Systemic liberation of interleukin-8 in the perioperative phase of liver transplantation

Serum levels of interleukin-8 (IL-8) were investigated in the perioperative phase of liver transplantation (LTx) in order to help determine whether this cytokine might serve as a parameter for preservation injury. In a study of 45 patients undergoing LTx, systemic IL-8 was estimated at the end of the anhepatic phase, at 30, 60, and 120 min after reperfusion of the graft, and 24 h and 7 days after LTx. A maximum mean concentration of 665 ± 135 pg/ml was seen 60 min after LTx. The minimum was found on the 1st postoperative day (POD 1): 328 ± 33 pg/ml. Significant changes were found between 60 min and PODs 1 and 7, as well as between 120 min and POD 1. Differences in cold ischemia time were not found to be significant. We conclude that monitoring of systemic IL-8 levels is not useful in the development of new liver preservation concepts. Received: 12 September 1996 Received after revision: 27 February 1997 Accepted: 3 March 1997     

A post-preservation vascular flush removes significant populations of donor leukocytes prior to lung transplantation

BackgroundDonor leukocytes are intrinsically involved in acute lung allograft rejection, via self-presentation of donor antigens to recipient leukocytes. Therapeutic modalities to remove donor leukocytes are currently unavailable. We evaluated if a vascular flush immediately following preservation can be used for this purpose.MethodsA post-preservation flush was performed with STEEN solution in n = 6 porcine lungs following static cold storage. The first 500 ml effluent from the left atrium was collected and an inflammatory profile performed.ResultsA total of 1.17 billion (±2.8 × 10⁸) viable leukocytes were identified within the effluent. T cells were the dominant cell population, representing 82% of the total mobilised leukocytes, of which <0.01% were regulatory T cells. IL-18 was the most abundant cytokine, with a mean concentration of 84,216 pg (±153,552 pg). In addition, there was a mean concentration of 8819 ng (±4415) cell-free mitochondrial DNA.ConclusionThere is an immediate transfer of donor leukocytes, cytokines and damage-associated molecular patterns following reperfusion. Such a pro-inflammatory donor load may enhance alloantigen presentation and drive recipient alloimmune responses. A post-preservation flush may therefore be an effective method for reducing the immune burden of the donor lung prior to transplantation.     

Origins of the Knife: Early Encounters with the History of Surgery

We evaluated the production of the interleukins (ILs) IL1- &;#103 , IL-6, and IL-10 in both the vasculature and pulmonary tissue before and after 24 h of lung preservation. The cardiopulmonary blocs of 21 Balb-c mice were divided into three study groups (7 mice/group) and were flushed through the pulmonary artery with Krebs-Henseleit buffer (K-Hb) at 4°C at a rate of 0.2 mL/min as follows: Group 1, lung washout: lungs were flushed until pulmonary effluent was clear. Group 2, perfusion: After lungs were flushed until pulmonary effluent was clear, lungs were perfused during 30 min. Group 3, preservation: Lungs were flushed until pulmonary effluent was clear, and the cardiopulmonary bloc was preserved immersed into (K-Hb) at 4°C. After 24 h of preservation, lungs were reperfused during 30 min. In all study groups the caudal lobe from the left lung was taken for microscopical study; all other lobes were homogenized with (K-Hb) and the supernatant was obtained. IL-1 &;#103 , IL-6, and IL-10 production in lung effluents (washout, perfusion, and reperfusion) and in lung tissue were measured by enzyme-linked immunosorbent assay (ELISA). In the lung effluent, there was no statistical difference between IL-1 &;#103 and IL-6 concentrations. In all study groups, IL-10 production was significantly higher than IL-1 &;#103 and IL-6 levels. IL-10 level was lowest in the 24-h preservation group when it was compared to the other groups. In group 1, there was a negative correlation ( r =-.599, p < .05) between IL-1 &;#103 and IL-10. In pulmonary tissue, IL-1 &;#103 was higher in group 2 when compared to groups 1 ( p = .001) and 3 ( p = .002), and it was significantly lower in group 3. IL-10 was lower in group 1 when compared to groups 2 ( p = .001) and 3 ( p = .004). In groups 1 and 2, IL-1 &;#103 was significantly higher than IL-6 and IL-10. In group 3, IL-10 was higher than IL-1 &;#103 ( p = .0001) and IL-6 ( p = .0001). Correlation of effluent/tissue index with histological findings showed a negative correlation between IL-10 effluent/tissue relation and inflammation ( r =-.68, p < .01). In conclusion, the main cytokine found in lung effluents was IL-10, followed by IL-6 and IL-1 &;#103 . On the other hand, cytokine concentration in lung tissue homogenates was mainly due to the presence of IL-1 &;#103 . However, this cytokine shows a significant reduction in lung tissue after prolonged preservation.     

The impact of liver preservation in HTK and UW solution on microcirculation after liver transplantation

Severe microcirculatory disturbances due to endothelial cell damage and leukocyte adherence during reperfusion of transplanted livers are considered to contribute to early graft failure. Since the degree of reperfusion injury after liver transplantation depends on the length of preservation time and the solution used for preservation, the aim of our study was to assess three solutions with respect to microvascular perfusion and leukocyte adhesion. Therefore, rat livers were stored up to 24 h in Euro-Collins (EC), University of Wisconsin (UW), or histidin-tryphtophan-ketoglutarate (HTK) solutions prior to orthotopic transplantation. The livers were studied in situ 60 min postoperatively using intravital fluorescence video microscopy. Using simple syringe flushing (10 ml), sinusoidal perfusion decreased below 50% in EC preserved livers after 8 h preservation, in HTK preserved livers after 16 h preservation, and remained higher than 70% in livers preserved in UW up to 24 h. Permanent adhesion of leukocytes was increased more rapidly in organs after 1, 8, 16, and 24 h preservation in HTK (16%, 15%, 34%, and 49.7% ± 4.7%) compared to those preserved in UW (15%, 18%, 17%; and 32.7% ± 3.3%; P < 0.05). Using a 10-fold volumn of the organ weight of HTK solution during the harvesting procedure, with an 8 min equilibration period, sinusoidal perfusion (39.6 ± 4.7%) and leukocyte adhesion (42.7 ± 3.1%) were not improved after 24 h. In contrast, equilibration with a volumn of approximately 40-times the liver weight improved sinusoidal perfusion (70.8% ± 2.7%; P < 0.01) and leukocyte adhesion (24.9% ± 3.1%; P < 0.01) significantly. Thus, using HTK solution, simple flushing prior to long-term cold storage resulted in microcirculatory disturbances when compared to UW solution. Larger volumns of HTK solution with an additional equilibration period of 8 min, however, reduced leukocyte adhesion and improved sinusoidal perfusion to a similar degree as UW solution.     

Adding Pulsatile Vascular Stimulation to Venous Systemic Oxygen Persufflation of Liver Grafts

The effect of adding pulsatility to gaseous oxygen persufflation during liver preservation was studied in an isolated rat liver model. Livers from male Wistar rats were retrieved 30 min after cardiac arrest of the donor and subjected to 18 h of cold storage. Some grafts were subjected to nonpulsatile or pulsatile gaseous oxygen persufflation. Graft viability was assessed thereafter upon warm reperfusion in vitro (n = 5 per group). Pulsatile persufflation significantly improved parenchymal integrity (enzyme release, bile flow) upon reperfusion, with respect to nonpulsatile persufflation or cold storage (CS) (e.g., max. release of alanine aminotransferase: 44 ± 10 vs. 178 ± 29 vs. 345 ± 100 U/L; pulsatile vs. nonpulsatile persufflation vs. CS).The effect was associated with the prevention of the ischemic decline in gene and protein expression of the vasoprotective Krüppel‐like factor 2, increased perfusate levels of the endogenous vasodilator nitric oxide, and reduced portal vascular resistance upon reperfusion, while nonpulsatile persufflation was less effective (e.g., vascular resistance: 1235 ± 108 vs. 1607 ± 155 vs. 2215 ± 208 Pa s/mL; pulsatile vs. nonpulsatile persufflation vs. CS). In conclusion, pulsatile mechanostimulation of the hepatovasculature seems a genuine protective mechanism, affecting early graft recovery upon reperfusion.     

Cyclosporine overcomes cold preservation/reperfusion injury of liver graft: Chemokine release and liver ultrastructure

There is a growing body of evidence that the cytokine, tumor necrosis factor-α (TNF-ga), plays an important role in the development of hepatic ischemia/reperfusion injury. We found that the immunosuppressants, cyclosporine-A (CsA), azathioprine, and FK506, have protective effects on such injury. The purpose of the present study was to elucidate mechanisms involved in these beneficial effects of the immunosuppressant, CsA, on liver injury following cold preservation and transplantation, with special reference to the suppression of TNF-α release. Rat livers were stored in Euro-Collins solution (EC) at 4°C for 6h and orthotopically transplanted. The animals allotted to two groups: group A (untreated controls) and group B (CsA pretreatment of recipients). CsA (10 mg/kg, p.o.) was given for 3 consecutive days preoperatively. CsA pretreatment of the recipients significantly improved the 2-week survival rate (0/6 for group A, 3/6 for group B;P<0.05) and this was associated with a significant decrease in serum TNF-α levels 2h posttransplantation (group A, 69.8±15.7 pg/ml; group B, 22.8±6.8; mean±SEM;n=12 each;P<0.05) and amelioration of sinusoidal endothelial injury, assessed by electron microscopy. Plasma endotoxin levels following reperfusion of the grafts were not altered by the CsA therapy. Morphologically, CsA pretreatment of the recipients did not alter activation of Kupffer cells. CsA pretreatment of the recipient aids in preventing cold preservation/reperfusion injury of the liver graft, possibly by modulating effects of TNF-α.     

Bone-Resorbing Cytokines from Peripheral Blood Mononuclear Cells after Hormone Replacement Therapy: A Longitudinal Study

Conflicting results have been reported in several cross-sectional studies measuring cytokine production from adherent monocytes in pre- and postmenopausal women. Furthermore, the target cells for the action of estrogen are still debated. We therefore assessed in a longitudinal manner the cytokine production from different fractions of peripheral blood mononuclear cells (PBMC) cultured for 48 h. PBMC were obtained from 30 postmenopausal women before and after 6 months of hormone replacement therapy (HRT). Women were randomly allocated to two groups: an adherent PBMC group (n= 20) and a total PBMC group (n= 9). After 6 months of treatment, urinary pyridinoline levels were markedly decreased in both groups (353 ± 24 vs 114 ± 13 μg/mmol creatinine and 325 ± 35 vs 164 ± 31 μg/mmol creatinine respectively, p<0.01). Culture supernatants were assayed for interleukin 1β (IL-1β), interleukin 6 (IL-6), soluble IL-6 receptor (IL-6rs) and tumor necrosis factor alpha (TNF-α). In the adherent PBMC group, HRT induced a nonsignificant trend toward decreased levels of IL-1β (35 ± 10 vs 13 ± 5 pg/ml), TNF-α (333 ± 58 vs 222 ± 30 pg/ml) and IL-6 (115 ± 70 vs 17 ± 10 pg/ml). In contrast, in the total PBMC group, HRT induced a consistent and dramatic decrease in levels of IL-1β (104 ± 22 vs 25 ± 8 pg/ml), IL-6 (5950 ± 1041 vs 1011 ± 361 pg/ml), IL-6rs (148 ± 33 vs 35 ± 12 pg/ml) (p<0.01) and TNF-α (1468 ± 315 vs 585 ± 207 pg/ml, p= 0.05). We then evaluated whether HRT had the same effect in vitro. Adherent or total PBMC of 8 postmenopausal women were cultured with or without 10⁻⁸M 17β-estradiol or tibolone for 48 h. Production of IL-1β, TNF-α, IL-6 and IL-6rs was not affected by the presence of 17β-estradiol or tibolone in cultures of these cell fractions. In conclusion, our data indicate that non-adherent PBMC could mediate the response to HRT. HRT may exert its action indirectly via noncirculating cells, as suggested by the absence of an in vitro effect. Received: 11 July 2000 / Accepted: 15 January 2001     

The role of plasma coating on the permeation of cytokine-inducing substances through dialyser membranes

We studied the effects of coating of dialyser membranes withplasma proteins on the permeation of bacteria-derived cytokine-inducingsubstances (CIS). An in vitro dialysis circuit using polysulphone(PS) or modified cellulose triacetate (mCT) dialysers was used.Precoating of the dialysers was performed by recirculation of10% normal human plasma for 30 min in the blood compartmentand subsequent rinse with pyrogen-free saline. Samples fromthe blood compartment were tested for induction of interleukin-1(IL-1), interleukin-1ß (IL-1ß) and tumournecrosis factor (TNF) at various time points after challengingthe dialysate with sterile culture supernatants from Pseudomonasaeruginosa. Contamination of the dialysate resulted in the appearanceof CIS in the blood compartment of both polysuphone modifiedcellulose triacetate (IL-1: PS, time 0: 81±11 pg/ml,time 60min: 4747±1822 pg/ml, P<0.05; mCT, time 0:235±141 pg/ml, time 60 min: 1632±531 pg/ml, P<0.05).The plasma protein layer reduced the penetration of CIS significantlyonly for polysulphone (IL-1: PS, time 60: 4747±1822 versus880±525 pg/ml, P<0.05; modified cellulose triacetate,time 60 min: 1632±531 pg/ml versus 930±326 pg/ml).Samples from the blood compartment contained <6 pg/ml LAL-reactivematerial at all time points. We conclude that plasma coatingof polysulphone dialysers reduces the permeability for CIS derivedfrom Pseudomonas, either by reducing the effective pore sizeor by adsorption of proteins that bind CIS. When precoated dialyserswere used, transfer of CIS through both dialysers was comparable,suggesting that under the conditions of in vivo haemodialysisthere may be no difference between the dialysers regarding thepenetration of CIS from the dialysate.     

Elevation of cytokines during open heart surgery with cardiopulmonary bypass: participation of interleukin 8 and 6 in reperfusion injury

Myocardial ischaemia is one of the major causes of low output syndrome during open heart surgery. Injury associated with ischaemia and reperfusion has been considered to result, in part, from the action of neutrophils, the interaction of neutrophils with vascular endothelial cells, and the effects of cytokines which are mediators that induce and modify reactions between these substances. We investigated cell injury in relation to the concentrations of interleukins 6 and 8 (IL-6 and IL-8), which have recently received attention as neutrophil activators. Neutrophil counts, granulocyte elastase (GEL), IL-6, IL-8, tumour necrosis factor-α (TNF-α), CK, and CK-MB concentrations were determined serially in 11 patients undergoing open heart surgery with cardiopulmonary bypass (CPB). Neutrophil counts (mean ±SD 2717 ±2421 μl^?1 preoperatively) peaked 60 min after declamping the aorta at 7432 ±4357 μl^?1 (P < 0.01) and remained elevated 7136 ±5194 μl^?1 at 180 min (P < 0.01). Plasma GEL level (168 ±71 μg sd L^?1 preoperatively) peaked at 1134 ±453 μg · L^?1 120 min after declamping of the aorta (P < 0.01) and remained elevated, 1062 ±467 μg · L^?1, after 180 min (P < 0.01). Serum IL-6 level (118 ±59 pg · ml^?1 preoperatively) peaked at 436 ±143 pg · ml^?1 60 min after declamping of the aorta (P < 0.01) and remained elevated, 332 ±109 pg · ml^?1, after 180 min. Serum IL-8 level (37 ±44 pg · ml^?1 preoperatively) peaked at 169 ±86 pg · ml^?1 at 60 min after declamping of the aorta (P < 0.001) and remained elevated at 113 ±78 pg · ml^?1 180 min after declamping of the aorta. Serum TNF-α was decreased at 60 min after aortic occlusion but otherwise did not change. Plasma GEL concentrations correlated with serum IL-8 levels (R = 0.7, P = 0.001) and the IL-6 and IL-8 concentrations correlated with the duration of aortic clamping (R = 0.64, P = 0.01, R = 0.7, P = 0.01). We conclude that the increases of IL-6 and IL-8 occur as a result of ischaemia, and suggest that these cytokines participate in reperfusion injury by activating neutrophils.     

Liberation of vasoactive substances and its prevention with thromboxane A2 synthase inhibitor in pig liver transplantation

There are multiple causes of liver graft nonfunction in the early post-transplant period. Since a severe microcirculatory disturbance based on ischemia-reperfusion liver injury is considered to be the main underlying pathophysiology, it is suspected that various vasoactive substances are liberated after reperfusion of the graft. In order to investigate this matter, we conducted an experimental study with pig liver allotransplantation. Two groups of animals received donor grafts with or without thromboxane synthase inhibitor (sodium ozagrel), 1.25 mg/kg body weight intravenously, given at the time of liver harvesting. All of the recipient animals in the treatment group (n=10) survived longer than 7 days whereas three of ten animals in the control group died within 7 days. Serum lactate dehydrogenase (LDH) in the recipient serum at 1 h after reperfusion was significantly lower in the treatment group (915.1±167.3 U/l) than in the control group (1264.4±134.7 U/l). Serum thromboxane B₂ (2261.7±1055.7 pg/ml) and endothelin-1 (6.3±2.2 pg/ml) after reperfusion in the treatment group were significantly lower than those in the control group (4220.0±1711.0 pg/ml and 11.2±3.1 pg/ml, respectively). Although serum angiotensin II after reperfusion tended to be lower in the treatment group than in the controls serum renin activity was less than 3 ng/ml in both groups of animals. There were no differences in the plasma endotoxin levels between the two groups. We conclude that the administration of sodium ozagrel to the donor animals provided better graft function in recipients than no such treatment. We speculate that the inhibition of thromboxane A₂ production suppresses the liberation of other vasoconstrictive substances, preventing microcirculatory disturbance and, thereby, contributing to improved graft function after liver transplantation.     

Influence of postoperative acute-phase response on angiogenesis and tumor growth: Open vs. laparoscopic-assisted surgery in mice

Inflammatory responses and tumor growth are increased after laparotomy compared with laparoscopy in some animal models. Proinflammatory cytokines interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) upregulate the expression of vascular endothelial growth factor (VEGF). Our aim was to investigate the influence of postoperative inflammatory responses on angiogenesis and tumor growth. 5 Χ 10⁶ B51LiM cells were injected into the cecal wall of Balb/c mice. After 2 weeks, the animals were randomized into the following three groups: open cecectomy (OC), CO₂-laparoscopic-assisted cecectomy (LC), and helium-laparoscopic-assisted cecectomy (LH). On postoperative day 12, the mice were killed. Tumor load scores and weight were significantly greater after laparotomy than after laparoscopy. Serum IL-6 levels 6 hours after surgery (OC: 4157 ± 1297 pg/ml vs. LC: 2514 ± 1417 pg/ml vs. LH: 2255 ± 1714 pg/ml) and VEGF levels on postoperative day 12 (OC: 231 ± 125 pg/ml vs. LC: 45 ± 9 pg/ml vs. LH: 49 ± 8 pg/ml), measured by enzyme-linked immunosorbent assay, were significantly higher in the laparotomy group. Microvessel density was also significantly higher in the OC group (OC: 34.3 ± 11.5 vs. LC: 15.5 ± 12.5 vs. LH: 18.5 ± 11.9). There was a positive correlation between IL-6 and VEGF postoperative serum levels (rho = 0.67; P < 0.001). We concluded that increased systemic levels of proinflammatory cytokines and VEGF are associated with increased angiogenesis and tumor growth after laparotomy compared to laparoscopy in mice. Presented at the Fifty-Seventh Annual Sessions of the Owen H. Wangensteen Surgical Forum, The American College of Surgeons Clinical Congress, San Francisco, California, October 6–10, 2002; and published as an abstract in Journal ofthe American College of Surgeons 2002; 195:S69. Supported by an International Fellowship Grant from the American Society of Colon and Rectal Surgeons (M.P.) and by a Postdoctoral Grant (EX2001-35105008) from the Ministry of Education and Culture of Spain.     

Reduction in nonparenchymal cell injury and vascular endothelial dysfunction after cold preservation of the liver by gaseous oxygen

Abstract  Reintroduction of oxygen to previously anoxic tissue may result in severe cell injury (oxygen paradox) and contribute to the so-called reperfusion damage of is-chemic organs. Our study investigated the influence of simple gas eous oxygen supply during ischemia on nonparenchymal cell alterations upon reperfusion of the liver. Livers from male Wistar rats were isolated, rinsed blood-free and stored for 48 h at 4 °C in UW-preservation solution (group 1; n = 6). Gaseous oxygen was insufflated into a second group of livers (group 2; n = 6) during the storage period via the inferior caval vein at a pressure limited to 18 mmHg. To simulate the period of slow rewarming of the organ during surgical implantation in vivo, all livers were incubated at 25 °C in saline solution for 30 min prior to reperfusion. Reperfusion was carried out in vitro in a recirculating system with Krebs-Henseleit buffer. A control group was perfused immediately after harvest. The technique of aero bic storage (group 2) resulted in normal vascular perfusion charac teristics without elevation of portal venous pressure (PVP) above control values, in contrast to group 1 livers which showed a significantly elevated PVP, averaging between 1.5 and 2 times the values of the control. Hepatic efflux of NO (nmol/ml) after 10 min of reperfusion was massively increased in group 1, while only low concentrations were found in group 2 and in control livers. Kupffer cell activation after ischemia was shown by a huge increase in acid phosphate release upon reperfusion compared with the control, with significantly lower values in group 2 after 10 min of reperfusion than in group 1. Thus, aerobic ischemia by gaseous oxygen persuf-flation seems an appropriate tool for long-term organ preservation, pre venting vascular and parenchymal dysfunction upon reperfusion.     

Measurement of the vasoconstrictive substances endothelin,angiotensin II,and thromboxane B2 in cold storage solution can reveal previous renal ischemic insults

In a rat model, the left kidney was subjected to 60 min of normothermic ischemia followed by 15 min of reperfusion, whereas the right kidney, serving as a paired control, was not rendered ischemic. Both kidneys were then perfused in situ with either Euro-Collins (EC) solution (n=12) or University of Wisconsin (UW) solution (n=6) for 10 min. Each kidney was then harvested and stored at 4°C in its respective solution. After 24 and 48 h of cold storage, the following vasoactive substances were measured in the preservation media: endothelin (ET), angiotensin II (A-II), thromboxane (B₂) (TxB₂), and prostaglandin I₂ (PGI₂). After 24 h in EC solution, left kidneys uniformly produced significantly higher concentrations of each vasoactive substance than right kidneys: ET 1.64±0.3 pg/ml vs 0.82±0.1 pg/ml (P0.009); A-II 20.8±6.2 pg/ml vs 7.75+2.3 pg/ml (P0.007); TxB₂ 100.8±17.7 pg/ml vs 40.1±11.7 pg/ml (P0.04); PGI₂ 638.3±41.1 pg/ml vs 318.3±36.4 pg/ml (P0.001), respectively. At 48 h, a similar pattern of results was obtained as the kidney continued to produce TxB₂ and prostacyclins during the 24–48 h period. In the UW solution, basal levels of ET and A-II were lower than those in EC solution, but similarly increased after initial ischemia. At 24 h, the concentrations produced by the left and right kidneys were as follows: ET 0.66±0.1 pg/ml vs 0.48±0.1 pg/ml (P0.14); A-II 10.36±3.7 pg/ml vs 2.14±0.7 pg/ml (P0.006); TxB₂ 178±53 pg/ml vs 52±23.1 pg/ml (P0.001); and PGI₂ 448.3±49 pg/ml vs 323±44.3 pg/ml (P0.01), respectively. After 48 h, the range of concentrations of each substance was similar to that obtained after 24 h. In further studies, the concentrations of ET and A-II were measured in solution previously used to preserve human kidneys (n=7). The mean concentration of ET and A-II in these samples was 3.82±1.14 pg/ml and 21.3±9.2 pg/ml, respectively, whereas in control media both substances were below the limits of detection. These results demonstrate that vasoconstrictive substances can be measured in the preservation media after a kidney has been stored cold and that higher concentrations are found when the organ has been subjected to prior normothermic ischemia. The measurement of these vasoactive substances before transplantation may reveal that the kidney has been subjected to previous ischemic events. Moreover, these vasoactive substances could be involved in the early recovery of renal function after kidney transplantation.     

Pretreatment of Donors with Endothelin Receptor Antagonist TAK-044 Improves Cardiac Functional Recovery Following Preservation with University of Wisconsin Solution

Objective : Previous studies suggest that endothelin-1 (ET-1) plays a role in myocardial ischemia/reperfusion injury. Although administration of an endothelin receptor antagonist to the recipient has been shown to improve myocardial function after ischemia/reperfusion in a rat heart transplantation model, the effect of administering an endothelin receptor antagonist to the donor has not yet been examined. This study was designed to investigate the effects of pretreating donors with an ET A /ET B endothelin receptor antagonist (TAK-044) on myocardial function after cold preservation of a rat heart. Design : Male rats were pretreated with normal saline (control group, n = 8), TAK-044 (TAK group, n = 8, 1 mg/kg). Following cardiac arrest using cardioplegia, we washed out the coronary vascular beds with cold University of Wisconsin solution followed by 6-h preservation. After preservation, the hearts were mounted on a Langendorff apparatus to estimate aortic flow (AF), coronary flow (CF), cardiac output (CO), systolic pressure (SP), heart rate (HR), and rate-pressure product (RPP: HR &#50 SP). The concentration of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) within the coronary perfusate during reperfusion was measured. Results : AF, SP, and CO were significantly greater in the TAK group than in the control group ( p = 0.0045, 0.004, and 0.0295, respectively). Conclusion : Pretreatment of donors with a nonselective endothelin receptor antagonist (TAK-044) improved cardiac functional recovery following preservation and may be beneficial for prolonged myocardial preservation.     

Lower Limb Ischemia-Reperfusion Injury Triggers a Systemic Inflammatory Response and Multiple Organ Dysfunction

Restoration of blood flow to an acutely ischemic lower limb may, paradoxically, result in systemic complications and unexpected mortality. We investigated the effect of acute ischemia-perfusion of the lower limb on cytokine production and end organ function. Plasma concentrations of tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) were determined in five groups of male Wistar rats: control, 3 hours of bilateral hind limb ischemia alone, and 3 hours of bilateral hind limb ischemia followed by 1 hour, 2 hours, or 3 hours of reperfusion, respectively. In a second experiment, the effect of lower limb ischemia-reperfusion on remote organs (lung, liver, and kidney) was assessed biochemically and histologically. There was a significant increase in plasma concentrations of TNF-a in plasma of animals subjected to 3 hours of bilateral hind limb ischemia followed by 1 hour of reperfusion, 40.1 +/- 4.4 pg/ml, when compared with controls, 22.6 +/- 4.4 pg/ml, or animals in the ischemia-alone group, 16.3 +/- 5.2 (p <0.05). Plasma concentration of IL-6 increased progressively and significantly in animals subjected to bilateral hind limb ischemia followed by 1 hour of reperfusion, 720 +/- 107 pg/ml; 2 hours of reperfusion, 1987 +/- 489 pg/ml; or 3 hours of reperfusion, 6284 +/- 1244 (p <0.0001), compared with controls, 104 +/- 43 pg/ml; or animals in the ischemia-alone group, 140 +/- 55 pg/ml. In the study comparing portal and systemic concentrations of IL-6, systemic concentrations of IL-6, 967 +/- 184 pg/ml were significantly higher than those in the portal circulation 577 +/- 127 pg/ml (p <0.05). There was a significant increase in plasma concentrations of urea, creatinine, aspartate transaminase, alanine transaminase, and lactic dehydrogenase in reperfused animals compared with controls (p <0.001). Morbidity and mortality following reperfusion of the acutely ischemic limb may be a manifestation of multiple organ dysfunction caused by a systemic inflammatory response triggered by reperfusion of the ischemic extremities.     

Predictive value of liver tissue flow in assessment of the viability of liver grafts after extended preservation in pigs

The crucial damage in cold storage of liver allografts is to the hepatic sinusoidal lining (microcirculation). Using different solutions, we studied whether determinations of graft tissue flow were valuable in estimating the viability of liver grafts. Twenty-three pairs of female pigs underwent orthotopic liver transplantation and were assigned to five groups according to the cold preservation time or solutions used: in group I the liver grafts were stored in Euro-Collins solution (EC) for 4 h (n = 3), in group II the grafts were stored in EC for 12 h (n = 5), in group HI the donor was pretreated with azathioprine (AZA), 1 mg/kg per day, orally (PO) for 3 days before harvesting and the graft was implanted after 12 h cold storage with EC (n = 6), in group IV the graft was stored in modified University of Wisconsin solution (mUW) for 4 h (n = 3), and in group V the graft was stored in mUW for 24 h (n = 6). Liver tissue blood flow (LTBF) was measured, using a laser doppler device, at 60 min after recirculation of the graft. In the case of EC preservation, LTBF (ml/100 g of liver tissue per min) correlated well with 4-day survival: 21.2 ± 3.0 ml/100 g of tissue per min mean ± SD, in group I (3/3, 100%); 10.0 ± 2.8 ml/100 g of tissue per min in group II (0/5, 0%); and 19.1 ± 3.4 m1/100 g of tissue per min in group III (5/6, 83.3%) (P < 0.05, group II vs I and III). All grafts with LTBF of more than 15 ml/100 g tissue per min functioned well. However, changes in microcirculation of the mUW-stored livers did not correlate with early function of the graft: 23.0 ± 2.3 m1/100 g of tissue per min in group IV (4-day survival; 3 of 3, 100%) and 23.5 ± 9.1 m1/100 g of tissue per min in group V (0 of 6, 0%). This was accompanied by graft dehydration during storage and an increased number of erythrocytes in the hepatic sinusoids post-recirculation. We concluded that assessment of liver tissue flow by LDF was very helpful and easy to apply in predicting liver graft failure in the case of preservation with Euro-Collins solution. However, LTBF should be carefully evaluated as a marker of liver graft viability when the liver graft is preserved with mUW.     

Glucose Intolerance Modifies the Inflammatory Response After Intestinal Ischemia-Reperfusion

Streptozotocin administration in newborn rats (nSTZ-rats) leads to adults with mild insulin deficiency and normoglycemia, and is accepted as a model of type 2 diabetes. We examined possible differences in the production of inflammatory mediators between healthy and nSTZ-rats after ischemia-reperfusion (I-R). Two-month-old control and nSTZ-rats were randomly separated into control and intestinal I-R groups. After reperfusion, samples were obtained from the portal vein (PV) infrahepatic cava vein (ICV), suprahepatic cava vein (SCV), jejunal wall, and pancreas. Nitric oxide (NO), lipid hydroperoxides (LPO), tumor necrosis factor alpha (TNF-α), 60 kDa receptor (sTNF-R1), 80 kDa (sTNF-R2), and intercellular adhesion molecule-1 (ICAM-1), were determined. After I-R, nSTZ-rats showed increased plasma concentrations of LPO, NO, ICAM-1 (0.5141 ± 0.083 vs 0.024 ± 0.003, ICV; 0.574 ± 0.075 vs 0.023 ± 0.003, SCV; 0.528 ± 0.067 vs 0.027 ± 0.003 PV; ng/ml), TNF-α (42.4 ± 5.7 ICV, 248.4 ± 28.2 SCV, and 33.6 ± 4.0 PV. In n STZ-rats, vs 4.36 ± 0.57, 4.74 ± 0.77, and 3.16 ± 0.32, respectively, in control rats; pg/ml), and sTNF-R1. Both TNF-α and NO plasma levels were higher in SCV than in ICV and PV after I-R. In addition, after I-R, jejunal wall of nSTZ-rats showed an increase of TNF-α IL-1, and IL-10 levels. A pre-existing state of glucose intolerance intensifies the inflammatory response after intestinal I-R.     

Cold preservation of the small intestine with the new Celsior-solution

The aim of the present study was to evaluate the potential of Celsior, a recently developed cardioplegic and heart storage solution, to protect the small bowel during ischemic storage. Small bowel segments were isolated from rats, flushed with either UW or Celsior solution, and cold-stored for 18 h at 4 °C in the respective solution. After ischemic storage, some preparations were freeze-clamped for analysis of tissue metabolites while other preparations were tested for structural and functional integrity by isolated perfusion in vitro using a previously validated model. After 18 h of ischemic storage no significant differences were seen between Celsior and UW with regard to the development of edema, energy charge, or creatine phosphate, but lactate accumulation was significantly reduced in the Celsior group, although glucose catabolism was not inhibited. Histological evaluation of the cold-stored organs showed no differences with regard to structural integrity between the two groups. Total vascular resistance upon reperfusion was significantly lower in the Celsior group (666 ± 126 vs 827 ± 88 MPa s m^{–3 *}), as was the intestinal release of LDH (9.7 ± 4.4 vs 18.2 ± 4.6 U/l *). Carbohydrate absorption from the intestinal lumen amounted to venous effluent concentrations of 0.58 ± 0.24 vs 0.18 ± 0.15 mg% ^* of galactose in the Celsior and UW groups, respectively. Within the limits of this in vitro pilot study, Celsior provided better postischemic recovery of the small bowel than UW in terms of vascular perfusion characteristics, enzyme release, and carbohydrate absorption and may, thus, be considered a suitable alternative for intestinal organ preservation. Received: 13 June 1997 Received after revision: 19 September 1997 Accepted: 8 October 1997     

Failure of IL-8 to assess early reperfusion injury following lung transplantation of cardiac death donor pigs

Although interleukins (IL) 8 and 10 predict lung viability in lung transplantation from heart beating donors (HBD) and IL-1β is a marker of ex vivo performance from after cardiac death donors (ACDD), IL expression in the recipient remains unknown. This study assessed IL-1β, IL-8 and IL-10 as indicators of functional performance in single-lung transplantation from ACDD pigs. Animals were divided into: (i) HBD: immediate lung excision; (ii) ACDD: fibrillation, 30 min warm ischemia and 3 h topical cooling. Left lungs of both groups were then flushed with Perfadex and stored at 3–4 °C for 3 h. IL in bronchoalveolar lavage fluid (BAL) and hemodynamic and graft function were measured in the donor and during the 2 h reperfusion period in the recipient. Myeloperoxidase, nuclear factor kappa beta, wet/dry weight ratio and a histologic injury score were assessed from biopsies in basal conditions in the donor and at the end of reperfusion. Despite similar pulmonary function and histologic markers of injury in both groups and higher IL-1β in the donor of ACDD, IL-8 during reperfusion was significantly lower in ACDD (119 ± 33% of basal) than in HBD (306 ± 238%, P  < 0.05) recipients. The paradoxical behavior of IL-8 makes it an unreliable predictor of ACDD early outcome in this transplantation model.     

大鼠心跳停搏供肝在原位肝移植术中损伤的预防

目的：探讨预防和减轻大鼠心跳停搏供肝在原位肝移植术中的损伤，以提高手术成功率。方法：雄性SD大鼠随机分为心跳停搏热缺血30min(N-30)和45min(N-45)两组;，每组分别行原位肝移植术30只次。同时，根据是否对供体手术方法进行改进又分为常规组和改良组。结果：(1)常规组和改良组的冷缺血时间分别为(70.04±1.48)和(70.36±1.42)min(P>0.05)，无肝期均为(16.40±0.73)min，肝下下腔静脉阻断时间均为(22.75±1.16)min，受体手术时间均为(90.58±3.76)min。(2)N-30和N-45常规组分别有5和9只受体术后死于原发性移植肝无功能，而改良组仅为1和2只(40%∶12%,P<0.05);(3)N-30和N-45组因术中分别出现供肝损伤致再灌注后供肝大量渗血、无肝期过长、切除受体肝脏时麻醉过深，而各有5和7，2和1，2和2只受体术后死亡。(4)N-30和N-45组术后1周存活率分别为50%和30%(P<0.05)。结论：预防心跳停搏供肝游离时损伤、供肝再灌注后渗血、无肝期过长和切除受体肝脏时麻醉过深是大鼠心跳停搏供肝原位肝移植手术成功的关键。