Rosemary Hickman

Animal models of fulminant hepatic failure

The six requirements for a satisfactory animal model of fulminant hepatic failure are reversibility, reproducibility, death from liver failure, a therapeutic window, a large animal model, and minimal hazard to personnel. Different models may be required to evaluate the various types of liver failure seen in man. The available models include surgical anhepatic and devascularization procedures, as well as hepatotoxic drug administration using agents such as carbon tetrachloride, acetaminophen, or galactosamine. Currently combined surgical and drug models appear to provide the best model but the search for the ideal models continues.The six requirements for a satisfactory animal model of fulminant hepatic failure are reversibility, reproducibility, death from liver failure, a therapeutic window, a large animal model, and minimal hazard to personnel. Different models may be required to evaluate the various types of liver failure seen in man. The available models include surgical anhepatic and devascularization procedures, as well as hepatotoxic drug administration using agents such as carbon tetrachloride, acetaminophen, or galactosamine. Currently combined surgical and drug models appear to provide the best model but the search for the ideal models continues.

Energy charge as an indication of liver viability : a comparison of changes in livers that remained intact with those subjected to autografting

As efforts to assess the viability of liver grafts continue, the recent description of noninvasive measurement by fluorimetry or magnetic resonance spectrometry of adenine nucleotides has brought energy charge into focus again as an index of viability. Many previous studies have been conducted in human donor livers that have clinical relevance but which cannot be standardized, or in rats in which the hepatic artery is not anastomosed. In the present study, pig livers were definitively rendered ischemic for 1 or 2 hr. In one group the livers were then revascularized (intact) while in the other, the livers were removed during the final 20-30 min of the ischemic period and were subjected to autograft. There was a marked difference in survival between the intact and the autograft groups. One hour of ischemia in the intact group was associated with survival comparable to that of autograft controls (8-100 days); 2 hr of ischemia caused shortened survival, ranging from 2 to 18 days. In the recipients of autografts, survival after 1 hr of ischemia ranged from 3 to 16 days; after 2 hr of ischemia no autograft recipient survived overnight. The energy charge returned to the preoperative level after 2 hr of ischemia in both intact and autograft groups. The concentrations remained depressed after 2 hr of ischemia in autografted animals, thus being associated with survival. However, the patterns of total adenine nucleotide and adenosine triphosphate were not always similar to those of energy charge. The concentrations of aspartate aminotransferase were similarly elevated in all ischemic groups irrespective of duration or subsequent survival. There was, however, a close association between euglobulin lysis times (ELT) and survival. In the autograft recipients of livers subjected to 2 hr of ischemia that did not survive overnight the ELT remained significantly shortened. It is concluded that adenine nucleotide metabolism is important as an index of viability, but that concentrations of total and individual adenine nucleotides and the energy change all need to be computed. There does, however, appear to be an absolute relationship between survival and euglobulin lysis time that would be clinically useful in patients undergoing liver transplantation or hepatic vascular exclusion.

Fragmentation pattern of mucins in normal and diseased gastric mucosae : A glycoprotein fractionates with gastric mucins purified from mucosal scrapings of cancer and peptic ulcer patients

Background/Aims: Gastric cancer, a fatal malignancy, is prevalent in the Western Cape region of South Africa. The aim of this study was a biochemical characterisation of gastric mucins in this disease, compared with gastric ulceration and controls from transplant donors. Methods: Mucins were extracted in a denaturing medium (to prevent endogenous proteolysis) and purified by caesium chloride density gradient ultracentrifugation. Analysis of mucin was by gel filtration, SDS-PAGE and Western blotting methods. Results: All samples of mucin when analysed by gel filtration were found to contain polymeric glycoprotein together with varying amounts of lower-molecular-weight glycoprotein. SDS-PAGE and Western blot analysis showed that diseased stomachs had glycopeptides of a wider range in size and antigenicity with a greater number of smaller fragments immunoreactive to monoclonal antibodies 2–12M1 and 9–13M1. We identified by SDS-PAGE a glycoprotein which co-fractionates in a caesium chloride density gradient with mucins isolated from gastrectomy specimens resected for carcinoma and peptic ulceration and which was absent from mucins of the transplant donor control group. This neuraminidase-sensitive glycoprotein resisted dissociation from mucin during purification in a 3.5 M CsCl density gradient but was partially separable by Sepharose 2B gel chromatography and heat treatment (100°C, 2.0 min) in SDS. Chemical analysis of the glycoprotein by HPLC favours it being an N-linked glycoprotein. Its non-ideal electrophoretic properties make its exact size estimation difficult and we ascribe to it a broad size range of Mr ∼55–65 kD. Conclusion: We conclude that mucins from diseased stomachs were more degraded than those from donors and that the diseased mucosa reproducibly secretes a Mr ∼55–65 kD glycoprotein, the role of which needs to be established.

FUNCTION OF KIDNEY GRAFTS FROM BRAIN-DEAD DONOR PIGS: THE INFLUENCE OF DOPAMINE AND TRIIODOTHYRONINE

There are conflicting reports about the effects of administration of dopamine to brain-dead donors upon posttransplant organ function. This study compared the survival and serum creatinine levels in pigs that received renal grafts from untreated controls, from controls in which either the donor or donor and recipient received dopamine or from animals rendered brain dead for 16 hr by acute elevation of intracranial pressure, and given standard supportive treatment. In two additional groups, brain-dead donors were given dopamine or dopamine with triiodothyronine. Recipients of grafts from control animals or from brain-dead donors survived the 7-day period of study and showed minimal changes in serum creatinine. Recipients of grafts from brain-dead donors given dopamine however showed reduced survival and progressive increase in serum creatinine. This did not occur in the group given triiodothyronine concurrently with dopamine. It is suggested that if administration of dopamine is essential to treat donor hypotension, concurrent use of triiodothyronine may preserve posttransplant renal function.

Increased serum immunoglobulin levels following portacaval shunt in the normal rat

Normal rats subjected to end-to-side portacaval shunt showed decreased survival and weight gain, a progressive fall in serum albumin and reciprocal rise in serum gamma globulin when compared with sham-operated controls for 12 weeks. Antibacterial lipopolysaccharide antibody was detected in significant titre at the sixth and twelfth weeks. It is suggested that the elevated levels of gamma globulin and reversal of albumin/globulin ratios noted in these animals may represent an immune response to bacterial lipopolysaccharides released into the systemic circulation as a result of the portacaval shunt. The hyperglobulinaemia of cirrhosis in human subjects may have a similar aetiology.

Tumour necrosis factor levels during acute rejection and acute tubular necrosis in renal transplant recipients.

Plasma tumour necrosis factor levels were measured serially in 16 patients following renal transplantation, and in 10 patients on haemodialysis and in 12 patients on peritoneal dialysis. The patients on peritoneal dialysis had lower plasma TNF levels than the patients on haemodialysis. There was a decrease in TNF levels immediately following renal transplantation; this is probably related to the bolus doses of methylprednisolone administered intra-operatively. Patients with acute rejection had higher levels of TNF than non-rejecting patients. The increase in TNF levels in rejecting patients was observed 2 days before the clinical manifestation of acute rejection. There was a marked decrease in TNF levels in rejecting patients in response to treatment with steroids. Patients with delayed graft function had higher levels of TNF on the first post-operative day compared to patients with immediate function. These changes in plasma TNF levels following renal transplantation have important clinical and therapeutic implications.

The effect of prostaglandins, branched-chain amino acids and other drugs on the outcome of experimental acute porcine hepatic failure.

Prostaglandins, N-acetyl-cysteine, cholestyramine and essential phospholipids have all been shown to protect experimental animals from severe hepatic damage in various models when used prophylactically. Silibinin has been used in the treatment of Amanita phalloides poisoning. Branched-chain amino acids have been recommended in acute hepatic failure. We have used all these forms of therapy at the time of initiation of hepatic failure in a reliable pig model. Of the above, only prostaglandins have been shown to reverse the effects of the hepatic insult in terms of prolonged survival and histological changes. Although conventional liver function tests and plasma amino acids in prostaglandin-treated animals are not improved, cerebrospinal fluid amino acids remain normal, in contrast to the other groups of untreated and treated hepatic failure animals.

Hepatic blood flow during reduced liver grafting in pigs : a comparison of controls and recipients of intact allografts

Intraoperative changes in portal venous and hepatic arterial flow were compared in porcine recipients of reduced liver grafts with recipients of intact grafts and sham-operated controls. Control animals showed no significant changes in hepatic blood flow (measured with perivascular ultrasonic cuffs), heart rate, mean arterial pressure, cardiac output, acid/base balance, plasma sodium, potassium, glucose, or catecholamines. Recipients of intact or reduced grafts showed hypotension, reduced cardiac output, tachycardia, and increased systemic vascular resistance during the anhepatic phase, which lasted approximately 30 min. These changes returned to normal in recipients of intact grafts but in recipients of reduced grafts, levels returned only to 50–60% of baseline. After intact grafting, total liver blood flow and the portal and arterial components returned to baseline within 2 hr of revascularization, but after reduced grafting, hepatic arterial flow values remained depressed to 50–60% of baseline. Plasma epinephrine and norepinephrine were unaltered during control operation but increased 4- to 20-fold in recipients of all grafts. These returned towards baseline in all except recipients of reduced grafts, in which norepinephrine levels remained significantly elevated for the 4 hr of postoperative study. These data highlight persistent elevation of plasma norepinephrine after reduced liver grafting, which may have contributed to the diminished hepatic arterial flow. These results need to be confirmed in adult recipients of split liver grafts in whom grafts are comparatively small. In such patients receiving donor livers which have undergone prolonged storage, the effects of increased plasma norepinephrine levels upon donor agonal arterial spasm may be significant.

Comparison of in vivo and ex vivo porcine liver function using the same liver

In vivo and ex vivo liver function was compared using the same livers to exclude interanimal variation in hepatic function. Six male pigs were anesthetized, and catheters and perivascular flow probes placed for transhepatic sampling and hepatic arterial and portal venous flow measurement. After a 2-h in vivo study period, the livers were resected and studied immediately afterwards for a further 2 h ex vivo as an isolated perfused preparation (Experiment A). Hepatic function in a further 6 pig livers (Experiment B) was studied ex vivo only for comparison with the ex vivo livers from Expt. A to determine whether the prior in vivo study had affected hepatic function. Despite using the same livers with similar total hepatic blood flows, (0.91 +/- 0.16 ml.g-1 x min-1) in vivo and (0.84 +/- 0.03 ml.g-1 x min-1) ex vivo, hepatic oxygen consumption (6.5 +/- 0.9 vs 2.6 +/- 0.2 ml O2 x 100 g-1), adenosine-5-triphosphate content (5.22 +/- 0.62 vs 4.14 +/- 0.71 microM.g liver-1) and bile flow (15.1 +/- 1.2 vs 6.0 +/- 1.0 ml.h-1) were initially less ex vivo and remained so throughout the study, while perfusate potassium (initially) (3.7 +/- 0.1 vs 6.4 +/- 0.3 meq.l-1), and aspartate aminotransferase (50 +/- 9 vs 76 +/- 5UL-1) was consistently higher than in vivo values. Initial hepatic energy charge (0.620 +/- 0.034 vs 0.552 +/- 0.061) and total adenine nucleotides 12.49 +/- 0.60 vs 11.66 +/- 0.62 microM.g liver-1) were not different and remained so subsequently.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of thyroid hormone replacement in a porcine brain death model

This study was conducted to determine whether the administration of tri-iodothyronine (T3) to brain-dead donor pigs would improve hemodynamic instability, serum levels of thyroid hormones, or the outcome of transplantation of donor livers. Brain death was caused in young pigs (25–38 kg) by rapid inflation of an intracranially implanted balloon catheter. The animals were maintained on a ventilator and frequent measurements of acid/base balance, electrolytes, and glucose were made. At the end of 16 hr, livers were removed and implanted into prepared recipients. Serum-free tri-iodothyronine fell to zero at the end of 16 hr, and there was a 4–6-fold decline in free thyroxine (T4). The levels of serum reverse T3 (rT3) however, increased up to 6-fold. In animals treated with tri-iodothyronine 2 Mg/hr, the serum levels of free T3 and T4 were not changed but the levels of serum reverse T3 (rT3) increased further. There were no apparent correlations between any hemodynamic parameter and serum thyroid hormone levels in the donors. After the liver transplants, recipients could be divided into those that survived longer than 6 days and those that did not. Although there were significant differences in the plasma levels of alanine amino-transferase, aspartate aminotransferase, and alkaline phosphatase, there was no correlation between survival and whether the donor had received tri-iodothyronine. Although other hormones, including insulin and cortisol, may also be necessary, there is no indication from these studies that the administration of tri-iodothyronine to brain-dead donors of liver grafts benefits the serum hormone levels in the donors or the subsequent survival of the recipients.