The combination of mitomycin-induced blood cells with a temporary treatment of ciclosporin A prolongs allograft survival in vascularized composite allotransplantation.

BACKGROUND: Vascularized composite allotransplantation (VCA) is a rapidly expanding field of transplantation and provides a potential treatment for complex tissue defects. Peripheral blood mononuclear cells (PBMCs) shortly incubated with the antibiotic and chemotherapeutic agent mitomycin C (MMC) can suppress allogeneic T cell response and control allograft rejection in various organ transplantation models. MMC-incubated PBMCs (MICs) are currently being tested in a phase I clinical trial in kidney transplant patients. Previous studies with MICs in a complex VCA model showed the immunomodulatory potential of these cells. The aim of this study is to optimize and evaluate the use of MICs in combination with a standard immunosuppressive drug in VCA. METHODS: Fully mismatched rats were used as hind limb donors [Lewis (RT11)] and recipients [Brown-Norway (RT1n)]. Sixty allogeneic hind limb transplantations were performed in six groups. Group A received donor-derived MICs combined with a temporary ciclosporin A (CsA) treatment. Group B received MICs in combination with a temporarily administered reduced dose of CsA. Group C served as a control and received a standard CsA dose temporarily without an additional administration of MICs, whereas Group D was solely medicated with a reduced CsA dose. Group E received no immunosuppressive therapy, neither CsA nor MICs. Group F was given a continuous standard immunosuppressive regimen consisting of CsA and prednisolone. The endpoint of the study was the onset of allograft rejection which was assessed clinically and histologically. RESULTS: In group A and B, the rejection-free interval of the allograft was significantly prolonged to an average of 23.1 ± 1.7 and 24.7 ± 1.8 days compared to the corresponding control groups (p < 0.01). Rejection in groups C, D, and E was noted after 14.3 ± 1.1, 7.8 ± 0.7, and 6.9 ± 0.6 days. No rejection occurred in control group F during the follow-up period of 100 days. No adverse events have been noted. CONCLUSION: The findings of this study show that the combination of MICs with a temporary CsA treatment significantly prolongs the rejection-free interval in a complex VCA model. The combination of MICs with CsA showed no adverse events such as graft-versus-host disease. MICs, which are generated by a simple and reliable in vitro technique, represent a potential therapeutic tool for prolonging allograft survival through immunomodulation.

Local administration of Mitomycin-C-Treated peripheral blood mononuclear cells (PBMCs) prolongs allograft survival in vascularized composite allotransplantation.

BACKGROUND: VCA offers a potential treatment for extensive tissue defects. First results of systemic administration of Mitomycin C-treated PBMCs in VCA demonstrated a significant prolongation of allograft survival. The aim of this study is to evaluate if local administration of MMC-PBMCs prolongs allograft survival in allogeneic hind limb transplantations of the rat. METHODS: Sixty allogeneic hind limb transplantations in the rat were performed in six groups. Lewis rats (LEW) were used as hind limb donors and Brown-Norway rats (BN) as recipients. Animals in group A received donor-derived MMC-treated PBMCs locally (i.m.). Group B received no immunosuppressive therapy, group C received a standard immunosuppressive regime consisting of FK506 and Prednisolon, group D (BN to BN) comprised isograft transplantations without immunosuppressive treatment, group E received non-treated PBMCs (i.m.) and group F received phosphate buffered saline (PBS) without cells. The transplanted hind limbs were assessed for color, edema, skin, hair condition, and consistency of the thigh every 8 hours. RESULTS: Rejection in group A was delayed to an average of 7.2 ± 0.6 days. Survival times were significantly prolonged (P < 0.01) compared to control groups B, E, and F (5.5 ± 0.7, 5.8 ± 0.7, and 5.7 ± 0.5 days). Control groups C and D showed no signs of rejection. CONCLUSION: The findings of this study show that local administration of MMC-PBMCs has no side effects and significantly extends allograft survival. Further experiments with MMC-PBMCs treatments repeated at different time-points and being added to low dose immunosuppressive protocols need to be performed to improve experimental and eventually clinical outcome after VCA. © 2015 Wiley Periodicals, Inc. Microsurgery 36:417-425, 2016.

Targeting of pancreatic and prostate cancer stem cell characteristics by Crambe crambe marine sponge extract.

Cancer stem cells (CSCs) are suggested as reason for resistance of tumors toward conventional tumor therapy including pancreatic and advanced prostate cancer. New therapeutic agents are urgently needed for targeting of CSCs. Marine sponges harbor novel and undefined compounds with antineoplastic activity but their potential to eliminate CSC characteristics is not examined so far. We collected 10 marine sponges and one freshwater sponge by diving at the seaside and prepared crude methanolic extracts. The effect to established pancreatic and prostate CSC lines was evaluated by analysis of apoptosis, cell cycle, side population, colony and spheroid formation, migratory potential in vitro and tumorigenicity in vivo. While each sponge extract at a 1:10 dilution efficiently diminished viability, Crambe crambe marine sponge extract (CR) still strongly reduced viability of tumor cells at a dilution of 1:1,000 but was less toxic to normal fibroblasts and endothelial cells. CR inhibited self-renewal capacity, apoptosis resistance, and proliferation even in gemcitabine-selected pancreatic cancer cells with acquired therapy resistance and enhanced CSC characteristics. CR pretreatment of tumor cells diminished tumorigenicity of gemcitabine-resistant tumor cells in mice and totally abolished tumor take upon combination with gemcitabine. Our data suggest that CR contains substances, which render standard cancer therapy more effective by targeting of CSC characteristics. Isolation of bioactive metabolites from CR and evaluation in mice are required for development of new CSC-specific chemotherapeutic drugs from a marine sponge.

Glycine and taurine equally prevent fatty livers from Kupffer cell-dependent injury: an in vivo microscopy study.

BACKGROUND: IRI still is a major problem in liver surgery due to warm ischemia and organ manipulation. Steatosis is not only induced by diabetes, hyperalimentation, alcohol and toxins, but also chemotherapy given before resection. Since steatotic livers are prone to Kupffer cell-dependent IRI, protection of steatotic livers is of special interest. This study was designed to compare the effect of taurine and glycine on IRI in steatotic livers. MATERIALS AND METHODS: Steatosis was induced with ethanol (7 g/kg b.w.; p.o.) in female SD rats. Ten minutes after inactivation of Kupffer cells with taurine or glycine (300 mM; i.v.), left liver lobes underwent 60 minutes of warm ischemia. Controls received the same volume of valine (300 mM; i.v.) or normal saline. After reperfusion, white blood cell-endothelial interactions and latex-bead phagocytosis by Kupffer cells were investigated. Liver enzymes were measured to estimate injury. For statistical analysis, ANOVA and Student's t-test were used. RESULTS: Glycine and taurine significantly decreased leukocyte- and platelet-endothelium interactions and latex-bead phagocytosis (p < 0.05). Liver enzymes were significantly lower after glycine and taurine (p < 0.05). CONCLUSIONS: This study shows that preconditioning with taurine or glycine is equally effective in preventing injury to fatty livers most likely via Kupffer cell-dependent mechanisms.

Celecoxib enhances radiation response of secondary bone tumors of a human non-small cell lung cancer via antiangiogenesis in vivo.

PURPOSE: Cyclooxygenase-2 (COX-2) inhibitors mediate a systemic antitumor activity via antiangiogenesis and seem to enhance the response of primary tumors to radiation. Radiosensitizing effects of COX-2 inhibition have not been reported for bone metastases. Therefore, the aim of this study was the investigation of the radiosensitizing effects of the selective COX-2 inhibitor celecoxib in secondary bone tumors of a non-small cell lung carcinoma in vivo. MATERIALS AND METHODS: Human A549 lung carcinomas were implanted into a cranial window preparation in male SCID mice (n = 24). Animals were treated with either celecoxib or radiation (7 Gy single photon dose) alone or a combination of celecoxib and radiation, respectively. Untreated animals served as controls. The impact of radiation and COX-2 inhibition on angiogenesis, microcirculation, and tumor growth was analyzed over 28 days by means of intravital microscopy and histological methods. RESULTS: Monotherapies with radiation as well as celecoxib had significant antitumor effects compared to untreated controls. Both therapies reduced tumor growth and vascularization to a similar extent. The simultaneous administration of celecoxib and radiation further enhanced the antitumor and antiangiogenic effects of single-beam radiation. With the combined treatment approach, tumor vascularization and tumor size were decreased by 57% and 51%, respectively, as compared to monotherapy with radiation. CONCLUSION: The combined application of radiation therapy and COX-2 inhibition showed synergistic effects concerning the inhibition of tumor growth and tumor angiogenesis. Therefore, the combination of radiation with COX-2 inhibitor therapy represents a promising approach to improve the therapeutic efficacy of radiotherapy of bone metastases.

Optimal timing of extracorporeal shock wave treatment to protect ischemic tissue.

Enhancement of flap survival through extracorporeal shock wave treatment (ESWT) is a promising new technique; however, no attempt has been made to define the optimal time point and frequency of ESWT to optimize treatment with ESWT for ischemic indications. Twenty-eight male Wistar rats were randomized into 4 groups and an oversized, random-pattern flap was raised and reattached in place in each animal. ESWT was applied 7 days before (group E7) or immediately after the surgical intervention (group E0). The third group was treated with ESWT 7 days before and additionally immediately after the operation (group E7/0). The fourth group served as a control group and did not receive any ESWT (group C). Seven days after flap harvest the results of flap survival, perfusion, microvessel density, and vascular endothelial growth factor concentrations were assessed. Flap survival was significantly increased in all ESWT groups as compared with the control group. The groups (E7 and E0) that received ESWT pre- or postoperatively showed a significant increase in flap perfusion and microvessel density. Combined pre- and postoperative ESWT application (group E0/E7) did not demonstrate a cumulative effect in any evaluation. In this study, we were be able to prove the effectiveness of ESWT in the protection of ischemic tissue flaps. This study suggests that single postoperative application is the most efficacious protocol for clinical applications of ESWT in the treatment of ischemic tissue.

Induction of HSP70 shows differences in protection against I/R injury derived by ischemic preconditioning and intermittent clamping.

BACKGROUND: Ischemic preconditioning (IP) and intermittent clamping (IC) increase the ischemic tolerance of the liver. The underlying mechanisms are not completely understood. Heat shock proteins protect cellular integrity in stress and have been discussed as mediators in preconditioning. IP and IC in rat livers were compared with respect to HSP induction and postischemic microcirculation. METHODS: All animals were exposed to 70min of partial warm liver ischemia. Different clamping protocols were used: in control animals (C) 70min continuous ischemia was applied. IP was performed by 5min ischemia and 10min reperfusion before the 70min ischemia time. In IC-groups, ischemia time of 70min was divided into four intervals. Each group included 21 animals with 3 different reperfusion intervals; either 30min, 12 or 36h. Intravital microscopy was performed after 30min of reperfusion. AST-levels and HSP induction were analysed 90min, 12 and 36h after reperfusion. RESULTS: IP and IC significantly improved sinusoidal perfusion (IP: 83.4±2.8%; IC: 84.4±4.6% vs. C: 60.4±3.9%; p<0.001) and leucocyte adherence in sinusoids (IP: 51.9±12.0, IC: 40.9±4.7 vs. C: 90.1±17.7/mm(2) liver surface; p<0.001) and postsinusoidal venules. AST-levels were minimized in IP and IC compared to controls (12h after reperfusion: IP: 969±934U/l, IC: 675±562U/l vs. C: 2373±792U/l; p=0.004). In the course of reperfusion HSP70 protein expression doubled between 90min and 12h in IC (0.529±0.227 vs. 0.992±0.246; p<0.05) and control-groups (0.572±0.314 vs. 1.106±0.309; p<0.05) whereas it remained unchanged in the IP-group (0.437±0.383 vs. 0.412±0.439; n.s.). CONCLUSION: Microcirculation is similarly preserved by IP and IC. The early protection derived by IP prevents further induction of HSP70 in opposite to IC. Therefore, IP may offer a more comprehensive protection against I/R on a cellular and transcriptional level.

Fundamental efforts toward the development of a therapeutic cocktail with a manifold ameliorative effect on hepatic ischemia/reperfusion injury.

OBJECTIVE: Ischemia/reperfusion injury is mediated by various mechanisms. The present study was designed to evaluate the effect of a multiple pharmacological approach toward ischemia/reperfusion injury reduction. METHODS: The left liver lobe of Sprague-Dawley rats underwent normothermic ischemia for 90 minutes after a cocktail (glycine, taurine, alanine, arginine, and prednisolon) intravenous administration. Controls received normal saline. Liver injury (transaminases, histology) and cellular activation [Kupffer cell phagocytosis, production of tumor necrosis factor-alpha (TNFalpha), and prostaglandin E-2 (PGE(2))], as well as microcirculation and leukocyte-endothelial interaction (in vivo microscopy), were assessed. RESULTS: Whereas in controls a substantial increase of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase to 2,341+/-477, 1,442+/-262, and 1,973+/-73 U/L, respectively, was measured eight hours after reperfusion, the cocktail significantly reduced the increase of enzymes by 33-80% (P<0.05). Further, necrosis, index of liver damage, and index of leukocyte infiltration significantly decreased after the cocktail (P<0.05). Moreover, the cocktail improved acinar and sinusoidal perfusion, while the sinusoidal diameters, leukocyte-endothelial interaction, Kupffer cell phagocytic activity, and TNFalpha/PGE(2) serum levels were significantly reduced, the latter by 86% and to 1.64-fold, respectively. CONCLUSIONS: This study depicts that multifaceted pharmacological tackling of ischemia/reperfusion injury is feasible and protects rat liver tissue from warm ischemia/reperfusion injury.

Quantification of liver perfusion by dynamic magnetic resonance imaging: experimental evaluation and clinical pilot study.

Changes in liver microcirculation are considered essential in assessing ischemia-reperfusion injury, which in turn has an impact on liver graft function and outcome following liver transplantation (LTx). The aim of this study was to introduce dynamic magnetic resonance imaging (dMRI) as a new technique for overall quantification of hepatic microcirculation and compare it to perfusion measured by laser Doppler flowmetry (LDF; hepatic artery/portal vein) and thermal diffusion (TD). The study included 3 groups, measuring hepatic blood flow and microcirculation with the help of TD, LDF, and dMRI. In group I (9 landrace pigs; 26 +/- 5 kg), the native liver before and after partial portal occlusion was studied; in group II (6 landrace pigs; 25.5 +/- 4.4 kg), the liver 24 hours after LTx was studied; and in group III (14 patients), the liver on days 4 to 7 following LTx was studied. A close correlation was found between dMRI measurements and TD (r = 0.7-0.9, P < 0.01) in 4 defined regions of interest. Portal blood flow and partial occlusion of the portal vein were accurately detected by LDF flowmetry and correlated well with dMRI (r = 0.95, P < 0.01). In the clinical setting, representative TD measurements in segment 4b of the transplanted liver correlated well with dMRI analysis in other segments. Quantification of the portal blood flow and imaging of the whole liver could be performed simultaneously by dMRI. In conclusion, dMRI has been proved to be a sensitive modality for the quantification of liver microcirculation and hepatic blood flow in experimental and clinical LTx. It allows for a synchronous, noninvasive assessment of macrocirculation and microcirculation of the liver and could become a valuable diagnostic tool in advanced liver surgery and transplantation.

Effects of hemodilution with a hemoglobin-based oxygen carrier (HBOC-201) on ischemia/reperfusion injury in a model of partial warm liver ischemia of the rat.

BACKGROUND: Ischemia/reperfusion injury is an unavoidable complication in liver surgery and transplantation. Hemodilution with colloids can reduce postischemic injury but limits oxygen transport. Hemoglobin-based oxygen carriers have been evaluated as blood substitute and provide a plasma-derived oxygen transport. It was the aim of our study to evaluate the combined benefits of hemodilution with a better oxygen supply to reperfused liver tissue by the use of HBOC-201 (Hemopure). MATERIAL AND METHODS: A model of partial warm liver ischemia in the rat was used. One group served as untreated control, the other groups were hemodiluted either with Ringer's lactate, Dextran-70, HBOC-201 or a mixture of Dextran and HBOC-201. After reperfusion, intravital microscopy studies were done and tissue pO(2) levels and transaminases measured. Statistical analysis was done by one- and two-way ANOVA, followed by pairwise comparison. RESULTS: Hemodilution with Ringer's lactate did not show any improvement compared to the control group. Dextran and HBOC group were superior to the Ringer and control animals in all parameters studied. Leucocyte adherence in postsinusoidal venules improved from 569.03+/-171.87 and 364.52+/-167.32 in control and Ringer group to 131.68+/-58.34 and 68.44+/-20.31/mm(2) endothelium in Dextran and HBOC group (p<0.001). Concerning tissue pO(2) levels, HBOC (23.4+/-5.0 mmHg) proved to be superior to Dextran (7.9+/-4.4 mmHg; p=0.007). CONCLUSION: HBOC was equivalent to Dextran in reducing I/R injury in the liver, but improved oxygenation of postreperfusion liver tissue.

P-selectin inhibition reduces severity of acute experimental pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is characterized by disturbed pancreatic microcirculation with tissue necrosis. Platelet and leukocyte activation contributes to microcirculatory disorders and inflammatory tissue infiltration. P-selectin mediates the adhesion of both activated platelets and leukocytes to the vessel wall. The aim of this study was to investigate the effect of P-selectin inhibition by monoclonal antibodies (AB) on experimental AP. METHODS: AP was induced in rats by glycodeoxycholic acid (GDOC) intraductally and by cerulein infusion. Animals were divided into 4 groups: (1) severe AP (GDOC); (2) severe AP + platelet inhibition (GDOC + selectin AB); (3) control (Ringer); (4) control + platelet inhibition (Ringer + selectin AB). 24 h after AP induction, histology and serum (amylase, thromboxane A2) were evaluated (6 animals per group). In additional 12 animals of each group, platelet and leukocyte activation as well as erythrocyte flow patterns were evaluated by intravital microscopy 12 h after AP induction. RESULTS: AP induction caused significant tissue inflammation and necrosis with increased amylase and thromboxane levels. Prophylactic inhibition of P-selectin reduced tissue inflammation and necrosis significantly. Severe AP led to significantly more adherent platelets and leukocytes in capillaries and venules. In contrast, antibody-treated animals showed significantly reduced platelet-endothelium interaction compared with untreated AP animals. Antibody application in control animals without AP did not induce any changes compared with healthy control animals. CONCLUSION: Inhibition of P-selectin reduces tissue damage in experimental AP. This is associated with a reduction in platelet- and leukocyte-endothelium interaction and an improvement in pancreatic microcirculation.

Alcohol pretreatment increases hepatic and pulmonary injury in experimental pancreatitis.

BACKGROUND: Systemic complications including pancreatitis-associated lung injury (PALI) are critical factors that determine the outcome of severe necrotizing pancreatitis (SNP). The aim of the present study was to evaluate the role of chronic alcohol exposure on the development of PALI. METHODS: 48 rats were fed either a Lieber deCarli control or alcohol diet for 6 weeks. After completion, SNP was induced by intraductal infusion of bile salt followed by intravenous infusion of cerulein over 6 h. Control animals received i.v. Ringer's solution. Intravital microscopy of the liver was performed 6 h after induction of SNP to evaluate hepatic perfusion and leukocyte adhesion. Serum parameters, edema, inflammation, and histological changes were evaluated at 12 h. IL-6 levels were evaluated in portal venous and systemic blood as well as in pancreatic tissue homogenates. RESULTS: Alcohol pretreatment did not affect pancreatic injury in SNP. PALI was aggravated after alcohol ingestion. These animals showed increased hepatic microcirculatory disturbances, compared to SNP alone. IL-6 showed peak levels in SNP with alcohol pretreatment, although they were also elevated in SNP alone. Systemic levels of IL-6 were higher than in the portal vein. CONCLUSION: In SNP, alcoholic pretreatment increases pulmonary damage, while pancreatic injury is identical. The liver seems to participate in this effect by increased hepatic cytokine release.

Protective effects and anti-inflammatory pathways of exogenous calcitonin gene-related peptide in severe necrotizing pancreatitis.

BACKGROUND: Microcirculatory disturbances are known to play a pivotal role in the pathogenesis of severe necrotizing pancreatitis (SNP). Calcitonin gene-related peptide (CGRP) is a vasodilatatory neuropeptide with potential anti-inflammatory effects. This study characterizes the protective effects and the anti-inflammatory pathway of exogenous CGRP in SNP. METHODS: SNP was induced in rats using the glycodeoxycholic acid model. CGRP was injected prophylactically before or therapeutically after initiation of the disease. Pancreatic damage was assessed using intravital microscopy, histology, NF-kappaB p50/p65 electrophoretic mobility shift assay, serum cytokine assay and ICAM-1 immunohistochemistry at 6 or 12 h after the onset of disease. RESULTS: Pancreatic microcirculatory disturbances, nuclear NF-kappaB levels and pancreatic ICAM-1 expression were increased in SNP compared to controls. After CGRP application, microcirculatory disturbances, NF-kappaB levels and pancreatic ICAM-1 expression were attenuated compared to pancreatitis alone. Moreover, pancreatic morphologic damage was significantly reduced by both prophylactic and therapeutic application of CGRP. CONCLUSIONS: CGRP is a neuropeptide that ameliorates the development of SNP in rats and may provide new treatment options. Its anti-inflammatory effects appear to be mediated by the modulation of pancreatic microcirculation and the inflammatory cascade.

Danshen protects liver grafts from ischemia/reperfusion injury in experimental liver transplantation in rats.

Reperfusion injury remains one of the major problems in transplantation. Free radicals and disturbance of microcirculation are the supposed main contributors. Recent evidence shows that Danshen, a traditional Chinese drug used in vascular diseases, can scavenge radicals and improve microcirculation. This study investigates its effect on liver transplantation (LTx). Before organ recovery, female Sprague-Dawley rats (210-240 g) received intravenous Danshen or the same volume of Ringer solution as control. LTx was performed after 1 h of cold storage. Microperfusion, leukocyte-endothelium interaction and latex-bead phagocytosis were evaluated with in vivo microscopy. Survival, transaminases and histology were assessed. Immunohistology was used for TNF-alpha levels. anova and Fisher's exact test were employed for statistical analyses as appropriate. Survival increased from 60% in controls to 100% (P < 0.05). AST and LDH decreased from 3969 +/- 1255 U/l and 15444 +/- 5148 U/l in controls to 1236 +/- 410 U/l and 5039 +/- 1594 U/l, respectively (P < 0.05). In vivo microscopy revealed decreased leukocyte-adherence and increased blood flow velocity in sinusoidal zones after administration of Danshen (P < 0.05), while latex-bead phagocytosis was found in 60% of controls (P < 0.05). The TNF-alpha index decreased from 2.08 +/- 0.09 in controls to 1.09 +/- 0.09 (P < 0.05). This study clearly demonstrates hepatoprotective effects after experimental LTx, which can be explained via anti-oxidative effects, improved microcirculation and decreased Kupffer cell activation.

The efficiency of heart protection with HTK or HTK-N depending on the type of ischemia.

We investigated isolated guinea pig hearts (n = 121) in an ischemia/ reperfusion model with the aim to compare the efficiency of the cardioplegic solution HTK with its novel replacement HTKN. Following consolidation with Tyrode's solution, ischemia started either immediately or after preceding cardioplegia with HTK, HTKN, or modified HTK enriched with Ca. Ischemia lasted either 80 min at 30 °C, or 360 min at 5 °C, or 81 min at 30 °C with intermittent cardioplegic perfusion. During ischemia we measured intracellular calcium (iCa++) and the time of gap junction uncoupling (t-in). During reperfusion we measured the reestablishment of cell coupling (t-ret), left ventricular developed pressure (LVDP), and heart rhythm (VC-RR). In 5 °C groups, iCa++ at t-in was significantly higher than before ischemia, and longest t-in, shortest t-ret, and best VC-RR were observed after HTK-protection. Of all 30 °C groups, the intermittent group with modified HTK showed shortest t-ret, best VC-RR, and the highest LVDP. At 5 °C, HTK groups had higher LVDP than HTK-N groups, but not at 30 °C. The data suggest that the higher calcium level in the HTK-N solution improves reperfusion after short ischemia at 30 °C but for long lasting ischemia at 5 °C it is beneficial to use the HTK solution.

The effect of melatonin on hearts in ischemia/reperfusion experiments without and with HTK cardioplegia.

Our aim was to investigate if the cardioplegic solution HTK can be improved by the addition of the ROS scavenger melatonin. 158 guinea pig hearts without (UI80) or with HTK protection (HTK80) were investigated in ischemia/reperfusion experiments. Ischemia lasted 80 min at 30 °C. Melatonin was given before ischemia (UI80 + M1, HTK80 + M1) or before and after ischemia (UI80 + M2, HTK80 + M2). We measured the left ventricular developed pressure (LVDP), diastolic pressure (LVPmin), cardiac rhythm (VC-RR), time of electrical cell uncoupling (t-in) and recovery (t-ret), intracellular Ca++ [Ca++], and postischemic ROS. After 45 min reperfusion, LVDP in UI80 was significantly higher than in HTK80 (p < .01). Compared to UI80, the postischemic ROS burst was slightly smaller in HTK80 and significantly smaller in HTK80 + M1 and HTK80 + M2 (p < .05). Melatonin had no effect on LVPmin, t-in, t-ret, [Ca++], and on LVDP in groups UI80 + M1 and HTK80 + M1, improved slightly VC-RR (n. s.) but significantly decreased LVDP in the groups UI80 + M2 and HTK80 + M2 (p < .01). With melatonin we were able to attenuate the postischemic ROS burst, but the tissue damage by ROS seemed to be less important for the chosen ischemia condition because melatonin was unable to improve the functional recovery during reperfusion of HTK protected hearts.

Reduced glutathione in the liver as a potential viability marker in non-heart-beating donors.

Although the use of non-heart-beating donors (NHBD) is the oldest type of organ transplantation, the results were and still are disappointing. To consider using a liver from NHBD, it is of importance to assess the graft viability. Our aim was to assess the role of reduced liver glutathione (rGSHL) as a potential predictive marker of liver function before transplantation. Autotransplanted livers were subjected to 0, 60, and 90 minutes of ischemia in 20 pigs. We analyzed systemic cardiocirculatory parameters, bowel ischemia by endotoxin, endotoxin-neutralizing capacity, oxidative stress, hepatic perfusion parameters, liver enzymes, local bowel ischemia, and liver oxidative stress (rGSHL and oxidized glutathione in the liver). Autotransplantation was comparable to donor explantation/recipient transplantation with respect to systemic and hepatic parameters. Liver ischemia for 0, 60, and 90 minutes resulted in survival in 100% (NHBD-0), 71% (NHBD-60), and 57% (NHBD-90) of animals. Of all parameters, only hepatic microperfusion, pHi of the sigmoid colon, and bowel ischemia by endotoxin in the NHBD-90 group showed significant changes compared to NHBD-60 and control animals. Although systemic endotoxin-neutralizing capacity and total glutathione in erythrocytes levels were mainly influenced by cold perfusion, hepatic oxidative stress increased with ischemia time. The cut-off value of 11.5 ng/mmol of rGSHL could distinguish survivors from nonsurvivors, independent of the ischemia time. In conclusion, rGSHL has the potential of becoming an important viability marker, as it could predict survival in autotransplantation NHBD model regardless of the ischemia time. Further investigation to declare reasons for differing rGSHL levels within the liver is required.

The influence of selenium substitution on microcirculation and glutathione metabolism after warm liver ischemia/reperfusion in a rat model.

Ischemia/reperfusion (I/R) injury is a variable yet unavoidable complication in liver surgery and transplantation. Selenium-dependent glutathione-peroxidases (GPx) and selenoproteins function as antioxidant defense systems. One target in preventing I/R injury is enhancing the capacity of endogenous redox defense. It was the aim of this study to analyze the effects of selenium substitution on liver microcirculation, hepatocellular injury and glutathione status in a model of partial warm liver ischemia in the rat. Sodium selenite was administered in three different dosages i.v.: 0.125 microg/g, 0.25 microg/g and 0.375 microg/g body weight and compared to an untreated control group (each n=10). Intravital microscopy was performed after 70 min of partial warm liver ischemia and 90 min of reperfusion. Liver tissue and plasma samples were taken at the end of the experiment for laboratory analysis. Microcirculation improved significantly in all therapy groups in contrast to control animals. ALT levels decreased significantly whereas malondialdehyde levels remained unchanged. In liver tissue, selenium supplementation caused an increase in the amount of total and reduced glutathione without changes in oxidized glutathione. This effect is likely mediated by selenite itself and selenoprotein P rather than by modulating GPx activity. We were able to show that selenite substitution has an immediate protective effect on I/R injury after warm hepatic ischemia by acting as a radical scavenger and preserving the antioxidative capacity of the liver.

Signs of reperfusion injury following CO2 pneumoperitoneum: an in vivo microscopy study.

BACKGROUND: During laparoscopic surgery, pneumoperitoneum is generally established by means of carbon dioxide (CO(2)) insufflation which may disturb hepatic microperfusion. It has been suggested that the desufflation at the end of the procedure creates a model of reperfusion in a previously ischemic liver, thus predisposing it to reperfusion injury. METHODS: To study the effects of pneumoperitoneum on hepatic microcirculation, Sprague-Dawley rats underwent pneumoperitoneum with an intraabdominal pressure of 8 or 12 mmHg for 90 min. Subsequently, in vivo microscopy was performed to assess intrahepatic microcirculation and transaminases were measured to index liver injury. RESULTS: A CO(2) pneumoperitoneum of 8 mmHg did not change serum transaminases; however, further increase of intraperitoneal pressure to 12 mmHg significantly increased AST, ALT, and LDH measured after desufflation to almost 1.5 times as much as control values of 49 +/- 5 U/L, 31 +/- 3 U/L, and 114 +/- 12 U/L. In parallel, in all subacinar zones the permanent adherence of both leukocytes and platelets to the endothelium increased by about sixfold and threefold, respectively. Furthermore, Kupffer cells labeled with latex beads as an index for their activation were significantly increased compared to controls. CONCLUSION: This in vivo observation demonstrated traces of reperfusion injury in liver induced by the insufflation and desufflation of CO(2 )pneumoperitoneum. The clinical relevance of this finding and the issue of using hepatoprotective substances to prevent this injury should be further investigated.

Hearts during ischemia with or without HTK-protection analysed by dielectric spectroscopy.

OBJECTIVE: We investigated canine hearts during ischemia after aortic cross clamping (UI, n = 20) and after HTK-cardioplegia (HTK, n = 24) at 35 °C, 25 °C, 15 °C, and 5 °C with the aim to compare tissue changes caused by the activity of anaerobic metabolism(AAM), cell membrane destruction(CD), and gap junction uncoupling(GJU). APPROACH: We measured continuously the complex dielectric spectrum(DS), ATP- and lactate content, extracellular pH, and rigor contracture. To identify changes in DS caused by AAM, CD, and GJU we performed additional experiments on the gap junction-free skeletal muscle. We used heart model simulations to calculate the effect of temperature. MAIN RESULTS: AAM affected the DS at 10 MHz and we found a strong correlation between DS and the proton concentration with a maximum of DS at 10 mmol g-1 dry weight in ATP-concentration. The time of GJU was detected by a characteristic increase in DS and CD by a characteristic decrease at 13 kHz. In comparison to UI, GJU, AAM and CD were delayed by HTK and by hypothermia, indicating a minimization of energy consumption and an improved preservation of tissue structure. SIGNIFICANCE: The novel findings were that in UI at 5 °C GJU occurred earlier and AAM remained constant, indicating a less effective preservation in UI by deep hypothermia in contrast to HTK.