SEARLE AND B. FLAKS Front the Department of Pathology, Mtedical,School, University of Bristol, Bristol, England Received for publication April 15, 1976 Summary.-The effects of hyperbaric oxygenation and ambient pressure oxygenation on the viability of pig liver stored in the Vickers' transportable organ storage unit were assessed by electron microscopy. It was found that hyperbaric oxygenation resulted in marked liver damage in contrast to ambient pressure storage. Although electron microscopy proved to be a sensitive method for detecting damage induced by unsuitable storage protocols consideration is given to the possibility that other changes, which may not be detectable by electron microscopy, limit the success of liver storage. SINCE the earliest attempts at liver transplantation (Welch, 1955), it has been clear that the extreme sensitivity of the liver to ischaemia is a major factor limiting the success of this procedure. Several methods of liver storage have been developed (Marshall, 1971; Spilg and Terblanche, 1971, for reviews). The more successful of these have involved the use of simple hypothermia (Abouna et al., 1971; Hadjiyannakis et al., 1971; Schalm et al., 1969; Spilg et al., 1971) and continuous hypothermic perfusion either at ambient pressure (Belzer et al., 1970; Hobbs et al., 1968; Turner and Alican, 1970) or combined with hyperbaric oxygenation (Brettschneider et al., 1965). In addition, attempts to utilize a transportable organ storage unit have been made (Hinchliffe et al., 1970). This would have the advantage of making it possible to transport the donor liver to the most closely matched, but perhaps distant, recipient. In order to develop effective methods of liver storage, it is first necessary to have a reliable means of assessing the viability of the stored livers prior to transplantation. Various liver function tests have been employed for this purpose (Bombeck et al., 1968) but there are doubts concerning their predictive value (Abouna et al., 1969; Drapanas, Zemel and Vang, 1966; van Wyk and Eiseman, 1966). The use of morphological criteria as an index of liver viability has not been extensively investigated. A few histopathological studies have been reported (Bombeck et al., 1968; Hobbs et al., 1968) but the findings were generally difficult to correlate with the fate of the stored livers after transplantation. On the other hand, electron microscopy has been shown to be a sensitive and potentially reliable method of evaluating liver damage (Bombeck et al., 1968), but has hitherto received scant attention. In the present study electron microscopy has been used to assess the effects of various storage procedures on the pig liver, using a transportable organ storage unit. MATERIALS AND METHODS Animals.-The animals used in the liver storage programme were Landrace x Large WVhite hybrid pigs, weighing from 28 to 32 kg. Liver Storage.-The technique of pig liver storage using the Vickers' (England) transportable organ storage unit has been described previously (Hinchliffe et al., 1970) and will therefore only be described briefly. During hepatectomy the pig liver was core-cooled by perfusion through the portal vein and the

2 516 R. F. SEARLE AND B. FLAKS FIG. 1.-Electron micrograph of porcine hepatic tissue. Detail of hepatic cell cytoplasm after hyperbaric storage showing an autophagic vacuole (AV). x 12,000. FIG Low-power electron micrograph of hepatic tissue after hyperbaric storage. The hepatocytes in the top and bottom right-hand side of the field contain a vesiculated granular endoplasmic reticulum (ger) and a dilated Golgi zone (G). The hepatocytes on the left-hand side of the field show severe structural derangement and contain swollen mitochondria which are clumped into closely packed aggregates (*). The perisinusoidal hepatic cell surface of these latter hepatocytes is freely bathed by the sinusoidal contents (S). x 7,200. FIG. 3. Electron micrograph of porcine hepatic tissue after hyperbaric storage. An erythrocyte (arrow) is present within the peripheral cytoplasm of the hepatocyte containing a vesiculated granular endoplasmic reticulum. x 10,800. hepatic artery with either chilled (4 C) balanced salt solution, or chilled, diluted heparinized fresh pig blood direct from the Vickers' Unit. In the first series, 7 pig livers were stored in the Vickers' unit for periods up to 13 h with hyberbaric oxygenation (3 atmospheres absolute, 100% oxygen). Continuous perfusion was carried out, at 10 C, using either stored homologous heparinized blood, diluted with an equal volume of diluent, or fresh, heparinized pig blood, one part of which was diluted with 2, 3 or 4 parts diluent. In a second group, 7 pig livers were stored for periods up to 12 h using oxygenation at ambient pressure. Continuous perfusion was carried out at 10 C, using either fresh homologous plasma diluted with an equal volume of diluent, stored heparinized blood diluted with an equal volume of diluent or fresh heparinized blood diluted with 2 parts of diluent.

3 LIVER VIABILITY AFTER EXTRACORPOREAL STORAGE 517 FIG. 4.-Electron micrograph of hepatic sinusoid (S) after hyperbaric storage. The Kupffer cells (K) lie free in the lumen. Note that the limiting membrane is disrupted (*). x 8,575. FIG. 5. Low-power electron micrograph of porcine hepatic tissue after ambient pressure storage. The cell contents appear to be essentially normal in morphology. x 6,425. Samples of hepatic tissue were removed from the edge of the right central pig liver lobe (1) after initial perfusion with either chilled balanced salt solution or fresh heparinized blood; (2) immediately following liver storage in the Vickers' unit. Hepatic tissue from 6 normal young adult pigs served as control tissue. 34 The hepatic tissue was finely minced in icecold cacodylate-buffered 4% glutaraldehyde, ph 7-2, fixed for 4 h, and then washed for not less than 16 h in cacodylate-buffered 025M sucrose at 0 to 4 C. After this the hepatic tissue was post-fixed for 2 h in phosphatebuffered 1% osmium tetroxide, dehydrated in a

4 518 R. F. SEARLE AND B. FLAKS graded ethanol series and embedded in Epon 812. Sections were cut on a Sorvall MT-1 Porter-Blum ultramicrotome, using either glass or Ge-Fe-Ri diamond knives. They were mounted on washed copper grids, stained with lead tartrate and examined in either a Hitachi HS7 or a Philips EM 300 electron microscope. RESULTS Effect of Hyperbaric Storage After hyperbaric storage, most hepatocytes contained increased numbers of autophagic vacuoles (Fig. 1) and exhibited severe glycogen depletion. Control " washout" liver samples showed a normal fine structure. Other regions of stored hepatic tissue contained hepatocytes which displayed more conspicuous fine structural alterations (Fig. 2). These regions were widely distributed throughout the hepatic tissue and it was not possible to relate their distribution to any particular area of the liver lobule. The altered hepatocytes characteristically possessed a vesiculated granular endoplasmic reticulum and dilated Golgi zones. Further, the continuity of the perisinusoidal hepatic cell surface may have been disrupted in some cells, since the peripheral cell cytoplasm frequently contained free erythrocytes (Fig. 3). In addition, a minority of hepatocytes displayed more severe morphological derangement, with swollen mitochondria which were clumped together (Fig. 2). The hepatic sinusoids were generally similar in appearance to those of the " washout " livers. Occasionally, however, the Kupffer and endothelial cell plasma membrane was disrupted, with some release of the cytoplasmic contents into the sinusoidal lumen (Fig. 4). These cells frequently gave the impression of lying free in the sinusoid and, in those regions of the hepatic tissue where the sinusoidal lining was lacking, interruptions in the perisinusoidal hepatic cell surface were noted. The sinusoidal lining was also commonly lacking above those hepatocytes which displayed severe derangement (Fig. 2). Effect of Ambient Pressure Storage The fine structure of porcine hepatic tissue examined after ambient pressure storage, using either dilute blood or fresh plasma, closely resembled that of the control " washout " liver samples. There was no evidence of fine structural damage to either the hepatocytes or the sinusoidal lining (Fig. 5). DISCUSSION One of the most successful methods so far reported for liver storage employs the use of hyperbaric oxygenation. This method was used by Brettschneider and co-workers (1968) who obtained survival of dog recipients of orthotopic liver transplants when the livers had been stored for 8 to 12 h beforehand. Electron microscopy of pig livers stored under hyperbaric oxygenation showed extensive liver damage. Most hepatocytes showed severe glycogen depletion and increased autophagic activity. Additional evidence of hepatic cell damage after hyperbaric storage, however, was afforded by the occurrence of hepatocytes characterized by a vesiculated granular endoplasmic reticulum. Similar findings have been described in hepatic tissue subject to hypoxia (Ashford and Burdette, 1965) or ischaemia (Bassi and Bernelli-Zazzera, 1964). It is well known that vesiculation of the granular endoplasmic reticulum is a common non-specific response to cellular injury of many kinds (Trump and Ericsson, 1965). Focal interruptions in the cell surface of these hepatocytes, indicated by the presence in the peripheral hepatic cytoplasm of red blood cells, would necessarily affect the ionic transport mechanism at the cell surface, and this in turn could have resulted in cellular oedema and vesiculation of the granular endoplasmic reticulum. Other hepatocytes were seen which closely resembled the " Councilman " bodies (Klion and Schaffner, 1966) which appear during hepatic cell necrosis, and probably represent the terminal and irreversible

5 LIVER VIABILITY AFTER EXTRACORPOREAL STORAGE 519 consequences of severe cellular oedema. There was also some evidence of disruption of the integrity of the sinusoidal lining after only short periods of storage with hyperbaric oxygenation. Similar damage has been reported after prolonged periods of storage with oxygenation at ambient pressure (Belzer et al., 1970). This results in platelet deposition on the damaged surface after transplantation (Perkins, May and Belzer, 1970), and may be the cause of the lethal abnormal bleeding in the present study. Sinusoidal damage has been attributed to localized hypoxia during storage (Belzer et al., 1970). It is likely that hyperbaric oxygenation per se is also detrimental, since this effect was not observed in pig livers stored with ambient pressure oxygenation. In contrast, liver storage with ambient pressure oxygenation was not associated with morphological evidence of liver damage. It may be concluded, on the basis of electron microscope examination alone, that the optimal protocol for the preservation of pig livers, for up to 12 h in the Vickers' organ storage unit, consists of ambient pressure storage. In contrast, hyperbaric oxygenation is clearly contraindicated. However, the failure of those pig livers which had been stored even under the " optimal " protocols to support life could not be correlated with their apparently well preserved fine structure. It is possible that critical damage to the ionic transport mechanisms at the hepatic cell surface was incurred during storage and this may not have been detectable by electron microscopy. That such damage may indeed have taken place as a result of hypothermic storage is indicated by the report that the energy-dependent ionic transport system of the liver, unlike that of the kidney, is highly cold-sensitive, so that it is severely impaired at levels of hypothermia of 10 C (Martin et al., 1972). Future methods of evaluating the adequacy of liver storage for liver transplantation cannot therefore rely solely on morphological criteria, even though electron microscopy has proved to be a sensitive test for detecting liver damage. We are indebted to A. Hinchliffe and his co-workers of the Department of Surgery, University of Bristol, for their co-operation in providing the tissue sample for this investigation. This work was carried out during the tenure of a Medical Research Council (London) Scholarship for Training in Research Methods No. G77/3319 by one of us (R.F.S.). REFERENCES ABOUNA, G. M., ASHCROFT, T., HULL, C., HODSON, A., KIRKLEY, J. & WALDER, D. N. (1969) The Assessment of Function of the Isolated Perfused Porcine Liver. Br. J. Surg., 56, 289. ABOUNA, G. M., Koo, C. G., HOWANITZ, L. F., ANCARANI, E. & PORTER, K. A. (1971) Successful Orthotopic Liver Transplantation after Preservation by Simple Hypothermia. Transpl. Proc., 3, 650. ASHFORD, T. P. & BURDETTE, W. J. (1965) Response of the Isolated Perfused Hepatic Parenchyma to Hypoxia. Ann. Surg., 162, 191. BASSI, M. & BERNELLI-ZAZZERA, A. (1964) Ultrastructural Cytoplasmic Changes of Liver Cells after Reversible and Irreversible Ischaemia. Expt. mol. Pathol., 3, 332. BELZER, F. O., MAY, R. E., BERRY, M. N. & LEE, J. C. (1970) Short Term Preservation of Porcine Livers. J. surg. Res., 10, 55. BOMBECK, C. T., BIAVA, C., LONDON, R. E. & NYHUS, L. M. (1968) Parameters of Normal Liver. Function in Isolated, Perfused Bovine Liver. In Organ Perfusion and Preservation, ed. J. C. Norman, New York: Appleton-Century Crofts. p BRETTSCHNEIDER, L., DALOZE, P. M., HUGIJET, C., PORTER, K. A., GROTH, C. G., KASHIWAGI, N., HUTCHISON, D. E. & STARZL, T. E. (1968) Use of Combined Preservation Technique for Extended Storage of Orthotopic Liver Homografts. Surg. Gynec. Obstet., 126, 263. DRAPANAS, T., ZEMEL, R. & VANG, J. 0. (1966) Haemodynamics of the Isolated Perfused Pig Liver; Metabolism According to Routes of Perfusion and Rates of Flow. Ann. Surg., 164, 522. HADJIYANNAKIS, E. J., CALNE, R. Y., MARSHALL, V. C. & DAVIS, D. R. (1971) Successful Preservation of Pig's Livers for 5 to 8 Hours by Simple Ice Storage. Br. J. Surg., 58, 835. HINCHLIFFE, A., IMMELMANN, E. J., BOWES, J. B., HUNT, A. C., WHITE, H. J. O., JOHNSON, M. G., GOLBY, M., PEACOCK, J. H. & RIDDELL, A. G. (1970) A Transportable Apparatus for Liver Preservation. Europ. surg. Res., 2, 427. HOBBS, K. E. F., HUNT, A. C., PALMER, D. B., BADRICK, F. E., MORRIS, A. M., MITRA, S. K., PEACOCK, J. H., IMMELMAN, E. J. & RIDDELL, A. G. (1968) Hypothermic Low Flow Liver

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