Gert Engelbrecht

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.

Another method to prevent venous thrombosis in microsurgery: an in situ venous catheter.

Free-flap failure is in the order of 4 to 10 percent. Heparin is more effective at preventing venous thrombosis than arterial thrombosis. This study was undertaken to investigate the efficacy of delivering heparin at a high dose locally but low dose systemically (heparin infusion via a catheter placed proximal to the venous anastomosis) to prevent venous thrombosis in microsurgery. A model of venous thrombosis was first established by a venous inversion graft in the rat femoral vein (this was performed in seven animals and resulted in 100 percent thrombosis). Saline and heparin were delivered proximal to the inverted vein graft to assess the effect of each in preventing venous thrombosis. Flow/patency distal to the inverted vein graft was assessed by observation under the microscope, the milk test, and rate of flow (flowmeter). Saline infused via a catheter proximal to the venous inversion graft resulted in 100 percent thrombosis in 10 animals. Heparin (100 U/ml at 2 to 3 ml/hour) infused through a catheter for 2 hours proximal to the anastomosis resulted in flow in all 10 animals during the infusion. Blood was also taken before beginning the procedure (control) and after the heparin infusion distal to the anastomosis (local partial thromboplastin time) as well as in the contralateral femoral vein (systemic). The control for all animals that received heparin was <3 minutes. The systemic partial thromboplastin time after heparin infusion was <3 minutes in seven animals, 3.3 minutes in two animals, and >7 minutes in one animal. The local partial thromboplastin time distal to the inverted vein graft was >10 minutes in nine animals and 3.7 minutes in one animal. The study also had a clinical component, in which a catheter was placed in a vein of the free flap, and heparin was infused over 5 days. This technique has been used in 83 consecutive free flaps. In three recent free flaps performed on the limbs, the local partial thromboplastin time (close to the anastomosis) was raised but the systemic time was normal. This technique offers a method in preventing venous thrombosis in microsurgery. It is simple to implement and is not associated with the systemic complications of heparin.

Non-toxicity of IV Injected Perfluorocarbon Oxygen Carrier in an Animal Model of Liver Regeneration Following Surgical Injury

Lethal dose experiments in animals have demonstrated that second-generation perfluorocarbon oxygen carriers are remarkably non-toxic. However, this non-toxicity has not previously been demonstrated in a liver failure scenario. A surgical liver damage and regeneration model in rats was selected using a well-controlled cross tabulated study design. A large number of physiological, biochemical, and hematological parameters were measured. No indications were found that intravenously injected perfluorooctyl bromide emulsion was toxic at the concentrations employed, in either healthy or severe liver injury scenarios. Neither was there any significant impact on the rate of liver regeneration following the injuries. Bearing in mind prior human clinical studies, it is therefore safe to assume that perfluorocarbon emulsions are also non-toxic in bioartificial liver treatments.

Amino acid imbalance following portal diversion in the rat. The relevance of nutrition and of hepatic function.

End-to-end portacaval transposition has previously been shown to produce less hepatocellular dysfunction than end-to-side portacaval shunt in the rat. Liver weight is also significantly reduced after portacaval shunt compared to portacaval transposition and these differences are not abolished by pair-feeding. Histological evidence of CNS damage is also reduced in transposed rats compared to shunted animals. This study examines the amino acid and hormone changes in these models. The characteristic amino acid changes of chronic liver disease (decreased branched-chain and elevated aromatic amino acids) are reproduced in portacaval shunt rats, but not in portacaval transposition. The differences between these groups in the branched-chain amino acids, but not those in the aromatic amino acids, are reduced by pair-feeding. Insulin and glucagon are elevated to a similar extent in both groups. These findings add further support to a role for peripheral amino acid imbalance in the pathogenesis of portal-systemic encephalopathy. Normal liver function, maintained by replacement of portal inflow with systemic blood, appears to minimize both CNS damage and amino acid changes.

Vaccinia Virus Complement Control Protein (VCP) Improves Kidney Structure and Function Following Ischemia/Reperfusion Injury in Rats

Renal transplantation is often confronted with ischemia reperfusion (I/R) injury that accounts for a delayed recovery of the graft. This surgically and biologically induced injury often results in activation of the complement system. The vaccinia virus complement control protein (VCP) down-regulates both the classical and alternative complement pathways by preventing the formation of C3b, a component where both pathways converge. The aim of the study was to investigate the effect of VCP on renal I/R injury. Long Evans rats were subjected to laparotomy, mobilization of the right kidney in unilateral ischemia, and both kidneys in bilateral ischemia. The renal arteries were clamped for 60 min followed by 24 h reperfusion time. The animals were randomly allocated to receive recombinant VCP (rVCP), natural VCP, and humanized recombinant VCP (hrVCP) combination, vehicle (PBS), or sham group. Blood samples were collected for biochemical studies, and the kidneys were removed for histopathologic and immunohistochemical studies. The biochemical studies in the bilateral ischemia showed that the PBS group displayed 1.5-fold and 5-fold increases in the urea and creatinine concentrations, respectively, compared with the VCP/hrVCP groups. In both models, the histopathologic study revealed focal necrosis of the tubular epithelial cells in the rVCP or VCP/hrVCP treated animals compared with the diffuse and markedly elevated field scores in the PBS controls. The immunohistochemical study showed significant C3 deposition in the renal tubules of the PBS controls compared with the rVCP or VCP/hrVCP groups, suggesting that rVCP, VCP/hrVCP reduced I/R injury by inhibiting the biosynthesis of C3.

The effect of administration of FK506 on delayed regeneration in flushed partially hepatectomized livers

It has been shown previously that liver regeneration after partial hepatectomy in rats is delayed if the liver is subjected to either concurrent ischaemia, flushing with cold solution, or grafting. We have shown recently that treatment with CsA preoperatively overcomes the suppressive effect of flushing and returns the regenerative response to a normal time scale. The present study was designed to investigate whether administration of FK506 would also return the observed delayed regenerative response to normal. Long-Evans rats weighing 250-350 g were subjected to standard 68% partial hepatectomy. Group 1 had no further treatment; in group 2, the liver remnant was flushed with 10 ml cold (4 degrees C) Ringers lactate solution, and in group 3, FK506 (1 mg/kg/day) was administered by intramuscular injection for 3 days before the partial hepatectomy and flushing as in group 2; a final dose was given after completion of the procedures. Animals were killed in sets of 6 per group at 4, 24, 48, 72, and 96 hr after surgery and blood samples were taken for measurement of plasma aspartate amino-transferase. Liver biopsies were analyzed for measurement of thymidine kinase and ornithine decarboxylase activity and for counting of mitotic figures. While the highest recorded thymidine kinase activity occurred in group 1 at 24 hr, this was delayed to 48 hr in both group 2 and 3 and counts remained high up to 96 hr in group 3. Mitotic indices were only significantly elevated (compared with group 1 at 96 hr), while ornithine decarboxylase activity did not correlate with these changes being significantly lower than in groups 2 and 3 at 4 hr and in group 3 also at 24 hr. Plasma aspartate aminotransferase was also significantly higher in group 3. It is concluded that the administration of FK506 preoperatively to rats subjected to partial hepatectomy and flushing did not restore the delayed regenerative response to normal but enhanced the response (as measured by thymidine kinase but not by mitotic indices) which commenced at 48 hr and was still present at 96 hr.

The effect of intravenous versus enteral infusions of lipid emulsions (Intralipid) on bacterial translocation and immune function in a rat model of resuscitated haemorrhagic shock and trauma

Abstract This study aimed to elucidate the influence of the route of administration of exogenous lipid emulsions (Intralipid, Kabivitrum, Stockholm, Sweden) on bacterial translocation and immune suppression in a shocked and resuscitated rat trauma model. Sixty-five male Long-Evans rats were divided into three groups, viz. controls, enterally fed and intravenously fed rats. Experimental rats were traumatized by laparotomy with manipulation of bowel, creation of subcutaneous and femoral catheterization tunnels, and shocked by controlled exsanguination for twenty minutes before resuscitation by reinfusion of shed blood. While the experimental groups received 20% Intralipid infusions, post-operatively, the control group received standard rat chow and water ad libitum . Lipid infusions were administered at a rate of 70ml/kg/day, either intravenously via a central line, or enterally, via a gastro-jejunostomy line. Twenty four hours later, at sacrifice, bacterial translocation rates into mesenteric lymph nodes (MLN), blood and organs were studied. In addition, mitogen-stimulated lymphocyte transformation rates (LTR), as an index of immune suppression were assessed. The greatest rates of translocation were to the mesenteric lymph nodes, with only insignificant translocation to peripheral blood, liver and spleen occurring. Both experimental groups showed significantly more translocation than the uninjured controls (P

Does liver transplantation in the rat cause a regenerative response. The effect of arterialisation of the graft.

This study was conducted to determine the pattern of early regenerative response to orthotopic intact liver transplantation in the rat and to investigate whether the response differed in grafts with or without revascularisation of the arterial bed. Outbred male Long Evans (LE-LE allogeneic, non rejector) rats weighing 300–350g were subjected to orthotopic intact liver allograft using a “sleeve” anastomosis for the hepatic artery. Total warm ischaemia ranged from 19 to 34 minutes and no storage was employed. Comparison was made with a group of control rats which were subjected to 25 minutes total inflow occlusion and regeneration was measured with tissue thymidine kinase (TK) and mitotic figures. Samples were taken at 1,2,4,7,10 and 20 days post-operatively. Plasma aspartate aminotransferase (AAT) and light microscopy were used to evaluate hepatocyte necrosis. There was a brief sharp increase in TK and AAT in the first 24 hours after sham operation but no appearance of mitotic figures. A similar but more prolonged increase in TK occurred in the arterialised transplant group with the highest levels recorded on day 4. The level remained significantly elevated above pre-operative until 10 days and declined within 20 days. Mitotic figures appeared at 2 days, reached significance at 7 and 10 days and had disappeared by 20 days. The pattern of changes was accentuated in animals in which the artery was not reanastomosed and the increases in TK and AAT were still significant at 20 days. Whilst similar degrees of peri-portal cellular infiltrate occurred in both groups of rats, bile duct proliferation was most obvious in non-arterialised animals. As compared with a previously prepared group of partially hepatectomised animals, the regenerative response after liver transplant was delayed and prolonged especially in the non-arterialised group. It is concluded that a regenerative response occurred in liver allografts in rats soon after operation, which was slightly prolonged if the hepatic artery was not anastomosed and that the response seemed to be related to hepatocyte damage which occurred as part of the procedure. The relevance of these findings to clinical liver transplantation is discussed.

Effect of mesentericocaval shunt upon globulin levels in the rat

Rats develop hyperglucagonemia after end-to-side portacaval shunt and also after splenocaval shunt. In this study, animals with mesocaval shunt were shown not to develop hyperglobulinemia or increased titers of lipopolysaccharide antibodies. It is currently thought that the hyperglobulinemia of cirrhosis results from diversion of immunogens past the liver into the systemic circulation with stimulation of sites of globulin synthesis. Since all procedures result in development of immune complexes in the systemic circulation, an alternative hypothesis was sought. It is proposed that the liver may regulate antibody production by the spleen but that this mechanism only operates if splenic venous blood passes through the liver. Portacaval shunt and splenocaval shunt result in diversion of splenic venous blood past the liver and, consequently, failure of regulation, while after mesocaval shunt, splenic venous blood still enters the liver and thus the regulatory mechanism could operate.

One-stage total hepatectomy in the rat using microvascular anastomoses

A method is described for one‐stage total hepatectomy in the rat using microvascular techniques. The operation consists of creation of a side‐to‐side mesocaval shunt performed just distal to the renal veins, total removal of the liver with ligation of the portal vein, hepatic arteries, and the bile duct. The vena cava is reconstructed with a segment of vena cava taken from a donor animal. The procedure takes 32 ± 5 min to complete. Blood glucose concentration was maintained by infusions of balanced salt solution containing from 0.625% to 2.5% dextrose. Mean survival time was 10.5 hr (range, 5.5 to 21.5 hr).