Xenogeneic Perfusion Research Articles

Effect of TNF-alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys.

Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system. Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1. Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group. We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.

Thrombin Generation and Platelet Activation in a Xenogenic Lung Perfusion Model Determine Survival of GalTKO.hCD46 Lungs

Purpose The development of GalTKO pigs with additional expression of hCD46 has contributed to significantly improved outcome of lungs in an ex vivo xenogenic perfusion setting. However, “survival” of those lungs varies from a very early failure to an elective termination after 4h of perfusion. Here we evaluate factors that correlate with early GalTKO.hCD46 lung failure. Methods and Materials 33 transgenic GalTKO.hCD46 pig lungs were perfused with heparinized fresh human blood until failure (by oxygenation or PA flow and pressure criteria) or elective termination at 4h. Based on the survival time, experiments were divided into 3 groups (group 1: survival Results Median survival time was 171’ with 11 lungs surviving Conclusions Results of this analysis demonstrate that intensity of initial coagulation cascade and platelet activation (but not complement activation or anti-non-Gal IgM level) correlates with the outcome of xenogenic lung perfusion. We conclude that targeting coagulation and platelet activation, rather than complement or antibody, are required to improve survival of xenogenic lungs.

Effect of TNF‐alpha blockade on coagulopathy, survival and endothelial activation in xenoperfused porcine kidneys

Background: Following pig to primate kidney transplantation, endothelial cell activation and xenogenic activation of the coagulation (XAC) system of the recipient eventually leading to organ dysfunction and disseminated intravascular coagulation (DIC) can be observed (1). In this study we examined the effect of a TNF‐Receptor‐Fusionprotein (TNF‐RFP) on XAC and endothelial cell activation utilizing an appropriate ex‐vivo perfusion system.Methods: Using an ex‐vivo perfusion circuit based on C1‐Inhibitor (C1‐Inh) and low dose heparin administration, we have analysed XAC following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with HTK organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10) or with addition of TNF‐RFP (n = 5). After perfusion tissue samples were obtained for QPCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E‐Selectin, ICAM‐1 and VCAM‐1 in QPCR.Results: Kidney survival during organ perfusion with human blood, CI‐Inh, heparin but without any further pharmacological intervention was 126 ± 78 min. XAC was observed with significantly elevated concentrations of d‐Dimer, thrombin antithrombin complex (TAT), resulting in formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E‐Selectin and VCAM‐1. In contrast, pharmacological intervention with TNF‐RFP reduced the consumption of antithrombin and fibrinogen and prolonged organ survival to 240 min (±0). Additionally, formation of microtrombi was reduced compared to the xenogenic control without pharmacological intervention. Remarkably, also endothelial cell activation was significantly reduced in the TNF‐RFP group.Conclusions: We conclude that TNF‐RFP can interfere with XAC and is able to suppress endothelial cell activation in this ex vivo perfusion model, thus representing a potential candidate as an adjuvant immunosuppressive drug.Reference: 1. BÜhler L, Basker M, Alwayn IPet al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000; 9: 1323–1331.

Expression of Inflammation and Coagulation Genes During GalTKO.hCD46 Xenogenic Lung Perfusion

Expression of Inflammation and Coagulation Genes During GalTKO.hCD46 Xenogenic Lung Perfusion

GalTKO.hCD46 Transgenic Pig Lungs: 4-Year Experience in a Xenogenic Perfusion Model

Purpose: Perfusion of wild type pig lungs with human blood leads within minutes to hyperacute rejection that is associated with the immediate sequestration of circulating platelets and neutrophils and the activation of the coagulation and complement cascade. The generation of GalTKO pigs has significantly improved survival, however, the described reactions are still present even though at a lower level only. To further control the activation of the complement cascade, a human complement regulatory protein (hCPRP) gene was inserted on the basis of a GalTKO-pig. Those GalTKO.hCD46 transgenic organs have been use in xenotransplantation for several years now. Here we present the results of our 4-year experience with the use of GalTKO.hCD46 pig lungs in a xenogenic lung perfusion model. Methods: Wildtype (n=17), GalTKO (n=16) and GalTKO.hCD46 transgenic pig lungs (n=31) were perfused with heparinized fresh human blood until failure or elective termination after 4 hours. At prespecified time points, blood and tissue samples were collected. Pulmonary artery (PA) flow and pressure were recorded online. Results: Median survival time (MST) in the wild type group was 10‘, in the GalTKO cohort 120‘. The additional expression of hCD46 showed a trend to higher MST of 180′ (p=0.21). Pulmonary vascular resistance was not improved in the GalTKO.hCD46 group when compared to GalTKO alone. Platelet sequestration was significantly reduced in the early perfusion phase (% initial platelets remaining at 5‘: GalTKO.hCD46 51±34 vs. GalTKO 26±14, p=< 0.05) but not at the later time points. Complement activation measured by C3a level was lower at all time points in the GalTKO.hCD46 group with statistically highly significant lower level after 2h of perfusion (GalTKO.hCD46 237±175 vs. GalTKO 873±1002, p= 0.009). Conclusion: Complement activation is better controlled in GalTKO pig lungs genetically modified to express hCD46, and presumably contributes to the trend toward longer survival of those organs. However, various other rejection mechanisms like activation of the coagulation cascade, platelet sequestration, and/or non-Gal antibody-mediated rejection lead to organ injury. Additional interventions targeted to these mechanisms will very likely be necessary to further advance lung xenotransplantation toward clinical application.

Quercetin glucuronide preserves cardiac function in porcine hearts during perfusion with human whole blood by inhibiting platelet/neutrophil activation and preserving venular barrier function

Quercetin glucuronide (QG) reportedly inhibited the activation of blood cells of the acute immune system and stabilized venular wall models (cells of human origin) in presence of inflammatory mediators. We hypothesized that QG would protect isolated pig hearts during acute activation of the immune system (xenogenic perfusion with human whole blood). 12 hearts of non‐transgenic landrace pigs were explanted (organ preservation solution supplemented with 50 μM QG) and immediately cirumfused for 180 min in an ex vivo working‐mode with human blood in the absence (control group, n=6) or presence of 50 μM QG in the blood (n=6). Hemodynamic function was evaluated and inflammatory interactions were histologically investigated. All hearts maintained mandatory perfusion pressure of &gt;55 mmHg throughout the whole study period. Cardiac function was significantly improved in the QG‐group and very similar to that of pig hearts perfused with autologous blood. Stroke work index (mean ± SEM) was 4272±434 erg/g in the QG‐group vs. 2790±558 erg/g in controls without QG (P&lt;0.05) after 30 min and was still significantly higher at the end of the perfusion period (QG: 3145 ± 160 vs. control: 1570 ± 300 erg/g; p&lt;0.05). Venulothrombosis, venulitis, and excessive accumulation of leucocytes could not be detected in the QG‐group after specific staining and microscopic examination of ventricular myocardium. Supported by a grant of the DFG.

Efficacy of double-filtration plasmapheretic cross-circulation with a high-permeability membrane using canine harvested liver in porcine fulminant hepatic failure model

Introduction The use of bioartificial liver devices requires. A sufficient liver cell mass to provide adequate metabolic support, reduction of xenogeneic immune reactions, and avoidance of viral transmission. We have developed a plasmapheresis system using a semipermeable membrane combined with canine whole liver perfusion (PMCWLP). In this study, we investigated the efficacy of our system in a porcine fulminant hepatic failure (FHF) model. Methods The porcine FHF model was established by intraportal administration of alpha-amanitin (0.1 mg/kg) and lipopolysaccharide (1 μg/kg). Nine hours after drug injection, xenogenic perfusion treatment was performed twice within 6 hours ( n = 5). As the plasmapheresis device, we used a hollow-fiber module with cellulose diacetate porous fibers (pore size, 0.05 μm, surface area, 2 m 2). The canine whole liver was perfused with modified Krebs solution, which is commonly used in many laboratories, containing albumin (2 g/dL) and glucose (300 mg/dL). Control pigs ( n = 10), had the circuit not connected to the whole canine liver. Results The survival of FHF pigs was significantly increased by the treatment (58.9 ± 21.8 hour) compared with the controls (22.3 ± 8.1 hour). Mean blood ammonia levels and intracranial pressure during treatment were significantly lower compared with control groups. Conclusion Treatment of FHF pigs with the system significantly increased survival time, suggesting that this method may have applications as a clinical liver assist device.

No functional benefit for hDAF-transgenic rat livers despite protection from tissue damage following perfusion with human serum.

Currently, xenogeneic extracorporeal liver perfusion is used in the treatment of acute liver failure. In order to determine whether transgeneity for human regulatory proteins could improve the functional outcome of the ex-vivo liver in relation to the histopathological changes, we studied the effect of the humoral mechanism in xenogeneic isolated rat liver perfusion in normal and transgenic rat livers. Isolated rat liver perfusion was performed for 2 h in normal rat livers with Krebs Henseleit (KH) and human serum (HS), and in livers transgenic for human decay accelerating factor (hDAF; Tg HS). Function of the liver was established by measurement of liver enzymes and bile production, and clearance of bromosulphophthalein (BSP). Tissue specimens taken after perfusion were analysed by routine histology and immunofluorescence staining for C3c deposition. No change in release of liver enzymes could be established throughout the perfusion period. In the 2nd hour, a higher level of bile production was seen for the transgenic group than for the HS group. The transgenic rat livers outperformed the normal livers perfused with HS, when BSP concentration in the bile was measured; however, clearance of BSP from the perfusate was not significantly different. Haematoxylin-eosin (HE) staining of the liver tissue showed evidence of hyperacute rejection in the HS group. There was only mild tissue injury in the transgenic liver. High-intensity fluorescent staining for C3c deposition was seen in the normal livers perfused with HS, and significantly less in the transgenic livers. Although histologically less tissue damage and less C3c deposition was shown, no significantly improved function of the livers transgenic for hDAF was established. These results suggest that for short-term extracorporeal liver perfusion transgenesis offers no functional benefit.

Adenovirus-mediated gene transfer of triple human complement regulating proteins (DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation model. Part II: xenogeneic perfusion of the porcine liver in vivo

Abstract In this study, the adenovirus-mediated gene transfer of triple human complement regulating proteins was investigated in xenogeneic pig liver perfusion. The porcine liver was perfused in situ at 4 °C under a pump-driven veno-venous shunt of the portal vein and inferior vena cava, with 5 to 15×1011 plaqueforming units (pfu) of adenovirus vector (group 1: AxCALacZ; 2: AxCACD59; 3: AxCACD59 + AxCADAF; 4: AxCACD59 + AxCADAF + AxCAMCP) for 1 h (for each, n=3). The livers were harvested 24 h after gene transfer and then were reperfused ex-vivo with fresh human blood for 2 h. In immunohistochemical staining, each complement regulating protein (CRP) showed a distribution similar to that of the LacZ expression. The C3 levels in the perfusate were also maintained at higher levels in group 4 from 60 to 120 min after reperfusion (C3: 85% to 95% of the initial level) than in groups 1 to 3 (C3: 80% to 90% of the initial level) from 60 to 120 min after reperfusion. The complement deposition on the porcine liver [C3, membrane attack component (MAC)] decreased significantly more in group 4 than in groups 1 to 3. In conclusion, the adenovirus-mediated multiple gene trasnfer of human CRPs (hCRPs) was found to effectively suppress the complement activation in xenogeneic pig liver perfusion.

Adenovirus-mediated gene transfer of triple human complement regulating proteins (DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation model. Part II: xenogeneic perfusion of the porcine liver in vivo.

In this study, the adenovirus-mediated gene transfer of triple human complement regulating proteins was investigated in xenogeneic pig liver perfusion. The porcine liver was perfused in situ at 4 degrees C under a pump-driven veno-venous shunt of the portal vein and inferior vena cava, with 5 to 15x10(11) plaque-forming units (pfu) of adenovirus vector (group 1: AxCALacZ; 2: AxCACD59; 3: AxCACD59 + AxCADAF; 4: AxCACD59 + AxCADAF + AxCAMCP) for 1 h (for each, n=3). The livers were harvested 24 h after gene transfer and then were reperfused ex-vivo with fresh human blood for 2 h. In immunohistochemical staining, each complement regulating protein (CRP) showed a distribution similar to that of the LacZ expression. The C3 levels in the perfusate were also maintained at higher levels in group 4 from 60 to 120 min after reperfusion (C3: 85% to 95% of the initial level) than in groups 1 to 3 (C3: 80% to 90% of the initial level) from 60 to 120 min after reperfusion. The complement deposition on the porcine liver [C3, membrane attack component (MAC)] decreased significantly more in group 4 than in groups 1 to 3. In conclusion, the adenovirus-mediated multiple gene transfer of human CRPs (hCRPs) was found to effectively suppress the complement activation in xenogeneic pig liver perfusion.

Efficacy of Nonwoven Fabric Bioreactor Immobilized with Porcine Hepatocytes for Ex Vivo Xenogeneic Perfusion Treatment of Liver Failure in Dogs

We have developed a new bioartificial liver bioreactor filled with porcine hepatocytes immobilized on polyester nonwoven fabric (NWF). In this study, we investigated the efficacy of our hybrid bioartificial liver system incorporating the NWF bioreactors and an immunoglobulin adsorbent column for perfusion treatment in a canine liver failure model. Xenogeneic perfusion treatment for operative canine liver failure models were performed for 3 h, and survival time, intracranial pressure, and blood and cerebrospinal fluid data were documented. Treatment was carried out without obstruction by immunological rejection when immunoglobulin adsorbent columns were used with the NWF bioreactors in combination. Dogs treated with this system exhibited a restricted increase of intracranial pressure and significant compensatory effects on blood and cerebrospinal amino acid imbalances as shown by a significant improvement of Fischer's ratio. On the other hand, relatively low capacity for ammonia elimination was shown as compared with homologous direct hemoperfusion.

Improved hepatic microcirculation by human soluble urinary thrombomodulin in the xeno-perfused porcine liver.

Both the protein C/thrombomodulin system and the heparin/anti-thrombin III system are major physiological anticoagulant systems, which may also play a major role in preserving the hepatic microcirculation in xenogeneic liver transplantation. To compensate for the functional incompatibilities of the porcine thrombomodulin (TM)-cofactor activity beyond species for human thrombin, soluble human TM protein was tested in xenogeneic perfusion of the porcine liver. The livers were harvested from adult female pigs and perfused through the portal vein (PV) and hepatic artery (HA) for 2 hr, with fresh human blood in group 1 (n=5), fresh porcine blood (10 units/ml) in group 2 (n=5), and fresh human blood with TM (50,000 units/1.5 l) in group 3 (n=5). The tissue PO2 level, tissue blood flow, PV and HA pressures were all continuously monitored. Circulating perfusate and liver tissue samples were periodically obtained for blood chemistry and histologic analyses. The activated protein C (aPC) level was significantly elevated in the TM-treated group 3 (47.5%+/-3.5% at preperfusion and 51%+/-2.8% after 120 min of perfusion) in comparison to group 1 (32.3%+/-7.2% and 35.3+/-12.0%). The hepatocyte enzyme release of aspartate aminotransferase (AST) was suppressed significantly more in group 3 (238.2+/-107 IU/l), than in group 1 (672.3+/-160 IU/l) at 2 hr after reperfusion. In group 3, the tissue PO2 levels and tissue blood flow also remained significantly higher throughout the perfusion. The platelet counts in the perfusate remained significantly higher in group 3 (37.1% to 74.3% of the preperfusion level) than in group 1 (4.4% to 14.7%), after 0 to 80 min of perfusion. According to the histologic findings, the degree of interlobular hemorrhaging and congestion decreased remarkably more in group 3 than in group 1. These findings thus indicated that soluble thrombomodulin protein extracted from human urine remarkably improved hepatic microcirculation in the xenoperfused porcine liver. The thrombomodulin/protein C system might, thus, play an important role in restoring the physiological anticoagulant system in the xenoperfused porcine liver.

Efficacy of Nonwoven Fabric Bioreactor Immobilized with Porcine Hepatocytes for Ex Vivo Xenogeneic Perfusion Treatment of Liver Failure in Dogs

Efficacy of Nonwoven Fabric Bioreactor Immobilized with Porcine Hepatocytes for Ex Vivo Xenogeneic Perfusion Treatment of Liver Failure in Dogs

The Use of Ex Vivo Xenogeneic Whole Liver Perfusion as a Bridge to Liver Regeneration or Liver Transplantation

The Use of Ex Vivo Xenogeneic Whole Liver Perfusion as a Bridge to Liver Regeneration or Liver Transplantation

Bile acids in xenogeneic ex-vivo liver perfusion: function of xenoperfused livers and compatibility with human bile salts and porcine livers.

In recent years, hepatic support systems using xenogeneic cells have been developed to support patients in fulminant hepatic failure. The extent to which xenogeneic hepatocytes metabolize and excrete human organic anions is unclear. In these studies we examined the ability of the ex vivo porcine liver to clear human bile acids during extracorporeal liver perfusion (ELP). Four patients with fulminant hepatic failure underwent extracorporeal liver perfusion with 9 porcine livers. The venovenous circuit was designed as previously described (NEJM,1994,331:234) as were the immunologic features (Transplantation 1994,58:1162). Bile from the porcine liver and serum samples were collected hourly during perfusion. Three bile acids (glycocholic, glycodeoxycholic, taurodeoxycholic acid) were selected as markers for human bile and three (glycohyocholic, glycohyodeoxycholic, and glyco-3alpha-hydroxy-6-oxo-5beta-cholanoic acid) for markers of pig bile. Bile acids from both serum and bile were processed and analyzed through high performance liquid chromatography. The Students' t test was used for statistical analysis. The mean duration of perfusions was 4.1+/-1.5 hr. The mean total bile acid clearance from serum (243+/-44 micromol/h) was similar to the total bile acid biliary excretion (286+/-84 micromol/hr, P = 0.06). After 1 hr of perfusion, bile samples demonstrated a predominance of pig bile salts (65%). After 3 hr of perfusion, human bile acids made up 85% of total biliary bile acids. Pig bile acids appeared in patients' sera after 1 hr of perfusion, and after 3 hr, 35% of serum bile salts were pig-specific. Porcine livers perfused with human blood can clear the serum of potentially toxic human bile acids and excrete them into bile. Simultaneously, the percentage of pig-specific bile acids in patient serum increases during xenogeneic perfusion for unknown reasons. The relative hepatic uptake of bile acid from serum is similar to bile acid excretion in bile. Further development of systems using porcine livers or hepatocytes is warranted.

Impact of immunoadsorption on xenogeneic extracorporeal pig liver perfusion: assessment of organ function during autologous reperfusion.

The shortage of liver grafts has resulted in an intensive search for alternative strategies of liver support in acute liver failure. Extracorporeal perfusion of pig livers (ECLP) is not a new concept, but recent improvements in cardiopulmonary bypass technology and new knowledge about xenogeneic immunologic mechanisms have made it a potential therapeutic modality. In previous suboptimal experimental and clinical models, no statements about the beneficial effect of long-lasting periods of ECLP were made. The aim of this study was to evaluate the capacity of pig liver for metabolic regeneration after xenogeneic ECLP under largely physiologic conditions. To delay hyperacute rejection (HAR), we utilized immunoadsorption (IA) of naturally preformed xenogeneic antibodies (XNAbs). This led to a stable xenogeneic ECLP for 45 min. However, IA was not able to prevent completely organ damage or loss of function resulting from insufficient autologous reperfusion. The inability to regenerate during autologous reperfusion provides a new measure of the function of a xenogeneic perfused pig liver. In addition, our results implicate that patients with fulminant liver failure will benefit from short perfusions with several fresh organs rather than from long perfusion with a single pig liver.

Human complement regulatory proteins protect swine lungs from xenogeneic injury

Background. Pulmonary xenotransplantation is not possible because of hyperacute lung injury, the pathogenesis of which is unknown. This study evaluates complement-dependent pathways of pulmonary injury during heterologous perfusion of swine lungs. Methods. Lungs from unmodified swine and swine expressing human decay-accelerating factor and human CD59 (hDAF/hCD59 swine) were perfused with either human plasma or baboon blood. Pulmonary vascular resistance and static pulmonary compliance were measured serially, and swine lung tissue were examined by light microscopy. Complement activation was assessed by serial measurements of baboon plasma C3a-desArg concentrations. Results. Perfusion of unmodified swine lungs with human plasma and baboon blood resulted in hyperacute lung injury within minutes of perfusion. However, function was preserved in swine lungs expressing human decay-accelerating factor and human CD59. In both study groups, xenogeneic perfusion with baboon blood resulted in at least a sevenfold increase in plasma C3a-desArg levels suggesting transient activation of complement. Conclusions. Lungs from swine expressing human decay-accelerating factor and human CD59 were resistant to injury during perfusion with human plasma and baboon blood, indicating that complement mediated some of the features of xenogeneic acute lung injury.

Intravital microscopic investigation of xenogeneic microcirculation and impact of complement depletion by cobra venom factor.

Discordant xenografts are hyperacutely rejected within minutes. Disturbances in the microcirculation are considered to be the central mechanisms of hyperacute xenogeneic rejection (HXR). In this study intravital fluorescence microscopy was applied to investigate the dynamics of microcirculatory alterations in a setting in which HXR was inhibited by complement (C) depletion. Blood flow was measured as rat livers were perfused with isogeneic rat or xenogeneic human blood to assess the pattern of either physiological isogeneic hemoperfusion or in the course of HXR. Next, the complement system of the perfusate was inactivated by cobra venom factor (CVF) in order to inhibit HXR. Liver sinusoids of the isogeneic group were homogeneously perfused (sinusoidal perfusion rate 93.6+/-0.3%), whereas in the xenogeneic group the sinusoidal perfusion rate dropped to 67.1+/-3%. The perfusion in the periportal zone of an acinus was significantly lower ( 59.0+/-3.3%) than in the pericentral zone (76.2+/-3.1%). Treatment with CVF improved the sinusoidal perfusion to a value of 85.6+/-2.3%, physiological perfusion, however could not be reached. In contrast to the isogeneic group, massive white blood cell (WBC) and platelet accumulation was found in the xenogeneic group, especially in the terminal portal vessels and in the periportal zone of liver acini. WBC and platelet counts show that the adherence of these cells appears rapidly in the first 5 min after reperfusion as firm adherence. CVF was not able to inhibit WBC and platelet accumulation, indicating that WBC endothelial interactions do not require an intact complement system. Bile flow, a parameter of liver function, decreased only slightly during isogeneic perfusion. The addition of CVF to the rat blood reduced the bile flow to one half of the untreated isogeneic flow, indicating a hepatotoxic side-effect of CVF. In xenogeneic perfusion the bile flow dropped to 62.6% and with the addition of CVF to 37.5% in the first 15 min after reperfusion. The bile flow of the CVF treated groups recovered during the perfusion but could not reach isogeneic values.

Monitoring of microhemodynamic changes during ex vivo xenogeneic liver perfusion using intravital microscopy

Monitoring of microhemodynamic changes during ex vivo xenogeneic liver perfusion using intravital microscopy

Analysis of the microcirculation during xenogeneic liver perfusion in the guinea pig--rat model. The contribution of leukocytes to the rejection process.

Since the main feature of hyperacute rejection is a disturbance of the xenograft's microcirculation, we analyzed microhemodynamic parameters during xenogeneic hemoperfusion of the guinea pig (GP) liver and investigated the contribution of leukocytes to the rejection process using intravital fluorescence microscopy. Isolated GP livers were hemoperfused via the portal vein in a recirculating system with a constant flow of 1 ml/min per g liver. In contrast to isogeneic perfusion with heparinized GP blood, a disturbance in the microcirculation was observed during xenogeneic perfusion using heparinized rat blood, with significantly higher values of perfusion pressure, reduced sinusoidal perfusion rates, and a larger number of stagnant leukocytes. A complete breakdown of the microcirculation, with the highest values of perfusion pressure and the smallest perfusion index, was associated with 100% accumulated leukocytes when rat blood was anticoagulated with sodium citrate. Almost isogeneic perfusion values were obtained when fucoidin, which inhibits L-selectin-dependent cell interaction, was added to heparinized rat blood. These data indicate that leukocyte-endothelial cell interaction contributes to xenogeneic rejection.