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.

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.

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.

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.

Acute alcohol-induced pancreatic injury is similar with intravenous and intragastric routes of alcohol administration.

OBJECTIVES: Five percent of alcoholics develop an acute pancreatitis (AP). The mechanism leading to pancreatic injury is not yet understood. Microcirculatory disorders seem to play a pivotal role. The objective of this study was to compare alcoholic pancreatic injury in response to intravenous and intragastric routes of alcohol administration. METHODS: Alcohol was applied in rats intravenously (IV) or gastric via a surgical implanted feeding tube (IG). Serum alcohol concentration was maintained between 1.5‰ and 2.5‰. Four subgroups (n = 6/group) were examined in the IV/IG arm and compared with healthy controls. Pancreatic microcirculation, enzyme levels, and morphological damage were assessed after 3, 6, 12, and 24 hours. RESULTS: Microcirculatory analysis showed significantly disturbed pancreatic perfusion and increased adherent leukocytes in IV and IG animals. In IV and IG groups, serum amylase was increased without morphological signs of AP compared with healthy controls. CONCLUSIONS: Alcohol application does not induce AP in rodents, but impairs pancreatic microcirculation irrespectively of the application route. Intravenous application is commonly used and shows no disadvantages compared with the physiological intragastric application form. Therefore, the intravenous route offers a valid model, which mimics the physiological process for further studies of the influence of acute alcohol intoxication on the pancreas.

Adipose derived stem cells protect skin flaps against ischemia-reperfusion injury.

BACKGROUND: Advances in the treatment of ischemia- reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. We examined the capability of adipose derived stem cells (ADSCs) to protect tissue against IRI using an extended inferior epigastric artery skin flap as a flap ischemia- reperfusion injury (IRI) model. METHODS: ADSCs were isolated from Lewis rats and cultured in vitro. Twenty- four rats were randomly divided into three groups. Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and group III (ADSCs treatment) underwent 3 h of ischemic insult. During reperfusion group III was treated by intravenous application of ADSCs and group II was left untreated. Five days postoperatively, flap survival and perfusion were assessed. Microvessel density was visualized by immunohistochemistry and semi- quantitative real-time polymerase chain reaction addressed differential gene expression. RESULTS: Treatment with ADSCs significantly increased flap survival (p<0.001) and flap perfusion (p<0.001) when compared to the control group II. Microvessel- density in ADSCs treated group was not significantly increased in any group. No significant differences showed the comparison of the experimental group III and the sham operated control group I. ADSCs treatment (Group III) was accompanied by a significantly enhanced expression of pro-angiogenic and pro-inflammatory genes. CONCLUSION: Overall, our study demonstrates that ADSCs treatment significantly enhances skin flap survival in the aftermath of ischemia to an extent that almost equals surgical results without ischemia. This effect is accompanied with a pronounced and significant angiogenic response and an improved blood perfusion.

Combined effects of chronic and acute ethanol on pancreatic injury and microcirculation.

OBJECTIVES: Aim of the study was to investigate pancreatic microcirculatory and histopathological changes in rats after chronic ethanol liquid diet feeding. METHODS: To investigate the influence of chronic alcohol exposition (CAE) on the pancreas, rats were fed with either Lieber-DeCarli (LDC) control diet or LDC alcohol diet for 2, 4, or 6 weeks and received additionally an acute ethanol administration (AEA) for 90 minutes. Intravital microscopy was performed at baseline, 45 minutes, and 90 minutes after starting AEA. Pancreatic perfusion and leukocyte adhesion were assessed, and pancreatic damage was evaluated by histology. RESULTS: Capillary perfusion was reduced in all animals after AEA. After previous CAE, there was a significant increase in leukocyte adhesion compared to control groups (P < 0.05). Most importantly, leukocyte adhesions were already increased at baseline after CAE and before the acute bolus was infused (P < 0.05). Moreover, only animals that received LDC alcohol diet developed mild histological changes consisting of pancreatic edema and vacuoles, whereas those that received AEA alone did not. Histological changes and cytokine levels correlated with the duration of prior CAE. CONCLUSIONS: Long-term alcohol intake activates endothelium and sensitizes the pancreas for inflammatory reactions leading to an increased likelihood of a clinically evident episode of acute pancreatitis.

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.

Extracorporeal shock wave treatment protects skin flaps against ischemia-reperfusion injury.

Advances in the treatment of ischemia-reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. Using an extended inferior epigastric artery skin flap as a flap ischemia-reperfusion injury (IRI) model, we examined the capability of extracorporeal shock wave treatment (ESWT) to protect tissue against IRI in a rat flap model. Twenty-four rats were used and randomly divided into three groups (n=8 for each group). Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and Group III (ESWT) underwent 3h of ischemic insult. During reperfusion Group III was treated with ESWT and Group II was left untreated. Histological evaluation was made to investigate treatment induced tissue alterations. Survival areas were assessed at 5d postoperatively. Skin flap survival and perfusion improved significantly in the ischemic animals following ESWT (p<0.001, respectively). The tissue protecting effect of ESWT resulted in flap survival areas and perfusion data equal to non-ischemic, sham operated flaps. In line with the observation of better flap perfusion, tissue from ESWT-treated animals (Group III) revealed a significantly increased frequency of CD31-positive vessels compared to both the ischemic (Group II; p=0.003) and the non-ischemic, sham operated control (Group I; p<0.005) and an enhanced expression of pro-angiogenic genes. This was accompanied by a mild suppression of pro-inflammatory genes. Our study suggests that ESWT improves flap survival in IRI by promoting angiogenesis and inhibiting tissue inflammation. The study identifies ESWT as a low-cost and easy to use technique for surgical techniques that aim at reducing ischemia-reperfusion-induced tissue injury.

Shock wave treatment in composite tissue allotransplantation.

INTRODUCTION: Composite tissue allotransplantation is a newly emerged field of transplantation. Shock wave technology has already been used in the treatment of urologic and orthopedic disorders. Recent studies demonstrated a suppression of the early proinflammatory immune response. METHODS: 50 allogeneic hindlimb transplantations were performed on rats in 5 different groups. Group A (n = 10), (Lewis → Brown-Norway) received 500 impulses of extracorporeal shock wave. Groups B, C, D, and E served as control groups with group B (n = 10) receiving no immunosuppression, group C (n = 10) receiving FK506 and prednisolone, group D (n = 10) receiving no immunosuppression with isograft transplantations (Brown-Norway → Brown-Norway) and group E receiving 500 impulses of extracorporeal shock wave on the contralateral hindlimb. RESULTS: Rejection of the allogeneic hindlimb occurred on average 7.12 days after transplantation in group A (extracorporeal shock wave). Rejection was significantly delayed compared to the control groups B (no immunosuppression) and E (contralateral hindlimb), where rejection of the allogeneic hindlimb occurred on average 5.49 and 5.6 days after transplantation (t test, P < .01). No rejection was seen in groups C and D. CONCLUSIONS: For the first time, shock waves have been applied in a composite tissue allotransplantation model and resulted in a significant immunosuppressive effect. These promising first results have showed that shock wave treatment is clinically relevant in composite tissue allotransplantation and justify subsequent research to improve the experimental and clinical outcome.

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.

Comparison of extracorporal shock wave pretreatment to classic surgical delay in a random pattern skin flap model.

BACKGROUND: Extracorporal shock wave therapy has a significant positive effect on rescuing the ischemic zone of flap tissue if applied immediately after surgical intervention. The purpose of this study was to determine the potential preoperative effect of noninvasive extracorporal shock wave therapy to precondition flap tissue compared with the well-established surgical delay procedure. METHODS: Thirty-two male Wistar rats were randomized into four groups, and an oversized, random-pattern flap was raised in each animal. In group D7, a surgical delay was carried out 1 week before full flap harvest. In group E7, the whole flap area was treated with extracorporal shock wave therapy to induce mechanical delay. Group E7D7 was treated preoperatively with a combination of surgical delay and extracorporal shock wave therapy. Group C constituted the control group, in which the skin flap was harvested without any prior intervention. Seven days after flap harvest, flap survival, perfusion, microvessel density, and vascular endothelial growth factor concentration were assessed. RESULTS: Flap survival, perfusion, and microvessel density were significantly increased in the delay group (group D7) and the extracorporal shock wave therapy group (group E7) compared with the control group (group C). Combining both pretreatments (group E7D7) did not have a favorable cumulative effect. Vascular endothelial growth factor expression was not significantly increased in any group. CONCLUSIONS: Although not superior to surgical delay, the authors see many advantages of extracorporal shock wave therapy; it is noninvasive, easily applicable, less time- consuming, and less expensive. Thus, it may constitute an alternative procedure in clinical situations that warrant a noninvasive, fast, and easily applicable treatment.

Preoperative shock wave treatment enhances ischemic tissue survival, blood flow and angiogenesis in a rat skin flap model.

INTRODUCTION: Extracorporeal shock wave treatment (ESWT) has recently been shown to enhance skin flap survival. However, the bio-mechanisms operating during preoperative ESWT remain unclear. The aim of our study was to investigate whether preoperative ESWT can improve blood flow in ischemic skin flaps and to elucidate its possible mechanisms. METHODS: 14 male-rats were randomized into two groups and an oversized ventral random-pattern flap was raised. Experimental group received extracorporeal shock-wave treatment (ESWT) with an energy of 500 mJ/mm(2) seven days prior to total flap elevation, while control group received no treatment prior to total flap elevation. Seven days postoperatively, surviving flap area, perfused flap area, microvessel density and VEGF concentration were measured. RESULTS: Surviving flap area (59.43 ± 14.72 % to 42.71 ± 10.75 %, p = 0.026), perfused flap area (62.00 ± 8.58 % to 45.14 ± 10.50 %, p = 0.007), microvessel density (18.13 ± 5.11 to 11.09 ± 1.12, p = 0.016) and VEGF to total protein ratio (0.2107 ± 0.0935 to 0.0123 ± 0.0069, p = 0.008) were significantly elevated in the ESWT group. CONCLUSION: Preoperative ESWT can improve skin flap survival through enhanced topical blood perfusion and neovascularization via elevation of angio-active factors.

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.

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.

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.

Effects of gadolinium chloride and glycine on hepatic and pancreatic tissue damage in alcoholic pancreatitis.

OBJECTIVE: Systemic complications in alcoholic pancreatitis are supposed to be aggravated by inflammatory liver damage. Resident macrophages including hepatic Kupffer cells play a pivotal role in mediating systemic complications in severe necrotizing pancreatitis (SNP). The aim of this study was to evaluate the effects of Kupffer cell inhibition on the inflammatory liver damage in experimental alcoholic pancreatitis. METHODS: Rats were fed with either alcohol or control diet for 6 weeks before induction of SNP. Animals were allocated into 4 groups: healthy controls, controls with SNP, SNP with gadolinium chloride or glycine (permanent vs temporary inhibition of hepatic Kupffer cells) prophylaxis. Hepatic microcirculation and morphologic damage of the liver and pancreas were assessed. RESULTS: Alcohol feeding and SNP increased hepatic and pancreatic injury compared with SNP alone. Gadolinium chloride and glycine improved hepatic microcirculation. In contrast, pancreatic and hepatic morphological damage was reduced by gadolinium chloride but not by glycine. CONCLUSIONS: Alcohol exposure aggravates hepatic and pancreatic injury in SNP. Gadolinium chloride reduces both microcirculatory and morphological damage, whereas glycine did not improve histological damage.

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.

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.